Molecular characterization of foreign and domestic isolates of Rhizoctonia solani for the biological
control of leafy spurge Euphorbia esula
by Deanna Lynne Nash
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in
Microbiology
Montana State University
© Copyright by Deanna Lynne Nash (1998)
Abstract:
Fungal isolates belonging to the genus Rhizoctonia are often very diverse and difficult to characterize.
The most recognized method for identification and characterization is anastomosis testing, and more
recently molecular characterization using regions of ribosomal RNA genes. The present study sought to
determine the phylogenetic relationship among foreign and domestic Rhizoctonia isolates being
screened as possible biocontrol agents of Euphorbia esula. Restriction enzyme analysis (REA) of a 28s
rDNA region, and sequence data of internal transcribed spacer (ITS) regions were used to infer
phylogeny. The results obtained from REA and from the sequence data of ITS regions are similar, and
the results of both methods indicate that several foreign isolates are very similar to domestic isolates. MO LECULAR CHARACTERIZATION O F FOREIGN A N D DOMESTIC ISOLATES OF
RHIZ0CT0N1A SOLANI F O R TH E BIOLOGICAL C O N T R O L O F LEAFY SPURGE
EUPHORBIA ESULA
by
Deanna Lynne Nash
A thesis submitted in partial fulfillment
o f the requirements fo r the degree
of
Master o f Science
, in
Microbiology
M O N T A N A STATE UNIVERSITY - BO ZEM AN
Bozeman, Montana
May 1998
APPROVAL
O f a thesis submitted by
Deanna Lynne Nash
This thesis has been read by each m em ber o f the thesis committee and has been found to
be satisfactory regarding content, English usage, format, citations, bibliographic style, and con­
sistency, and is ready fo r submission to the college o f graduate studies.
Dr. Joan M. Henson
(Signature)
(Date)
Approved fo r the Department o f Microbiology
Dr. Keith Cooksey
(Signature;
Approved fo r the College o f Graduate Studies
Dr. Joseph J. Fedock
(Date)
STATEMENT OF PERMISSION T O USE
In presenting this thesis in partial fulfillment o f the requirements fo r a masters degree at
Montana State University -Bozeman, I agree that the library shall make it available to bor­
rowers under the rules o f the library.
If I have indicated my intention to copyright this thesis by including a copyright notice
page, copying is allowable only fo r scholarly purposes, consistent w ith “fair use" as prescribed
in the U. S. Copyright Law. Request for permission fo r extended quotation from o r repro­
duction o f this thesis in whole o r in parts may be granted only by the copyright holder.
Date
/9?y
iv
I dedicate this work to my family,
for their patience, because some things...
arrive in their Own mysterious hour,
on their own mysterious terms.
ACKNO W LEDGEM ENTS
I graciously thank Dr. Joan Henson fo r inviting me into her lab and fo r sharing her
knowledge w ith me. This cooperative project, funded by the USDA1w ould not have been
possible w ith o ut her o r w ithout the support from Dr. Tony Caesar, Dr. RC. Quimby, and Dr.
Nina Zidack o f the USDA. Results o f the anastomosis grouping and all o f the Rhizoaonia iso­
lates used in this study w ere kindly provided by Dr. Tony Caesar.
I am grateful fo r the good company I kept in the lab: Dr. Barb Frederick, Dr. Tresa
Goins, Dr. TheCan Caesar, .Anastassia Litvintseva, Dr. Bill Edens, Cathy Raven, Kathy
Sheehan, and last but not least, W hisper Alexander.
I extend my gratitude to Dr. Cliff Bond, Bridgid Irvine, and my friends from insect
pathology: N orm a Irish, Elaine O m a1Annie Peckham-DeMeijl Jennie Birdsall, Victoria
Golding, Mary Swan, Ellen Palmer, Rhona M arietta, Suzanne W lson, and Ty Engle.
Many thanks to the Microbiology staff and graduate students: Kathy Billings-Sandoval,
Heather Edens, Maria Kohler, Robin W iliams, W endy Cochran, Karen X u, Mike Ferris, Ace
Baty, Eric Kern, Stacey Anderson, Dave Davidson, David Walker, Fintan Van O m m en Kloeke1
Elinor Pulcini, Thane Fbpke, Maiko Papke, Dm itri Kazmin, Scott Kobayashi, and Tom Foubert.
Special thanks to Chuck, Katie, Heidi, M om and Dad, Jeanne, Rick and Chantelle,
Rickey, Ashley, Huckleberry, Georgia, Boone, Rosie, Tucker, Spike, Wally, Molly, and Slim fo r
being there.
vi
TABLE OF C O N TE N TS
Page
Chapter
1.
2.
3.
4.
M O LECULAR CHARACTERIZATION OF RHIZOCTONIA
SOLANI FO R T H E BIOLO G ICAL C O N T R O L OF LEAFY
SPURGE EUPHORBIA ESU LA.................................................................
The Plant Euphorbia e s u la .................................................................
Conventional C ontrol o f Euphorbia e s u la .......................................
Rhizoctonia solani as a Biological C ontrol A g e n t.............................
Classification o f Rhizoctonia solani Com plex ..................................
Hyphal Anastomosis Grouping ........................................................
Systematics o f Rhizoctonia.................................................................
C O LLE C TIO N OF FOREIGN A N D DOMESTIC RHIZOCTONIA
ISOLATES FOR MOLECULAR CHARACTERIZATION ...................
In tro d u ctio n ............................................................ ............................
Field Materials and Methods ............................................................
Results .................................................................................................
RESTRICTION ENZYME ANALYSIS T O CREATE A RESTRICTION
FRAGMENT LENGTH POLYMORPHISM LIBRARY ........................
In tro d u ctio n ....................................................................................... ..
Materials and M e th o d s ......................................................................
Fungal Cultures ...........................................................................
D N A P re pa ra tio n ........................................................................
Polymerase Chain R eaction........................................................
Restriction Enzyme Analysis o f PCR Products ........................
Results .................................................................................................
D iscussion............................................................................................
2
3
4
5
7
IO
IO
IO
II
14
14
15
15
15
16
16
22
22
PHYLOGENETIC RELATIONSHIPS INFERRED FROM SEQUENCES
O F INTERNAL TRANSCRIBED SPACER REGIONS A N D .
Rl BOSOMAL D N A ......................................................................................................26
In tro d u ctio n .................................................................................. ; ..................... 26
Materials and M e th o d s .............. ...........................................................................26
Fungal Cultures ............................................................
26
D N A P re pa ra tio n ............................................................................................ 27
Polymerase Chain R eaction..................................................
27
Automated Sequencing.................................................................................. 27
A ignm ent o f Sequences .................................................................................28
Bootstrapping.............................................................................
28
Results .................................................................................................................... 29
D iscussion............................................................................................................... 30
vii
TABLE OF C O N TE N TS (continued)
C hapter
EPILOGUE
Page
............................................................................................................................34
REFERENCES C I T E D ............................................................................................................... 35
APPENDICES ......................................................................
Appendix A - RFLP Fingerprints.................................................................................. 40
Appendix B - Collection o f Distance Matrix C alculations....................................... 47
Appendix C - REA Distance Matrix V a lu e s ..............................................................115
Appendix D - Aigned Sequences ............................................................................ 120
Appendix E - Matrix Depicting the N um ber o f Nucleotide Matches
Between Each Isolate O u t o f 400 Alignable Sites ......................... 139
viii
LIST OF TABLES
Table
1.
Page
Classification o f hyphal anastomosis reactions using the criteria o f
MacNish et al. to delimit anastamosis groups (AG) in Rhizoctonia solani ..........
6
Oligonucleotides prim er sequences used to amplify rR N A gene
o f Rhizoctonia solani Isolates . ................................................................................
8
3.
Identification and features o f domestic isolates o f Rhizoctonia solani .................
12
4.
Identification and features o f foreign isolates o f Rhizoctonia s o la n i......................
13
5.
Genotype classes and restriction phenotypes o f the domestic isolates ............
19
6.
Genotype classes and restriction phenotypes o f the foreign is o la te s ....................20
2.
ix
LIST OF FIGURES
Figure
Page
1.
Schematic representation o f the internal transcribed spacer regions...................
2.
RFLP fingerprints produced by the digestion o f LROR1 LR? amplified
rD N A w ith restriction enzyme Hha I analyzed on a 4% agarose gel.................. 17
3.
Calibration curve based on the I kb D N A ladder fo r the RFLP fingerprint
shown in Figure 2 ........................................................................................................
9
18
4.
Diagram o f the calculation cell used to evaluate the Distance Matrix.................. 21
5.
PCR products o f rD N A amplified by restriction enzymes LROR and LR?.
6.
N eighbor joining phenogram o f R. solani based on restriction enzyme
analysis..............................................................................................................................25
7.
Neighbor joining free fo r Rhizoctonia solani inferred from 400 alignable sites
o f rD N A sequence data.................................................................................................32
8.
Bootstrap consensus tree generated from 100 bootstrap replications o f
400 alignable sites o f rD N A sequence data................................................................33
. . 24
ABSTRACT
Fungal isolates belonging to the genus Rhizoctonia are often very diverse and difficult
to characterize. The most recognized method fo r identification and characterization is anas­
tomosis testing, and m ore recently molecular characterization using regions o f ribosomal
R N A genes. The present study sought to determine the phylogenetic relationship among
foreign and domestic Rhizoctonia isolates being screened as possible biocontrol agents o f
Euphorbia esula. Restriction enzyme analysis (REA) o f a 28s rD N A region, and sequence
data o f internal transcribed spacer (ITS) regions w ere used to infer phylogeny. The results
obtained fro m REA and fro m the sequence data o f ITS regions are similar, and the results o f
both methods indicate that several foreign isolates are very similar to domestic isolates.
CHAPTER I
M OLECULAR CHARACTERIZATION OF RHIZOCTONIA SOLANI FO R TH E
BIOLO G ICAL C O N T R O L OF LEAFY SPURGE EUPHORBIA ESULA: IN T R O D U C T IO N
Economic losses based on the infestation o f the com m on weed Euphorbia esula (E.
esula) alone are estimated at m ore than $ I OO million in the United States (5). Due to the
fact that conventional methods fo r controlling E. esula (leafy spurge) are extrem ely costly and
that it is difficult to achieve long te rm control, w e are looking at alternative solutions such as
biological control. The biological control agent that w e are particularly interested in is the
soil-borne fungus Rhizoctonia solani (R. solani) due to its ability to decrease stands o f E. esula
(8). In o rd e r to consider the release o f this agent as a biological control agent, it must be
better characterized using molecular tools.
The Plant Euphorbia esula
The perennial plant leafy spurge, E. esula, belonging to the family Euphorbiacea, is a
plant species native to Eurasia. The introduction o f E. esula to the United States from
Eurasia, occurred in approximately 1827 through the importation o f impure seed (37). Leafy
spurge is present on all continents with the. exception o f Australia (23), but only in N o rth
America, in a region encompassing southern Canada and the north central United States, is
the presence o f E. esula considered a serious problem. In these areas alone, nearly 2.5
2
million acres are infested by £. esula (23,37). The infestation chokes o u t native plant and
grass species, thereby depleting valuable grazing areas that wildlife and livestock depend on
fo r survival. £. esula is considered to be an irritant foodstuff fo r animals due to a white milky
latex that is present in all parts o f the plant. The latex, which seals any wounds that the plant
may endure, can cause blisters and dermatitis in both man and cattle, as well as serve as a
m outh and digestive tract irritant leading to scours and death in cattle (23, 28). Economic
losses due to the infestation o f £. esula are estimated at more than $ 100 million in the affect­
ed states (5).
The flowers o f this plant are small, yellowish-green and arranged in bountiful small
clusters. The plant itself may reach three feet in height and reproduce by either ro o t stalks
o r seed. The seeds o f £. esula are oblong shaped, grayish to purple in co lo r and are con­
tained in a tri-compartmental capsule that explodes when dry, projecting seeds as far away as
fifteen feet from the plant. Once established, £. esula is very difficult to control due to it’s
extensive ro o t system, w ith ro o t depths exceeding fourteen feet, and extrem ely hardy seeds,
able to germinate after lying dorm ant in soils fo r up to eight years (23,28,37).
Conventional Control o f Euphorbia esula
It is extremely difficult to achieve long-term control o f £. esula. The most appropri­
ate control o f £. esula is assessed on a case by case basis. The options include chemicals,
cultivation, sheep grazing, insects and pathogens, o r a combination o f these techniques used
over a long period o f time. The tw o most com m only used chemicals to control £. esula are
the herbicides 2,4-D , dicamba (brand name BanveI(D) and picloram (brand name
lb r d o n " ^ ) (23), Both broadleaf herbicides w o rk well, but application is prohibited if £.
3
esula occurs around water. Cultivation practices, such as cropping, can also diminish £. esula
populations, but may not be a practical choice fo r pasture and rangeland situations. The use
o f sheep grazing does not eradicate £. esula, but provides a reduction in stands in addition to
being a well suited control measure along waterways. The use o f insects and pathogens fo r
the biological control o f £. esula is another measure that is effective, but generally takes
longer to obtain visible results ( 5 ,16,23,28). The te rm biological control agent encompasses
any native biotic factors that may suppress the population o f another organism, in this case,
£. esula. Currently the trend is to implement ecologically sound biological control measures
to achieve long-term control o f £. esula.
Rhizoctonia solani as a Biological Control Agent
In the search fo r biological control agents, an increasing num ber o f fungi are being
collected and screened fo r their potential use against plant pathogens and weeds. However,
before the release .of such agents can be considered, a responsible assessment o f the risks
involved must be made. For imported pathogens, quarantine guidelines, such as extensive
host range, pathogenicity testing and the development o f genetic markers, which allow char­
acterization and monitoring o f the released biological control agent, must be met (32).
Strains o f Rhizoctonia solani (R. solani) are among the few pathogens in the U.S. that
are capable o f reducing £. esula populations naturally and through field applications, as a bio­
logical control agent. Reld observations, where R. solani is involved in abating populations o f
£. esula, confirm that surrounding grasses are largely unaffected by strains o f R. solani that are
pathogenic to leafy spurge (8). Observations by Caesar et al. (IO ) are that diseased plants o f
Euphorbia in Eurasia are pervasive and may provide a larger pool o f highly virulent, well
adapted, and narrow host range strains fo r the control o f £. esula. Greenhouse tests
■4
conducted by Caesar et al. ( I I) using Fusarium species show that the onset o f symptoms
was in general m ore rapid using foreign strains than domestic strains. As found earlier, the
m ost virulent domestic strain's pathogenic to £. esu/o w ere in association w ith ro o t damage
caused by insect biological control agents. Because Fusarium spp and Rhizoctonia spp both
reduce stands o f £, esula, and insect associated Fusarium spp collected in Eurasia are m ore
effective at causing disease due to a m ore rapid onset o f symptoms, insect associated foreign
strains o f R solani may also be excellent candidates as biological control agents o f this noxious
weed (1 0 ,1 I). The ideal R solani candidate selected fo r biological control is one that exhibits
aggressiveness towards £. esula while maintaining a narrow host range.
Classification o f the Riizoctonia solani Complex
Fungi named R solani occur w orld-w ide, are economically im portant plant
pathogens, and may actually include several different species (21). These plant pathogens
have an extended host range that infect over 150 plant species, including agriculturally im por­
tant crops as well as weed species. R solani causes a variety o f diseases including ro o t rot,
cankers, damping-off, fruit decay and foliage disease. These fungi do not produce spores,
and therefore must be identified on the basis o f their mycelial characteristics (2,26).
Historically, fungi classified as Rhizoctonia species have been lumped together and
classified as such due to their lack o f distinctive taxonomic features (24,34). W ith this classifi­
cation scheme, it is not surprising that Rhizoctonia species are extrem ely diverse and may
belong to several different orders o f basidiomycetes as well as ascomycetes. Because o f the
importance o f numerous Rhizoaonia species as plant pathogens, an assortment o f practical
approaches have been developed fo r identifying these species. These approaches are need­
ed because conventional taxonom ic measures are difficult to apply to Rhizoaonial due to its
5
high levels o f phenotypic variation and the inability o f most strains to fruit in culture (34).
Isolates o f R. solani vary greatly in their cultural appearance, growth characteristics and patho­
genicity towards plants as well as virulence (20). Tb date, the most unambiguous method fo r
the characterization o f Rhizoctonia is based on the occurrence o f hyphal cell wall fusion
between related isolates, and is called anastomosis grouping..
Hvphal Anastomosis Grouping
In atypical anastomosis reaction test, tw o different fungal isolates are placed on agar
near each oth e r but not touching. Isolates belonging to the same anastomosis group (AG)
are generally attracted to each other, evident by hyphal growth tow ard each other, that leads
to hyphal fusion and the killing reaction (35). Isolates belonging to different AGs typically do
not undergo hyphal fusion w ith each other, and isolates belonging to the same AG do not
always anastomose w ith each other at equal frequencies. Some isolates cannot be assigned
to an AG because they have not been observed to anastomose with any representatives o f
known AGs (24).
A t least 12 AGs have been delineated within the Rhizoctonia solani com ­
plex, and in practice, when an isolate is unable to anastomose w ith existing representa­
tives, it is assigned to a new AG. Table I lists the classification criteria o f hyphal anastomosis
reactions o f MacNish et al. Even with such straightforward criteria to follow, determining the
anastomosis group o f an R. solani isolate can be very difficult. Environmental conditions such
as humidity, temperature, light, and nutritional requirements need to be perfect fo r hyphal
fusion to occur (13).
An intraspecific group is a further subdivision o f isolates within an AG that anasto­
mose w ith each other more frequently, o r else possess other unique, distinguishing charac­
ters. Such distinguishing characteristics may include host-range specificity, nutrient utilization
6
o r genetic differences based on molecular markers (20,34).
Though the AG classification scheme described allows a method fo r dividing R. solani
isolates into m ore distinct groups, it does not offer other significant pieces o f information such
as distinguishing pathogenic isolates from nonpathogenic isolates. AG grouping as an indica­
to r o f genetic relatedness has been substantiated by D N A /D N A hybridization (34), though
the mechanisms o f anastomosis are not fully understood (24), which perpetuates an illdefined Rhizoctonia solani complex.
Table I , Classification o f hyphal anastomosis reactions using the criteria o f MacNish et al. to
delimit anastamosis groups (AG) in Rhizoctonia solani ( 13).
Reaction
Phenotype
Nature o f genetic relationship
between isolates
CO: N o interaction
Hyphae grow past each other,
no recognition
Isolates have no genetic relation­
ship and belong to different AG
C I : Hyphal
contact only
N o evidence o f wall o r m em ­
brane contact, reaction may o r
may not be accompanied by cell
death
Isolates have a distant genetic
relationship and belong to either
the same o r different AG
C2: Killing reaction
Wall fusion (anastomosis) evi­
dent, w ith cell death o f anasto­
mosing and adjacent cells,
somatic incompatibility response
often macroscopically visible
Isolates represent genetically dis­
tinct individuals that belong to
the same AG
C3: Perfect fusion
Wall and membrane fusion evi­
dent, point o f anastomosis not
Isolates are genetically identical
o r closely related, individuals
belong to the same AG and may
represent clones
clearly visible, cell death absent
7
Svstematics o f Rhizoctonia
The te rm systematics is the comparative study o f biotic diversity at any level ( 18).
Systematics o f Rhizoctonia is hindered by the ambiguity and shortage o f credible, m orphologi­
cal characteristics to soundly characterize this genus. Therefore molecular approaches offer a
new avenue fo r the characterization o f Rhizoctonia. Looking at molecular data can yield
much information fo r establishing classification systems'and phylogenetic relationships at the
species level (34). In particular, conserved evolutionary markers, such as the genes coding
fo r ribosomal RNA, appear to be appropriate fo r comparisons o f evolution at the species
level because the rR N A genes evolve at a slower rate than other parts o f the genome
(1,6,35).
Several oth e r properties o f the rR N A gene make this gene a useful launching point
fo r comparative analysis. Ribosomal D N A genes in basidiomycetes are tandemly repeated
multigene families containing both genic and nongenic, o r spacer regions. Each repeat con­
tains a copy o f the 18s, 5.8s, and 28s ribosomal subunit RNAs in addition to tw o spacer
regions (internal transcribed spacer o r ITS). As shown, in Figure I , the 5.8s rD N A gene is
typically flanked by ITS I and ITS2, which separates the 5.8s rD N A fro m the 18s and 28s
genes (31). This gene in particular possesses highly conserved gene regions that are valuable
in identifying organisms, as well as exhibiting regions such as the ITS regions that are consid­
erably variable. Foremost, there is a homolog o f this gene in every living cell, making it a
well suited gene region as a basis fo r molecular comparison. In addition, genetic exchange
between organisms has not been shown to occur in the rRN A operon, strengthening the
rationale fo r using this region (38). Examinations o f the variable ITS regions can often reveal
differences present among separate isolates o f the same species that may not present them ­
selves when only examining the ribosomal subunit regions (15,20,29).
8
Having chosen the ribosomal subunit genes and the ITS regions to base o u r molecu­
lar comparisons, w e elected to amplify them using the polymerase chain reaction (PCR).
T he PCR allowed us to exponentially amplify the rD N A region o f interest. The specific p o r­
tion to be amplified was governed by the choice o f the oligonucleotide primers. One advan­
tage that exists by using PCR is that it enabled us to amplify homologous gene segments in
each o f the isolates fo r comparison. A second advantage o f using the PCR is the relatively
short length o f tim e needed to complete the reaction. Typically, each PCR run using the
thermal cycler was under fo u r hours and w e w ere able to use crude D N A samples.
Theoretically, the PCR is sensitive enough to detect a single target molecule fo r amplification
w ith o u t having to use other potentially harmful methods that, fo r example, may involve
radioactivity ( 17).
The PCR was the initial step fo r each o f the tw o methods w e chose to analyze o u r
isolates. T he first method was the creation o f a restriction fragment length polymorphism
(RFLP) library using restriction enzyme analysis (REA), and the second method was sequence
analysis. Both PCRs w ere identical except fo r the oligonucleotide primers chosen to amplify
different portions o f the r RNA gene (see Table 2).
Table 2.
Oligonucleotides prim er sequences used to amplify rR N A gene o f Rhizoctonia
solani isolates.
Primer
Nucleotide sequence 5'—3 ’
■LR7
LROR
ITS4
ITS5
TACTACCACCAAG ATC T
ACCCG CTG AACTTAAG C
T C C T C C G C T T A n -GATATGC
GGAAGTAAAAGTCGTAACAAGG
9
ITS 5
LROR
LR7
ITS 4
Figure I . Schematic representation o f the internal transcribed spacer regions. The boxes
represent the ribosomal subunits. The arrows indicate the direction o f the
oligonucleotide primers (4).
IO
CHAPTER 2
C O LLE C T IO N OF FOREIGN A N D DOMESTIC RHIZOCTONIA ISOLATES FOR
MOLECULAR CHARACTERIZATION
Introduction
O u r objective in collecting both foreign and domestic isolates fo r molecular charac­
terization stems from the fact that E. esula originated from Eurasia. There, £ esula popula­
tions are kept in check by a natural complex o f insect and plant pathogens, including R. solani
(8). W e w ere looking fo r foreign and domestic isolates that appeared to be closely related.
Ideally, w e wanted to find foreign isolates that are related to isolates that already exist in the
U.S., that show aggressiveness towards £. esula, and exhibit a narrow host range. Release o f
such isolates as biocontrol agents, in essence, would not be as risky as releasing an isolate o r
a new pathogen that does not already exist in the U.S. Equally as interesting is the basic
information gained and possible future correlations that can be made by n ot only comparing
the isolates to each other, but by using all the information w e can gather to better character­
ize R. solani.
Field Materials and Methods
Surveys fo r disease within stands o f Euphorbia spp. w ere undertaken in Eurasia 1993,
1994, 1995 and 1996, Entire plants were uprooted, placed in plastic bags and stored in an
insulated container, then w ere cultured as soon as possible. Samples o f Euphorbia which
exhibited wilting o r stem necrosis w ere examined fo r decay in crow n tissue o r dead ro o t
buds, then washed thoroughly. The sample was then vigorously wiped w ith sterile cotton
saturated w ith 0.5 % N aO C I. Tissue from the sample was excised w ith a sterile scalpel and
placed on acidified potato dextrose agar ( PDA) fo r isolation. The cultures obtained w ere ini­
tially determined to be Rhizoctonia spp. based on microscopic characteristics, such as hyphal
branching at 90° angles, and constriction at the septa, as well as cultural characteristics,
including rapid growth, colony morphology, and sclerotia formation. Apparent Riizoctonia
spp. w ere hyphal-tipped and transferred to agar again (8).
T he Rhizoctonia isolates from the diseased plants were evaluated fo r their ability to
anastomose w ith thirteen isolates representing R. solani, Field isolates and tester strains w ere
coupled on 2 % w ater agar plates at a distance o f 2.5 centimeters apart and assessed fo r
anastomosis. To better visualize anastomosis reactions, hyphae w ere stained with a Methyl
blue/lactophenol solution. The nuclear condition o f the isolates was determined by vegeta­
tive cell staining w ith a solution o f 4, 6-diamidino-2-phenyl indole (DAPI) (8).
Results
Tables 3 and 4 summarize information obtained fo r the 4 1 isolates collected. The
tables list the R. solani sample, the original host o f isolation, the geographic location, the iden­
tity determined by anastomosis testing and staining, and w hether o r not insects were pre­
sent. Pathogenicity and virulence testing on all the isolates is incomplete (data not shown).
Preliminary results indicate that isolates 0 1 and 06 may be good biological control candidates
because they exhibited high aggressiveness to roots o f E. esula (7).
12
Table 3. Identification and Features o f Domestic Isolates o f Rhizoctonia solani
Sample # ' Designation
Identity o f
Rhizoctonia spp
Insect
Presence
Host
Location
Ol
94-Lyman
A G -4
Euphorbia esula
Yes
02
94-WSS-M
A G -4
Euphorbia esula
No
03
94-Miss-M
A G -4
Euphorbia esula
No
Lyman Creek
Bozeman, M T
W hite Sulfur
Springs, MT
Missoula, M T
04
94-C C #2JL
A G -4
Euphorbia esula
No
Cabin Creek, M T
05
06
07
08
09
10
II
94-6J Rhiz.
93-2
93-1
94-20
94-1 I
95-3
95-6
A G -4
A G -4
A G -4
bi nucleate
multinucleate
bi nucleate
multinucleate
Euphorbia
Euphorbia
Euphorbia
Euphorbia
Euphorbia
Euphorbia
Euphorbia
No
No
No
No
No
12
13
14
15
tester isolate
95-5
95-10
95-9
AG -4-HG I
bi nucleate
bi nucleate
bi nucleate
16
17
18
19
20
21
tester isolate
KVAM 4
KVA M 2
A G -4
multinucleate
n/a
Euphorbia esula
Euphorbia esula
' Centaurea mac­
ulosa
n/a
Euphorbia esula
Euphorbia esula
Euphorbia esula
Euphorbia esula
Euphorbia esula
Fort Benton, M T
Sidney, MT
Sidney, MT
Two D ot, MT
Devil's Tower, W Y
Stillwater, M T
Knutson Creek,
Theodore
Roosevelt National
Fbrk (TRNP)1 N D
n/a
TRNR N D
Two Dot, M T
Hill Co, M T
KMM 5X01
96-2
9 6 Jo h n o 3
multinucleate
binucleate
binucleate
multinucleate
esula
esula
esula
esula
esula
esula
esula
No
' No
No
No
No
No
Yes
Yes
Yes
Ys
n/a
Lambert, MT
Lambert, MT
Lambert, M T
Fremont Co, W Y
Savage, M T
13
Table 4. Identification and Features o f Foreign Isolates o f Rhizoctonia solani
Sample #
Designation
22
93f-23C
23
Identity o f
Rhizoaonia spp
Host
Insect
Presence
Location
£. esula/virgata
No
Volgograd, Russia
9 3 f-16c
multinucleate/
A G -4
binucleate
£. stepposa
No
24
25
26
27
93f-23A
9 4 f-15
94F-38
9 5 f-l4
multinucleate
binucleate
binucleate
binucleate
£.
£.
£.
