Utilizing lethal translocation homozygotes of barley (Hordeum vulgare L.)
by Daniel Reid Biggerstaff
A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in
Crop and Soil Science
Montana State University
© Copyright by Daniel Reid Biggerstaff (1988)
Abstract:
Seed of F1 translocation heterozygotes in barley (Hordeum vulgare L.) from crosses between male
steriles and selected translocation homozygotes was treated with the chemical mutagen diethylsulphate.
Translocations were selected to represent breakpoints in the 14 barley chromosome arms, with the
breakpoints as far from the centromere as possible. The male sterile genes selected were located near
the centromere of one chromosome involved in the translocation.
Mg head rows were selected on the basis of aberrant ratios of plants with respect to fertility. The
expected ratio of plants in a row is 1 fertile:2 semisterile: 1 male sterile; rows selected had the aberrant
ratio of 0:2:1, representing possible lethal translocation homozygotes. Twenty-three lethal translocation
homozygote mutants have been recovered. At least one breakpoint in each of the 14 barley
chromosome arms, and all but six of the possible chromosome translocation combinations are
represented.
Maximum likelihood formulae for estimating recombination values from genetic studies using lethal
translocation homozygotes were derived and incorporated in a Microsoft R fortran program. A
spontaneous male sterile mutant (Hector msg,,ec) was assigned to the short arm of chromosome 2,
using F2 data from crosses made with a lethal translocation homozygote tester set. The advantages and
efficiency of this model in genetic studies are described.
Transfer of genes conditioning resistance to barley scald, incited by Rhynchosporium secalis (Oud.)
Davis, with appropriate lethal translocation homozygotes is demonstrated. This approach allows
establishment of populations homozygous for resistance genes without the necessity of screening for
resistance in each cycle of selection. Such populations will be heterozygous and variable for other
agronomic characters. UTILIZING LETHAL TRANSLOCATION HOMOZYGOTES
OF BARLEY (HORDEUM VULGARE L.)
by
Daniel Reid Biggerstaff
A thesis submitted in partial fulfillment
of the requirements for the degree
of
Doctor of Philosophy
in
Crop and Soil Science
j
MONTANA STATE UNIVERSITY
Bozeman, Montana
March 1988
ii
APPROVAL
of a thesis submitted by
Daniel Reid Biggerstaff
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and has been found to be satisfactory regarding content, English
usage, format, citations, bibliographic style, and consistency, and is
ready for submission to the College of Graduate Studies.
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STATEMENT OF PERMISSION TO USE
In
presenting
requirements
for
this
thesis
in
a doctoral degree
partial
fulfillment
of
the
at Montana State University,
I
agree that the Library shall make it available to borrowers under
rules of the Library.
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prescribed in the U.S. Copyright Law.
or
reproduction
Microfilms
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this
thesis
Requests for extensive copying
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right to reproduce and distribute by abstract in any format."
Signature__
Date
/f,
V
ACKNOWLEDGEMENTS •
I wish to express my sincere appreciation to the following:
Professor R.F.,Eslick, for giving me the opportunity to learn the
art of plant breeding while gaining professional training and work
experience as his Research Assistant;
Drs. E.A.
Hockett, R.L. Ditterline, L.E. Wiesner, D.E. Mathre,
E.L. Sharp, and R.B. Walker, for unselfishly giving of their time and
energy in support of my education;
The many Department of Plant and Soil Science faculty, staff, and
graduate students, especially the members of the "barley crew," who
have helped me so often;
Dr. R.T. Ramage, for providing the necessary barley translocation
stocks and performing mutagen treatment on the original Fg material;
Dr. M.E. Bjarko, for conducting the scald reaction screening in
the gene transfer portion of this research; .
My parents, Ronald and Mildred, who instilled in me the value of
education and encouraged my intellectual growth for over four decades.
Above all, I express my sincere gratitude to Sherry for her love,
sacrifice,
and
seemingly endless
patience.
Let
me also
extend
a
special thanks to my favorite young people, Kim, Tracey, and Darcie.
Thank you all for your total support.
This
research
was
supported
Research Grant 5901-0410-9-0-359-0.
in
part
by
USDA/SEA
Competitive
vi
TABLE OF CONTENTS
Page
APPROVAL................................... ,........ ..........
STATEMENT OF PERMISSION. TO USE... ...........
VITA..........
ACKNOWLEDGEMENTS.......................................
TABLE OF CONTENTS.........................
ii
iii
iv
v
vi
LIST OF TABLES................................................ . 'viii
LIST OF FIGURES.........................................
ix
ABSTRACT...............................................
x
INTRODUCTION.................................................. ;
I
LITERATURE REVIEW..............................
MATERIALS AND METHODS.........................................
Developing LethalTranslocation Homo zygotes.................
Linkage Studies......................
Gene Transfer............................
RESULTS AND DISCUSSION.................
Developing LethalTranslocation Homozygotes..........
Linkage Studies..............................
Gene Transfer....................
Summary....................................................
LITERATURE CITED......................
on co «=1" inm m
Genetic Male Sterility..........
Chemical Mutagenesis............
Translocations..................
Recombination Values (Linkage)...
Lethal Translocation Homozygotes.
Rhynchosporium Secalis Resistance
3
7
7
10
13
15
15
19
25
35
36
vii
TABLE OF CONTEMTS— Continued
Page
APPENDICES
A.
B.
Treatment Procedures for Mutagen Treatments
on Barley Used at Washington State University,
Pullman , Washington................. ..................
42j
Computer Programs and Tables to Facilitate
the Calculation of Recombination Values............. ... ..
49
viii
LIST OF TABLES
Table
1.
2.
3•
4.
5.
6.
7.
8.
9.
10.
/
Page
Description of translocations utilized
to establish tester stocks............................
,8
Description of genetic male steriles
utilized to establish tester stocks............ ......
9
Scald resistant cultivars selected as
possible donors of genes conditioning
resistance to Rhynchosporium secalis.............. .. . .
14
Number of head rows and lines selected
following mutagenesis of balanced male
sterile-translocation stocks...... . ............. .
16
Ratios obtained using selfed lethal trans­
location homozygote tester stocks... ..................
18
.Expected genotypic frequencies in the Fg
of selfed translocation heterozygotes.............. .
20
Derivation of the factor to be divided by
/I to obtain the standard error of p ................ .
21
Ratios and recombination estimates obtained
in crosses of Hector msg,,ec and an LTH
tester set........................................ ..;.
24
Seedling reaction to several isolates of
Rhynchosporium secalis for progeny of
crosses between LTH tester stocks and
scald resistant cultivars...... •..... ................
28
Values to facilitate the use of the product
method when using F 2 data from LTH crosses.... ...... .
.51
ix
LIST OF FIGURES
Figure
1.
2.
3•
4.
5.
6.
7.
8.
Page
Diagrammatic representation of the expected
F 2 segregation (chromosomal and genetic)
from a cross between a genetic male sterile
and a translocation homozygote...............
11
Diagrammatic representation of the expected
segregation (chromosomal and genetic) in the
progeny of a translocation heterozygote having
a recessive lethal gene in the interstitial
segment, and a recessive male sterile gene
proximal to the centromere on the normal
chromosome...................................
12
Diagrammatic representation of the expected
F 1 segregation (chromosomal and genetic) from
a cross between a homozygous scald resistant
plant and a translocation heterozygote........
27
Diagrammatic representation of the expected
F i segregation (chromosomal and genetic) from
a cross between a genetic male sterile and a
translocation heterozygote...................
34
Basic program to calculate and print bc/ad,
b/a, and appropriate factors to be divided
by the square root of N for LTH tester stock
progeny......................................
50
Microsoft® fortran program to calculate
maximum likelihood estimates of recombination
values.......................................
52
Data entry instructions and sample printout
for maximum likelihood program.......... ............. .
60
Combined maximum likelihood estimate of p for
three data sets that indicated linkage between
msg,,ec and breakpoints of chromosome 2 ............. .
61
X
ABSTRACT
Seed of Fi translocation heterozygotes in barley (Hordeum vulgare
L.) from crosses between male steriles and selected translocation
homozygotes was treated with the chemical mutagen diethylsulphate.
Translocations were selected to represent breakpoints in the 14 barley
chromosome arms , with the breakpoints as.far from the centromere as
possible.
The male sterile genes selected were located near the
centromere of one chromosome involved in the translocation.
Mg head rows were selected on the basis of aberrant ratios of
plants with respect to fertility.
The expected ratio of plants in a
row is I fertile: 2 semisterile:I male sterile; rows selected.had the
aberrant ratio of 0 :2 :1 , representing possible lethal translocation
homozygotes.
Twenty-three lethal translocation homozygote mutants
have been recovered. At least one breakpoint in each of the 14 barley
chromosome arms, and all but six of the possible chromosome transloca­
tion combinations are represented.
Maximum likelihood formulae for estimating recombination values
from genetic studies using lethal translocation homozygotes were
derived and incorporated in a Microsoft^)fortran program. A spontane­
ous male sterile mutant (Hector msg,,ec) was assigned to the short arm
of chromosome 2 , using Fg data from crosses made with a lethal
translocation homozygote tester set. The advantages and efficiency of
this model in genetic studies are described.
Transfer of genes conditioning resistance to barley scald,
incited by Rhynchosporium secalis (Oud.) Davis, with appropriate
lethal translocation homozygotes is demonstrated.
This approach
allows establishment of populations homozygous for resistance genes
without the necessity of screening for resistance in each cycle of
selection.
Such populations will be heterozygous and variable for
other agronomic characters.
I
INTRODUCTION
Barley (Hordeum vulgare L.) is one of the first crops domesti­
cated by man and is grown throughout the world as a major cereal crop.
It is also used extensively for genetic studies, in mutagen experi­
ments , and as a laboratory organism by physiologists, biochemists,
and others.
Since barley is not only an important commercial crop,
but is also of great interest to scientists from many disciplines, a
novel use of barley in a genetic study may be used as a genetic model
in other species.
A
translocation
heterozygote
having
a
lethal
gene
on
the
interstitial segment of the translocated chromosome and a recessive
male
sterile
gene
near
the
centromere
of
the
non-translqcated
chromosome would provide barley breeders with a useful genetic tool.
This
lethal
translocation
homozygote
(LTH)
would
facilitate
gene
transfers, increase the speed and precision of linkage studies, and
give breeders another way to maintain recessive genes, including newly
mutated lethal genes, in a recognizable heterozygous stock,
in this
attempt
would
also
Success
demonstrate the potential of combining
mutagenesis and cytogenetics to obtain mutants at specific loci.
The purpose of this study was to develop the LTH stock described
above
and
transfers.
demonstrate
its
use
in
genetic
studies, including gene
This method of gene transfer would be cost effective when
transfering genes conditioning traits (such as nutrient value, malting
2
quality,
or disease resistance)
that are difficult or ■expensive to
measure in a segregating population.
Transfer of genes conditioning
resistance to barley scald (incited by Rhynchosporium secalis [Oud.]
Davis) from a resistant cultivar to a breeding population would be
particularly
valuable
considering
the
disease in many areas of the world.
study
would
allow
establishment
crop
losses
caused
by. this
The approach proposed by this
of
populations
homozygous
for
resistance genes without the necessity of screening for resistance in
each cycle of selection.
Such populations would, be heterozygous and
variable for other agronomic characters.
3
LITERATURE REVIEW
Genetic Male Sterility
Genetic male sterility in barley .was first reported by Suneson
(1940).
Since then over 280 different male sterile mutational events
in spring barley have been reported and 39 have been determined tp, be ■
non-allelic
(Foster
al. ■, 1983;
et
Hockett, 1972,
Hockett et al., 1968; Hockett and Reid, 1981).
1984
and '1988;
Numerous workers'.have
reported chromosomal location of these mutants (Eslick, 1976; Eslick
,
.
•
■
'
.
et al., 1974; Jarvi and Eslick, 1967; Ramage and Eslick,■1975; Tuleen, .
1971).
Eslick
(1971) , Haus
(1984), Foster and Fothergill • (1,981) ,
Hockett and Eslick (1971), and Roath and Hockett (1971) have presented
additional
information about origin, inheritance, pollen and anther
morphology, and possible uses of genetic male sterile mutants.
Chemical Mutagenesis
Barley has been used
. . .
extensively in studies of chemical, muta­
genesis (Constantin, 1975; Nilan et al., 1963; Nilan, 1964).
Probably
more information is available on mutation induction in barley than in
any other crop (Nilan, 1974 and 1981).
Diethylsulphate has. been used
successfully in barley mutation work because it. induces a high number
of
point
mutations
(Constantin,
1975;
with
Nilan
relatively
few
et al., 1963)•
chromosomal
Techniques
aberrations
for utilizing
diethylsulphate and other mutagens have been described in the Manual
1»
on Mutation Breeding (1977) •
Scholz (1971) and Milan (1981) present
reviews of the utilization of induced mutations in barley.
( 1 981 ) also
reviews
the
extensive
Milan
research on sodium azide
(NaNg)
mutagenesis conducted at Washington State University.
Translocations
A
chromosomal
homologous
chromosomal
aberration
chromosomes
interchange, a
translocation.
Burnham
discussions
the
Zecevic
have
of
where
exchanged
reciprocal
(1962) and
behavior
)
and
terminal
segments
positions
is
known
translocation, or
Ramage
of
nonas
a
simply
a
(1963) provide excellent
.
.
■
utilization
'
of
translocations.
(1975) presents an extensive literature review on early work
with barley translocations.
A recent review of barley translocations
is found in Sogarrd and von Wettstein-Knowles (1987).
Following
anaphase
I,
adjacent
disjunction of the chromosomes
involved in a translocation will result in spores that abort because
of chromosomal duplications and deficiencies.
