Development of 14 microsatellite markers in the Queensland koala




Дата канвертавання25.04.2016
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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*
1Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801 USA

2Leibniz Institute for Zoo and Wildlife Research, 10315, Berlin, Germany
*Corresponding Author: roca@illinois.edu
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) . However, because koala range is continuous across state borders, this variation may be clinal rather than discrete . 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) . Morphological characters, such as fur color and thickness, muzzle shape and body size, are known to vary between northern and southern populations . 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 . 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 . 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 . A threat to koalas in this region is habitat conversion and fragmentation . 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 . 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 .

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 . 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 . This difference in genetic variability was expected since koalas in Victoria have suffered from bottlenecks, founder effects and translocations . 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 . This established that the two island populations have low genetic diversity compared to northern koala populations that did not suffer from bottlenecks .

Most population genetic studies of Queensland koalas have relied on sequencing the mitochondrial control region . 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 . A recent study using museum samples showed that low levels of mtDNA diversity in koala populations had been present prior to recent population decline . A study conducted on five southeast Queensland populations suggested that there has been female-mediated gene flow historically (based on adjacent populations sharing haplotypes) . However, the limited distribution of mtDNA haplotypes would also indicate that barriers to gene flow have existed among populations .

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 was used to identify microsatellite repeat motifs by screening the sequences for di-, tri-,tetra- and penta- nucleotide motifs, with a minimum of 10 repeats each. MSATCOMMANDER interfaces with PRIMER 3 software , 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) .

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. . 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 . 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 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 . 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 and GENEPOP . Deviations from Hardy-Weinberg equilibrium were calculated using GENEPOP, v 4.0 . Linkage disequilibrium between pairs of loci was calculated with FSTAT, v. 2.9.3.2 .


Details of the PCR setup and PCR algorithm

PCR Components

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

a2.5 mM of each dNTP (dATP, dCTP, dGTP, and dTTP) blend (ABI, N8080260)

bAmpliTaq Gold DNA Polymerase with Buffer II and MgCl2 solution (ABI, N8080249)

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

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


References for supplementary information

Supplementary Table S1. Roche 454 sequences for 14 koala microsatellite loci

Locus

Sequences (STR, flanks, primer targets, and sequence surrounding the target region)

Phci2

ATTAGCATGCTCAATCAGCATATATTCTCCTACCTCCTACCACATTGAGCCAGCTGCAGAAGCAAAAATCCGTGTGTGTGTGTGTGTGTGTGTGTTGAATGAAGATGAAAATCACTGACAAAATGATTTAAAGTAGTTGTGGAATTTGTCACTCCCTAATTAATTCTGTGACCAAAGTATTTTGGAGACCTTCAAACTGAATTTTCTTCAAGGGCCTAATGAATATCAATTCTGGGAAAACATTGTATTACATTTCTGCTTTTAATTGATTCTAGTCCCTTGGTGAAAACTAATTCTGATCCCTAGTCTGTCCCTTCCTAGATTTCAGATCTCAAGTTTGTTTTTCAATAATGACTTTT

Phci5

AGGAGAATATGATAGATAAGAGCTTTTCACTTTGTTTTTGGACCTCTAATAGGTGGCACACTGCTACAAATAAGACGAATGCTCAATAAATGCCTACTAACTATAACAGGTGCAACCCGCCCATTTTAGATTTGAGAAAATCAAGGTCTATAAAGATTGAGTGACTTGACCAATGTTACACAGGTGATAAGCAGAAGAACCAGAGTTAGAATCCAGATATCTGACTCCAGGGTCAGTGCTCATTTCACTATACCATAATAATGGGAAAATGAAAAAGGACCAAGGGATAGGGGATGAAGTTGAGGTGGTGCAGCAAGAGGTCTCAAAAGCACATAGCCTGGATTCATTCATTCATTCATTCATTCATTCATTCATTCATTCATTCATCAACCATTTATTAAGCCCTTTCTATGTGAGAGCCCTGTACCAGGTACTATTCCTGAGAAACTCTGAAATCTGGCTATGGCTACAGGTAGAGATGACAAAAGAAACAGTTACCAGTTACTTGGAAAACACCCAGCAGGGTTAATTCCAAATGTAGCTATTTCCATATGTTATTCTACTACAGAAGCACTAGATTCAGTAACAACTAGTCTATTAAGGACTCCTAATAATACTTAAAAAAGAAAAGAAAAGAAAACCTTTTGGGAATGTTAAAGGGAACTCACATTGCTCTCTTCACCTTCTATGAAGAGAAGACAGGGAAGGTGAAAGGGGCAGATAATTAGATTAAAAATCAGTGATTGAAACATGAAAAGCTATGTATATGTCTCAAAGAGACTTGTGCTTTCTAGAAGAACTGACAGTTCTTTCCCTCTTTAAGTCAAGTTGACAGTGACTGTACTGTATATAGAAAATTGCTGTAAGTTGCCCTTTGCAAACCTGAGGCTGTTAAGACTTCCGAAGAGGAA

