中国农业科学 ›› 2017, Vol. 50 ›› Issue (7): 1282-1293.doi: 10.3864/j.issn.0578-1752.2017.07.011

• 园艺 • 上一篇    下一篇

基于CAPS标记的西瓜果实与种子相关性状QTL分析

迟莹莹,高鹏,朱子成,栾非时,李桂英,于鹏   

  1. 东北农业大学园艺学院/农业部东北地区园艺作物生物学与种质创制重点实验室,哈尔滨150030
  • 收稿日期:2016-08-22 出版日期:2017-04-01 发布日期:2017-04-01
  • 通讯作者: 栾非时,E-mail:luanfeishi@neau.edu.cn
  • 作者简介:迟莹莹,Tel:0451-55191317,E-mail:chiyingying1234@163.com。
  • 基金资助:
    国家西甜瓜产业技术体系(CARS-26-02)、哈尔滨市科学技术局应用技术研究与开发项目(2013AE6AW061)

The QTL Analysis of Fruit and Seed Associated Traits in Watermelon Based on CAPS Markers

CHI YingYing, GAO Peng, ZHU ZiCheng, LUAN FeiShi, LI GuiYing, YU Peng   

  1. Horticulture College, Northeast Agricultural University/Key Laboratory of Biology and Genetic Improvement of Horticultural Crops, Ministry of Agriculture, Harbin 150030
  • Received:2016-08-22 Online:2017-04-01 Published:2017-04-01

摘要: 【目的】基于西瓜全基因组重测序数据,挖掘SNP位点并将其转变为CAPS标记,构建遗传连锁图谱,对西瓜果实与种子相关性状进行QTL分析,为西瓜果实与种子相关性状主效基因精细定位及克隆奠定理论基础。【方法】以普通栽培西瓜品系(Citrullus lanatus ssp. vulgaris)‘W1-1’和黏籽西瓜品系(Citrullus lanatus ssp. mucosospermus)‘PI186490’为亲本杂交获得F1代,以‘W1-1’为轮回亲本,构建BC1P1群体。采摘成熟果实,对每个西瓜果实中心及边缘可溶性固形物、中心及边缘果肉硬度、种子百粒重、种皮底色进行调查及数据分析。对亲本材料进行覆盖度约为20×的全基因组重测序,用BWA、SAMTOOLS及VCFTOOLS等软件比对并检测亲本材料在全基因组范围内的SNP位点。运用SNP2CAPS软件选择7种在西瓜基因组上具有丰富酶切位点的限制性内切酶,对包含SNP位点的序列进行酶切位点分析,转化CAPS标记。选取全基因组范围内均匀分布的450个CAPS分子标记,筛选多态性CAPS标记,对BC1P1群体内225株分别进行基因分型,使用QTL IciMapping及Windows QTL CartographerV2.5等软件进行遗传图谱的构建和西瓜果实与种子相关性状的QTL分析。【结果】在两亲本间共获得SNP位点751 532个,根据7种限制性内切酶位点信息共开发了450个CAPS分子标记,筛选出其中具有多态性的200个CAPS标记,在225株BC1P1群体构建一张包含11个连锁群(分别对应11条染色体)的遗传连锁图谱,覆盖长度1 376.95 cM,标记间的平均遗传距离为6.88 cM。QTL分析定位到与果实与种子相关性状QTL位点15个,其中包括中心可溶性固形物含量相关位点3个(CTSS2.1CTSS2.2CTSS8.1);边缘可溶性固形物含量相关位点1个(ETSS2.1);中心果实硬度相关位点3个(CFF6.1CFF6.2CFF8.1);边缘果实硬度相关位点2个(EFF6.1EFF6.2);种皮底色相关位点4个(SCC8.1SCC8.2SCC8.3SCC8.4);种子百粒重相关位点2个(SHW6.1SHW9.1)。15个QTL位点的贡献率范围为5.25%—74.59%,贡献率大于15%的位点有5个(CTSS8.1SCC8.1SCC8.2SCC8.3SCC8.4)。【结论】共开发出SNP位点751 532个,QTL分析检测到西瓜果实与种子相关性状QTL位点15个,其中主效QTL位点5个,分别为果实中心可溶性固形物及种皮底色相关位点(CTSS8.1、SCC8.1SCC8.2SCC8.3SCC8.4),为进一步精细定位及克隆西瓜果实与种子优良性状基因奠定了基础。

关键词: 西瓜, 遗传连锁图谱, SNP, CAPS, QTL

Abstract: 【Objective】 Cleaved amplified polymorphic sequence (CAPS) markers are useful tools for detecting single nucleotide polymorphisms (SNPs). The aim of this study is to convert SNP sites into CAPS markers based on high-throughput re-sequencing data in watermelon for linkage map construction and quantitative trait locus (QTL). 【Method】 A genetic map was constructed using a BC1P1 segregating population consists of 225 individuals, which F1 was obtained to backcross with P1 from female parent (garden female parent) and male parent (PI186490). The center and edge brix, center and edge flesh firmness, 100-seed-weight and seed coat color were investigated, respectively, then the obtained data were analyzed by SPSS19. Two inbred lines which are female and male parents, were re-sequenced and analyzed by Perl self-compiled script for CAPS marker development. It was found that 90% and 88% of the assembled sequences of the two parental materials could map to the reference watermelon genome, respectively. With the published genome as a reference, the obtained data were assembled with BWA, and explored for the SNP by SAMTOOLS. The sequence of the SNP loci was extracted by in house perl script and then input into SNP2CAPS to transform into CAPS markers. The 450 CAPS restriction sites were selected evenly on the markers whole-genome. CAPS primers were designed 100-500 bp around the mutation by Primer Premier 6.Screening polymorphic CAPS markers for each plant within 225 BC1P1 populations were genotyped. Finally QTL analysis of fruit and seed associated traits in watermelon was done by using QTL IciMapping and Windows QTL CartographerV2.5 softwares. 【Result】 A total of 751 532 SNPs were detected, 450 pairs of CAPS were designed with 7 restriction enzymes among which 200 pairs of primers were polymorphic. An initial CAPS-based genetic linkage map was constructed with the BC1P1 population which spanned 1 376.95 cM with 11 linkage groups, 15 QTLs were detected and the rate of contribution explained 5.25%-74.59%. Among the additive loci there were 3 loci for center brix (CTSS2.1, CTSS2.2, CTSS8.1), 1 for edge brix (ETSS2.1), 3 for center flesh firmness (CFF6.1, CFF6.2, CFF8.1), 2 for edge flesh firmness (EFF6.1, EFF6.2), 4 for seed coat color (SCC8.1, SCC8.2, SCC8.3, SCC8.4), and 2 for 100-seed-weight (SHW6.1, SHW9.1). 【Conclusion】 A total of 751 532 SNPs were detected. Phenotypic contribution rate of 15% or more has five QTL (CTSS8.1, SCC8.1, SCC8.2, SCC8.3, SCC8.4) which are center brix and seed coat color. The research will provide a scientific basis for further fine positioning and cloning the genes controlling superior traits of fruit in watermelon.

Key words: watermelon, genetic linkage map, SNP, CAPS, QTL