ZHU Chuankun (朱傳坤) PAN Zhengjun (潘正軍) WANG Hui (王輝) CHANG Guoliang (常國亮) DING Huaiyu (丁懷宇)

1Jiangsu Engineering Laboratory for Breeding of Special Aquatic Organisms,Huaiyin Normal University,Huai’an 223300,China

2Jiangsu Collaborative Innovation Center of Regional Modern Agriculture&Environmental Protection,Huaiyin Normal University,Huai’an 223300,China

Received Jan. 13, 2017; accepted in principle Feb. 27, 2017; accepted for publication Apr. 25, 2017

AbstractThe Ussuri catfishPseudobagrus ussuriensisis a promising aquaculture fi sh with sexual dimorphism that males grow 2-3 times faster than females. Therefore, all-male breeding could increase production of this fi sh, and sex-linked markers would be useful during the breeding process. In this study,a male-associated marker namedPuGT54was isolated inP.ussuriensisfrom a previously constructed microsatellite library. A total of 185P.ussuriensisindividuals containing 95 females and 90 males were used to test the usage of this maker. And results showed that sexes of all individuals were successfully identified, indicating the high reliability and accuracy of this marker for sex identifi cation. Furthermore,the male-Specific fragment amplified by this maker was cloned and sequenced, which was 118 base pairs in length. As homologous genes of this fragment was not found through BLASTn, detailed information of the male-Specific fragment is unknown presently. Nevertheless, this marker would be helpful in conservation,breeding and ecological study ofP.ussuriensis.

Keyword: Pseudobagrus ussuriensis; male-associated marker; microsatellite

1　INTRODUCTION

significant sexual dimorphisms, especially on growth rate and body size have been reported in many fishes(Kobayashi et al., 2012; Mei and Gui, 2015). However,in most fishes it is diffcult to identify male and female fry morphologically (Kobayashi et al., 2012). Recently,molecular markers have been proved to be effcient for sex identifi cation and successfully used in many fishes(Wang et al., 2009, 2013; Dan et al., 2013; Agawa et al.,2015; Xu et al., 2015; Zhang et al., 2016).

Ussuri catfishPseudobagrusussuriensis(Siluriformes: Bagridae), which distributes from the Heilongjiang River to the Zhujiang (Pearl) River (Chu et al., 1999) in China, is a promising aquaculture species with growth rate of males being 3 times faster than that of females (Pan et al., 2015). However, its natural resources have sharply declined in recent years because of human activities (Tao et al., 2012). In our previous study, a male-Specific SCAR (sequence characterized amplified regions) marker has been isolated using the AFLP (amplified fragment length polymorphism) genotyping method (Pan et al., 2015).In this study, we intended to develop reliable sexassociated markers from a different approach which was to screen these markers from a newly-constructed microsatellite library of our laboratory (Zhu et al.,2017). This study will enrich the types and number of sex-associated markers which would increase the reliability and accuracy of sex identifi cation inP.ussuriensis, meanwhile, provide useful tools for allmale breeding, aquaculture management and natural resource conservation in this fi sh.

Fig.1　Electrophoretic results of the sex-associated marker PuGT54 in female and male P. ussuriensisThe male-Specific locus was marked with an arrow.

2　MATERIAL AND METHOD

Totally, 185 individuals ofP.ussuriensiscontaining 95 females and 90 males identified by examination of gonads were collected from the Huai’an Fisheries Technical Guidance Station. Fin clips were sampled and stored in anhydrous ethanol at 4°C until DNA extraction. Total genomic DNA was extracted following the phenol-chloroform protocol (Sambrook and Russell, 2001). Qualities of DNA samples were tested by 1% agarose gel electrophoresis and concentrations were measured using a Nanodrop 2000 UV spectrophotometer (Thermo, USA).

