西瓜遗传图谱构建及果实相关性状QTL分析

doi:10.3864/j.issn.0578-1752.2014.14.012

摘要/Abstract

摘要： 【目的】利用CAPS及SSR标记构建西瓜遗传图谱，对西瓜果实相关性状进行QTL分析，为西瓜果实性状改良、主效基因精细定位及克隆奠定基础。【方法】授粉后40 d对母本PI186490、父本LSW-177以及两者杂交获得的F2群体的果实进行采摘，对每个果实的果形指数、中心和边缘可溶性固形物、中心和边缘果肉硬度、果皮硬度、种子长度、种子宽度、种子厚度以及种子百粒重进行调查，将所得数据用软件SPSS19进行统计分析。通过Illumina HiSeq 2000高通量测序平台对两亲本材料进行基因组重测序，每样品产出10 G数据量，覆盖西瓜基因组20×以上，所得数据以已经发布的基因组数据为参考基因组，用bwa软件进行基因组组装，组装后利用Samtools软件进行SNP发掘，利用perl语言自编脚本提取SNP位点前后1 000 bp的序列，将SNP及其侧翼序列输入软件SNP2CAPS以转化为CAPS标记。在每条染色体上平均选取20个CAPS酶切位点，利用Primer Premier 5软件在突变位点上下游100-500 bp左右设计CAPS引物，进行PCR扩增和酶切检验，酶切产物用1%琼脂糖凝胶电泳检测。SSR引物来源于前人发表文献，PCR扩增产物用聚丙烯酰胺凝胶电泳检测。对所有分子数据进行卡方检验，在其中选择符合1﹕2﹕1比例的标记用于构建遗传连锁图谱。利用Mapmaker/Exp version 3.0软件构建遗传连锁图谱，用Group命令对标记进行连锁分组，标记数目少于8的连锁群用Compare命令进行排序优化，标记数多于8的连锁群用Try命令排序。绘制遗传图谱使用Map Chart 2.1软件。QTL分析运用QTL Network 2.0软件，利用置换测验做1 000次重复，临界阈值为P=0.005，采用复合区间作图法，在每条染色体上以1.0 cM步行速度在全基因组范围内扫描，分析QTL加性效应和上位效应。【结果】本遗传连锁图谱共包含16个连锁群，涉及CAPS标记87个，SSR标记9个，覆盖基因组1 484.3 cM，平均图距15.46 cM。利用QTL Network 2.0分析，检测到6个西瓜果实相关性状的8个QTL位点和1对上位效应位点，其中包括果形指数QFSI 1、中心可溶性固形物QCBR、中心果肉硬度QCFF、边缘果肉硬度QEFF、种子长度QSL各1个，种子宽度QSWD 1、QSWD 2、QSWD 3 3个；上位效应位点包括果形指数FSI 2、FSI 3。表型贡献率大于等于10%的QTL有6个，可解释11.7%—18.8%的遗传变异。【结论】以CAPS标记为主要标记构建西瓜遗传图谱，并且定位了控制西瓜果实相关性状的8个加性QTL与1对上位性QTL，可用于进一步精细定位与克隆西瓜果实优良性状基因。

关键词: 西瓜 , 遗传连锁图谱 , CAPS , QTL

Abstract: 【Objective】The purpose of this study is to construct a molecular genetic map and map the QTL of the fruit-associated traits with CAPS and SSR markers in watermelon, which will provide a theoretical basis for traits improving, gene fine mapping and gene cloning.【Method】Fruits of female parent PI186490, male parent LSW-177 and F2 population derived from the cross between the two watermelon strains were picked in 40 days after pollination. Fruit shape index, center and edge brix, center and edge flesh firmness, rind hardness, seed length, seed width, seed thickness and 100-seed-weight were investigated correspondingly, then the obtained data were analyzed by SPSS19. Both parent materials genomes were resequenced by Illumina HiSeq 2000 platform for high-throughput sequencing, outputed 10G each data sample, covered more than 20× of watermelon genome. With the published genome as a reference, the obtained data were assembled with bwa, and explored for the SNP by Samtools. The sequence 1 000 bp around the SNP loci was extracted by self perl script and then inputed into SNP2CAPS to transform into CAPS markers. Twenty CAPS restriction sites were selected evenly on the 11 chromosomes. CAPS primers were designed 100-500bp around the mutation by Primer Premier 5 for PCR amplification and digestion detection. 1% agarose gel electrophoresis was used to detect the digestion products. SSR markers in this experiment were come from the published literature. The products of SSR-PCR were detected by polyacrylamid gel electrophoresis. All the molecular data were tested by chi-square. Markers which were confirmed the proportion 1﹕2﹕1 were chosen for the genetic linkage map. The genetic linkage map was constructed by Mapmaker/Exp version 3.0. The markers were grouped with the order ‘Group’. The number of the markers in the group which was less than 8 was sequenced faultlessly with the order ‘Compare’, which was more than 8 was ordered with the order ‘Try’. Map Chart 2.1 was used for drawing this genetic linkage map. QTL Network 2.0 was used for QTL analysis. 1 000 times repeats were done with the replacement testing, the critical threshold was P=0.005, and the method of constructing the map was composite interval mapping. The whole genome was scanned on every chromosome with 1 cM walking speed. QTL additive effect and epistatic effect were analyzed by the software.【Result】This genetic linkage map contained 16 linkage groups and included 87 CAPS markers and SSR markers. The map was 1 484.3 cM and the average distance between two makers was 15.46 cM. Mapping the QTL of the fruit-associated traits and analyzed by software QTL Network 2.0, and a total of 8 additive QTL and one pair of epistatic QTL were detected. Among the additive loci, 1 is for fruit shape index(QFSI 1), 1 for center brix (QCBR), 1 for center flesh firmness(QCFF), 1 for edge flesh firmness(QEFF), 1 for seed length(QSL), and 3 for seed width(QSWD 1, QSWD 2, QSWD 3). The epistatic loci, FSI 2 and FSI 3 are for fruit shape index. Phenotypic contribution rate of 10% or more have six QTL, which explained 11.7% -18.8% of the genetic variation. All of the QTL explained 7.12%-18.8% of the phenotypic variation.【Conclusion】A molecular genetic linkage map composed mainly of CAPS markers was constructed. Eight additive QTL and one pair of epistatic QTL, which control the fruit-associated traits of watermelon were located, thus providing a scientific basis for further fine positioning and cloning the genes controlling superior traits of fruit of watermelon.

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

刘传奇, 高鹏, 栾非时. 西瓜遗传图谱构建及果实相关性状QTL分析[J]. 中国农业科学, 2014, 47(14): 2814-2829.

LIU Chuan-Qi, GAO Peng, LUAN Fei-Shi. Construction of a Genetic Linkage Map and QTL Analysis of Fruit-Associated Traits in Watermelon[J]. Scientia Agricultura Sinica, 2014, 47(14): 2814-2829.

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