基于SLAF-seq技术的黑棘鲷微卫星标记开发及其在鲷科鱼类中的通用性研究

黑棘鲷(Acanthopagrusschlegelii)是广泛分布于西北太平洋的暖温性中下层经济鱼类,也是我国沿海重要的海洋捕捞鱼类和增养殖对象。然而,目前有关黑棘鲷的微卫星标记研究报道较少,难以对其种质资源状况作出精确评估。本研究采用SLAF-seq技术测序共获得22489个二至六碱基重复的黑棘鲷微卫星序列,短重复序列(二、三碱基)占总微卫星序列的90.8%,长重复序列(四至六碱基)占有9.2%。经过157对随机合成引物的多态性筛选,开发出49个高多态性的黑棘鲷微卫星标记,其中短重复序列位点有25个,长重复序列位点有24个。每个位点的等位基因数(Na)为2—20(均值为8.3),观测杂合度(Ho)和期望杂合度(He)分别为0.097—0.938和0.122—0.922(均值分别为0.663和0.701),多态信息含量(PIC)为0.118—0.897(均值为0.655)。经Bonferroni校正后,有47个位点符合哈迪-温伯格平衡(HWE),各位点间未检测到连锁不平衡现象,仅2个位点偏离HWE。结果表明,所开发的大部分微卫星标记具有高多态性,蕴含的遗传信息含量较为丰富,能够为黑棘鲷的种群遗传资源评估提供数量充足、类型多样的有效分子标记。跨物种扩增结果显示,有43个黑棘鲷微卫星标记可在9种鲷科鱼类中成功扩增,其中28个标记在太平洋棘鲷(Acanthopagruspacificus)、黄鳍棘鲷(Acanthopagruslatus)和澳洲棘鲷(Acanthopagrusaustralis)中具有较好的通用性,2个标记在平鲷(Rhabdosargussarba)、蓝点赤鲷(Pagruscaeruleostictus)、真赤鲷(Pagrusmajor)、二长棘犁齿鲷(Evynnis cardinalis)及黄牙鲷(Dentex hypselosomus)中具有通用性。这些通用性标记可为阐明鲷科属、种间的系统进化关系和棘鲷属鱼类的群体遗传学分析提供新的标记来源和研究角度。

DEVELOPMENT OF MICROSATELLITE MARKERS FOR ACANTHOPAGRUS SCHLEGELII BASED ON SLAF-SEQ TECHNOLOGY AND GENERALITY IN THE FAMILY SPARIDAE

The black porgy (Acanthopagrus schlegelii) (Perciformes:Sparidae) is a meso-demersal warm-water fish and widely distributed in the western Pacific coastal waters. It has become an important target species of mariculture in China. However, studies on the microsatellite markers of A. schlegelii are scarce. To make an accurate assessment of its current germplasm resources, based on specific-locus amplified fragment sequencing (SLAF-seq) technology, we identified 22 489 di- to hexanucleotide repeats microsatellites from the genomic DNA of A. schlegelii. The shorter repeats motifs (2-3 mers) accounted for 90.8%, while the longer repeat motifs (4-6 mers) for 9.2% of the total microsatellite sequences. Finally, 49 high polymorphic microsatellite markers were developed from 157 pairs of randomly synthesized primers, among which 25 are shorter repeats motifs loci and the other 24 are longer repeat motifs loci. The allele number per locus ranges from 2 to 20 (mean 8.3), and the observation and expected heterozygosity varies from 0.097 to 0.938 (mean 0.663), and 0.122 to 0.922 (mean 0.701), respectively. The polymorphism information content (PIC) ranges from 0.118 to 0.897 (mean 0.655). Only two microsatellite loci are deviated from the Hardy-Weinberg equilibrium (HWE) after the Bonferroni correction, while the other 47 loci are consistent with HWE and no linkage disequilibrium was detected among these 47 loci. The results show that most of the microsatellite markers are highly polymorphic, and contained abundant genetic information. These polymorphic microsatellite markers provide abundant and various effective molecular markers to assess the population genetic resources. In addition, as shown in cross-species amplification, 43 microsatellite markers could be successfully amplified in nine species of family Sparidae. Among them, 28 are transferable in Acanthopagrus pacificus, Acanthopagrus latus, and Acanthopagrus australis; 2 are transferable in Rhabdosargus sarba, Pagrus caeruleostictus, Pagrus major, Evynnis cardinalis, and Dentex hypselosomus. The transferable markers provide new analytical tools for elucidating phylogenetic relationships among species and genus of the family Sparidae, and for understanding the population genetic of Acanthopagrus species in the future.