£.
esula/virgata
esula/virgata
esula/virgata
esula/virgata
No
No
Yes
28
9 5 f-l3
multinucleate
£. esula/virgata
Yes
29
95f-20
multinucleate
£. esula/virgata • Yes
30
9 4 f-18
multinucleate
C diffusa
Novdmarieskaya,
Russia
Volgograd, Russia
Spakovskoe, Russia
Cavai IIon, France
Fuzesabony1
Hungary
Puspokladany,
Hungary
Izsa1Slovak
Republic
Novomarieskaya,
31
32
33
94f-38a
94f-38b
94f-3 N
binucleate
binucleate
binucleate
34
35
9 5 f-18
9 5 f-16
binucleate
binucleate
36
9 5 f-19
binucleate
37
9 5 f-10
38
39
40
41
Ys
No
No
Yes
Russia
Cavaillon, France
Cavaillon1 France
Spakovskoe Sniish1
Russia
Vesprem1 Hungary
Vesprem1 Hungary
multinucleate
£. esula/virgata
£. esula/virgata
Centaurea
maculosa
£. stepposa
Centaurea
maculosa
Centaurea
maculosa
£. esula/virgata
Ys
96f-IO
binucleate
£. esula/virgata
No
9 6 f-l4
binucleate
£. esula/virgata
Yes
Inner Mongolia,
Yes
No
China
Bautou1China
Inner Mongolia,
China
9 6 f-l7
9 6 f-12
multinucleate
binucleate
£. esula/virgata
£. esula/virgata
No
Ys
Yes
Izsa1Slovak
Republic
Balmasurvos,
Hungary
X ilin h ot1 China
14
CHAPTER 3
RESTRICTION ENZYME ANALYSIS T O CREATE A RESTRICTION FRAGMENT
LENGTH POLYMORPHISM LIBRARY
Introduction
The first approach w e employed to characterize R. solani involved the P C R to ampli­
fy regions o f rD N A to create o u r restriction fragment length polymorphism (RFLP) finger­
print library using restriction enzyme analysis (REA). REA is based on the capability o f restric­
tion enzymes to identify D N A sequences that are 4 to 8 bases in length and. cleave the dou­
ble helix at these sites. The num ber o f restriction sites that exist fo r each isolate determines
the num ber o f fragments generated fo r each isolate and the distance between each restric­
tion site governs the size o f the fragments produced by these cuts. The products o f these
restriction digests are separated by standard agarose gel electrophoresis. The banding pat­
terns produced by these restriction enzyme cuts are reproducible but usually vary somewhat
between gels. Banding pattern's produced by this method are known as the RFLP finger­
prints. The banding pattern o r RFLP fingerprint will change if changes in the D N A occur
such as a point mutation, that would result in the loss or gain o f a restriction site, o r chrom o­
somal rearrangement (38). By comparing the RFLP fingerprints, w e can estimate the genetic
similarity o f the isolates (30,36).
.
15
Materials and Methods ■
Fungal cultures
A to ta l o f 41 foreign and domestic Rhizoctonia solani isolates, representing anastomo­
sis group 4, intraspecific groups I and 2, w ere analyzed. A l isolates w ere maintained on
potato dextrose agar at 4°C. Each isolate was grown in 250 ml potato dextrose broth at
ro o m tem perature w ith shaking fo r seven days.
D N A preparation
Mycelia from each isolate was sterile filtered and dehydrated on a Iyopholizer fo r 24
hours. The dehydrated isolates w ere then ground w ith 12.5 grams o f non-sterile sand to a
fine p ow de r in a sterilized m ortar w ith a pestle. The D N A was prepared using the "Sand
Prep " method ( 14). The powder-sand mixtures w e re transferred to polypropylene centrifu­
gation tubes containing nine ml o f extraction buffer and gently mixed by rolling the tubes back
and forth on the bench top. The tubes w ere placed on their sides and gently rocked on an
electric rocker fo r at least one hour at 60° C. After a one hour incubation, 6 ml o f chloroform -octanol (2 4 :1) w ere added to each tube. Each tube was then gently rolled on its side
fo r 5 minutes and centrifuged fo r ten minutes at 4000 rpm. The aqueous layer o f each tube
was poured into new tubes each containing I ml 10% CTAB (56 ml H 2 0 , 10 g CTAB1 33ml
2 .SM NaCI), then balanced w ith the addition o f at least 4 ml o f chloroform -octanol and cen­
trifuged fo r ten minutes at 4000 rpm. Following centrifugation, the aqueous layer o f each
sample was pipetted into centrifugation tubes containing 6 ml isopropanol. The D N A in
these tubes w ere allowed to precipitate fo r 30 minutes and then centrifuged fo r 20 minutes
at 7000 rpm. The aqueous layer o f each sample was discarded and the pellets that
16
remained w e re left to dry on the bench top overnight. The dried D N A pellets w ere then
suspended in I ml TE buffer and stored at -20°C until later use.
Polymerase Chain Reaction
PCR reactions w ere performed using the oligonucleotide primers LROR and LR7
(see Figure I and Table 2) with template D N A obtained from the Sand Prep D N A extraction
m ethod. The D N A samples w ere diluted I : I OO before performing PCR in o rd er to obtain
the greatest yield o f PCR products. Thirty PCR cycles w ere performed on an automated
thermal cycler, using the following parameters: I minute denaturation step at 94°C, I minute
annealing step at 50°C, and I minute prim er extension step at 7 2°C follow ed by a final 7
minute incubation step at 72°C to ensure complete polymerization o f any remaining PCR
products (36). PCR products w ere examined on a 2 % ethidium bromide-stained agarose
gel over an ultraviolet light transilluminator, and photographed.
Restriction Enzvme Analysis o f PCR Products
Restriction enzyme analysis was performed directly on the PCR products according
to the manufacturer’s directions (N e w England Biolabs). The restriction enzymes used to
digest the PCR products are as follows: Avail, Hha I , H palll Msp I and Taq I . The RFLPs
w ere analyzed by separation o f the fragments using electrophoresis on a 4 % agarose gel.
Figure 2 shows an example o f RFLP banding patterns obtained from restriction enzyme
Hha I . Four gels per restriction enzyme were run to accommodate all 4 1 isolates, and pho­
tos o f these gels are found in Appendix A. The I kb D N A ladder standard was run w ith each
group o f isolates fo r each restriction enzyme. The fragments o f the standard are o f known
size, so the fragment size was plotted against the distance travelled fro m the origin. An
17
example o f a calibration curve o f the I kb D N A ladder is shown in Figure 3 fo r the H hal gel
shown in Figure 2.
A calibration curve fo r each gel and fo r each restriction enzyme was
plotted. This allowed us to determine the size o f the fragments produced fo r each isolate by
each restriction enzyme.
S H 22 23 24 25 26 27 28 29 30 3 1 H S
Figure 2.
RFLP fingerprints produced by the digestion o f LROR1 LR7 amplified rD N A w ith
restriction enzyme Hha I analyzed on a 4% agarose gel. Isolate numbers and
ladder identification are shown. The I kb D N A ladder is labeled "S" and the
H n d lll lambda ladder is labeled "H".
18
t
2.6
M
2.4
Distance from origin (mm)
Figure 3.
Calibration curve based on the I kb D N A ladder for the RFLP fingerprint shown
in Figure 2 (1 8 ).
Letter designations fo r RFLP banding patterns w ere assigned to all isolates fo r each
restriction enzyme used. Isolates with identical RFLP banding patterns w ere placed in the
same letter group. Ifth e patterns o f the lettered groups were the same fo r all six restriction
enzymes, the isolates w ere placed in the same class. This is summarized in Tables 5 & 6.
19
Table 5.
Genotype classes and restriction phenotypes o f the domestic isolates.
Restriction phenotype o f fragm ent digested w ith:
Sample #
Ol
02
03
04
05
06
07
08
09
10
II
12
13
14
15
16
17
18
19
20
21
Class
I .
I
I
I
I
I
Il
III
IV
III
I
V
Vl
Vll
Vlll
IX
I
X
Xl
X ll
X lll
Avail
A
A
A
A
A
A
• B
A
A
A
A
A
C
A
D
A
A ■
A
D
A
E
Hha \
Hpall
/Vlspl
Sau3AI
A
A
A
A
A
A
B
A
A
A
A
C
C
D
E
F
A
A
F
A
G
A
A
A
A
A
A
B
C
A
C
A
D
E
F
G
H
A
A
I
C
A
A
A
A
A
A
B
C
A
C
A
D
E
F
G
H
A
A
I
C
J
J
A
A
A
A
A
A
B
C
C
C
A
C
D
E
B
F
A
A
K
G
H
.
.
.
Tdql
A
A
A
A
A
A
A
A
A
A
A
B
C
A
D
D
A
E
D '
A
F
20
Table 6.
Genotype classes and restriction phenotypes o f the foreign isolates.
Restriction phenotype of fragment digested with:
Sample #
Class
Avail
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
X IV
XV
XVI
XVII
XVIII
III
I
X IX
XX
XXI
X XII
Vlll
X XIII
X X IV
Vlll
X
A
A
A
A
G
A
A
A
A
D
D
D
C
D
D
A
A
• A
F
A
XXV
XXV
XXVI
XXVII
Hha\
Hpall
Mspl
Sau3AI
Taql
A
N
A
C
O
C
A
N
P
G
G
G
K
G
G
A
C
C
L
M
A
N
A
C
O
• C
A
N
P
G
G
G
K
G
G
A
C
C
L
M
A
C
A
C
L
C
A
C
K
I
I
B
A
A
A
A
C
C
D
E
F
A
A
F
A
• G
F
E
H
F
E
A
A
A
I
'
J
J
K
B
A
C
C
C
C
J
A
A
A
D
K
G
D
H
D
D
E
E
E
I
A
A distance matrix was calculated by using the equation
D = I-I(Z n xy)Z(Zn^Jny),
w here D represents the distance to be entered into the matrix; nx represents the num ber
o f fragments found in the first o f the tw o isolates being compared; ny represents the num ber
o f fragments found in the second o f the tw o isolates being compared; and
num ber o f restriction fragments the tw o isolates have in common. The
represents the
sign indicates the
summation o f the values fo r each restriction digest ( 19,36). The RFLP banding patterns and
classes aided in the construction o f a distance matrix. Figure 4 is an example o f one "cell"
used in calculating the matrix. Appendix B contains the entire collection o f cell calculations in
21
matrix form , and Appendix C contains the distance, o r D, values only. This matrix is the dis­
tance matrix manipulated by the phylogenetic software to infer phytogeny.
Neighbor, fro m Felsenstein's Phylip version 3.572 software package, was the first step
towards inferring phytogeny. This used the Neighbor Joining method to create a phylogenet­
ic tree fro m the distance matrix data. The Neighbor Joining method is an estimation proce­
dure to make the best estimate o f evolutionary history based on the data set. It uses an
algorithm, o r specific sequence o f steps, that leads to a phylogenetic tree. This program
to o k the raw data provided by the distance matrix and drew an initial tree. It then adjusted
the nodes on the tree based on the average divergence from all the o ther nodes by con­
structing a modified distance matrix. From here, a phenogram using Felsenstein's Drawgram
was produced to give us a visual representation o f the data set.
Number o f fragments produced
for each restriction enzyme
respectively
(Ol) (41)
Isolates being
compared «
Restriction Enzymes
Ol v 41
Ava Il
Hha I
Hpa Il
Msp I
Sau 3Al
Taq I
0.20
t
nx
4
4
3
3
3
5
22
♦
ny
4
6
5
6
2
5
28
where D = I -2(XnXy )/(In x + Z n y)
This is the value entered into the
REA distance matrix
Figure 4.
nxy "
4
4
3
3
I
5
20
Number of fragments
the tw o isolates have
in common for each
restriction enzyme
Summation (%) of each respective
column to be used in solving for D
Diagram o f the calculation cell used to evaluate the Distance Matrix. The
num ber o f fragments produced fo r each restriction enzyme was visually
determined from RFLP fingerprints.
22
Results
Two different bands w ere produced during the PCR reaction using the primers
LROR and LR7 (Figure 5). O u t o f 4 1 isolates, 27 isolates produced a 1.4 kilobase (kb) band,
11 isolates produced a 1.8 kb band, and 3 isolates presented both the 1.4 and 1.8 kb bands.
These findings are in agreement w ith results obtained by Cubeta et al, 1991 (12). They
reported that when using the primers LROR and LR7, they also observed a 1,4 kb band, a
1.8 kb band, o r both.
Occasionally, their isolates containing both the 1.4 and 1.8 kb frag­
ments produced an additional 1.6 kb band, w e did not observe the production o f this addi­
tional 1.6 kb band. They theorized that “The intermediate 1.6 kb band may be the result o f
heteroduplex formation between D N A strands o f different lengths, and that these bands may
represent different length variants o f the rR N A gene that individual isolates can possess (34)."
They could not explain the similarity o f restriction patterns from isolates that produced differ­
ent length variants o f rD N A , which is consistent w ith o u r results and RTLP banding patterns.
These length variants did not appear to be artifacts o f PCR due to the fact that they are con­
sistent and repeatable, and they did not cluster together on the REA phenogram (Figure 6).
Discussion
Five groups o f isolates presented identical RFLP fingerprints and w ere placed in RFLP
classes I, III, VIII, X , and X X V (see Tables 5 & 6). It is interesting to note that each o f four o f
the classes (I, III, VIII, and X ) include at least one foreign isolate. RFLP classes I, III, and X
included domestic isolates from Montana and foreign isolates from Hungary. Isolates from
Montana, Russia, and Slovak Republic (samples 15, 33, and 36 respectively) all possessed
identical banding patterns and w ere placed in class VIII. Class X X V includes isolates 38 and
23
39, both isolated in China, but from tw o very different geographic areas, located hundreds o f
miles apart. These class assignments suggest relatedness o f the isolates but it is important to
keep in mind that the assignment is based solely on banding patterns fro m the RFLP analysis.
This assessment method does not supply information on a mutational basis and could yield
erroneous estimates o f genetic similarity (35). If, fo r example, a new restriction site evolves
due to mutation between tw o pre-existing restriction sites, one large fragment may disap­
pear resulting in the creation o f tw o new, shorter fragments. This may be a serious problem
because even though tw o isolates may share tw o o u t o f three restriction sites, they no
longer have fragments in com m on (18). It is also noted that insertions, possibly o f transposable elements, and deletions are difficult to handle when using REA and that gaining a restric­
tion site is less likely than losing one. RFLP interpretation is also complicated by isolates that
may contain m ore than one rD N A length variant ( 12). Nevertheless, some argue that if you
have a substantially large data set such as this, RFLP data can still be useful in determining
relatedness (18).
Figure 5.
RCR products o f rD N A amplified by restriction enzymes LROR and LR7. This
depicts different length variants o f 1.4 kb o r 1.8 kb bands. Three o f the isolates
produced both 1.4 kb and 1.8 kb bands. Isolate numbers and ladder
identification are shown. The I kb D N A ladder is labeled “S".
25
II
17
95-6
North Dakota
KVAM 4
Montana
28
95f-13
01
94-Lyman
Hungary
Montana
. 07
93-1
Montana
96f-17
China
08
94-20 Montana
___ 38
96f-10
China
27
95f-14
Hungary
— 20
96-2 Wyoming
10
95-3
Montana
---- 39
96f-14
China
— 25
94f-15
Russia
----- 12
------ 13
tester
95-5
15
36
33
35
-3 1
2
North Dakota
95-9
Montana
95f-l9
Slovak Republic
94f-3N
Russia
Hungary
95f-16
94f-38a
94f-38b
France
France
-34
95f-18
tester
-------------21
-----------19
KVAM 5X01
__ 30
94f-18
Russia
95-10
Montana
14
23
29
41
93f-16c
95f-20
96f-12
Hungary
Montana
96Johno 3
94f-38
France
----- 26
Montana
Russia
Slovak Republic
China
Wyoming
L - 09
94-11
Russia
22
93f-23C
Russia
24
93f-23A
Montana
18
KVAM 2
Hungary
37
95f-10
H
02
03
94-WSS-M
94-Miss-M
04
94-CC#2JL
05
94-6J Fthiz
06
93-2
Figure 6.
Montana
Montana
Montana
Montana
Montana
Neighbor joining phenogram o f R solani based on restriction enzyme analysis.
26
CHARTER 4
PHYLOGENETIC RELATIONSHIPS INFERRED FROM SEQUENCES OF INTERNAL
TRANSCRIBED SPACER REGIONS A N D RIBOSOMAL D N A
Introduction
■For further characterization w e elected to sequence rD N A and ITS regions o f R.
solani. W hile RFLPs can often differentiate strains, data on sequences o f D N A can also be
used to infer phylogeny (38). The most practical approach is to analyze sequences o f
homologous segments. The rR N A gene is the best studied sequence used in phylogenetic
comparisons evident by resources such as GenBank and the Ribosomal RNA Database
Project that offer free alignment services by e-mail. Sequence data may confirm previous
findings, contradict previous findings, o r provide new insight. Sequencing ultimately provides
us w ith a wealth o f information and will undoubtedly play an increasing role in taxonomy.
Obtaining sequence data fo r o u r isolates allowed us to infer phylogeny using Felsenstein’s
Phylip software package.
Materials and Methods
Fungal cultures
See Chapter 3 Materials and Methods.
Tl
D N A preparation
See Chapter 3 Materials and Methods.
Polymerase Chain Reaction
The polymerase chain reaction (PCR) was performed using the same thermal cycler
parameters as described in Chapter 2 (p. 15). The reaction differed only by the region o f
the ribosomal RNA gene amplified using the primers ITS4 and ITS5 (Figure I). The primers
fro m this reaction w ere removed from the PCR products prior to sequencing by using a
Qiagen Q IAquick PCR purification kit according to manufacturers’ instructions.
Concentrations o f the samples w ere determined by reading A ^ Q w ith a spectrophotometer.
Automated Sequencing
D N A sequencing was conducted by Joan Strange at the University o f Montana,
Missoula, w ith an automated sequencer. This method is termed “automatic" in that one
does not have to manually record the results. Rather than radioactively labeled D N A frag­
ments, the D N A fragments are fluorescently labeled and detected by a laser during elec­
trophoresis. The sequence is directed to a com puter and recorded in chromatograph form .
From this point, the chromatograph can be translated into a D N A sequence either visually o r
by com puter software, Sequence data from these reactions were aligned "on-line" using the
BLASTN database, producing 100% homology w ith several Rhizoctonia solani in the
sequence database. This confirmed that w e in feet had amplified and sequenced the 5.8s
rD N A , ITS region that w e had targeted.
28
Alignment o f Sequences
The sequences obtained did not allow fo r complete alignment o f all 700 nucleotides.
Therefore, a combined sequence data set o f 400 nucleotides was used fo r the alignment.
Alignment o f the isolates’ sequences was accomplished using the Align Plus, version 2 soft­
ware package w ith isolate " 0 1" serving as the reference molecule. The aligned sequences
are found in Appendix D.
I b compare the isolates, I chose the Distance Method, which creates a distance
matrix fro m the aligned sequence data. Distance comparison methods calculate the percent
changes fo r the data set, taking each nucleotide position and determining simply w hether it is
the same o r different fo r all the isolates in the data set. This is unlike Maximum Likelihood
Methods that weight transversion and transition nucleotide changes differently. Neighbor, uti­
lizing the N eighbor Joining method was used to create a phylogenetic tree, which was then
used by Drawgram to produce a visual representation o f the data set (Figure 7) (18).
Bootstrapping
The application o f bootstrap methods is used to create a reliable phylogenetic tree
by decreasing stochastic effects that may influence the branching o f the tree. In situations
w here resampling is impractical o r impossible, bootstrapping creates pseudoreplicate data
sets by randomly sampling the original matrix to create new matrices. W e used 100 boot­
strap replications w ith bootstrap proportions listed to the left o f the branches (Figure 8). The
bootstrap proportion represents the frequency w ith which a given branch is found by analysis
o f the pseudoreplicate data sets ( 18). Again, Felsenstein's Phylip software was used. First
Seqboot created 100 randomized sequence alignments, and then DNADist created distance
matrices fo r each bootstrap replication. The Neighbor Joining method follow ed using
29
Neighbor and the phenogram was constructed using Drawgram.
Results
PCR products obtained using ITS 4 and ITS 5 oligonucleotide primers were approxi­
mately 700 base pairs in length (data not shown). Researchers claimed that they were able
to distinguish the ir R, solani isolates according to virulence, based on differences in the size o f
the PCR product using the same primers (4). Due to the fact that o u r virulence testing has
not been completed, w e are unable to make such claims at this point in time'. However, if in
the future w e are able to make the same claim o f distinguishing virulence based on the size
o f the PCR product, it would definitely hasten the screening process o f choosing appropriate
isolates fo r biocontrol. Figure 7 shows the phenogram constructed fro m the sequence data.
Figure 8 shows the phenogram constructed fro m the bootstrap data. Branches on the tree
that have bootstrap proportions greater than 50 have been bolded.
N o correlations fo r insect damaged isolates w ere detected by obvious clustering on
the Neighbor-Joining o r bootstrap consensus tree. O n the bootstrap consensus tree, iso­
lates 23 and 34 did not cluster together, and w ere the only tw o isolates to have £ stepposa
as a host. However, all four isolates, including one domestic and three foreign isolates, origi­
nating fro m the host plant Centaurea maculosa, cluster at the to p o f the bootstrapping
phenogram to the right o f a bootstrap proportion o f 63. Sixteen o u t o f nineteen o f the
domestic isolates collected originated in Montana and are distributed evenly over the tree.
Isolates from Hungary did not cluster together, n or did isolates from China, Hungarian iso­
late 28 and isolate I I fro m N o rth Dakota branch together with a bootstrap proportion o f 65
and differ by only one nucleotide. Though isolates 27 and 36 w ere isolated fro m different
countries (Hungary and Slovak Republic) and fro m different hosts, they group together on
30
the tree w ith a bootstrap proportion o f 100, and differ by only nine nucleotides. Two o u t o f
three isolates collected fro m a small field in France clustered together while the third isolate
branches by itself. This helps depict the diversity o f R solani isolates in a small geographical
area. Russian isolates 22 and 24 are identical to each other as are China isolates 38 and 39
which w ere isolated in separate regions o f Inner Mongolia.
The nucleotide differences referenced above w ere obtained by using the Align Plus
software package to help interpret the trees. The matrix depicting the num ber o f nucleotide
matches between each isolate is listed in Appendix E. Keep in mind that the phylogenetic
tree is just a visual representation o f the sequence data and that the interpretation o f the tree
is subjective. Fbired isolates 38 and 39 have identical sequences matching 400 o ut o f 400
nucleotides. Given that, one might interpret isolates 23 and 34 to be identical o r isolates 2 1
and 26 to be identical but they differ by 138 and 133 nucleotides respectively. This is not to
say that the inferred trees are not informative, just that caution should be taken in the inter­
pretation. Slight differences in the num ber o f nucleotide matches arise each tim e a different
reference molecule is selected. Regardless o f which molecule is chosen as the reference
molecule, the obvious clustering o f isolates shown in Figure 8 remain fairly constant. O ne
mistake to avoid in interpreting the bootstrap consensus tree, given by Hillis et al, is not to
focus on individual branches, but to look at the tree as a whole.
Discussion
O u r study revealed that although R. solani isolates display a high level o f sequence
variation in the rD N A ITS regions, some foreign and domestic strains are identical. This is
not surprising considering that most o f the isolates w ere collected from the same host
species E. esula. Recently, Boysen et al. (1996) reported that phylogenetic analysis showed
31
that the A G -4 isolates they examined could be divided into three subgroups that correlate
w ith habitat and virulence. Following that study, Kuninaga et al. ( 1997) evaluated the genetic
diversity within and between anastomosis groups in R. solani. They w ere able to distinguish
hybridization groups A G 4-H G -I and A G 4-H G -II as well as correlate the information to tw o
o f the three subgroups o f Boysen et al. ( 1996). The hypovirulent subgroups o f Boysen et al.
evaluated by Kuninaga et al. showed lo w hom ology to subgroups HG-1 and HG-II but did
show high related ness to a binucleate Rhizoctonia isolate belonging to the group w ith a
Ceratobasidium sexual state. This is intriguing due to the fact that o u r sample collection con­
sists o f both multinucleate and binucleate isolates. Perhaps o u r virulence tests will yield simi­
lar results and w e may find that o u r multinucleate and binucleate strains m erit separate
species. O ne o f the advantages o f having used sequence analysis here is that when o u r viru­
lence tests are complete, w e can compare the results o f different studies to o u r ow n w ith o u t
performing the experiments.
The findings in this study show that several o f the foreign isolates are identical to the
domestic isolates, suggesting that a larger survey is needed. A consistent relationship o f soilborne pathogens w ith insect ro o t damage has been observed (9), and w e need to apply vir­
ulence and insect data to the rD N A , ITS sequence data. Tb do this, a critical mass o f foreign
isolates is needed to assess AG and insect association, The determination, by sequence
analysis, that some foreign and domestic isolates are identical should qualify these foreign iso­
lates as possible release agents. This information proves that these fungi already exist in U.S.
soil and, in essence, a foreign isolate may simply be a more effective pathogen and a better
adapted biocontrol agent.
32
02
94-WSS-M
Montana
•05
94-6| Rhiz
Montana
11
28
17
18
03
95-6
North Dakota
95f-13
KVAM 4
KVAM 2
Hungary
Montana
Montana
94-Miss-M
04
Montana
94-CC#2JL
Montana
95-10
Montana
r 14
94f-3N
Russia
1.41
96f-12
China
------ 15
95-9
Montana
95f-14
Hungary
95f-19
Slovak Republic
n r 33
4S
31
32
94f-38a
94f-38b
France
France
— 13
95-5
North Dakota
— 35
95f-16
Hungary
— 08
94-20
Montana
95-3
Montana
I ' 0
96-2
Wyoming
r b 20
I
38
I 39
25
12
09
E
19
96f-IO
96f-l4
China
China
94f-15
Russia
tester
94f-18
Russia
------ 23
93f-l6c
Russia
•34
95f-18
Hungary
29
95f-20
Slovak Republic
94-1 I
Wyoming
KVAM 5X01
tester
Montana
93-1
Montana
--------21
96 Johno 3
94f-38
96f-17
■ 37
06
95f-10 Hungary
93-2
Montana
24
01
93f-23A Russia
94-Lyman
Montana
22
93f-23C
Figure 7.
Montana
France
China
Russia
Neighbor joining tree for Rhizoctonia solani inferred from 400 alignable sites o f
rD N A sequence data.
33
95-10
Montana
-3 3
94f-3N
Russia
-41
96f-12
China
15
95-9
Montana
36
95f-19
Slovak Republic
27
95f-14
Hungary
94-CC#2JL
Montana
94f-38a
France
94f-38b
France
95f-16
Hungary
95-5
North Dakota
94f-15
Russia
-------- 20
96-2
Wyoming
100 ,
-3 9
96f-14
China
-3 8
10
95-3
Montana
96f-10
Montana
China
tester
'34
95f-18
Hungary
-23
93f-16c
Russia
'29
95f-20
Slovak Ftepublic
'09
94-1 I
Wyoming
19
KVAM 5X01
Montana
■30 94f-18
Russia
tester
-26
94f-38
France
- 2 1 96 Johno 3
Montana
■07 93-1
Montana
■40
■37
-06
■03
— 01
271
' 17
95f-10
94-Lyman
93f-23C
95f-13
95-6
Montana
Russia
Hungary
North Dakota
94-WSS-M
Montana
KVAM 2
Montana
94-6J Rhiz
Figure 8.
Hungary
'2 4
93F-23A
Russia
KVAM 4
Montana
'28
— 02
— 18
China
93-2
Montana
94-Miss-M
Montana
------- 22
98
96f-17
Montana
Bootstrap consensus tree generated from 100 bootstrap replications o f 400
alignable sites o f rD N A sequence data.
34
EPILOGUE
Results o f the sequence data reinforce some o f the results obtained by REA data,
T he results are not identical, and this may be due to some o f the problems associated w ith
REA, such as not being able to detect mutational changes as the result o f a loss o r gain o f a
restriction site due to insertions o r deletions. In addition, the regions o f the rD N A that w e
used fo r REA and fo r sequence analysis differ, which may also explain some slight variance in
the results. This same variance, however strengthens the evidence fo r similarity o f isolates
that appear to be the same using both methods. Small errors, such as the incorporation o f
the w ro n g nucleotide, is also to be expected when using the PCR. Given these possible
avenues fo r error, it is still obvious that several foreign and domestic isolates appear to be
very similar using both methods. These comparative results, along w ith the pathogenicity
and virulence testing, will aid in the selection o f the appropriate isolates fo r biocontrol o f E.
esula.
In comparing both o f the methods used, I would choose sequencing over REAfor
m ost applications. In the past few years, sequencing has become relatively cheap and easy
to obtain. The tim e required fo r sequencing is shorter than that fo r REA and it ultimately
gives m ore information. Given the sequence data, restriction sites can be found visually o r
by using com puter software packages. These software packages are capable o f searching fo r
restriction sites, displaying the position o f the sites, and determining the exact size o f the
bands that are produced given a particular restriction enzyme. This capability aids in the
selection o f restriction enzymes that are informative which saves tim e and money.
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12.
Cubeta, M.A., E. Echandi, T. Abernethy and R. Vilgalys. 1991. Characterization o f
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19.
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20.
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2 1.
Keijer, J., P.M. Houterman, A M. Dullemans and M G. Korsman. 1996. Heterogeneity
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Kuninaga, S., XNatsuaki, T. Talteuchi and R. Yokosawa. 1997. Sequence variation o f
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37
23.
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Lui, Z.L., L.L. Domier and J.B.Sinclair. 1995. Polymorphism o f genes coding fo r
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39
APPENDICES
40
APPENDIX A
RFLP FINGERPRINTS
41
S H Ol 02 03 04 05 06 07 08 09 IO II H S
■
SS
12 13 14 15 16 17 18 19 20 21 S S
S
# ^ # # # #
•**
m m m #
S
H 22 23 24 25 26 27 28 29 30 3 1 H S
S S 32 33 34 35 36 37 38 39 40 41 S S
m m
m ^
^ m m
...
