MICROSATELLITE LETTERS

Development of polymorphic microsatellite loci isolatedfrom the Ancherythoculter nigrocauda

Yanhong Sun • Guiying Wang • Dongmei Zhu •

Jian Chen • Pei Li • Qing Li

Received: 12 May 2014 / Accepted: 2 June 2014

� Springer Science+Business Media Dordrecht 2014

Abstract Twelve polymorphic microsatellite markers

from Ancherythoculter nigrocauda were developed using

the fast isolation by AFLP of sequences containing repeats

(FIASCO) protocol. These loci were characterized by

genotyping 48 individuals. The observed number of alleles

per locus ranged from 4 to 14 with an average of 9.4. The

expected and observed heterozygosities ranged from 0.185

to 0.907 and from 0.083 to 0.952, respectively. Among

these polymorphic microsatellite loci, four of the loci

(HWB01, HWB04, HWB12 and HWB16) significantly

deviated from Hardy–Weinberg equilibrium after Bonfer-

roni correction (P \ 0.05). All of the microsatellite loci

were in linkage equilibrium. These microsatellite markers

would be useful for investigating the genetic diversity and

population structure of A. nigrocauda.

Keywords Ancherythoculter nigrocauda �Microsatellite � Polymorphism

Ancherythroculter nigrocauda, which belongs to subfamily

Culterinae, family Cyprinidae, and order Cypriniformes

(Luo 1998), is a fish endemic to the upper reaches of the

Yangtze River in China. Recently, this species has faced

the problem of the population being decreased noticeably

due to long-term overfishing and habitat degradation

(Xiong et al. 2006). To protect and maintain sustainable

utilization of A. nigrocauda fishery resource, it is necessary

to make clear the genetic information of this species.

Microsatellite markers are powerful tools for the analysis

of genetic diversity and genetic structure, but until now

none of species specific microsatellite have been developed

for A. nigrocauda.

In this study, 12 informative microsatellite loci were

isolated and the essential characterization of them were

also studied. An enriched partial genomic library for the

repeat motif (GT)n was constructed essentially following

the fast isolation by AFLP of sequences containing repeats

(FIASCO) method (Zane et al. 2002) with slight modifi-

cations (Zhu et al. 2005). Enriched fragments ranging from

400 to 1,000 bp were inserted into pMD18-T vector (Ta-

KaRa, Dalian) and transformed into Escherichia coli DH5acompetent cells. Positive clones were amplified using M13

universal primers and visualized by agarose gel

electrophoresis.

One hundred clones with different length of insert

fragments were sequenced, 87 of them contained simple

sequence repeats. Subsequently, 16 pairs primer were

designed using the Primer Premier V. 5.0 from the

sequences containing five or more repeats. All of the 16

pairs of primers were tested using 48 A. nigrocauda indi-

viduals from Wu Lake in Wuhan, Hubei province of China.

A total of 25 ll reaction volume contained 50–100 ng

genomic DNA, 5 lM forward primer (50 modified with

FAM, HEX or ROX), 5 lM reverse primer, 10 mM

dNTPs, 109 Taq buffer (15 mM MgCL2) and 0.5 unit Taq

polymerase (Takara). PCR amplifications were carried out

with the following profile: 95 �C for 5 min; 35 cycles

comprising 94 �C for 30 s, the optimized annealing

Y. Sun � G. Wang � D. Zhu � J. Chen � P. Li � Q. Li

Wuhan Aquaculture Science Research Institute, Wuhan 430207,

China

Y. Sun

Wuhan Academy of Agricultural Science Technology,

Wuhan 430071, China

Y. Sun � G. Wang � D. Zhu � J. Chen � P. Li � Q. Li (&)

Wuhan Xianfeng Aquaculture Technology Co. Ltd,

Wuhan 430207, China

e-mail: xfsckj@163.com

123

Conservation Genet Resour

DOI 10.1007/s12686-014-0241-5

temperature (Table 1) for 30 s and 72 �C for 30 s; a final

extension of 72 �C for 7 min. The PCR products were

genotyped on an ABI 3730 DNA sequencer (ABI) and

analyzed using Genescan 3.7 (ABI).

