Development of 14 microsatellite markers in the Queensland koala (Phascolarctos cinereus
adustus) using next generation sequencing technology
Supplementary Material
Christina T. Ruiz-Rodriguez1, Yasuko Ishida1, Alex D. Greenwood2 and Alfred L. Roca1*
1
Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801
USA
2
Leibniz Institute for Zoo and Wildlife Research, 10315, Berlin, Germany
*Corresponding Author: roca@illinois.edu
1
Supplementary background and methods
The koala (Phascolarctos cinereus), is an arboreal marsupial for which as many as three
subspecies have traditionally been recognized, each corresponding to the range of the koala
within an Australian state: Queensland (P.c. adustus), South Australia (P.c. cinereus) and
Victoria (P.c. victor) (Lee and Martin 1988). However, because koala range is continuous across
state borders, this variation may be clinal rather than discrete (Houlden et al. 1999; Lee and
Martin 1988; Troughton 1941). Genetic analyses using mitochondrial DNA have suggested that
the three subspecies do not correspond perfectly to three distinct evolutionary significant units
(ESUs), but rather that koala populations may be considered to represent a single ESU consisting
of multiple management units (MUs) (Houlden et al. 1999). Morphological characters, such as
fur color and thickness, muzzle shape and body size, are known to vary between northern and
southern populations (Lee and Martin 1988). In recognition of this variation, zoos currently
manage northern Australian koalas from Queensland as a separate stock from southern
Australian koalas, which include koalas from New South Wales and Victoria. We here follow the
convention adopted by zoos of using the traditional subspecies nomenclature for the Queensland
koala (P.c. adustus).
In the past century, the koala has been subject to severe population decline and local
extirpation. During the early 1900s, millions of koalas were hunted for their pelts. The
exploitation of koalas for their fur, together with the reduction of Eucalyptus forests, brought the
species to the brink of extinction. Koalas were extirpated in the state of South Australia and were
nearly wiped out in the state of Victoria (Lee and Martin 1988; Worthington-Wilmer et al. 1993).
2
In an effort to conserve the koala in southern Australia, koalas were translocated to off-shore
islands and these insular populations were later used to restock the southern Australian mainland
(Houlden et al. 1996b). While Queensland koalas were not as strongly impacted by the fur trade,
their populations still suffered from hunting, habitat fragmentation and disease. Today,
populations in Queensland are patchily distributed (Gordon et al. 2008). A threat to koalas in this
region is habitat conversion and fragmentation (Fowler et al. 2000; Lee et al. 2010). In 2002, the
U.S. Fish and Wildlife listed the koala as ‘threatened’ under the Endangered Species Act, while
the species is listed as ‘vulnerable’ by the government of Australia (Department of the
Environment 2013). A major threat to all koala populations in Australia is a koala retrovirus
associated with leukemia and lymphoma, which appears to be responsible for susceptibility to
secondary infections such as Chlamydia (Simmons et al. 2012).
Microsatellite markers have been previously developed using DNA from a southern
Australian koala. Initially, six polymorphic microsatellite primer pairs were designed and
published in a study of paternity and pedigree analysis of koalas from southern Australia
(Houlden et al. 1996a). In a follow up study, using the same six microsatellite loci to investigate
genetic diversity in southern koala populations, the loci revealed low levels of genetic variability
in koalas from southeastern Australia when compared to koala populations from northeastern
Australia (Houlden et al. 1996b). This difference in genetic variability was expected since koalas
in Victoria have suffered from bottlenecks, founder effects and translocations (Houlden et al.
1996b). Since then, 11 other microsatellite markers have been developed using DNA from a
southern Australian koala. These were combined with 5 of the previously published
microsatellite markers, and used to genotype koalas from French Island and Kangaroo Island
reporting an average of 3.8 and 2.4 alleles per locus, respectively (Cristescu et al. 2009). This
3
established that the two island populations have low genetic diversity compared to northern
koala populations that did not suffer from bottlenecks (Cristescu et al. 2009).
