中国农业科学 ›› 2023, Vol. 56 ›› Issue (5): 935-950.doi: 10.3864/j.issn.0578-1752.2023.05.010

• 园艺 • 上一篇    下一篇

‘鲁丽’ב红1#’苹果杂交群体全基因组KASP标记开发及验证

郑文燕1(), 常源升1, 何平1, 何晓文1, 王森1, 高文胜2, 李林光1(), 王海波1()   

  1. 1 山东省果树研究所,山东泰安 271000
    2 山东省农业技术推广中心,济南 250100
  • 收稿日期:2022-04-26 接受日期:2022-07-14 出版日期:2023-03-01 发布日期:2023-03-13
  • 通信作者: 王海波,E-mail:wanghaibo992@126.com。李林光,E-mail:llg6536@163.com
  • 联系方式: 郑文燕,E-mail:1587493148@qq.com。
  • 基金资助:
    山东省自然科学基金(ZR2021MC045); 山东省农业良种工程项目(2021LZGC024); 国家自然科学基金(31901976); 泰山产业领军人才工程项目(LJNY202026); 国家现代农业产业技术体系建设专项资金(CARS-27)

Development and Validation of KASP Markers Based on a Whole- Genome Resequencing Approach in a Hybrid Population of Luli × Red No. 1

ZHENG WenYan1(), CHANG YuanSheng1, HE Ping1, HE XiaoWen1, WANG Sen1, GAO WenSheng2, LI LinGuang1(), WANG HaiBo1()   

  1. 1 Shandong Institute of Pomology, Tai’an 271000, Shandong
    2 Shandong Agricultural Technology Extension Center, Ji’nan 250100
  • Received:2022-04-26 Accepted:2022-07-14 Published:2023-03-01 Online:2023-03-13

摘要:

【目的】利用‘鲁丽’ב红1#’亲本及杂交后代,开发苹果叶片性状竞争性等位基因特异PCR(kompetitive allele-specific PCR,KASP)标记,为苹果光能高效利用育种提供参考。【方法】以‘鲁丽’和I型红肉苹果‘红1#’及其134个杂交后代的幼嫩叶片为材料,调查叶片性状表型(包括叶片长度、宽度、叶绿素含量、花青苷含量、光合参数和叶绿素荧光参数等)数据。采用Illumina HiSeq 2500测序平台进行全基因组重测序,通过短序列比对软件将测序序列(reads)回贴到苹果参考基因组,利用GATK软件工具包检测单核苷酸多态性(single nucleotide polymorphisms, SNP)标记。【结果】‘鲁丽’和‘红1#’及其134个杂交后代重测序得到有效reads数分别为164 776 660、149 482 876和3 927 370 200。与苹果参考基因组比对率分别为98.77%、98.89%和97.79%。共检测到6 445 766个SNP。经过滤后获得94 208个特异性强,准确性高的SNP位点。对每个SNP进行KASP引物设计,最终开发了5 802个苹果全基因KASP标记。这些KASP标记的多态性信息含量(polymorphism information content,PIC)平均值为0.31,PIC大于0.35的占30.13%。Simpson遗传多样性指数在0.01—0.67,平均每个位点遗传多样性指数为0.53。观测杂合度平均值为0.32。KASP标记所在基因的KEGG功能聚类分析显示,“叶酸介导的一碳代谢途径”是差异基因富集最显著的途径。通过对‘鲁丽’ב红1#’杂交群体24个叶片相关性状进行表型调查,结果显示亲本‘鲁丽’和‘红1#’在叶片净光合速率、气孔导度(Gs)等性状的表型值均存在显著差异。杂交后代表型变异幅度较大,各性状分离广泛。随后对杂交群体叶片性状表型值与KASP标记基因型进行相关性分析,结合KASP标记注释,筛选得到21个与叶片大小、Gs和色素含量性状显著相关的KASP标记。【结论】利用‘鲁丽’和‘红1#’及其134个杂交后代全基因组重测序数据开发了5 802个苹果全基因组KASP标记,获得21个与叶片性状显著相关的KASP标记,为开展标记辅助苹果光能高效利用育种提供了理论参考。

关键词: 苹果, 全基因组重测序, 叶片性状, KASP标记

Abstract:

【Objective】 A series of kompetitive allele-specific PCR (KASP) markers for traits related to apple leaves were developed to provide a reference to effectively utilize light in apple breeding using a Luli ×Red No. 1 hybrid population as materials. 【Method】This study investigated the leaf trait phenotypic data (including leaf length, width, contents of chlorophyll and anthocyanin, photosynthetic rate and chlorophyll fluorescence parameters) of Luli, a type I red-fleshed apple, Red No. 1, and 134 hybrids. Whole-genome resequencing was conducted on an Illumina HiSeq 2500 platform. The clean sequencing reads were mapped to the apple reference genome using BWA software. GATK software was used to identify single nucleotide polymorphisms (SNPs). 【Result】A total of 164 776 660, 149 482 876, and 3 927 370 200 clean reads were obtained by whole-genome resequencing from Luli, Red No. 1 and 134 hybrid individuals, respectively. The data were aligned to the reference genome sequences with mapping rates of 98.77%, 98.89%, and 97.79%, respectively. A total of 6 445 766 SNPs were identified. The SNPs were then filtered, and 94 208 accurate and high-quality SNPs were selected for subsequent KASP primer design. Finally, 5 802 KASP markers were developed based on a high-throughput whole-genome resequencing approach. The average polymorphism information content (PIC) of these KASP markers was 0.31, and there were 30.13% PICs>0.35. The Simpson genetic diversity index ranged from 0.01 to 0.67, with an average value of 0.53. The mean observed heterozygosity was 0.32. A Kyoto Encyclopedia of Genes and Genomes enrichment analysis of the genes for KASP marker revealed that “one carbon pool by folate” was the most significant pathway. An analysis of the phenotypic data of leaf-related traits in both the parents and individuals from the Luli ×Red No. 1 cross showed that there were significant differences in the phenotypic values of leaf net photosynthetic rate, stomatal conductance (Gs) and other traits between the parents Luli and Red No. 1. The F1 generation exhibited a wide phenotypic variation, and the most traits segregated extensively. Α detailed study of the phenotypic data of leaf traits of the hybrid population with the genotype and annotations of the KASP markers finally resulted in the development of 21 KASP markers, which was correlated with leaf-related traits, including chlorophyll content, Gs and leaf length and width.【Conclusion】Use of the whole-genome resequencing data of Luli, Red No. 1 and 134 hybrid individuals resulted in the development of 5 802 KASP. A total of 21 of these KASP markers significantly correlated with leaf traits, which provided a theoretical reference for the high utilization of light energy in apple breeding.

Key words: apple, whole-genome resequencing, leaf-related traits, KASP marker