The number of alleles (Na), observed heterozygosity

(HO), expected heterozygosity (HE), exact test of Hardy–

Weinberg Equilibrium (HWE) and linkage disequilibrium

were analyzed using ARLEQUIN version 3.0 (Excoffier

et al. 2005).

Among 16 microsatellite markers (see Appendix), 12 of

them produced polymorphic DNA products (Table 1). The

number of alleles per locus ranged from 4 to 14 with an

average of 9.4. The expected and observed heterozygosities

ranged from 0.185 to 0.907 and from 0.083 to 0.952.

Among these polymorphic microsatellite loci, four loci

(HWB01, HWB04, HWB12 and HWB16) showed signifi-

cant deviations (P \ 0.05) from HWE after sequential

Bonferroni correction. No evidence for linkage

disequilibrium among loci at a 5 % significance level.

These polymorphic microsatellite markers may be used as

powerful tools for investigating genetic diversity and

genetic structure of A. nigrocauda.

Acknowledgments This research is funded by Wuhan Chenguang

Project for Youth Scholar (No. 201372304010832).

Appendix: Nucleotide sequences of 16 microsatellites

for Ancherythoculter nigrocauda

[HWB01

TCAGTGAAAGTGTTGATCCGTGCTGCAAAACTA

TATTGCAAGAGAATGGCATTGAAGTGACGGAGAA

ACCGCAGATGACTAAAGAAGAGTTGATTGCAGAG

ATTCAGGTAAGTGATTCTTCTGACTCAAGTTCTGC

ACCCGAATATCAAGTGCCCTGATTTGAATCGCCGC

CAAGCTAATGGCGTCCTCCAATGCAGCAACAGTA

Table 1 Isolation and characterization of polymorphic microsatellite loci from the Ancherythoculter nigrocauda

Locus Primer sequence (50–30) Repeat

motif

Ta

(�C)

NA Size

range (bp)