Most population genetic studies of Queensland koalas have relied on sequencing the
mitochondrial control region (Worthington-Wilmer et al. 1993; Houlden et al. 1999; Fowler et al.
2000; Lee et al. 2010). Genetic studies of koalas in Queensland have shown only moderate to
low levels of mitochondrial DNA (mtDNA) variability within populations and higher levels of
mtDNA variation across populations (Worthington-Wilmer et al. 1993; Houlden et al. 1999). A
recent study using museum samples showed that low levels of mtDNA diversity in koala
populations had been present prior to recent population decline (Tsangaras et al. 2012). A study
conducted on five southeast Queensland populations suggested that there has been femalemediated gene flow historically (based on adjacent populations sharing haplotypes) (Fowler et al.
2000). However, the limited distribution of mtDNA haplotypes would also indicate that barriers
to gene flow have existed among populations (Lee et al. 2010).
We report initial development of novel microsatellite markers and statistics for
polymorphism using DNA from ten unrelated Queensland koalas from the San Diego Zoo. DNA
was extracted from blood samples using a DNeasy Blood and Tissue Kit (QIAGEN), following
the recommended protocol. One Queensland koala DNA sample (Pci-SN404; “SN” refers to the
North American Regional Studbook number) was subjected to shotgun sequencing using a
Roche 454 GS FLX Titanium Rapid Library Preparation Kit. A DNA library was prepared and
sequenced on 1/16 of a plate at the University of Illinois at Urbana-Champaign (UIUC) Core
Sequencing Facility. The software MSATCOMMANDER 1.0.8 (Faircloth 2008) was used to
identify microsatellite repeat motifs by screening the sequences for di-, tri-,tetra- and pentanucleotide motifs, with a minimum of 10 repeats each. MSATCOMMANDER interfaces with
4
PRIMER 3 software (Rozen and Skaletsky 2000), and was modified to allow for the design of
primers to amplify target loci with a size range between 100-250 bp, and an optimal melting
temperature of 60.0°C (range 58°C to 65°C). All other settings in MSATCOMMANDER were
kept at default values (e.g., primer length, GC content, GC clamp, self and pair complementarity,
and maximum end stability) (Brandt et al. 2013).
An initial output of 286 suitable primer pairs was searched against the entire 454
generated sequence database to ensure the uniqueness of the primer target sequences, thereby
avoiding repeat elements. To minimize the risk of amplifying non-target loci, each primer
sequence was also queried against the non-redundant NCBI sequence database using NCBI
Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). Additionally, primer
sequences were screened using UCSC In-Silico PCR (http://genome.ucsc.edu/cgi3bin/hgPcr) to
ensure that primers would not target human DNA sequences. Thirty-four primer pairs were
designed for loci with di-, tri- and tetra-nucleotide motifs (mainly dinucleotide motifs). Those
that failed to amplify product by PCR were dropped from further testing.
