柿属EST-SSR引物设计及遗传多样性研究

Study on EST-SSR primer design and genetic deversity of Diospyros L.

利用从NCBI上下载的9 474条EST(Expressed Sequence Tags)序列,设计并开发EST-SSR引物,用于柿属种质资源的遗传多样性研究。下载的初始序列拼接成1 390条重叠序列和4 097条无重复序列。用SSRHunter软件搜索到601条EST序列中含有540个SSR。SSR出现频率为10.96%,其中以2核苷酸为主。依据检出的SSR-ESTs序列设计SSR引物100对,对9个柿属(Diospyros L.)种质资源样品进行了PCR扩增,其中42对引物有扩增带,占设计引物的42%,其中30对引物有多态性,占可扩增引物的71.4%。同时发现引物15是美洲柿(雄)的特异引物;引物30是青化北野柿的特异标记;引物69和引物80为美洲柿的特异引物;引物99是无核软枣的特异引物。

The 9 474 ESTs of Diospyros L.in the database of NCBI were downloaded,then were used to design EST-SSR makers.The obtained makers were used to analyze diversity of 9 germplasm resources（Diospyros L.）.The 9 474 ESTs after some redundants and some low qualities were removed,finally assembled into 1 390 contigs and 4 097 singles.Then by using SSRHunter software,540 SSRs in 601 ESTs were found,accounting for 10.96% of ESTs,and most of them were dinucleotides.One hundred primer pairs were designed for polymorphism analysis in 9 genotypes（Diospyros L.）.Forty two primer pairs showed the amplification,accounting for 42% of designed primers,and 30 primer pairs showed polymorphisms,accounting for 71.4% of primers available.Meanwhile,P15 was special primer of D.virginiana（male）,P30 was special primer of north Qinhua-D.spp.,P69 and P80 were special primers of D.virginiana,P99 were special primers of D.lotus.