...........
.....
RFLP fingerprints produced by the digestion o f LROR1 LR7
amplified rD N A with restriction enzyme Ava 11 analyzed on a
4 % agarose gel. Isolate numbers and ladder identification are
shown. The I kb D N A ladder is labeled "S" and the Hzndlll
lambda ladder is labeled "H".
42
S H Ol 02 03 04 05 06 07 08 09 10 I I H S
S H
12 13 14 15 16 17 18 19 20 21 H S
:: : * * # # #
«8
%
, .
■
>
Si B ■ ...
: Si
m *« x«
# # : :# # :# W W %%!#
::
'
S H 22 23 24 25 26 27 28 29 30 3 1 H S
SH
32 33 34 35 36 37 38 39 40 41
HS
ii
Mm
♦ HI
—
^ ^
... A;s::
<
-
It
m
y??
W?
:::
S y..
Si*
:
Bis
RFLP fingerprints produced by the digestion o f LROR1 LR7
amplified rD N A with restriction enzyme H hal analyzed on a 4 %
agarose gel. Isolate numbers and ladder identification are
shown. The I kb D N A ladder is labeled “S” and the Hmdlll
lambda ladder is labeled "H".
43
SS Ol 02 03 040506 07 08 09 10 11 S S
'
S S 12 13 14 15 16 17 18 19 20 21 S S
' <X>
i t
#* # '
Q
. .
S S 22 23 24 25 26 27 28 29 30 3 1 S H
# #
# # #
I
■
SS
■
. ..
Jpl
,
.
32 33 34 35 36 3738 39 40 41
RFLP fingerprints produced by the digestion o f LROR1 LR7
amplified rD N A with restriction enzyme Hpoll analyzed on a
4 % agarose gel. Isolate numbers and ladder identification are
shown. The I kb D N A ladder is labeled “S" and the Hfndlll
lambda ladder is labeled "H".
SH
44
S S Ol 02 03 04 05 0607 08 09 10 I I S S
S S 12 13 14 15 16 17 18 19 20 21 SS
RFLR fingerprints produced by the digestion o f LROR1 LR7
amplified rD N A with restriction enzyme Mspl analyzed on a 4%
agarose gel. Isolate numbers and ladder identification are
shown. The I kb D N A ladder is labeled “S" and the Hfndlll
lambda ladder is labeled “H".
45
RFLP fingerprints produced by the digestion o f LROR1 LR7
amplified rD N A with restriction enzyme Sou3AI analyzed on a
4% agarose gel. Isolate numbers and ladder identification are
shown. The I kb D M A ladder is labeled “S" and the H n d lll
lambda ladder is labeled "H".
46
RFLR fingerprints produced by the digestion o f LROR1 LR7
amplified rD N A with restriction enzyme Taq\ analyzed on a 4%
agarose gel. Isolate numbers and ladder identification are
shown. The I kb D N A ladder is labeled “S" and the Hfndlll
lambda ladder is labeled “H ” .
47
APPENDIX B
C O LLE C TIO N O F DISTANCE MATRIX CALCULATIONS
48
Ol v 41
Ava Il
Hha\
HpaW
Msp\
Sau 3Al
Taql
0.20
02 v 41
Avoll
Hhal
Hpall
Mspl
Sou3AI
Taql
0.20
03 v 41
Avoll
Hhal
Hpall
Mspl
Sau3A\
Taql
0.20
0 4 v 41
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.20
05 v 41
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.20
nx
4
4
3
3
3
5
22
ny
4
6
5
6
2
5
nxy
4
4
3
3
I
5
28 20
nx
4
4
3
3
3
5
22
ny
4
6
5
6
2
5
28
nxy
4
4
3
3
I
5
Avail
Hhal
Hpall
Mspl
Sau3Al
Taql
4
4
3
3
3
5
20
0.43
22
nx
4
4
3
3
3
5
22
ny
4
6
5
6
2
5
28
nxy
03 v4 0 nx
4
4
3
3
I
5
20
Avail
Hhal
HpaW
Mspl
Sau3A\
Taql
0.43
4
4
3
3
3
5
nx
4
4
3
3
3
5
22
ny
4
6
5
6
2
5
nxy
4
4
3
3
I
5
28 20
04 v 40
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
nx
4
4
3
3
3
5
0.43
22
nx
4
4
3
3
3
5
22
ny
4
6
5
6
2
5
05 v 40
Avail
Hhal
HpaW
Mspl
Sau3Al
Taql
0.43
nx
4
4
3
3
3
5
nxy
4
4
3
3
I
5
28 20
01 v 40
Avail
Hhal
Hpall
Mspl
Sau 3Al
Taql
0.43
nx
4
4
3
3
3
5
ny
5
5
3
3
2
2
22 20
02 v 40 nx
nxy
3
2
2
2
I
2
12
01 v 39
Avail
Hhal
Hpall
Mspl
Sau 3AI
Taql
0.26
nx
4
4
3
3
3
5
ny
4
4
4
4
2
3
22 21
nxy
4
4
2
2
I
3
16
nx
4
4
3
3
3
5
nxy
4
4
2
2
I
3
16
ny
5
5
3
3
2
2
20
nxy
3
2
2
2
0 2 v 39
Avail
Hhal
Hpall
Mspl
I
5au3AI
2
12
0.26
ny
4
4
4
4
2
3
22 21
ny
5
5
3
3
2
2
22 20
nxy
3
2
2
2
03 v 39
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
nx
4
4
3
3
3
5
0.26
ny
4
4
4
4
2
3
22 21
nxy
4
4
2
2
I
3
16
nx
4
4
3
3
nxy
4
4
2
2
nxy
4
4
2
2
I
3
16
22
I
2
12
Taql
ny
5
5
3
3
2
2
20
nxy
3
2
2
2
I
2
12
0 4 v 39
Avail
Hhal
Hpall
Mspl
0.26
ny
4
4
4
4
3 2
5
3
22 21
ny
5
5
3
3
2
2
20
nxy
3
2
2
2
I
2
12
0 5 v 39
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
nx
4
4
3
3
3
5
0.26
22
5au3AI
Taql
ny
4
4
4
4
2
3
21
I
3
16
49
Ol V 38
Avoll
Hha\
Hpoll
Mspl
Sou3AI
Toql
0.26
nx
4
4
3
3
3
5
22
ny
4
4
4
4
2
3
21
nxy
4
4
2
2
I
3
16
Ol v 37
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.05
nx
4
4
3
3
3
5
ny
4
4
3
3
3
3
22 20
nxy
4
4
3
3
3
3
20
02 v38
Avoll
Hhal
HpaW
Mspl
5ou3AI
Taql
0.26
nx
4
4
3
3
3
5
22
ny
4
4
4
4
2
3
21
nxy
4
4
2
2
I
3
|6
02v37
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.05
nx
4
4
3
3
3
5
ny
4
4
3
3
3
3
22 20
nxy
4
4
3
3
3
3
20
02v36
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.50
nx
4
4
3
3
3
5
22
ny
5
4
4
4
2
3
22
nxy
4
2
I
I
I
2
II
03v38
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.26
nx
4
4
3
3
3
5
22
ny
4
4
4
4
2
3
21
nxy
4
4
2
2
I
3
16
03v37
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.05
nx
4
4
3
3
3
5
ny
4
4
3
3
3
3
20
nxy
4
4
3
3
3
3
20
03v36
Avail
Hhal
Hpall
Mspl
Sau3A\
Taql
0.50
nx
4
4
3
3
3
5
22
ny
5
4
4
4
2
3
22
nxy
4
2
I
I
I
2
II
04v38
Avail
Hhal
Hpall
Mspl
Sau3AI
Toql
0.26
nx
4
4
3
3
3
5
22
ny
4
4
4
4
2
3
21
nxy
4
4
2
2
I
3
16
04 v 37
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.05
ny
4
4
3
3
3
3
22 20
nxy
4
4
3
3
3
3
20
04 v 36
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql .
0.50
nx
4
4
3
3
3
5
22
ny
5 •
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02 v 21
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5
22
04 v 08
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.20
nx
4
4
3
3
3
5
22
ny
4
4
4
4
2
5
23
nxy
4
4
2
2
I
5
18
04 v 07
Avail
Hhal
Hpall
Mspl
SauSAl
Taql
0.43
nx
4
4
3
3
3
5
ny
4
3
3
3
2
5
22 20
nxy
2
•
0
2
2
I
5
12
04v06
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql •
0.00
nx
4
4
3
3
3
5
22
ny
4
4
3
3
3
5
22
nxy
4
4
3
3
3
5
22
05v08
Avail
Hhal
HpaW
Mspl
5au3AI
Taql
0.20
nx
4
4
3
3
3
5
22
ny
4
4
4
4
2
5
23
nxy
4
4
2
2
I
5
18
05 v 07
Avail
Hhal
Hpall
Mspl
SauSAl
Taql
0.43
nx
4
4
3
3
3
5
nxy
2
0
2
2
I
5
12 ■
05 v 06
Avail
Hhal
Hpall
Mspl
SauSAl
Taql
0.00
nx
4
4
3
3
3
5
22
ny
4
4
3 ’
3
3
5
22
nxy
4
4
3
3
3
5
22
22
ny
4
3
3
3
2
5
20
ny
4
3
3
3
2
5
22 20
60
01 v 05
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.00
nx
4
4
3
3
3
5
22
ny
4
4
3
3
3
5
22
nxy
4
4
3
3
3
5
22
01 v 04
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.00
nx
4
4
3
3
3
5
22
ny
4
4
3
3
3
5
22
nxy
4
4
3
3
3
5
22
01 v 03
Avail
Hhal .
HpaW
Mspl
Sau3AI
Taql
0.00
nx
4
4
3
3
3
5
22
ny
4
4
3
3
3 .
5
22
nxy
4
4
3
3
3
5
22
02 v 05
Avail
Hhal
Hpall
Mspl
Sau3A\
Taql
0.00
nx
4
4
3
3
3
5
22
ny
4
4
3
3
3
5
22
nxy
4
4
3
3
3
5
22
02v04
Avail
Hhal
HpaW
Mspl
Sau3Al
Taql
0.00
nx
4
4
3
3
3
5
22
ny
4
4
3
3
3
5
22
nxy
4
4
3
3
3
5
22
02 v 03
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql ■
0.00
nx
4
4
3
3
3
5
22
ny
4
4
3
3
3
5
22
nxy
4
4
3
3
3
5
22
03v05
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.00
nx
4
4
3
3
3
5
22
ny
4
4
3
3
3
5
22
nxy
4
4
3
3
3
5
22
03v04
Avail
Hhal
HpaW
Mspl
Sau3A\
Taql
0.00
nx
4
4
3
3
3
5
22
ny
4
4
3
3
3
5
22
nxy
4
4
3
3
3
5
22
03 v 03
Avail
Hhal
HpaW
Mspl
Sau3A\
Taql
0.00
nx
4
4
3
3
3
.5
22
ny
4
4
3
3
3
5
22
nxy
4
4
3
3
3
5
22
04 v 05
Avail
Hhal
HpaW
Mspl
Sau3A\
Taql
0.00
nx
4
4
3
3
3
5
22
ny
4
4
3
3
3
5
22
nxy
4
4
3
3
3
5
22
04 v 04
Avail
Hhal
HpaW
Mspl '
Sau3A\
Taql
0.00
nx
4
4 •
3
3
3
5
22
ny
4
4
3
3
3
5
22
nxy
4
4
3
3
3
5
.
22
05 v 05
Avail
Hhal
Hpall
Msp I
Sau3AI
Taql
0.00
nx
4
4
3
3
3
5
22
ny
4
4
3
3
3
5
22
nxy
4
4
3
3
3
5
22
61
Ol V 02
Avoll
Hhal
Hpall
Mspl
Sou3AI
Taql
0.00
nx
4
4
3
3
3
5
22
ny
4
4
3
3
3
5
22
nxy
4
4
3
3
3
5
22
02v02
Avail
Hhal
Hpall
Mspl
Sau3A\
Taql
0.00
nx
4
4
3
3
3
5
22
ny
4
4
3
3
3
5
22
nxy
4
4
3
3
3
5
22
01 vO I
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.00
nx
4
4
3
3
3
5
22
ny
4
4
3
3
3
5
22
nxy
4
4
3
3
3
5
22
62
06 v 4 l
Avoll
Hha\
HpaW
Msp\
SoiSAI
Taq\
0.20
nx
4 '
4
3
3
3
5
22
ny
4
6
5
6
2
5
28
nxy
4
4
3
3
I
5
20
06 v 40
Avail
Hhal
HpaW
Mspl
SoiSAI
Taql
0.43
07 v 41
Avail
Hha\
HpaW
Mspl
SaiSAI
Taql
0.50
nx
4
3
3
3
2
5
20
ny
4
6
5
6
2
5
28
nxy
2
0
2
2
I
5
12
08 v 41
Avail
Hhal
HpaW
Mspl
SaiSAI
Taql
048
nx
4
4
4
4
2
5
23
ny
4
6
5
6
2
5
28
09 v 41
Avail
Hhal
HpaW
Mspl
SaiSAI
Taql
0.14
nx
4
4
3
3
2
5
21
10 v 41
Avail
Hhal
HpaW
Mspl
SaiSAI
Taql
0.18
nx
4
4
4
4
2
5
23
nx
4
4
3
3
3
5
ny
5
5
3
3
2
2
20
nxy
3
2
2
2
I
2
12
06v39
Avail
Hhql •
Hpall
Mspl
SaiSAI
Taql
0.26
nx
4
4
3
3
3
5
22
ny
4
4
4
4
2
3
21
nxy
4
4
2
2
I
3
16
07 v 40
Avail
Hhal
HpaW
Mspl
SaiSAI
Taql
OSO
ny
5
5
3
3
2 2
5
2
20 20
nxy
2
I
2
2
I
2
10
07v39
Avail
Hhal
HpaW
Mspl
SaiSAI
Taql
0.61
nx
4
3
3
3
2
5
20
ny
4
4
4
4
2
3
21
nxy
2
0
I
I
I
3
8
nxy
4
4
3
3
2
5
21
08 v 40
Avail
Hhal
HpaW
Mspl
SaiSAI
Taql
0.40
nx
4
4
4
4
ny
5
5
3
3
2
2
20
nxy
3
2
2
2
2
2
13
08v39
Avail
Hhal
HpaW
Mspl
SaiSAI
Taql
005
nx
4
4
4
4
2
5
23
ny
4
4
4
4
2
3
21
nxy
4
4
4
4
2
3
21
ny
4
6
5
6
2
5
28
nxy
4
4
3
3
2
5
21
09 v 4 0
Avail
Hhal
HpaW
Mspl
SaiSAI
Taql
0S7
nx
4
4
3
3
ny
5
5
3
3
2
2
20
nxy
3
2
2
2
2
2
13
09v39
Avail
Hhal
HpaW
Mspl
. SaiSAI
Taql
0.19
nx
4
4
3
3
2
5
21
ny
4
4
4
4
2
3
21
nxy
4
4
2
2
2
3
17
ny
4
6
5
6
2
5
28
nxy
4
4
3
3
2
5
21
10 v 40
Avail
Hhal
HpaW
Mspl
SaiSAI
Taql
0.40
ny
5
5
3
3
2
2
20
nxy
3
2
2 .
2
2
2
13
I 0 v 3 9 nx
Avail
4
Hhal
4
HpaW
4
Mspl
4
SaiSAI 2
5
Taql
0.05
23
ny
4
4
4
4
2
3
21
nxy
4
4
4
4
2
3
21
22
nx
4
3
3
3
2
5
23
2
5
21
nx
4
4
4
4
2
5
23
63
nxy
4
4
2
2
I
3
16
06v37
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.05
07v38
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.61
nx
4
3
3
3
2
5
20
ny
4
4
4
4
2
3
21
nxy
2
0
I
I
I
3
8
07v37
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.50
nx
4
3
3
3
08v38
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.05
nx
4
4
4
4
2
5
23
ny
4
4
4
4
2
3
21
nxy
4
4
4
4
2
3
21
08v37
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.26
nx
4
4
4
4
09v38
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.19
nx
4
4
3
3
2
5
21
ny
4
4
4
4
2
3
21
nxy
4
4
2
2
2
3
17
09v37
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.12
nx
4
4
3
3
nx
4
4
4
4
2
5
23
ny
4
4
4
4
2
3
21
nxy
4
4
4
4
2
3
21
I0 v 3 7
Avail
Hhal
Hpall
Mspl
SauSAl
Taql
0.26
nx
4
4
4
4
>
ny
4
4
4
4
2
3
21
OO
PO
nx
4
4
3
3
3
5
22
O
06v38
Avail
Hha\
HpaW
Msp\
Sau3AI
Taq\
0.26
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.05
nx
4
4
3
3
3
5
ny
4
4
3
3
3
3
22 20
nxy
4
4
3
3
3
3
20
06v36
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.50
nx
4
4
3
3
3
5
22
ny
5
4
4
4
2
3
22
nxy
4
2
I
I
I
2
11
ny
4
4
3
3
2 3
5
3
20 20
nxy
2
0
2
2
I
3
10
07v36
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.57
nx
4
3
3
3
2
5
20
ny
5
4
4
4
2
3
22
nxy
2
I
I
I
2
2
9
ny
4
4
3
3
3
3
20
nxy
4
4
2
2
I
3
16
08v36
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.42
nx
4
4
4
4
2
5
23
ny
5
4
4
4
2
3
22
nxy
4
2
2
2
I
2
13
ny
4
4
3
3
3
3
20
nxy
4
4
3
3
I
3
18
09v36
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.49
nx
4
4
3
3
2
5
21
ny
5
4
4
4
2
3
22
nxy
4
2
I
I
I
2
II
ny
4
4
3
3
3
3
20
nxy
4
4
2
2
I
3
16
10 v 36
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.42
nx
4
4
4
4
2
5
23
ny
5
4
4
4
2
3
22
nxy
4
2
2
2
I
2
13
2
5
23
2
5
21
2
5
23
64
06 v 35
Avail
Hha\
HpaW
Mspl
SauSAl
Taql
0.50
nx
4
4
3
3
3
5
22
ny
5
4
4
4
2
3
22
nxy
4
3
I
I
0
2
11
07 v 35
Avail
Hhal
Hpall
Mspl
SaulAl
Taql
nx
4
3
3
3
2
5
nxy
2
0
I
I
0.67
20
ny
5
4
4
4
2
3
22
08v35 nx
Avail
Hhal
HpaW
Mspl
SaulAl
Taql
0.38
4
4
4
4
2
5
23
09v35 nx
ny
5
4
4
4
2
3
22
I
2
7
nxy
4
3
2
2
I
2
14
Avail
Hhal
HpaW
Mspl
SaulAl
Taql
0.44
4
4
3
3
2
5
21
ny
5
4
4
4
2
3
22
I
I
2
12
10 v 35
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
nx
4
4
4
4
2
5
23
ny
5
4
4
4
2
3
22
nxy
4
3
2
2
I
2
14
0.38
nxy
4
3
I
06 v3 4 nx ny
Avail
Hhal
HpaW
Mspl
SaulAl
Taql
0.50
4
4
3
3
3
5
22
4
4
4
4
3
3
22
07 v3 4 nx ny
Avail
Hhal
HpaW
Mspl
SaulAl
Taql
0.67
4
3
3
3
4
4
4
4
2 3
5
3
20 22
08v34 nx ny
Avail
Hhal
HpaW
Mspl
SaulAl
Taql
0.47
4
4
4
4
2
5
23
4
4
4
4
3
3
22
09v34 nx ny
Avail
Hhal
Hpall
Mspl
SaulAl
Taql
0.49
10 v 34
Avail
Hhal
HpaW
Mspl
SaulAl
Taql
0.47
4
4
3
3
2
5
21
4
4
4
4
3
3
22
nx
4
4
4
4
2
5
23
ny
4
4
4
4
3
3
22
nxy
2
3
2
2
I
I
11
nxy
I
I
2
2
0
I
7
nxy
3
3
2
2
06y33 nx
Avail
Hhal
HpaW
Mspl
SaulAl
Taql
0.50
07 v 33
• Avail
Hhal
HpaW
Mspl
SaulAl
Taql
0.57
ny
5
4
4
4
2
3
22
nx
4
3
3
3
2
5
ny
5
4
4
4
2
3
20 22
08v33 nx ny
I
I
Avail
Hhal
HpaW
Mspl
SaulAl
Taql
12
0.42
nxy
2
3
2
2
I
I
II
Avail
Hhal
HpaW
Mspl
SaulAl
Taql
0.49
nxy
3
3
2
2
4
4
3
3
3
5
22
nxy
4
2
I
I
I
2
II
nxy
2
I
I
I
2
2
9
nxy
4
2
2
2
4
4
4
4
2
5
23
5
4
4
4
2
3
22
09v33 nx
ny
5
4
4
4
2
3
22
I
I
2
II
ny
5
4
4
4
2
3
22
nxy
4
2
2
2
I
2
13
4
4
3
3
2
5
21
I0v33 nx
I
I
Avail
Hhal
HpaW
Mspl
SaulAl
Taql
12
0.42
4
4
4
4
2
5
23
I
2
13
nxy
4
2
I
65
nx
4
4
3
3
3
5
22
ny
5
4
4
4
4
3
24
07v32
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.64
nx
4
3
3
3
2
5
20
08v32
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.36
nxy
4
3
I
I
2
2
13
06v3l
Avail
Hhal
HpaW
Mspl
Sau3A\
Taql
0.49
nx
4
4
3
3
3
5
ny
5
4
4
4
4
4
22 25
nxy
4
2
I
I
2
2
12
06v30
Avail
. Hhal
HpaW
Mspl
Sau3Al
Taql
■ 0.35
nx
4
4
3
3
3
5
22
ny
4
4
7
7
2
3
27
nxy
4
4
3
3
0
2
16
ny nxy
5
2
0
4
4
I .
4
I
4
2
3 ' 2
24 8
07 v 3 1
Avail
Hhal
HpaW
Mspl
Sau3A\
Taql
0.56
nx
4
3
3
3
2
5
ny
5
4
4
4
4
4
20 25
nxy
2
2
I
I
2
2
10
07 v 30
Avail
Hhal
HpaW
Mspl
Sau3Al
Taql
0.62
nx
4
3
3
3
2
5
20
ny
4
4
7
7
2
3
27
nxy
2
0
2
2
I
2
9
nx
4
4
4
4
2
5
23
ny
5
4
4
4
4
3
24
nxy
4
3
2
2
2
2
15
08 v 3 1
Avail
Hhal
HpaW
Mspl
Sau3A\
Taql
0.42
nx
4
4
4
4
2
5
23
ny
5
4
4
4
4
4
25
nxy
4
2
2
2
2
2
14
08v30
Avail
Hhal
■ HpaW
Mspl
Sau3AI
Taql
0.32
nx
4
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10 v 32
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06v28
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06v26
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07v26
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08 v 24
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09v26
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06 v 19
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Taql
0.45
nx
4
4
4
4
2
5
23
ny
4
4
5
5
3
3
24
nxy
4
3
I
2
I
2
13 .
08 v 15
Avail
Hhal
Hpall
Mspl
Sau3Al
Taql
0.42
nx
4
4
4
4
2
5
23
ny
5
4
4
4
2
3
22
nxy
4
2
2
2
I
2
13
09 v 17
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.07
nx
4
4
3
3
2
5
21
ny
4
4
3
3
3
5
22
nxy
4
4
3
3
I
5
20
09 v 16
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.47
nx
4
4
3
3
2
5
21
ny
4
4
5
5
3
3
24
nxy
4
3
I
I
I
2
12
09 v 15
Avail
Hhal
. Hpall
Mspl
Sau3A\
Taql
0.49
nx
4
4
3
3
2
5
21
ny
5
4
4
4
2
3
22
nxy
4
2
I
I
I
2
II
IO v 17
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.20
nx
4
4
4
4
2
5
23
ny
4
4
3
3
3
5
22
nxy
4
4
2
2
I
5
18
IO v 16
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.45
nx
4
4
4
4
2
5
23
ny
4
4
5
5
3
3
24
nxy
4
3
I
2
I
2
13
IO v 15
Avail
Hhal
Hpall
Mspl
Sau3Al
Taql
0.42
nx
4
4
4
4
2
5
23
ny
5
4
4
4
2
3
22
nxy
4
2
2
2
I
2
13
nx
4
4
3
3
3
5
71
06 v 14
Avail
Hha\
HpaU
Mspl
Sau3AI
Taql
0.27
nx
4
4
3
3
3
5
22
ny
4
5
5
5
3
5
27
nxy
4
3
2
2
2
5
18
0 6 v 13
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.28
07 v 14
Avail
Hhal
Hpall
Mspl
SauSAl
Taql
0.49
nx
4
3
3
3
2
5
20
ny
4
5
5
5
3
5
27
nxy
2
I
I
I
2
5
12
07 v 13
Avail
Hhal
HpaW
Mspl
Sau3A\
Taql
0.51
nx
4
3
3
3
2
5
ny
4
4
4
4
2
7
20 25
08 v 14
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.24
nx
4
4
4
4
2
5
23
ny
4
5
5
5
3
5
27
nxy
4
3
2
3
2
5
19
08 v 13
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.29
nx
4
4
4
4
2
5
23
09 v 14
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.25
nx
4
4
3
3
2
5
21
ny
4
5
5
5
3
5
27
nxy
4
3
2
2
2
5
18
09 v 13
Avail
Hhal
HpaW
Mspl
Sau3A\
Taql
0.26
IO v 14
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.24
nx
4
4
4
4
2
5
23
ny
4
5
5
5
3
5
27
nxy
4
3
2
3
2
5
19
IO v 13
Avail
Hhal
HpaW
Mspl
Sau3Al
Taql
0.29
nx
4
4
3
3
3
5
ny
4
4
4
4
2
7
25
06 v 12
Avail
Hhal
HpaW
, Mspl
Sau3A\
Taql
0.28
nx
4
4
3
3
3
5
22
ny
4
4
4
4
2
7
25
nxy
4
3
2
2
I
5
17
nxy
I
I
2
2
0
5
II
07 v 12
Avail
Hhal
HpaW
Mspl
Sau3A\
Taql
0.42
nx
4
3
3
3
2
5
20
ny
4
4
4
4
2
7
25
nxy
2
I
2
2
I
5
13
ny
4
4
4
4
2
7
25
nxy
3
3
2
3
I
5
17
08 v 12
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.17
nx
4
4
4
4
2
5
23
ny
4
4
4
4
2
7
25
nxy
4
3
3
3
2
5
20
nx
4
4
3
3
2
5
21
ny
4
4
4
4
2
7
25
nxy
3
3
2
3
I
5 '
17
09 v 12
Avail
Hhal
HpaW
Mspl
Sau3A\
' Taql
0.22
nx ny
4
4
4
4
3 ■4
3
4
2
2
5
7
21 25
nxy
4
3
2
2
2
5.
18
nx
4
4
4
4
2
5
23
ny
4
4
4
4
2
7
25
nxy
3
3
2 .
3
I
5
17
. IO v 12
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
. 0.17
nx
4
4
4
4
2
5
23
nxy
4
3
3
3
2
5
20
22
nxy
3
3
2
3
I
5
.
17
ny
4
4
4
4
2
7
25
72
06 v 14
Avail
Hha\
HpaW
Msp\
Sau3A\
Taql
0.27
nx
4
4
3
3
3
5
22
n/
4
5
5
5
3
5
27
nxy
4
3
2
2
2
5
18
06 v 13
Avail
Hhal
HpaW
Mspl
SauSAI
Taql
0.28
nx
4
4
3
3
3
5
ny
4
4
4
4
2
7
22 25
nxy
3
3
2
3
I
5
17
06 v 12
Avail
Hhal
HpaW
Mspl
Sau3Al
Taql
0.28
nx
4
4
3
3
3
5
22
ny nxy
4
4
3
4
2
4
4
2
2
I
7 .5
25 17
07 v 14
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.49
nx
4
3
3
3
2
5
20
ny
4
5
5
5
3
5
27
nxy
2
I
I
I
2
5
12
07 v 13
Avail
Hhal
HpaW
Mspl
Sau3Al
Taql
0.51
nx
4
3
3
3
2
5
ny
4
4
4
4
2
7
20 25
nxy
I
I
2
2
0
5
II
07 v 12
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.42
nx
4
3
3
3
2
5
20
ny
4
4
4
4
2
7
25
nxy
2
I
2
2
I
5
13
08 v 14
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.24
nx
4
4
4
4
2
5
23
ny
4
5
5
5
3
5
27
nxy
4
3
2
3
2
5
19
08 v 13
Avail
Hhal
HpaW
Mspl
Sau3A\
Taql
0.29
nx
4
4
4
4
2
5
23
ny
4
4
4
4
2
7
25
nxy
3
3
2
3
I
5
17
08 v 12
Avail
Hhal
HpaW
Mspl
Sau3Al
Taql
0.17
nx
4
4
4
4
2
5
23
ny
4
4
4
4
2
7
25
nxy
4
3
3
3
2
5
20
09 v 14
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.25
nx
4
4
3
3
2
5
21
ny
4
5
5
5
3
5
27
nxy
4
3
2
2
2
5
18
09 v 13
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.26
nx
4
4
3
3
2
5
21
ny
4
4
4
4
2
7
25
nxy
3
3
2
3
I
5
17
09 v 12
Avail
Hhal
HpaW
/Vlspl
Sau3AI
Taql
0,22
nx
4
4
3
3
2
5
21
ny
4
4
4
4
2
7
25
nxy
4
3
2
2
2
5
18
IO v 14
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.24
nx
4
4
4
4
2
5
23
ny
4
5
5
5
3
5
27
nxy
4
3
2
3
2
5
19
IO v 13
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.29
nx
4
4
4
4
2
5
23
ny
4
4
4
4
2
7
25
nxy
3
3
2
3
I
5
17 '
IO v 12
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.17
nx
4
4
4
4
2
5
23
ny
4
4
4 .
