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从NCBI数据库下载了287349条萝卜EST序列,经预处理后得到无冗余EST序列58105条,全长38 22.476kb。利用MISA搜索SSR位点,得到含有SSR位点的EST序列3523条,共3718个SSR,平均每 10.39kb就出现1个SSR。分析发现萝卜EST序列中存在265种重复基元类型,其中二、三、六核苷酸重复是主导的重复基元类型,二核苷酸中以比例高达91.34%的AG/CT重复基元为主;三核苷酸中主导的重复基元类型是AAG/CTT,比例为35.86%;六核苷酸中,存在两种主要的重复基元,分别为AAGGAG/CCTCTT和AAGAGG/CCTTCT,所占比例为12.78%。这些结果表明,萝卜 EST-SSRs出现频率较高,类型丰富,具有良好的多态性潜能和开发利用价值。使用primer3.0 批量设计,并以不同主导重复基元类型初步合成183对EST-SSR引物,以12份典型萝卜种质、1对亲本及其F1的基因组DNA为模板,对其有效性进行验证,筛选出有清晰扩增产物的引物159 对,其中具多态性引物64对;在试验亲本及其F1中表现多态性的引物30对,共显性引物27对。
287349 ESTs of radishes were downloaded from the NCBI database. 58105 ESTs without any redundant ESTs were obtained after pretreatment, which was 38 22.476 kb in length. Using MISA to search SSR loci, 3523 ESTs containing SSR loci were obtained, with a total of 3718 SSRs, with an average of one SSR per 10.39 kb. The results showed that there were 265 types of repeat motifs in the EST sequence of radish. Among them, two, three and six nucleotide repeats were the dominant repeat type. The AG / CT repeat motifs with up to 91.34% The main repeat type of trinucleotide was AAG / CTT with a ratio of 35.86%. There were two major repeat units in hexanucleotide, AAGGAG / CCTCTT and AAGAGG / CCTTCT The ratio is 12.78%. These results show that the radish EST-SSRs appear more frequently, with abundant types, and have good polymorphism potential and development and utilization value. Using primer3.0 batch design, 183 pairs of EST-SSR primers were preliminarily synthesized based on different dominant repeat motifs. The validity of the primers was verified by using 12 typical radish germplasms, 1 parental and F1 genomic DNA as template , And 159 pairs of primers with clear amplification products were screened out, of which 64 pairs were polymorphic; and 30 pairs of primers showed polymorphism in test parents and F1, and 27 pairs of codominant primers.