大黄鱼微卫星标记的开发及其遗传方式分析
ISOLATION AND GENETIC ANALYSIS OF MICROSATELLITE MARKERS FOR LARIMICHTHYS CROCEA
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摘要: 采用FIASCO方法构建大黄鱼(AC)n微卫星富集文库, 从文库中随机挑选90个白色克隆, 经过菌液PCR筛选得到60 (66.7%)个阳性克隆进行测序, 其中有56个克隆(93.3%)含有 CA/GT 重复数大于5的微卫星序列。56个微卫星序列中, 二核苷酸微卫星51个(91.1%), 三核苷酸微卫星5个; 二核苷酸重复中有48个为(AC)n重复, 占二核苷酸总数的94.1%。根据Weber的微卫星分类规则, 完美型占75.0%, 非完美型占8.9%, 复合型微卫星占16.1%。共设计引物52对, 在1个大黄鱼家系中35对引物所在位点具有多态性, 28个(80.0%)位点子代基因型为1:1:1:1(AB CD/AB AC )分离类型, 6个位点属1:1分离类型, 1个位点属1:2:1 (AB AB)分离类型。35个位点中有32个位点的分离符合孟德尔分离比(P0.05), 另外3个位点(LYC0137、LYC0139、LYC0152)明显偏离1:1或者1:1:1:1的孟德尔分离比(P0.05)。本研究开发的微卫星标记为大黄鱼微卫星遗传连锁图谱构建以及群体遗传学、分子进化和系统发育等研究提供了有用的分子工具。Abstract: We constructed an (AC)n-microsatellite-enriched library for large yellow croaker Larimichthys crocea(Richardson 1846) using the method of FIASCO. Ninety clones were randomly selected for further colony amplification. The rate of positive clones reached to 66.7%, and 56 of the 60 positive clones sequences contained one or more SSR. By the analysis of repeat motif, dinucleotide repeats were the most dominant (91.1%), followed by trinucleotide repeats (8.9%). Among dinucleotides, (AC)n repeats were the most frequent (94.1%). Using Webers classification rules, the sequences were divided into three categories, the percentage of perfect repeat sequences, imperfect repeat sequences, and compound repeat sequences was 75.0%, 8.9%, and 16.1, respectively. Fifty two microsatellites primer pairs were designed and synthesized. The result showed that 35 SSRs were polymorphic in 46 F1 generation individuals. Twenty eight (80.0%) loci exhibited a segregation ratio of 1:1:1:1 (AB CD/AB AC), serving as the most useful markers segregating in co-dominant fashion. Six loci exhibited a segregation ratio of 1:1, and one loci exhibited a segregation of 1:2:1. Polymorphism analysis showed that 32 SSRs were consisted with Mendelian segregation ratio and could be used to construct the linkage map, while the other 3 SSRs (LYC0137, LYC0139, LYC0152) were departure from the expected Mendelian segregation patterns. These SSRs should be available for genetic linkage mapping, population genetics studies, molecular evolution and phylogenetic study of large yellow croaker.
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