4
2
7
25
nxy
4
3
3
3
2
5
20
73
06 v I I
Avail
Hhal
HpaW
Mspl
Sau3A\
Taql
0.00
nx
4
4
3
3
3
5
22
ny
4
4
3
3
3
5
22
nxy
4
4
3
3
3
5
22
06 v 10
Avail
Hhal
HpaW
Mspl
Sau3AI
Tdql
0.20
nx
4
4
3
3
3
5
ny
4
4
4
4
2
5
22 23
nxy
4
4
2
2
J
5
18
06v09
Avail
Hhal
HpaW
Mspl
SauSAl
Tdql
0.07
nx
4
4
3
3
3
5
22
ny
4
4
3
3
2
5
21
nxy
4
4
3
3
I
5
20
07 v I I
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.43
nx
4
3
3
3
2
5
20
ny
4
4
3
3
3
5
22
nxy
2
0
2
2
I
5
12
07 v 10
Avail
Hhal
HpaW
Mspl
Sau3AI
Tdql
0.53
nx
4
3
3
3
ny
4
4
4
4
2 2
5
5
20 23
nxy
2
0
I
I
I
5
10
07v09
Avail
Hhal
HpaW
Mspl
SauSAI
Taql
0.41
nx
4
3
3 ■
3
2
5
20
ny
4
4
3
3
2
5
21
nxy
2
0
2
2
I
5
12
08 v I I
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.20
nx
4
4
4
4
2
5
23
ny
4
4
3
3
3
5
22
nxy
4
4
2
2
I
5
18
08 v 10
Avail
Hhal
HpaW
Mspl
Sau3AI
Tdql
0.00
nx
4
4
4
4
2
5
23
ny
4
4
4
4
2
5
23
nxy
4
4
4
4
2
5
23
08v09
Avail
Hhal
HpaW
Mspl
SauSAI
Taql
0.14
nx
4
4
4
4
2
5
23
ny
4
4
3
3
2
5
2 1.
nxy
4
4
2
2
2
5
19
09 v I l
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.07
nx
4
4
3
3
2
5
21
ny
4
4
3
3
3
5
22
nxy
4
4
3
3
I
5
20
09 v 10
Avail
Hhal
HpaW
Mspl
Sau3AI
Tdql
0.14
nx
4
4
3
3
2
5
21
ny
4
4
4
4
2
5
23
nxy
4
4
2
2
2
5
19
09v09
Avail
Hhal
HpaW
Mspl
SauSAl
■ Tdql
.0
nx
4
4
3
3
2
5
21
ny
4
4
3
3
2
5
21
nxy
4
4
3
3
2
5
21
IO v I I
Avail
Hind fl
Hhal
HpaW
Mspl
Sau3AI
Tdql
0.20
nx
4
0
4
4
4
2
5
23
ny
4
0
4
3
3
3
5
22
nxy
4
0
4
2
2
I
5
18
IO v 10
Avail
Hind fl
Hhal
HpaW
Mspl
SauSAI
Tdql
0.00
nx
4
0
4
4
4
2
5
23
ny
4
O
4
4
4
nxy
4 .
O
4
4
4
2
2
5
23
5
23
74
06v08
Avoll
Hhal
HpaW
Mspl
SauSAl
Taql
0.20
nx
4
4
3
3
3
5
22
ny
4
4
4
4
2
5
23
nxy
4
4 •
2
2
I
5
18
06v07
Avail
Hhal
Hpall
Mspl
SauBAI
Taql
0.43
nx
4
4
3
3
3
5
22
ny
4
3
3
3
2
5
20
nxy
2
0
.
2
2
I
5
12
07v08
Avail
Hhal
Hpall
Mspl
SauBAI
Taql
0.53
nx
4
3
3
3
2
5
20
ny
4
4
4
4
2
5
23
nxy
2
0
I
I
I
5
10
07v07
Avail
Hhal
HpaW
Mspl
SauBAI
Taql
0.00
nx
4
3
3
3
2
5
20
ny
4
3
3
3
2
5
20
nxy
4
3
3
3
2
5
20
08v08
Avail
Hhal
Hpall
Mspl
SauBAI
Taql
0.00
nx
4
4
4
4
2
5
23
ny
4
4
4
4
2
5
23
nxy
4
4
4
4
2
5
23
06vQ 6
Avail
Hhal
HpaW
Mspl
SauBAI
. Taql
0.00
nx
4
4
3
3
3
5
22
ny
4
4
3
3
3
5
22
nx
4 '
4
3
3
3
5
22
75
I I v 41
Avail
Hha\
HpaW
Msp\
Sau3A\
Taql
0.20
nx
4
4
3
3
3
5
22
ny
4
6
5
6
2
5
28
nxy
4
4 ■
3
3
I
5
20
I I v 40
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.43
12 v 41
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.28
nx
4
4
4
4
2
7
25
ny
4
6
5
6
2
5
28
nxy
4
3
3
2
2
5
19
13 v 4 l
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.25
nx
4
4
4
4
2
7
25
ny
4
6
5
6
2
5
28
14 v 41 nx
Avail
4
Hhal
5
HpaW
5
Mspl
5
SauSAl 3
Taql
5
27
0.13
15 v 41
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.32
nx
5
4
4
4
2
3
22
nx
4
4
3
3
3
5
22
ny
5
5
3
3
2
2
20
nxy
3
2
2
2
I
2
12
I I v39
Avail
Hhal
. Hpall
Mspl
Sau3AI
Taql
0.26
nx
4
4
3
3
3
5
22
ny
4
4
4
4
2
3
21
nxy
4
4
2
2
I
3
16
12 v 40
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.33
nx
4
4
4
4
2
7
25
ny
5
5
3
3
2
2
20
nxy
3
2
3
3
2
2
15
. 12 v 39
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.22
nx
4
4
4
4
2
7
25
ny
4
4
4
4
2
3
21
nxy
4
3
3
3
2
3
18
nxy
3
3
4
4
I
5
20
13 v 40
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.51
nx
4
4
4
4
2
7
25
ny
5
5
3
3
2
2
20
nxy
2
2
2
2
I
2
II
I3 v 3 9
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.35
nx
4
4
4
4
2
7
25
ny
4
4
4
4
2
3
21
nxy
3
3
2
3
I
3
15
ny
4
6
5
6
2
5
28
nxy
4
5
4
4
2
5
24
. 14 v 40
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.40
nx
4
5
5
5
3
5
27
ny
5
5
3
3
2
2
20
nxy .
3
3 '
2
2
2
2
14
I4 v 3 9
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.29
nx
4
5
5
5
3
5
27
ny
4
4
4
4
2
3
21
nxy
4
3
2
3
2
3
17
ny
4
6
5
6
2
5
28
nxy
4
4
3
3
I
2
17
15 v 40
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.62
nx
5
4
4
4
ny
5
5
3
3
2
2
20
nxy
3
I
I
I5 v 3 9
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.40
nx
5
4
4
4
2
3
22
ny
4
4
4
4
2
3
21
nxy
4
2
2
2
I
2
13
2
3
22
I
8
76
I I v 38
Avoll
Hhal
Hpoll
Aispl
Sou3AI
Toql
0.26
nx
4
4
3
3
3
5
22
ny
4
4
4
4
2
3
21
nxy
4
4
2
2
I
3
16
I I v 37
Avail
Hhal
Hpall
Aispl
Sau3AI
Taql
0.05
ny
4
4
3
3
3
3
22 20
nxy
4
4
3
3
3
3
20
11 v 36
Avail
Hhal
HpaW
Mspl
Sau3A\
Taql
0.50
nx
4
4
3
3
3
5
22
ny
5
4
4
4
2
3
22
nxy
4
2
I
I
I
2
II
12 v 38
Avail
Hhal
Hpall
Mspl
Sau3A\
Taql
0.22
nx
4
4
4
4
2
7
25
n/
4
4
4
4
2
3
21
nxy
4
3
3
3
2
3
18
I2 v 3 7
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.33
nx
4
4
4
4
ny
4
4
3
3
3
3
20
nxy
4 .
3
2
2
I
3 .
15
I2 v 3 6
Avail
Hhal
Hpali
Mspl
Sau3AI
Toql
0.45
nx
4
4
4
4
2
7
25
ny
5
4
4
4
2
3
22
nxy
4
3
I
I
I
3
13
I3 v 3 8
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.35
nx
4
4
4
4
2
7
25
ny
4
4
4
4
2
3
21
nxy
3
3
2
3
I
3
15
13 v 3 7
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.33
nx
4
4
4
4
7
25
ny
4
4
3
3
3
3
20
nxy
3
3
2
3
I
3
15
I3 v 3 6
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
. 0.40
nx
4
4
4
4
2
7
25
ny
5
4
4
4
2
3
22
nxy
4
3
2
2
0
3
14
14 v 38
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.29
nx
4
5 '
5
5
3
5
27
ny
4
4
4
4
2
3
21
nxy
4
3
2
3
2
3
17
14 v 37
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.32
nx
4
5
5
5
3
5
27
ny
4
43
3
3
3
20.
nxy
4
3
2
2
2
3
16 '
I 4 v 3 6 nx
Avail
4
Hhal
5
Hpall
5
Aispl
5
' Sau3AI 3
Taql
5
0.18
27
ny
5
4
4
4
2
3
22
nxy
4
4
4
4
2
2
20
15 v 38
Avail
Hhal
Hpall
Aispl
Sau3AI
Taql
0.40
nx
5
4
4
4
2
3
22
ny
4
4
4
4
2
3
21
nxy
4
2
2
2
I
2
13
15 v 37
Avail
Hhal
HpaW
Mspl
Sau3A\
Taql
0.48
nx
5
4
4
4
ny
4
4
3
3
3
3
20
nxy
4
2
I
I
I
2
II
ny
5
4
4
4
2
3
22
nxy
5
4
4
4
2
3
22
nx
4
4
3
3
3
5
2
7
25
2
2
3
22
• . 15 v 36
Avail ■
Hhal
HpaW
Mspl
Sau3AI
Taql
0.00
nx
5
4
4
4
2
3
22
77
I I v 35
Avail
Hha\
HpaW
Msp I
Sau3AI
Taq\
0.50
nx
4
4
3
3
3
5
22
ny
5
4
4
4
2
3
22
nxy
4
3
I
I
0
2
II
I l v 34
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.50
ny
4
4
4
4
3
3
22 22
nxy
2
3
2
2
I
I .
11
I l v 33
. Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.50
12 v 35
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.49
nx
4
4
4
4
2
7
25
ny
5
4
4
4
2
3
22
nxy
4
2
I
I
I
3
12
12 v 3 4
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.66
nx
4
4
4
4
ny
4
4
4
4
3
3
22
nxy
2
2
I
I
I
I
8
13 v 3 5
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.40
nx
4
4
4
4
2
7
25
ny
5
4
4
4
2
3
22
nxy
4
2
2
2
I
3
14
13 v 3 4
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.40
nx
4
4
4
4
7
25
ny
4
4
4
4
3
3
22
14 v 35
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.27
nx
4
5
5
5
3
5
27
ny
5
4
4
4
2
3
22
nxy
4
3
4
4
I
2
18
14 v 34
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.47
nx
4
5
5
5
3
5
27
15 v 35
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.09
nx
5
4
4
4
2
3
22
ny
5
4
4
4
2
3
22
nxy
5
3
4
4
I
3
20
15 v 34
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.32
nx
5
4
4
4
nx
4
4
3
3
3
5
2
7
25
2
2
3
22
nx
4
4
3
3
3 •
5
22
ny
5
4
4
4
2
3
22
nxy
4
2.
I
I
I
2
II
12 v 33
Avail
Hhal
HpaW
• Mspl
5au3AI
Taql
0.45
nx
4
4
4
4
2
7
25
ny
5
4
4
4
2
3
22
nxy
4
3
I
I
I
3
13
nxy
4
2
2
3
I ■
2
14
13 v 3 3
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.40
nx
4
4
4
4
2
7
25
ny
5
4
4
4
2
3
22
nxy
4
3 .
2
2
0
3
14
ny
4
4
4
4
3
3
22
nxy
2
4
3
2
I
I
13
14 v 33
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0,18
nx
4
5
5
5
3
5
27
ny
5
4
4
4
2
3
22
nxy
4
4
4
4
2
2
20
ny
4
4
4
4
3
3
22
nxy
4
3
3
3
0 .
2
15
15 v 33
Avail
Hhal
HpaW
Mispl
SauSAl
■ Taql
0.00
nx
5
4
4
4
2
3
22
ny
5
4
4
4
2
3
22
nxy
5
4
4
4
2
3
22
78
I I v 32
Avoll
Hhal
HpaW
Mspl
Sau3A\
Taql
0.43
nx
4
4
3
3
3
5
22
n/
5
4
4
4
4
3
24
nxy
4 •
3
I
I
2
2
13
II v 31
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.49
ny
5
4
4
4
4
4
22 25
nxy
4
2
I
I
2
2
12
11 v 30
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.35
nx ny
4
4
4
4
3
7
3
7
3 ■2
5
3
22 27
nxy
4
4
3
3
0
2
16
12 v 32
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.51
nx
4
4
4
4
2
7
25
ny
5
4
4
4
4
3
24
nxy
4
2
I
I
2
2
12
12 v 3 1
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.48
nx
4
4
4
4
ny
5
4
4
4
4
4
25
nxy
4
3
I.
I
2
2
13
12 v 3 0
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.42
nx
4
4
4
4
2
7
25
ny
4
4
7
7
2
3
27
nxy
4
3
2
2
I
3
15
13 v 3 2
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.43
nx
4
4
4
4
2
7
25
ny
5
4
4
4
4
3
24
nxy
4
2
2
2
I
3
14
13 v 3 l
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.36
nx
4
4
4
4
7
25
ny
5
4
4
4
4
4
25
nxy
4
3
2
2
I
4 .
16
13 v 3 0
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.31
nx
4
4
4
4
2
7
25
ny
4
4
7
7
2
3
27
nxy
3
3
4
4
I
3
18
14 v 32
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.22
nx
4
5
5
5
3
5
27
ny
5
4
4
4
4
3
24
nxy
4
•
3
4
4
3
2
20
14 v 3 1
Avail
Hhal ■
HpaW
Mspl
SauSAl
Taql
0.23
nx
4
5
5
5
3
5
27
ny
5
4
4
4
4
4
25
nxy
4
3
4
4
3
2
20
14 v 30
Avail
Hhdl
' Hpall
Mspl
Squ3AI
Taql
0,44
nx
4 '
5
5
5
3
5
27
ny
4
4
7
7
2
3
27
nxy
4
3
3
2
I
2
15
15 v 32
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.13
nx
5
4
4
4
2
3
22
ny
5
4
4
4
4
3
24
nxy
5
3
4
4
2
2
20
15 v 3 1
Avail
Hhal
HpaW .
Mspl
Sau3AI
Taql
0.15
nx
5
4
4
4
ny
5
4
4
4
4
4
25
nxy
5
3
4
4
2
2
20
15 v 30
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.43
nx
5
4
4
4
2
3
22
ny
4
4
7
7
2
3
27
nxy
4
2
2
2
I
3
14
nx
4
4
3
3
3
5
2
7
25
2
2
3
22
79
I I v 2 9 nx
Avail
4
H hal
4
HpaW
3
M spl
3
Sau3AI 3
Taql
5
0.24
22
ny
4
4
4
4
2
5
23
nxy
4
3
2
2
I
5
17
I I v 2 8 nx
Avail
4
4
H hal
3
H p a ll
3
M spl
Sau3AI 3
Taql
5
22
0.00
ny
4
4
3
3
3
5
22
nxy
4
4
3
3
3
5
22
I I v 27 nx
Avail
4
H hal
4
H p a ll
3
M spl
3
Sau3AI 3
Taql
5
22
0.20
ny
4
4
4
4
2
5
23
nxy
4
4
2
2
I
5
18
I 2 v 2 9 nx
Avail
4
H hal
4
H p a ll
4
M spl
4
S au3A \
2
Taql
7
0.29
25
ny
4
4
4
4
2
5
23
nxy
4
2
2
2
2
5
17
I 2 v 2 8 nx
Avail
4
4
H hal
4
H p a ll
M spl
4
Sau3AI 2
Taql
7
25
0.28
ny
4
4
3
3
3
5
22
nxy
4
3
2
2
I
5
17
12 v 27 nx
Avail
4
H hal
4 '
HpaW
4
M spl
4
Sau3A\
2
7
Taql
0.17
25
ny
4
4
4
4
2
5
23
nxy
4
3
3
3
2
5
20
13 v 29 nx
Avail
4
H hal
4
H p a ll
4
M spl
4
Sau3AI 2
Taql
7
25
0.29
ny
4
4
4
4
2
5
23
nxy
3
2
3
3
I
5
17
I 3 v 2 8 nx
Avail
4
4
H hal
H p a ll
4
4
M spl
Sau3AI 2
7
Taql
25
0.28
ny
4
4
3
3
3
5
22
nxy
3
3
2
3
I '
5
17
13 v 27 nx ny
Avail
4
4
4
4
. Hhal
HpaW
4
4
M spl
4
4
Squ3A\
2
2
Taql
7
5
0.29
25 .23
nxy
3
3
2
3
I
5
17
14 v 29 nx
Avail
4
H hal
5
H p a ll
5
M spl
5
S au3A \
3
Taql
5
046
27
ny
4
4
4
4
2
5
23
nxy
4
3
3
4
2
5
21
I 4 v 2 8 nx
. Avail
4
5
Hhal
H p a ll
5
M spl ■ 5
Sau3AI 3
5
Taql
27
0.27
ny
4
4
3
3
3
5
22
nxy
4
3
2
2
2
5
18
14 v 27 nx
Avail
4
Hhal
5
HpaW
5
-M spl
5
Sau3A\
3
Taql
5
27
0.24
ny
4
4
4
4
2
5
23
nxy
4 ■
3
2
3
2
5
19
15 v 29 nx
Avail
5
H hal
4
H p a ll
4
M spl
4
Sau3A! 2
Taql
3
22
0.47
ny
4
4
4
4
2
5
23
nxy
4
3
I
I
I
2
12
15 v 28 nx
Avail
5
Hhal
4
4
H p a ll
M spl
4
ny
4
4
3
3
3
5
22
nxy
4
2
I
I
I
2
II
15 v 2 7 nx
Avail
5
Hhal
4
HpaW
4
M spl
4
Sau3AI 2
Taql
3
22
0.42
ny
4
4
4
4
2
5
23
nxy
4
2
2
2
I
2
13
Sau3A \
2
Taql
3
0.50
22
80
I I v 26 nx
Avail
4
Hhal
4
H p a ll
3
M spl
3
Sau3AI 3
Taql
5
0.65
22
n / nxy
2
2
4
3
2
0
2
0
3
I
5
I
18 7
I 2 v 2 6 nx
Avail
4
H hal
4
H p a ll
4
M spl
4
Sau3AI 2
Taql
7
25
0.67
ny
2
4
2
2
3
5
18
I 3 v 2 6 nx
Avail
4
H hal
4
HpaW
4
M spl
4
Sau3AI 2
Taql
7
25
0.63
11 v 2 5 nx ny
Avail
4
4
H hal
4
4
4
H p a ll
3
M spl
3
4
3
Sau3A \
2
5
Taql
5
22 23
0.29
nxy
4
2
2
2
I
5
16
11 v 2 4 nx ny
Avail '
4
4
Hhal
4
5
H p a ll
3
3
M spl
3
3
Squ3A\
3
3
Taql
5 -5
22 23
. 0.07
nxy
4
3
3
3
3
5
21
12 v 25 nx
Avail
4
H hal
4
4
H p a ll
M spl
4
Sau3AI 2
Taql
7
25
0.17
ny
4
4
4
4
2
5
23
nxy
4
3
3
3
2
5
20
12 v 24 nx
Avail
4
H hal
4
■ H p a ll
4
M spl
4
Sau3A\
2
Taql
7
0.25
25
ny
4
5
3
3
3
5
23
nxy
4
4
2
2
I
5
18
ny nxy
2
2
4
2
2
0
2
2
3
0
5
2
18 8
13 v 2 5 nx
4
Avail
4
H hal
4
H p a ll
M spl
4
ny
4
4
4
4
2
5
23
nxy
3
3
2
3
I
5
17
13 v 24 nx
Avail
4
Hhal
4
H p a ll
4
M spl
4
Sau3AI 2
Taql
7
0.25
25
ny
4
5
3
3
3
5
23
nxy
3
4
2
3
I
5
18
14 v 26 nx
Avail
4
H hal
5
H p a ll
5
M spl
5
Sau3AI 3
Taql
5
27
0.73
ny
2
4
2
2
3
5
18
nxy
2
3
0
0
0
I
6
14 v 25 nx
Avail
4
5
H hal
H p a ll
5
5
M spl
Sau3AI 3
Taql
5
27
0.20
ny nxy
4
4
4
4
4
2
4
3
2
2
5 .5
23 20
14 v 24 nx
Avail
4
H hal
5
5
H p a ll
M spl
5
' SauSAI 3
■ Taql
5
0.20
27
ny
4
5
3
3
3
5
23
nxy
4
5
2
2
2
5
20
15 v 26 nx
Avail
5
H hal
4
H p a ll
4
M spl
4
Sau3AI 2
Taql
3
0.80
22
ny nxy
2
I
4
3
2
0
2
0
3
0
5
0
18 4 .
15 v 25 nx
5
Avail
H hal
4
H p a ll
4
M spl
4
Sau3AI 2
3
Taql
22
0.33
ny
4
4
4
4
2
5
23
15 v 24 nx
Avail
5
H hal
4
H p a ll
4
• Mspl
4
Sau3AI 2
Taql
3
22
0.42
ny nxy
4
4
5
4
3
I
3 ' I
3
I
2
5
23 13
nxy
2
2
0
0
0
3
7
-
Sau3A \
Taql
0.29
2
7
25
nxy
4
4
2
2
I
2
15
■
81
I l v 23 nx
Avail
4
Hha\
4
HpaW
3
M spl
3
S au3A \
3
Taql
5
22
0.24
ny
4
4
4
4
2
5
23
nxy
4
3
2
2
I
5
17
I I v 22 nx ny
4
4
Avail
4
4
H hal
3
3
H p a ll
3
M spl
3
3
Sau3AI 3
5
5
Taql
22 22
0.05
nxy
4
3
3
3
3
5
21
11 v 21 nx
Avail
4
H hal
4
H p a ll
3
M spl
3
Sau3A\
3
Taql
5
22
0.62
ny
4
4
5
5
3
4
25
nxy
2
2
I
2
I
I
9
12 v 23 nx
Avail
4
H hal
4
H p a ll
4
M spl
4
S au3A \
2
Taql
7
0.21
25
ny
4
4
4
4
2
5
23
nxy
4
4
2
2
2
5
19
12 v 22 nx
4
Avail
4
H hal
4
H p a ll
4
M spl
Sau3AI 2
7
Taql
25
0.23
ny
4
4
3
3
3
5
22
nxy
4
4
2
2
I
5
18
12 v 2 l
Avail
nx
4
4
4
4
2
7
25
ny
4
4
5
5
3
4
25
nxy
2
3
2
2
I
I
II
13 v 2 3
Avail
ny
4
4
4
4
2
5
23
nxy
3
4
3
3
I
5
19
13 v 2 2 nx
4
Avail
4
H hal
4
H p a ll
4
M spl
7
25
ny
4
4
3
3
3
5
22
nxy
3
4
2
3
I
5
•18
13 v 21 nx
Avail
4
4
H hal
H p a ll
4
. M spl
4
Sau3AI 2
Taql
7
25
0.56
ny
4
4
5
5
3
4
25
nxy
3
3
I
I
I
2
II
14 v 23 nx
Avail
4 .
5
H hal
H p a ll
5
M spl
5
Sau3AI 3
Taql
5
0.12
27
ny
4
4
4
4
2
5
23
nxy
4
4
3
4
2
5
22
14 v 22 nx
4
Avail
5
H hal
5
H p a ll
5
M spl
3
S au3A l
5
Taql
27
0.22
ny
4
4
3
3
3
5
22
nxy
4
4
2
2
2
5
19
14 v 2 l
Avail
nx
4
5
5
5
3
5
27
ny
4
4
5
5
3
4
25
nxy
2
3
3
3
2
I
14
15 v 23 nx
Avail
5
H hal
4
H p a ll
4
M spl
4
S au3A l
2
Taql
3
0.47
22
ny
4
4
4
4
2
5
23
nxy
4
3
I
I
'
I
2
12
15 v 22 nx
5
Avail
H hal
4
4
H p a ll
4
M spl
5au3AI 2
3
Taql
0.45
22
ny
4
4
3
3
3 "
5
22
nxy
4
3
I
I
I
2
12
15 v 2 l
Avail
nx
5
Hhal
4
H p a ll
4
M spl
4
Sau3A\
2
Taql
3
0.45 ■ 22
ny
4
4
5
5
3
4
25
nxy
3
3
3
3
I
0
13
Hhal
H p a ll
M spl
Sau3AI
Taql
0.21
nx
4
4
4
4
2
7
25
S au3A l
Taql
0.23
2
Hhal
H p a ll
M spl
Sau3AI
Taql
0.56
Hhal
H p a ll
M spl
Sau3A\
Taql
0.46
82
I I v 2 0 nx
Avail
4
H hd
4
Hpall
3
Wlspl
3
Sau3AI 3
Taql
5
0.22
22
nY
4
4
4
4
3
5
24
nxy
4
4 '
2
2
I
5
18
I l v 19 nx ny
Avail
4
5
H hal
4
4
3
H p a ll
3
M spl
3
3
2
SauSAl
3
Taql
3
5
22 20
0.38
nxy
4
3
2
2
0
2
13
I l v 18 nx
■ Avail
4
Hhal
4
H p a ll
3
M spl
3
SauSAl
3
5
Taql
0.05
22
ny
4
4
3
3
3
3
20
nxy
4
4
3
3
3
3
20
12 v 20 nx
Avail
4
H hd
4
Hpdl
4
M spl
4
SauSAl
2
Taql
7
0.18
25
ny
4
4
4
4
3
5
24
nxy
4
3
3
3
2
5
20
12 v 19 nx
4
Avail
H hal
4
H p a ll
4
M spl
4
ny
5
4
3
3
2
3
20
nxy
4
2
I
I
I
3
12
12 v 18 nx
Avail
4'
Hhal
4
H p a ll
4
Mspl
4
Sau3AI 2
Taql
7
. 0.33
25
ny
4
4
3
3
3
3
20
nxy
4
3
2
2
I
3
15
I 3 v 2 0 nx
Avail
4
H hal
4
H p a ll
4
M spl
4
SauSAl
2
Taql
7
0.31
25
ny
4
4
4
4
3
5
24
nxy
3
3
2
3
I
5
17
13 v 19 nx
4
Avail
H hal . 4
4
• H p a ll
M spl
4
7
25
ny
5
4
3
3
2
3
20
nxy
4
2
2
2
I
3
14
13 v 18 nx
Avail
4
H hal
4
H p a ll
4
M spl
4
SauSAl
2
Taql
7
0.33
25
ny
4
4
3
3
3
3
20
nxy
3
3
2
3
I
3
15
14 v 20 nx
Avail
4
H hal
5
H p a ll
5
M spl
5
SauSAl
3
Taql
5
0.25
27
ny
4
4
4
4
3
5
24
nxy
4
3
2
3
2
5
19
I 4 v 19 nx
Avail
4
H hal
5
HpaW .
5
■Mspl
5 '
Sau3AI 3
5
Taql
27
0.49
ny
5
4
3
3
2
3
20
nxy
4
3
I
I
I
2
12
14 v 18 nx
Avail
4
H hal
5
H p a ll
5
M spl
5
SauSAl
3
5
T a q l.
