| 摘要: |
| 【目的】开发适用于花椒（Zanthoxylum bungeanum）和竹叶花椒（Zanthoxylum armatum）的EST-SSR引物，为花椒属物种的鉴定及遗传多样性探究提供分子标记。【方法】对花椒和竹叶花椒分别进行转录组测序(RNA-seq)，基于得到的EST序列开发SSR引物。在2种花椒的EST-SSR引物中分别随机挑选60对引物，用12份材料（4份花椒和8份竹叶花椒）为样本，利用琼脂糖凝胶电泳验证其特异性。从有特异性条带的引物中，再随机选取花椒和竹叶花椒SSR引物各15对，选取6个（2份花椒和4份竹叶花椒）样本，利用聚丙烯酰胺银染电泳进行多态性检测。【结果】花椒共有64 944条Unigene，碱基对长度共54 073 890 bp，含有12 746个SSR位点，分布在10 595条Unigene上，SSR位点出现频率为19.63%，平均分布距离4.24 kb。竹叶花椒Unigene共75 669条，碱基对长度58 975 053 bp，共15 096个SSR位点，分布在12 612条Unigene上。SSR位点出现频率为19.95%，平均分布距离为3.91 kb。60对花椒引物中，有46对成功扩增出特异性条带，扩增效率76.67%；60对竹叶花椒引物中，有41对扩增出特异性条带，扩增效率68.33%。花椒和竹叶花椒的特异性条带大小在100~300 bp，主要集中在150~250 bp。多态性验证试验中，15对花椒引物中有12对产生了多态性条带，多态率80.00%；15对竹叶花椒引物中则有14对产生了多态性条带，多态率93.33%。【结论】基于花椒和竹叶花椒转录组高通量测序结果开发的EST SSR分子标记引物，具有较明显的特异性和多态性。 |
| 关键词: 转录组测序 花椒 EST-SSR 引物开发 |
| DOI： |
| 分类号: |
| 基金项目:四川省农业科技成果转化项目（16NZ0067） |
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| Development of EST-SSR markers of Zanthoxylum species based on RNA sequencing |
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DENG Yangchuan,XIANG Li,SU Yanyan,et al
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| Abstract: |
| 【Objective】To develope suitable primers for Zanthoxylum bungeanum and Zanthoxylum armatum,which is aim to provide molecular markers for genetic diversity and species identity of Zanthoxylum.【Method】Sequences of Z. bungeanum and Z. armatum were determined by RNA-seq and SSR primers were developed based on obtained EST sequences.Randomly 60 pairs primers were selected from the EST-SSR primers of two species and 12 samples (4 Z. bungeanum and 8 Z. armatum) were used to detect specificity by agarose gel electrophoresis.Then,15 pairs of primers with specificity strands were randomly selected from the two species and polymorphism of 6 samples of Z. bungeanum and Z. armatum was detected by polyacrylamide gel electrophoresis (PAGE).【Result】In Z. bungeanum,there were 64 944 Unigene in total and length of base pair was 54 073 890 bp including 12 746 SSR loci on 10 595 Unigene.The occurrence frequency of SSR was 19.63% with average distance of 4.24 kb.In Z.armatum,here were 75 669 Unigene including 15 096 SSR loci on 12 612 Unigene.The length of base was 58 975 053 bp,occurrence frequency of SSR was 19.95%,and average distance was 3.91 kb.There were 46 pairs of primers of Z. bungeanum and 41 pairs of primers of Z. armatum with specific bands and the ratio was 76.67% for Z. bungeanum and 68.33% for Z. armatum.The lengths of bands were 100-300 bp and mainly distributed in 150-250 bp.A total of 12 pairs of primers of Z. bungeanum produced polymorphic bands with polymorphism ratio of 80.00%,and 14 pairs of Z.armatum produced polymorphic bands with polymorphism ratio of 93.33%.【Conclusion】The screened EST-SSR primers for Z. bungeanum and Z. armatum based on RNA seq showed significant specificity and polymorphism. |
| Key words: RNA-sequencing Zanthoxylum bungeanum EST-SSR primer development |
