TECHNICAL NOTE

Isolation and characterization of polymorphic microsatellite locifrom the endangered Tarim schizothoracin (Schizothoraxbiddulphi Gunther)

Xiaoling Gong • Zhongkai Cui • Chenghui Wang

Received: 19 March 2012 / Accepted: 2 April 2012 / Published online: 11 April 2012

� Springer Science+Business Media B.V. 2012

Abstract Tarim schizothoracin (Schizothorax biddulphi

Gunther) is an extremely endangered freshwater fish in

China. In this paper, 15 polymorphic microsatellite loci

were characterized in this species. The number of alleles

ranged from 4 to 14, the observed heterozygosities (HO)

were from 0.7609 to 1.0000, and the expected heterozy-

gosities (HE) ranged from 0.7273 to 0.9194. The poly-

morphism information content was from 0.6684 to 0.9020.

Only two loci deviated significantly from the Hardy–

Weinberg equilibrium, and none of the loci combinations

showed significant linkage disequilibrium. These micro-

satellite loci are useful in investigating the genetic vari-

ability in Tarim schizothoracin.

Keywords Schizothorax biddulphi � Microsatellite �Genetic variation

Tarim schizothoracin (Schizothorax biddulphi Gunther), an

extremely endangered freshwater species, is distributed

only in the Tarim River drainage of Xinjiang Autonomous

Region (province) in China. In 1967, more than 1,200 tons

of Tarim schizothoracin were harvested in the Bosten Lake

(Zhang et al. 2007), the biggest lake in the Tarim River

drainage. However, the population declined sharply since

the end of 1960s because of combined forces of overfish-

ing, exotic species invasion, and environmental changes. In

the 1990s, this species has nearly become extinct in the

Bosten Lake (Zhang et al. 2007). In 1998, Tarim schizo-

thoracin was listed in the ‘‘China Red Data Book of

Endangered Animals–Fish Volume’’ (Yue and Chen 1998)

and in the Xinjiang Provincial Second-Class Protected

Animals in 2004. At present, only a few Tarim schizo-

thoracin are found in the Aksu River, Weigan River,

Yarkand River, and Hotan River (Yang et al. 2011). Thus,

conducting genetic conservation and implementing man-

agement programs for this species are urgently necessary.

However, very little knowledge on this fish is available,

including biology and genetics. Developing molecular

genetic markers is the first step in conducting genetic

diversity investigation and resource conservation strategies

for this endangered species. This paper is the first to report

on the isolation and characteristics of polymorphic micro-

satellite markers in the endangered Tarim schizothoracin.

Forty-seven individuals were collected from the Cherchen

River in 2010 (Qiemo County, Xinjiang Province of China).

A small piece of the caudal fin in each individual was

removed and stored in 95 % ethanol. Genomic DNA was

extracted using Phenol/Chloroform procedure (Sambrook

and Russell 2002). Pooled genomic DNA from three indi-

viduals was digested with the restriction enzyme Sau3AI.

Fragments of 300–1,000 bp were separated, purified, and

then ligated into short adapters (Micr-A: 50-GAT CGT CGA

CGG TAC CGA ATT CT-30, Micr-B: 50-GTC AAG AAT

TCG GTA CCG TCG AC-30). Pre-enrichment polymerase

chain reaction (PCR) was performed in a 50 ll volume

containing 2 ll DNA templates (about 100–150 ng), 25 ll

PCR mix (0.5 lM Taq DNA Polymerase, 0.4 lM dNTPs,

3 lM MgCl2), 0.8 ll primers (0.5 lM), and 22.2 ll distilled

water. PCR conditions consisted of 94 �C for 3 min, then 20

X. Gong � Z. Cui

Key Laboratory of Exploration and Utilization of Aquatic

Genetic Resources Certificated by Ministry of Education,

Shanghai Ocean University, Shanghai 201306, China

C. Wang (&)

Key Laboratory of Freshwater Aquatic Genetic Resources

Certificated by Ministry of Agriculture, Shanghai Ocean

University, Shanghai 201306, China

e-mail: wangch@shou.edu.cn

123

Conservation Genet Resour (2012) 4:795–797

DOI 10.1007/s12686-012-9646-1

cycles at 94 �C for 1 min, 60 �C for 1 min, 72 �C for 2 min,

and finally 72 �C for 10 min (Chen et al. 2009; Hu et al.