27
0.32
ny
4
4
3
3
3
3
20
nxy
4
3
2
2
2
3
16
15 v 20 nx
Avail
5
H hal
4
H p a ll
4
M spl
4
SauSAl
2
Taql
3
22
0.43
ny
4
4
4
4
3
5
24
nxy
4
2
2
2
I
2
13 '
15 v 19 nx
5
Avail
H hal
4
H p a ll
4
M spl
4
ny
5
4
3
3
2
3
20
nxy
5 •
3
I
I
I
3
14
15 v 18 nx
Avail
5
H hal
4
H p a ll
4
Mspl
4
Sau3AI 2
Taql
3
22
0.48
ny
4
4
3
3
3
3
20
nxy
4
2
I
I
I
2
II
SauSAl
Taql
0.47
SauSAl
Taql
0.38
2
7
25
2
SauSAl
Taql
2
0.33
22
3
83
I l v 17 nx
Avail
4
Hha\
4
HpaW
3
M sp\
3
S au3A \
3
Taql
5
0.00
22
ny
4
4
3
3
3
5
22
nxy
4
4
3
3
3
5
22
I l v 16 nx
Avail
4
H hal
4
3
H p a ll
M spl
3
3
S au3A l
Taql
5
0.43
22
ny nxy
4
4
4
3
5
I
5
I
3
2
3 .2
24 13
I l v 15 nx
Avail
4
4
H hal
H p a ll
3
M spl
3
S au3A l
3
Taql
5
0.50
22
ny
5
4
4
4
2
3
22
nxy
4
2
I
I
I
2
II
12 v 17 nx
Avail
4
4
H hal
H p a ll
4
M spl
4
S au3A \
2
Taql
7
25
0.28
ny
4
4
3
3
3
5
22
nxy
4
3
2
2
I
5
17 '
12 v 16 nx
Avail
4
Hhal
4
H p a ll
4
M spl
4
7
25
ny
4
4
5
5
3
3
24
nxy
4
2
I
I
I
3
12
12 v 15 nx
Avail • 4
H hal
4
H p a ll
4
M spl
4
SauSAI 2
Taql
7
0.45
25
ny
5
4
4
4
2
3
22
nxy
4
3
I
I
I
3
13
13 v 17 nx
Avail
4
H hal
4
H p a ll
4
M spl
4
Sau3Al
2
Taql
7
25
0.28
ny
4
4
3
3
3
5
22
nxy
3
3
2
3
I
5
17
13 v 16 nx
Avail
4
H hal
4
4
H p a ll
M spl
4
SauSAI 2
Taql '
7
25
0.43
ny
4
4
5
5
3
3
24
nxy
3
2
2
3
I
3
14
13 v 15 nx
Avail
4
H hal
4
H p a ll
4
Mspl
4
SauSAI 2
. Taql
7
25
0.40
ny
5
4
4
4
2
3
22
nxy
4
3
2
2
0
3
14
14 v 17 nx
Avail
4
H hal
5
Hpal I
5
Mspl
5
Sau3AI 3
Taql
5
27
0.27
ny
4
4
3
3
3
5
22
nxy
4
3
2
2
2
5
18
14 v 16 nx
Avail
4
H hal
5
H p a ll
5
M spl
5
Sau3A \
3
Taql
5
0.33
27
ny
4
4
5
5
3
3
24
nxy
4
3
3
3
2
2
17
14 v 15 nx
Avail
4
H hal
5
5
H p a ll
M spl
5
Sau3AI 3 .
Taql
5
27
0,18
ny
5
4
4
4
2
3 .
22
nxy
4
4
4
4
2
2
20
15 v 17 nx
5
Avail
H hal
4
H p a ll
4
M spl
4
S au3A \
2
Taql
3
22
0.50
ny
4
4
3
3
3
5
22
nxy .
4
2
I
I
I
2
II •
15 v 16 nx
Avail
5
H hal
4
H p a ll
4
M spl
4
Sau3A \
2
Taql
3
0.26
22
ny
4
4
5
5
3
3
24
nxy
4
3
3
3
I
3
17
15 v 15 nx
Avail
5
H hal
4
H p a ll
4
M spl
4
S au3A l
2
Taql
3
22
0.00
ny
5
4
4
4
2
3
22
nxy
5
4
4
4
2
3
22
Sau3A \
Taql
0.51
2
84
I l v 14 nx
Avail
4
Hhal
' 4
HpaW
3
M sp\
3
3
S au3A \
Taql
5
22
0.27
ny
4
5
5
5
3
5
27
nxy
4
3
2
2
2
5
18
I l v 13 nx
Avail
4
■H h a l
4
3
H p a ll
3
M spl
3
SauSAl
5
Taql
22
0.28
ny
4
4
4
4
2
7
25
nxy
3
3
2
3
I
5
17
I l v 12 nx
Avail
4
H hal
4
HpaW
3
M spl
3
SauSAl
3
Taql
5
22
0.28
ny
4
4
4
4
2
7
25
nxy
4
3
2
2
I
5
17
12 v 14 nx
Avail
4
H hal
4
H p a ll
4
M spl
4
S auS A l
2
Taql
7
25
0.27
ny
4
5
5
5
3
5
27
nxy
4
4
2
2
2
5
19
12 v 13 nx
Avail
4
4
H hal
4
H p a ll
M spl
4
SauSAl
2
7
Taql
25
0.32
ny
4
4
4
4
2
7
25
nxy
3
4
2
2
I
5
17 •
12 v 12 nx
Avail
4
Hhal
4
HpaW
4
M spl
4
SauSAl
2
Taql
7
0.0,0 . 25
ny
4
4
4
4
2
7
25
nxy
4
4
4
4
2
7
25
13 v 14 nx
Avail
4
H hal
4
H p a ll
4
M spl
4
SauSAl
2
Taql
7
25
0.27
ny
4
5
5
5
3
5
27
nxy
3
4
.
3
3
I
5
19
13 v 13 nx
4
Avail
Hhal
4
4
HpaW
M spl
4
2
SauSAl
Taql
7
25
0.00
ny
4
4
4
4
2
7
25
nxy
4
4
4
4
2
7
25
I 4 v 14 nx
Avail
4
5
H hal
H p a ll
5
M spl
5
S auS A l
3
Taql
5
0.00
27
ny
4
5
5
5
3
5
27
nxy
4
5
5
5
3
5
27
Ilv ll
Avail
Hfial
Hpall
Mspl
5au3AI
Taql
0.00
nx
4
4
3
3
3
5
22
ny
4
4
3
3
3
5
22
nxy
4
4
3
3
3
5
22
86
16 v 41 nx
Avoll
4
H hal
4
H p a ll
5
Mspl
5
SooSAI 3
Taql
3
0.35
24
nY nxy
4
4
6
4
5
2
6
4
2
I
5
2
28 17
16 v 40 nx
Avail
4
4
Hhal
5
H p a ll
M spl
5
3
Sau3A \
Taql
3
24
0.64
17 v 41 nx
Avail
4
H hal
4
H p a ll
3
M spl
3
Sou3AI 3
5
Taql
0.20
22
ny
4
6
5
6
2
5
28
nxy
4
4
3
3
I
5
20
17 v 40 nx
Avail
4
4
H hal
3
H p a ll
3
M spl
Sau3AI 3
5
Taql
18 v 41 nx ny
Avail
4
4
H hal
6
4
Hpall
3
5
Mspl
3
6
Sau3AI 3
2
Taql
3
5
0.25
20 28
nxy .
4
4
3
3
I
3
18
18 v 40 nx
4
Avail
4
Hhal
3
H p a ll
M spl
3
3
SauSAl
3
Taql
19 v 4 l
Avail
nxy
4
4
2
2
I
2
15
19 v 40 nx
Avail
5
H hal
4
3
H p a ll
M spl
3
H hal
H p a ll
M spl
Sau3AI
Taql
0.38
nx
5
4
3
3
2
3
20
20 v 41 nx
Avail
4
H hal
4
H p a ll
4
M spl
4
Sau3AI 3
5
Taql
24
0.19
ny
4
6
5
6
2
5
28
ny
4
6
5
6
2
5
28
nxy
4
4
3
3
2
5
21
ny
5
5
3
3
2
2
20
nxy
3
I
I
I
I
I
8
ny
5
5
3
3
2
2
22 20
nx
4
4
5
5
3
3
24
ny
4
4
4
4
2
3
21
nxy
4
3
I
2
I
2
13
nxy
3
2
2
2
I
2
12
17 v 39 nx
Avail
4
Hhal
4
H p a ll
3
M spl
3
SauSAl
3
Taql
5
22
0.26
ny
4
4
4
4
2
3
21
nxy
4
4
2
2
I
3
16
nxy
3
2
2
2
I
2
12
I8 v 3 9
Avail
nx
4
H hal
4
3 ,
H p a ll
M spl • 3
Sau3AI 3
Taql'
3
20
0.22
ny
4
4
4
4
2
3
21
nxy
4
4
2
2
I
3
16
ny
5
5
3
3
2
2
20
nxy
3
I
I
I
I
I
8
I9 v 3 9
Avail
nx
5
4
3
3
2
3
20
ny
4
4
4
4
2
3
21
nxy
4
3
I
I
I
2
12
2 0 v4 0 nx ny
nxy
3
2
2
2
2
2
13
2 0 v 3 9 nx
Avail
4
Hhal
4
H p a ll
4
M spl
4
SauSAl
3
Taql
5
0.07
24
ny
4
4
4
4
2
3
21
nxy
4
4
4
4
2
3
21
0.43
0.40
ny
5
5
3
3
2
2
20 20
SauSAl
Taql
2
0.60
20
Avail
H hal
HpaW
M spl
Sau3AI
Taql
0.41
3
4
4
4
4
3
5
24
5
5
3
3
2
2
20
I6 v 3 9
Avail
H hal
H p a ll
M spl
SauSAl
Taql
0.42
Hhal
H p a ll
Mspl
Sau3AI
Taql
0.41
87
I6 v 3 8
Avoll
Hha\
HpaW
Msp\
Sou3AI
Taql
0.42
nx
4
4
5
5
3
3
24
n/
4
4
4
4
2
3
21
nxy
4
3
I
2
I
2
13
I6 v 3 7
Avail
Hhal
Hpall
Mspl
SauSAl
Taql
0.41
nx ny
4
4
4
4
5
3
5
3
3
3
3
3
24 20
nxy
4
3
I
I
2
■
2
13
17 v 38
Avoll
Hhal
Hpall
Mspl
Sou3AI
Taql
0.26
nx
4
4
3
3
3
5
22
ny
4
4
4
4
2
3
21
nxy
4
4
2
2
I
3
16
17 v 37
Avail
Hhal
Hpall
Mspl
SauSAl
Taql
0.05
nx
4
4
3
3
3
5
Tl
ny
4
4
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Avail
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16
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Avail
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0.26
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18
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0.35
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0.36
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0.42
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0.45
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0.24
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17
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16
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0.40
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23
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4
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0.15
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4
4
4
4
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24
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23
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0.22
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13
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0.44
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4
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12
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4
4
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nxy
2
2
3
3
2
0
12
17 v 23
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.24
nx
4
4
3
3
3
5
22
ny
4
4
4
4
2
5
23
nxy
4
3
2
2
I
5
17
. 17 v 22
Avail
Hhal
Hpall
Mspl
SauSAl
Taql
0.05
nx
4
4
3
3
3
5
ny
4
4
3
3
3
5
22
nxy
4
3
3
3
3
5
21
17 v 21
Avail
Hhal
Hpall
Mspl
SauSAl
Taql
0.62
nx
4
4
3
3
3
5
22
ny
4
4
5
5
3
4
25
nxy
2
2
I
2
I
I
9
18 v 23
Avail
Hhal
Hpall
Mspl
SauSAl
Taql
030
nx
4
4
3
3
3
3
20
ny
4
4
4
4
2
5
23
nxy
4
3
2
2
I
3
15
18 v 22
Avail
Hhal
Hpall
Mspl
SauSAl
Taql
0.10
ny
4
4
3
3
3
5
20 22
nxy
4
3
3
3
3
3
19
18 v 2 1
Avail
Hhal
Hpall
Mspl
SauSAl
Taql
0.60
nx
4
4
3
3
3
3
20
ny
4
4
5
5
3
4
25
nxy
2
2
I
2
I
I
9
I9 v 2 3
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.40
nx
5
4
3
3
2
3
20
ny
4
4
4
4
2
5
23
nxy
4
4
I
I
I
2
13
19 v 2 2
Avail
Hhal
Hpall
Mspl
SauSAl
Taql
0.43
nx
5
4
3
3
20
ny
4
4
3
3
3
5
22
nxy
4
2
2
2
0
2
12
19 v 2 1
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.69
nx
5
4
3
3
2
3
20
ny
4
4
5
5
3
4
25
nxy
3
2
I
I
0
0
7
20v23
Avail
Hhal
Hpall
Mspl
SauSAl
Taql
0.15
nx
4
4
4
4
3
5
24
ny
4
4
4
4
2
5
23
nxy
4
3
3
3
2
5
20
20v22
Avail
Hhal
Hpall
Mspl
SauSAl
Taql
0.26
nx
4
4
4
4
3
5
24
ny
4
4
3
3
3
5
22
nxy
4
3
2
2
I
5
17
20v2l
Avail
Hhal
Hpall
Mspl
SauSAl
Taql
0.59
nx
4
4
4
4
3
5
24
ny
4
4
5
5
3
4
25
nxy
2
2
2
2
I
I
10
22
nx
4
4
3
3
3
3
2
3
93
I 6 v 2 0 nx
4
4
5
5
3
3
24
n/
4
4
4
4
3
5
24
nxy
4
3
I
2
I
2
13
16 v 19 nx
Avail
4
Hhal
4
HpaW . 5
M spl
5
SauSAl
3
Taql
3
17 v 20 nx
Avail
4
H hal
4
H p a ll
3
M sp l
3
Sau3AI 3
Taql
5
22
0.22
ny
4
4
4
4
3
5
24
nxy
4
4
2
2
I
5
18
17 v 19 nx
Avail
4
Hhal
4
HpaW
3
M sp l
3
SauSAl
3
Taql
5
22
0.38
ny
5
4
3
3
2
3
20
18 v 20 nx
Avail
4
H hal
4
HpaW
3
M sp l
3
Sau3AI 3
Taql
3
nxy
4
4
2
2
I
3
16
18 v 19 nx
Avail
4
Hhal
4
HpaW
3
M spl
3
SauSAl
3
Taql
3
0.35
20
ny
5
4.
3
3
2
3
20
ny
4
4
4
4
3
5
24
19 v 20 nx
Avail
5
H hal
4
HpaW
3
M sp l
3
Sau3AI 2
Taql
3
0.45
20
ny
4
4
4
4
3
5
24
nxy
4
3
I
I
I
2
12
20 v 20 nx
Avail
4
Hhal
4
HpaW
4
M sp l
4
3
SauSAl
Taql
5
24
0.00
ny
4
4
4
4
3
5
24
nxy
4
4
4
4
3
5
24
Avail
Hhal
H p a ll
M sp l
SauSAl
Taql
0.46
0.27
0.32
ny
5
4
3
3
2
3
24 20
19 v 19 nx
Avail
'
H hal
HpaW
M sp l
SauSAl
Taql
0.00
16 v 18 nx
Avail
4
Hhal
4
HpaW
5
M spl
5
SauSAl
3
Taql
3
24
0.41
ny
4
4
3
3
3 ,
3
20
nxy
4
3
2
2
■
0
2
13
17 v 18 nx
Avail
4
Hhal
4
HpaW
3
M spl
3
SauSAl
3
Taql
5
22
0.05
ny
4
4
3
3
3
20
20
18 v 18 nx
Avail
4
H hal
4
3
HpaW
M sp l
3
SauSAl
3
Taql
3
20
0.00
ny
4
4
3
3
3
3
20
nxy
4
4
3
3
3
3
20
nxy .
4
3
2
2
0
2
20 13
ny
5
4
3
3
2
3
20 20
5
4
3
3
2
3
nxy
4
4
I
2
I
3
15
nxy
5
4
3
3
2
3
20
3
nxy
4
3
I
I
2
2
13
nxy
4
4
3
3
3
3
94
16 v 17
Avail
Hhal
HpaW
Msp\
Sau3AI
Taql
0.43
nx
4
4
5
5
3
3
24
ny
4
4
3
3
3
5
22
nxy
4
3
I
I
2
2
13
17 v 17
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.00
nx
4
4
3
3
3
5
22
ny
4
4
3
3
3
5
22
nxy
4
4
3
3
3
5
22
16 v 16
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.00
nx
4
4
5
5
3
3
24
ny
4
4
5
5
3
3
24
nxy
4
4
5
5
3
3
24
95
21 v 41
Avail
Hhal
Hpall
Aispl
Sau3AI
Taql
0.58
nx
4
4
5
5
3
4
25
ny
4
6
5
6
2
5
28
nxy
2
3
2
2
I
I
II
21 v 40
Avail
Hhal
Hpall
Mspl
Sau3A\
Taql
0.64
nx
4
4
5
5
3
4
25
22 v 41
Avail
Hhal
Hpall
Aispl
Sau3AI
Taql
0.24
nx
4
4
3
3
3
5
22
ny
4
6
5
6
2
5
28
nxy
4
3
3
3
I
5
19
nx
4
4
3
3
3
5
23 v 41
Avail
Hhal
Hpall
Mspl
Sau3A\
Taql
0.22
nx
4
4
4
4
2
5
23
ny
4
6
5
6
2
5
28
24 v 41
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
048
nx
4
5
3
3
3
5
23
25 v 41
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
048
nx
4
4
4
4
2
5
23
ny
5
5
3
3
2
2
20
nxy
I
2
I
2
I
I
8
21 v 39
Avail
Hhal
Hpall
Mspl
Sau3Al
Taql
0.57
nx
4
4
5
5
3
4
25
ny
4
4
4
4
2
3
21
nxy
2
2
2
2
I
I
10
22v40
Avail
Hhal
Hpall
Aispl
Sau3AI
Taql
0.43
ny
5
5
3
3
2
2
22 20
nxy
3
2
2
2
I
2
12
22v39
Avail
Hhal
Hpall
Aispl
Sau3AI
Taql
030
nx
4
4
3
3
3
5
22
ny
4
4
4
4
2
3
21
nxy
4
3
2
2
I
3
15
nxy
4
3
3
3
2
5
20
23 v 4 0
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.40
nx
4
4
4
4
5
23
ny
5
5
3
3
2
2
20
nxy
3
2
2
2
2
2
13
23v39
Avail
Hhal
Hpall
Mspl
Sau3A{
Taql
0.18
nx
4
4
4
4
2
5
23
ny
4
4
4
4
2
3
21
nxy
4
3
3
3
2
3
18
ny
4
6
5
6
2
5
28
nxy
4
5
3
3
I
5
21
24 v 40
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.40
nx
4
5
3
3
3
5
23
ny
5
5
3
3
2
2
20
nxy
3
3
2
2
I
2
13
24v39
Avail
Hhal
HpaW
Mspl
■Sau3AI
Taql
0.32
nx
4
5
3
3
3
5
23
ny nxy
4
4
4
3
4
2
2
4
2 ■I
3
3
21
15
ny
4
6
5
6
2
5
28
nxy
4
4
3
3
2
5
21
25 v 40
Avail
Hhal
Hpall
Mspl
Sau3Al
Taql
0.44 •
nx
4
4
4
4
ny
5
5
3
3
2
2
20
nxy
3
I
2
2
2
2
12
25v39
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.14
nx
4
4
4
4
2
5
23
ny
4
4
4
4
2
3
21
2
2
5
23
nxy
4
2
4
4
2
3
19
96
21 v 38
Avoll
Hhal
HpaW
Mspl
Sau3A\
Taql
0.57
nx
4
4
5
5
3
4
25
ny
4
4
4
4
2
3
21
nxy
2
2
2
2
I
I
IO
2 I v 37
Avail
Hhal
HpaW
Mspl
Sau3A\
Taql
0.60
nx
4
4
5
5
3
4
25
ny
4
4
3
3
3
3
20
nxy
2 .
2
I
2
I
I
9
21 v 36
Avail
• Hhal
HpaW
Mspl
Sau3A\
Taql
0.45
nx
4
4
5
5
3
4
25
ny
5
4
4
4
2
3
22
22v38
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
OJO
nx
4
4
3
3
3
5
22
ny
4
4
4
4
2
3
21
nxy
4
3
2
2
I
3
15
22v37
Avail
Hhal ■
HpaW
Mspl
Sau3AI
Taql
0.10
nx
4
4
3
3
3
5
ny
4
4
3
3
3
3
20
nxy
4
3
3
3
3
3
1.9
22v36
Avail
Hhal
HpaW
Mspl
SauSAI
Taql
0.45
nx
4
4
3
3
3
5
22
ny nxy
5
4
3
4
4 . I
4
I
2
I
3
2
22 12
23v38
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.18
nx
4
4
4
4
2
5
23
ny
4
4
4
4
2
3
21
nxy
4
3
3
3
2
3
18
23v37
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
OJO
5
23
ny
4
4
3
3
3
3
20
nxy
4
3
2
2
I
3
15
23v36
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.47
nx
4
4
4
4
2
5
23
ny
5 ■
4
4
4
2
3
22
24v38
Avail
Hhal
HpaW
Msp I
Sau3AI
Taql
0.32
nx
4
5
3
3
3
5
23
ny nxy
4 . 4
4
3
4
2
4
2
2
I
3 ■3
21 15
. 24v37
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.12
nx
4
5
3
3
3
5
23
ny
4
4
3
3
3
3
20
nxy
4
3
3
3
3
3
19
24v36
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.42
nx
4
5
3
3
3
5
23
ny nxy
5
4
4
4
4
I
I
4
2 ' I
3
2
22 13
25v38
Avail
Hhal
HpaW
Mspl
Sau3A\
Taql
0.14
nx
4
4
4
4
2
5
23
ny
4
4
4
4
2
3
21
25 v 37
Avail
Hhal
HpaW
Mspl
Sau3A\
Taql
0J5
nx
4
4
4
4
ny
4
4
3
3
3
3
20
nxy
4
2
2
2
I
3
14
25v36
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0J3
nx
4
4
4
4
2
5
23
ny
5
4
4
4
2
3
22
nxy
4
2
4
4
2
3
19
22
nx
4
4 ■
4
4
2
2
5
23
nxy
3
3
3
3
I
0
13
nxy
4
3
I '
I
I
2
12
nxy
4
4
2
2
I
2
15
97
21 v 35
A m ll
Hha\
HpaU
Msp\
Sau3A\
Taq\
0.53
nx
4
4
5
5
3
4
25
ny
5
4
4
4
2
3
22
nxy
3
2
3
3
0
0
II
21 v 34
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.49
nx
4
4
5
5
3
4
25
22 v 35
Avail
Hha\
HpaW
Mspl
Sau3AI
Taql
0.55
nx
4
4
3
3
3
5
22
ny
5
4
4
4
2
3
22
nxy
4
2
I
I
0
2
10
nx
4
4
3
3
3
5
23 v 35
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.51
nx
4
4
4
4
2
5
23
ny
5
4
4
4
2
3
22
24 v 35
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.51
nx
4
5
3
3
3
5
23
25 v 35
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.38
nx
4
4
4
4
2
5
23
ny
4
4
4
4
3
3
22
nxy
3
3
2
2
I
I
12
21 v 33
Avail
Hhal
Hpall
Mspl
SauSAl
Taql
0.45
nx
4
4
5
5
3
4
25
ny
5
4
4
4
2
3
22
nxy
3
3
3
3
I
0
13
22v34
Avail
Hhal
Hpall
Mspl
SaaSAI
Taql
0.55
ny
4
4
4
4
3
3
22 22
nxy
2
2
2
2
I
I
10
22 v 33
Avail
Hhal
Hpall
Mspl
SauSAl
Taql
0.45
nx
4
4
3
3
3
5
22
ny
5
4
4
4
2
3
22
nxy
4
3
I
I
I
2
12
nxy
4
2
I
I
I
2
II
23 v 34
Avail
Hhal
Hpall
Mspl
SauSAl
Taql
0.56
nx
4
4
4
4
5
23
ny
4
4
4
4
3
3
22
nxy
2
2
2
2
I
I
10
23 v 33
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.47
nx
4
4
4
4
2
5
23
ny
5
4
4
4
2
3
22
nxy
4
3
I
I
I
2
12
ny
5
4
4
4
2
3
22
nxy
4
3
I
I
0
2
II
24 v 34
Avail
Hhal
Hpall
Mspl
SauSAl
Taql
0.47
nx
4
5
3
3
3
5
23
ny
4
4
4
4
3
3
22
nxy
2
4
2
2
I
I
12
24v33
Avail
Hhal
Hpall
Mspl
SauSAl
Taql
0.42
nx
4
5
3
3
3
5
23
ny
5
4
4
4
2
3
22
nxy
4
4
I
I
I
2
13
ny
5
4
4
4
2
3
22
nxy
4
3
2
2
I
2
14
25 v 34
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.47
nx
4
4
4
4
ny
4
4
4
4
3
3
22
nxy
3
3
2
2
I
I
12
25 v 33
Avail
Hhal
Hpall
Mspl
SauSAl
Taql
0.33
nx
4
4
4
4
2
5
23
ny
5
4
4
4
2
3
22
nxy
4
4
2
2
I
2
15
2
2
5
23
98
21 v 32
Avail
Hha\
Hpall
Mspl
Sau3A\
Taql
0.43
nx
4
4
5
5
3
4
25
ny
5
4
4
4
4
3
24
nxy
3
2
3
3
2
I
14 '
21 v 3 1
Avail
Hhal
Hpall
Mspl
Sau3Al
Taql
0.36
nx
4
4
5
5
3
4
25
ny
5
4
4
4
4
4
25
nxy
3
4
3 .
3
2
I
16
21 v 30
Avail
Hhal
HpaW
Mspl
Sau3A\
Taql
0.69
nx
4
4
5
5
3
4
25
ny
4
4
7
7
2
3
27
nxy
2
2
2
2
0
0
8
22v32
Avail
Hhal
Hpall
Mspl
Sau3A\
Taql
0.48
nx
4
4
3
3
3
5
22
ny
5
4
4
4
4
3
24
nxy
4
2
I
I
2
2
12
22 v 3 1
Avail
Hhal
Hpall
Mspl
Sau3Al
Taql
0.45
nx
4
4
3
3
3
5
ny
5
4
4
4
4
4
25
nxy
4
3
I
I
2
2
13
22v30
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.39
.
nx
4
4
3
3
3
5
22
ny
4
4
7
7
2
3
27
nxy
4
3
3
3
0
2
15
23v32
Avail
Hhal
Hpall
Mspl
Sau3Al
Taql
0.49
nx
4
4
4
4
2
5
23
ny
5
4
4
4
4
3
24
nxy
4
2
I
I
2
2
12
23v3l
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.46
nx
4
4
4
4
5
23
ny nxy
5
4
4
3
4
I
4
I
4
2
4 .2
25 13
23v30
Avail
Hhal
HpaW
Mspl
Sau3A\
Taql
0.36
nx
4
4
4
4
2
5
23
ny
4
4
7
7
2
3
27
nxy
4
3
3
3
I
2
16
24 v 32
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.45
nx
4
5
3
3
3
5
23
ny
5
4
4
4
4
3
24
nxy
4
3
I '
I ■
2
2
13
24 v 3 1
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.46
nx
4
5
3
3
3
5
23
ny
5
4
4
4
4
4
25
nxy
4
3
I
I
2
2
13
24v30
Avail
Hhal
HpaW
. Mspl
Sau3AI
Taql
0.40
nx
4
5
3
3
3
5
23
ny
4
4
7
7
2
3
27
nxy
4
3
3
3
0
2
15
25 v 32
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.36
nx
4
4
4
4
2
5
23
ny
5
4
4
4
4
3
24
nxy
4
3
2
2
2
2
15
25 v 3 1
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.38
nx
4
4
4
4
ny
5
4
4
4
4
4
25
nxy
4
3
2
2
2
2
15
25 v 30
Avail
Hhal
HpaW
Mspl
Sau3A\
Taql
0.40
nx
4
4
4
4
2
5
23
ny
4
4
7
7
2
3
27
nxy
4
2
3
3
I
2
15
22
2
2
5
23
99
2 lv 2 9
Avoll
Hha\
HpaW
Mspl
Sau3A\
Taql
0.58
nx
4
4
5
5
3
4
25
n/
4
4
4
4
2
5
23
nxy
2
2
I
3
I
I
10
2 l v 2 8 nx
'4
Avail
Hhal
4
5
Hpall
5
Mspl
Sau3A\ 3
Taql
4
25
0.62
ny
4
4
3
3
3
5
22
nxy
2
2
I
2
I
I
9 .
