基于ddGBS的黑杨派杨树SNP位点挖掘

Development of SNP markers in section Aigeiros poplar based on ddGBS technology

【目的】基于双酶切测序基因分型(Double-digest genotyping by sequencing,ddGBS)技术,挖掘黑杨派特异性单核苷酸多态性(Single-nucleotide-polymorphism,SNP)位点信息,旨在为杨树SNP标记开发、种质指纹图谱构建、遗传多样性分析及重要经济性状关联分析等研究提供位点信息。【方法】以46份黑杨派无性系种质为材料,采用MseI+TaqaI双酶切基因组构建GBS文库并测序,使用Bowtie2将过滤后的序列数据比对到毛果杨参考基因组上,Stacks软件开发SNP位点,SnpEff软件注释SNP位点,R语言分析SNPs位点数目与染色体长度的相关性。【结果】共获得108 Gb数据,每个样本平均数据量为2.35 Gb;样本测序总序列数为218156221条,总碱基为62832509890 bp,样本序列数及其碱基长度的平均值分别为4742526条和1365924128 bp。Q30值为93.32%~94.44%,平均值为93.87%;GC含量为41.83%~44.96%,平均值为42.65%;碱基测序的平均错误率低于0.10%。这表明,GBS测序质量较高。序列比对至参考基因组的成功率为64.25%~73.53%,平均为69.89%;位点测序深度为12.40~24.70,平均值为18.40。过滤后共获得131194个SNP位点,主要位于基因上、下游和转录区,其中,C/T和A/G变异类型最多,转换类型明显高于颠倒类型。98.41%(129104)SNPs位点能成功定位到19条染色体上,位点平均分布密度为1/3188 bp;SNPs位点数目与染色体长度呈极显著线性正相关。【结论】GBS技术能够有效地挖掘SNP位点信息,可用于大规模杨树SNP标记开发,为进一步开展杨树种质指纹图谱构建、遗传多样性分析以及重要经济性状关联分析等研究奠定了基础。

【Objective】The specific single-nucleotide-polymorphism(SNP)markers in section Aigeiros poplar were excavated by Double-digest genotyping by sequencing(ddGBS)technology,in order to provide genetic information for the fingerprint construction,genetic diversity analysis and association analysis of important economic traits of Populus.【Method】The GBS library was constructed and sequenced after double enzyme digestion of MseI+TaqaI for 46 clonal samples.The filtered sequence data were aligned to the reference genome P.trichocarpa by using Bowtie2.Stacks was used to analyze the sequencing data and develop high-quality SNP markers.The SNP sites were annotated by SnpEff,and the correlation between the number of SNPs and chromosome length was analyzed by R language.【Result】A total of 108 Gb reads data were obtained,and the average data size of each sample was 2.35 Gb.The total number of sequenced fragments was 218156221,and the total base was 62832509890 bp.The average number and length of the sequenced fragments were 4742526 and 1365924128 bp,respectively.The Q30 value was from 93.32%to 94.44%,with an average of 93.87%.The content of GC was 41.83%~44.96%,with an average of 42.65%.The average error rate of base sequencing was less than 0.10%.This indicated that ddGBS can generate high-quality SNPs data.The rate of sequencing fragment alignment to the P.trichocarpa genome was 64.25%~73.53%,with an average of 69.89%.The sequencing depth was 12.40~24.70,with an average of 18.40.SNPs were mainly located within the upstream,downstream and transcriptional regions of genes.Among them,C/T and A/G variant types were the most,and the conversion type was significantly higher than the inversion type.98.41%(129104)SNPs were successfully located on 19 chromosomes,with an average distribution density of 1/3188 bp.The number of SNPs was extremely significantly correlated with chromosome length.【Conclusion】GBS technology could effectively develop SNP sites,which can be used for the large-scale development of Populus SNP markers,providing a foundation for further research on germplasm fingerprint construction,genetic diversity analysis and association analysis of important economic traits of Populus.