中国农业科学 ›› 2023, Vol. 56 ›› Issue (8): 1547-1560.doi: 10.3864/j.issn.0578-1752.2023.08.010

• 园艺 • 上一篇    下一篇

基于GBS技术开展柚类资源群体遗传评价并发掘酸含量相关基因

江东1(), 王旭1, 李仁静1, 赵晓东2, 戴祥生2, 柳正葳3   

  1. 1 西南大学柑橘研究所,重庆 400712
    2 井冈山农业科技园管理委员会,江西吉安 343016
    3 井冈山大学,江西吉安 343016
  • 收稿日期:2022-05-25 接受日期:2022-10-08 出版日期:2023-04-16 发布日期:2023-04-23
  • 基金资助:
    国家重点研发计划(2018YFD1000101); 国家重点研发计划(2019YFD1001401); 种质资源精准鉴定(19211142); 江西科技计划项目(20161BBF60048)

Population Genomic Structure of Pomelo Germplasm and Fruit Acidity Associated Genes Identification by Genotyping-by-Sequencing Technology

JIANG Dong1(), WANG Xu1, LI RenJing1, ZHAO XiaoDong2, DAI XiangSheng2, LIU ZhengWei3   

  1. 1 Citrus Research Institute, Southwest University, Chongqing 400712
    2 Jinggangshan Agricultural Science and Technology Park Management Committee, Ji’an 343016, Jiangxi
    3 Jianggangshan University, Ji’an 343016, Jiangxi
  • Received:2022-05-25 Accepted:2022-10-08 Published:2023-04-16 Online:2023-04-23

摘要:

【目的】弄清我国柚类种质资源的起源与演化、群体遗传结构和多样性水平,高效利用柚类自然资源群体发掘与果实重要品质性状相关的基因,为柚类种质资源群体的遗传结构研究、起源演化和柚类种质创新提供重要的理论及实践依据。【方法】利用国家果树种质重庆柑橘资源圃中保存的具有广泛遗传多样性和不同地理来源的282份柚类资源作为材料,采用EcoR I限制性内切酶消化基因组DNA后构建GBS文库,然后进行Illumina HiSeq PE150二代测序获得短读序列,通过BWA软件将序列映射到柚参考基因组上,利用SAMTOOLS软件鉴定SNP位点。依据SNP的基因分型结果,进行主成分分析和群体结构分析,并采用邻接法构建系统演化树,分析亚群的遗传多样性水平。选择23个低酸种质和32个高酸种质构成两个群体进行Fst、XP-CLR分析,同时利用282个柚类种质的基因型数据与果实可滴定酸含量的表型数据进行GWAS全基因组关联分析。【结果】利用GBS简化基因组测序技术对282份柚类种质进行测序,获得201.66 Gb的原始测序数据,每个样本的平均测序数据量为0.72 Gb,经过测序深度为5×次要等位基因频率>0.01、Miss0.2的筛选条件过滤,最终获得121 726个高质量的SNP位点。主成分分析、群体结构分析和聚类分析均表明282份柚类资源可被分为6个亚群,其中柚与‘清见’杂交群体、葡萄柚等橘柚杂种群体可明显区别于其他真正柚类种质。不同地理来源和特定类型的柚类种质在遗传水平上存在明显差异,来源于泰国、越南等东南亚国家的柚类资源形成了较为独特的类群,与国内的沙田柚类、文旦柚类、垫江柚类等群体能够明显区分开。在中国南方的大部分柚类种质中有来自于越南柚的基因渗入,表明越南是柚的原始起源中心。另外,一些来源不清的柚类种质也通过GBS技术得以准确鉴定。Fst、XP-CLR选择性清除分析发现在7号染色体上的强烈选择信号区域中包含有丙酮酸脱氢酶复合物二氢硫辛酰赖氨酸残基乙酰转移酶(PDH-E2)和铝激活苹果酸转运蛋白(ALMT9),涉及柠檬酸的合成和运输。GWAS全基因组关联分析在2号染色体上鉴定了一个与果实酸度高度关联的区域。【结论】GBS技术为研究柚的系统发育和演化提供了一种可靠和高效的方法。本研究表明人工杂交育种、长期人工选择和驯化、地理隔离是形成不同类型柚类种质的驱动力,同时也是导致柚类种质遗传分化和多样性增加的重要原因。通过Fst、XP-CLR选择性清除和全基因组关联分析鉴定了多个与柚类果实柠檬酸含量相关的候选基因,为柚类的遗传改良和育种提供了重要的基因资源。