21 v 27
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.58
nx
4
4
5
5
3
4
25
ny
4
4
4
4
2
5
23
nxy
2
2
2
2
I
I
10
22v29
Avail
Hhal
Hpall
Mspl
Sau3A\
Taql
0.29
nx
4
4
3
3
3
5
22
ny
4
4
4
4
2
5
23
nxy
4
2
2
2
I
5
16
22v28
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.05
nx
4
4
3
3
3
5
ny
4
4
3
3
3
5
22
nxy
4
3
3
3
3
5
21
22 v 27 nx
Avail
4
Hhal
4
Hpall ■3
Mspl
3
Sau3AI 3
Iaql
5
22
0.24
ny
4
4
4
4
2
5
23
nxy
4
3
2
2
I
5
17
23v29
Avail
Hhal
Hpall
Mspl
Sau3Al
Taql
0.09
nx
4
4
4
4
2
5
23
ny
4
4
4
4
2
5
23
nxy
4
2
4
4
2
5
21
23v28
Avail
Hhal
Hpall
Mspl
Sau3A\
Taql
0.24
nx
4
4
4
4
5
23
ny
4
4
3
3
3
5
22
nxy
4
■
3
2
2
I
5
17
23v27
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.13
nx
4
4
4
4
2
5
23
ny
4
4
4
4
2
5
23
nxy
4
3
3
3
2
5
20
2 4 v 2 9 nx
Avail
4
Hhal
5
Hpall
3
Mspl
3
Sau3AI 3
Taql
5
23
0.26
ny
4
4
4
4
2
5
23
nxy
4
3
2
2
I
5
17
24v28
Avail
Hhal
Hpall
Mspl
Sau3A\
Taql
0.07
nx
4
5
3
3
3
5
23
ny
4
4
3
3
3
5
22
nxy
4
3
3
3
3
5
21
24v27
Avail
Hhal
Hpall
M s p l,
Sau3AI
Taql
0.26
nx
4
5
3
3
3
5
23
ny
4
4
4
4
2
5
23
nxy
4
3
2
2
I
5
17
2 5 v 2 9 nx
Avail
4
Hhal
4
Hpall
4
Mspl
4
Sau3Al 2
Taql
5
0.13
23
ny
4
4
4
4
2
5
23
nxy
4
3
3
3
2
5
20
25v28
Aval:l
Hhal
Hpall
Mspl
Sau3AI
Taql
0.29
nx
4
4
4
4
ny
4
4
3
3
3
5
22
nxy
4
2
2
2
I
5
16
25v27
Avail
Hhal
HpaW .
Mspl .
Sau3AI
Taql
0.09
nx
4
4 .
4
4
2
5
23
ny
4
4
4
4
2
5
23
nxy
4
2
4
4
2
5
21
22
2
2
5
23
IOO
2lv26
Avail
Hha\
HpaU
Mspl
Sau3A\
Taql
0.53
nx
4
4
5
5
3
4
25
ny
2
4
2
2
3
5
18
nxy
2
2
I
I
I
3
10
21 v 25
Avail
Hhal
HpaW
Mspl
SaiSAI
Tdql
0.54
nx
4
4
5
5
3
4
25
22v26
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.70
nx
4
4
3
3
3
5
22
ny
2
4
2
2
3
5
18
nxy
2
2
0
0
I
I
6
nx
4
4
3
3
3
5
23v26
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.71
nx
4
4
4
4
2
5
23
ny
2
4
2
2
3
5
18
24v26
Avail
Hhal
HpaW
Mspl
SauSAI
Taql
0.66
nx
4
5
3
3
3
5
23
ny
2
4
2
2
3
5
18
25v26
Avail
Hind fl
Hhal
HpaW
Mspl
Sau3AI
Taql
0.71
nx
4
0
4
4
4
2
5
23
ny nxy
2
2
0
0
4
3
2
0
2
0
3
0
5
I ■
18 6
ny
4
4
4
4
2
5
23
nxy
2
3
2
2
I
I
II
21 v 24
Avail
Hhal
HpaW
Mspl
SaSAI
Tdql ’
0.58
nx
4
4
5
5
3
4
25
ny
4
5
3
3
3
5
23
22 v 25
Avail
Hhal
HpaW
Mspl
SaiSAI
Tdql
0.24
ny
4
4
4
4
2
5
22 23
nxy
4
3
2
2
I
5
17
22v24
Avail
Hhal
HpaW
Mspl
SaSAI
Tdql ;
0.02
nx
4
4
3
3
3
5
22
ny nxy
4
4
5 ■4
3
3
3
3
3
3
5
5
23 22
nxy
2
2
I
0
0
I
6
23 v 25
Avail
Hhal
HpaW
Mspl
SaSAI
Tdql
0.13
nx
4
4
4
4
5
23
ny
4
4
4
4
2
5
23
nxy
4
3
3
3
2
5
20
23 v 24
Avail
Hhal
HpaW
Mspl
SaSAI
Tdql
0.22
nx
4
4
4
4
2
5
23
ny
4
5
3
3
3
5
23
nxy
4
4
2
2
I
5
18
nxy
2
3
0
0
I
I
7
24 v 25
Avail
Hhal
HpaW
Mspl
SaSAI
Tdql
0.22
nx
4
5
3
3
3
5
23
ny
4
4
4
4
2
5
23
nxy
4
4
2
2
I
5
18
24 y 24
Avail
Hhal
HpaW
Mspl
SaSAI
Tdql
0.00
nx
4
5
3
3
3
5
23
ny
4
5
3
3
3
5
23
nxy
4
5
3
3
3
5
23
25 v 25
Avail
Hind fl
Hhal
HpaW
Mspl
SaSAI
Tdql
0.00
nx
4
0
4
4
4
ny
4
0
4
4
4
nxy
4
0
4
4
4
2
2
2
5
23
5
23
5
23
2
nxy
2
3
I
2
I
I
10
IOI
21 v 23
Avoll
Hhal
Hpoll
Msp I
SouSAI
Taql
0.54
nx
4
4
5
5
3
4
25
ny
4
4
4
4
2
5
23
nxy
2
3
I
3
I
I
11
21 v 22
Avail
Hhal
H pall
Mspl
Sau3AI
Taql
0.57
nx ny
4
4
4
4
5
3
5
3
3
3
4
5
25 22
nxy
2
3
I
2
I
I
10
2 2 v 2 3 nx
Avail
4
Hhal
4
Hpall
3
Mspl
3
SauSAI 3
Taql
5
22
0.20
ny
4
4
4
4
2
5
23
nxy
4
4
2
2
I
5
18
22v22
Avail
Hhal
Hpall
Mspl
SauSAl
Taql
0.00
nx
4
4
3
3
3
5
22
nxy
4
4
3
3
3
5
22
2 3 v 2 3 nx
Avail
4
Hhal
4
Hpall
4
Mspl
4
SauSAI 2
Taql
5
23
0.00
ny
4
4
4
4
2
5
23
nxy
4
4
4
4
2
5
23
ny
4
4
3
3
3
5
22
21 v 2 1
Avail
Hhal
Hpall
Mspl
SauSAl
Taql
0.00
nx
4
4
5
5
3
4
25
ny
4
4
5
5
3
4
25
nxy
4
4
5
5
3
4
25
102
26 v 41
Avail
Hha\
HpaW
Msp I
Sau3AI
Taq\
0.65
nx ny
2
4
4
6
2
5
2
6
3
2
5
5
18 28
nxy
2
4
0
I
0 . .
I
8
26 v 40
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.84
ny
5
5
4
2 3
2 3
3
2
5
2
18 20
27 v 41
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.18
nx
4
4
4
4
2
5
23
ny
4
6
5
6
2
5
28
nxy
4
4 '
3
3
2
5
21
27 v 40
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.40
nx
4 ’
4
4
4
28 v 41
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.20
nx
4
4
3
3
3
5
22
ny
4
6
5
6
2
5
28
nxy
4
4
3
3
I
5
20
29v4l
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.18
nx
4
4
4
4
2
5
23
ny
4
6
5
6
2
5
28
30 v 41
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.31
nx
4
4
7
7
2
3
27
ny
4
6
5
6
2
5
28
nxy
I
I
0
0
0
I
3
26v39
Avail
Hhal ■
HpaW
Mspl
SauSAl
Taql
0.69
nx
2
4
2
2
3
5
18
ny
4
4
4
4 '
2
3
21
nxy
2
3
0
0
0
• I
6
ny
5
5
3
3
2
2
20
nxy
3
2
2
2
2
2
13 '
27y39
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.05
nx
4
4
4
4
2
5
23
ny
4
4
4
4
2
3
21
nxy
4
4
4
4
2
3
21
28v40
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.43
ny
5
5
3
3
2
2
22 20
nxy
3
2
2
2
I
.
2
12 •
2 8 v 3 9 nx
Avail
4
Hhal
4
HpaW
3
Mspl
3
SauSAl 3
Taql
5.
22
0.26
ny
4
4
4
4 ■
2
3
21:
nxy
4
4
2
2
I
3
16
nxy
4
4
3
3
2
5
21
29v40
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.44
nx
4
4
4
4
nxy
4
3
3
3
2
3
18
nxy
4
4
4
4
I
2
19
30v40
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.53
nx
4
4
7
7
nx
2
2
5
23
nx
4
4
3
3
3 ■
5
2
5
23
2
3
27
ny
5
5
3
3
2
2
20
nxy
3
I
2
2
2
2
12 '
29v39
Avail
Hhal
HpaW
Mspl
SaaSAI
Taql
.0.18
nx
4
4 ■
4
4
2
5
23
ny
4
4
4
4
2
3
21
ny
5
5
3
3
2
2
20
nxy
3 •
2
2
2
I
I
II
30v39
Avail
H h a l.
HpaW
Mspl
Saa3AI
Taql
0.29
nx
4
4
7
7
2
3
27
ny nxy
4
4
4 .4
4
3
4
3
2
I
3
2
2.1 17
103
26v38
Avoll
Hha\
Hpoll
Mspl
Sou3AI
Taq\
0.69
nx
2
4
2
2
3
5
18
ny
4
4
4
4
2
3
21
nxy
2
3
0
0
0
I
6
26v37
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.63
ny
4
4
4
2 3
2 3
3
3
5
3
18 20
nxy
2
3
0
0
I
I
7
26v36
Avail
Hhal
Hpall
Mspl
■ Sau3AI
Taql
0.80
27v38
Avail
HPoI
Hpall
Mspl
Sau3A\
Ibql
0.05
nx
4
4
4
4
2
5
23
ny
4
4
4
4
2
3
21
nxy
4
4
4
4
2
3
21
27v37
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.26
nx
4
4
4
4
ny
4
4
3
3
3
3
20
nxy
4
4
2
2
I
3
16
27 v 36
Avail .
Hhal
Hpall
Mspl
Sau3AI
Taql
0.42
nx
4
4
4
4
2
5
23
ny
5
4
4
4
2
3.
22
nxy
4
2
2
2
I
2
13
28v38
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.26
nx
4
4
3
3
3
5
22
ny
4
4
4
4
2
3
21
nxy
4
4
2
2
I
3
16
28v37
Avail
Hhal
Hpall
Mspl
SauSAl
Taql
0.05
nx
4
4
3
3
3
5
ny
4
4
3
3
3
3
22 20
nxy
4
4
3
3
3
3
20
28v36
Avail
Hhal
Hpall
Mspl
SauSAl
Taql
0.50
nx
4
4
3
3
3
5
22
ny
5
4
4
4
2
3
22
nxy
4
2
I
I
I
2
II
29v38
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
048
nx
4
4
4
4
2
5
23
ny
4
4
4
4
2
3
21
nxy
4
3
3
3
2
3
18
29v37
Avail
Hhal
Hpall
Mspl
SauSAl
Taql
OJO
nx
4
4
4
4
ny
4
4
3
3
3
3
20
nxy
4
3
2
2
I ’
3
15
29v36
Avail
Hhal
Hpall
Mspl
■SauSAl
Taql
0.47
nx
4
4
4
4
2
5
23
ny
5
4
4
4
2
3
22
nxy
4
3
I
I
I
2
12
30v38
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.29
nx
4
4
7
7
2
3
27
ny
4
4
4
4
2
3
21
nxy
4
4
3
3
I
2
17
30v37
Avail
Hhal
Hpall
Mspl
SauSAl
Taql
0.32
nx
4
4
7
7
ny
4
4
3
3
3
3
20
nxy
4
4
3
3
0
2
16
30v36
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.43
nx
4
4
7
7
2
3
27
ny
5
4
4
4
2
3
22
nxy
4
2
2
2
I
3
14
nx
2
2
5
23
2
5
23
2
3
27
nx ny nxy
2
5
I
4
4 .3
2
4
0
2
4
0
3
2
0
5
3
0
18 22 4
104
26 v 35
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.75
nx ny
2
5
4
4
2
4
2
4
3
2
5
3
18 22
nxy
I
4
0
0
0
0
5
26v34
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.65
27v35
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.38
nx
4
4
4
4
2
5
23
ny
5
4
4
4
2
3
22
nxy
4
3
2
2
I
2
14
28v35
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.50
nx
4
4
3
3
3
5
22
ny
5
4
4
4
2
3
22
nxy
4
3
I
I
0
2 ■
II
29v35
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.42
nx
4
4
4
4
2
5
23
ny
5
4
4
4
2
3
22
nxy
4
4
I
I
I
2
13
29v34
Avail
Hhal
Hpall
Mspl
Sau3A\
Taql
0.51
nx
4
4
4
4
30v35
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.35
nx
4
4
7
7
2
3
27
ny
5
4
4
4
2
3
22
nxy
4
3
2
2
2
3
16
30v34
Avail
Hhal
Hpall
Mspl
Sau3A\
Taql
0.47
nx
4
4
7
7
nx
ny
4
4
4
2 4
2 4
3
3
5
3
18 22
nxy
2
3
0
I
I
0
7
26v33
Avail
Hhal
Hpall
Mspl
Sau3A\
Taql
0.80
nx ny
2
5
4
4
2
4
2
4
3
2
5
3
18 22
nxy
I
3
0
0
0
0
4
27v34
Avail
Hhal
Hpall
Mspl
■ Sau3AI
Taql
0.47
nx
4.
4
4
4
ny
4
4
4
4
3
3
22
nxy
3
3
2
2
I
I
12
27v33
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.42
nx ny
4
5
4 .4
4
4
4
4
2
2
5
3
23 22
nxy
4
2
2
2
I
2
13 .
28v34
Avail
Hhal
Hpall
Mspl
SaulAl
Taql
■ 0.50
nx
4
4
3
3
3
5
ny
4
4
4
4
3
3
22
nxy
2
3
2
2
I
I
Il
28v33
Avail
Hhal .
HpaW
Mspl
Sau3A\
Taql
0.50
nx
4
4
3
3
3
5
22
ny
5
4
4
4
2
3
22
nxy
4
2
I
I
I
2
II
ny
4
4
4
4
3
3
22
nxy
2
3
2
2
I
I
11
2 9 v 3 3 nx
Avail
4
Hhal
4
Hpall
4
Mspl
4
5au3AI 2
5
Taql
0.47
23
ny
5
4
4
4
2
3
22
nxy
4
3
I
I
I
2
12
ny
4
4
4
4
3
3
22
nxy
2
3
3
3
0
2
13
30v33
Avail
Hhal .
HpaW
Mspl
Sau3A\
Taql .
0.43
'
nx
4
4
7
7
2
3
27
ny
5
4
4
4
2
3
22
nxy
4
2
2
2
I
3
14
2
2
5
23
22
2
5
23
2
3
27
105
26v32
Avoll
Hhal
HpaW
Mspl
Sou3AI
Toql
0.76
nx
2
4
2
2
3
5
18
ny
5
4
4
4
4
3
24
nxy
I
4
0
0
0
0
5
26 v 3 1
Avail
Hhal
Hpall
Mspl
SaaSAI
Taql
0.86
ny
5
4
4
2 4
2. 4
3
4
5
4
18 25
nxy
I
2
0
0
0
0
3
26v30
Avail
Hhal
HpaW
Mspl .
SauSAI
Taql
0.69
nx
2
4
2
2
3
5
18
27 v 32
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.36
nx
4
4
4
4
2
5
23
ny
5
4
4
4
4
3
24
nxy
4
3
2
2
2
2
15
27 v 3 1
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.42
nx
4
4
4
4
ny
5
4
4
4
4
4
25
nxy
4
2
2
2
2
2
14
27 v 30
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.32
nx
4
4
4
4
2
5 .
23
28v32
Avail
Hhal
Hpall
Aispl
Sau3AI
Taql
0.43
nx
4
4
3
3
3
5
22
ny
5
4
4
4
4
3
24
nxy
4
3
I
I
2
2
13
28v3l
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.49
nx
4
4
3
3
3
5'
ny
5
4
4
4
4
4
25
nxy
4
2
I
■
I
2
2
12
28v30
Avail
Hhal
HpaW
Aispl
SauSAI
Taql
0.35
nx
4
4
3
3
3
5
22
ny
4
4
7
7
2
3
27
nxy
4
4
3
3
0
2
16
29v32
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.40
nx
4
4
4
4
2
5
23
ny
5
4
4
4
4
3
24
nxy
4
4
I
I
2
2
14
29v3l
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.50
nx
4
4
4
4
ny
5
4
4
4
4
4
25
nxy
4
2
I
I
2
2
12
29v30
Avail
Hhal
HpaW
Mspl
SauSAl
Taql
0.36
nx
4
4
4
4
2
5
23
ny
4
4
7
7
2
3
27
nxy
4
3
3
3
I
2
16
30v32
Avail
Hhal
Hpall
Aispl
Sau3AI
Taql
0.45
nx
4
4
7
7
2
3
27
ny
5
4
4
4
4
3
24
nxy
4
3
2
2
I
2
14
30 v 3 1
Avail
Hhal
Hpall
Aispl
Sau3AI
Taql
0.50
nx
4
4
7
7
ny
5
4
4
4
4
4
25
nxy
4
2
2
2
I
2
13
30 v 30
Avail
Hhal
HpaW
' Mspl ■
SauSAl
Taql
Q.00
nx
4
4
7
7
2
3
27
ny nxy
4
4
4
4
7
7
7 ■7
2
2
3
3
27 27
'
nx
2
2
5
23
22
2
5
23
2
3
27
ny
4
4
7
7
2
3
27
nxy
2
3
I
I
0
0
7
ny nxy
4
4
'4
4
7 .3
3
7
2
I
3
2
27 17
106
2 6 v2 9 nx
Avail
Hha\
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Sau3AI
Taql
0.61
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2
4
4
4
2
4
2
4
3
2
5
5
18 23
2 7 v2 9 nx
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.13
4
4
4
4
2
5
23
28 v2 9 nx
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.24
4
4
3
3
3
5
22
29 v2 9 nx
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
4
4
4
4
2
5
0.00
23
26 v2 8 nx ny nxy ,
Avail ' 2 4 2
nxy
2
4
I
0
0
I
8
4
3
3
3
5
22
3
0
0
I
I
7
0,71
ny
4
4
3
3
3
5
22
nxy
4
4
2
2
I
5
18
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
4
4
4
4
2
5
0.00
23
2 8 v2 8 nx ny
nxy
4
4
3
3
3
5
Hhal
Hpall
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Sau3A\
Taql
0.65
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4
3
3
3
2
5
20
Avail
Hhal
Hpall
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SauBAI
Taql
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4
4
4
4
2
5
23
nxy
4
3
2
2
I
5
17
Avail
Hhal
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Mspl
Sau3A\
Taql
ny
4
4
4
4
2
5
23
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4
4
4
4
2
5
23
4 .
2
2
3
5
18
2 7 v2 8 nx
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4
4
4
4
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5
23
0.20
0.00
.
26 v27 nx . ny
Avail
Hhal
Hpall
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Sau3AI
Taql
4
4
4
4
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5 '
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4
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3
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22
4
4
3
3
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5
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2
4
2 .
2
3
5
18
27 v27 nx
4
4
4
4
2
5
23
ny
4
4
4
4
2
5
23
nxy
2
3
0
0
0
I
6
nxy
4
4
4
4
2
5
23
26v26
Avail
Hha\
HpaW
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Sau3AI
Taql
0.00
nx
2
4
2
2
3
5
18
ny nxy
2
2
4
4
2
2
2
2
3
3
5
5
18 18
108
31 v 41
Avoll
Hha\
HpaW
Msp\
Sou3AI
Taql
036
nx
5
4
4
4
4
4
25
ny
4
6
5
6
2
5
28
nxy
4
3
3
3
2
2
17
31 v 4 0
Avail
Hhal
HpaW
Mspl
SaiSAI
Taql .
0.56
nx
5
4
4
.4
4
4
25
ny
5
5
3
3
2
2
20
nxy
3
2
I
I
2
I
10
31 v 3 9
Avail
Hhal
HpaW
Mspl
SaiSAI
Taql
0.43
nx
5
4
4
4
4
4
25
ny
4
4
4
4
2
3
21
nxy
4
2
2
2
2
I
13
32 v 41
Avoll
Hhal
Hpall
Mspl
SoiSAI
Taql
OSI
nx
5
4
4
4
4
3
24
ny
4
6
5
6
2
5
28
nxy
4
4
3
3
2
2
18
32 v 40
Avail
Hhal
Hpall
Mspl
SaiSAI
Taql
0.59
nx
5 •
4
4
4
4
3
24
ny
5
5
3
3
2
2
20
nxy
3
I
I
I
2
I
9
32v39
Avail
Hhal
HpaW
Mspl
SaiSAI
Taql
038
nx
5
4
4
4
4
3
24
ny
4
4
4
4
2
3
21
nxy
4
3
2
2
2
I
14
33 v 4 l
Avail
Hhal
Hpall
Mspl
SaiSAI
Taql
032
nx
5
4
4
4
2
3
22
ny
4
6
5
6
2
5
28
nxy
4
4
3
3
I
2
17
33v40
Avail
Hhal
HpaW '
Mspl
SaiSAI
Taql
0.62
nx
5
4
4
4
ny
5
5
3
3
2
2
20
nxy
3
I
I
I
I
8
33v39
Avail
Hhal
HpaW
Mspl
SaiSAI
Taql
0.40
nx
5
4
4
4
2
3
22
ny
4
4
4
4
2
3
21
nxy
4
2
2
2
I
2
13
34 v 41
Avail
Hhal
Hpall
Mspl
SaiSAI
Taql
0.40
nx
4
4
4
4
3
3
22
ny
4
6
5
6
2
5
28
nxy
3
4
3
3
I
I
15
34 v 40
Avail
Hhal
HpaW
Mspl
SaiSAI
Taql
0.71
ny
5
5
3
3
2
2
20
nxy
2
I
I
I
I
0
6
34 v 39
Avail
Hhal
HpaW
Mspl
SaiSAI
Taql
0.44
nx ny
4
4
4
4
4. 4
4
4
3 ■2
3
3
22 21
nxy
3
3
2
2
I
I
12
35 v 41
Avail
Hhal
Hpall
Mspl
SaiSAI
Taql
032
nx
5
4
4
4
2
3
22
ny
4
6
5
6
2
5
28
nxy
4
4
3
3
I
2
17
35 v 40
Avail
Hhal
HpaW
Mspl
SaiSAI
Taql
0.62
ny nxy
5
3
5 . I
3
I
3
2
I
2
I
20 8
35 v 39
Avail
Hhal
HpdW
Mspl
SaiSAI
Taql
035
nx
5 '
4
4
4
2
3
22
nxy
4
3
2
2
I
2
14
2
3
22
nx
4
4
4
4
3
3
22
nx
5
4
4
4
2
3
22
ny
4
4
4
4
2
3
21
109
3lv38
Avoll
Hha\
HpaW
Mspl
Sau3A\
Taql
0.43
nx
5
4
4
4
4
4
25
nY
4
4
4
4
2
3
21
nxy
4
2
2
2
2
I
13
31 v 3 7
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.51
nx
5
4
4
4
4
4
25
ny
4
4
3
3
3
3
20
nxy
4
2
I
I
2.
I
11
3lv36
Avail
Hhal
HpaW
Mspl
Sau3AI
Toql
0.15
nx
5
4
4
4 '
4
4
25
ny
5
4
4
4
2
3
22
32v38
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.38
nx
5
4
4
4
4
3
24
ny
4
4
4
4
2
3
21
nxy
4
3
2
2
2
I
14
32v37
' Avail
Hhal
Hpall
Mspl
Sau3Al
Taql
0.45
nx
5
4
4
4
4
3
24
ny
4
4
3
3
3
3
20
nxy
4
3
I
I
2
I
12
32v36
Avail
Hhal
HpaW
Mspl
Sau3A\
Taql
0.13
nx
5
4
4
4
4
3
24
ny ■nxy
5
5
4
3
4
4
4
4
2 .2
3
2
22 20
33v38
Avail
Hhal
Hpall
Mspl
Sau3A\
Taql
0.40
nx
5
4
4
4
2
3
22
ny
4
4
4
4
2
3
21
nxy
4
2 .
2 .
2
I
2
13
33 v 37
Avail
Hhal
Hpall
Aispl
Sau3AI
Taql
0.48
nx
5
4
4
4
ny
4
4
3
3
3
3
20
nxy
4
2
I
I
I
2
II
33v36
Avail
Hhal
HpaW
Aispl
5au3AI
Taql •
0.00
nx
5
4
4
4
2
3
22
ny
5
4
4
4
2
3
22
nxy
5
4
4
4
2
3
22
34v38
Avail
Hhal
Hpall
Mspl
Sau3A\
Taql
0.44
nx
4
4
4
4
3
3
22
ny
4
4
4
4
2
3
21
nxy
3
3
2
2
I
I
12
34v37
Avail
Hhal
Hpall
Mspl
Sau3A\
Taql
0.43
nx
4
4
4
4
3
3
ny
4
4
3
3
3
3
20
nxy
3
3
2
2
I
I
12
34v36
Avail
' Hhal
Hpall
Aispl
Sau3AI
Taql.
,0.36
nx
4
4
4
4
3
3
22
ny
5
4
4
4
2
3
22
nxy
4
2
3
3
0
2
14
35v38
Avail
Hhal
Hpall
Mspl
Sau3A\
Taql
0.35
nx
5
4
4
4
2
3
22
ny
4
4
4
4
2
3
21
nxy
4
3
2
2
I
2
14
35 v 37
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.48
nx
5
4
4
4
ny
4
4
3
3
3
3
20
nxy
4
3
I
I
0
2
11
35v36
Avail
Hhal
HpaW
Mspl
Sau3A\
Taql
0.09
nx
5
4
4
4
2
3
22
ny
5
4
4
4
2
3
22
nxy
5
3
4 '
4
I
3
20
2
3
22
22
2
3
22
nxy
5
3
4
4
2
2
20
I IO
31 v 3 5
Avail
Hha\
HpaW
Msp\
Sau3A\
Taql
0.23
nx
5
4
4
4
4
4
25
ny
5
4
4
4
2
3
22
nxy
5
2
4
4
I
2
18
31 v 3 4
Avail
Hhal
Hpall
Mspl
Sau3A\
Taql
0.28
nx
5
4
4
4
4
4
25
ny
4
4
4
4
3
3
22
nxy
4
3
3
3
2
2
17
31 v 3 3
Avail
Hhal
HpaW
Mspl
Sau3Al
Taql
0.15
ny
5
4
4
4
2
3
22
nxy
5
3
4
4
2
2
20
32v35
Avail
HTiaI
Hpall
Mspl
Sau3AI
Taql
0.13
nx
5
4
4
4
4
3
24
ny
5
4
4
4
2
3
22
nxy
5
4
4
4
I
2
20
32v34
Avail
Hhal
HpaW
Mspl
Sau3A\
Taql
0.26
nx
5
4
4
4
4
3
24
ny
4
4
4
4
3
3
22
nxy
4
3
3
3
2
2
17
3 2 v 3 3 nx ny
Avail
5
5
Hhal
4 .4
HpaW
4
4
Mspl
4
4
Sau3A\ 4
2
Taql
3
3
0.13
24 22
nxy
5
3
4
4
2
2
20
33 v 35
Avail
Hhal
HpaW
Mspl
Sau3A\
Taql
0.09
nx
5
4
4
4
2
3
22
ny
5
4
4
4
2
3
22
nxy
5
3
4
4
I
3
20
33v34
Avail
Hhal
HpaW
Mspl
SauSAI
Taql
0.32
nx
5
4
4
4
2
3
22
ny
4
4
4
4
3
3
22
nxy
4
3
3
3
0
2
15
33v33
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.00
34 v 35
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.32
nx
4
4
4
4
3
3
22
ny
5
4
4
4
2
3
22
nxy
4
3
3
3
0
2
15
34v34
Avail
Hhal
HpaW
Mspl
Sau3Al
Taql
0.00 .
nx
4
4
4
4
3
3
22
ny
4
4
4
4
3
3
22
nxy
4
4
4
4
3
3
22
35 v 35
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.00
nx
5
4
4
4
2
3
22
ny
5
4
4
4
2
3
22
nxy
5
4
4
4
2
3
22
nx
5
4
4
4
4
4
25
nx
5
4
4
4
2
3
22
ny
5
4
4
4
2
3
22
nxy
5
4
4
4
2
3
22
111
31 v 32
Avail
Hha\
HpaW
Msp\
SauSAl
Taql
0.10
nx
5
4
4
4
4
4
25
ny
5
4
4
4
4
3
24
nxy
5
2 .
