厚朴转录组SSR标记的开发及功能分析

Development and function analysis of SSR molecular markers in Magnolia officinalis transcriptome

目的分析厚朴转录组中的SSR位点,并设计引物初步验证,对含SSR的序列进行功能分析,为厚朴分子辅助标记育种和资源保护提供了有利工具。方法将通过厚朴高通量转录组测序获得的Unigene序列进行SSR位点挖掘,利用Primer.03进行引物设计,随机挑选45对引物进行PCR,并利用Blast软件对含有SSR的Unigene进行功能分析。结果在16 369条厚朴转录组序列中共获得8 635个SSR位点,出现频率为52.75%。SSR序列中单核苷酸、二核苷酸和三核苷酸为优势重复类型,重复比例最高为10次,主导重复基元类型为A/T(47.16%)、AG/CT(31.74%)、AAG/CTT(6.53%)。45对引物中22对(48.89%)可以扩增出预期大小的条带。含SSR的Unigene与能量和氧化还原等代谢过程,以及RNA转运、剪接体和植物激素信号转导等通路有关。结论厚朴高通量转录组序列的SSR位点具有类型丰富、特异性强和潜能高等特点,同时SSR序列的功能分析将为厚朴基因挖掘和分子标记辅助育种提供有利的工具。

Objective SSR markers, primers and unigene function in the transcriptome of Magnolia officinalis were analyzed in this study to provide powerful tools for molecular marker-assisted breeding and resources protection in this plant. Methods A total of 16 369 unigenes obtained by transcriptome sequencing were used to explore SSR. SSR primers were designed by Primer.0 3 and 45 pairs were randomly selected for PCR. The gene function analysis of SSR unigene were obtained by Blast. Results A total of 8 635 SSR were identified, accounting for 52.75%. Among them, mononucleotide, dinucleotide and trinucleotide type were the dominant types, and the type which repeated for 10 times was the most. Additionally, the dominant repeat types were A/T, AG/CT and AAG/CTT, and the distribution frequencies were 47.16%, 31.74%, and 6.53%. Twenty-two pairs of 45 primers produced fragments with expected molecular size. Unigenes containing SSR were annotated to the process of energy and redox reaction, RNA transport, spliceosome and plant hormone signal transduction metabolic pathway. Conclusion The SSR markers in the transcriptome of M. officinalis show rich type, strong specificity and high potential, which will benefit to the candidate gene mining and marker-assisted breeding.