Rapid development of microsatellite markers from the large yellow croaker (Pseudosciaena crocea) using next generation DNA sequencing technology
Rapid development of microsatellite markers from the large yellow croaker (Pseudosciaena crocea) using next generation DNA sequencing technology
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DOI：
10.1016/j.bse.2013.09.019
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发表时间：
2013-12-01
影响因子：
1.6
通讯作者：
Wang, Chaofeng
中科院分区：
文献类型：
--
作者：
Lu, Zhenming;Li, Hongmei;Wang, Chaofeng
利用Roche 454测序技术，从中国重要的经济鱼类刺鱼（Pseudosciaena crocea）中鉴定出大量的微卫星。共鉴定出2535个SSR序列，其中二核苷酸最多（59.61%），其次是四核苷酸（29.94%）和三核苷酸（7.06%）。在二核苷酸重复序列中，频率最高的是AC基序，占总数的79.15%。AAT（36.31%）和TCTA（39.66%）分别是三核苷酸和四核苷酸基序中最常见的基序。共有1068个微卫星位点具有适合设置聚合酶链反应（PCR）引物的侧翼序列。为了验证鉴定出的ssr，随机选取40对引物进行PCR。共有32个引物组（80.00%）产生了与预期大小匹配的强PCR产物，27个引物组（67.50%）具有多态性。利用27组引物对漳州海域34个野生个体进行分析，每个位点等位基因数为5 ~ 11个，平均等位基因丰富度为6.74。没有发现任何一对位点之间的连锁不平衡，表明标记是独立的。Hardy-Weinberg平衡检验显示，经过连续Bonferroni校正后，27个微卫星位点中有6个存在显著偏差。与传统方法相比，这种微卫星隔离方法具有成本效益和时间效益。(C) 2013 Elsevier Ltd.版权所有。
We have identified a large number of microsatellites from Pseudosciaena crocea, an economically important fish species in China, by taking advantage of the Roche 454 sequencing technology. A total of 2535 simple sequence repeats (SSR) motifs were identified among which dinucleotides were the most frequent (59.61%), followed by tetra- (29.94%) and trinucleotides (7.06%). Among dinucleotide repeats, the most frequent repeats were AC motifs, accounting for 79.15% of the total. AAT (36.31%) and TCTA (39.66%) motifs were the most frequent of the tri- and tetranucleotide motifs, respectively. A total of 1068 microsatellite loci had flanking sequences suitable for setting primers for polymerase chain reactions (PCR). To verify the identified SSRs, 40 primer pairs were randomly selected for PCR. In total, 32 primer sets (80.00%) produced strong PCR products matching their expected sizes, and 27 (67.50%) were polymorphic. In an analysis of 34 wild individuals from the Zhangzhou Sea area (Fujian Province, China) with the 27 primer sets, the number of alleles per locus ranged from 5 to 11 and the mean allelic richness was 6.74. Linkage disequilibrium was not observed between any pair of loci, indicating that the markers were independent. A Hardy-Weinberg equilibrium test revealed significant deviation in 6 of the 27 microsatellite loci after sequential Bonferroni corrections. This microsatellite isolation method is cost- and time-effective in comparison to traditional approaches. (C) 2013 Elsevier Ltd. All rights reserved.