Characterization and development of SSR markers from transcriptome sequence of Bretschneidera sinensis Hemsl.
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摘要: 基于伯乐树（Bretschneidera sinensis Hemsl.）转录组测序数据，采用生物信息学方法分析该物种转录组中SSR位点的分布特征，利用TP-M13-SSR （Simple sequence repeat with tailed primer M13）方法检测12株无亲缘关系样本SSR引物位点的多态性。结果显示，8772个SSR位点分布在6732条unigene序列上，SSR位点出现频率和分布密度分别为25.48%和1/4.39 kb。不同重复基元类型中，单核苷酸、2核苷酸和3核苷酸为主要SSR基元类型，SSR数分别占SSR总数的53.72%、29.42%和15.42%。其中，2核苷酸基元类型中，AG/CT基元出现的SSR位点最多，占总数的22.21%；3核苷酸基元类型中，AAG/CTT基元分布的SSR位点最为丰富，ACC/GGT和ATC/ATG次之。120对自主开发的SSR引物中，有68对扩增产物检测到目的片段，其中有49对引物位点呈现多态性，多态位点比例为72.06%；单个位点等位基因数目为2 ～ 7，平均单个位点等位基因数为3.55。研究结果表明基于转录组序列开发的伯乐树SSR位点多态性较高。Abstract: Bretschneidera sinensis Hemsl. is a model plant for research on angiosperm phylogeny, paleogeography, and paleoclimatology. To develop simple sequence repeat(SSR) markers for studies on population genetics of B. sinensis, candidate SSR loci were searched based on high-throughput sequencing and characterized using bioinformatics. Furthermore, SSR primer locus polymorphism in 12 genotypes without affinities was detected by simple sequence repeats with tailed primers. A total of 8772 SSR loci were obtained, which were distributed in 6732 unigenes. The appearance frequency and distribution density of SSR loci were 25.48% and 1/4.39 kb, respectively. In all SSR loci, single nucleotides, dinucleotides, and trinucleotides were the main repeat motif types, accounting for 53.72%, 29.42%, and 15.42% of total SSR loci, respectively. AG/CT was the most abundant dinucleotide repeat motif, accounting for 22.21% of total SSR loci. AAG/CTT was the most abundant trinucleotide repeat motif, followed by ACC/GGT and ATC/ATG. Of the 120 primers, the target fragment was detected in 68 pairs of primer products and 49 were successfully amplified in all tested genotypes and were verified as polymorphic, with the proportion of polymorphic loci being 72.06%. The number of alleles per locus ranged from 2 to 7 (average of 3.55). These results suggest that the SSR loci developed from on transcriptome sequence of B. sinensis were highly polymorphic. Analysis of the distribution characteristics of the high-throughput transcriptome sequence of B. sinensis could be helpful in gene mining, and these new polymorphic SSR markers should provide a valuable molecular tool for investigating the genetic diversity, population structure, phylogeographic history, and construction of core collection for ex situ conservation of this species.
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Keywords:
- Bretschneidera sinensis /
- Transcriptome /
- SSR marker /
- Polymorphism
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