In barley, the aborted
female spores serve as a phenotypic character, termed semisterility
(sterility in the spike), by which the translocation heterozygote can
be visually recognized
(Ramage, 1963).
When interstitial crossing
over is followed by alternate disjunction, the crossover chromatids
are in the spores that abort.
alternate
disjunction,
In barley, which exhibits an excess of
recovered
(Kramer and Blander, 1961).
crossovers
are
greatly
reduced
5
Recombination Values (Linkage)
Bateson and Punnett (1911) were the first to describe a method
for
measuring
additional
linkage
methods
for
intensity.
Numerous workers have presented
estimating
linkage
intensities
in
genetic
studies (Fisher and Balmukand, 1928; Immer, 1929; Immer and Henderson,
1943;
Kramer
extended
and
these
Burnham,
methods
1947;
to
Stevens, 1939),
include
crosses
and
involving
several have
chromosomal
translocations (Hanson, 1952; Hanson and Kramer, 1950; Joachim, 1947).
Allard (1956) discusses the relative advantages of several methods of
calculating recombination values and provides formulae and tables to
facilitate these calculations.
Lethal Translocation Homozygotes
The potential uses for the lethal translocation homozygote (LTH)
mutants being sought in this study were proposed by Eslick
(1972).
Initial work on this study was reported by Biggerstaff and Eslick
(1978).
Estimation of linkage intensities from genetic studies using
LTH was reported by Biggerstaff (1987).
Rhynchosporium Secalis Resistance
Mackie
(1929)
reported
resistance
to
scald
single recessive gene in an unspecified cultivar.
conditioned
by
a
Resistance to scald
has since been described in many cultivars (Hansen and Magnus, 1973i
Khan et al., 1984; Roane and Starling, 1952; Schein, I960; Webster et
al., 198 0 ), and the inheritance of resistance has been studied by
6
numerous
researchers
(All
et
al. , 1976;
Baker
and
Larter,. 1963;
Bockelman et al., 1977 and 1978; Dyck and Schaller, 1961a and 1961b;
.
Habgood and Hayes, 1971; Riddle and Briggs, 1950; Wells and.Skoropad,
1963).
Shipton et al. (1974) give a review of barley scald.
The symbol Rha for genes conferring resistance to R_. secalis was
used by Bryner
(1957) and later amended to Rh by Robertson (I960).
Moseman
proposed
(1972)
a
three-letter
symbol
and
a
number
for
resistance to pests, which is the currently accepted, symbolization—
the first letter being R , or r , indicating dominance or recessive
resistance, the second and third letters being abbreviations for the
genus and species of the pest.
7
MATERIALS AND METHODS
Developing Lethal Translocation Homozygotes
Selected translocation homozygotes were crossed to genetic male
steriles.
Translocations were selected to represent breakpoints in
the 14 barley chromosome arms, with the breakpoints as far from the
centromere as possible, as determined from the literature (Table I),
and/or in agreement with Finch and Bennett (1982), Hagberg (1986), and
Jensen (1971).
Seed of all the translocations used in this study were
provided by Dr. R.T. Ramage, University of Arizona, Tucson, Arizona.
Most
of the genetic
centromere
of
one
male sterile genes used are
of
the
chromosomes
involved
located near the
in
the
translocation (Table 2).
reciprocal
,
The F i progeny of these crosses were grown in a winter nursery at
Mesa, Arizona, to obtain maximum seed increase.
cross
was
treated
with
0.015
M
diethylsulfate
Fg seed from each
using
procedures
developed at Washington State University, Pullman, Washington (Nilan
et al., 1963)•
Treated
This procedure is outlined in Appendix A.
seed
(M-j seed, Fg generation)
were
space
planted
at
Bozeman, Montana, in rows 30 cm apart to obtain populations of plants
5 to 8 cm apart within rows.
Mg seed was harvested as individual
spikes taken at random in each population.
Mg head rows were planted
at Bozeman, Montana, as single, space planted rows 3 m in length, 30
cm apart,with plants 5 to 8 cm apart within the rows.
8
Table I. Description of translocations utilized to establish tester
stocks.
Trans­
location
Designation
Cultivar
BreakPoints*
Tl-Be
Tl -4e
Tl-Sf
Tl -6a
Tl -6e
T1-6J
T I-7c
T1-71
Tl-7k
T2-3a
T2-3c
T 2-4a
T2-4d
T2-5a
T2-5e
T3-4b
T3-4d
Bonus
Bonus
Bonus
Mars
Bonus
Bonus
Mars
Bonus, ert a
Bonus , ert a
Gull
Bonus
Mars
Bonus
Bonus
Bonus
Mars
Bonus
Sd
L
S?
S
L
L
S?
L
L
S?
Sd
Cen
S?
Sd
L
Cen
L
Ld
S
L?
Sat
S?
Sat
Sat
Sat
L
S
Sd
S
L
Ld
T4-5e
T4-7b
T5-6b
T5-7g
T 6-7c
T3-7c+
Bonus
Bonus
Bonus
Bonus
Bonus
Bonus
Bonus
Sd
L
L
L
S
Ld
S
LP
Sat
L
L
S
Ld
S
3-7d
*
— —
S
—
Authority
Linde-Laursen (1984)
Persson (I970)
Ramage et al. (1961)
Tuleen (1974)
Persson (I970)
Persson (1970)
Milan (1964)
Ramage (1971)
Persson (1970)
Kasha and Burnham (1965)
Linde-Laursen (1984)
Holm (1963); Eslick (1981)
Ramage et al. (1961)
Linde-Laursen (1984)
Eslick (1981)
Milan (1964)
Sogarrd and Wettstein-Knowles
(1987)
Linde-Laursen (I984)
Linde-Laursen (1984)
Hagberg et al. (I975)
Tuleen (1974)
Ramage and Suneson (1961)
Linde-Laursen (I984)
Linde-Laursen (I984)
= Interchange chromosome with lower number listed first, higher
number listed second.
S
= short arm
L
= long arm
Sat = satellite(short
arm)
?
= probably in that arm
= breakpoint not determined
Cen = centromere region
d
= distal
p = proximal
9
Table 2.
Description of genetic male steriles utilized to establish
tester stocks.
Cultivar
Cl
No.
Symbol
Gene
Location
References
Betzes
6398
msg Ica
near centro­
mere of 5
Ramage (I966)
Compana
5438
msg 2ax
near centro­
mere of 2
Ramage (I966)
Carlsberg II
10114
msg 5ce
near centro­
mere of 3
Hockett & Eslick (1971);
Eslick & McProud (1974)
Heines Hanna
9532
msg 6cf
near centro­
mere of 6
Eslick et al. (1974);
Falk & Kasha (1982)
Compana
5438
msg 10ay
near centro­
mere of I
Hockett & Eslick (1971);
Eslick (1976)
Betzes
6398
msgI 4cm
near centro­
mere of I
Hockett & Eslick (1971);
Eslick (1976)
Betzes
6398
msgl6co
on chromo­
some 7
Hockett & Eslick (1971)
Compana
5438
msg I8cq
on chromo­
some 7
Ramage & Eslick (1975);
Hockett (1981)
Intro.
(Russia)
14393
msgl 9cr
near centro­
mere of 7
Hockett & Eslick (1971)
Glacier/
Compana
10861
msg22 e
on chromo­
some I
Hockett (1972); Eslick
(1976)
Betzes
6398
msg 24 v
near centro­
mere of 4
Jarvi & Eslick (1967);
Wolfe (1984)
Betzes
6398
msg25r
near centro­
mere of 4
Eslick (1971)
Betzes
6398
msg32 w
near centro­
mere of I
Eslick (1976)
Betzes
6398
msg36 bk
on chromo­
some 6
Eslick et al. (1974);
Franchowiak & Hockett
(1987)
Betzes
6398
msg I8z
on chromo­
some 7
Eslick (1971); Hockett
(1981 )
10
At maturity each Mg head row was examined for the absence of
fully fertile plants.
The expected ratio is I fertile (translocation
homozygote) : 2 semisterile (translocation heterozygotes):I male sterile
genetic (normal homozygote)
(Figure I).
Semisterile plants, usually
four, from rows having no fertile plants were harvested individually.
Mg seed from these plants were planted as above at Bozeman, Montana.
Each plant within an Mg row was classified for spike fertility to
confirm
a
0
(Figure 2).
for
selected
fertile: 2
semisterile: I
male
sterile
genetic
ratio
This sequence was continued through the Mg generation
lines.
characteristics
Lines
in addition
were
selected
to goodness
for
superior
of fit to a
agronomic
0 :2:1
ratio.
Backcrosses between selected LTH and other male sterile genetic Betzes
stocks further improved agronomic characteristics.
Linkage Studies
Expected Fg genotypic frequencies for LTH were calculated and
formulae
for
the
application
likelihood method were derived.
of
the
product
method
and
maximum
A tester set of LTH was crossed to a
spontaneous male sterile genetic mutant collected from a commercial
field of the cultivar Hector.
Fg segregation ratios, from F 1 selfed
translocation heterozygotes, were used to estimate linkage intensity
between
the monofactorial recessive
breakpoints.
male
sterile and translocation
11
,Msg
,msg
,Msg
,msg
iXXXxkxxxxxxxxXxxXi
IX X X
K X X X X X X X)-----------
IKKKK » :K K K K *:K *:K K K K < |
#
(translocation homozygote)
(male sterile)
,msg
,Msg
HX x x xi
I X X x m xxTXXXXXXXXXI
(translocation heterozygote)
#
-#
----------------- I XXXXl
,msg____________
,msg__________
-------rnrrx,
V - s^------- DTTTXl
,msg__________
i x x x m - x X X X X ’ Xxxi-
I Fertile
Figure I.
IKKKK JK K K K K K K K K H
iXxxKK.xxxxxxXXxXxXI
IX X X^ P T a X X X X X X X X X X X a)
IXXXWKXXXXXXXXXxxxi
2 Semisterile
I Male sterile
Diagrammatic representation of the expected F 2 segregation
(chromosomal and genetic) from a cross between a genetic
male sterile and a translocation homozygote.
12
.msR Let
---r a
(semisterile)
V isg let---gonno
Sg let--- I
xxxs
jnsg Let
^ Msg let---Kxy-yi
Figure 2.
^msg Let
(XXX
g X X M X X X X X X X Xh-
0 (lethal)
msgLet
2 Semisterile
XXXxXXx
X x x x Xi
I Male sterile
Diagrammatic representation of the expected segregation
(chromosomal and genetic) in the progeny of a translocation
heterozygote having a recessive lethal gene in the inter­
stitial segment, and a recessive male sterile gene proximal
to the centromere on the normal chromosome.
Msg and let
represent hypothetical male sterile genetic and lethal
genes, respectively.
13
Gene Transfer
Selected
LTH were crossed to cuItivars reported to have genes
conditioning resistance to several isolates of Rhynchosporium secalis
(Oud.) Davis, incitant of barley scald (Table 3).
F-j progeny of these
crosses were grown in a winter nursery at Mesa, Arizona.
Semisterile
F 1 plants were harvested individually and Fg plant rows were space
planted, 12 m in length, 30 cm apart at Bozeman, Montana.
Approxi­
mately 50 individual spikes were harvested from fertile Fg plants (Fg
seed).
Fg seedlings from remnant seed and Fg head rows were tested
against several isolates of R_. secalis.
from
Lewistown
and
Culbertson,
Montana;
Turkey; Tal Hadia, Syria; and Morocco.
maintenance,
seedling
Isolates included selections
inoculation,
Davis,
California;
Izmir,
Methods of R. secalis culture
and
classification
reaction were as described by Bockelman et al. (1977).
for
scald
Table 3.
Scald resistant cultivars selected as possible donors of genes conditioning resistance to
Rhgnchosporium secalis.
Resistance Genes
Reported to Be
on Chromosome
Authority*
Cultivar
Cl
Number
Atlas 46
7323
Rrs (allelic series
w/Rrs3 and Rrs4)
3
2, 3
967
rrsl (alleliCyto rrs"’
and rrs )
rrs6
3
I, 3
4
I
Jet
Gene Designation
Other
Genes Reported
in Literature
Rrs2
Kitchin
1296
Rrs9
4
I
La Mesita
7565
Rrs4
3
3
RrslO
Modoc
7566
Rrs24
3
2
2
Rrs2, Rrs , rrsb
Osiris
1622
Rrs4
3
2, 3
rrs6, RrslO
936
Rrs
3
2
rrs6
14400
Rrs
3
3, 4
Rrs5, rrs6
Trebi
Turk
*Authority:
I
2
3
4
=
=
=
=
Bockelman et al., 1977
Dyck and Schaller, 1961b
Habgood and Hayes, 1971
Wells and Skoropad, 1963
15
. RESULTS AND DISCUSSION
Developing Lethal Translocation Homozygotes
The results obtained
in this study demonstrate that recessive
lethal genes can be induced with diethylsulfate and recovered with the
simple classification techniques outlined.
The number of head rows (or lines) selected and continued in each
generation is given in Table 4.
Out of 4933 M 2 head rows, 858 (17.5%)
appeared to be lacking the fertile (translocation homozygote) class.
Most of the M 2 head rows were grown from single spikes on two-rowed
cultivars,
thus the number of surviving plants per row was small.
Therefore, some lines lacked fertiles by chance alone, not because of
homozygous recessive lethal genes.
selected
Mg and Mjj lines
spike fertility.
0:2:1 ratio.
Approximately 70 percent of the
exhibited a 0 :2:1
ratio with respect to
Nearly 80 percent of the M 5 lines closely fit a
About 6 percent of the original M 2 head rows were found
to fit a 0 :2:1 ratio in the M 5 generation.
