该研究利用基于全基因组限制性酶切位点简化基因组测序技术(RAD-seq技术),开发濒危植物羊踯躅(Rhododendron molleG.Don)全基因组SSR标记,并对3个群体共63份羊踯躅材料进行验证鉴定,为进一步研究羊踯躅的遗传多样性和群体遗传结构以及保护利用提供技术支持.结果显示:(1)羊踯躅基因组测序获得原始数据7.653G bp,过滤后为7.513G bp;经组装发现,羊踯躅171.534Mbp的基因组分布在498 252 contigs中.(2)通过SSR检测,在11 961SSR位点中获得了11 687对SSR分子标记,并且二核苷酸为基序的重复类型最丰富,达51.76％.(3)随机选取128对SSR标记在6个羊踯躅株系中进行PCR扩增,获得20对高多态性的SSR标记.(4)用所选的20对多态性SSR标记对3个群体共63份羊踯躅材料进行验证分析发现,这些多态性SSR标记位点的等位基因数为4～16个,期望杂合度(He)为0.489～0.908.研究表明,羊踯躅的SSR丰度适中,且二核苷酸为羊踯躅中最丰富的重复序列,该实验进一步证明RAD-seq技术是一种经济有效的基因测序方法,实验中开发的SSR引物将有助于进一步研究羊踯躅和其他近缘种的群体结构和多样性.%The study set of simple sequence repeat (SSR) markers were developed using Restriction site Associated DNA (RAD) approach and Illumina paired-end sequencing to the whole genome of endangered plants Rhododendron molle G.Don.And it would provide technical support for further investigation of population structure and diversity of R.molle and other congener species from the evaluation of 63 individuals from three different populations.The results showed that:(1) the sequencing output generated a FASTQ file size of raw reads 7.653G bp,and the clean reads was to 7.513G bp.About 171.534 M bp of R.molle genome distributed over 498 252 contigs were obtained upon assembly of sequencing data.(2) After filtering and SSR detection,a final set of 11 687 simple sequence repeats with primers were obtained from 11 961 microsatellite loci.The di-nucleotides motif unit were the most abundant (51.76％) repeat sequences.(3) 128 microsatellite markers were selected randomly to detect the polymorphism in 6 different individuals of R.molle,from which 20 polymorphic primers were identified.(4) Then the characteristics and availability of polymorphic primers were further evaluated in 63 individuals from three different populations.The number of alleles per locus ranged from 4 to 16,expected heterozygosity (He) values ranged from 0.489 to 0.908,respectively.Our result showed that the abundance of SSR existed in R.molle was moderate and the di-nucleotides were the most abundan repeat sequences.It further confirms that the RAD-seq method is an efficient and cost-effective means for SSR discovery,and these polymorphic microsatellite markers developed in this study will be useful for further investigation of population structure and diversity of R.molle and other congener species.
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