Genealogies of koalas from the San Diego Zoo were examined in the 2008 North
American Regional Studbook. Ten unrelated koala individuals were chosen for genotyping. The
PCR setup and algorithm were the same as used successfully by Ishida et al. (2012). Details of
the PCR setup and thermocycling are also listed in a protocol below. All 34 forward primers had
attached a tail consisting of M13 forward sequence (5’ TGT AAA ACG ACG GCC AGT), to
enable labeling with a fluorescent tag (Boutin-Ganache et al. 2001). Primer pairs were initially
tested by PCR performed in a 15 µl reaction mixture that consisted of a final concentration of
200 µM of each dNTP, 1x PCR buffer II, 2 mM MgCl2, 0.04 units/µl of AmpliTaq Gold
Polymerase along with 1.2 µL of primer mix (primer mix recipe is listed below) and 0.5 µl of
5
template DNA. Touchdown PCR was used with the following algorithm: initial 95°C for 10 min;
with cycles of 15 sec at 95°C; followed by 30 sec at 60°C, 58°C, 56°C, 54°C, 52°C (2 cycles
each), or 50°C (last 30 cycles); and 45 sec at 72°C; with a final extension of 30 min at 72°C (see
below). An aliquot of each PCR product was examined on a 1.5 to 2% agarose gel with ethidium
bromide. Amplicons were then diluted depending on the intensity of the image in the gel (a 15X
dilution for dimmer bands and a 20X dilution for brighter bands) and electrophoresed on an ABI
3730XL capillary sequencer at the UIUC Core Sequencing Facility. Microsatellite fragments
were viewed and scored with Genemapper Version 3.7 software (Applied Biosystems) and
binned using Allelogram v2.2 (Morin et al. 2009). Among the successful primers, fourteen pairs
were chosen for further analysis based on quality of initial genotyping results, such as the
absence of artifactual peaks. These fourteen primer pairs were used for genotyping to determine
marker variability in the ten koala samples (Table S1). Allelic diversity, observed heterozygosity
and expected heterozygosity were calculated using the MS Tool v3 (Parks 2001) and GENEPOP
(Raymond and Rousset 1995). Deviations from Hardy-Weinberg equilibrium were calculated
using GENEPOP, v 4.0 (Raymond and Rousset 1995). Linkage disequilibrium between pairs of