Use of a recessive marker gene in coupling with the translocation
breakpoint is a refinement of the general procedure.
TI-7k translocation homo zygotes used
zygous for the ert-a dense ear locus.
The TI-TiI and
in this study were also homo­
Ert-a has been reported to be
very near the centromere of chromosome I (Persson, I 970 ); therefore,
the absence of erectoides mutants in M 2 head rows was evidence of the
lack of translocation homozygotes (fertiles).
Table 4.
Number of head rows and lines selected following mutagenesis of balanced male steriletranslocation stocks.
Male Sterile/
Translocation
msglO/Tl-3e
msglO/Tl-4e
msglO/Tl-5f
msglO/Tl-6a
msglO/Tl-6e
msglO/Tl-6j
msglO/Tl-7c
msglO/Tl-7i
msglO/Tl-7k
msg 2/T2-3a
msg 2ll2-2>c
msg 2/T2-4a
msg 2/T2-4d
msg 2/T2-5a
msg 2/T2-5e
msg 5/T3-4b
msg 5/T3-4d
msg l/T4-5e
msgl9/T4-7b
msg l/T5-6b
msg l/T5-7g
msg 6/T6-7c
msgl8/T3-7c+
3-7d
M2
Head
Rows
216
216
216
216
290
216
252
216
252
138
200
216
216
216
216
216
97
216
216
252
216
216
212
M2
Head Rows
without
Fertiles
Selected
M 0 Lines
Continued
M„ Lines
with
0:2:1
Ratio*
Selected
Mg Lines
Continued
76
12
44
37
28
36
26
36
39
25
26
32
56
45
27
24
20
68
20
50
29
49
53
18
12
16
15
18
15
15
15
16
12
12
16
18
18
14
12
14
18
12
18
13
18
24
8
3
10
9
12
13
13
7
16
11
12
14
11
16
13
6
14
14
10
10
9
14
10
8
3
9
9
9
9
9
7
9
9
9
9
9
9
9
6
9
9
9
9
9
9
9
*Fertile:Semisterile:Male sterile
M, Lines
with
0:2:1
Ratio
5
2
7
7
4
7
9
3
8
9
8
3
7
5
5
4
7
7
9
4
5
7
6
Selected
M^ Lines
Continued
M
Lines
with
0:2:1
Ratio
5
2
7
7
4
7
9
3
8
9
8
3
7
5
5
4
7
7
6
4
5
7
6
4
2
6
5
4
6
8
3
6
8
6
3
5
5
5
I
6
7
3
2
3
7
2
17
Some of the induced lethals are listed in Table 5.
The most
frequent and readily identified mutants were albinos and non-heading
dwarfs.
All dwarfs listed were easily distinguished in the tillering
stage and none produced seed in our field environments.
Under certain
field conditions, some dwarf mutants are short lived.
Further study
is suggested
to determine the nature of the other lethal mutants.
Each mutant family within a translocation designation was assigned a
single
letter
designator
to
aid
in
data
grouping
and
seed
stock
to
allow
labeling.
Sufficient
selection
of
lines
the
designation.
were
best
carried
mutant
in
family
each
within
generation
each
translocation,
For example, because too many fertile plants were being
found in T3-4b, mutant family b, and T5-6b, mutant family c, these
were
replaced
respectively
in
later
(Table 5).
generations
by
mutant
families
e
and
I,
Eight of the 10 fertile plants in T5-7g,
mutant family h, were counted in one generation; no fertiles have been
found in subsequent generations.
Twenty-three LTH stocks have been developed, including one with a
double
translocation,
T3-7c+3-7d
(Table
5).
These
mutant
stocks
represent at least one breakpoint in each of the 14 barley chromosome
arms, except possibly the short arm of chromosome 5 , and include all
but
six
of
(Table I).
the
21
possible
chromosome
translocation
combinations
This makes them an ideal tester set for linkage studies or
from which to select a smaller set.
Since this study was initiated,
improved Giemsa N-banding techniques
(Singh and Tsuchiya, 1982) and
use
of
Giemsa
C-banding
(Linde-Laursen, 1984;
Kunzel, 1987)
have
Table 5.
Ratios obtained using selfed lethal translocation homozygote tester stocks.
Translocation Heterozygote Ratios,
F , Numbers
--------------------------------Additional
X-over
X for
Male
SemiMale
LTH
Class,
Fit to
Steriles
Mutant Fertile sterile Sterile 2:1 ratio T
Used?
2
Current Tester Stock
msglO/Tl-3e/*2 Betzes
msgl4/Tl-4e/*2 Betzes
msgl4/Tl-5f/*3 Betzes
msglO/Tl-6a/*2 Betzes
msg32/Tl-6e/*3 Betzes
msg32/Tl-6j/*2 Betzes
msgl4/Tl-7c/*2 Betzes
msg32/Tl-7i/*2 Betzes
msglO/Tl-7k/*3 Betzes
msg 5/T2-3a/* Betzes
msg 5/T2-3c/* Betzes
msg25/T2-4a/*2 Betzes
msg24/T2-4d/*3 Betzes
msg l/T2-5a/*2 Betzes
msg l/T2-5e/*3 Betzes
msg 5/T3-4b/* Betzes
msg 5/T3-4b/* Betzes
msg 5/T3-4d/* Betzes
msg l/T4-5e/*3 Betzes
msgl8/T4-7b/*3 Betzes
msg l/T5-6b/*2 Betzes
msg36/T5-6b/*3 Betzes
msg l/T5-7g/*2 Betzes
msg36/T6-7c/*2 Betzes
msg 5/T3-7c+3-7d/*Betzes
^SemisterilerMale sterile
Mutant
Family
d
a
f
C
a
b
k
d
C
b
h
C
Identifiable
LTH
Mutant
2
dwarf
74
albinos
49
albinos
dwarf
albinos
albinos
albinos
78
75
81
62
q
V
d
b
e
O
m
b
C
I
h
I
C
albinos
dwarf
albinos
44
dwarf
29
3
0
4
0
3
0
0
I
0
0
0
4
I
0
I
11
2
0
0
6
29
I
10
3
3
319
255
266
137
286
272
230
292
327
164
193
168
249
212
154
224
59
145
196
397
232
104
389
354
157
175
140
132
90
162
167
165
146
156
99
105
90
180
125
64
77
34
87
92
261
120
62
321
173
104
.94
.77
.01
3.78
1.55
4.13
11.56
.00
.24
2.08
.47
.27
13.10
2.07
1.66
9.50
.42
1.72
.26
11.03
.09
1.14
40.44
.06
4.62
none
msgl0,msg22
msgl0,msg32
msgl4
msgl0,msgl4
msglO
msglO
msglO
none
msg 2
msg 2
msg 2
msg 2
msg 2
msg 2
msg24
msg24
msg24
msg25
msgl6,msgl9
msg36
msg I
none
msg 6
msgl8
^ Male sterile stocks used in crosses to obtain combined observed ratios
19
shown
that
incorrect.
several
of
the
previously
published
breakpoints
were
Subsequent studies may require further refinement of the
LTH tester set.
Linkage Studies
In an Fg linkage test involving a monofactorial character pair
(Aa) and semisterility due to chromosomal translocations (Bb), the Aa
pair segregates in a 3:1 ratio, while the Bb pair segregates in a 1:1
ratio
of semisteriles
(Bb)
to
fertiles
(BE or bb).
If p is the
recombination value in repulsion , the four classes of male and female
gametes are:
The
■
AB
Ab
aB
-E_
IrR
IrR
2
2
2
sum of the
expected
genotypes is shown in Table 6 .
ab
2
frequencies for each of the four Fg
In the special case of an Fg of an
■
•
LTH, one-quarter of the progeny are marked or removed by lethality.
To determine the recombination percentage from Fg data, the bc/ad
ratio can be used.
To facilitate this calculation for LTH studies,
the bc/ad ratios corresponding to recombination values from 0 to 50
percent are printed in Table 10. (Appendix B).
Table 10 also has the
factor to be divided by /5" to obtain the standard error of p . Figure
5 (Appendix B) is the basic computer program to calculate values in
Table 10 (Appendix B).
20
Table 6 .
Expected genotypic frequencies in the F 2 of selfed
translocation heterozygotes.
a
b
c
d
Semisterile
Fertile
Semisterile
Fertile
A__Bb
A BB
A__bb
aaBb
aaBB
aabb
Phenotypic Class
Fertile v Semisterile
Genotypes
Totals (Normal)
2-2p+2p2
~^r~
I+2p-2p2
4
2p-2p2
4
1-2p+2p2
4
Totals (LTH)
2-2p+2p2
3
2p-p2
3
2p-2p2
3
1-2p+p2
3
The
standard
error
formula for
values of p
is after
Joachim
(19^7) and is as follows:
SE =
/
V
I
nS(i)
where S(i) is the average amount of information per individual in the
F 2 population, and n the number of F 2 individuals.
Therefore, /l/S(i)
is a factor which can be divided by the /n to obtain the standard
error.
This factor is derived for an LTH in Table 7.
Since a translocation homo zygote in the F 2 from a test cross
between a semisterile of an LTH stock and an identifiable gene is
self-roguing
(or marked), the average linkage intensity information
per classified F 2 individual is greater than if normal translocation
stocks are used.
At complete linkage with the translocation break­
point (p=0), the F 2 ratio of the four phenotypes a:b:c:d is 2:1:0:I in
the F 2 of a normal translocation heterozygote and is 2:0:0:I in the F 2
21
of a translocation heterozygote from an LTH tester set.
Therefore,
each classified F g individual in phenotypic class b or c (recombin­
ants) corresponds to a greater change in phenotypic ratios in the Fg
of LTH crosses than in the Fg of normal translocation crosses.
As a
numerical example, at p=.2 the average information per classified Fg
individual in the Fg of a normal translocation heterozygote is 2.141
(Joachim, 1947) and is 5.489 in the Fg of an LTH (Table 10, Appendix B
[/1/5.489 = .4208]).
The same amount of information may be obtained
from an LTH by mutant population less than half the size of a normal
translocation by mutant population.
Table 7.
Derivation of the factor to be divided by /iTto obtain
the standard error of p .
S(i) = S
Fg
Class
a
m
2 - 2p+ 2p 2
3
b
2p-p 2
d
-2+4p
3
WT
dm \ 2
,dp j
4(l-4p+4p2)
9
X
I
m
=
I each class
3
=
4 l-4p+4p2
3 2-2p+2p2
2 -2p+2p 2
3
4(l-2p+p2)
9
3
2-4p
3
4(l-4p+4p2)
9
2p-2p 2
l-2p+p 2
3
- 2 +2p
3
4(l-2p+p2)
9
3
l-2p+p2
3
c
dm
dp
lx
m
2p- 2p 2
2 -2p
4 l-2p+p2
2p-p2
3
2p-p2
3
4 l-4p+4p2
3 2p-2p 2
3
=
4
3
*m is the expected proportion of the total; dm/dp is the derivative of
m with respect to p.
22
In a special case where the semisterility character cannot be
classified in all progeny (for example, aa is a recessive genetic male
sterile), the ratio b/a will give an estimate of linkage intensity.
To facilitate this calculation for LTH, the b/a ratios corresponding
to recombination values from 0 to 50 percent are printed in Table 10
(Appendix B), with factors to be divided by \Zn to obtain the standard
error of p.
The
most
useful
method
of
estimating
combined
values in heredity is the method of maximum likelihood.
recombination
This method
may be applied to any genetic data providing information about linkage
and can be used if more than one type of data is available.
Formulae
to facilitate the maximum likelihood estimation of linkage intensity
between translocation breakpoints in the LTH tester stocks and other
gene pairs were derived using the method described by Allard (1956).
Estimation equations and information equations for F 2 data from selfed
translocation heterzygotes following such crosses are shown below.
When phenotypic classes a, b , c and d are separated:
Estimation Equation...
= 0
Information Equation...
(a+b+c-td) (4/3)
1-4p+4p2
2 (1 -p + p 2 )
When phenotypic classes a and b are separated and classes c and d
are combined:
23
Estimation Equation...
a
' 2p-1
+ b
I-P+P 2
2 (l-p)
2p-p2
= 0
Information Equation...
These formulae have been incorporated into a Microsoft^ fortran
program (Equation sets 45 and 46, Figure 6 , Appendix B) in addition to
all equations in Hanson and Kramer
(1950) and Allard (1956).
Data
entry using any word processing program or line editor is possible
(see
data
entry
instructions
and
resultant
p
solution
printout,
Figure 7, Appendix B).
A special case linkage problem demonstrates the application of an
LTH tester set in genetic studies.
An apparent genetic male sterile
mutant was collected in a commercial field of the cultivar Hector.
ratios of progeny from open pollination —
(x2
fit
to
3:1 ratio
pollination —
=
.46)
and
Fg
81 fertile:23 male sterile
F-j ratios
of
progeny
from sib
11 fertile: 8 male sterile (x2 fit to 1:1 ratio = .47)
(Biggerstaff, 1981 ) suggested a monofactorial recessive male sterile
mutant.
This mutational event was assigned the designation Hector
msg, ,ec (Hockett, 1984).
Ratios and recombination estimates obtained
in crosses of Hector msg,,ec.and an LTH tester set can be found in
Table 8 .
The highly significant homogeneity x2 for a single p indicates
these nine data sets cannot be considered homogeneous and should not
be combined.
An excess of fertiles in data set 7 gives a poor fit to
Table 8.
Ratios and recombination estimates obtained in crosses of Hector msg,,ec and
an LTH tester set.
