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本研究对大菱鲆EST数据库中12 434条序列进行微卫星搜索,共搜索出831条EST-SSR序列,其中l6条为明确标注免疫相关功能的基因序列.根据此l6条EST-SSR序列设计引物,经过PCR扩增检测和反应条件的优化,最终筛选出9对扩增效果理想的EST-SSR标记对大菱鲆减数分裂雌核发育二倍体子代进行遗传变异检测,结果表明:在Scoph-2-1、Scoph-7-1、Scoph-10-1微卫星位点上大菱鲆亲本和雌核发育二倍体子代均为纯合基因型,其余6个微卫星位点(Psetta-1-1、Psetta-1-2、Psetta-3-1、Psetta-3-2、Scoph-4-1、Scoph-6-1)在大菱鲆雌核发育二倍体仔鱼群体中的观测杂合度≥0.700,期望杂合度>0.460,平均期望杂合度为0.503.大菱鲆减数分裂雌核发育二倍体还显示出较高的重组率,平均重组率为89%;综合上述结果推断,9个免疫相关EST-SSRs标记可有效地用于分析大菱鲆减数分裂雌核发育二倍体子代的遗传变异;6个位点的重组率计算结果能够为今后对大菱鲆的着丝粒进行精确定位提供依据.
In this study, 12 434 EST sequences of the turbot EST database were searched for microsatellites, and 831 EST-SSR sequences were searched out, of which 16 were gene sequences that were clearly labeled with immune-related functions.According to these 16 EST-SSR sequences After PCR amplification and optimization of reaction conditions, 9 pairs of EST-SSR markers were selected to test the genetic variation of meiosis diploid progeny of turbot. The results showed that: At Scoph-2-1, Scoph-7-1 and Scoph-10-1 microsatellite loci, the diploid progeny and gynogenetic diploid progeny of the turbot were homozygous, and the other six microsatellite loci (Psetta-1-1, Psetta-1-2, Psetta-3-1, Psetta-3-2, Scoph-4-1, Scoph-6-1) The observed heterozygosity≥0.700, the expected heterozygosity> 0.460 and the average expected heterozygosity was 0.503. The diploid of meiosis of meiosis of turbot (Scophthalmus maximus) also showed higher recombination rate with the average recombination rate of 89% The results infer that nine immune-related EST-SSRs markers can be effectively used to analyze the genetic variation of meiosis diploid progeny of meiosis in turbot; 6-locus recombination The results can provide a basis for centromere of turbot precise positioning.