loci was calculated with FSTAT, v. 2.9.3.2 (Goudet 1995).
6
Details of the PCR setup and PCR algorithm
PCR Components
a
Volume (µl)
Distilled and deionized water
9.28
10X PCR buffer II
1.50
dNTP mix (10 mM) a
1.20
MgCl2 (25 mM)
1.20
Primer mix (see recipe below)
1.20
AmpliTaq Gold Polymerase b
0.12
Template DNA
0.50
Total volume
15.0
2.5 mM of each dNTP (dATP, dCTP, dGTP, and dTTP) blend (ABI, N8080260)
AmpliTaq Gold DNA Polymerase with Buffer II and MgCl2 solution (ABI, N8080249)
b
Primer mix
Volume (ul)
20 µM reverse primer
20
20 µM M13 tailed forward primer
1.5
100 µM fluorescent labeled M13 forward primer
4
TLE (10mM Tris-HCL, 0.1 mM EDTA)
21.5
PCR algorithm
Touchdown
10 min at 95°C
2 cycles of 15 sec at 95°C, 30 sec at 60.0°C, 45 sec at 72°C
2 cycles of 15 sec at 95°C, 30 sec at 58.0°C, 45 sec at 72°C
7
2 cycles of 15 sec at 95°C, 30 sec at 56.0°C, 45 sec at 72°C
2 cycles of 15 sec at 95°C, 30 sec at 54.0°C, 45 sec at 72°C
2 cycles of 15 sec at 95°C, 30 sec at 52.0°C, 45 sec at 72°C
30 cycles of 15 sec at 95°C, 30 sec at 50.0°C, 45 sec at 72°C
30 min final extension at 72°C
Hold at 4°C
8
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Supplementary Table S1. Roche 454 sequences for 14 koala microsatellite loci
Locus
Phci2
Phci5
Phci9
Sequences (STR, flanks, primer targets, and sequence surrounding the target region)
ATTAGCATGCTCAATCAGCATATATTCTCCTACCTCCTACCACATTGAGCCAGCT
GCAGAAGCAAAAATCCGTGTGTGTGTGTGTGTGTGTGTGTTGAATGAAGATGAA
AATCACTGACAAAATGATTTAAAGTAGTTGTGGAATTTGTCACTCCCTAATTAA
TTCTGTGACCAAAGTATTTTGGAGACCTTCAAACTGAATTTTCTTCAAGGGCCTA
ATGAATATCAATTCTGGGAAAACATTGTATTACATTTCTGCTTTTAATTGATTCT
AGTCCCTTGGTGAAAACTAATTCTGATCCCTAGTCTGTCCCTTCCTAGATTTCAG
ATCTCAAGTTTGTTTTTCAATAATGACTTTT
AGGAGAATATGATAGATAAGAGCTTTTCACTTTGTTTTTGGACCTCTAATAGGT
GGCACACTGCTACAAATAAGACGAATGCTCAATAAATGCCTACTAACTATAAC
AGGTGCAACCCGCCCATTTTAGATTTGAGAAAATCAAGGTCTATAAAGATTGAG
TGACTTGACCAATGTTACACAGGTGATAAGCAGAAGAACCAGAGTTAGAATCC
AGATATCTGACTCCAGGGTCAGTGCTCATTTCACTATACCATAATAATGGGAAA
ATGAAAAAGGACCAAGGGATAGGGGATGAAGTTGAGGTGGTGCAGCAAGAGG
TCTCAAAAGCACATAGCCTGGATTCATTCATTCATTCATTCATTCATTCATTCAT
TCATTCATTCATCAACCATTTATTAAGCCCTTTCTATGTGAGAGCCCTGTACCAG
GTACTATTCCTGAGAAACTCTGAAATCTGGCTATGGCTACAGGTAGAGATGACA
