利用SLAF-seq技术开发带鱼(Trichiurus japonicus)微卫星标记以及跨物种扩增检测

带鱼(Trichiurus japonicus)是广泛分布于东亚大陆架海域的暖温性近底层鱼类,长期位居我国单鱼种渔获量第一位。自上世纪70年代以来,持续过度捕捞和海洋环境变化导致中国近海带鱼资源基础和遗传多样性受到严重影响,但有关带鱼种群的微卫星DNA标记研究却较为缺乏,不利于该物种遗传资源评估和保护。为此,本研究采用基于高通量测序平台的SLAF-seq技术,从带鱼的195 308个SLAF标签中识别出25 704个二至六碱基重复微卫星位点。经过引物扩增验证,最终筛选出36个具有多态性的微卫星标记。各位点等位基因数4～35,均值14.47。观测杂合度0.214～1.000,期望杂合度0.456～0.979,均值分别为0.620和0.803。所有位点的多态信息含量值均大于0.25。经Bonferroni校正,21个位点符合哈迪-温伯格平衡,且各位点间不存在连锁不平衡。这21个多态性微卫星标记可为带鱼的种群遗传资源研究提供新的有效分子标记和技术支撑。Bottleneck分析结果表明,宁波近海带鱼群体未检测到近期的遗传瓶颈效应,这可能与目前东海区带鱼野生群体数量还比较庞大、遗传变异仍然较为丰富有关。跨物种扩增结果显示,分别有12、16、4和3个带鱼微卫星标记在带鱼属(Trichiurus)、沙带鱼(Lepturacanthus savala)、窄颅带鱼(Tentoriceps cristatus)及小带鱼(Eupleurogrammus muticus)中具有较好的通用性,这些微卫星标记为今后带鱼科相应属、种的系统进化关系研究提供新的分析手段和契机。

Development of Microsatellite Loci for Hairtail (Trichiurus japonicus) by Using SLAF-seq Technology and Cross-species Amplification Tests

The hairtail(Trichiurus japonicus), widely distributed in the tropical and warm-temperate waters of the east Asian continental shelf, has long been the first annual catch among various marine fishes in China. Since the1970 s, the sustained overfishing and changes in marine environment have severely impacted the resource base and genetic diversity of T. japonicus population in the Chinese coastal waters. However, there are few available microsatellite markers isolated in T. japonicus for its population genetics studies, which is not conducive to the resource assessment and conservation of this species. Therefore, in this study, by using the specific-locus amplified fragment sequencing(SLAF-seq) technology, which were based on the high throughput sequencing platform, we have identified 25 704 microsatellites loci of di-to hexanucleotide repeats sequences from 195 308 SLAFs in T.japonicus. After primers screening, 36 microsatellite markers were finally developed, which the alleles number were at a range of 4 to 35(mean 14.47). The observed and expected heterozygosity ranged from 0.214 to 1.000(mean0.620) and 0.456 to 0.979(mean 0.803), respectively. Polymorphism information contents of all loci are greater than 0.25. Twenty-one microsatellite loci were consistent with the Hardy-Weinberg equilibrium after Bonferroni correction, and there was no linkage disequilibrium been detected among them. These 21 polymorphic microsatellite markers can be served as useful molecular tool and technical support for the population genetic resources study of T. japonicus. The result of Bottleneck analysis showed no evidence of a recent bottleneck in the T. japonicus population from Ningbo coastal waters. This may be related to the large numbers of wild populations and high levels of genetic variations of T. japonicus in the East China Sea. In addition, cross-species amplifications revealed that transferability of 12, 16, 4 and 3 microsatellite markers of T. japonicus was positive across Trichiurus,Lepturacanthus savala, Tentoriceps cristatus and Eupleurogrammus muticus, respectively. These microsatellite markers would provide new analytical tools and opportunities for the phylogenetic relationship study of corresponding genus and species of Trichiurus japonicus in the future.