HE HO GenBank

Accession no

HWB01 F: GATGTGCTAAAGTTTGCGTG (TG)5 55 4 174–180 0.684 0.200a KJ755528

R: AGAATACTGTACTGGGGGGA

HWB02 F: GTGCTTTGTCATTTCTTTAT (GA)6 58 6 274–312 0.185 0.083 KJ755529

R: ACTTCCTGTTGGGTCTTT

HWB03 F: GCGAAACAGGTTGTAATAA (TC)13 58 10 178–204 0.887 0.735 KJ755530

R: TCTGAACTCTGAGCCGTC

HWB04 F: TGTGTGTATCTGTGTCCCC (TC)5 55 14 125–345 0.907 0.820a KJ755531

R: ATCGTCATCCACTCGTTC

HWB05 F: TCCAGAAGCGTCTCCATCCT (GT)5 55 11 219–279 0.511 0.444 KJ755532

R: CGAAACCCTCACCAGCACC

HWB06 F: GTAAGTGGTGATTGATAGTGG (GT)20 55 14 187–271 0.749 0.851 KJ755533

R: TTTTACTGGGATGATACAAGA

HWB07 F: AGGAGGAGAGTGGAGAAATGGG (GT)7 52 12 191–237 0.827 0.875 KJ755534

R: GGCTTGGAGAGGGGGAAAA

HWB08 F: AGGTGTTTATCTGCTCTTCC (AG)6 52 6 256–278 0.372 0.244 KJ755535

R: TCTCTGTCACTCGTATTTAGG

HWB10 F: TGTTTATTTGGTTAGGTGCGAGT (GT)6N(TG)8 55 8 131–153 0.595 0.659 KJ755537

R: GGCTGTAGGTGGAAGAGTGAGAC

HWB11 F: TCAGCAGGACATCAGGTAAGT (CA)6 50 11 141–207 0.511 0.383 KJ755538

R: CATTGTCTCTGTGCGTTTCTA

HWB12 F: AACAACATAAACAAGCGGTGA (GT)8CT(GT)3 58 5 328–336 0.669 0.869a KJ755539

R: GAAGAGAGACAGCAGGTGTAGAC

HWB16 F: TTATGAAGGTGTCGTAAATGCCAG (GT)15 58 12 220–286 0.864 0.952a KJ755543

R: CCAGATGTATTCTCTCCCCAGGT

Ta, Annealing temperatures; NA, Number of alleles; HE, Expected heterozygosity; HO, Observed heterozygositya Significant deviation from the Hardy–Weinberg Equilibrium after Bonferroni correction

Conservation Genet Resour

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AAAATGATAAACTATTAGCATGTGGATGTGTGCC