2010). Microsatellite-containing fragments were coupled

selectively with a biotin-labeled (CA)15 probe, and then the

hybridized target fragments were captured by streptavidin-

coated magnetic beads (Dynabeads M-280 Streptavidin).

The captured DNA fragments were purified and ligated to

pMD-19T vector, and then transformed into competent

Escherichia coli cells. The positive clones were sequenced

on automated ABI 3700 DNA Sequencer. One hundred forty

clones were sequenced and 91 (65 %) were found containing

microsatellite motifs.

According to adequate flanking regions, forty primers

were designed using an online software web primer (http://

www.yeastgenome.org/cgi-bin/web-primer). The gradient

thermal cycler (Eppendorf, Germany) optimized the PCR

conditions with an annealing step of 53–63 �C to select the

optimum annealing temperature. PCR was performed in a

volume of 10 ll containing 1 ll genomic DNA (0.5 lM),

5 ll PCR mix (the same components as above), 1 ll primers

(0.5 lM each), and 3 ll distilled water. PCR conditions

included an initial denaturation for 5 min at 95 �C, followed

by 30 cycles for 30 s at 94 �C, 30 s at optimal annealing

temperature (Table 1), 30 s at 72 �C, and lastly, 10 min at

72 �C. The PCR products were visualized by the QIAxcel

multicapillary gel electrophoresis system (Qiagen, Ger-

many). Fifteen microsatellite loci displayed high degree of

amplified polymorphism (Table 1), which were deposited in

the GenBank (Accession Nos. JN574731–JN574750).

Population genetics parameters were expressed as allele

number (NA), observed heterozygosity (HO) and expected

heterozygosity (HE). The polymorphism information con-

tent (PIC) was analyzed using PopGen32 (Yeh et al. 1999).

Linkage disequilibrium for all loci was tested using

Table 1 Characteristics of 16 polymorphic microsatellite loci for Tarim schizothoracin

Locus Primer sequences(50–30) Repeat motif NA Ta(�C) Size

range(bp)

PIC HO HE PHWE Genbank

accession

no.

