基于简化基因组测序技术的油茶SNP标记开发及指纹图谱构建

Development of SNP markers and construction of DNA fingerprint in teaoil Camellia germplasms based on simplified genome sequencing

【目的】基于简化基因组,挖掘油茶单核苷酸多态性(SNP)位点,筛选可用于油茶种质鉴定的简化SNP组合位点,构建SNP指纹图谱,建立一种快速准确鉴定广西油茶主栽良种苗木的SNP分子标记方法。【方法】以12个油茶无性系种质的两个重复共24份油茶标准样本为材料,采用ddRADseq流程进行文库构建,使用BWA将过滤后的测序数据比对到已发布的南荣油茶Camellia oleifera var.“Nanyongensis”参考基因组上,利用GATK进行SNP位点筛选,ANNOVAR软件进行SNP位点注释,STRUCTURE软件进行群体结果分析,使用PLINK进行主成分分析;利用R语言,使用条件随机筛选法(CRS),筛选能够区分出油茶种质的最简SNP组合,绘制指纹图谱。【结果】测序数据质量良好,可用于SNP分子标记位点的开发筛选。与参考基因组比对后,共获得622064个为SNP标记位点,其中无基因型缺失的SNP位点40147个,多态性信息含量(PIC)大于0.35的SNP位点2094个,前后60 bp碱基保守无变异的SNP位点共184个。最终筛选出可以将12个油茶无性系种质区分开的15个核心SNP标记位点组合,并以此绘制出SNP指纹图谱。【结论】基于简化基因组,建立了广西主要栽培油茶种质的SNP标记开发和指纹图谱绘制的方法,为苗期油茶种质的快速准确鉴定提供了理论依据和技术指导,助力规范油茶苗木市场,促进油茶产业健康发展。

【Objective】To establish a rapid and accurate method for identification of the major-cultivated tea-oil seedlings in Guangxi,we screened the single nucleotide polymorphism(SNP)sites,developed the SNP markers and drew the fingerprint map based on simplified genome sequencing.【Method】Two replicates of 12 tea-oil Camellia germplasms were used as samples.The library was constructed using double digest restriction site-associated sequencing(ddRADseq)and the filtered data was mapped to the released reference genome data of Camellia oleifera var.“Nanyongensis”using BWA.GATK was used for SNP site screening,ANNOVAR software was used for SNP site annotation,STRUCTURE software was used for population analysis,and PLINK was used for principal component analysis.Using R language,conditional random screening(CRS)was used to select the simplest SNP combinations that could distinguish tea-oil Camellia germplasms.【Result】The sequencing data was of good quality and could be used for the development and screening of SNP molecular marker sites.After sequence alignment with the reference genome,a total of 622064 SNP sites were obtained,among which 40147 SNP sites were without genotype deletion.Furthermore,there were 2094 SNP sites with polymorphism information content(PIC)value greater than 0.35,184 of which were with no base variations from 60 bp upstream to 60 bp downstream of the SNP sites.Finally,15 core SNP sites that could distinguish 12 tea-oil Camellia germplasms were screened out,and the fingerprint map was figured out.【Conclusion】The method about SNP marker development and fingerprint illustration for Guangxi main tea-oil Camellia germplasms was established based on simplify genome sequencing,providing theoretical and technical assistance for distinguishing the germplasms fast and accurately,ultimately normalizing the tea-oil Camellia seedling market and promoting the sound development of tea-oil Camellia industry.