AGTTGGATGTGCTAAAGTTTGCGTGTGACTGTGTG

TGTGTCACCCCTCCTCCTCCTCTTCAAGAGTCTGAT

GCAGCAGCAGCCTGTATCTGCACCATATGAATCAT

TTCAATCGATTCACAACGCATGGTTTTATTAGACC

TCAAACAGTAAATATGATTTTTTCCCCCCAGTACA

GTATTCT

[HWB02

GACGGTCAGTTTGTCTGGATTGCTGTCTGTGGC

CAAGTGTTTTTGTATAAACAATACTGATGTTCTCA

CATACTTTCCAGAAAGGCGTTTTGGAACCACATAC

AGTCAACTGTGATTGTGAGATCTGCCAAGAACTG

GATCAATACCTAGTAAGACATAATCGCTTGCAATT

ACACAGTCATGTCCAAACATCAGCATGACCATCG

AAATTCATCACAAAAATCTGAAATTATTTCTAAAA

TGTGCTTTGTCATTTCTTTATGAACTCTTGTCTCAG

CAGAATGATGACGTGTGTTTCGTGTGTGAGTCTGA

GGGAGATCTTGTGTGTTGTGACGAGTGTCCACGAGC

TTTTCATTCACACTGTCATCTACCAGCTGCAGACG

GAGACTCACTTGGGTGAGAGAGAGAGAAATCATG

CTGGTTTAGACAAACTAAACAGTTGACTTTGTGTA

ATATTGTGTGTTTTGTCTGTACAGAAGCCAGTGGA

GCTGCACATTCTGTGTGATGAAGAATATGAAAGA

TTCCAGTCAAAAGACCCAACAGGAAGTTTTGAGC

AGTCCAGTCTCTCAGTACACACTGGTTTGTGCAAA

ACTTTTTGTCCTTACAATGGAAGTCAATGGGCTCC

TAAATAACATTTATTTTACCATATAGATGAAAAAT

TTCAACTTTTTGTGTGAACTAACACTTACAGGTAT

GTTTGTTTCTTTTTTCTCTTTCAGCACTGCCAGTGT

TTGCTGTTACACTTGCTACATGAGAACATGACTGA

CCCCTGCACCAATGTAAGTATCATCTTTAGCATCT

TCATCTGTCTGTT

[HWB03

GGGAGTCTGAATACTTTCCGTATCCACTGTATT

TATGCATATCTCCCTGCTGAAGATCCTGAAGCCAC

GTAGACCTAAATATGAGGACACGGTTCACACACG

TACACACACAGACAGATGTGATGGATATAGCAAG

ATGGCTGTATCAGCGAAACAGGTTGTAATAACAT

CTCACACTGTACCTGATATAGATCAAAATGTCCCT

GTGTATCTCTCTCTCTCTCTCTCTCTCTCTCTCTAT

CTTTGTCTCTGATTTCAGCTGGGTGAGCTGGATGG

GGTGGAGAAACCACAGCCGCACTGGTTTCACACG

CTGCAGGTGCGACGGCTCAGAGTTCAGAGGGGCC

GCAGTAACAGCGATCCTATGGGAGGAAAGAGCTT

CCAGCAAGAGTTCCAGTGGGTGAGGGACACACTA

ATACAACTCAGACATACACCAATACACAACATGT

GATT

[HWB04

GAATTACTTCCAATCCTAGAGAATGTCTGAGAA

AGGTAAGAACTTTAGGAAGTGTGCTGATCCGTGT

CCGAGATTCTTGACACCGGAATACACGCACATCCT

ATGTGTTCATATGCCTTACAAACGTGCAAGAGAGT

GTGTGTATCTGTGTCCCCATGTTTGACACTTGATA

TACACATTTTTCTAGGTGTTTTCTGTCTGGAGAGG

AGCGCGCATAGACGGTGTTTGAGGGCGCTGTCTG

TGTGTTTGTTTGTTGTTTACTGTGAGCGTCACCCTA

