论文部分内容阅读
本研究以宁薯4号叶、根和匍匐茎茎尖为材料构建链特异性文库进行转录组测序并对测序数据重新拼接。应用SSR检索软件从74.8 Mb测序数据中(51 006 Unigenes)发掘到1 867个SSR位点,平均发生频率为1/41 kb。重复基序中以三核苷酸、六核苷酸和二核苷酸为主,分别占总SSR位点数的58.2%、12.8%和10.7%;56种三核苷酸重复基序中以(GAA/TTC)n为主,占总SSR重复基序比例的4.1%;160种六核苷酸重复基序中以(AGCCAC/GTGGCT)n、(AGATGA/TCATCT)n、(GCAGGT/ACCTGC)n、(AAACCC/GGGTTT)n和(GGTGGA/TCCACC)n为主,分别占总SSR重复基序比例的0.2%。对1 867个SSR位点序列设计了1 692对EST-SSR引物,从中抽取100对引物进行有效性和多态性验证。结果表明,76对(76%)引物能扩增出预期目标片段;40对能扩增出目标片段的EST-SSR引物中有19对引物在分析47份马铃薯种质资源遗传多样性中表现出多态性差异,多态性引物比例为47.5%;共检测到80个等位基因,平均每对引物检测到4.2个等位基因;多态性信息含量(PIC)值变化在0.343~0.819之间,平均为0.658,属于高多态位点。本研究可为分析马铃薯种质资源遗传结构、构建精细遗传图谱、克隆定位功能基因和分子标记辅助育种等研究提供了标记基础。
In this study, we constructed a strand-specific library of leaves, roots and stolons of Ningshu 4 for transcriptome sequencing and reassembled the sequencing data. A total of 1 867 SSR loci were found from the 74.8 Mb sequencing data (51 006 Unigenes) using SSR search software, with an average frequency of 1/41 kb. The repeat motifs were dominated by trinucleotide, hexanucleotide and dinucleotide, accounting for 58.2%, 12.8% and 10.7% of the total number of SSR loci, respectively. Of the 56 trinucleotide repeat motifs, GAA / TTC) n, accounting for 4.1% of the total SSR repeat motifs. Among the 160 hexanucleotide repeat motifs, AGCCAC / GTGGCT n, (AGATGA / TCATCT) n, (GCAGGT / ACCTGC) n , (AAACCC / GGGTTT) n and (GGTGGA / TCCACC) n, respectively, accounted for 0.2% of the total SSR repeat motifs. A total of 1 692 EST-SSR primers were designed for the 1 867 SSR loci. 100 pairs of primers were selected for validation and polymorphism. The results showed that 76 pairs (76%) of the primers could amplify the expected target fragment. Among the 40 pairs of EST-SSR primers that could amplify the target fragment, 19 pairs of primers showed the genetic diversity of 47 potato germplasms Polymorphism, the proportion of polymorphic primers was 47.5%; 80 alleles were detected, with an average of 4.2 alleles per primer pair; polymorphism information content (PIC) value of 0.343 ~ 0.819 Between, an average of 0.658, belonging to high polymorphic loci. This study can provide a basis for the analysis of genetic structure of potato germplasm resources, construction of fine genetic map, cloning and location of functional genes and molecular marker-assisted breeding.