[关键词]
[摘要]
目的 分析野三七Panax vietnamensis var. fuscidiscus(PVF)转录组中的SSR位点信息,并设计简单重复序列(SSR)引物,以期为野三七分子标记辅助育种提供有力工具。方法 利用MISA工具筛选了野三七转录组测序获得的126 758条unigenes,对其SSR位点信息进行了分析;在此基础上利用Primer3设计SSR引物,并随机选择30对SSR引物对13株不同来源的野三七进行多态性扩增分析。结果 在野三七的转录组中,共搜索到21 320个SSRs,分布于17 780条unigenes,SSR位点出现频率为16.82%。SSRs位点中二核苷酸重复是主要类型,占总SSRs的52.52%;其次是三核苷酸重复基序(28.08%)。SSRs所包含的重复基元中,二核苷酸重复基元AG/CT和AT/AT是优势重复基元类型,占总SSRs的46.25%。利用Primer3共设计出39 336对SSR引物。随机选择30对引物进行PCR扩增,其中29对(96.67%)扩增出清晰、可重复的条带,15对(50.00%)扩增条带表现出多态性。利用UPGMA作图,将13个材料分为2类。结论 野三七转录组中SSR位点出现频率高,类型丰富;大量的SSR为其遗传多样性分析和遗传图谱构建提供了丰富的候选分子标记。
[Key word]
[Abstract]
Objective The SSR loci information in the transcriptome of Panax vietnamensis var. fuscidiscus (PVF) was analyzed in this study to provide more powerful tools for molecular marker-assisted breeding in this plant. Methods Simple sequence repeats (SSR) loci were searched in all 126 758 unigenes by using MISA. SSR loci information was analized and SSR primers were designed by Primer3. Furthermore, 30 pairs of primers were randomly selected for the polymorphic analysis on 13 PVF plants collected from different habitats. Results A total of 21 320 SSRs were found in the transcriptome of PVF, distributed in 17 780 unigenes with the distribution frequency of 16.82%. Di-nucleotide repeat was the main type, accounted for as much as 52.52% of all SSRs, followed by tri-nucleotide repeat motif (28.08%). The dinucleotide repeat motifs of AG/CT and AT/AT were the predominant repeat types (46.25%). Using Primer3, a total of 39 336 pairs of SSR primers were designed. For validating the availability of those SSR primers, we randomly selected 30 pairs of primers for PCR amplification. Among them, 29 pairs of primers (96.67%) produced clear and reproductive bands, 15 pairs of primers (50.00%) showed polymorphism, and 13 PVF plants were divided into two groups by UPGMA. Conclusion There are numerous SSRs in PVF transcriptome with high frequency and various types, this will provide the abundant candidate molecular markers for genetic diversity study and genetic map for this plant.
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[基金项目]
国家自然科学基金项目(81303164);国家“十二五”支撑计划课题(2011BAI13B01)