Translocation Heterozygote
________ Ratios,
________
<---- Phenotypic Class--- >
b
a
c+d
SemiMale
Fertile sterile Sterile
x2 for
fit to
1:2:1
Ratio*
Homogeneity
x 2 for
Single p
Recombination Estimates
Data
Set
Cross
Mutant
Family
I
Tl-3e
d
47
81
67
9.70
4.04
2
Tl-6 j
b
49
91
54
1.00
6.81
3
Tl-7 i
d
42
77
39
.16
8.18
4
T2-3c
h
I
57
23
25.39
14.11
1.7 + 1.7
1.7 + 1.8
5
T2-4d
q
4
119
68
54.45
32.36
3.3 + 1.7
3.3 + 1.6
6
T2-5a
V
3
109
71
57.22
33.84
2.7 + 1.6
2.7 + 1.5
7
T3-4b
b
73
85
46
12.81
43.80
8
T5-7 g
h
54
94
137
81.36
.07
9
T6-7c
I
12
35
16
1.28
.09
Total
*1 Fertile:2 Semisterile:! Male sterile
143.30
b/a
Maximum
Likelihood
25
a I fertile:2 semisterile:I
male sterile.
T3-4b, mutant family b ,
shows an excess of fertiles in Table 5 as well, indicating possible
crossovers between the breakpoint and lethal gene.
An excess of male
steriles in data set 8 also gives a poor fit to a 1:2:1 ratio.
i
This
relationship can also be seen in Table 5, T5-7g, mutant family h.
These data suggest possible gametic selection.
Data sets I, 2, 3 and
9 are homogeneous and support the hypothesis of independence.
Note
that the disturbances of data sets 7 and 8 are not in a direction that
would indicate linkage.
Data sets
4, 5 and 6 are all deficient in the fertile class,
indicating linkage between msg,,ec and the breakpoints of T2-3c, T2-4d
and T2-5a, respectively.
Since the only translocated chromosome in
common is chromosome 2 , and the short arm of chromosome 2 in all cases
(Table I), these data indicate the probable location of msg,,ec is
near the centromere or on the short arm of chromosome 2 .
The maximum likelihood or product method is used to calculate
the recombination values between msg,,ec and the breakpoints of T2-3c,
T2-4d, and
T2-5a,
respectively
(Table
8 ).
The maximum likelihood
method can be used to calculate the combined recombination value of
2.8 + .9 (see Figure 8 , Appendix B).
This is the recombination value
between msg,,ec and the centromere if msg,,ec is on the long arm of
chromosome 2 .
Gene Transfer
If the appropriate semisterile from an LTH stock is crossed with
a normal plant, those genes on the normal chromosome region opposite
26
the interstitial segment
for recombination.
(Figure 3) will have a reduced opportunity
Selecting a semisterile plant in the F-| 0f such a
cross, and selecting.only fertile plants from its Fg progeny, will
effectively transfer those genes opposite the interstitial segment.
The scald resistant cultivars shown in Table 3 were crossed with
LTH tester
sets
transfer.
Selected progeny from thesecrosses were tested against
several
isolates
to demonstrate the use of LTH’s in effecting gene
of
Rhynchosporium
secalis.
Fg
and
Fg
seedling
reactions are summarized in Table 9As a precaution, .Fg spikes were only harvested in Fg rows that
segregated
for the identifiable LTH mutant
(where one is expected,
Table 5). This guards against selecting Fg plants where the lethal (or
marker) mutant may be a recombinant.
12 LTH stocks,
This option is not available in
nor is the mutant always sufficiently viable to be
read.
Data summarized
over 60 Fg
9000
head
in Table 9 include seedling reaction data from
families tested against two or three isolates, and over
rows
tested
against
single
isolates.
Although
some
populations are small, there were several noteworthy trends in the
reaction types.
In crosses involving T2-3c and T3-4d (Table 9), data sets 13, 15,
and 43 have the greatest shift toward fully resistant Fg *s, with all
three isolates, indicating linkage between scald resistance genes and
the
interstitial segment
of these
two translocations.
Since most
workers have reported scald resistance genes associated with chromo­
somes 3 and 4, this would support their findings.
Strong linkage is
27
Rrs Msg Let
q
Rrs Msg Let
^ rrs Msg Ietnmnn
g
rrs msft Let
♦♦♦♦♦♦♦♦ ♦♦
S
(fertile)
(semisterile)
#
0 Rrs Msg Let
Rrs Msg Let
^ rrs Msg Let
rrs Msg let
>:
:<
I Fertile
Figure 3-
nnnno
I Semisterile
Diagrammatic representation of the expected F-j segregation
(chromosomal and genetic) from a cross between a homozygous
scald resistant plant and a translocation heterozygote.
Rrs, msg and let represent hypothetical scald resistance,
male sterile genetic and lethal genes, respectively.
All
genes are depicted as being allelic.
Table 9.
Seedling reaction to several isolates of Rhynchosporiunt secalis for progeny of crosses
between LTH tester stocks and scald resistant cultivate.
___________________Rhynchosporium secalis Isolates___________________
Data
Set
I
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Resistant
Cultivar
Atlas 46
Turk
LTH
Tl-3e
Tl-4e
Tl-ba
Tl-be
Tl-7c
T2-3c
T2-5a
T3-4d
T4-7b
Tl-7c
Tl-71
Tl-7k
T2-3c
T2-4d
T3-4d
T4-5e
T5-6b
T5-7g
T6-7c
T3-7c, 3-7d
Mutant
Family
d
a
C
a
k
h
v
O
b
k
d
C
h
q
O
m
i
g
i
C
<----- Lew B 77—81 ————— >
<------ -Mor 25 ———---——>
<——81 Izmir
------ —3---- —
Res Seg Susc*
------ —3-----Res Seg Susc
-------3-----Res Seg Susc
19
I
7
3
14
42
13
26
20
21
11
11
10
29
7
16
6
28
7
14
7
6
7
I
10
I
3
30
10
24
53
19
76
44
36
6
20
13
18
28
43
I
28
I
67
44
21
26
26
4
15
10
0
14
0
4
13
4
6
5
----2---Res Susc
—
—
31
42
31
16
—
—
57
54
16
12
—
—
29
96
10
12
——
— —
60
69
58
75
89
53
69
60
63
39
15
24
0
13
10
20
17
18
18
12
56
0
0
0
22
23
0
3
54
30
7
9
6
43
60
24
24
29
12
18
9
8
34
20
26
33
15
15
20
22
36
16
48
14
12
17
19
11
9
33
16
I
22
2
22
13
26
19
27
0
6
5
0
12
0
4
11
2
7
10
----2---Res Susc
5
5
52
28
51
13
12
56
52
7
— —
__ •—
56
56
49
53
70
50
54
60
54
19
8
20
0
22
5
I
8
6
7
7
10
12
17
52
14
50
23
20
15
14
20
11
20
20
0
27
0
25
12
18
21
27
3
18
12
0
10
0
3
14
17
15
10
Table 9— continued.
___________________ Rhynchosporium secalis Isolates___________________
Data
Set
Resistant
Cultivar
21
22
23
24
25
26
27
28
29
30
31
Jet
32
33
34
35
36
37
38
39
40
Kitchin
LTH
Tl-4e
Tl-5f
Tl-6a
Tl-6e
Tl-6 j
T2-3a
T2-4a
T2-4d
T3-4d
T4-7b
T3-7c, 3-7d
Tl-4e
Tl-7c
Tl-71
T2-4a
T2-4d
T4-5e
T4-7b
T5-7g
T3-7c, 3-7d
Mutant
Family
a
f
C
a
b
b
C
q
O
b
C
a
k
d
C
q
m
b
8
C
<----- Lew B 77-81a------>
<-------- Cal^--------- >
<— 81 Davise->
------ —3------Res Seg Susc*
------ -3------------2---Res Seg Susc Res Susc
------ -3-----Res Seg Susc
----2---Res Susc
12
18
5
7
2
11
3
3
13
7
0
29
9
9
29
31
33
15
29
17
26
15
9
I
7
11
11
6
11
7
4
17
27
21
18
6
11
58
48
18
12
15
17
9
39
34
21
27
10
13
29
67
62
66
67
2
10
6
10
7
38
3
5
14
7
2
30
17
9
31
28
11
15
27
18
23
24
18
I
6
9
9
0
10
14
I
11
18
7
12
9
8
9
22
12
14
10
8
11
10
11
5
13
8
9
8
12
16
23
25
16
7
18
4
6
9
2
5
23
16
10
10
11
20
4
14
3
9
26
22
20
32 ■
13
21
24
34
43
28
21
33
12
22
15
14
2
19
43
47
23
55
39
60
17
14
22
5
14
8
8
8
6
13
16
9
10
14
14
5
10
12
7
20
17
23
22
28
17
25
19
16
17
20
15
20
13
6
12
7
16
11
11
7
12
16
16
20
27
I
55
78
13
12
36
7
20
17
12
9
3
22
17
10
13
22
23
18
13
10
14
21
7
15
16
10
0
22
25
Table 9— continued.
___________________Rhynchosporium secalis Isolates___________________
Data
Set
Resistant
Cultivar
41
42
43
44
45
La Mesita
LTH
Tl-3e
Tl-7k
T2-3c
T2-4a
T2-5a
Mutant
Family
d
C
h
C
V
<----- Lew B 77-81*----- >
<-------- Cald--------- >
<— 81 Culbf- >
------—3------Res Seg Susc*
-------3-----Res Seg Susc
-------3-----Res Seg Susc
30
9
43
22
6
4
6
7
20
20
16
9
0
9
19
---—2---Res Susc
70
22
39
32
12
7
23
4
11
61
26
9
42
17
0
4
12
9
19
I
18
4
0
8
46
---—2---Res Susc
32
28
24
24
6
13
11
4
6
41
30
9
43
19
I
4
10
7
14
2
14
4
0
21
39
-81 TH 8—
— -
>
F1
J
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Osiris
Tl-3e
Tl-4e
Tl-5f
Tl-6a
Tl-7c
Tl-71
Tl-7k
T2-3a
T2-4d
T2-5a
T2-5e
T3-4b
T3-4d
T4-7b
T3-7c , 3-7d
d
a
f
C
k
d
C
b
q
V
d
e
O
b
C
49
21
39
18
15
28
25
48
16
22
15
15
49
31
16
0
2
11
9
3
18
23
0
14
3
8
7
I
28
16
0
I
0
2
I
4
2
0
2
0
I
6
0
6
4
150
66
53
54
56
53
83
58
71
73
65
84
70
55
53
2
0
3
2
4
9
3
I
6
2
0
6
6
0
0
36
9
13
15
11
15
13
46
8
9
10
9
47
17
14
6
11
26
20
7
25
26
2
18
30
35
8
3
21
22
8
4
9
15
I
10
9
0
12
10
15
12
0
12
13
26
26
21
25
29
24
28
32
31
35
19
28
30
19
32
8
3
4
9
0
4
9
0
14
4
11
10
17
10
11
Res
Seg
Susc
27
11
17
17
2
12
13
13
8
22
13
14
26
26
14
21
11
10
24
7
15
27
18
18
23
21
11
15
24
20
2
3
11
8
10
7
10
8
11
3
4
3
9
4
20
Table 9— continued.
Rhynchosporium secalis Isolates
Data
Set
Resistant
Cultivar
61
62
63
64
65
66
Trebi
67
68
69
70
71
72
73
74
Modoc
LTH
Tl-3e
Tl-Sf
Tl-71
T2-3a
T2-4a
T4-5e
Tl-3e
T2-4d
T2-5a
T2-5e
T3-4d
T5-7g
T6-7c
T3-7c, 3-7d
Mutant
Family
d
f
d
b
C
m
d
q
V
d
O
8
I
C
<------ -- Lew B 77--81a-- ———^
F3
Fo
J
Susc*
Res
Susc
Res
Seg
15
90
12
75
5
25
26
11
27
25
13
30
9
0
11
0
4
0
41
—
46
56
33
36
46
15
I
17
11
27
18
13
8
10
14
13
32
8
12
19
23
17
7
5
9
0
7
7
10
58
36
41
53
51
38
51
44
14
19
8
13
15
31
22
40
—
20
5
28
11
*Resistant, segregating, susceptible
^Isolate
Isolate
^Isolate
Isolate
^Isolate
Isolate
^Isolate
from
from
from
from
from
from
from
Lewistown, Montana, reselected Bozeman 1977-81
Morocco, 1976
Izmir, Turkey, 1981
Davis, California, 1978
Davis, California, 1981
Culbertson, Montana, 1981
Tal Hadia, Syria, 1981
< ---------
-Cald
F3
Res
Seg
10
10
12
30
6
4
24
30
27
19
12
20
---- —^
<— 81 Culb f- >
F3
Susc
Res
Seg
13
31
5
39
5
18
12
14
13
7
7
10
Fo
Susc
16
10
11
0
4
0
Res
27
3
9
16
19
10
5
5
26
19
17
19
Susc
25
0
31
3
10
8
32
also indicated in data sets 6 , 8 , 5 8 , and 71 with these same trans­
locations,
but
not
with
all
isolates.
This
virulence types among the various isolates.
indicates
different
For example, in data set
8 , the Atlas x TS-1Id Fg progeny are quite resistant to the Lew B 77-81
isolate and quite susceptible to the Mor 25 isolate.
Linkage is also indicated between Tl-Se, TI-1Ie, and T2-3a and
scald resistance genes in Osiris (data sets 46, 47, and 53), and T2-3&
and scald resistance genes in Trebi (data set 64).
Again, this would
support previous findings, especially Bockelman et al. (1978).
The Fg seedling reactions to isolate Lew B 77-81 in data set 48
(Osiris x T1-5f) and in data set 62 (Trebi x Tl-5f) indicate.linkage
and would
literature.
