文冠果转录组SSR特征分析及EST-SSR标记开发

摘要

[目的]利用高通量测序技术分析文冠果转录组中SSR位点分布类型与特征,并设计开发EST-SSR标记,为文冠果的遗传多样性分析提供标记资源.[方法]利用MISA软件筛选文冠果51 867条unigenes中的SSR(2～6 bp)位点,统计分析SSR位点的分布特征、发生频率和平均分布距离.利用Prime 3在线软件随机设计合成60对EST-SSR标记,用我国7个省份的16份文冠果材料对所设计的标记进行多态性筛选;用Popgene 32软件分析标记的等位基因数(NA)、观测杂合度(HO)、期望杂合度(HE)和多态性信息含量(PIC)等.[结果]从文冠果unigenes中筛选出6 707个SSR位点,其平均发生频率为12.93％,平均分布距离5.38 kb,平均长度17.30 bp,其中2、3核苷酸重复单元的发生频率分别为6.24％和4.93％.设计的60对标记中有47对能扩增出与目的片段长短相符的产物,其中14对多态性较高,共扩增到52个等位基因,Ho和HE分别为0～0.813和0.353～0.762,PIC为0.283～0.701.[结论]文冠果转录组SSR重复单元以2、3核苷酸重复为主;新开发的14个EST-SSR标记可作为文冠果种质资源多样性分析的标记资源.%[Objective] High-throughput sequencing technology was used to analyze the distribution types and characteristics of SSRs in transcriptome of Xanthoceras sorbifolia and EST-SSR markers were developed to provide basis for genetic diversity and evolutionary origin analysis of X.sorbifolia.[Method] The number,distribution characteristics,frequency of occurrence and mean distribution distance of SSRs screened by MISA from 51 867 unigenes of X.sorbifolia were counted and analyzed.Sixty EST-SSR primers were designed by on line software Prime 3 and 16 germplasms of X.sorbifolia from seven provinces of China combined with 6％ modified polyacrylamide gel electrophoresis were used for polymorphism screening of these markers.The number of alleles,observed heterozygosity and expected heterozygosity of these markers were analyzed using Popgene 32.[Result] A total of 6 707 SSRs were identified from the X.sorbifolia unigenes.The average frequency of occurrence,distribution distance and average SSR length of these SSRs were 12.93％,5.38 kb and 17.30 bp.The frequencies of occurrence of dinucleotide and trinucleotide repeats were 6.24％ and 4.93％,respectively.Among the 60 markers,47 amplified the targeted fragments and 14 markers appeared to be polymorphic with a total of 52 alleles,the observed and expected heterozygosity ranged from 0 to 0.813 and 0.353 to 0.762,respectively.The PIC values varied from 0.283 to 0.701.[Conclusion] The SSR repeat units in transcriptome of X.sorbifolia were mainly dinucleotide and trinucleotide repeats and the 14 EST-SSR markers developed in this work are useful for genetic diversity a-nalysis in X.sorbi folia.