TCGGGATGCTCCGTTCTCGTTTGACACTCTTCTCA

AGGGAAGGGGTGTCTACATCTGTGCCTGGTGATTC

TGGCCGGGTTATGCCGAGGCGAAACCGTGCCTAC

AATCATAGGGCTCGCAGATGAGCTTGTTGGGAGT

TGTGAGGGAGTTGTATTCTCTCTCTCAACTTCCTT

GGGGTTGGCGAGAACGAGTGGATGACGATGACGT

TCGTTTTTGACGTCATCGCGTCTGGTGAGCGCTCC

TCTGGCTTGTGCATGCAAGCCGCTGTGTGTTTGGG

ACGCGCCTCTCGGCGGGCTGCAGCTGCCGTGAAA

GCACGTTAACTCTTGACAAGGCACACCTGTGAATT

GAAAACCATTCCAAGTGACCACCTCGTGAATCTG

ATGAGAGAATGCCACGCTGTCATCAAAGCTAAAT

GTAGCTACTTTGGACAATCTAAAATATAAAACAC

ATCCTGGTTGTT

[HWB05

TAAAGGGGTACAGATAGGTAACGGCAGCGAGC

AGATACTGTTCCAGAAGCGTCTCCATCCTTCAGTC

ACCTCGGTCTACACTTTCCGCCCACAGAGACTCGC

ACCAGCGTGTCATTTTCAGCACACTGGCAGAGTG

AGAAAGACTGTGTGTCTGTGTGCACATATGTGTGT

GAGCCTGTCATTTGGACCCCATGGGACCCAGTAA

GTTTACAGCAAGTGATTTACTCAAACTCAGTGCAG

CATGTTGTTTGCCAGATGGGCACCAAACTGGCAA

GTGTGTGTGTACATATCAAGGCTGGTGCTGGTGAG

GGTTTCGTGCGGTGGGTGAACGTTCTGGTCTTGCT

GCTCAGACTGCCAGCCTCTTGGGGTGGGCAGATG

AGAGGGATCCATAAGGCAGAACTCCTCCAAAATG

TTGTTTTCTGCATCAGGTCTTTTCCTGTCCTGTCAC

AGATACAGCTTTCTCCCCCAGTCTCTGAATAAAAC

ACAAGTACACACTTTTGATAATCACAGCATAGT

[HWB06

TAAAATTAGTGTAAGTGGTGATTGATAGTGGAT

AATAATATATGTATAATGTACTGACTTATGTAAAA

TGTACTCACATTTTAGACACAATATTGTTTCTTTAC

TAAATGATTCAGCATTTTGAATGAATCAGCTGAGT

CAAAGATTCAATGACCCATTCATGTGTGTGTGTGT

GTGTGTGTGTGTGTGTGTGTGTGTGTGTATCTCTG

CGTGTGTGTAGTGAGTGTGTGTGTCTGTTTTGTAC

TCTTGTATCATCCCAGTAAAATGCATGTTTGAGAT

GTTTTAACTAAAAGCAACTTGCACTGACATATCT

[HWB07

TAAATACACACTCAAACACCTATTCACAATGGA

CAATGGTTTCGTCAGCTTCCGGTTGCGCTGCGCCA

GCGCGACCAGGCCATGACGTCATAAGCAGCTGCC

GCTCGATCGTTCGGTTTGGCCAGCGCAGCGTTTCC

CACCAGAAGACTGACTACGCCCACATCGACAAAC

AGCACAGAGTCGAAACAAGATGACAAGTGCAGTG

TAGGTGTATGAAATGACCGGGAAATGGATATGGA

ATAGATGACAGGAGGAGAGTGGAGAAATGGGAG

AATGTCCATGTGCTATCGTGATCGTGTGTGCACCC

Conservation Genet Resour

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GCTACTACGATAGCGGCAACGGCTGGGAGAAACG

