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基于转录组序列的龙脑樟EST−SSR标记开发及通用性分析

Development of EST−SSR Markers and Generalizability Analysis of Cinnamomum officinarum Based on Transcriptome Sequence

  • 摘要: 利用MISA软件对通过转录组测序获得的187095条龙脑樟unigenes进行SSR位点检测,探讨SSR的分布规律及组成特征并设计引物,进行引物多态性和通用性分析。结果表明:在187095条unigenes中共搜索到90590个SSR位点,SSR出现频率为48.42%,平均1.99 kb出现1个SSR位点,含有SSR位点的unigenes共计68573个,SSR发生频率为36.65%。龙脑樟EST−SSR重复类型中除单核苷酸外,二核苷酸重复类型出现频率最高,其次是三核苷酸。在检测到的21777个二核苷酸重复SSR中,重复单元AG/CT(16.06%)数量最多,为优势重复单元,其次是AT/AT(5.37%)、AC/GT(2.53%)、CG/CG(0.09%)。三核苷酸中AAG/CTT(4.37%)出现频率最高,为优势重复单元。利用Primer3.0设计并合成118对SSR引物,有96对扩增成功(81.36%),其中14对呈现多态(11.86%),在5种不同化学类型中的平均多态性信息含量(PIC)为0.378,其中有3对引物属于高多态性位点。开发的14对EST−SSR引物在同属的油樟、牛樟、黄樟、阴香、猴樟等5个物种中的通用性为100%。综上所述,基于龙脑樟转录组开发的EST−SSR标记具有较高的多态性和通用性,该方法是可行的。

     

    Abstract: SSR loci were detected in 187095 unigenes of Cinnamomum officinarum obtained by transcriptome sequencing using MISA software to explore the distribution pattern and compositional characteristics of SSRs and design primers for primer polymorphism and generalization analysis. The results showed that 90590 SSR sites were found in 187095 unigenes, and the frequency of SSR occurrence was 48.42%, with an average of 1 SSR site in 1.99 kb. The total number of unigenes containing SSR sites was 68573, and the frequency of SSR occurrence was 36.65%. Cinnamomum officinarum EST-SSR repeat types had the highest frequency of occurrence of dinucleotide repeat types, followed by trinucleotide, in addition to mononucleotide. Among the 21,777 dinucleotide repeat SSRs detected, repeat unit AG/CT(16.06%) had the highest number and was the dominant repeat unit, followed by AT/AT(5.37%), AC/GT(2.53%), and CG/CG(0.09%). The trinucleotide AAG/CTT (4.37%) had the highest frequency of occurrence and was the dominant repeating unit. Primer3.0 was used to design and synthesize 118 pairs of SSR primers, 96 pairs were amplified successfully(81.36%), of which 14 pairs showed polymorphism(11.86%), and the average polymorphism information content(PIC) was 0.378 in 5 different chemotypes with 3 pairs of primers associated with highly polymorphic sites. The 14 pairs of EST−SSR primers developed in this study showed 100% generalizability in 5 species of the same genus, including C. longepaniculatum, C. kanehirae, C. parthenoxylon, C. burmanni and C. bodinieri. In conclusion, the EST-SSR markers developed based on the transcriptome of C. officinarum have high polymorphism and generalizability, and the method is feasible.

     

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