Phci9

GAAAGGCTCCCAGGTCAGGGGAGTGCATATATCATGGTTGTCTCCGGCAGGGGGAAAAGCCACACTATCCTCATTTGTCAGATGACCTCACTTCATACCTTTACTGGCCCTCATTATGAAGTGGGTGTTTGGCTTAAGGTCCCCCTCCATCTCTCACCCTAACCCTACTGAAGGACAAGCTCTGGGAGGCAGGGGCAGTGTTCCATCTAATCGAAACTCACAGGTGCCTCACAGATCCTCCTGGAACTGAACTGGCTGGAGTCTCTTGTTATGCTAAATTGGCCACAGACAGCTGCACTTGCTTGGGAAAGTTAGATTCCTTCCATGCAGGGGCATTGCCAAGGACACTGTACTGGGCTCATAGGAAGAAGAGGTGGCGGCAGCAGCAGCAGAAAGAGCAGTAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGAAGCATGGTAGCAACAGCAGCAGGAGCAGGAACAGAAGCATGGTAGCAGCAGGGGCAGCAGCA

Phci10

TATACAAAGTTATTTCAAGAGCAGGAGCACCCTCATAATTGGAGAGTGTGGGTGAGGCAGCAAAGGCCTCCATTAACAATAACAAACAAACAACAATAGCTAGCATTTAGATATTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTAATCTATCTATCATCTGTCTTTTTATTGGCAGCTATAGACTCTAGAGTGCTGAACCTGGAATCAGGAAGAGCTGACTTTAAATCCAACCTCAGACACTAGCTATATGACCCCGGACAAGTCACTTAATCTCTGCCTGACTCACTTTCTTCAACTGTAAAATGAGGATAATAATAGCACCTCCTGCCCAATGTTACTGTGAAGATCGAATAAGATGATATTTGTAAAGTGCTCAGTGCAGTTCCTGGCACAAAACAATTTATTTATAGCACTTGAAGGTTTTCAAAGCACTTTACAAATATCATCTCATATGATTCTCAGAACCCTGAAAGGTAGGTGCTATTGTTATCCTCATTGGGCAATAGTGTATGAATTGTAGGAAAAGTGAGGATGCCAAAAGATGGTGAGGAGGATGCAATGCATTTTGGTGTAGGATTCAGCCAGTGCAAAGACGGAATGTCATGCATGCAGAACAACCAGTCATCCTGTTTGGACCAAAAGCAGAGAGTCCACCAAGGGGAATACAGCTAAGACTGGAGGGGCACATGGGAACCGGCATGAGAAAGGATTTCAATGCACGGCTGAGTTCAAGCTTGAGATCCGAGGGCTTGTTCAATTAGAAGTCATGTTAATGTCAGCACAGTGCTGGTGTTTATGGACGCTTCTCTTCTGGTCACACACCTCTGGTATTTCTGAGGAGTAATGGAGAGCTCTGATGAGACCTCTCAGCAACAGGCAGAGAGTTCTG

Phci12

AGGTACACACCTAACATCCAAGTAGAAATCTATGGTAGATAGCTGCTGATATGGGACTAGATAGCTCAGGGGAGAGACTGGGGCTGGATATGCAGATGTGGGAGTCATCTGCATAGGGACAGTAACTGAACCCAAGGAAGCAGATGAGATTATAGTAGAGGAAGAGAACTGGGAGGTGACTACCCTAAGGAGGCAGGAGATAGAGGTGATGACCTAATGAAACAGAATGAGAGAGACGAGTGTCATGGAAGCCACAGGAGGAGAGAGAATATAGAAGAAGGTAGTAGCAACAGTGTCAGCCTACAGAATCCCCTTCTGTGCTAATCAGTGGACAAAGGAGGATATGCTCAGGGCTCAACAATTATAATGGCCTCCTAATAACTCTTCCTGCCTTCAATCTCCCCTCTTCAATACCTTTTCCACATAGCTGCCAAAGTGATATTCTTATAGTACGTTTGAACATGTCATTCACCTAGGAGGGCAGGAAGGATAGGCCAGACTAACAGAAGGACCTCTCAAGGCTCTTCACCCTTGGTCAATTGACACCATCTGGATACTCACCAACAACACTAATGATAATAATGATGATGACGACGACGACGACGACGACGACGACGACGACGACGACGATAATGGTAACAACAAGTAACAGGGGGAAAGGAACTGAACCGAGGGAGTTTGGAACACAGGGAACTTGTAGAGTAAGGAAACTCTCTGTCAATGAGAAGTCTCTGCAACTTACAATCTTAGAGAGTTGCCTAGAGCACTGAGAGGTTAA