A total of 61 markers developed from an enriched microsatellite library in our previous study (Zhu et al.,2017) were applied to screen sex-associated markers using 5 male and 5 female individuals. Candidate markers were further confi rmed in the remaining 90 females and 85 males. PCR was performed in a reaction mixture of 12.5 μL, which contained 1.3 μL of 10×reaction buffer, 0.5 μL of dNTP (2.5 mmol/L),50 ng of template DNA, 0.5 μL of forward and reverse primer mix (2.5 μmol/L), 1 U of Taq polymerase(CWBIO, China) and 9.2 μL sterile water. All reactions were performed by a 96 well thermal cycler (T100,BioRad) following this program: an initial denaturation at 95°C for 5 min, followed by 36 cycles of denaturation at 95°C for 45 s, annealing at optimal temperature for 45 s, extension at 72°C for 50 s, and a fi nal extension at 72°C for 8 min. PCR amplicons were separated through electrophoresis in 10% non-denaturing polyacrylamide gels and visualized using ethidium bromide (EB)staining with the pBR322/MspI DNA marker (TianGen,China) being applied as the standard DNA ladder.

In order to obtain their sequence information, sexSpecific loci were cloned and sequenced. Brief l y,confi rmed potential sex-associated markers were amplified in a male and female respectively, and products were separated by 1.5% agarose gel electrophoresis. Then target fragments were retrieved and purified using the Gel Extraction Kit (CWBIO,China). Purified PCR products were ligated to the PMD18-T vector (TaKaRa, Japan) and translated into DH5α competent cells. Plasmids of positive clones were extracted and sequenced in I-congene Biological Science and Technology Co. Ltd., Wuhan, China.

3　RESULT AND DISCUSSION

Out of the 61 screened microsatellites, one marker namedPuGT54(GenBank accession number:KX809660; annealing temperature: 54°C) with forward and reverse primer sequences of 5′-ACGGCGAAAGGTGAGTAGC-3′ and 5′-TTTGAGAGCACACGCAGGT-3′, respectively, was a potential male-Specific marker. The band pattern of this marker showed that in females only one locus was amplified, while in males an additional Specific locus was also amplified. Then this marker were further examined in the remaining 90 female and 85 male individuals, and the results displayed that all males had a Specific band comparing to females (Fig.1), indicating the confi rmation of this sex-associated marker. Compared to the male-Specific marker developed in our previous study (Pan et al.,2015), this marker could amplify a common locus shared by both males and females, therefore, inaccurate identifi cation of false-positive females caused by emergence of null allele in males could be avoided.

Then the male-Specific locus was cloned and sequenced, which was 118 base pairs (bp) in length.While comparing to the common locus shared by both sexes, the male-Specific locus was 12 bp shorter which was 6 times of the core repeat unit (CA), while nucleotide differences in fl anking sequence were not detected (Fig.2). Orthologous sequences of this fragment were searched in the GenBank through BLASTn, however, no informative blast hits were detected. Moreover, one of our previous studies has reported a male-Specific marker and extended it by genome walking (Pan et al., 2015), and native BLAST was performed throughout this sequence, but the result showed that the male-Specific locus developed in this study did not belong to it. Therefore, further studies are needed to understand the location in chromosomes and function of this male-Specific locus.

Fig.2　Sequence information of the male-Specific fragment amplified by the sex-associated marker in P. ussuriensisBlack lines marked locations of primers, and short bars stand for absent nucleotides in male-Specific locus compared to the common one.

In fact, as the sex determination mechanism is quite complex in fi sh (Gui, 2007), although sexSpecific markers have been reported in many fi sh species, detailed information of most markers was still unclear at the present time (Agawa et al., 2015;Xu et al., 2015). In spite of this, sex-Specific markers are still useful tools in ecology and conservation studies of fishes. The marker developed in this study will be useful for sex ratio determination of natural populations ofP.ussuriensisin which sexes ofindividuals were hard to identify morphologically.Furthermore, through combining usage of this marker and the previous SCAR marker, the reliability and accuracy of sex identifi cation will be increased and would be helpful in the management of artifi cial breeding especially all-male breeding of this species.

4　ACKNOWLEDGEMENT

The authors thank TONG Jingou, YU Xiaomu,WU Nan, ZHOU Fengjian, YU Xiangsheng, QIANG Xiaogang, and JIANG Gongcheng for technical assistance and sample collection.