AAAGAAACAGTTACCAGTTACTTGGAAAACACCCAGCAGGGTTAATTCCAAAT
GTAGCTATTTCCATATGTTATTCTACTACAGAAGCACTAGATTCAGTAACAACT
AGTCTATTAAGGACTCCTAATAATACTTAAAAAAGAAAAGAAAAGAAAACCTT
TTGGGAATGTTAAAGGGAACTCACATTGCTCTCTTCACCTTCTATGAAGAGAAG
ACAGGGAAGGTGAAAGGGGCAGATAATTAGATTAAAAATCAGTGATTGAAACA
TGAAAAGCTATGTATATGTCTCAAAGAGACTTGTGCTTTCTAGAAGAACTGACA
GTTCTTTCCCTCTTTAAGTCAAGTTGACAGTGACTGTACTGTATATAGAAAATTG
CTGTAAGTTGCCCTTTGCAAACCTGAGGCTGTTAAGACTTCCGAAGAGGAA
GAAAGGCTCCCAGGTCAGGGGAGTGCATATATCATGGTTGTCTCCGGCAGGGG
GAAAAGCCACACTATCCTCATTTGTCAGATGACCTCACTTCATACCTTTACTGGC
CCTCATTATGAAGTGGGTGTTTGGCTTAAGGTCCCCCTCCATCTCTCACCCTAAC
CCTACTGAAGGACAAGCTCTGGGAGGCAGGGGCAGTGTTCCATCTAATCGAAA
CTCACAGGTGCCTCACAGATCCTCCTGGAACTGAACTGGCTGGAGTCTCTTGTT
ATGCTAAATTGGCCACAGACAGCTGCACTTGCTTGGGAAAGTTAGATTCCTTCC
ATGCAGGGGCATTGCCAAGGACACTGTACTGGGCTCATAGGAAGAAGAGGTGG
CGGCAGCAGCAGCAGAAAGAGCAGTAGCAGCAGCAGCAGCAGCAGCAGCAGC
AGCAGCAGCAGCAGCAGCAGCAGCAGCAGAAGCATGGTAGCAACAGCAGCAG
GAGCAGGAACAGAAGCATGGTAGCAGCAGGGGCAGCAGCA
11
Phci10
Phci12
Phci15
TATACAAAGTTATTTCAAGAGCAGGAGCACCCTCATAATTGGAGAGTGTGGGTG
AGGCAGCAAAGGCCTCCATTAACAATAACAAACAAACAACAATAGCTAGCATT
TAGATATTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCT
ATCTAATCTATCTATCATCTGTCTTTTTATTGGCAGCTATAGACTCTAGAGTGCT
GAACCTGGAATCAGGAAGAGCTGACTTTAAATCCAACCTCAGACACTAGCTAT
ATGACCCCGGACAAGTCACTTAATCTCTGCCTGACTCACTTTCTTCAACTGTAAA
ATGAGGATAATAATAGCACCTCCTGCCCAATGTTACTGTGAAGATCGAATAAGA
TGATATTTGTAAAGTGCTCAGTGCAGTTCCTGGCACAAAACAATTTATTTATAG
CACTTGAAGGTTTTCAAAGCACTTTACAAATATCATCTCATATGATTCTCAGAA
CCCTGAAAGGTAGGTGCTATTGTTATCCTCATTGGGCAATAGTGTATGAATTGT
AGGAAAAGTGAGGATGCCAAAAGATGGTGAGGAGGATGCAATGCATTTTGGTG
TAGGATTCAGCCAGTGCAAAGACGGAATGTCATGCATGCAGAACAACCAGTCA
TCCTGTTTGGACCAAAAGCAGAGAGTCCACCAAGGGGAATACAGCTAAGACTG
GAGGGGCACATGGGAACCGGCATGAGAAAGGATTTCAATGCACGGCTGAGTTC
AAGCTTGAGATCCGAGGGCTTGTTCAATTAGAAGTCATGTTAATGTCAGCACAG
TGCTGGTGTTTATGGACGCTTCTCTTCTGGTCACACACCTCTGGTATTTCTGAGG
AGTAATGGAGAGCTCTGATGAGACCTCTCAGCAACAGGCAGAGAGTTCTG
AGGTACACACCTAACATCCAAGTAGAAATCTATGGTAGATAGCTGCTGATATGG
GACTAGATAGCTCAGGGGAGAGACTGGGGCTGGATATGCAGATGTGGGAGTCA
TCTGCATAGGGACAGTAACTGAACCCAAGGAAGCAGATGAGATTATAGTAGAG
GAAGAGAACTGGGAGGTGACTACCCTAAGGAGGCAGGAGATAGAGGTGATGA
CCTAATGAAACAGAATGAGAGAGACGAGTGTCATGGAAGCCACAGGAGGAGA