Phci15

AATAAACAACAAAAAAACCCCTTTGTACTTCAAATAAATGATCTCAAAGTCCTTCATGTCACCTTTCAGGTACTGTGACTTATGGCTGGTCCTGTGAAATAGTCAGCACCCAGGAATGGGCAATATATACATCAAGGGCCTGGACCCATTTGTCACTCCACACCAGAGGGTGCTTTGGGGCATCTGCCAAACTTCCCATGACTTTGTGAGGTTCTCTGAGAGCTGACTGCTATTCTTCCCCTGTACTAGACCCTTTTGTGTGCGGTGTCATTTTATCTCTCTTAGCCTGGGCTGGGCTGGTAAGGCCTATCCAATCAGATTATCAGCTAGTAGCCCCCATCCTTCTGTTCCCTCTTTTGGATTCCTCATCTCTGTCTGTGTTCCTTAAAGTTCTCATAAAGGTAGGCTTGCAGAGGGATCAAGAGAGGGGCAGACCTATGATTTAACTGTATGGAGAACACCCAGATGAGAAAACTCCCTATANTAATGCAGGTCAGCACATTCTCTGCAATTTCTAATTCTTTGAGCGTCGCCCAGGGCACTGAGTGGTTAAGTAACTTGCCCAAGATCATGAAGCCAACATGCGTCACAGATAGGATTTGAACACAGGTCTTCCTGCTCTTTTTATCTATAGATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTGTCAAGGCCAGTTTTCTATCCACTATGCCACACCAGTTCTTGTG

Phci16

CATCTCAGTCCAGTGGCAAGATAAAGATCAGGCCAACTGGAGATGGCCCTGGATGCAGTGAGAGACCTTGACCTTTTTAAACTAAGGTCTTCAACAGGTCTCAGTTTGACTGAGGCAACACCCATTCAGTAAGAAATGAGGCAAAATATGGCCTAGGTTTTGAGTGTATAAGGACACAAAAACACATCTTGGACCTGGAGTAGCTATTAAGAAGGTTCCATGTGTGAGACTTGGGTATTTTAGGTGCTACATGTAGAAGGAAATGCTCTGTGTGTGTGTGTGTGTGTGTTAGGAACATGAAAAGTATTGTGGTCTCAAGAGTCAGGGTAAATGATAACAAAGGCTGAGTGCTAGAAGAGTTCATGTATTTATAAGCTTTTACCACCTTGGAGTCCTTCTATTTCTTGAGTTCTGCTTAAGAGAAGATAAGGAACAACTTGCTGGTCTATGAATGTTTTTATGCAGATACTCTGAAAACCTTTCCTATAACTCATACCTCAAACTTCCCACATGATTATCCTTTTGCCCATACAAAAAAAGTCAAGGGAGCCTAATTCACACACACACACACACACACACACACACACACACACACACACAC

Phci17

ACCTCTATCCCAATACCTCTCCAGTTCACCCACACAGGCACACGTGCGTGCACACACACACACACACATACATACATACATACATACATACACACACACACACACACACACACACACACACACATATTTCAGGCCCCTTTTACCTCATGCCTGGAGTCTAGGGGATGTCTCCTATTTCACCTCCCTGGTCCCCCCCCAAGCCAACCAAGCAAGTTTTCCCATGTGTCTGAACGTGCCATCTCACCCAACCTTGACAGGGATGGGCAATGTAGCATAGTAGAGCTGTCCATGTGGCAGAGGGACTATGGTGTAGGAGACAGAGCTGGCCTCAGAGCCAGCAGAAGCTGGGTTCCAGTCATCCATCTGACCCATACTGGCAGCGTGACCTGACTTCTCCGTCAGCTATGAGACAGTTATGTCGGCATGTAAGCTGCAGAGAAGGAGGTCCTAGTCCTGTCTTCTTCCCTAGTTCTCTCCTCCTTTGCCGTCCATAGGCTACGCCTCTGCCCTCTTCCTCACACCTTCTTAACCCAATCTACAACCTGGCTTTCCTTCTCGACATCCCACCCAAATGGCCTTCTCCAAGGTGACCTTGGTCTCTTCTCTTGCCCTTCCTCCATGACCTCTCTGTGGCTCACCCTGGTTCTCCCCGCTCCTTCACACCCCTTCCTAGGGCTCCATTCACACCCCTTCCTAGGCTCCATTCCCTGCATCTCTGGGCCATGG

