Development and characterization of EST-SSR markers in the eastern oyster Crassostrea virginica
Development and characterization of EST-SSR markers in the eastern oyster Crassostrea virginica
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DOI：
10.1007/s10126-007-9011-7
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发表时间：
2007-08-01
影响因子：
3
通讯作者：
Guo, Ximing
中科院分区：
文献类型：
--
作者：
Wang, Yongping;Guo, Ximing
从东方牡蛎（Crassostrea virginica）的表达序列标签（est）中开发出简单序列重复（SSR）标记。从GenBank下载东部牡蛎的ESTs，筛选至少具有8个二核苷酸单位或5个三、四、五和六核苷酸重复单位的SSRs。9101条ESTs筛选出127条（1.4%）含ssr序列。共设计了88条含有ssr的ESTs引物，具有良好且充足的侧翼序列。聚合酶链反应（PCR）扩增成功71对引物，其中19对（27%）引物扩增片段比预期长度长，可能是由于内含子。用聚丙烯酰胺凝胶法对3个种群的5对牡蛎中66对短于800 bp的片段进行多态性筛选，其中53对（80%）具有多态性。通过自动测序仪对来自3个种群的30只牡蛎的53个多态SSRs进行了标记和基因分型。其中5个SSRs在每个牡蛎中扩增了两个以上的片段，表明位点重复。其余48个ssr个体有2个等位基因，其中11个为零等位基因。在分析的30只牡蛎中，SSRs平均每个位点有9.3个等位基因，范围从2到24。在一个拥有100个后代的家庭中分离出43个位点，其中9个位点与孟德尔比率有显著偏差（3个经过Bonferroni校正）。70%的基因座在C. rhizophorae中成功扩增，34%在C. gigas中成功扩增。本研究表明，est是开发东部牡蛎SSR标记的宝贵资源，est衍生的SSR标记比基因组SSR标记更具跨种可转移性。
Simple sequence repeat (SSR) markers were developed from expressed sequence tags (ESTs) in the eastern oyster (Crassostrea virginica). ESTs of the eastern oyster were downloaded from GenBank and screened for SSRs with at least eight units of dinucleotide or five units of tri-, tetra-, penta-, and hexa-nucleotide repeats. The screening of 9101 ESTs identified 127 (1.4%) SSR-containing sequences. Primers were designed for 88 SSR-containing ESTs with good and sufficient flanking sequences. Polymerase chain reaction (PCR) amplification was successful for 71 primer pairs, including 19 (27%) pairs that amplified fragments longer than expected sizes, probably due to introns. Sixty-six pairs that produced fragments shorter than 800 bp were screened for polymorphism in five oysters from three populations via polyacrylamide gels, and 53 of them (80%) were polymorphic. Fifty-three polymorphic SSRs were labeled and genotyped in 30 oysters from three populations via an automated sequencer. Five of the SSRs amplified more than two fragments per oyster, suggesting locus duplication. The remaining 48 SSRs had 2 alleles per individual, including 11 with null alleles. In the 30 oysters analyzed, the SSRs had an average of 9.3 alleles per locus, ranging from 2 to 24. Forty-three loci segregated in a family with 100 progeny, with nine showing significant deviation from Mendelian ratios (three after Bonferroni correction). Seventy percent of the loci were successfully amplified in C. rhizophorae and 34% in C. gigas. This study demonstrates that ESTs are valuable resources for the development of SSR markers in the eastern oyster, and EST-derived SSRs are more transferable across species than genomic SSRs.