MICROSATELLITE LETTERS

Development and characterization of 20 polymorphicmicrosatellite loci for the Lhasa schizothoracin Schizothoraxwaltoni

Xiang-Zhao Guo • Gui-Rong Zhang •

Kai-Jian Wei • Wei Ji • Rui-Bin Yang •

Jonathan P. A. Gardner • Qi-Wei Wei

Received: 21 November 2013 / Accepted: 25 November 2013

� Springer Science+Business Media Dordrecht 2013

Abstract Schizothorax waltoni is a valued economic fish

endemic to Tibet, China. However, there is little informa-

tion on genetic population structure and genetic diversity

for this species. Twenty polymorphic microsatellite loci

were isolated and characterized in 42 individuals collected

from the Yarlung Tsangpo River in Tibet. The number of

alleles per locus ranged from 9 to 28 with an average of

18.45. The expected heterozygosity and Shannon-Wiener

diversity index ranged from 0.716 to 0.942 and from 1.675

to 3.038, respectively. These microsatellite loci are cur-

rently being used to evaluate the genetic diversity and

genetic population structure to contribute to conservation

and management of S. waltoni.

Keywords Schizothorax waltoni � Microsatellites �Genetic diversity � Schizothoracinae � Tibet

The Lhasa schizothoracin (Schizothorax waltoni Regan,

1905), (Cyprinidae: Schizothoracinae) is an endemic tet-

raploid fish that is mainly distributed in the middle reaches

of the Yarlung Tsangpo River in Tibet, China (Fisheries

Bureau of Tibet Autonomous Region 1995; Wu et al.

1999). It is a valued economic fish in this region and

characterized by low growth rate and late sexual maturity

as adaptation to the high altitude and cold weather. For the

past few decades, stocks of S. waltoni have been declining

rapidly due to overexploitation and biological invasion of

exotic fishes. The main captured fishes are now 200–300 g

per individual, compared with more than 500 g in the

1970s (Fisheries Bureau of Tibet Autonomous Region

1995). Despite its ecological and economic importance,

little information is known about genetic population

structure and genetic variability among populations.

Microsatellites are highly informative molecular markers

and widely used in population genetic studies. However,

microsatellites have not been developed in S. waltoni

except for limited information on its chromosome, phy-

logeny, mitogenome and biology. In this study, twenty

polymorphic microsatellite loci were first isolated and

characterized in S. waltoni to study genetic diversity and

genetic population structure among populations.

Genomic DNA was extracted from pectoral fin tissue

using phenol–chloroform method. Microsatellite loci were

developed using the fast isolation by AFLP of sequences

containing repeats (FIASCO) method (Zane et al. 2002)

with minor modification. The methods for constructing

(AGAT)n-microsatellite enriched libraries and screening

positive clones followed Xu et al. (2013). A total of 150

Xiang-Zhao Guo and Gui-Rong Zhang have contributed equally to

this work.

X.-Z. Guo � G.-R. Zhang � K.-J. Wei (&) � W. Ji �R.-B. Yang � J. P. A. Gardner

Key Laboratory of Freshwater Animal Breeding, Ministry of

Agriculture, College of Fisheries, Huazhong Agricultural

University, Wuhan 430070, People’s Republic of China

e-mail: kjwei@mail.hzau.edu.cn

X.-Z. Guo � G.-R. Zhang � K.-J. Wei � W. Ji � R.-B. Yang �J. P. A. Gardner

Freshwater Aquaculture Collaborative Innovation Center of

Hubei Province, Wuhan 430070, People’s Republic of China

J. P. A. Gardner

School of Biological Sciences, Victoria University of

Wellington, P O Box 600, Wellington 6140, New Zealand

Q.-W. Wei

Key Laboratory of Freshwater Biodiversity Conservation,

Ministry of Agriculture, Yangtze River Fisheries Research

Institute, Chinese Academy of Fishery Sciences, Wuhan 430223,

People’s Republic of China

123

Conservation Genet Resour

DOI 10.1007/s12686-013-0106-3

positive clones were sequenced using an ABI PRISM 3730

sequencer. Thirty-six primer pairs were designed using

Primer Premier 5.0.

Each primer pair was detected for reliable amplification

using 42 individuals of S. waltoni collected from the

Xaitongmoin section of the Yarlung Tsangpo River in

Tibet. PCR amplifications were carried out in a 10 ll

volume containing 19 Taq reaction buffer (Fermentas),

0.5 lM of each primer, 1.5 mM of MgCl2, 0.5 U of Taq

DNA polymerase (Fermentas), 200 lM of each dNTP, and

Table 1 Characterization of 20 microsatellite loci for S. waltoni

Locus Accession

no.