Phci18

ACAAGCCACTCACTCGGTGTGGTGACACTCAGGCAGTTCTGGGAGCCAGAGACAGCCTGGTACCAAAATTTCTAATAAAATGGAGGTAATGGCTTAAACTACCTTAATGCTTCAGTTTCCCCTCCCCCATCCTCATACCCCATAGCTACTCTCTATGACTGTCATCTCTTAGCTGGAATTTAACCACTTTTGTTTCTGAGGCTCATCCTTAAATGATGACCTTTCCTGCCTGACTGCCCCATATCACGGGGACCTTTTCCTCCTCTTATCTTCCTATGGTTTGCACTCCTCATTGGGTATGAAACTCAGACTAGACTGCCTGCTTCTGTTAGTTATATTGTCATGTGCCCTGCCTCTCCAACTAGACTGTCAACTTTTGGAGGGCAGGGAATGCGTTTTCTTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTCGCCTTTACAGTGCCTAACACATAGCTGGAAACACAGTAAGGGCTCAGAAAATAGTTGCTGATTGACTGAATGACTGGTTAGATCAGTGAACTAACAAAATACATTCATCATCTCAGAAGCTCTCTCAGTCAGACCCCCTGGTGGATTCCCCAGGGGCCAAACTGTTTTTCAATGTCCAGAGGATGGACTGAAGGCTTCCAAGTCAAAGAATGACGGAGCTGAAAGGAACCTTACATACCATCTAGTCAAATCCCCTCTTTTGCAGGTCAAAGGAACTGAGGCCACGACATGCCCGCTGTCACATAGCGAGTTGGTGGCAAAGCTGCGTCTAGAACCCAGATCTCAGTTCCGGTTCAGTGCTCCTTTCGCAGAGCCAGAGCA

Phci19

AGTTCACAAACTCATCAACATTGCATTAATGTCTCATTTTTCCCACATCCCCTCCACCATTTGTAATTTACTTTTCTGTCCTATTAGTCAATATAATAGGTATGAGGTAGTACCTCAGGATTCTTTTAATTTGCATTTTTCCAATCAATAGTGAGTACGAGTATGTTTCATAAGGCTGTACATAGTTTTGATTAATTCATCTGTTCATGAAAACTGTTCATAACTTTTGATCATTTATCAACTGAGGAAGGGCTTATATTTATAAATTTGACTCATTTCTCTATATGTTTTAGAAATGACACCTGTATCAGAGAAACTTACTTCAATTTTTTTCAGTTGCTATTTGCCTTTCTCTCAGTGCATTCCTCTCTCACCCCTTAATTTTATACACACACACACACACACACACACACACACACACACAACACACACATCATCCTCATATCAACTCACATCTGTGCCCTCTGTCTATATATGCTCCTTCCAACTGCCCTTATAATGAAAAAGTTCTTATGAGTTACAAATACATCTTCTCATATAGGAATGGAAACAGTTTAACCTTATTAAAT

Phci22

GAATTGTCTGCCTTAGGAGGCAACGGATTGCCCTTTTTTGGAGGTCTTTAAGCAAAGATTGGATGACCATTTGTCAGGTATGTTGTAATAAAGATTCTTTTCTCTGGTATAGGTTGGACTAAGCAGCTTTTGAGATCCTTTGCAATTCTGAAATTCTGTGATTCTCACCTAAACTATTGTAATAGTCTCCTAATCAGTCTTCCTGCCTCCAGTCTATGTTCTTCCCAAGTCCATCTCCTCTTCAACCATCCTCTAGTCACCAGAGTACGTCTTCCCATGAACTGCTCTGATCATATCATGCCTCTTTTCAACAACCTTAAGAAGCTCCTTATTGCTTACTGAAACACACACACACACACACACACACACACACACACTCTCTGATGCTTGCAGTTCAAGGTCACCCACACTCTGGCTCCACTTTCCCTCTCCAACATTCCCTAAAATTTTACCTTTTCACATTCTCTCTATTCCCAACAAAGTGAGCCACTCCCCATCCTCCTGCCC

