基于SLAF-seq技术的白皮松SNP分子标记开发

Development of SNP molecular markers of Pinus bungeana based on SLAF-seq technology

【目的】在白皮松全基因组范围内开发大量特异性SNP分子标记,为白皮松关键基因定位、分子标记辅助选择和种质资源评价提供足够多的分子标记资源。【方法】本研究以5个群体的共52份白皮松资源为材料,选择火炬松基因组为参考基因组,利用特异性位点扩增片段测序技术(SLAF-seq),在多态性SLAF标签上开发大量特异性SNP位点,并过滤出一批高质量SNP位点用于白皮松不同群体的遗传多样性分析。【结果】通过序列对比分析,共获得23597049个SLAF标签,其中具多态性的SLAF标签有370659个,共开发得到1291290个白皮松群体SNP。在缺失率小于20%、次要等位基因频率(MAF)大于5%的条件下,对所有SNP位点进行过滤,共得到346840个高一致性的白皮松群体SNP,占SNP总量的26.9%,其中包含9个仅在北京鹫峰(JF)群体中存在变异的SNP位点、148个仅在陕西蓝田(LT)群体中存在变异的SNP位点、425个仅在甘肃麦积山(MJS)群体中存在变异的SNP位点、1466个仅在陕西午子山(WZS)群体中存在变异的SNP位点、4个仅在山西柏洼山(BWS)群体中存在变异的SNP位点。基于过滤后的346840个SNP分子标记在5个白皮松群体中进行的遗传多样性分析表明,白皮松不同群体间的遗传多样性存在显著差异,其中MJS和WZS群体的遗传多样性水平相对较高,JF群体的遗传多样性水平相对较低。【结论】研究结果表明,利用SLAF-seq技术可以实现全基因组范围内大量SNP标记位点的开发,且开发的SNP标记在白皮松不同群体中表现出较为丰富的遗传多态性,为白皮松种质资源鉴定、QTL定位、遗传连锁图谱构建以及重要性状的关联分析等研究奠定了基础,对今后白皮松种质资源保护和分子标记辅助选择育种具有重要意义。

[Objective]This paper aims to develop a large number of specific SNP molecular markers in the whole genome of Pinus bungeana,and provide enough resources of molecular markers for key gene mapping,marker assisted selection and germplasm resource evaluation of P.bungeana.[Method]In this study,52 P.bungeana resources from five populations were used as materials,and the genome of P.taeda was selected as reference genome.A large number of specific SNP sites were developed on polymorphic SLAF tags using specific length amplification fragment sequencing(SLAF-seq),and a batch of high-quality SNP sites were filtered out for genetic diversity analysis of different populations of P.bungeana.[Result]Through sequence comparative analysis,23597049 SLAF tags were obtained,including 370659 polymorphic SLAF tags and 1291290 SNPs of P.bungeana population were developed.Under the condition of missing rate was less than 20%and minor gene frequency(MAF)was greater than 5%,a total of 346840 SNPs with high consistency were obtained,accounting for 26.9%of the total SNPs,including 9 SNP loci with mutation only in Jiufeng,Beijing(JF)population,148 SNP loci with mutation only in Lantian,Shaanxi(LT)population,425 SNP loci with mutation only in Maiji Mountain,Gansu(MJS)population,1466 SNP loci with mutation only in Wuzi Mountain,Shaanxi(WZS)population,4 SNP loci with mutation only in Baiwa Mountain,Shanxi(BWS)population.Genetic diversities of 5 P.bungeana populations were analyzed based on 346840 SNPs,and the results showed that there were significant differences in genetic diversity among different populations of P.bungeana,among which the level of genetic diversity was relatively high in MJS and WZS population,and relatively low in JF population.[Conclusion]The results showed that SLAF-seq technology can be used to develop a large number of SNP markers in the whole genome,and the developed SNP markers showed abundant genetic polymorphisms in different populations of P.bungeana.The results of this study lay a foundation for the identification of germplasm resources,QTL mapping,construction of genetic linkage map and association analysis of important traits of P.bungeana,and are of great significance for the protection of germplasm resources and molecular marker assisted breeding of P.bungeana in the future.