基于高通量测序的青石斑鱼基因组微卫星开发及评价

Development and Population Genetic Diversity Analysis of Microsatellite Markers in Epinephelus awoara

本研究采用基因组高通量随机测序的方法对青石斑鱼(Epinephelus awoara)微卫星标记进行了发掘,结果共获得青石斑鱼(Epinephelus awoara)基因组原始数据2.860G。采用MISA(MIcroSAtellite)软件进行微卫星位点搜索,获得967 657条微卫星序列。随机选取了96条微卫星序列设计引物并利用8尾青石斑鱼个体进行多态性位点筛选,共获得60个具有多态的微卫星标记。利用其中16个微卫星标记对一个青石斑鱼亲鱼群体的种群遗传学特征进行评价,共检测到129个等位基因,单个标记的表观等位基因数(NA)分布范围为2~14,平均为7.167;表观杂合度(HO)分布范围为0.422~1.000,平均为0.678 6;期望杂合度(HE)分布范围为0.481~0.893,平均为0.676 8。有5个标记的等位基因频率不符合"哈迪-温伯格平衡",其中EAW-55596、EAW-21755、EAW-30334 3个标记表现为杂合子显著缺失(P<0.05),而EAW-33674和EAW-52520 2个标记表现为杂合子显著过剩(P<0.05)。连锁不平衡检测表明各位点间无连锁不平衡现象。多态信息含量(PIC)分布范围为0.362 3~0.872 2,平均PIC=0.639 7>0.5,说明该亲鱼群体的遗传多样性比较高。而这些微卫星标记将对于青石斑鱼的遗传变异分析、亲缘关系鉴定提供了技术支持,这也将为青石斑鱼的分子遗传育种奠定了基础。

In this study,The microsatel[ites of Epinephelus awoara were isolated, and the genetic diversi- ty were analysed. Firstly, High throughput sequencing technology was used to develop microsatellites of Epinephelus awoara, and 2. 860 Gbp raw data was obtained. Microsatellites were identified by screening the genome sequences using software MISA（MIcroSAtellite）, and 967 657 different kinds of repeat motif types of microsatellite sequences were discovered. Ninety-six pairs of primers were designed according to unique microsatellite flanking sequences for amplification by polymerase chain reaction （PCR） with the software Primer 5.0, among which 60 loci were clearly amplified and shown polymorphic. In this paper, 16 polymorphic microsatellite loci were characterized to evaluate the genetic diversity of parent population （48 individuals included） of Epinephelus awoara. A total of 129 alleles were detected, the number of al- leles per loci ranged from 4 to 14 and effective number of alleles ranged from 1. 905 9 to 8. 295 3 with the average of 7. 166 7 and 4. 241 1, respectively. The observed heterozygosities （Ho） were from 0. 422 to 1. 000 （mean=0. 678 6）, and the expected heterozygosities （HE） ranged from 0. 481 to 0. 893 （mean= 0. 676 8）. Five loci deviated significantly from the Hardy-Weinberg equilibrium. Three markers indicated significant heterozygote deficiency including EAW-55596, EAW-21755 and EAW-30334. Otherwise, EAW-33674 and EAW-52520 indicated significant heterozygote excess. And none of the loci combinations showed significant linkage disequilibrium. The polymorphism information content （PIC） was from 0. 362 3 to 0. 872 2 （mean=0. 639 7〉0. 5）, and this result indicated that the genetic diversity of popula- tion was at high level. Thus, these microsatellite loci first presented useful molecular markers in the anal- ysis of the genetic variability and in the investigation of genetic relations of Epinephelus awoara, which will provide a basis for genetic breeding.