Tl-7c).
suggest
a gene position not
previously reported
in
the
Similarly, linkage is indicated in data set 50 (Osiris x
These data are probably not sufficient to suggest a resist­
ance gene on chromosome I.
Data in several instances suggest the transfer of genes condi­
tioning susceptibility to R_. secalis, including data sets 2 , 3» 4, and
7 to Mor 25, data sets 27, 28, and 31, to Lew B 77-81 and Mor 25, data
set 40 to three isolates (Lew B 77-81, Mor 25, and 81 Davis) and data
set 45 to three isolates (Lew B 77-81, Mor 25, and 81 Cul).
If there
has not been a recovered double crossover or a mistake in crossing,
these data are difficult to explain in terms of the proposed LTH gene
transfer scheme.
Although research reported here is from the preliminary screening
cycle
and
some
populations
these data indicate that
:
'
have been developed to facilitate transfer of
.
suitable LTH stocks
.
are
.
small,
33
certain scald resistance genes.
determine
if
the
Tl-5
or
Further screening is suggested to
Tl-7. translocations
show, linkage
to
a
previously unassigned scald resistance gene.
Other difficult genetic problems can also be facilitated with LTH
stocks.
For example, rapid allelism tests can be accomplished using
the concepts depicted in Figure 4.
A cross between a plant having a
known gene homozygous and the appropriate LTH will produce semisterile
progeny with the general genetic and chromosomal configuration of Pg
in Figure 4.
If the homozygous recessive gene in P<| is allelic to the
gene
normal
on
the
chromosome
of
Pg,
the F-j will
be
50 percent
semisterile and 50 percent homozygous (male sterile in this case) for
the
recessive
gene
being
allele
tested, and
semisteriles will segregate 1:1 for that trait.
the
progeny
of
the
If the genes are non­
allelic and independent of the interstitial segments of both translo­
cated chromosomes, all F i plants will be heterozygous for the trait
and their progeny will segregate 9 : 7 for that character.
An appropriate LTH stock could facilitate selecting the balanced
repulsion phase of two phenotypically similar genes (for example, male
sterile genetic genes).
If a homozygous
male sterile genetic
is
crossed to the appropriate LTH, having a non-allelic male sterile gene
tightly linked to the interstitial segment of the same chromosome on
which the other gene is located
(non-allelic example above), the F^
will segregate I fertile:I semisterile.
Plants in the fertile class
will have the two male sterile genetic genes in a balanced, repulsion
phase condition.
34
0
msK Let
^ __msg Let_______
0
mag Let
• — Mss let—
onma
■Hal
(male sterile)
(semisterile)
*
>
msR Let
0
msg Let
msg Let_______
0
— #—
let---IYTTH
KXXMXXXXXXX
ixxxmx:
I Male sterile
Figure 4.
:
I Semisterile
Diagrammatic representation of the expected F segregation
(chromosomal and genetic) from a cross between a genetic
male sterile and a translocation heterozygote. Msg and let
represent hypothetical male sterile genetic and lethal
genes, respectively.
Ms& genes are depicted as being
allelic.
1
35
Summary
This study demonstrates that mutagenesis and simple cytogenetic
screening techniques can be used to induce and recover useful mutants
at specific loci.
A much higher than expected rate of success was
achieved.
LTH
chromosome
tester
sets
can
location for new
be
used
to
rapidly
determine
probable
(or previously unassigned) genes.
The
efficiency of LTH tester stocks in estimates of recombination values
approaches that of a backcross.
The transfer of genes conditioning resistance to barley scald is
demonstrated.
This approach allows the establishment of populations
homozygous for resistance genes without the necessity of screening for
resistance in each cycle of selection.
heterozygous
and
variable
for
other
(Such populations would be
agronomic
characters.)
The
frequency of alleles for resistance in these populations would be very
high and repeated cycles of LTH crosses would keep their frequency
very high.
36
LITERATURE CITED
37
LITERATURE CITED
All, S.M., A.H. Mayfield, and B.G. Clare. 1976. Pathogenicity of 203
isolates of Rhynchosporium secalis on 21 barley cultivars.
Physiol. Plant Pathol. 9: 135-143.
Allard, R.W. 1956. Formulas and tables to facilitate the calculation
of recombination values in heredity. Hilgardia 24: 235-278.
Baker, R.J. and E.N. Barter.
1963.
The inheritance of
resistance in barley. Can. J. Genet. Cytol. 5: 445-449.
scald
Bateson, ¥. and R.C. Punnett.
1911.
On gametic series involving
reduplication of certain terms. J. Genet. I: 293-302.
Biggerstaff, D.R.
1981.
Unpublished data.
Biggerstaff, D.R.
1987.
Abstract: Using interchange homozygote
lethals to estimate linkage. Barley Newsl. (in press).
Biggerstaff, D.R. and R.F. Eslick.
1978.
cytogenetics and mutagenesis to recover
specific loci. Barley Newsl. 22: 13.
Abstract: Combining
unusual mutants at
Bockelman, H.E., E.L. Sharp, and R.F. Eslick.
1977.
Trisomic
analysis of genes for resistance to scald and net blotch in
several barley cultivars. Can. J. Bot. 55: 2142-2148.
Bockelman, H.E., E.L. Sharp, and R.F. Eslick.
1978.
Observed
recombination between T3 translocation stocks and scald resis­
tance loci. Barley Genet. Newsl. 8 : 17-20.
Bryner, C.S. 1957. Inheritance of scald resistance in barley.
Thesis, Penn. State Univ. Diss. Abstr. 17: 2752.
Ph.D.
Burnham, C.R.
1962.
Interchanges,
pp. 66-116. In.: Discussions in
Cytogenetics. Burgess Publishing Co., Minneapolis.
Constantin, M.J.
1975.
Mutations for chlorophyll-deficiency in
barley: Comparative effects of physical and chemical mutagens.
Barley Genet. Ill: 96-112.
Proc. Third. Int. Barley Genet.
Symp., Garching.
Dyck, P.L. and C.W. Schaller. 1961a.
Inheritance of resistance in
barley to several physiologic races of the scald fungus. Can; J.
Genet. Cytol. 3: 153-164.
38
Dyck, P.L. and C.W. Schaller. 196lb.
scald resistance with a specific
Genet. Cytol. 3: 165-169.
Association of two genes for
barley chromosome.
Can. J.
Eckhoff, J.L. and R.T. Ramage.
1984.
Assignment of a short awn
mutant to chromosome 4. Barley Genet. NewsI. 14: 20-21.
Eslick, R.F. 1971. Balanced male steriles and dominant pre-flowering
selective genes for use in hybrid seed production. Barley Genet.
II: 292-297. Washington State Univ. Press, Pullman. .
Eslick, R.F.
1972. Barley genetics— Rolls Royce, Model T, Chrysler,
or Ford. Barley Newsl. 15: 69-70.
Eslick, R.F.
1976.
Male sterile genes on chromosome I.
Genet. Newsl. 6 : 14-20.
Eslick, R.F.
1981.
Barley
Unpublished data in personal communication.
Eslick, R.F. and W.L. McProud. 1974.
Positioning of male sterile 5
(msg5) on chromosome 3. Barley Genet. Newsl. 4: 16-23.
Eslick, R.F., R.T. Ramage, and D.R. Clark.
1974. . Two genetic male
steriles, msg 6 and msg,,bk, assigned to chromosome 6 . Barley
Genet. Newsl. 4: 11-15.
Falk,
D.E. and K.J. Kasha.
1982.
Registration of a shrunken
endosperm, male-sterile germplasm to facilitate hybridization in
barley. Crop Sci. 22: 450.
Finch, R.A. and M.D. Bennett. 1982.
The karyotype of Tuleen 346
barley. Theor. Appl. Genet. 62: 53-58.
Fisher, R.A. and B. Balmukand. 1928. The estimation of linkage from
the offspring of selfed heterozygotes. J. Genet. 20: 79-92.
Foster, C.A. and M. Fothergill. 1981.
Breeding F-| hybrid barley.
Barley Genetics IV: 766-771.
Proc. Fourth Int. Barley Genet,
Symp., Edinburgh.
Foster, C.A., M. Fothergill, and A.D. Hale.
1983. The WPBS genetic
male sterile barley collection. Barley Genet. Newesl. 13: 6 - 8 . .
Franckowiak, J.D. and E.A. Hockett.
1987.
Allelism tests for the
genetic male sterile msg,,bk. Barley Genet. Newsl. 17: 77?
Habgood, R.M. and J.D. Hayes. 1971. The inheritance of resistance to
Rhynchosporium secalis in barley. Heredity 27: 25-37.
39
Hagberg, A.
1986.
Induced structural rearrangements.
pp. 17-36.
In: Genetic Manipulation in Plant Breeding.
¥. Horn, C.J.
Jensen, W. Odenbach, and 0. Schieder, eds.
Walter de Gruyter,
Berlin.
,
Hagberg, G., L. Lehman, and P. Hagberg. 1975. Segmental interchanges
in barley:
I. Translocations involving chromosomes 5 and 6.
Hereditas 80: 73-82.
Hansen, L.R. and H.A. Magnus. 1973.
Virulence spectrum of Rhynchosporium secalis in Norway and sources of resistance in barley.
Phytopathol. Z. 76: 303-313. .
Hanson, W.D.
1952.
An interpretation of the observed amount of
recombination in interchange heterozygotes of barley.
Genetics
37: 90-100.
Hanson, W.D. and H.H. Kramer.
1950.
The determination of linkage
intensities from F g and Fg genetic data involving chromosomal
interchanges in barley. Genetics 35: 559-569Haus, T.E. 1984. The use of male sterile stocks in linkage analysis.
Barley Genet. Newsl. 14: 55-56.
Hockett, E.A. 1972. Coordinator's report on the genetic male sterile
barley collection. Barley Genet. Newsl. 2: 139-144.
Hockett, E.A. 1984. Coordinator's report on the genetic male sterile
barley collection. Barley Genet. Newsl. 14: 70-75.
Hockett, E.A.
1988.
Unpublished data on the genetic male sterile
barley collection.
Hockett, E.A. and R.F. Eslick. 1971.
Genetic male-sterile genes
useful in hybrid barley production.
Barley Genet. II: 298-307.
Washington State Univ. Press, Pullman.
Hockett, E.A., R.F. Eslick, D.A. Reid, and G.A. Wiebe. 1968. Genetic
male sterility in barley.
II.
Available spring and winter
stocks. Crop Sci. 8: 754-755Hockett, E.A. and D.A. Reid.
1981.
Spring and winter genetic malesterile barley stocks. Crop Sci. 21: 655-659»
Holm,
G.
1963Gene mapping of chlorophyll mutations in barley.
Barley Genet. I: 186-188. Proc. First Int. Barley Genet. Symp.,
Wageningen.
Immer, F.R.
1930.
Formulae and tables
intensities. Genetics 15: 81-98.
for
calculating
linkage
40
Immer, F.R. and MLT. Henderson.
Genetics 28: 419-440.
Jarvi, A.J. and R.F. Eslick. 1967.
4. Barley Newsl. 11: 17.
1943.
Linkage studies in barley.
•(
;
A male sterile gene on chromosome
Jensen, J. 1971. Mapping of 10 mutant genes for necrotic spotting in
barley by means of translocations.
pp. 213-219In_: Barley
Genetics II. Proc . Second Inti. -Barley Genet. Symp. R.A. Nilan,
ed. Washington State Univ. Press, Pullman.
Joachim, G.S.
1947.
The product method of calculating linkage from
F 2 data involving semisterility; and its application to a barley
translocation. Genetics 32: 580-591•
Kasha, K.J. and C.R. Burnham.
1965.
The location of interchange
breakpoints in barley. I. Linkage studies and map orientation.
Can. J. Genet. Cytol. .7: 62-77. .
Khan, T.N., P.A. Portmann, and R . McLean.
1984. Evaluating resist­
ance of barley breeding lines to scald in field plots. Euphytica
33: 897-901.
Kramer, H.H. and B.A.S. Blander. 1961. Orienting linkage maps on the
chromosomes of barley. Crop Sci. I: 339-342.
Kramer, H.H. and C.R. Burnham.
1947.
Methods of combining linkage
intensity values from backcross, Fg and Fg genetic data.
Genetics 32: 379-390.
Kunzel, G.
1987.
Giemsa banding of barley chromosomes in meiosis
improves identification of chromosome arms involved in transloca­
tions. Barley Genet. Newsl. 17: 83-86.
Linde-Laursen, I . 1984.
Breakpoints localized to chromosome arm or
region in 26 translocation lines of barley using Giemsa
C-banding. Barley Genet. NewsI. 14: 12-13.
Manual on Mutation Breeding. 1977. Technical Reports Series No. 119,
2nd ed. International Atomic Energy Agency, Vienna.
Moseman, J.G. 1972. Report on genes for resistance to pests.
Genet. Newsl. 2: 145-147.
Barley
Nilan, R.A.
1964.
The cytology end genetics of barley, 1951-1962.
Monographic Supplement No. 3. Research Studies 32(1). Washington
State University, Pullman. 278 pp.
41
Nilan, R.A. 1974. Barley (Hordeum vulgare). pp, 93-110. In: Hand­
book of Genetics. Vol. 2: Plants, Plant Viruses, and Protists.
R.C. King, ed. Plenum Press, New York.
Nilan, R.A.
1981.
Recent advances in barley mutagenesis.
Barley
Genetics IV: 823-831.
Proc. Fourth Int. Barley Genet. Symp.,
Edinburgh.
Nilan, R.A., C.F. Konzak, R.E. Heiner, and E.E. Froese-Gertzen. 1963.
Chemical mutagenesis in barley.
Barley Genet. I: 35-54.
Proc.