ATACAGAGAATGAATGAGGTAATTAGATGGGTGT

GTGTGTGTGTGAGCGCGTGCGCGTGCATGCCAGT

GCTAATCTAAAATGATGCTTCCCCAGTGGTGGCAG

CTAATGTGACTTTATCACATCATTTTCCCCCTCTCC

AAGCCACGTACTGGCCGGAAACCTGGACAGGTAG

TTGGGTACCACATTGGTGCCGTATCT

[HWB08

GATACATGGTTGTGGAGTGCAATGCTGTACCAC

AACTCCTCTACCATTGAACGGCATCTTCACACACG

CTCACGTTGACAAACTCACACAAACACACATCTG

CTGATGTGAATCATTAGAGCCAGTGCTGAGGCTG

GTCTTTGAGGGTGCTCTGCTCAAGCTAGCTGGAAA

GCCTAATGAGCTAGCAAATAACTGCAGAGACCAA

GGAAACACACAAAAGCTGCACACACATTCGTGCT

CGCGGCCGTGCGTGGTGGTCTGCGGGCCTCATGC

GGCGTCTGCGAGAATCAGAAGAGACAGAGGTGTT

TATCTGCTCTTCCCTCTGCTGCACATGGCCAAATC

AAACGAGAGGAGAAAGTTTCCTATTCTATAAAAG

CGCTTTAGATCAATTGCGAGCATCCAGCCTACATC

AAACCTGTTTTACACAGAGAGAGAGAGAAAGCAA

GAAAGAGGAGGAGGGAACAAAAAGTACAGGAGG

AGACTACAATTTGGCAGCACGAGAAATGCTGCTT

TCAATCTGCCATTGGGCGGCACAGGCCTAAATAC

GAGTGACAGAGAAATAAATGCAAAGTTCATCCCA

ACAGAGCAATGCTGAAAAGTATTTGATCAAATGG

AATTGAGGGAGAAGATAGGAGGTTTTACTTTTTAT

TGTGTTTGTTTGTTTCTTTTCAATGAGTAGATATCA

GCACTAAATGTTATAATATT

[HWB09

AAAGTGTTACCAATATTGGTTCAGTACTGATAC

TAATACTTCTAATTAGGGGCAGTTGTGGCCCAATG

GTTTTGTAACCCAAAGGTCGCAGGTTCGATTCTCA

ACACCAGCAGGAAACGACCTTGAGCAAGGCACCG

AGCCCCTAATCGCTCCGCGGGCGCTGCAATGAAT

GGCCTGCCTCTGGGTGTGTGTGTCACGGTTTGCAG

TGTGTGTGTGTGTGTGTGTGCATGTGCAGATTGTG

CACTAACTTGGATGGGTTAATACATTATGAATAA

TCATACATTGATTACTTATGTAGTAGTTTGTGTGT

GTGTGTGTGTGTGTGTGTGCAACATTGCTTGGCTC

TTTCTGTAGGTTTGCTGTTTGTCTGTGTGTTATGTA

CAGGGTCATGCACAACATTTGCCT

[HWB10

ACTGAGCCATATTGAGCAGAAGAGACTTCTTAC

GGTAGTGAAATATAATGCAGGCTTTAGATAGATG

TTATATAAGGAAACTGGAAGTGTGTGTGTGTTTA

TGTGAGCATTTGTTGCAATTGTGTTGTGTTGATAT

ATGTCAAAACGTCGGTATGTGTTTGAAGCAAGTGT

TTATTTGGTTAGGTGCGAGTTAGCCAACATGTGTG

TGTTTCGGATGTGTTTCTGAGCTCTTGTGTGTGTG

TGTGTGTATAAAGTTGATGAGAGAGACCTTTCCC

CCCTCGCCTCAGTCTCACTCTTCCACCTACAGCCG

TGTTTTTTCAGGCTTCGGACCAGAAGTAAACACTT

TGTCCCGGCTTTTGTTGTACGTGTGTGTGTATGCG

TTTGACTCTTGGCGCTCGGTGGTGAAATAGGAAA

GCCCTTCCTTCACAT

[HWB11

CAGGATGTTTTTTGAACACAACGTTGGGTCAGC

AGGACATCAGGTAAGTAAACTGTGCAATGATACT

TTAGCAGAGAGAACGAGGATAATGATATCAGTCT

TTATCCAGACTGATATCATTATCATATCTGAATCA

TTATCAGGACTAGGATATCGTGGATGACAGATGA

TTGGAGGAAGCACACACACACACTTGCAGTTTGG

TAGAAACGCACAGAGACAATGGAGGAATAGGATG

AGGAGAACACACTGTAGAATCACAAGAGATAAGT

ACAGAATCGACAATGGAGAACTTGCTTGGAGAAT

TCGTAGGAGAAACTGGAAATGAGTCCTTTACGTG

TGAACGAAACCGGACACTGGAGTGAGGTAAGGTG

TGTGCTTTTGAAGCCAGCGTGATGACTGTGCTGAT

GAGTGGCATGTGCTTGTGATCAGTACTCTGGTGAG

GAAGTGCGCTGTGATTGGCTGATGTGGGAGCCTG

GCTGATCTGTGACACCCCTGTCGAGCGCATTGCTG

GAAGATGTGTGTTGAACATCCACTTTGCATT

[HWB12

AAATAGGCCTAACTGTTTTCTCGGGATCAAAGC

TTGCACAAATCGAACAACATAAACAAGCGGTGAC

ACCCGACTTGTGAGTAACTGATCTCTTGCACTCCC

ATCTGTGTCTGCATGAGGGTCACTCCCATGGCAGA

CATCTCTCGCCTTTTAGAGTTGCTCCCTGTGACCT

