基于NCBI香葱转录组数据库的SSR标记开发

Development of SSR Markers in Allium fistulosum L.var.caespitosum Makino Based on NCBI Transcriptome Database

为了明确香葱转录组中SSR的总体分布特点,开发香葱EST-SSR标记用以香葱遗传多样性研究,本研究从NCBI数据库上获取葱属转录组数据(SRR3144570)并利用MISA软件筛选香葱转录组测序,共获得94805条转录本序列,检测出9511个SSR位点,SSR出现频率为10.03%。在搜索的6种SSR位点类型中,1~3个核苷酸重复类型为香葱SSR重复的主要类型(占98.63%),其中单核苷酸占55.80%、二核苷酸占21.32%、三核苷酸重复类型占21.51%。SSR重复基元主要有41种,其中以一核苷酸重复基元类型A/T(55.01%)、二核苷酸重复基元类型AC/GT(8.11%)和AT/AT(7.95%)频率最高。分析得到转录组SSR重复序列的长度在10~158 bp之间,平均长度为53.23 bp。根据这些SSR位点,利用Primer Premier 3.0进行引物开发,共设计出9492对SSR引物。随机选取200对引物,其中有197对能进行有效扩增,占选取SSR引物对的98.5%,同时37对引物在6份表型差异显著的香葱种质资源中呈现多态性。采用该37对引物对56份香葱种质鉴定多样性,发现在相似系数为0.68时,可将56份香葱种质分为4个类群,聚类结果与表型结果一致。以上结果表明,基于NCBI数据库香葱转录组测序开发的SSR标记丰富了香葱的分子标记数量,为香葱遗传多样性分析、种质资源鉴定提供充足可靠的标记来源。

In order to clarify the overall distribution of SSRs in transcriptome of Allium fistulosum L.var.caespitosum Makino,EST-SSR marker was developed to study the genetic diversity of it.In this study,chives transcriptome data(SRR3144570) were obtained from the NCBI database,and 94 805 transcriptome sequences were obtained by MISA software.A total of 9 511 SSR sites were detected,with the frequency of SSR being 10.03%.Among the 6 SSR types,1~3 nucleotide repeats played a dominant role,accounting for 98.63% of the total SSRs,while the single-nucleotide,dinucleotide and trinucleotide repeats were 55.80%,21.32% and 21.51% respectively.There were 41 types of SSR repeats,and among which the frequency of A/T(55.01%),AC/GT(8.11%),AT/AT(7.95%) were the highest.The length of repeated sequences ranged from 10 to 158 bp,with an average length of53.23 bp.According to these SSR sites,primers were developed by Primer Premier 3.0,and a total of 9 492 pairs SSR primers were designed.Among 200 randomly selected primers,197 pairs(98.5%) could be amplified effectively,among which 37 pairs showed polymorphism in 56 germplasm chives resources.The 37 pairs of primers were used to identify 56 chives,and it was found that 56 germplasm chives could be divided into 4 groups when the similarity coefficient was 0.68.The above results indicated that SSR markers developed based on NCBI database transcriptome sequencing of chives enriched the number of molecular markers,and provided sufficient and reliable markers for genetic diversity analysis and germplasm resource identification of chives.