4
4
4
3
22
32v32
Avail
Hhal
Hpall
Mspl
SauSAl
Taql
0.00
nx
5
4
4
4
4
3
24
ny
5
4
4
4
4
3
24
nxy
5
4
4
4
4
3 '
24
31 v 3 1
Avail
Hhal
Hpall
Mspl
SauSAl
Taql
0.00
nx ny
5
5
4
4
4
4
4
4
4
4
4
4
25 25
nxy
5
4
4
4
4
4
25
112
3 6 v 4 l nx
n/
4
6
5
6
2
5
nxy
Avail
Hha\
HpaW
Msp\
SauSAI
Taql
0.32
5
4
4
4
2
3
22
4
4
3
3
I
2
28 17
37 v 41
Avail
Hhal
HpaW
Mspl
SauSAI
Taql
0.25
nx
4
4
3
3
3
3
20
ny
4
6
5
6
2
5
28
38v4l
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
nx
4
4
4
4
2
3
21
ny
4
6
5
6
2
5
nxy
4
4
3
3
I
3
18
36v40 nx
Avail
Hhal
HpaW
Mspl
SauSAI
Tdql
0.62
37 v 40
Avail
Hhal
HpaW
Mspl
Sau3AI
Tdql
0.40
nxy
3
I
I
I
I
I
8
ny
5
5
3
3
2
2
20 20
nxy
3
2
2
2
I
2
12
Avail
Hhal
HpaW
Mspl
Sau3AI
Tdql
0.22
38v39
Avail
Hhdl
HpaW
Mlspl
■Sau3AI
Taqi
0.14
nx
4
4
3
3
3
3
nxy
38V40
nx ny
Avail
Hhal
HpaW
Mspl
Sau3AI
Tdql
28
4
4
3
3 .
2
3
19
4
4
4
4
2
3
21
5
5
3
3
2
2
20
nxy
3
2
2
2
2
2
13
ny
4
6
5
6
2
5
28
nxy
4
4
3
3
2.
3
19
39v40 nx
0.22
nx
4
4
4
4
2
3
21
40 v 41
Avail
Hind fl
Hhal
HpaW
Mspl
Sau3AI
Taql
0.50
nx
5
0
5
3
3
2
2
20
ny
4
0
6
5
6
2
5
28
nxy
3
0
2
2
2
2
I
12
0.22
39 v 4 l
Avail
Hhal
HpaW
Mspl
Sau3AI
Taql
0.37
Avail
Hhal
HpaW
Mspl
SauSAI
Tdql
0.37
4
4
4
4
2
3
21
ny
5
5
3
3
2
2
20
nxy
3
2
2
2
2
2 '
13
40 v 40
Avail
Hind fl
Hhal
HpaW.
Mspl
Sau3AI
Tdql
nx
5
0
5
3
3
2
2
20
ny
5
0
5
3
3
2
2
20
nxy
5
0
5
3 ,
3
2
2
20
0.00
36v39 nx
ny
5
5
3
3
2
2
20
5
4
4
4
2
3
22
ny
4
4
4
4
2
3
21
nxy
4
2
2
2
I
2
13
4
4
3
3
3
3
20
ny
4
4
4
4
2
3
21
nxy
4
4
2
2
I
3
16
nx
4
4
4
4
2
3
21
ny
4
4
4
4
2
3
21
nxy
4
4
4
I
2
3
18
. 39v39 nx
ny
4
4
4
4
2
3
21
nxy
4
4
4
4
2
3
21
Avail
Hhal
HpaW
Mspl
Sau3AI
Tdql
0.40
5
4
4
4
2
3
22
37v39 nx
Avail
Hhal
HpaW
Mspl
Sau3AI
Tdql
0,00
4
4
4
4
2
3
21
I 13
36v38
Avail
Hha\
HpaW
Mspl
Sau3AI
Taql
0.40
nx
5
4
4
4
2
3
22
ny
4
4
4
4
2
3
21
nxy
4
2
2
2
I
2
13
36v37
Avail
Hhal
Hpall
Mspl ■
Sau3A\
Taql
0.48
ny
4
4
3
3
2 3
3
3
22 20
nxy
4
2
I
I
I
2
II
37v38
Avail
Hhal
Hpall
Mspl
Sau3AI
Iaql
0.22
nx
4
4
3
3
3
3
20
ny
4
4
4
4
2
3
21
nxy
4
4
2
2
I
3
16
37 v 37
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.00
nx
4
4
3
3
3
3
ny
4
4
3
3
3
3
nxy
4
4
3
3
3
3
38v38
Avail
Hhal
Hpall
Mspl
Sau3AI
Taql
0.00
nx
4
4
4
4
2
3
21
ny
4
4
4
4
2
3
21
nxy
4
4
4
4
2
3
21
nx
5
4
4
4
20 20 20
36v36
Avail
Hhal
Hpall
Mspl
SauSAl
Taql
0.00
nx
5
4
4
4
2
3
22
ny
5
4
4
4
2
3
22
nxy
5
4
4
4
2
3
22
I 14
41 v 41
Avail
Hha\
Hpall
Mspl
SauSAI
Taql
0.00
nx
4
6
5
6
2
5
28
ny
4
6
5
6
2
5
28
nxy
4
6
5
6
2
5
28
APPENDIX C
REA DISTANCE MATRIX VALUES
116
isolate #
I
V
01
02
03
04
05
06
07
08
09
10
II
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
01
02
0.00
0.00
0.00
0.00
0.00
0.00
0.43
0.20
0.07
0.20
0.00
0.28
0.28
0.27
0.50
0.43
OOO
0.05
008
0.22
0.62
005
0.24
0.07
0.29
0.65
0.20
0.00
0.24
0.35
0.49
0.43
0.50
0.50
0.50
0.50
005
0.26
0.26
0.43
0.20
0.00
OOO
0.00
0.00
OOO
OOO
0.43
0.20
0.07
0.20
OOO
0.28
0.28
0.27
0.50
0.43
0.00
0.05
0.38
0.22
0.62
0.05
0.24
007
0.29
0.65
0.20
OOO
0.24
0.35
0.49
0.43
0.50
0.50
0.50
0.50
0.05
0.26
0.26
043
0.20
03
04
0.00 0.00
0.00 0.00
OOO 0.00
0.00 0.00
0.00 0.00
0.00 0.00
0.43 0.43
0.20 0.20
0.07 0.07
0.20 0.20
0.00 0.00
0.28 0.28
0.28 0.28
0.27 0.27
0.50 0.50
0.43 0.43
0.00 0.00
0.05 0.05
0.38 0.38
0.22 0.22
0.62 0.62
0.05 0.05
0.24 0.24
0.07 0.07
0.29 0.29
0.65 0.65
0.20 0.20
0.00 0.00
0.24 0.24
0.35 0.35
0.49 0.49
0.43 0.43
0.50 0.50
0.50 0.50
0.50 0.50
0.50 0.50
0.05 0.05
0.26 0.26
0.26 0.26
0.43 0.43
0.20 0.20
05
06
0.00
0.00
0.00
0.00
0.00
0.00
0.43
0.20
0.07
0.20
0.00
0.28
0.28
0.27
0.50
0.43
0.00
0.05
0.38
0.22
0.62
0.05
0.24
0.07
019
0.65
010
0.00
014
015
0.49
0.43
0.50
0.50
010
0.50
0.05
016
016
0.43
010
0.00
0.00
0.00
0.00
0.00
0.00
0.43
010
0.07
010
0.00
018
018
017
0.50
0.43
0.00
0.05
018
0.22
0.62
0.05
014
0.07
019
015
010
0.00
014
0.35
0.49
0.43
0.50
0.50
0.50
0.50
0.05
016
016
0.43
010
. 07
0.43
0.43
0.43
0.43
0.43
0.43
0.00
0.53
0.41
013
0.43
0.42
0.51
0.49
0.57
0.68
0.43
0.50
0.55
0.55
0.64
018
0.49
0.40
0.49
0.79
0.53
0.43
0.53
0.62
016
0.64
0.57
017
0.67
0.57
0.50
O il
O il
010
0.50
08
09
IO
II
010
0.20
010
0.20
010
010
0.53
0.00
0.14
0.00
010
0.17
019
014
0.42
0.45
010
016
0.44
0.02
018
014
0.13
016
0.09
0.71
0.00
010
0.13
012
0.42
016
0.42
0.47
018
0.42
016
0.05
0.05
0.40
0.18
0,07
0.07
0.07
0.07
0.07
0.07
0.41
0.14
0.00
0.14
0.07
012
0.26
0.25
0.49
0.47
0.07
0.12
0.32
0.16
O il
0.12
0.18
0.14
013
019
0.14
0.07
0.18
019
0.48
0.42
0.49
0.49
0.44
0.49
0.12
0.19
0.19
017
0.14
O lO
O lO
O lO
O lO
O lO
O lO
0.53
0.00
0.14
0.00
O lO
0.17
019
0.24
0.42
0.45
O lO
016
0.44
0.02
018
0.24
0.13
016
0.09
0.71
0.00
O lO
0.13
012
0.42
016
0.42
0.47
018
0.42
016
0.05
0.05
0.40
0.18
0.00
0.00
0.00
0.00
0.00
0.00
0.43
O lO
0.07
O lO
0.00
018
0.28
0.27
O lO
0.43
0.00
0.05
018
0.22
0.62
0.05
014
0.07
019
015
O lO
0.00
014
0.35
0.49
0.43
0.50
O lO
O lO
O lO
0.05
016
016
0.43
O lO
117
sample #
I
V
Ol
02
03
04
05
06
07
08
09
10
II
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
12
13
0.28
0.28
0.28
0.28
0.28
0.28
0.42
0.17
0.22
0.17
0.28
0.00
0.32
0.27
0.45
0.51
0.28
0.33
0.47
0.18
0.56
0.23
0.21
0.25
0.17
0.67
0.17
0.28
0.29
0.42
0.48
0.51
0.45
0.66
0.49
0.45
0.33
0.22
0.22
0.33
0.28
0.28
0.28
0.28
0.28
0.28
0.28
0.51
0.29
0.26
0.29
0.28
032
0.00
0.27
0.40
0.43
0.28
0.33
0.38
0.31
0.56
0.23
0.21
0.25
0.29
0.63
0.29
0.28
0.29
0.31
0.36
0.43
0.40.
0.40
0.40
0,40
0.33
0.35
035
0.51
0.25
14
15
0.27 0.50
0.27 0.50
0.27 • 0.50
0.27 0.50
0.27 0.50
0.27 0.50
0.49 0.57
0.24 0.42
0.25 0.49
0.24 0.42
0.27 0.50
0.27 0.45
0.27 0.40
0.00 0.18
0.18 0.00
0 3 3 0.26
0.27 0.50
0 3 2 0.48
0.49 0 3 3
0.25 0.43
0.46 0.45
0.22 0.45
0.12 0.47
0.20 0.42
0.20 0 3 3
0.73 0.80
0.24 0.42
0.27 0.50
0.16 0.47
0.44 0.43
0.23 0.15
0.22 0.13
0.18 0.00
0.47 0 3 2
0.27 0.09
0.18 0.00
032 048
0.29 0.40
0.29 0.40
0.40 0.62
0.13 0 3 2
16
17
0 4 3 0.00
0.43 0,00
0.43 0.00
0.43 0.00
0.43 0.00
0.43 0.00
0.68 0.43
0.45 0.20
0.47 0.07
0.45 0.20
0 4 3 0.00
0,51 0.28
0.43 0.28
0 3 3 0.27
0.26 0.50
0.00 0.43
0.43 0.00
0.41 0.05
032 038
0.46 0.22
0.51 0.62
0.48 0.05
0 3 6 0.24
0 4 5 0.07
0.45 0.29
0.57 0.65
0.45 0 .2 0
0.43 0.00
0.28 0.24
0.29 0.35
0.35 0 4 9
0.25 0.43
0.26 0.50
0.43 0.50
0.22 0.50
0.26 0.50
0.41 0.05
0.42 0.26
0.42 0.26
0.64 0 4 3
0.35 ■ 0.20
18
19
0.05
0.05
0.05
0.05
0.05
0.05
0.50
0.26
0.12
0.26
0.05
033
0.33
032
0.48
0.41
0.05
0.00
0.35
0.27
0.60
0.10
030
0.12
035
0.63
0.26
0.05
0.30
032
0.51
0.45
0.48
0.48
048
048
0.00
0.22
0:22
0.40
0.25
038
0.38
038
038
038
038
0.55
0.44
032
0.44
038
0.47
038
049
033
0.32
038
0.35
0.00
0.45
0.69
0.43
0.40
0.40
0.44
0.63
0.44
038
0.40
0.28
0.47
036
033
038
0.24
033
0.35
0.41
0.41
0.60
038
20
21
0.22 0.62
0.22 0.62
0.22 0.62
0.22 0.62
0.22 0.62
0.22 0.62
0.55 ■ 0.64
0.02 0.58
0 4 6 0.61
0.02 0.58
0.22 0.62
0.18 0.56
0.31 0.56
0.25 0.46
0.43 0.45
0 4 6 0.51
0.22 0.62
0.27 0.60
0.45 0.69
0.00 0.59
0.59 0.00
0.26 0.57
0.15 0.54
0.28 0.58
0.1 I 0.54
0.71 0.53
0.02 0.58
0.22 0.62
0.15 0.58
0 3 3 0.69
0.47 . 0 3 6
0.42 0.43
0.43 0.45
048 049
0 3 9 0.53
043 045
0.27 . 0.60
0.07 0.57
0.07 0.57
0.41 0.64
0.19 0.58
22
0.05
0.05
0.05
0.05
0.05
0.05
038
0.24
0.12
0.24
0.05
0.23
0.23
0.22
0.45
048
0.05
0.10
043
0.26
0.57
0.00
0.20
0.02
0.24
0.70
0.24
0.05
0.29
039
0.45
0.48
0.45
0.55
0.55
0.45
0.10
0.30
030
043
0.24
118
sample #
I
V
Ol
02
03
04
05
06
07
08
09
10
II
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
-*>
23
24
25
26
27
28
29
30
31
32
33
0.24
0.24
0.24
0.24
0.24
0.24
0.49
0.13
0.18
0.13
0.24
0.21
0.21
0.12
0.47
0.36
0.24
OJO
0.40
0.15
0.54
OJO
0.00
0.22
0.13
0.71
0.13
0.24
0.09
0.36
0.46
0.49
0.47
0.56
0.51
0.47
OJO
048
048
0.40
0.22
0.07
0.07
0.07
0.07
0.07
0.07
0.40
0.26
044
0.26
0.07
0.25
0.25
OJO
0.42
0.45
0.07
042
0.40
0J8
0.58
0.02
0.22
0.00
0.22
0.66
0J6
0.07
0J6
0.40
046
0.45
0.42
0.47
0.51
0.42
0.12
0J2
0.32
0.40
048
0J9
0J9
0J9
0J9
0J9
0.29
0.49
0.09
0J3
0.09
0J9
047
0J9
OJO
0J3
0.45
0J9
0.35
0.44
04 1
0.54
0J4
043
0.22
0.00
0.71
0.09
0J9
043
0.40
0J8
0J6
0J3
0.47
0J8
0J3
0.35
044
044
0.44
048
0.65
0.65
0.65
0.65
0.65
0.65
0.79
0.71
0.69
0.71
0.65
0.67
0.63
0.73
0.80
0.57
0.65
0.63
0.63
0.71
0.53
0.70
0.71
0.66
0,71
0.00
0.71
0.65
0.61
0.69
0.86
0.76
0.80
0.65
0.75
0.80
0.63
0.69
0.69
0.84
0.65
0.20
OJO
0.20
OJO
OJO
OJO
0.53
0.00
044
0.00
OJO
047
0J9
0J4
0.42
0.45
OJO
0J6
0.44
0.02
0.58
0.24
043
0J6
0.09
0.71
0.00
OJO
0.13
0J2
0.42
0J6
0.42
0.47
0J8
0.42
0J6
0.05
0.05
0.40
048
0.00
0.00
0.00
0.00
0.00
0.00
0.43
OJO
0.07
OJO
0.00
0J8
0J8
0.27
0.50
0.43
0.00
0,05
0J8
0.22
0.62
0.05
0J4
0.07
0J9
0.65
OJO
0.00
0.24
0J5
0.49
0.43
0.50
0.50
OJO
OJO
0.05
0J6
0J6
0.43
0.20
0.24
0.24
0J4
0.24
0J4
0J4
0.53
043
04 8
043
0J4
0J9
0J9
04 6
0.47
0J8
0J4
OJO
0.40
045
0.58
0J9
0.09
0J6
043
0.61
043
0.24
0.00
0J6
OJO
0.40
0.47
O JI
0.42
0.47
OJO
04 8
048
0.44
048
0.35
0.35
0.35
0.35
0.35
0.35
0.62
0.32
0J9
0J2
0.35
0.42
O JI
0.44
0.43
0J9
0.35
0J2
0J8
0J3
0.69
0J9
0J6
0.40
0.40
0.69
0J2
0.35
0J6
0.00
OJO
0.45
0.43
0.47
0.35
0.43
0J2
0J9
0J9
0.53
0.31
0.49
0.49
0.49
0.49
0.49
0.49
0J6
0.42
0.48
0.42
0.49
0.48
0J6
0J3
04 5
0.35
0.49
O JI
0.47
0.47
0J6
0.45
0.46
0.46
0J8
0.86
0.42
0.49
OJO
OJO
0.00
040
04 5
0J8
0J3
04 5
O JI
0.43
0.43
0J6
0J6
0.43
0.43
0.43
0.43
0.43
0.43
0.64
0J6
0.42
0J6
0.43
O JI
0.43
0J2
043
0.25
0.43
0.45
0J6
0.42
0.43
0.48
0.49
0.45
0J6
0.76
0J6
0.43
0.40
0.45
040
0.00
043
0J6
043
043
0.45
0J8
0J8
0J9
0.31
0.50
OJO
0.50
OJO
OJO
OJO
0.57
0.42
0.49
0.42
0.50
0.45
0.40
04 8
0.00
0J6
0.50
0.48
0J3
0.43
0.45
0.45
0.47
0.42
0J3
0.80
0.42
OJO
0.47
0.43
045
043
0.00
0J2
0.09
OJO
0.48
0.40
0.40
0.62
0.32
J
I 19
sample #
I
V
Ol
02
03
04
05
06
07
08
09
10
II
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
-*>
34
35
36
37
38
0.50
0.50
0.50
0.50
0.50
0.50
0.67
0.47
0.49
0.47
0.50
0.66
0.40
0.47
0.32
0.43
0.50
0.48
0.38
0.48
0.49
0.55
0.56
0.47
0.47
0.65
0.47
0.50
0.51
0.47
0.28
0.26
0.32
0.00
0.32
0.36
0.43
0.44
0.44
0.71
0.40
0.50
0.50
0.50
0.50
0.50
0.50
0.67
0.38
0.44
0.38
0.50
0.49
0.40
0.27
0.09
0.22
0.50
0.48
0.24
0.39
0.53
0.55
0.51
0.51
0.38
0.75
0.38
050
0.42
0.35
0.23
0.13
009
0.32
0.00
0.09
0.48
0.35
0.35
0.62
052
0.50
0.50
0.50
050
050
0.50
0.57
0.42
0.49
0.42
050
0.45
0.40
0.18
0.00
0.26
0.50
0.48
0.33
0.43
0.45
0.45
0.47
0.42
0.33
0.80
0.42
050
0.47
0.43
0.15
0.13
0.00
0.36
0.09
0.00
0.48
0.40
0.40
0.62
0.32
0.05
0.05
0.05
0.05
0.05
0.05
050
0.26
0.12
0.26
0.05
0.33
0.33
052
0.48
0.41
0.05
0.00
0.35
0.27
0.60
0.10
050
0.12
055
0.63
0.26
0.05
050
052
0.51
0.45
0.48
0.43
0.48
0.48
0.00
0.22
0.22
0.40
0.25
0.26
0.26
0.26
0.26
0.26
0.26
0.61
0.05
0.19
0.05
0.26
0.22
055
0.29
0.40
0.42
0.26
0.22
0.41
0.07
0.57
050
0.18
052
0.14
0.69
0.05
0.26
0.18
0.29
0.43
058
0.40
0.44
0.35
0.40
0.22
0.00
0.14
057
0.22
. 39
0.26
0.26
0.26
0.26
0.26
0.26
0.61
0.05
0.19
0.05
0.26
0.22
0.35
0.29
0.40
0.42
0.26
0.22
0.41
0.07
0.57
050
0.18
052
0.14
0.69
0.05
0.26
0.18
0.29
0.43
058
0.40
0.44
055
0.40
0.22
0.14
0.00
057
0.22
. 40
0.43
0.43
0.43
0.43
0.43
0.43
0.50
0.40
057
0.40
0.43
053
0.51
0.40
0.62
0.64
0.43
0.40
0.60
0.41
0.64
0.43
0.40
0.40
0.44
0.84
0.40
0.43
0.44
0.53
0.56
0.59
0.62
0.71
0.62
0.62
0.40
057
0.37
0.00
0.50
41
0.20
0.20
0.20
0.20
0.20
0.20
0.50
0.18
0.14
0.18
0.20
0.28
0.25
0.13
052
0.35
0.20
0.25
058
0.19
0.58
0.24
0.22
0.18
0.18
0.65
0.18
0.20
0.18
0 .3 1
056
0.31
052
0.40
0.32
052
0.25
0.22
0.22
0.50
0.00
[
j
APPENDIX D
ALIG NED SEQUENCES
121
G UIDE T O ALIG NED SEQUENCES
"A "
Adenine
"C "
Cytosine
"G "
Guanine
//IJ IZ Z
N"
/ / _ / /
" ."
Thymine
A 1C1G1o r T
Deletion
Match to Reference Molecule
Isolate 0 1 is the reference molecule
122
Is o la te
Seq
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
t — c — c - g t a g g t g a a - c - t - g c g g a a g g a t c a t t a t t g a a - - t t -------. fc —
- . c . - ,
.t “
- .c .
. fcg------•t “
. tfc
. --t — c -------. fc”
g -------. fc"
.t t t . fc"
g----. fc —
. fc —
t — .t t c t
. fc —
g— ■
—
. fcg -------, fc —
g ---• fc —
.t—
* fc”
. fc —
25
26
27
g --------
28
29
30
31
32
33
34
35
36
37
♦fc•g . --, --*t —
* ——
♦fc —
• ---
c
-c a
g --------
.t —
-c a
*t . fc —
• fc —
. ---
g --------
. --- 1
• fc —
. ---
g-----
* --- <
. fc —
. fc —
.t-
. t -----.t t - g --------
g --------n t
nn
n--
n ------g -------g --------
38
39
40
.t-
g -------g --------
1"
a --------
41
• fc ™
g --------
. fc —
1
123
Isolate
Sequence
01
a a t g t a g a g t ----t t g g t —t g t a g c t g g c t c c t a ------- a t t a a a —c t t
02
03
04
. c t . g . . . -------. c . . . — . . . c
g . c t t . —t
05
06
07
OS
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
g . c c . ---------------------- . —c . g g t . . a t . . t . . c -------t . c . g c —t g .
. . . t a . . . . .................... —
—. . . t ............
. . . . a .......... ....................— ...................... •c . ------------g—t . . . .ta. . . . —
:
.......... — . . . t . . . . .
. . . ag. . . . - - - . ga. . —. . . t
.................. ......................— . . . c
. c t . g . . . —:— . c . . . — . . . c
. c t . g . . . -------. c . . . — . . .-c
. c t . . gggg-. . . . n t t t . . c
. c t . . -------------------1 —ngc
. . -------‘------- g . c t t . —t . .
. . ---------- - - g . c t t . —t . .
— . . eg-------. . . t . . —t a .
. . . a ...............................— ...................... . c . a . . —
■
— t . c . t . —t . .
. . t a . . . . .....................— . . . t ...........
--------------------------. t . — . a c . a . .cc
c . n c g ------g a n . n . —t
. . t a . . . . .....................— . . . t . . . .
-----------------------------c— . . g . a . g c
. c t -. g. . . -------. c . . . — . . . c . . . .
. . . a .......... a t t ........... — .................
. . a .......... .......... ........... — . . . c . . .
. . a ...................... . . . . — . . . c . . .
. c t . g . . . -------. c . . . — . . . c . . ;
n c a . g . n . . -----n g . .n —g t .g n . n
..................a ----- g . t . . g c . . . c . . .
. c t . g . . . -------. c . .
. .c . . .
. . t a . . . . .................... — . . . t
. . t a . . . . .................... — . . . t
c . a . g . t . .c — . . . .c— . . . .
. c t . g . . . -------. c . . . — . . . c
c . . a g —.—g e n e . c —
. --------------g . c t t . —t
n . .cngag-. . n n . t — c
c . ------------ -— . t t — a .
----------------- CO
-------------------c c
g . c t t . —t . .
an
. g g g . t g g g ..........t —t . c
. ------------- g . c t t . —t
g . c t t . —t g
124
Isolate
Sequence
01
02
03
04
- —g-gg—g g c a - t g - t g c --------- a c a c - c t t t c —t c t t t c a t c —c
-----— . . . . . . . . . . . — - —
— . . . . . . . . . . — ............................... — .
. —. . — . . . . . . . . . . . — ---—
.......... —
.............. — .
. —. c —a . . . - . . —. . . ---------- . . g . —. . . c t —c t .................— .
07
-----. - c . —c t t t - . . c . . . ------;-----. . g . - t g . . g —a a . . . . ------------
10
-----c c . . —
14
15
21
23
—. . n ---------- —. . . —
7" . ; . n . . . . -----------------.
. —.c —a
. -----------. . g . —. . . c t —c t ....................—.
. —.c —a . . . —.. —. . . ---------- . . g . —. . . c t —c t ....................—.
-----------:-------c
. a — r----- . . . t a . c . a . — . g . . g . t . . g g .
e t c . - . . — . . g g - g . - . . n -----------t n . . - . - n c . — . . c . c . . .
.
25
26
27
-----cc • . — . . . . . . . . ------—
— ...........................
-----. - a a —a n . c - . t - . . t g a a c t t . t . . - . . g a . — . g . n g . c . . —g
. —.c —a
. -----------. . g . —. . . c t —c t ....................—.
29
30
n g g . - . . — . .n t - n . - . .. -----------n . n . - . - . c . — . . n n . . -nn— .
-----1—
t . — . . . . —. . —. . . -----------. . . . — . . . g n t . t ...............— .
33
34
35
36
-----. - . c —a
------------. . g . - . . . c t —c t ...............— .
-----. - . . — . . a n - a t - . . n------------. n g . - t . c . . — . . . c c a c c t — .
-----t - . t t c . . . . . . . . . ----------- . . g . - . - . g . — ........... t e a t — .
-----. —.c —a
. ------------. .g. - . . . c t —c t ................— .
40
41
-----c - . . —a. . . a . . . -------- - . . g . - . c g c . —a t . . . t . . . ---------. - . c —a
. ------------. . g . —. . . c t —c t ................—
—.
T
125
Isolate
01
02
03
04
05
06
07
OS
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
Sequence'
- c a t a c a c a c c t g t g c a c c -----t -g t g ag a ca g -
. t ----
,c .
. t -----
. C.
C.
.t -----
-----.-------. . c . . . .C......................t------------ .C.................................. t• *C»• • • C********* t
a ---------- . . c . . . . c ......................t------------ . . c .................................. -
,t . gagaccggtaa
.............a.***----.......................t ----- g g g . t t g c . . c . g . * g . . ----- c . . . t . c . c c . . c n .n .n . . n c .n a t . n ----n - c . n . t . nna
------------ . . c ..................................t -----gcgca cg . e g g . c . c . a g . g . * c . . ----- - c g . . a . . .
a ---------- . . c . . . . c ..................... t
. c . . . . n ..........n a n . .
. c . . . . n n . . . . a a n n . ----. c ................................ t ----. C .....................................t ----*C * * * *C * * * * * * * * * t
. c . * . . n n . . . . a a n . t ----. c .................................. t ----. C . . . . C ....................... t ----c
,
c ............
■.t t c . . .
cn .. . c ,
. t ----. t ----.. gac
.t'—
sl-
.... g n g . . ............... g .
................ g.
eng.
.« a g . t . t .
126
Is o la te
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
Sequence
-----a ----------------------t g --------------——----------t t t t g t -------ggggg
—
. -----------------— —
--------------- —
—
—
____ ................ ............
______------------------ . . ----------- ------------------- -----------— - ............
--------------------------------------------------------------------. g . a --------. a c c .
-----. ----------------------. t —:--------------------------- a .................................
— . ----------------------. . ----------- —------------------------------------------------------------------------- a c ------------------ :------------ . . e g . . --------t . t a .
---------------------------------------------- :------------------ ---f-------------------- .
-----. ----- :---------------- . . g g g a a t t t a c t t g t t g c . ., . \ t g --------. a a t a
---------------------------------------------- :------ ^----------------------------------■
—
-----. -----------------------. . ---------- -----------------.-----. . . i . . ......................
-----t a a g g t t -----------. c ----------- ------------ >-------. . . . t . ------- t . . . .
. ----------------------g . c c t t -----.-------------- — . . ..c t . -------t c a c .
---------------------------------------------- ----------------------- . g . a --------. a c c .
a a a . --------------------- a . ------=---------------------- . c . . c a --------. t c t .
. ---------------------- a . ---------------------------------. -------------------------.
— . ----------------------.