First Int. Barley Genet. Symp., Wageningen. *
Persson, G.
19-70.
An attempt to find suitable genetic markers for
dense ear loci in barley II. Hereditas 63: 1-28;
Ramage, R.T.
1963. Chromosome aberrations and their use in genetics
and breeding— translocations.
Barley Genet. I: 99-115.
Proc.
First Int. Barley Genet. Symp., Wageningen."
Ramage, R.T.
1966.
Techniques
Barley Newsl. 10: 44-49.
for
mapping
barley
chromosomes.
Ramage, R.T.
1971.
Translocations and balanced tertiary trisomics.
Barley Genet. NewsI. I: 74-80.
Ramage, R.T., C.R. Burnham, and A. Hagberg.
1961.
A summary of
translocation studies in barley. Crop Sci. I: 227-279.
Ramage, R.T. and R.F. Eslick. 1975.
Translocation linkage tests—
T2-7a x male sterile genes. Barley Genet. Newsl. 5: 46-48.
Ramage, R.T. and C.A. Suneson. 1961. Translocation-gene linkages on
barley chromosome 7- Crop Sci. I: 319-320.
Riddle, O.C. and F.N. Briggs. 1950.
Inheritance of resistance to
scald in barley. Hilgardia 20: 19-27.
Roane, C.W. and T.M. Starling.
1952. Scald resistance in Oldambster
barley. Plant Dis. Rep. 36: 212-213Roath, W.W. and E.A. Hockett. 1971.
Pollen development in genetic
male-sterile barley.
Barley Genet. II: 308-315.
Washington
State Univ. Press, Pullman.
Robertson, D.W. I960. Summary of linkage studies in barley, 1956-60.
Fourth Barley Improvement Conf. North Dakota Agr. Coll. Mimeo
Abstr., 25-31•
42
Schein, R.D.
1960.
Physiologic and pathogenic
Rhynchosporium secalis.
Penn. State Univ.
Bull. 664. 29 pp.
specialization of
Agric. Exp. Sta.
Scholz, F. 1971. Utilization of induced mutations in barley. Barley
Genet. II: 94-105. Washington State Univ. Press, Pullman.
Shipton, W.A., W.J.R. Boyd, and S.M. All.
Annu. Rev. Plant Pathol. 53: 839-861.
1974.
Scald of barley.
Singh, R.J. and T. Tsuchiya..
1982.
An improved Giemsa N-banding
technique for identification of barley chromosomes. J-. Hered.
73: 227-229.
Sogarrd, B. and P . von Wettstein-Knowles. 1987.
Barley: Genes and
chromosomes. Carlsberg Res. Commun. 52: 123-196.
Stevens, W.L.
1939.
Tables of the recombination fraction estimated
from the product ratio. J. Genet. 39: 171-180.
Suneson, C.A.
1940.
A male sterile character in barley: A new tool
for plant breeders. J. Hered. 31: 213-214.
Tuleen, N.A.
1971. Translocation-gene linkages from Fg seedlings in
barley.
Barley Genet. II: 208-212.
Washington State Univ.
Press, Pullman.
Tuleen, N.A.
1974.
Unpublished data in personal communication to
R.T. Ramage, dated April 1974.
Webster, R.K., L.F. Jackson, and C.W. Schaller. 1980.
Sources of
resistance in barley to Rhynchosporium secalis. Plant Dis. 64:
88-90.
Wells, S.A. and W.P. Skoropad.
1963.
Rhynchosporium secalis in barley.
187.
Inheritance of reaction to
Can. J. Plant Sci. 43: 184-
Wolfe, H.I. 1984. Unpublished data in personal communication to T.E.
Haus. Barley Genet. NewsI. 14: 55-56.
Zecevic, L.J., A. Popovic, and D . Maksimovic. 1975.
A reciprocal
translocation in barley after gamma irradiation of dry seeds.
Barley Genet. Ill: 293-302.
Proc. Third Int. Barley Genet.
Symp., Garching.
APPENDICES
APPENDIX A:
TREATMENT PROCEDURES FOR MUTAGEN TREATMENTS ON
BARLEY USED AT WASHINGTON STATE UNIVERSITY
PULLMAN, WASHINGTON
45
TREATMENT PROCEDURES FOR MUTAGEN TREATMENTS ON
BARLEY USED AT WASHINGTON STATE UNIVERSITY
PULLMAN, WASHINGTON
These treatments have been successfully used on hulless Himalaya
barley. Adjustments should be made for use on a different organism or
on a bulled variety of barley.
(1)
Presoak: The seeds should be flooded With water at O0C for
at least 12 hours to remove any growth inhibitors and to
completely hydrate the system.
The same effect can be
obtained by soaking about 6 hours at 20oc. Allow about 1 ml
of distilled water per seed and either use a running water
wash or change the water periodically.
(2)
Pretreatment: This step is designed to get the mutagen into
the system without much metabolic activity.
The seeds are
soaked in the mutagen solution prepared in 0.1 M pH 7
phosphate buffer for 6 hours at Ooc. The concentrations and
approximate amount of seedling injury which will be obtained
for the 3 common mutagens are given below:
iPMS - 0.025 M gives about 5% seedling injury and
increases gradually up to 0.050 M which gives
about 50% seedling injury.
DES
- 0.005 M gives about 5% seedling injury and
increases gradually up to 0.010 M.
Increases
to 80% injury at 0.020 M.
EMS
- 0.05 M gives about 5-10% seedling injury which
increases to about 50% seedling injury at 0.25
M.
To prepare the mutagen solution, the mutagen is pipetted
into a flask and the buffer is added. This mixture must be
shaken very vigorously to get the mutagen into solution. It
should be prepared and immediately put on the seeds since
the mutagens hydrolyze in the buffer.
About 0.5 to 1.0 ml
of mutagen solution should be allowed per seed.
(3)
Treatment: The pretreatment solution should be poured off
and a fresh mutagen solution added to the seeds. The seeds,
are then kept in the mutagen solution for 2 hours at 20OC.
The same solution concentration is used that was used in the
pretreatment.
.
.
46
(4)
Aftertreatment:
The seeds are rinsed 3 to 4 times with
distilled water and then soaked in distilled water for 12
hours at Ooc. This will leach out any unreacted mutagen
which would otherwise increase the physiological damage. A
running water aftersoak, or changing the water during.the
aftertreatment time is helpful.
(5)
The seeds may be planted immediately either in the lab or
the field. Adequate survival in field experiments can be
achieved when seedling injury is 30% or less. The seeds may
also be dried and planted at a more convenient time. In
this case, an additional 10-15% damage can be expected.
These times and concentrations of mutagens must be varied to fit your
needs. These are approximations of damage obtained from our results,
but they may not hold true under different conditions.
47
Chemical Constants of Mutagens
IPMS - molecular weight - 138.19; density at
Hydrolysis half-life
EMS
0°C
IO0C
20°C
3O0C
-
I •163
I .152
I .140
I .129
g/ml
g/ml
g/ml
g/ml
- 38 hours
- 8.5 hours
- 130 minutes
- 35 minutes
- molecular weight - 124.16; density at
Hydrolysis half-life
DES
0°C
IO0C
20°C
30°C
Ooc
IQOc
20°C
30°C
-1716
- 379
- 93
- 26
5°C - I .220 g/ml
25°C - I .203 g/ml
hours
hours
hours
hours
- molecular weight - 154.19; density at 25°C - I .180 g/ml
Hydrolysis half-life
0°C
IO0C
20°C
30°C
- 59
- 13
-3*3
I
hours
hours
hours
hour
Phosphate buffer solution:
Dissolve 13.92 g of K 2HPOij plus 2.72 g of KH2POij in
distilled water and make up to. I liter with distilled
water.
0.01 M solution of DES:
154.19
x .01
molecular weight of DES
molar concentration desired
1.5419 v 1.18 (density of DES at 25°C)
= 1.31 ml made up to I liter
48
Specific Mutagen Procedures
Day 1 . 5:00 PM
Place I kg of seed in a 2-liter plastic jar. Fill
with distilled water at about Ooc.
Place in
refrigerator and hold at about Oop.
7:00 PM
and 10:00 PM
Pour off water and replace with distilled water at
about Oop. Replace in refrigerator.
Day 2 . 8:00 AM
Rinse with distilled water at about OOP. Replace
rinse water with a 0.01 M solution of DES at about
O0C. Place in refrigerator.
2:00 PM
Pour off DES solution and replace with a 0.01 M
solution of DES at room temperature.
4:00 PM
Pour off DES solution and rinse four or five times
with distilled water. Replace rinse water with
distilled water at about 0°p. Place in refriger­
ator.
6:00 PM
and 8:00 PM
and 10:00 PM
Pour off water and replace with distilled water at
about 0°P. Replace in refrigerator. -
Day 3 , 8:00 AM
Pour off water and rinse two or three times with
distilled water.
Pour off rinse water.
Plant
immediately or dry seeds for later, planting.
APPENDIX B:
COMPUTER PROGRAMS AND TABLES TO FACILITATE
THE CALCULATION OF RECOMBINATION VALUES
50
Figure 5.
Basic program to calculate and print bc/ad, b/a, and
appropriate factors to be divided by the square root of N
for LTH tester stock progeny.
DEFSNG A~Z
fmt$ = " //.//#
PRINT " P
////.//#////
IH fJ tm
bc/ad
SQRT(N) for SE
FOR p = .01 TO .5 STEP .01
a =(2 - 2 * p +2 * (p
* 2)) / 3
b =(2 * p - 2 M p ' 2)) / 3
c = (2*
p-p
2) / 3
d =(l - 2 * p + p 2) / 3
f = (b * c) / (a * d)
h = (2 * p - p
2) / 2
g = (2 - 2 * p + 2 * p ~ 2) / 2
i = h / g
j = I - Aftp + 4 *
k = 2 - 2 * p + 2 *
I = I - 2 * p + P "
m= 2 * p - p
2
n = 2 * p - 2 * P '
o = 4/3
q = o * (j / k)
r = o * (I / m)
s = o * (j / n)
t = o + q + r +
u = I / t
v = SQR(u)
e = 2 * (j / k)
p
2
p * 2
2
2
z = 2 * (I / m)
w = e + z
x = I / w
y = SQR(x)
PRINT USING fmt$; p, f , v, i, y
NEXT
M JH H H f
b/a
M J H H tu
SQRT(N) for SE"
51
Table 1 0 .
Values to facilitate the use of the product method when
using F 2 data from LTH crosses.
P
bc/ad
SQRT(N) for SE
b/a
SQRT(N) for SE
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.10
0.11
0.12
0.13
0.14
0.15
0.16
0.17
0.18
0.19
0.20
0.21
0.22
0.23
0.24
0.25
0.26
0.27
0.28
0.29
0.30
0.31
0.32
0.33
0.34
0.35
0.36
0.37
0.38
0.39
0.40
0.41
0.42
0.43
0.44
0.45
0.46
0.47
0.48
0.49
0.50
0.0002
0.0008
0.0019
0.0034
0.0054
0.0079
0.0109
0.0144
0.0185
0.0232
0.0285
0.0344
0.0410
0.0482
0.0561
0.0648
0.0742
0.0844
0.0953
0.1071
0.1198
0.1333
0.1478
0.1631
0.1795
0.1968
0.2152
0.2346
0.2551
0.2767
0.2994
0.3233
0.3485
0.3749
0.4025
0.4315
0.4619
0.4936
0.5268
0.5614
0.5976
0.6353
0.6746
0.7156
0.7584
0.8029
0.8492
0.8975
0.9477
0.0869
0.1234
0.1518
0.1760
0.1976
0.2174
0.2359
0.2534
0.2700
0.2860
0.3014
0.3164
0.3311
0.3454
0.3594
0.3732
0.3869
0.4003
0.4136
0.4268
0.4399
0.4529
0.4658
0.4786
0.4914
0.5041
0.5167
0.5292
0.5417
0.5541
0.5664
0.5785
0.5906
0.6025
0.6143
0.6258
0.6372
0.6483
0.6592
0.6697
0.6799
0.6898
0.6992
0.7081
0.7166
0.7245
0.7319
0.7386
0.7446
0.7500
0.0100
0.100
0.0202
0.0304
0.0408
0.0512
0.0617
0.0723
0.0829
0.0936
0.1044
0.1152
0.1261
0.1371
0.1480
0.1590
0.1701
0.1811
0.1922
0.2032
0.2143
0.2253
0.2364
0.2474
0.2583
0.2692
0.2801
0.2909
0.3016
0.3122
0.3228
0.3332
0.3436
0.3538
0.3638
0.3738
0.3836
0.3932
0.4027
0.4120
0.4211
0.4300
0.4387
0.4471
0.4554
0.4635
0.4713
0.4788
0.4861
0.4932
0.5000
0.142
0.175
0.202
0.227
0.250
0.271
0.291
0.311
0.329
0.347
0.365
0.382
0.399
0.416
0.433
0.450
0.467
0.484
0.501
0.518
0.536
0.553
0.571
0.589
0.608
0.627
0.646
1.0000
0.666
0.686
0.707
0.728
0.750
0.773
0.796
0.819
0.844
0.869
0.895
0.921
0.949
0.977
1.006
1.035
1.066
1.096
1.128
1.160
1.192
1.225
52
Figure 6.
Microsoft!® fortran program to calculate maximum likelihood
estimates of recombination values.
C««******»» LINKAGE ***** (LINKLMN) *****
C
*
C
MAXIMUM LIKELIHOOD ESTIMATION OF P VALUES.