GAGAATATAGAAGAAGGTAGTAGCAACAGTGTCAGCCTACAGAATCCCCTTCT
GTGCTAATCAGTGGACAAAGGAGGATATGCTCAGGGCTCAACAATTATAATGG
CCTCCTAATAACTCTTCCTGCCTTCAATCTCCCCTCTTCAATACCTTTTCCACATA
GCTGCCAAAGTGATATTCTTATAGTACGTTTGAACATGTCATTCACCTAGGAGG
GCAGGAAGGATAGGCCAGACTAACAGAAGGACCTCTCAAGGCTCTTCACCCTT
GGTCAATTGACACCATCTGGATACTCACCAACAACACTAATGATAATAATGATG
ATGACGACGACGACGACGACGACGACGACGACGACGACGACGATAATGGTAA
CAACAAGTAACAGGGGGAAAGGAACTGAACCGAGGGAGTTTGGAACACAGGG
AACTTGTAGAGTAAGGAAACTCTCTGTCAATGAGAAGTCTCTGCAACTTACAAT
CTTAGAGAGTTGCCTAGAGCACTGAGAGGTTAA
AATAAACAACAAAAAAACCCCTTTGTACTTCAAATAAATGATCTCAAAGTCCTT
CATGTCACCTTTCAGGTACTGTGACTTATGGCTGGTCCTGTGAAATAGTCAGCA
CCCAGGAATGGGCAATATATACATCAAGGGCCTGGACCCATTTGTCACTCCACA
CCAGAGGGTGCTTTGGGGCATCTGCCAAACTTCCCATGACTTTGTGAGGTTCTC
TGAGAGCTGACTGCTATTCTTCCCCTGTACTAGACCCTTTTGTGTGCGGTGTCAT
TTTATCTCTCTTAGCCTGGGCTGGGCTGGTAAGGCCTATCCAATCAGATTATCAG
CTAGTAGCCCCCATCCTTCTGTTCCCTCTTTTGGATTCCTCATCTCTGTCTGTGTT
CCTTAAAGTTCTCATAAAGGTAGGCTTGCAGAGGGATCAAGAGAGGGGCAGAC
CTATGATTTAACTGTATGGAGAACACCCAGATGAGAAAACTCCCTATANTAATG
CAGGTCAGCACATTCTCTGCAATTTCTAATTCTTTGAGCGTCGCCCAGGGCACT
GAGTGGTTAAGTAACTTGCCCAAGATCATGAAGCCAACATGCGTCACAGATAG
GATTTGAACACAGGTCTTCCTGCTCTTTTTATCTATAGATCTATCTATCTATCTAT
CTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTGTCAAGGCCAGTTT
TCTATCCACTATGCCACACCAGTTCTTGTG
12
Phci16
Phci17
Phci18
CATCTCAGTCCAGTGGCAAGATAAAGATCAGGCCAACTGGAGATGGCCCTGGA
TGCAGTGAGAGACCTTGACCTTTTTAAACTAAGGTCTTCAACAGGTCTCAGTTT
GACTGAGGCAACACCCATTCAGTAAGAAATGAGGCAAAATATGGCCTAGGTTT
TGAGTGTATAAGGACACAAAAACACATCTTGGACCTGGAGTAGCTATTAAGAA
GGTTCCATGTGTGAGACTTGGGTATTTTAGGTGCTACATGTAGAAGGAAATGCT
CTGTGTGTGTGTGTGTGTGTGTTAGGAACATGAAAAGTATTGTGGTCTCAAGAG
TCAGGGTAAATGATAACAAAGGCTGAGTGCTAGAAGAGTTCATGTATTTATAA
GCTTTTACCACCTTGGAGTCCTTCTATTTCTTGAGTTCTGCTTAAGAGAAGATAA
GGAACAACTTGCTGGTCTATGAATGTTTTTATGCAGATACTCTGAAAACCTTTCC
TATAACTCATACCTCAAACTTCCCACATGATTATCCTTTTGCCCATACAAAAAA
AGTCAAGGGAGCCTAATTCACACACACACACACACACACACACACACACACAC
ACACACACAC
ACCTCTATCCCAATACCTCTCCAGTTCACCCACACAGGCACACGTGCGTGCACA
CACACACACACACATACATACATACATACATACATACACACACACACACACAC
ACACACACACACACACATATTTCAGGCCCCTTTTACCTCATGCCTGGAGTCTAG
GGGATGTCTCCTATTTCACCTCCCTGGTCCCCCCCCAAGCCAACCAAGCAAGTT
TTCCCATGTGTCTGAACGTGCCATCTCACCCAACCTTGACAGGGATGGGCAATG
