Liu Jun-Jun, Sniezko Richard A, Sturrock Rona N, Chen Hao
BMC Plant Biol. 2014 Dec 30;14:380. doi: 10.1186/s12870-014-0380-6.
Western white pine (WWP, Pinus monticola Douglas ex D. Don) is of high interest in forest breeding and conservation because of its high susceptibility to the invasive disease white pine blister rust (WPBR, caused by the fungus Cronartium ribicola J. C. Fisch). However, WWP lacks genomic resource development and is evolutionarily far away from plants with available draft genome sequences. Here we report a single nucleotide polymorphism (SNP) study by bulked segregation-based RNA-Seq analysis.
A collection of resistance germplasm was used for construction of cDNA libraries and SNP genotyping. Approximately 36-89 million 2 × 100-bp reads were obtained per library and de-novo assembly generated the first shoot-tip reference transcriptome containing a total of 54,661 unique transcripts. Bioinformatic SNP detection identified >100,000 high quality SNPs in three expressed candidate gene groups: Pinus highly conserved genes (HCGs), differential expressed genes (DEGs) in plant defense response, and resistance gene analogs (RGAs). To estimate efficiency of in-silico SNP discovery, genotyping assay was developed by using Sequenom iPlex and it unveiled SNP success rates from 40.1% to 61.1%. SNP clustering analyses consistently revealed distinct populations, each composed of multiple full-sib seed families by parentage assignment in the WWP germplasm collection. Linkage disequilibrium (LD) analysis identified six genes in significant association with major gene (Cr2) resistance, including three RGAs (two NBS-LRR genes and one receptor-like protein kinase -RLK gene), two HCGs, and one DEG. At least one SNP locus provided an excellent marker for Cr2 selection across P. monticola populations.
The WWP shoot tip transcriptome and those validated SNP markers provide novel genomic resources for genetic, evolutionary and ecological studies. SNP loci of those candidate genes associated with resistant phenotypes can be used as positional and functional variation sites for further characterization of WWP major gene resistance against C. ribicola. Our results demonstrate that integration of RNA-seq-based transcriptome analysis and high-throughput genotyping is an effective approach for discovery of a large number of nucleotide variations and for identification of functional gene variants associated with adaptive traits in a non-model species.
西部白松（WWP，Pinus monticola Douglas ex D. Don）因其对入侵性疾病白松疱锈病（WPBR，由真菌Cronartium ribicola J. C. Fisch引起）高度敏感，在森林育种和保护方面备受关注。然而，西部白松缺乏基因组资源开发，且在进化上与已有基因组草图序列的植物相距甚远。在此，我们报告一项基于混合分离群体RNA测序分析的单核苷酸多态性（SNP）研究。
利用一批抗性种质构建cDNA文库并进行SNP基因分型。每个文库获得约3600万至8900万个2×100碱基对的读段，从头组装生成了首个梢尖参考转录组，共包含54,661个独特转录本。生物信息学SNP检测在三个表达的候选基因组中鉴定出超过100,000个高质量SNP：松树高度保守基因（HCG）、植物防御反应中的差异表达基因（DEG）和抗性基因类似物（RGA）。为评估电子SNP发现的效率，利用Sequenom iPlex开发了基因分型检测方法，其揭示的SNP成功率为40.1%至61.1%。SNP聚类分析始终揭示出不同的群体，每个群体由西部白松种质收集中通过亲本溯源确定的多个全同胞种子家族组成。连锁不平衡（LD）分析确定了六个与主基因（Cr2）抗性显著相关的基因，包括三个RGA（两个NBS-LRR基因和一个类受体蛋白激酶-RLK基因）、两个HCG和一个DEG。至少一个SNP位点为跨西部白松群体的Cr2选择提供了优良标记。
西部白松梢尖转录组和那些经过验证的SNP标记为遗传、进化和生态研究提供了新的基因组资源。那些与抗性表型相关的候选基因的SNP位点可作为定位和功能变异位点，用于进一步表征西部白松对C. ribicola的主基因抗性。我们的结果表明，基于RNA测序的转录组分析与高通量基因分型相结合是发现大量核苷酸变异以及鉴定与非模式物种适应性性状相关的功能基因变异的有效方法。