C
USER SHOULD REFER TO ALLARD;HILGARDIA 24:235-278 (1956) AND
C
HANSON & KRAMER;GENETICS 35:559-569 (1950)
C
*
C
C Modified: 02/22/88 by Ric Roche for MS-DOS Microcomputers
C
Microsoft Fortran version 4.01
C
C READ LOOP
DIMENSION LI(20),L2(20),X(20,14),V(14),RCHISQ(20)
CHARACTER*80 HEADER
CHARACTER*24 INFILE
LOGICAL EOF
C
WRITE(*,8000)
8000 FORMAT(
1 * MAXIMUM LIKELIHOOD ESTIMATION OF P VALUES.'/,
2' USER SHOULD REFER TO ALLARD;HILGARDIA 24:235-278 (1956) &',/,
3' HANSON & KRAMER;GENETICS 35:559-569 (1950)',///,
4' Adapted by: Georgia Ziemba & D . Biggerstaff',//
5' Equations 45 & 46 Written by D . Biggerstaff',/
6' Converted to MS-DOS 02/22/88 by Ric Roche',//,
7' Enter input file name: ',\)
READ(*,'(A)')INFILE
0PEN(105,FILE=INFILE)
OPEN(108,FILE='LPT1')
190 READ(105,'(A)',END=I99)HEADER
WRITEd 08,' (6X ,A80)')HEADER
ILOOP=O
P=.500
KFIN=O
DO 77 K=I,20
91
READ(105,*,END=78)LI (K),L2(K),(X(K,I),1=1 ,14)
IF(LKK).EQ.0)GOT072
KFIN=KFIN+1
WRITEd 08,89)L1 (K) ,L2(K) ,(X(K ,I) ,1 =1 ,14)
89
FORMAT(6X,13,13,14F5.0)
77 CONTINUE
GOTO 72
78 EOF=.TRUE.
C INITIALIZE LOOP
72 TSCORE=O-O
TINFO=O.0
TCHISQ=O-O
53
Figure 6— continued.
SSCORE=O-O
SINFO=O-O
SCHISQ=O-O
C COMPUTE LOOP
DO 97 I=I,KFIN
222
97
115
560
116
DO 222 J=I,14
V(J)=X(I1J)
CONTINUE
CALL COMP(L1(I)1V 1P 1SSCORE1SINFO)
IF(SINFO-EQ-0.0)THEN
SCHISQ=O-O
ELSE
SCHISQ=(SSCORE*SSCORE)/ABS(SINFO)
ENDIF
RCHISQ(I)=SCHISQ
TSCORE=TSCORE + SSCORE
TINFO=TINFO + ABS(SINFO)
TCHISQ=TCHISQ + SCHISQ
CONTINUE
IL00P=IL00P+1
IF(TINFO.EQ.O)THEN
CF=O-O
ELSE
CF=TSCORE/TINFO
ENDIF
IF (ABS(CF)-LT-O-OOI)THEN
P=ABS(P+CF)
GOTO 560
ENDIF
IF (ILOOP.GE.20) GOTO 204
IF(ABS(CF).GE.0.001)P=ABS(P+CF)
IF(P.GT.0.60) GOTO 201
IF(P.LT.0.001) GOTO 560
GOTO 72
IF(TINFO-EQ-O)THEN
CHISQT=O-O
ELSE
CHISQT=(TSCORE«TSCORE)/TINFO
ENDIF
HCHISQ=TCHISQ-CHISQT
IF(TINFO-EQ-0.0)THEN
STDERR=O-O
ELSE
STDERR=SQRTd ./TINFO)
ENDIF
WRITEd 08,116 )CF
FORMAT(6X ,'CF=',F6-3)
WRITE(IOS1In)P
Figure 6— continued.
C
C
C
C
C
117 F0RMAT(6X, 'P=\F6.3)
WRITE(108,118)STDERR
118 FORMAT(6X,'STDERR=',FT-Bf/)
WRITE(108,777) (LI(I),L2(I),RCHISQ(I),1=1,KFIN)
777 FORMATf 11X,12,IX,12,2X,'CHISQR=',F7.3)
WRITE(I08,778)HCHISQ
778 FORMATf17X,'HCHISQR=',F7.3,////)
IF(EOF)THEn
GOTO 199
ELSE
GOTO 190
ENDIF
201 W R I T E d 08,202) P
202 FORMAT(6X ,'P GREATER THAN 0.50, PROBLEM STOPPED.P=',F6.4////)
IF(EOF)THEn
GOTO 199
ELSE
GOTO 190
ENDIF
204 WRITE (108,205) P
205 FORMAT(6X,'TOO MANY ITERATIONS-STOP P= •,F6.4////)
IF(.NOT.EOF)GOTO 190
199 C L O S E d 05)
CLOSE(IOB)
END
*
** THIS PROGRAM CAN BE MODIFIED BY ADDING OR SUBTRACTING
EQUATIONS FROM THE SUBROUTINE (THE GO TO NUMBERS MUST BE
CHANGED ACCORDINGLY).
*
SUBROUTINE COMP(LEVEL,G ,P ,SSCORE,SINFO)
DIMENSION G d 4)
GO TO (I,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,
&20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,
&38,39,40,41,42,43,44,45,46),LEVEL
1 SSCORE=((G(5)+G(8)+G(14))*2./P)+((G(6)+G(7)+G(11)+G(13))*(1.12.*P)/(P*(1.-P)))+((G(9)+G(10)+G(12))*2./(P-1.))
SINF0=(G(5)+G(6)+G(7)+G(8)+G(9)+G(10)+G(11)+G(12)+G(13)+G(14)
I)*2./(P*(I.-P))
RETURN
2 SSCORE=((G(1)+G(4))/P)+((G(2)+U(3))/(P-1.))
SINFO=(G(1)+G(2)+G(3)+G(4))/(P*(1.-P))
RETURN
3 SSCORE=(Gf1)/P)+((G(2)+G(3)+G(4))/(P-2.))
SINFO=(G(1)+G(2)+G(3)+G(4))*(1./(P*(2.-P)))
RETURN
4 SSC0RE=((G(5)+G(14))*2./P)+((G(6)+G(7)+G(11)+G(13))*(1.-2.*P)
1/(P*(1.-P)))+((G(10)+G(12))*2./(P-1.))+((G(8)+G(9))*2.*(2.*P1.)/(21.-2.*P+2.*P**2))
55
Figure 6— continued .
SINF0=(G(5)+G(6)+G(7)+G(8)+G(9)+G(10)+G(11)+G(12)+G(13)+G(14)
1)*2.*(1.-3.*P+3.*P**2)/(P*(1.-P)*(1._2.*P+2.*P**2))
RETURN
5 SSCORE=((G(5)+G(6))*(2.-2.*P)/(2.*P-P#*2))+((G(7)+G(8)+G(9))*
1(2.*P-1.)/(I.-P+P**2))+((G(10)+G(11))*(-2.*P)/(1.-P**2))+
(G(12)*2.2/(P-1.))+(G(13)*(1.-2.*P)/(P-P**2))+(G(14)*2./P)
SINFO=(G(5)+G(6)+G(7)+G(8)+G(9)+G(10)+G(11)+G(12)+G(13)+G(14)
&)*(2.+((1.-P)**2/(P*(2.-P)))+((1.-2.*P)**2/(2.*(I.-P+P**2)))+
(P**2&/(1.-P**2))+((1.-2.*P)**2/(2.*P*(1.-P))))
RETURN
6 SSCORE=(G(I)*2.*P/(2.+P**2))+((G(2)+G(3))*(-2.*P)/(1.-P#*2))+
I(G(4)*2./P)
SINFO=(G(1)+G(2)+G(3)+G(4))*2.*(1.+2.*P**2)/((2.+P**2)*(I.-P*
1*2 ))
RETURN
7 SSCORE=(G(3)*(-2.*P)/(I.-P**2))+(G(4)*2./P)
SINFO=(G(I)+G(2)+G(3)+G(4))/(I.-P**2)
RETURN
8 SSC0RE=((G(1)+G(2)+G(4))«2.*P/(3.+P**2))+(G(3)*(-2.*P)/(1.-P«
1*2 ))
SINFO=(G(I)+G(2)+G(3)+G(4))*(4.*P**2)/((3-+P**2)*(I.-P**2))
RETURN
9 SSCORE=(G(I)*2.*P/(2.+P**2))+(G(2)# (-2.*P)/(1.-P**2))
SINFO=(G(I)+G(2)+G(3)+G(4))*(3•*P**2)/((2.+P**2)*(1.-P**2))
RETURN
10 SSCORE=((G(1)+G(2)+G(3))*(-2.*P)/(4.-P**2))+(G(4)*2./P)
SINFO=(G(I)+G(2)+G(3)+G(4))*4./(4.-P**2)
RETURN
11 SSCORE=(G(1)*2.*P/(2.+P**2))+((G(2)+G(3)+G(4))*(-2.*P)/(2.-P
1**2 ))
SINFO=(G(1)+G(2)+G(3)+G(4))*4.*P**2/((2.+P**2)*(2.-P**2))
RETURN
12 SSCORE=(G(10)*2./(P-1.))+(G(11)*(1.-2.*P)/(P*(1.-P)))-((G(10
1)+G(11))*(-2.*P)/(1.-P**2))
SINFO=(G(10)+G(11))*2./(P*(1.+P)*(1.-P**2))
RETURN
13 SSCORE=((G(5)+G(8))*2./P)+((G(6)+G(7))*(1.-2.*P)/(P*(1.-P)))
1+(G(9)*2./(P-1.))-((G(5)+G(6)+G(7)+G(8)+G(9))*2.»P/(2.+P**2))
SINF0=(G(5)+G(6)+G(7)+G(8)+G(9))*4.*(2.+2.*P-P**2)/(P*(1.-P)
1*((2.+P**2)**2))
RETURN
14 SSC0RE=(G(5)*2./P)+((G(6)+G(7))*(1.-2.*P)/(P*(1.-P)))+((G(8)
I+G(9))*2.*(2.*P-1.)/(1.-2.*P+2.*P**2))-((G(5)+G(6)+G(7)+G(8)+
G(9))2*2.*P/(2.+P**2))
SINFO=(G(5)+G(6)+G(7)+G(8)+G(9))*4.*(2.-6.*P+3.*P**2+4.*P**3
I)/(P*(1.-P)*((2.+P**2)**2)*(I.-2.*P+2.*P**2))
RETURN
15 SSCORE=(G(8)*2./P)+(G(9)*2./(P-1.))-((G(8)+G(9))*2.»(2.*P-1.
1)/(1.-2.*P+2.*P**2))
56
Figure 6— continued.
SINF0=(G(8)+G(9))*4./((1.-2.*P+2.*P**2)**2)
RETURN
16 SSCORE=(G(12)*2./(P-1.))+(G(13)*(1.-2.*P)/(P*(1.-P)))+(G(14)
1*2./P)
SINFO=(G(12)+G(13)+G(14))/(2.*P*(1.-P))
RETURN
17 SSC0RE=((G(5)+G(6))*2.*(1.-P)/(P*(2.-P)))+((G(7)+G(8)+G(9))*
I(2.*P-1.)/(I.-P+P**2))+((G(10)+G(11))*(-2.*P)/(1.-P**2))
SINFO=(G(5)+G(6)+G(7)+G(8)+G(9)+G(10)+G(11))*((P**2/(1.-P**2))+
4((1.-2.*P)**2/(2.*(1.-P+P**2)))+((I.-P)**2/(P*(2.-P))))
RETURN
18 SSCORE=((G(5)+G(6))*2.*(1.-P)/(P*(2.-P)))+((G(7)+G(8)+G(9))*
1(2.*P-1.)/(1.-P+P**2))+(G(10)*2./(P-1.))+(G(11)*(1.-2.*P)/(P*
(1.-P2)))
SINFO=(G(5)+G(6)+G(7)+G(8)+G(9)+G(10)+G(11))*(1.+((1.-P)**2/(P*
&(2.-P)))+((1.-2.*P)**2/(2.*(1.-P+P**2)))+((1.-2.*P)**2/(2.*P*
(1.-4P))))
RETURN
19 SSC0RE=(G(5)*2./P)+((G(6)+G(7)+G(11))*(1.-2.*P)/(P*(1._P)))+
1((G(8)+G(9))*2.*(2.*P-1.)/(I.-2.*P+2.*P**2))+(G(10)*2./(P-1.))
SINF0=(G(5)+G(6)+G(7)+G(8)+G(9)+G(10)+G(11))*(3.-10.*P+10.*P
1**2)/(2.*P*(1.-P)*(1.-2.*P+2.*P**2))
RETURN
20 SSCORE=O.0
SINFO=O-O
SCHISQ=O-O
RETURN
21 SSCORE=((G(5)+G(8)+G(l4))*2./(P-1.))+((G(6)+G(7)+G(11)+G(13)
1)*(1.-2.*P)/(P-P**2))+((G(9)+G(10)+G(12))*2./P)
SINF0=(G(5)+G(6)+G(7)+G(8)+G(9)+G(10)+G(11)+G(12)+G(13)+G(14
I))*2./(P*(I.-P))
RETURN
22 SSCORE=((G(1)+G(4))/(P-1.))+((G(2)+G(3))/P)
SINFO=(G(1)+G(2)+G(3)+G(4))/(P*(1.-P))
RETURN
23 SSCORE=(G(I)/(P-1.))+((G(2)+G(3)+G(4))/(1.+P))
SINFO=(G(I)+G(2)+G(3)+G(4))/((P**2)-1.)
RETURN
24 SSCORE=C(G(5)+G(14))*2./(P-1.))+((G(6)+G(7)+G(1I)+G(13))*(1.
1-2.*P)/(P-P**2))+((G(10)+G(12))*2./P)+((G(8)+G(9))*2.*(2.*P-1.)
