引用本文:	吕振明,侯龙,龚理,刘立芹,陈永久,郭宝英,董迎辉,吴常文.基于de novo高通量测序的曼氏无针乌贼(Sepiella japonica)ESTs中微卫星位点筛选与特征分析.海洋与湖沼,2017,48(4):877-883.

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*基于de novo高通量测序的曼氏无针乌贼(Sepiella japonica)ESTs中微卫星位点筛选与特征分析*
吕振明¹, 侯龙¹, 龚理¹, 刘立芹¹, 陈永久¹, 郭宝英¹, 董迎辉², 吴常文¹
1.浙江海洋大学海洋生物种质资源发掘与利用国家地方联合工程实验室舟山 316022;2.浙江万里学院浙江省水产种质资源高效利用技术研究重点实验室宁波 315100

摘要:

对均一化转录组测序获得的47604个曼氏无针乌贼的微卫星序列进行分析，结果表明，乌贼转录组中微卫星位点丰富，每1402nt的EST中就有一段不小于12nt长度的微卫星序列。单碱基重复是EST微卫星序列的主要形式（38.69%），其次依次为三碱基（31.14%）、二碱基（26.35%）、四碱基（3.29%）、五碱基（0.38%）、六碱基（0.14%）重复，短序列类型占微卫星总量的96.18%。同碱基类型的微卫星序列组成又存在差异，AC（54.51%）和AG（31.22%）是最常见的二碱基重复序列；而AGC（16.37%）和AAC（14.06%）是最常见的三碱基重复序列。通过引物设计和体系优化，共筛选到了24对多态性微卫星位点，对来自福建漳州海域的35只野生曼氏无针乌贼（Sepiella japonica）个体进行群体遗传学检测，结果表明，每个位点检测到的等位基因数3-10个不等（均值5.9个）；平均杂合度观测值（H_o）和期望值（H_e）分别为0.518和0.681。除2个位点外所有位点的多态信息含量（PIC）都大于0.5。Hardy-Weinberg平衡检测表明，仅4个位点有显著偏离（P<0.05），未检测到连锁不平衡现象。这表明转录组高通量测序技术适于乌贼微卫星位点的开发，获得的SSR标记可用于今后乌贼资源遗传评估和科学有效管理过程。

关键词: 曼氏无针乌贼转录组 EST序列微卫星

DOI：10.11693/hyhz20170300079

分类号:Q789;S931

基金项目:国家自然科学基金项目，41406138号；浙江省自然科学基金项目，LY130190001号；浙江省水产种质资源高效利用技术研究重点实验室开放课题，KF201-6008号。

附件

ISOLATION AND ANALYSIS ON EST MICROSATELLITES OF SEPIELLA JAPONICA BY DE NOVO HIGH-THROUGHPUT TRANSCRIPTOME SEQUENCING

LÜ Zhen-Ming¹, HOU Long¹, GONG Li¹, LIU Li-Qin¹, CHEN Yong-Jiu¹, GUO Bao-Ying¹, DONG Ying-Hui², WU Chang-Wen¹

1.National and Provincial Joint Laboratory of Exploration and Utilization of Marine Aquatic Genetic Resources, Zhejiang Ocean University, Zhoushan 316022, China;2.Zhejiang Key Laboratory of Aquatic Germplasm Resources, Zhejiang Wanli University, Ningbo 315100, China

Abstract:

A total of 47604 microsatellite sequences from Sepiella japonica transcriptome was obtained and analyzed by next-generation sequencing. The results show that there were abundant SSR sequences in transcriptome of S. japonica and, in average, 1402nt EST sequence would contain a SSR in length of no less than 12 nucleotides. The mono-nucleotides SSR is the main type of microsatellites (38.69%), followed by tri-nucleotide (31.14%), di-nucleotide (26.35%), quad-nucleotide (3.29%), penta-nucleotide (0.38%), and hexa-nucleotide (0.14%) microsatellites. The shorter repeats accounted for 96.18% of the total quantity of the microsatellite sequences. Among di-nucleotide repeats, AC (54.51%) and AG (31.22%) were most common, while among tri-nucleotide repeats, AGC (16.37%) and AAC (14.06%) were most frequent. Twenty-four polymorphic microsatellites were isolated and tested in 35 cuttlefish individuals collected from a natural population in Zhangzhou sea area, Fujian province. The results show that the number of alleles per locus ranged from 3 to 10 and the mean allelic richness was 5.9; the mean observed heterozygosities (H_o) and expected heterozygosities (H_e) were 0.518 and 0.681, respectively. The polymorphic information content (PIC) values of all loci, except for two, were above 0.5. A Hardy-Weinberg equilibrium test revealed significant deviation in 4 of the 24 microsatellite loci after sequential Bonferroni corrections (P<0.05). Linkage disequilibrium was not observed between any pair of loci, indicating that the markers were independent. Therefore, this microsatellite isolation method is cost and time effective in comparison to traditional approaches. The SSR markers isolated will be useful in future's genetic evaluation process of this species, which is essential for a sound management of cuttlefish fishery resources.

Key words: Sepiella japonica transcriptome EST sequence microsatellites