TAGCATAGTAGAGCTGTCCATGTGGCAGAGGGACTATGGTGTAGGAGACAGAG
CTGGCCTCAGAGCCAGCAGAAGCTGGGTTCCAGTCATCCATCTGACCCATACTG
GCAGCGTGACCTGACTTCTCCGTCAGCTATGAGACAGTTATGTCGGCATGTAAG
CTGCAGAGAAGGAGGTCCTAGTCCTGTCTTCTTCCCTAGTTCTCTCCTCCTTTGC
CGTCCATAGGCTACGCCTCTGCCCTCTTCCTCACACCTTCTTAACCCAATCTACA
ACCTGGCTTTCCTTCTCGACATCCCACCCAAATGGCCTTCTCCAAGGTGACCTTG
GTCTCTTCTCTTGCCCTTCCTCCATGACCTCTCTGTGGCTCACCCTGGTTCTCCCC
GCTCCTTCACACCCCTTCCTAGGGCTCCATTCACACCCCTTCCTAGGCTCCATTC
CCTGCATCTCTGGGCCATGG
ACAAGCCACTCACTCGGTGTGGTGACACTCAGGCAGTTCTGGGAGCCAGAGAC
AGCCTGGTACCAAAATTTCTAATAAAATGGAGGTAATGGCTTAAACTACCTTAA
TGCTTCAGTTTCCCCTCCCCCATCCTCATACCCCATAGCTACTCTCTATGACTGT
CATCTCTTAGCTGGAATTTAACCACTTTTGTTTCTGAGGCTCATCCTTAAATGAT
GACCTTTCCTGCCTGACTGCCCCATATCACGGGGACCTTTTCCTCCTCTTATCTT
CCTATGGTTTGCACTCCTCATTGGGTATGAAACTCAGACTAGACTGCCTGCTTCT
GTTAGTTATATTGTCATGTGCCCTGCCTCTCCAACTAGACTGTCAACTTTTGGAG
GGCAGGGAATGCGTTTTCTTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTG
TGTCGCCTTTACAGTGCCTAACACATAGCTGGAAACACAGTAAGGGCTCAGAA
AATAGTTGCTGATTGACTGAATGACTGGTTAGATCAGTGAACTAACAAAATACA
TTCATCATCTCAGAAGCTCTCTCAGTCAGACCCCCTGGTGGATTCCCCAGGGGC
CAAACTGTTTTTCAATGTCCAGAGGATGGACTGAAGGCTTCCAAGTCAAAGAAT
GACGGAGCTGAAAGGAACCTTACATACCATCTAGTCAAATCCCCTCTTTTGCAG
GTCAAAGGAACTGAGGCCACGACATGCCCGCTGTCACATAGCGAGTTGGTGGC
AAAGCTGCGTCTAGAACCCAGATCTCAGTTCCGGTTCAGTGCTCCTTTCGCAGA
GCCAGAGCA
13
Phci19
Phci22
Phci27
AGTTCACAAACTCATCAACATTGCATTAATGTCTCATTTTTCCCACATCCCCTCC
ACCATTTGTAATTTACTTTTCTGTCCTATTAGTCAATATAATAGGTATGAGGTAG
TACCTCAGGATTCTTTTAATTTGCATTTTTCCAATCAATAGTGAGTACGAGTATG
TTTCATAAGGCTGTACATAGTTTTGATTAATTCATCTGTTCATGAAAACTGTTCA
TAACTTTTGATCATTTATCAACTGAGGAAGGGCTTATATTTATAAATTTGACTCA
TTTCTCTATATGTTTTAGAAATGACACCTGTATCAGAGAAACTTACTTCAATTTT
TTTCAGTTGCTATTTGCCTTTCTCTCAGTGCATTCCTCTCTCACCCCTTAATTTTA
TACACACACACACACACACACACACACACACACACACAACACACACATCATCC
TCATATCAACTCACATCTGTGCCCTCTGTCTATATATGCTCCTTCCAACTGCCCT
TATAATGAAAAAGTTCTTATGAGTTACAAATACATCTTCTCATATAGGAATGGA
AACAGTTTAACCTTATTAAAT
GAATTGTCTGCCTTAGGAGGCAACGGATTGCCCTTTTTTGGAGGTCTTTAAGCA
AAGATTGGATGACCATTTGTCAGGTATGTTGTAATAAAGATTCTTTTCTCTGGTA
TAGGTTGGACTAAGCAGCTTTTGAGATCCTTTGCAATTCTGAAATTCTGTGATTC
TCACCTAAACTATTGTAATAGTCTCCTAATCAGTCTTCCTGCCTCCAGTCTATGT
TCTTCCCAAGTCCATCTCCTCTTCAACCATCCTCTAGTCACCAGAGTACGTCTTC
