利用转录组测序开发甘蔗SNP分子标记

SNP molecular marker development based on sugarcane transcriptome sequencing

【目的】利用转录组测序开发甘蔗SNP分子标记,为甘蔗建立一种高效和低成本的分子标记开发方法。【方法】以40个甘蔗栽培品种为试验材料,使用高通量测序平台获得其转录组数据,经质控后将其匹配到参考基因组,然后使用GATK软件检测和过滤SNP分子标记,并使用snpEff对SNP进行注释及统计分析。利用这些SNP分子标记进行系统发生分析、主成分分析和群体结构分析。最后,开发KASP标记并随机验证其有效性。【结果】转录组数据经质控后平均每个样本获得6.5 Gb的序列。通过变异分析和多重过滤筛选后,共获得220397个注释到染色体上的双等位基因SNP位点,平均密度为3 SNP/kb。SNP类型及分布特征分析结果表明,编码区错义突变与沉默突变的比值(N/S)为1.05,转换类型和颠换类型的比值(Ts/Tv)为1.89,杂合SNP占38.66%~49.91%,位于转录本的SNP比例为44.74%。系统发生分析、主成分分析和群体结构分析结果均表明,甘蔗栽培品种遗传来源较为单一,但群体分化较大。开发了11176个KASP分子标记,并随机合成25组KASP引物进行多态性验证,共有23组(92%)引物能检测到扩增产物,7组(28%)在40个甘蔗材料中呈现多态性,进一步验证了这些标记有效性。【结论】与传统SSR分子标记和基于GBS(Genotyping-by-sequencing)简化基因组测序的SNP分子标记开发方法相比,基于转录组测序的甘蔗SNP分子标记开发方法在保证质量和数量的情况下能获得更大比例的有效SNP,更有利于发掘功能SNP位点,为甘蔗分子SNP标记的开发提供了新的途径。

【Objective】To develop SNP molecular marker of sugarcane(Saccharum officinarum spp.)based on transcriptome sequencing,so as to establish an efficient and low-cost molecular marker development method.【Method】Forty sugarcane varieties were taken as experiment materials. Transcriptomes of these materials were sequenced by high throughput sequencing platform,and they were mapped to reference genomes after quality control. GATK software was used to identify and filter SNP molecular marker,and SNP annotation and statistical analysis were performed using snpEff.Phylogenetic analysis,principal component analysis and population structure analysis were conducted with the SNP markers. Finally,KASP marker was developed and its validity was verified.【Result】After quality control of transcriptome data,6.5 Gb clean reads per sample were generated on average. Through variation analysis and multiple filter screening,a final set of 220397 biallelic SNP on chromosome were identified,with an average density of 3 SNP/kb. SNP type and distribution characteristics analysis showed that the overall missense/silent(N/S)ratio was 1.05,transition/transversion(Ts/Tv)ratio was 1.89,heterozygous SNP was 38.66%-49.91% and 44.74% of them distributed in transcripts. Phylogenetic,principal component analysis and population structure analysis showed that there were great differences in sugarcane genome among individuals,nevertheless its genetic background was narrow. 11176 KASP primers were designed,and 25 of them were used to test the polymorphism and authenticity of identified SNP,23(92%)primers were able to amplify the target products,and 7(28%)were polymorphic in 40 sugarcane materials,which verified the validity of these markers for sugarcane diversity analysis.【Conclusion】Compared with conventional SSR molecular marker or SNP marker development method based on genotyping by sequencing(GBS),sugarcane SNP identified from transcriptome can generate a larger proportion of effective SNPs with good quality and quantity. Therefore,it is better to identify functional SNP sites,providing a new way for developing SNP markers of sugarcane.