SB-5 F: GCACCAATCACAGCCTTGTTC

R: GCGGTTCAGCAGGGTCATAA

(CA)11N30(CA)15 9 58 178–276 0.8330 0.8864 0.8597 0.4129 JN574732

SB-12 F: TCACCGAGCAGGTTGCTAAG

R: GCTTGGGCTAAATCTGTGCG

(CA)23 7 60 382–445 0.8165 0.9574 0.8467 0.0642 JN574734

SB-13 F: CGAAAGCATTGGCCTGGT

R: GTGCTTCGTCTGCCTTGCAT

(TG)32(AG)23 10 60 328–420 0.8372 1.0000 0.8619 0.0566 JN574735

SB-14 F: TTGCTCGCAGCTATAGTGGC

R: TTACTCTGGGACGGCAGAAC

(GT)24N6(GT)9 12 60 233–280 0.9013 0.8936 0.9183 0.5514 JN574736

SB-17 F: ACACAGACTGGCGGCAAA

R: CAAGGGAGAGAAGTACCGTCCA

(CA)18 13 61 209–260 0.8905 0.9362 0.9085 0.1175 JN574738

SB-18 F: TCTGAATCATTCCAGTGTGACA

R: CTACTTCAGTCTGTGTGCATTG

(TG)34 14 61 238–300 0.9020 0.9556 0.9194 0.0586 JN574739

SB-22 F: GATCAGATGCGTAAGGTCAGG

R: GCTCAGTCCAGTTTCGCATG

(CA)19 4 59 228–275 0.6801 0.9787 0.7369 0.0000 JN574741

SB-23 F: CCCACCAATGTCACTGATGC

R: CAGTACATACCCACCTGTGGA

(TG)27 9 60 123–190 0.7809 0.7609 0.8122 0.0766 JN574742

SB-24 F: GCTTCATAATTCAGCAAGCAA

R: TCCCTGCAGAGTCATTACTCA

(TG)20 6 60 217–290 0.6801 0.8936 0.7335 0.0613 JN574743

SB-27 F: CGCTCCATTAAGGGTCAGGA

R: TGTGTGGATGACAGCTACGG

(AC)21N(CA)6 13 58 201–385 0.8602 0.8936 0.8824 0.0664 JN574745

SB-29 F: CTGGCCTTGATTTAGTGCCTG

R: ACGCACTGGCACAGATGTTC

(TG)33 4 60 300–340 0.6684 1.0000 0.7273 0.0000 JN574746

SB-33 F: TGTTTGACATCATGTGACGCC

R: AGCCTGACCTCCAAGGTTAAA

(GT)32 11 62 170–210 0.8888 0.9565 0.9078 0.0538 JN574748

SB-34 F: TCAAGGAAACATGGGGGTAA

R: TCCGTATCTGTCCGTCTCACA

(TG)17N(GT)9 12 61 238–355 0.8535 0.7826 0.8765 0.1252 JN574749

SB-35 F: ATAAGGCCGCAGCGAGAA

R: AAACCGGCGCGATATACG

(AC)19 12 59 241–465 0.8516 0.9778 0.8747 0.0578 JN574750

SB-40 F: CCACTCACCGAAAAACACG

R: CCCTTTAAACCTCCAGCTGC

(AC)5N18(AC)13 7 61 242–309 0.8048 0.8936 0.8362 0.1440 JN574731

NA number of alleles, Ta annealing temperature, PIC polymorphic information content, HO observed heterozygosity, HE expected heterozygosity, PHWE P value for

Hardy–Weinberg equilibrium test

796 Conservation Genet Resour (2012) 4:795–797

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FSTAT2.9.4 (Goudet 1995). The deviation from Hardy–

Weinberg equilibrium (HWE) in each locus was estimated

using GENEPOP 3.4 (Raymond and Rousset 1995), and

corrected by a sequential Bonferroni correction (Rice

1989).

The number of alleles (NA) ranged from 4 to 14

(mean=9.5333), HO ranged from 0.7609 to 1.0000 (mean=

0.9178), and HE ranged from 0.7273 to 0.9194 (mean=

0.8468). PIC ranged from 0.6684 to 0.9020 (mean=0.8166).

Two loci, SB-22 and SB-29, were discovered to have

deviated significantly from HWE. Meanwhile, none of the

loci combinations showed significant linkage disequilib-

rium after a sequential Bonferroni correction in multiple

tests. Thus, these microsatellite loci first presented useful

molecular markers in the analysis of the genetic variability

of Tarim schizothoracin, and in the investigation of genetic

relations of Schizothorax.

Acknowledgments The authors are grateful to Mr. Jianguo Yin for

his help in collecting the fish samples. The authors would also like to

thank Hu Juan and Zhang Xiaoyi for their kind assistance in the

experiment. This work was supported by the combined projects of

Shanghai Ocean University and Xinjiang Bohu Reed Industry Co.,

Ltd. (Grant No. D-8006-10-0047), the Shanghai Leading Academic

Discipline Project (Grant No. Y1101), and the Shanghai Science &

Technology Committee Project (10dz1200804).

References

Chen SQ, Liu QG, Ren SJ, Gong XL (2009) Polymorphic microsat-

ellite loci isolated from the topmouth culter (Culter alburnusBasilewsky). Conserv Genet Resour 1:337–339

Goudet J (1995) FSTAT (version 1.2): a computer program to

calculate F-statistics. J Hered 86:485–486

Hu J, Ren SJ, Fu CZ, Gong XL (2010) Isolation and characterization of

polymorphic microsatellite markers for the ariake icefish (Salanxariakensis kishinouye). Conserv Genet Resour 3:315–318

Raymond M, Rousset F (1995) GENEPOP (version 1.2): population

genetics software for exact tests and ecumenicism. J Hered 86:

248–249

Rice WR (1989) Analyzing tables of statistical tests. Evolution

43:223–225

Sambrook J, Russell DW (2002) Molecular cloning: a laboratory

manual, 3nd edn. Cold Spring Harbor Laboratory Press, New

York, p 479–483

Yang TY, Zhang RM, Guo Y, Meng W, Hai S, Ma YW (2011)

Comparative study on partial mitochondrial COI gene of

Aspiorhynchus laticeps and Schizothorax biddulphi. J Hydroecol

32(1):45–50 (In Chinese with English abstract)

Yeh F, Boyle T, Rongcai Y, Ye Z and Xian J (1999) POPGENE version

1.32. http://www.ualberta.ca/*fyeh/popgene_download.html (Nov

19, 2009)

Yue PQ, Chen YY (1998) China red databook of endangered animals

(Pisce). Science Press, Beijing, p 153–155

Zhang RM, Guo Y, Ma YW, Turxun (2007) A survey on the resource

and distribution of Schizothorax biddulphi Gunther. Freshw Fish

37(6):76–78

Conservation Genet Resour (2012) 4:795–797 797

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