CCATGAACTGCTCTGATCATATCATGCCTCTTTTCAACAACCTTAAGAAGCTCCT
TATTGCTTACTGAAACACACACACACACACACACACACACACACACACTCTCTG
ATGCTTGCAGTTCAAGGTCACCCACACTCTGGCTCCACTTTCCCTCTCCAACATT
CCCTAAAATTTTACCTTTTCACATTCTCTCTATTCCCAACAAAGTGAGCCACTCC
CCATCCTCCTGCCC
TTTAGAGAAAGAACAGAACTAACATTTAAAGGCAATTTGCATTTAATTTCCTTT
GAATTTTCAAGATAATACCCTTCTTGGTCTTCTAGTTTTACTTACTTCATATCCCC
AGCATTCCTGTTTCCATATTTAAGATGCATTGCTGTGCAATTTTATACTGCAGTA
ATATAGGGAGAGACTAGATTTCTGTGGTCTAAGAATCTCCTGGGTAAAGAAACT
CCCTCTACTGATGCAGATAGCATCTTCGCTACAATTTATAGCCTCAGAGAGCTG
CCTAGAGTACTGAGAAGGTAAATGATTTGCCCAGGATCACACAGTCAGTATGTT
GGCCGAAGTAGGATTTGAATCCATGTCTTCTTGACTTTGAGGCCATTTGTCTATC
TACTATATGATACTGCTTCTGTAGGAACACACACACACACACACACACACACAC
ACATCACATAGACACATACATGTGTATATCATGCTGGGTGAGTTAAGGTGTTGT
ATTGTAACCTTTGGAGGAGGGGTTTAGTGAATGTGAACTAATGAACAAAATTTT
GACTGCTAGTGTTTCATGCCATTATGAGTTGCAAGAAACTGATTTTGCTGGACA
GCAGCAAAAATTCATCCAATCCTTCAAGCTCATGTTCTAAACTNCAGAAGTGAA
TACCCTTAGGAAAGATACAGACTAAATGAAACTGAAAATATACTCATCTTTCCT
TCTTACTTCTCTCTGCTTTCATCCCTGAAATGCTGAAAGATTCTTTAGCTCTGATT
TTGGTAACTGTCAATAGGCCAGTCCTGTTTGCTCTCTGTGATTGACAGAT
14
Phci28
Phci31
CATTCATTCACCCCATGTTAGCTACACTTCTGCTGTGGGCTATCTGAAGCTCCCT
CACTCCCTTCCTATGTGGACCATAGATTAAATGGCCAACAAAATACCCCTCTCC
TCTAGTGGAGTCAGGGGCAAGGGTATCTGGGGAAAAACAGGATAGTATTGCGT
AAGAGGATATAGAAAAGAATACCTCACCTTTTTTTCTAGTTTCAGCACATGACC
AGGTCACCATTATCTTACTGCAATAGGGATATGGAGGAAAAGAATATTTTCCCT
TAAGCTTAAGGACAGGAATGGAGTGAAACCAGTTTAGGCTATTCTGGGCTACGT
CTTGGGCTACTCTGGCAGAATCCAAGATATCCTGGACCTAGAAACAAGGTTCCA
TTTGGAAGTGGCAGTTCCCTCCACACACACACACACACACACACACACACACAC
ACACACACACACAGAGATTTAATTCTCGCTACACAAGAGATCAGCTGTTCCCTT
GCTGTGATTCCGGCNTTGACAAAAGCACTGTAAAAAACCTTTCAGAGCTCTTGC
CCTTTATTATTCTTATGGAGCACAGAATTCCTGCTTTATTAATAAAGGAGGTTGT
CACAAAGAACAGCTTTGTGGAA
GCACTATATACTTATATGTGTACTTGGCTCCCCAACCAAAAGGGAAGATCTTTG
AGAGTAGGGACTGTTTCATTATTTGTACTTGAATTCCCAGGGCCAACAGAGATA
CACATACAGACATGTGTGTGCAAGCACACACACACACACACACACACACACAC
ACACTCTTAAATAAATGCTTGTTGATTGACTGGGGGAGGATTATTGGGGGAAGA
GAATAATCAGATTAGACTGAGTACTAACGTTTCACTTCTGCCTACCACCCCTTC
AGTACTGGGTATGTTCTCCTCTTGGCTAAAGAACAGAACCATAGCCTTCTGAAG
AGTTTCTTAGCAGTTCCAAATTTTGTCTGCAGATGGCCAAAAAAGATGATCGGT
CTTTAGAAAGACCTGAACATTAAAAAGTGATGTTATCCAAGAATATTACAAACA
CAGGTCCTAGGTATCTCTATAGCAACTATCACCATAGCACTAGGGTGCTCTGAA
AA
15