Repeat motif Primer sequence (50–30) Ta

(�C)

Size range

(bp)

NA HE H0

Schw01 KF857555 (ATCT)6…(TCTG)6…(TCTA)6 F: ATTGACCGAATGTTCAGATG

R: CTAGCATGTCTTAGGCTGTTG

56 203–287 21 0.928 2.824

Schw02 KF857556 (ATCT)10 F: CTTGTTCACTGTTTGCCCTTGT

R: GAATCTTGGGATGGCTTGGT

56 146–191 15 0.872 2.235

Schw03 KF857557 (TATC)19 F: GCGTGATCTTTCAGGCATAT

R: GTTACGGCGACTCAGAAGG

56 122–194 23 0.935 2.871

Schw04 KF857558 (ATCT)14…(TCCA)4 F: TATTTCCCCCATCAACACT

R: GCAACATTTATCAAGACCAAC

56 164–199 12 0.849 2.123

Schw05 KF857559 (TCTA)12 F: GGCTCTGGACGCTTTGAC

R: GGTTGCCGCTTCCTTATT

56 170–285 25 0.936 2.904

Schw06 KF857560 (ATCT)13 F: CCGTTGTTGGTTCTTTCG

R: GATTTGGCTTGATGTCTGC

56 150–202 13 0.858 2.166

Schw07 KF857561 (ATAG)5…(AGAT)11 F: GCTTTCCTTACTTTTACGGTCT

R: GGGAGCCCTGTTTCTTGAT

56 253–381 28 0.942 3.038

Schw08 KF857562 (AC)13 F: GCTGAAACATTCGGTCTG

R: TAGTCTGAAAAGTAAACGGC

56 90–118 11 0.850 2.072

Schw09 KF857563 (CTAT)15 F: TTACCAGATGGCAGCAGAG

R: CACGATGTGTGACAATAAAGAG

58 86–149 18 0.900 2.533

Schw10 KF857564 (ATCT)8 F: TCACACACACCTGCTCAAG

R: GACGGATGGATAAATGGA

56 158–247 22 0.931 2.823

Schw11 KF857565 (ATCT)12 F: ATCTGCTTACGCCCCAT

R: TTGTCTATTGCTGCTCATCA

58 115–195 17 0.844 2.186

Schw12 KF857566 (CTAT)15…(TA)4 F: ATTAGTCCTTGACATCTGC

R: CTTCGCTACTTGACACCT

56 183–310 25 0.937 2.931

Schw13 KF857567 (CTAT)14…(ATCT)10…(ATCT)6 F: GATGGCAGCAGAGTGAATA

R: AAGACAGTCCAGAACTTTGG

48 317–391 13 0.845 2.110

Schw14 KF857568 (ATCT)25 F: ACACACACAAGGACAGAATC

R: GATGAGCCTGAAGTTTGAA

56 193–280 20 0.931 2.806

Schw15 KF857569 (GTCT)4(TCTA)4 F: GGCAAAATCACGGCGACT

R: GACTTGGACTTCTCACCCCTTC

56 136–195 9 0.716 1.675

Schw16 KF857570 (TCTA)15 F: GTAACCTCCTGTCTGCTG

R: GCTGGACTATGACTCACTGT

56 142–211 18 0.910 2.642

Schw17 KF857571 (ATCT)8 F: CATTTAGGTTTGAGAGGAC

R: GATAGAACGATAGACAGACTG

56 92–178 23 0.939 2.941

Schw18 KF857572 (TCTA)14 F: CTGTATGTTTGTCCGTCC

R: CTGAAAGAGTTGAATAGAGG

56 104–176 17 0.906 2.514

Schw19 KF857573 (TATC)11 F: TCCGTCCATAAGTAGCAAGA

R: GGAGGGAGGCAAGGTAAT

56 142–186 14 0.896 2.409

Schw20 KF857574 (CTAT)15 F: TTGGGAGGAAATAAGGAG

R: ACAGTTTTTATGGACAGTGC

56 313–132 25 0.924 2.832

Ta annealing temperature, NA number of alleles, HE expected heterozygosity, H0 Shannon–Wiener diversity index

Conservation Genet Resour

123

30 ng of genomic DNA. PCR amplification conditions

were as follows: an initial denaturation at 94 8C for 4 min,

followed by 30 cycles of 94 8C for 30 s, locus-specific

annealing temperature (see Table 1) for 30 s, 72 �C for

45 s, and a final extension at 72 �C for 10 min. PCR pro-

ducts were separated on 8 % non-denaturing polyacryl-

amide gel and visualized by silver staining. A 50 bp DNA

ladder (Takara) was used as a standard to score allele size.

The number of alleles, expected heterozygosity and

Shannon-Wiener diversity index for each locus were cal-

culated using ATETRA 1.2 (Van Puyvelde et al. 2010).

Twenty out of 36 primer pairs designed were tested to be

polymorphic in 42 individuals and produced expected PCR

products. Up to four alleles at a single locus in S. waltoni

individuals were detected in this study, indicating that this

species is tetraploid. The number of alleles per locus ran-

ged from 9 to 28 with an average of 18.45 (Table 1). The

expected heterozygosity ranged from 0.716 to 0.942

(average 0.892). The Shannon–Wiener diversity index

ranged from 1.675 to 3.038 (average 2.532).

These newly developed microsatellite markers are cur-

rently being used for assessing the genetic diversity and

genetic population structure of S. waltoni, and will help to

effectively conserve this species.

Acknowledgments We thank Jian-Hui Qin, Bin Huo and Hui-Juan

Zhang for help with sample collection. This research was supported

by the Special Fund for Agro-scientific Research in the Public Interest

(Grant No. 201203086-13), the Scientific Research Foundation for the

Returned Overseas Chinese Scholars, State Education Ministry, and

the Open Project Program of Key Lab of Freshwater Biodiversity

Conservation, Ministry of Agriculture (Grant No. LFBC0803).

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