Phci27

TTTAGAGAAAGAACAGAACTAACATTTAAAGGCAATTTGCATTTAATTTCCTTTGAATTTTCAAGATAATACCCTTCTTGGTCTTCTAGTTTTACTTACTTCATATCCCCAGCATTCCTGTTTCCATATTTAAGATGCATTGCTGTGCAATTTTATACTGCAGTAATATAGGGAGAGACTAGATTTCTGTGGTCTAAGAATCTCCTGGGTAAAGAAACTCCCTCTACTGATGCAGATAGCATCTTCGCTACAATTTATAGCCTCAGAGAGCTGCCTAGAGTACTGAGAAGGTAAATGATTTGCCCAGGATCACACAGTCAGTATGTTGGCCGAAGTAGGATTTGAATCCATGTCTTCTTGACTTTGAGGCCATTTGTCTATCTACTATATGATACTGCTTCTGTAGGAACACACACACACACACACACACACACACACATCACATAGACACATACATGTGTATATCATGCTGGGTGAGTTAAGGTGTTGTATTGTAACCTTTGGAGGAGGGGTTTAGTGAATGTGAACTAATGAACAAAATTTTGACTGCTAGTGTTTCATGCCATTATGAGTTGCAAGAAACTGATTTTGCTGGACAGCAGCAAAAATTCATCCAATCCTTCAAGCTCATGTTCTAAACTNCAGAAGTGAATACCCTTAGGAAAGATACAGACTAAATGAAACTGAAAATATACTCATCTTTCCTTCTTACTTCTCTCTGCTTTCATCCCTGAAATGCTGAAAGATTCTTTAGCTCTGATTTTGGTAACTGTCAATAGGCCAGTCCTGTTTGCTCTCTGTGATTGACAGAT

Phci28

CATTCATTCACCCCATGTTAGCTACACTTCTGCTGTGGGCTATCTGAAGCTCCCTCACTCCCTTCCTATGTGGACCATAGATTAAATGGCCAACAAAATACCCCTCTCCTCTAGTGGAGTCAGGGGCAAGGGTATCTGGGGAAAAACAGGATAGTATTGCGTAAGAGGATATAGAAAAGAATACCTCACCTTTTTTTCTAGTTTCAGCACATGACCAGGTCACCATTATCTTACTGCAATAGGGATATGGAGGAAAAGAATATTTTCCCTTAAGCTTAAGGACAGGAATGGAGTGAAACCAGTTTAGGCTATTCTGGGCTACGTCTTGGGCTACTCTGGCAGAATCCAAGATATCCTGGACCTAGAAACAAGGTTCCATTTGGAAGTGGCAGTTCCCTCCACACACACACACACACACACACACACACACACACACACACACACAGAGATTTAATTCTCGCTACACAAGAGATCAGCTGTTCCCTTGCTGTGATTCCGGCNTTGACAAAAGCACTGTAAAAAACCTTTCAGAGCTCTTGCCCTTTATTATTCTTATGGAGCACAGAATTCCTGCTTTATTAATAAAGGAGGTTGTCACAAAGAACAGCTTTGTGGAA

Phci31

GCACTATATACTTATATGTGTACTTGGCTCCCCAACCAAAAGGGAAGATCTTTGAGAGTAGGGACTGTTTCATTATTTGTACTTGAATTCCCAGGGCCAACAGAGATACACATACAGACATGTGTGTGCAAGCACACACACACACACACACACACACACACACACTCTTAAATAAATGCTTGTTGATTGACTGGGGGAGGATTATTGGGGGAAGAGAATAATCAGATTAGACTGAGTACTAACGTTTCACTTCTGCCTACCACCCCTTCAGTACTGGGTATGTTCTCCTCTTGGCTAAAGAACAGAACCATAGCCTTCTGAAGAGTTTCTTAGCAGTTCCAAATTTTGTCTGCAGATGGCCAAAAAAGATGATCGGTCTTTAGAAAGACCTGAACATTAAAAAGTGATGTTATCCAAGAATATTACAAACACAGGTCCTAGGTATCTCTATAGCAACTATCACCATAGCACTAGGGTGCTCTGAAAA




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