关键词: GBS, 柚系统演化, 果实酸度调控基因, GWAS

Abstract:

【Objective】 To reveal the phylogeny, population genetic structure and diversity level of pomelo (Citrus grandis (L.) Osbeck) germplasm, and to efficiently utilize them to explore genes related to important fruit quality traits, this research provided an insight into the population genetic structure and phylogeny of pomelo germplasm and facilitated the pomelo varieties innovation.【Method】 A total of 282 pomelo accessions including landraces from different geographical regions and hybrid offspring of kiyomi tangor and pomelo were contained in this study. GBS library was constructed with genomic DNAs digested by EcoR I restriction endonuclease and sequenced on Illumina HiSeq PE150 platform, the clean short reads were then mapped to pomelo reference genome by BWA, and SNPs were called out with SAMTOOLS pipeline. Based on 121 726 SNPs genotyping data, principal component analysis (PCA) and population genetic structure analysis were carried out and phylogenetic trees were constructed with Neighbor-joining method. Furthermore, two sub-populations containing high-acid accessions (32) and low-acid accessions (23) were used to identify candidate genes related to fruit acidity by Fst and XP-CLR selective sweeping analysis. Meanwhile, the genotype data of 282 pomelo accessions and the phenotypic data of titratable acid content in fruit were used for GWAS.【Result】 A total of 201.66 Gb original reads were generated from 282 pomelo germplasm by GBS approach, in average each sample produced 0.72 Gb reads. After the screening conditions of sequencing depth of dp5, the miss less than 0.2 and minor alleles frequency (MAF)>0.01, and a total of 121 726 SNPs were selected out for subsequent analysis. The PCA, structure and phylogenetic analysis all supported that the 282 pomelo germplasm could be divided into 6 subgroups, among which pomelo and kiyomi hybrid population, grapefruits and other pomelo hybrid populations could be obviously different from true-to-type pomelo populations, pomelos originated from different geographical region displayed unique genetic feature. The pomelo germplasm from Thailand and Vietnam formed a relatively unique group different from other domestic groups, such as ShaTian pomelo, Wen Dan pomelo, and Dian Jiang pomelo in China. The genetic introgression from Vietnam pomelos were exhibited in most pomelo germplasm in southern China, suggested that Vietnam was the origin center for pomelo. In addition, some pomelo germplasm with unknown origin have been identified accurately by GBS technology. This study showed that different geographical distribution and artificial selection pressure had great effect on the genomic composition of pomelo. Besides, Fst, XP-CLR selective sweeping analysis revealed a strong selection signal region on chromosome 7 contained genes annotated as dihydrolipoyl transacetylase (DLT-E2) of pyruvate dehydrogenase complex (PDC) and aluminum-activated malate transporter (ALMT9), which involved in the synthesis and transportation of citric acid. In additional GWAS genome-wide association analysis identified another region on chromosome 2, which was also highly associated with fruit acidity. 【Conclusion】GBS technology provided reliable and efficient method for studying the phylogeny and evolution of pomelo. The study showed that artificial cross breeding, long-term artificial selection, geography isolation and domestication were the major driving forces for the formation of different types of pomelo germplasm. In addition, it clearly showed that Southeast Asian was primary center for pomelo origin and China mainland was secondary evolutionary center. Several candidate genes related to citric acid content in pomelo fruits were identified by Fst, XP-CLR selective sweeping and GWAS. This study provided important gene resources for the further genetic improvement and breeding of pomelo fruits.

Key words: GBS, pomelo phylogeny, genes related to fruit acidity, GWAS