------------------------— ....................................
— . ----------------------. . -------------------------------- -----------—— ............
. ---------------------- . .gggaa------------------------. . . a c . — t . c t .
— ---------------------------— — --------=
--------------------------------------------g------------ :-------- ga---------=-------- -------------. c a g . c --------. ccc .
-----. ----:------ ------------ . . ----------------- -----------------------------------------------1 ---------'------------ a . -----------;-------------- -— . -------------------- n. .
— . ----------------------. . ----------------------------------------------------------------------------------------------- ---------------r--------------------------------------g----------------------n a ----------- ,——
—
----------- g c a g . c ------- a c . c c
— :--------------------------------- --------------------- . g . a --------. a c c .
-----. -----------------------. . ----------:-------------- ..................... .......... ............
— --------------------------1------------------1------------. g . . n . .......................
------------------------------------------------ --T--------------------: . ------- a. . . .
. -------------------------------------T
— —---------------------------- -. .acc
. ------------------------------------ --------------------------------------- . . acc
-----------------------------------------------------------------------. . g------- a . acc
. ----------------------. n -----------"— ---------------c . .nnCcccc...........
------------------------------------------------ --------------------- . . g ------ . .acc
--------------------------------------------------------------------- . g . a ------- . a c c .
— . -------------------------------------------------- —
-r-----------------------------gatacctctcgtc.c
a -------. a . . c
c
---------'----------------------g-------a. acc
—
127
Is o la te
01
02
03
04
05
06
07
08
09
10
Sequence
erg--------------------- aacf—g--------------a a —-ct----t t ------------- a ------. . -------------------- . . . — . ---------- . . — . . --------. . -------------- . ------. . ______________ . . . __. ____________________. . _________. __
. t —:------------------. . a —a ------ ------- . . —a Cf------. n --------------c ------. . ------------------------------- . ----------------------------------------.. . -------------------------------------------------------------- . . ------------- . -------c t ------------------------ . eft—t ------.— . . — . . ------- c . -----------. g e t Cf
----. . ----------------------n . . — . ------------ cfc— . . -------c c ------------n —
c a ----------------------. . . —
—.-------------c . - —
a . a g g . . ---------------. -----. . ------------------- -g . . — . --------------gn—n . ------ n . ------------- . ------
11
. . ----------------- .___ . . . __ . _________. . __ . .,___ . . _________. _____
12 .
13
14
15
16
17
18
19
. . -----------------------------------------------=—- . c ------ . . ------------- g------a . -----:-------------- - . g . — . ------------- g g - - g c ----. . --------------c -----. t ------------ ---------. . a —a —•— ;— . . —a a —
—g . ------------- c ------c t ----------------------. . a —t -------------c c —a . -------a . ------------- . -----. . ------------r-------—. c t —t t t a a t t c . . —t . ----- . a ------------- . -------. . ______________ . . . __. ________. . __. . __-------------------. _____
. . ----------------------. ---------------------------------------------------------. -------c t t t t t g a a a t a c a . . . — . ------------1 . —a . a g g . . --------- . --------
22
23
24
. . -----------------------. ---------------------------------------------------------. -------. . ------------------------. g . — . ------:—
---------------- ------------n ------. . --------------------- . . . — . -----------------------------------------. -------------
27
28
29
30
31
32
33
34
35
3 6
37
38
39
40
41
. t -----------------------. . a —a --------------. . —ag----- . n -------------c -----. . ----------------------. . . — . ------------- ---------------- . . ------------- . -------. ---------------------------------------- =-----— .n -----------n . -------------g------. . ----------------------. . t t t . -------------t g — . g------ n . ------------- . -----. t -----------------------. . a —a ---- :— —. . —g . -----c . t c c g t c t . —
-----. t -----------------------. . a —a ---- ------- . . —g . -----c. t c c g t c t . -------. . ---------------------- 1 . a —a --------------. . —a a ---g . —
----------- c ------. . ---------------------- g . t —c --------------. g — . c ---cn------------- g------. t ------------------------. . a —a ----- -— . . g t . . ------ . c -----------. --------. t ------------------------. . a —a --------------. . —ag----- . n -------------c -----. . ----------------------- . . a — . ------------—
n . —n . ----. . —
------------- .—
----. . ---------------------g . . — . ------------ g c— . . ------ . c ------------- . —----. . ---------------------g . . — . ----------rgc—
— . c ------------- . -------, . ----------------------. t . — ------------- ----------------------—----------------. . ---------------------- t . a —a --------------. . —a a -----g . ------------ c ------
128
Is o la te
S equence
01
------------- t t g g a c c t t ----------- c t - ----- a c t c -
-cccct
02
03
04
. c c a t n . g g -----------
a.n-
.nnn
07
08
09
c t t c c g c . . t t . . a . . — ------- t a ------- . . a a ------------- . g . . n t t . . —
c ------- n g n n -
. .aag
.n n n .
10
11
. n t t . . tccngt
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
c . . --------- . -----------c g t c t g . c a -----.----. . c a g t . . g ----------c a a a c . . a . ----------. .t . . .c
05
06
. . c t t . tcctgt
a.tcttg.
------- . t . t
. . t ------------— -------a a t . .
. a t -------------- ---------a t a . .
a a t - - - -------------------. . a t a
a .t
gaa. «
g t c t . . . aatccacacaaac. . a a .
. . tt
c . --------. .
ccatn.gg
-ta.ac
. - -
a ........... n n a ------------ n ------- c . n n * * •t *
-ta.ac
- - c . .g—t t . n — .a.n-
- t .tnn
-t.ta.
a . c l. .
- . . nnn
n a ...........---------------------a . t n n
.tct.ta
ta.
■— ----------------n . ag g
. a a . . . a c --------- ac
.aa
. a a . . . a c --------- ac
.aa
*. . a .
. a a t — r-----.----------- . . a t a
. c a g t . . g-------- - . .
n t . — . n . —;------nn
n n . -------------- ----------. . a . .
■. c t . . t . a ---------- a .
. t a . ------------------------aaa . .
ccn tnngg---------n.
. a . . —^-----■
--------------.nnnn
. . . . a . n . --------- . c
**n .
.... c
. . . c t t . . t c c t g t . . -------. . . t ------------------------t a . ac
. . . c t t . . t c c t g t . .. ------. .. t ------------------------t a . ac
----------------—-------------------g . t . g
* **g'or^* ♦•
. . c a g t . . g----------------. a a t -------------- ;----------. . a t a
129
Isolate
Sequence
01
t g ------------
-cttctg-
- t c t ---------a c -------------
02
03
04
-acc.cn-
t . ———■
—
t ------------ a g a a . . nnncaann. c n t . t -
t . — -------
05
06
07
08
. a — r—
aaannnan
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
---------- . . c a . t a c -----------ca-----------.acaaaccc
ga-----------. t ------------
-.-a.aca.. . . 't
- t . . aat-tg .aa. - t . . t .c-
aa-
. .ca.t-
- n . . . atacanaatcaa
27
- . . ca.t- a c . . .n- a c c . cn-
28
29
n . -----’-------
- . . a . .t- . c c . .c- a . . gaa-ai. . g a a - t . .aat-n.ngan- t . .aat-acc.nn-a.. .n.- . .ca.t-
30
31
32
33
34
35
36
37
38
39
40
41
a a -----g a ------. . --------g
a . a --------c
g a a t ------
.ttacacaa
a a - - --------. c c ----------
26
a.tttaa
6.
* --------------------------------
..ta ttta..ta ttta. acaaaccc
c c ---------- c c --------------
. c c ---------a a -----------a a -----------. a -----------. anaaaccc
- ..ca.t -
-ggg..c :
- t . . aat-
a .ttaaa
aga—.----- naanacnn
. n --------1 .
. . nn----g . ----------------g
'
g a --------—a ; a-:------ n t
a a --------nn —
—
----t .ttaaa
t .ttaaa
. c ------—t .
g a - ------. ;
130
-
-
-
-
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
---------1 -----------------t a a ------- 1 ------------------------:------------------------ 1
---------, -----------------. -----------g ---------------------------— ---------------.
---------. ---------------- . . - T ------------------------------- ------------------------- .
---------. ----------------- . . ------- nngnangnta----------------'------------ .
-------- . -----------------. n . ---------------------— --------- :-----------------------.
- - - - - . --------------------------— . --------------------------------------------- .
---------. a c a a t a a a a . . . - -— c -------------------------------- -----------------a
g n t t g . ---------------- . . . --------. g n t g t a c t a a c a t c t g c -------------------.
---------. -----------------c . c -------a c a c c t g c ------------ =-----.---------------- .
---------. ---------------- g . . --------. g t n n t t n a t g t a n c a c t a a c a t c t g c .
---------. ----------------- . - n -------c --------------------------------------------------.
---------. -----------------. c . c a c g . -------------------------- —-------------------.
. ---------------g . . —-— . g t a a a g a t c c a c g t c t g c ---------------- .
.---------. -----------------g . . -------. g t a a g t c t g c ----------;--------------------.
. ----------------------------- _____----------------------------------------------------.
---------. -------------------- g------ c ------------------------------------------------- .
---------. -------------------------------------------------------- --------------------— .
---------. -----------------. . . -------------------—---------------—-----------------.
. ---------------c . c -------a c c t g c --------------------- :------------------ .
a --------------a . . -------n --------------------------------------------------.
. ---------------. t t ------- . -------------------------------------------------- a
---------. -----------------. . . ------- . --------------------------------■---------------- .
. ---------------. t . --------. ------------------------------.
. ---------------. . . ------- . —-----------------------— ------------------ .
-. ---------------g . . - :— . g t a a t t g a t g t a a c g c a t a c a t c t g c .
a a -----. -----------------g. . -------. ------------------------------------------------- a
- - . ---------------- . . . ------- nngnangn t ag t c t g c----------------------.
. ---------------- . n . --------c ------------------------------.
---------a ---------------- . . t -------a:-------- ------------------- =------------------ .
. ---------------c t n -------. n t a c a c c t g c --------------------------------.
. ---------------. g . --------. g t a a c c a c a g t c t g c —-------------------.
. ---------------. g . ------- . g t a a c a c a g t c t g c ------------------------ .
-----— . ---------------- g. . -------. g t a a a a c a g t c t g c —=-------------------- .
---------. -----------------. n . ------- . ntnngna------- -------- '------------------ .
. ---------------g . c -------a g t c t g c ---------------- ;—-------------------.
. --------------- n . . -------n n g a a n g t t a g t c t g c ----------------------.
---------n ----------------------------------------------------------------------------- - .
. -----------------g . . -------. g t a a t t g a t g t a a c g t a a c a t c t g c - .
. -----------------g . . — —. g t a a t t g a t g t a a c g t a a c a t c t g c - .
a ----------------a . t —:— . —T------------------------.
. -----------------g . . -------. g t a a g t n t g c ---------------.
-
01
02
03
04
05 •
06
07
OS
09
10
11
12
13
14
15
S equence
-
Is o la te
131
Is o la te
S equence
01
-cct-
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
gaatgtaattgatgtaacataacatctgct
aaataaataaaaa
aac
nntnt
naa
.a ^
132
Is o la te
S equence
01
--------------------- 1 ----------------------------------- t t g - t g c a t ----- 1 ----- a g - -
02
03
04
05
06
07
OS
10
-------------------- . c a t t g t g g t g g a t t t g g a . . . . . eg ------. ----- . a —
--------------------. ----------------------------------. a a a . t t . . ----- . -----. --------------------------------—.a.aa. 1 1 . . ------. ------ .
.—
-------------------- . ------------------ ----------------n. . —. . . n c ---- a ------. . —
. --------------------------------- - c aa a................... ------- . . -—
. ------------------------------- —.a a a . a a . . ------. ------ .
.—
--------------------. ---------------- ----------- .-----. a a a . a a . . ------. ------.
—
------------------ —. ----------------------------------. a a a . 1 1 . . ------. ------. . —
--------------------. -------------=--------------------. . c - a a . . c ------. ------c a t g
23
--------------------c ----------------------------------. . n - . t g g . n n a . ------
25
26
27
. ----------- ---------------- ------ . a a a . t . . . ------. ------ . . —
-------------- - - ' - c --------------------------- :-----. c a - . . . . c ------. --------------------------. --------------------------------- - . a a a . a a . . -----. ------. . —
29
t a c g g t n c t t n . ---------------------------------- .n. - ....................n ----- n. - -
31
32
33
34
35
36
. ----1----------------------------. a a a . a a . . ------. ------ . . —
. ---------------------------------. a a a . a a . . ------. ------ . . —
-------------------- . ------------ -—-----------------. aaa . a a . . ------. ----- . . —
. ---------------------------------. . . - g t . c . ------. n t a . a —
. ---------------------------------. a a a . 1 1 . . ------. ------ . . —
. ---------------------------- ---- . a a a . a a . . -----. -----
38
39
40
41
--------------------. ----------------------------:------. a a a . t t . . ------. ------. . —
- - - . -^-------------- ----------. a a a . t t . . ------. ----„ --------------------g--------------------------------c . c - . .g. . -----g -----t . g g
n ----------------------------------. a a a . a a . . ------. ------ . . - -
-
-
-
-
-
-
12
13
14
15
16
17
18
19
2 0
21
133
Is o la te
S equence
01
-ctgg— a t c t c a g t g t t a t g c t - - - — tgg-
02
03
04
05
06
07
- c . . a t c c . . a a J . . . aa. ******
08
tagcttgtgcttt
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
-Cl'
i^it ** **nn **********
—
....................... .....
-
—
a
.
.
. ----------------------
— ...................- a . . c a . . ---------— ...................- a . . ca. * . ----------
**** * * * * a.******
— g g . g t g ...........g g . g a ----------
ccc-
— n . t . . n . g . g . * n . gn-
26
t
27
28
.29
30
nn
31
32
33
34
35
— ................. —
a ............................
—g . g . t g . g n c a c . . . c -------- t t
— .................. -^a. . c a . . -------- —
—n . n n . n . n . .n . g . . .
--g
—
........—
a
—
------- ..................—
a . . . . . g--------- g t t
—..................—
a . . c a . . --------nn—n . a g g n . g n . . t . tanngaan
** ** *Cf*»a. *a. *ca * *
........- a . *c a . . ---------
36
37
38
39
40
41
ggtttgtg
— ...................—a ..............
—c.gc.tt.c.ggc.g,
******** a. * *c a * *
-ct
T
134
Is o la te
S equence
01
t c c a ----- c t c a - g c g t g a t a a g t -
02
03
04
05
06
07
.g-. .a..g .g t.a-
gtg
08
. g - ..............................-
09
. g - . . . ..............a . -
10
11
■g - .............................. -
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
41
a t . . ..tgc.cc.t.g,
a c ..............................-
ac.
ac.
•g •g ■g -
ac.
. . g - ..................... a . ---------------• . g - ................................................
a g .c c . . . a .g g .c .gccggcccc
----- a . n n - . g n n . g n . . n n —
. . g - ........................... g — g a a g - . . a g . c e n t . a. . g - .....................- -
----- n
.g
...g ..n ..,.
• g - ..............................
•g - .
• g-.
• g-.
n ----- a . n g - , g . n . g n . n .
.g-.
•g - .
38
39
40
-tatctatcgctgaggacac
. g - ............................... ■
. g - ............................... ■
c -----
,g . - a a t g e . . t . . c ■• g ~ ............................ -■
a c ..............................a a . c . a . t g . c . . . .c
t n . . t . — .n . . g g .n
a c . . . ......................t c .g t . g c . . .ct.g.
a c ..............................n t.n ..g .n .n ....
a
a
a
a
a
a
c
c
c
.
t
c
............................... ............................... ............................... .t . — . n . .gg.n
............................... ............................... -
ac.
ac.
g t . c a t a ...........c t . c g
. . a c ..............................-
135
Is o la te
S equence
01
cctgttaaa
a a g g g g t g g c c a a ------- g g t a a a t g c a g
02
03
04
05
06
07
t .
cc
t.c t.t
08
g
09
10
11
12
13
14
15
16
17
18
19
20
21
22
g
•
-g. • • .
------------------ . • — ......................................... g g . t . .
- . . . . --------------------------------- . . c a ......................................................
- . . . . a . . . ------------------------------------ ---------------------------- ---------- -------------
* • * *a
********
*g * *i * *a * * *
------------------------------------------------. t c ...............t c c t c t . c g . . g . a g t .
23
n n n *nn. . . ------------------------------- . *n. . . * c . -------- . *nn. . a . n n .
24
25
26
27
• • • •a ** *
• • • • *• «•
«g * ** *a. * * *
. -------------------------------------------- a. . a c c . . g . --------. c g c . — . t . .
28
29
30
31
32
33
34
35
36
37
n g . -----------------------------------. .an. . n . n . n . n --------. *n. . *g. *n.
g
g
nnn nn
n
ca
* I**L * ^9. * * *
• g c ................
38
39
40
41
eg—
•g—
• g c ................
. -----n. . g g .
t . . a * . g g tg tg a ta a tta tc t.c.cc
g * * * *a * * «
q * * * *a. * * *
. t c . --------g c ................
136
Iso la te
Sequence
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
a t g a a c c -----g c t t c t a a t a g t c c a t t g a c t t g g a c a a t a t — — c t a a -
—
_
___
_ _ . tier. a ...... ..................... c . . _ . r . .
—
Ir —
— ha . h. .......... -................r*.. r - -
. . . a _ _a .......... .............
— .act-
............... ......... . . . . C . . . . ..............
...............
..a tt..
.t .
g t.
p—
. . a . . g g . g . .cgacga.c .c c t .c .g t . . aaccc.c . . -
g n . g . . . ----- . g . . t . n .................n n . . . a n . . . n n n . n g a — - - a . n . a
A
A
t .a . . . . e t c . . . . . . gga. . g . . c .g .c g g ..cc.tg ccg — — n . .. a
♦ • •c ««
• • ♦ • fc>• ♦ • n. * 13,« » » • «g n . n . n . n . . . . a . a — — t a . . —
— ta . . -
. . . . g . . ...... ..................................
g a . g . n n ----- . g . . t . . . a c . . . . n n • U- • y Jil • • • JilllC • Cl •
—aa..a
n••
__aa
a
. a . a . . a . . . . — .. — — t a .t —
- - t a .t — . . c t t ...... ................... c c . . . . —
g . a a .................. c
— aa. . a
137
Isolate
Sequence
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
--------- ttt— atga-tc— tgatctcaaatcaggtaggactaccc
____________
____________
— ------- C ..— .ctC.C *t- ..gc........... ;.......
--------- c ..— tcc.-aggt •. .c---g .......... a ....
cccccaactc..g--.aa.-.t— *..c *.♦gg......... a ....
--------- trtt-t-
—
—
...C ....................
138
Is o la te
Sequence
01
gctgaacttaa
02
...........................
03
04
05
06
07
08
09
.........
.........
.........
.........
.......— .........
.........
10
...........................
11
12
13
14
15
16
17
18
19
.......
.........
....______
.........
.........
.........
.........
.........
20
...........................
21
22
23
24
...........................
...........................
25
.........
26
...........................
27
.........
28
29
30
......................
...........................
...........................
31
.........
32
...........................
33
34
35
..■.....—
.........
.........
36
...........................
37
......... ■
38
39
...........................
...........................
40
...........................
139
APPENDIX E
MATRIX DEPICTING TH E NUMBER OF NUC LEO TIDE MATCHES BETWEEN EACH
ISOLATE O U T OF 400 A U GNABLE SITES
140
This Appendix is in the form o f a matrix, where each isolate's sequence is compared to
every other isolate’s sequence over 400 alignable sites. The isolate # s across the to p are
the reference molecules used to obain the number of nucleotide matches with every other
isolate listed below the isolate # . For example, the first column used isolate 0 1 as a refer­
ence molecule in the Align Plus program. The numbers below it indicate h ow many
nucleotide matches each isolate # (to the left) had to the reference molecule out o f 400
alignable sites.
141
isolate #
24 '
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
O
L
01
02
03
04
05
06
07
08
09
10
II
12
13
14
15
16
17
18
19
20
21
22
23
400
397
398
334
396
397
231
308
341
31 I
396
327
308
311
310
339
399
399
344
312
213
400
253
400
316
202
310
397
277
329
321
318
307
223
310
307
385
315
315
243
308
02
03
04
05
397
400
399
334
396
399
231
305
345
31 I
396
328
313
310
31 I
340
398
398
345
313
214
397
252
397
318
201
31 I
398
399
400
335
397
399
230
305
343
31 I
398
323
313
310
311
340
399
399
345
313
203
398
251
398
315
204
31 I
397
278
329
320
318
309
222
312
312
386
315
336
336
337
400
336
336
237
314
316
310
334
31 I
315
351
354
326
336
336
321
308
207
336
242
336
314
204
376
335
396
396
397
334
400
396
397
280
331
321
319
309
224
311
312
386
315
315
245
315
242
307
307
260
315
327
324
349
226
323
367
336
309
309
238
350
07
10
11
343
309
345
31 I
345
31 I
314
308
315
344
345
31 I
238
237
321
357
400
322
324 . 400
344
312
324
310
313
329
316
327
315
328
353
319
343
309
343
309
387
323
324
380
222
216
343
309
255
249
.343
309
327
382
206
217
319
306
344
307
273
260
343
314
318
350
317
347
313
326
217
226
316
331
306
320
31 I
341
396
398
398
332
396
398
228
308
344
314
400
324
314
31 I
310
339
397
397
346
312
214
396
248
396
317
311
312
383
314
314
397
233
308
399
233
307
399
232
307
334
314
237
396
233
308
400
236
308
400
225
232
237
306
400
343
238
321
241
361
307.
234
398
308
328
237
299
314
244
316
310
318
256
31 I
313
257
340
242
308
235
398
308
398
235 . 308
345
243
323
311
253
366
214
216
21 I
233
397
308
252
245
195
397
233
308
317 ■ 254
361' .
201
215
21 I
313
244
326
310
397
233
278
187
254
328
224
308
320
249
336
318
248
331 .
307
257
308
225
189
225
31 I
247
321
312
242
328
224
387
308
314
256
365
314
365
256
325
325
383
383
330
320
318
304
221
312
307
385
315
315
242
246
247
307
256
310
246
308
326
312
TLl
306
344
315
396
324
312
31 I
31 I
339
397
397
345
31 I
216
396
253
396
318
201
313
397
277
328
321
318
306
222
06
258
258
08
243
313
09
207
310
399
277
142
isolate #
r
01
02
03
04
05
06
07
08
09
10
II
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
13
14 ■
15
16
17
18
19
20
21
312
313
313
356
311
313
259
321
317
328
312
304
336
399
400
327
313
313
319
331
212
312
244
312
341
342
342
330
341
342
244
310
353
323
343
330
323
326
327
400
343
343
352
399
398
399
334
397
398
231
306
343
309
397
324
308
310
31 I
399
398
399
334
397
398
231
306
343
309
397
324
308
310
31 I
339
400
400
346
312
214
399
253
399
316
204
311
342
343
343
323
345
343
231
321
387
319
344
323
319
318
321
352
344
344
400
321
212
342
257
342
322
209
312
343
272
346
323
318
319
215
314
313
313
306
31 I
313
247
366
324
380
312
313
331
329
331
320
314
314
323
400
222
314
247
314
389
215
319
31 I
259
313
354
353
331
225
317
31 I
341
335
321
309
395
395
207
202
203
209
202
202
216
207
214
214
208
222
21 I
209
208
226
202
202
206
222
400
207
189
207
222
267
207
199
199
203
207
217
215
179
216
202
202
221
221
12
323
323
323
308
322
326
241
299
324
308
324
400
31 I
298
298
330
322
322
323
31 I
208
323
255
323
312
206
289
328
271
317
301
300
296
222
309
294
322
312
312
233
298
312
313
313
313
312
314
240
316
315
331
314
31 I
400
335
336
319
314
314
319
331
215
312
237
312
336
216
321
314
261
307
339
335
334
231
31 I
312
312
353
313
312
256
312
316
329
31 I
304
335
400
399
326
312
312
318
329
213
31 I
243
31 I
331
212
377
31 I
257
305
353
350
394
230
354
375
316
333
214
380
312
258
306
354
351
395
322
321
312
335
335
261
332
332
250
230
355
376
317
334
334
251
336
394
395
324
230
341
257
341
324
210
317
342
277
340
326
324
325
221
329
317
340
326
326
247
322
339
400
400
346
312
214
399
253
399.
316
204
31 I
398
279
328
321
398
279
328
321
318
303
222
31 I
308
385
315
315
318
303
222
311
308
385
315
315
242
307
242
307
322
322
255
316
250
327
221
209
143
isolate #
r
01
02
03
04
05
06
07
08
09
10
II
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
23
24
25
26
27
28
29
251
400
397
252
398
251
334
244
396
249
397
250
231
193
243
308
341 ■ 255
31 I
247
396
248
253
327
235
308
31 I
235
310
236
339
255
399
249
399
249
255
344
312
245
213
183
400
251
253
400
400
251
316
245
202
199
310
234
397
250
277
275
329
249
321
244
318
242
231
307
223
262
310
244
235
307
385
249
315
246
315
246
400
397
398
334
396
397
231
308
341
31 I
396
327
308
31 I
310
339
399
399
344
312
213
400
253
400
316
202
310
397
277
329
321
318
307
223
310
307
385
318
318
313
312
204
205
206
206
205
203
219
209
208
215
207
314
315.
315
376
315
315
246
326
306
321
314
291
323
377
380
317
315
315
308
319
21 I
314
397
397
397
333
397
397
229
306
340
315
399
328
314
31 I
310
340
398
398
345
311
199
397
252
397
318
201
311
400
276
328
321
318
307
222
310
277
280
280
260
277
278
195
254
267
262
279
271
261
257
258
277
277
277
270
243
308
30
31
22
199
238
318
317
250
357
327
382
315
314
334
331
333
322
318
318
324
389
2 20
318
247
318
400
220
322
'3 1 8
210
216
208
208
210
206
206
207
217
271
204
197
204
220
400
207
205
182
201
214
218
238
314
324
207
400
313
258
317
358
354
329
223
332
320
310
202
340
373
238
341 '
391
314
315
315
394
394
218
218
320
320
305
384
314
314
243
248
331
226
246
375
242
308
308
210
184
212
208
210
256
304
343
259
199
277
279
277
260
184
258
278
400
271
263
263
254
226
268
257
277
260
260
210
261
331
321
321
329
329
320
315
325
321
. 330
330
320
224
247
308
336
343
320
314
350
330
320
315
297
335
307
351
309
310
352
338
326
321
330
330
321
321
344
315
350
211 . 2 1 1
331
321
255
248
331
321
315
356
201
214
339
304
330
321
273
265
31 I
400
. 315
400
312
394
307
350
235
218
302
347
338
303
318
331
311
311
353
353
249
306
259
349
144
isolate #
n
01
02
03
04
05
06
07
08
09
10
II
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
33
34
35
36
37
38
39
40
318
309
319
309
318
309
351
322
318
314
318
309
246 . 259
331
314
317
315
347
324
318
306
296
300
331
332
348
394
349
395
325
322
318
309
318
309
316
317
349
331
213
217
318
309.
244
239
318
309
352
329
214
214
338
373
318
309
265
254
308
303
394
352
223
224
224
230
222
225
197
227
217
232
221
226
237
238
234
221
222
222
217
227
169
223
258
223
227
190
310
315
312
321
313
317
245
319
312
327
314
31 I
322
352
353
327
313
313
31 I
333
216
310
31 I
312
312
367
312
312
242
326
306
318
311
290
321
375
376
317
312
312
309
321
385
386
386
334
383
387
226
308
341
31 I
385
324
308
310
311
336
385
385
339
309
204
385
253
315
313
313
309
312
314
250
365
323
383
313
312
333
332
334
326
315
315
324
395
217
315
315
313
313
309
312
314
250
365
323
383
313
312
333
332
334
326
315
315
324
395
217
315
.248
315
247
247
250
236
244
246
32
33
34
35
36
37
38
39
400
349
233
347
337
316
351
351
349
400
227
351
371
312
333
333
240
222
228
216
233
231
231
400
226
240
224
228
228
40
41
258
346
248
390
203
235
30
31
32
41
246
310
334
210
337
314
268
302
345
343
349
228
400
336
310
210
311
241
311
320
210
391
31 I
257
301
342
335
335
339
371
238
338
400
313
321
321
252
351
247
371
202
312
384
277
333
318
248
315
394
216
320
314
260
315
357
316
306
224
357
333
224
310
31 I
400
311
31 I
337
321
305
357
333
224
337
321
305
400
400
400
400
251
251
251
309
330
330
385
310
394
216
320
314
260
315
357
308
307
307
352
310
-3 0 9
258
264
249
321
312
246
244
326
251
308
233
300
267
334
248
394
249
395
239
322
250
309
250
309
249
314
252
329
225
213
247
308
203
246
247
308
250 . 331
224
212
242
375
248
308
216
261
245
302
259
353
256
2 44
209
256
243
249
253
253
400 .
248
350
. 390
237
351
371
31 I
332
332
248
400
MONTANA STATE UNIVERSITY LIBRARIES