基于SLAF-seq技术的不同类型越橘遗传变异和群体结构研究

Genetic variation and structure analysis of different type blueberries(Vaccinium spp.) based on SLAF-seq technology

【目的】利用越橘属栽培品种及野生资源在全基因组范围内筛选差异SNP分子标记,分析其遗传变异,了解不同类型群体间的遗传变异、基因交流、群体结构,为越橘种质创新提供遗传背景依据。【方法】以越橘属9个种的62份种质资源为材料,利用特异性位点扩增片段测序技术(SLAF-seq)获得的基因组SNP标记比对到品种Draper参考基因组上,进行供试材料遗传研究。【结果】测序获得169.87 Mb reads数据,平均Q_(30)值94.99%,包含172 513个多态性SLAF标签,筛选到高质量SNP标记4 133 595个,均匀分布在越橘12条染色体上。基于SNP标记的系统进化树准确反映了供试野生、北高丛和南高丛类群遗传差异以及品种间系谱遗传关系,反映出野生类型与栽培品种遗传差异显著,供试野生种具有独立的遗传背景且种间无基因交流发生,但野生种到栽培种存在单向的基因流;栽培品种类群存在明显的亚群结构,亚群间存在普遍的基因交流;半高丛品种北陆和南高丛品种军号与北高丛类型紧密聚类,矮丛品种美登、半高丛品种北村、黑珍珠遗传背景倾向于野生种。【结论】栽培品种近亲杂交和骨干亲本高频率使用导致品种间基因交流频繁、同质性高、遗传基础狭窄,野生种具有独立的遗传背景,但存在野生种到栽培种的单向基因流,应加大野生资源发掘利用以拓宽越橘遗传基础。

【Objective】Blueberry belongs to the genus Vaccinium of family Ericaceae,and has recently received much attention as blueberries have high contents of anthocyanins,flavonols,procyanidins and other types of phenolic compounds,which can improve night vision,increase anti-cancer activity and reduce the risk of heart disease.The consumption and cultivation acreage of blueberries is thus continually increasing worldwide.By 2020,the total cultivated area of blueberries in the world was 2 056 700hm^(2) and 66 400 hm^(2) in China,with a total production of 347 200 t.Blueberries are very rich in species,including the tetraploid highbush blueberries(Vaccinium corymbosum),lowbush blueberries(V.augustifolium) and hexaploid rabbiteye blueberries(V.ashei),and the diploid species V.uliginosum,V.vitisidaea,V.macrocarpon etc.However,their inter-or introgenetic background and phylogenetic relationship are still unclear,resulting in an unclear background of blueberry genetic diversity.The study could improve the understanding of the genetic basis of blueberries,by means of the genome-wide SNP markers to recognize the genetic variation and gene introgression between different blueberry populations.【Methods】In this study,a total of 62 germplasm resources of 9 blueberry species were used as materials.The specific amplified fragment sequencing technology(SLAF-seq) was used to facilitate sequencing and accompanied by the reference genome of southern highbush blueberry draper.The sequencing reads were compared with the reference genome using BWA software(v 0.7.17),and data quality was controlled using GATK(v 4.1.7.0) and SAM tools,respectively;the intersection of SNP markers obtained by the two methods was used as the final reliable SNP marker dataset.Based on the final reliable SNP,the vcf tools software(v 0.1.16) was used to remove markers with a deletion rate over 20% or a MAF less than 0.05.The SNP hylo software(v 20140701) was used to filter SNPs with a depth of less than 3 and a deletion rate of less than 20% to obtain SNPs for further data statistics and analysis.First,the phylogenetic tree was drawn using the obtained SNP markers,and the validity and accuracy of the markers were verified by combining the phylogenetic tree and the traditional classification of the test materials.Then,the genetic structure of the test materials was analyzed using the Admixture software to understand the genetic background and individual genetic composition.To display the genetic background differences and genetic relationships of the test materials more intuitively,the SNP Relate(v1.30.1) software package in R was used for Principal Component Analysis(PCA),and the ggplot2(v 3.3.6) was used for visualization.【Results】We harvested 169.87 MB reads data,with an average Q_(30) value of 94.99% and an average GC content of 40.24%,and detected a total of 319590 SLAF tags in 62 blueberry materials.172 513 polymorphic SLAF tags were verified by comparison with the reference genome.A total of 76 299 105 population SNP markers were detected using the GATK and SAM tools methods.A total of 4 133 595 population SNP markers were screened by integrity0.8 and minor allele frequency(MAF) 0.05,5.08% of the total SNP were evenly distributed among 12 chromosomes of blueberry.The phylogenetic tree was constructed based on the SNP markers and reflected the significant genetic differences among wild,northern highbush and southern highbush blueberry and the genetic relationships among cultivars,confirming the effectiveness and reliability of SNP markers in this study.The analysis of population structure based on SNP loci showed that there were significant genetic differences between wild types and cultivars,with the increase of K value,there was no subpopulation in wild types all the time,indicating that the tested wild types had independent genetic background and there was no gene exchange between the types,but there was unilateral gene flow from wild species to cultivars,suggesting that materials from wild species were used in the cultivation process of some cultivars,However,the cultivars had an obvious subgroup structure,suggesting that there was universal gene exchange between the subgroups.The classification of half-highbush blueberry Northland and southern highbush blueberry Reveille was closely clustered with the northern highbush type,The genetic background of the lowbush blueberry Blomidonand and half-highbush blueberry Northcountry and Blackpearl tended to be wild species at the genome-wide level.【Conclusion】The frequent use of backbone apparent varieties in breeding has increased the pattern of gene introgression between the bred cultivars,resulting in high genetic homogeneity in blueberries nowadays.The wild blueberry species are different in habitats and geographic distribution from the cultivars,and have an obviously different genetic background.