/(I2.-2.*P+2.*P**2))
SINFO=(G(5)+G(6)+G(7)+G(8)+G(9)+G(10)+G(11)+G(12)+G(13)+G(14
1))*2.*(1.-3.*P+3.*P**2)/(P*(1.-P)*(I.-2.*P+2.*P**2))
RETURN
25 SSC0RE=((G(5)+G(6))*(-2.*P)/(1.-P**2))+((G(7)+G(8)+G(9))*(2.
1*P-1.)/(1.-P+P**2))+((G(10)+G(11))*(2.-2.*P)/(2.*P-P**2))+(G
(12)*22./P)+(G(13)*(1.-2.*P)/(P-P*»2))+(G(14)*2./(P-1.))
57
Figure 6— continued .
SINF0=(G(5)+G(6)+G(7)+G(8)+G(9)+G(10)+G(11)+G(12)+G(13)+G(14))»
&(2.+((1 .-P)**2/(P*(2.-P)))+((1.-2.*P)*#2/(2.*(1-P+P**2))) +
&(P**2/(1.-P**2))+((1.-2.*P)**2/(2."P*(1.-P))))
RETURN
26 SSCORE=(G(I)*2.# (P-1.)/(3•-2.*P+P**2))+((G(2)+G(3))*2.*(I.-P
1)/(P*(2.-P)))+(G(4)*2./(P-1.))
SINF0=(G(1)+G(2)+G(3)+G(4))*(6.-(8.*P)+(4.*P**2))/((P**4)-(4
I.*P**3)+(7.*P**2)-(6.*P))
RETURN
27 SSCORE=(G(3)*2.*(1.-P)/(P*(2.-P)))+(G(4)*2./(P-1.))
SINFO=(G(3)+0(4))/(P*(P-2.))
RETURN
28 SSCORE=((G(1)+G(2)+G(4))*2.*(P-1.)/(4.-(2.#P)+P**2))+(G(3)*2
1.*(1.-P)/(P*(2.-P)))
SINF0=(G(1)+G(2)+G(3)+G(4))*(4.-8.*P+4.*P**2)/(P**4-4.*P"*3+
18.*P**2-8.*P)
RETURN
29 SSC0RE=(G(1)*2.*(P-1.)/(3.-2.*P+P**2))+(G(2)*2.*(1.-P)/(P*(2
I.-P)))
SINFO=(G(I)+G(2)+G(3)+G(4))*(3.-6.*P+3.*P**2)/(P**4-(4.*P**3
I) +(7.*P**2)-6.*P)
RETURN
30 SSC0RE=((G(1)+G(2)+G(3))*2.*(1.-P)/(3.+2.*P-P**2))+(G(4)*2./
I(P-1.))
SINFO=(G(1)+G(2)+G(3)+G(4))*4./(P**2-2.*P-3.)
RETURN
31 SSC0RE=(G(1)*2.*(P-1.)/(3.-2.*P+P**2))+((G(2)+G(3)+G(4))*2.*
1(1.-P)/(1.+2.*P-P**2))
SINF0=(G(1)+G(2)+G(3)+G(4))*(4.-8.*P+4.*P**2)/((P**4)-(4.*P*
1*3)+(6.*P**2)-(4.*P)-3.)
RETURN
32 SSCORE=(G( 10)*2./P)+(G(11)*(1.-2.*P)/(P<,(1.-P)))-( (G( 10)+G( I
II) )*2.*(1.-P)/(P*(2.-P)))
SINFO=(G(10)+G(11))*2./(P**4-5.*P**3+8.*P**2-4.*P)
RETURN
33 SSCORE=((G(5)+G(8))*2./(P-1.))+((G(6)+G(7))*(1.-2.*P)/(P*(1.
1-P)))+(G(9)*2./P)-((G(5)+G(6)+G(7)+G(8)+G(9))*2.*(P-1.)/(3.-2.
*P+P2**2))
SINF0=(G(5)+G(6)+G(7)+G(8)+G(9))*(12.-4.*P**2)/((P**6)-(5.*P
1**5)+(14.*P**4)-(22.*P**3)+(21.*P**2)-9.*P)
RETURN
34 SSC0RE=(G(5)*2./(P-1.))+((G(6)+G(7))*(1.-2.*P)/(P*(1._P)))+
(1(G(8)+G(9))*2.*(2.*P-1.)/(1._2.*P+2.*P**2))-((G(5)+G(6)+G(7)
+G(8)+2G(9))*2.*(P-1.)/(3.-2.*P+P**2))
SINFO=(G(5)+G(6)+G(7)+G(8)+G(9))*(16.*P**3-60.*P**2+48.*P-12
I .)/(9.*P-39.*P**2+82.*P**3-100.*P**4+77.*P"*5-39.*P**6+12.*P**
7-2.2*P**8)
RETURN
58
O O
Figure 6— continued.
C
C
C
35 SSC0RE=(G(8)»2./(P-1.))+(G(9)*2./P)-((G(8)+G(9))*2.«(2.»P-1.
1)/(1.-2.*P+2.*P**2))
SINF0=(G(8)+G(9))*4./((1.-2.*P+2.*P**2)**2)
RETURN
36 SSCORE=(G(12)*2./P)+(G(13)*(1.-2.*P)/(P*(1.-P)))+(G(14)*(2./
•(P-1.)))
SINFO=(G(I2)+G(13)+G(14))/(2.*P*(P-1.))
RETURN
37 SSCORE=((G(5)+G(6))*(-2.*P)/(1.-P**2))+((G(7)+G(8)+G(9))*(2.
1*P-1.)/(1.-P+P**2))+((G(10)+G(11))*2.*(1.-P)/(P*(2.-P)))
SINFO=(G(5)+G(6)+G(7)+G(8)+G(9)+G(10)+G(11))*((P**2/(I.-P**2))+
&((1.-2.*P)**2/(2.*(1.-P+P**2)))+((1.-P)**2/(P*(2.-P))))
RETURN
38 SSCORE=((G(5)+G(6))*(-2.*P)/(1.-P**2))+((G(7)+G(8)+G(9))*(2.
1*P-1.)/(1.-P+P**2))+(G(10)*2./P)+(G(11)*(1.-2.*P)/(P"(1.-P)))
SINFO=(G(5)+G(6)+G(7)+G(8)+G(9)+G(I0)+G(1I))•((P**2/(P*t2-1.
1))+((4.*P**2-4.*P+1.)/(2.*P-2.*P**2-2.))+((4.*P**2-4.*P+1.)/(2.
*P*2*2-2.*P))-1.)
RETURN
39 SSCORE=(G(5)*2./(P-1.))+((G(6)+G(7)+G(11))#(1.-2.*P)/(P*(1.1P)))+((G(8)+G(9))*2.*(2.#P-1.)/(I.-2.*P+2.*P**2))+(G(I0)*2./P)
SINF0=(G(5)+G(6)+G(7)+G(8)+G(9)+G(10)+G(11))»(3.-10.«P+10.»P
1**2)/(2.*P*(I.-P)*(I.-2.*P+2.*P**2))
RETURN
BREAKPOINT EQUATIONS
EQUATION 40 IS FOR F2, FOUR CLASSES
40 SSC0RE=(2.*G(2)/(1.+2.*P-2.*P**2))-(G(1)/(1.-P+P**2))+(G(3)/
1(P-P»*2))-(2.*G(4)/(1.-2.*P+2.*P**2))
SINFO=(G(I)+G(2)+G(3)+G(4))•((I./((2.*P-2.*P**2)#(1.-P+P**2)
1))+(2./(1.-4.*P**2+8.*P**3-4.*P**4)))
RETURN
EQUATION 41 IS FOR F2, TWO CLASSES DUE TO RECESSIVE LETHAL
41 SSCORE=(2.*G(2)/(I.+2.*P-2.*P**2))-(G(1)/(1.-P+P**2))
SINFO=(G(I)+G(2))*2./((1.-P+P**2)*(I.+2.*P-2.*P**2))
RETURN
EQUATION 42 IS FOR F2, SIX CLASSES DUE TO INCOMPLETE DOMINANCE
42 SSCORE=((G(3)+G(5)+G(8))/(P-P**2))-(2.*(G(4)+G(6)+G(7))/(1.12.*P+2.*P**2))
SINFO=(G(3)+G(4)+G(5)+G(6)+G(7)+G(8))*2./((P-P**2)*(1.-2.*P+
12.*P**2))
RETURN
EQUATION 43 IS FOR F3,FR0M SEMISTERILEDOMINANT F2
43 SSC0RE=(G(5)/(P-P**2))-(2.*G(6)/(1.-2.»P+2.*P«»2))+((G(5)+G(
16))/(1.-P+P**2))
SINFO=(G(5)+G(6))/((1.-2.*P+3.*P**2-2.1P**3+P,*4)*(P-3.*P**2
1+4.*P**3-2.*P**4))
RETURN
59
Figure 6— continued.
C
C
EQUATION 44 IS FOR F3 ,FROM NORMAL FERTILE DOMINANT F2
44 SSCORE=(2.*G(8)/(2.*P-2.*P**2))-(2.*G(7)/(1.-2.*P+2.*P**2))1(2.*(G(7)+G(8))/(1.+2.*P-2.*P**2))
SINF0=(G(7)+G(8))*4./((1.+4.*P-8.*P**3+4.*P**4)*(P-3.*P**2+4
I.*P**3-2.*P**4))
RETURN
EQUATION 45 IS FOR F2 DATA FROM LTH CROSSES
45 SSCORE=(G(1)*(2.*P-1.)/(1.-P+P**2))+(G(2)*2.*(1.-P)/(2.*P-P*
&*2))+(G(3)*(1.-2.*P)/(P-P**2))+(G(4)*2.*(P-1.)/(1.-P)**2)
SINF0=(G(1)+G(2)+G(3)+G(4))*(4./3.*(((1.-4.*P+4.*P**2)/(2
&.*(1.-P+P**2)))+((1.-2.*P+P**2)/(2.*P-P**2))+((1.-4.•P+4.
& * P * * 2 ) /( 2 .* P * (1 . - P ) ) )+1. ) )
C
RETURN
EQUATION 46 IS FOR F2 LTH DATA WHEN aa CLASSES COMBINED
46 SSCORE=G(1)*((2.*P-1.)/(1.-P+P**2))+G(2)*((2.-2.*P)/(2.*P&P««2))+(G(3)+G(4))»(-2.»P/(1.-P»»2))
SINF0=(G(1)+G(2)+G(3)+G(4))«(((8.»P«»2-8.»P+2.)/(3.-3.#P+3.
&eP*«2))+((4.-8.»P+4.*P**2)/(6.*P-3.#Pe#2))+(4.»P*»2/(3.-3.«
&P**2)))
RETURN
END
60
Figure 7•
Data entry instructions
likelihood program.
ALLARD'S CORRECT DATA
26 I 200. 57. 49. 30.
26 2 842. 234. 255. 126.
26 3 274. 71. 64. 45.
6 I 293. 107. 119. 35.
6 2 50. 21. 30.
0.
36 I
0.
0.
0.
0.
39 I
0.
0.
0.
0.
25 I
0.
0.
0.
0.
37 I
0.
0.
0.
0.
CF= .000
P= .402
STDERR=
.012
0.
0.
0.
0.
0.
0.
7.
16.
20.
0.
0.
0.
0.
0.
0.
13.
0.
0.
and
0.
0.
0.
0.
0.
0.
14.
43.
46.
sample printout for maximum
0.
0.
0.
0.
0.
0.
31.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
2.
27.
11.
0.
0.
0.
0.
0.
0.
10.
0.
0.
0.
0.
0.
0.
0.
8.
0.
5.
0.
0.
0.
0.
0.
0.
28.
0.
16.
0.
0.
0.
0.
0.
0.
36.
0.
12.
0.
26 I CHISQR=
.027
26 2 CHISQR= 1.139
26 3 CHISQR= 1.429
6 I CHISQR= 5.420
6 2 CHISQR= 6.236
36 I CHISQR= 22.359
39 I CHISQR=
.246
25 I CHISQR= 1.368
37 I CHISQR= 1.875
HCHISQR= 40.099
[See Allard, 1956, p. 255.]
Data Entry Instructions:
(1)
Line I is a header line (up to 80 characters).
(2)
Column I is the equation number for the data set.
Equations:
1-19
20
21-39
40-44
45-46
are after Allard (1956), Table 6.
is a dummy equation.
are the same as 1-19, but in coupling phase.
are after Hanson and Kramer (1950), Table 4.
are LTH equations.
(3)
Column 2 is the data set number or can be the number of times the
equation is used (Allard’s correct data example). Does not affect
the execution of the program — user information only.
(4)
Data columns 3-16, space or comma delineated. Must have a value.
Refer to Allard (1956), Hanson and Kramer (1950), and page 20 of
this thesis to determine appropriate phenotypic classes.
(5)
Put all zeros in columns 1-16 at the end of each file you wish to
run. Subsequent data set(s) can follow on next line; no break is
required.
61
Figure 8.
Combined maximum likelihood estimate of p for three data
sets that indicated linkage between msg,,ec and breakpoints
of chromosome 2.
msg ,,ec X LTH TESTER SET
Three linked
46
4
57.
I. 23. 0. 0. 0.
4. 68. 0. 0. 0.
46
5
119.
46
6
109.
3. 71. 0. 0. 0.
CF
= .001
P
= .028
STDERR = .009
46
46
46
4
5
6
CHISQR
CHISQR
CHISQR
HCHISQR
=
=
=
=
.198
.166
.000
•358
data sets
0. 0. 0
0. 0. 0
0. 0. 0
0. 0. 0. 0. 0.
0 . 0 . 0. 0 . 0.
0. 0. 0. 0. 0 .
MONTANA STATE
762 10051846 1