GTAACCTGCAAATTATCAATCCCTTTTCCCACTCT

TTATCAAAGCCTGCTTGCTCTCATCTTAGTGGCAA

AAGTTGCAAAGTTGTGTCCTGCTGAGCGGTGTTCA

GGTTGTGAGGTAGCTGTGGCAGAAAATGGTATCA

GGTGTGTGTGTGTGTGTCTGTGTGTATGGAGGTGT

CTGTGGAGCAGTCTACACCTGCTGTCTCTCTTCAG

TGCCCATTGGGGCTGGATTCCCAGAATGCCTTTCA

GGCAGCTGTTGACAGCCAGCACTGTGCCCTGGCC

CCCCAAACGGCTCTGGGGATCAGCTGCT

[HWB13

AAAATATAGCAATACCAAAATACAAACAATGT

AGAATAGCTTGAATACAGCGTGCGTCTCCCTCAG

ACTGTAAATAAAGCTCAGGCTCACTACATAACAC

GTCATCAGCGTCACACTCCGGAGCAGAAGGGGGC

GGTAATGCACCAATAAGCTGGATGCCAACCGCCG

TAAAACAGGAAAGAAGAAGAAGAAGAAGCGTGA

ACGCGAATTGACAACAGACCCGGAAGAGAAGAC

AATGCTGAATAAAATCGTAGTTTTTGTTATTTTTG

GACCAAAATGTATTTTCGATGCTTCAACAAATTCT

AACTAACCCACTGATGTCACATGGACTACTTTGAT

GATGTTTGTATTACCTTTCTGGACATGGACAGTAT

ACCGTACATACATTTTCAATGGAGGGACAGAAAG

CTCTCGGACTAAATCTAAAATATCT

[HWB14

ATGCCCTTCCCTCGCTAACTTCCCTCGGTCTCGT

TGACTCGGATGTACGTCATTGCTTACGTTGCACGA

GTGCCCACTTCTGGCGGAACCTTTGCAATGGGCTG

Conservation Genet Resour

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AACTGGAACGTCCTAAGCCCTTGATCACTTGGAAT

CCGCAATGGAGCTGATTTATTATTTATTTTTTCCTC

TTACAAATTTGTTTAATGTCAACTACTTATGTGTG

CGCGTCTACGTGTATGGCTGCATGTATAGTGTATG

TGTGTGTGTGTGTGTTGAGTGTGATCAGTGGAAA

GTGAATGAATAAATAATAGGGGCGGCAAACGGAG

GCAGCTGCTCTCTGTTGGCTTCTGAAGTGGCGCGG

GGTGAGGTGAGACAGGCGGCGGTGGAGAGTGCCG

TTTATTCACAACCAGGAGTCTGTTTACCGGAACAA

ACCAGTCGCCACAGCTACT

[HWB15

AACGAACCCTGGTGCAGTTGCTCGGTTAGTGCG

GTTCATTTGAACATATGTGAACGCTGCCATCCGAA

CCCTGGTGCACACCAAACAAACTCTGGTGATATAT

GAACGCAACACGGACCAAAGACATGTAACCGAAC

CAAAAACAGGAAGACGAGACCCTAAAAAGGAGA

GAATCCTCACGCATGTCGGTTTTTCTTGTCATAGG

CGCGAGTTTGCTCATCATCACAGGCATCAGACGC

GCGTCTCCACGCAGCAGATCGTTTGTGTGTGTGAC

GGATGGATTCCCGCCGGTGTTTTGACTACTTTACA

CATTTTATAAGCTCTTCACGAGTTCCCAACTGGCC

AAAATACCATCACATGCAGGCTACGCACCGCTAC

ACACACACAACGAGCACATTTACCTCAGCAAACA

GCATTGTTTTGGATGTTCGGTAAGTTCCGTCTCAA

AATAGGCAATACATCATAAAATCCGACCAATCAC

GTTGTGAATGTATCCCTATGCCT

[HWB16

CAAAGCTGGCGGACAGCAGTGCCCCGATTCTGT

TGGAGGTCCCTCTCTCCGGACCACTAGCTCATCCA

GGCAGTAAAAGAAGGCTGACAGGGATACAGATGG

TCTAGTATACATAGTATAAACAACAACTGGCTTAT

GAAGGTGTCGTAAATGCCAGGAGCAGCTGGCTAC

CAGAGCACAAAGTGGAACGAAAAGAAAAAGAGA

TATGATAAGATAGAGAGAAAGAACAGGGGAAATT

GTTTACATGTGTGCTTGGGTGTGGGCACATATGGG

TGTGTAGGGTTTGTGTGGGTGTGCAGGGTGTGTGT

GTGTGTGTGTGTGTGTGTGTGTTTTCACCCAGGGA

CCTGGGGAGAGAATACATCTGGTAGGACAGCTGT

TAGCGGTGTACAAGAGTATACTGATGGTGGTGAG

GAATGATAAATCACAGACTTTGCAGCATTGCAGA

CAGTGCACCAACATACCTGTGAGTGCAGGTACTC

AGGCACTGTCAGAAATGCTAAATAATCAATGACC

CTATCCAAACAACACAGCTACAACTGTCTAAATGT

GTCCGACTTGACAGTGACAACTGCGTAAAGCAAA

TTTGTCTCGAAATGAACACATT

Note: The repeat sequences for each microsatellite are

underlined.

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