| 西瓜果形基因图位克隆及分子标记开发 |
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| 引用本文： | 段雅如,高美玲,郭宇,梁晓雪,刘秀杰,徐洪国,刘继秀,高越,栾非时.西瓜果形基因图位克隆及分子标记开发[J].中国农业科学,2022,55(14):2812-2824. |
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| 作者姓名： | 段雅如 高美玲 郭宇 梁晓雪 刘秀杰 徐洪国 刘继秀 高越 栾非时 |
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| 作者单位： | 1.齐齐哈尔大学生命科学与农林学院,黑龙江齐齐哈尔 1610062.黑龙江省抗性基因工程与寒地生物多样性保护重点实验室,黑龙江齐齐哈尔 1610063.齐齐哈尔市农业技术推广中心,黑龙江齐齐哈尔 1610064.东北农业大学园艺园林学院,哈尔滨 150030 |
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| 基金项目： | 国家自然科学基金（31972437,31772334,31401891）； |
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| 摘 要： | 【目的】基于GBS（Genotyping-by-sequencing）高密度遗传图谱初步定位结果,通过图位克隆方法对西瓜果形基因进行精细定位,并开发功能性分子标记,有助于全面研究果形基因的功能及分子标记辅助选择育种。【方法】利用114份纯合西瓜品系全基因组重测序数据及其果形指数表型进行GWAS（genome-wide association study）分析,结合GBS高密度遗传图谱初步定位结果确定果形基因候选区段,以商业小型西瓜品种纯化所得品系K2（椭圆形、FSI=1.54±0.13）和L1（圆形、FSI=1.11±0.07）为材料构建F2群体,通过开发分子标记对果形基因ClFSI进行精细定位,根据西瓜参考基因组‘97103’v1注释信息确定候选基因,并通过实时荧光定量PCR（qRT-PCR）进行验证。【结果】利用1 152份F2群体,最终将ClFSI精细定位于3号染色体的FMFSI-1与FMFSI-2标记之间物理距离约63 kb区间内,共包含5个注释基因。其中Cla011257属于已报道与控制果实纵径和果形相关的SUN基因家族。经测序分析发现,西瓜椭圆形品系K2在该基因第3外显子上存在两个非同义突变位点Chr3：26846636 G-A和Chr3：26847041 G-A（‘97103’v1）,分别导致天冬酰胺（Asn）替换为天冬氨酸（Asp）和谷氨酸（Glu）替换为赖氨酸（Lys）,并利用Chr3：26847041突变位点开发功能性分子标记FSICAPS-2。qRT-PCR分析表明,K2（椭圆形）与Charleston Gray（细长形）中候选基因表达量无显著性差异,但均显著高于L1（圆形）。【结论】本研究将控制西瓜果形的ClFSI精细定位于3号染色体63 kb区间内,推测Cla011257为最终目的基因,Chr3：26847041和Chr3：26846636突变位点是导致果形不同程度伸长的重要位点,并开发了可以同时鉴定Cla011257多种突变类型的功能标记FSICAPS-2。
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| 关 键 词： | 西瓜 果形基因 图位克隆 分子标记 |
| 收稿时间： | 2021-11-03 |
Map-Based Cloning and Molecular Marker Development of Watermelon Fruit Shape Gene |
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| DUAN YaRu,GAO MeiLing,GUO Yu,LIANG XiaoXue,LIU XiuJie,XU HongGuo,LIU JiXiu,GAO Yue,LUAN Feishi.Map-Based Cloning and Molecular Marker Development of Watermelon Fruit Shape Gene[J].Scientia Agricultura Sinica,2022,55(14):2812-2824. |
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| Authors: | DUAN YaRu GAO MeiLing GUO Yu LIANG XiaoXue LIU XiuJie XU HongGuo LIU JiXiu GAO Yue LUAN Feishi |
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| Institution: | 1. College of Life Sciences, Agriculture and Forestry, Qiqihar University, Qiqihar 161006, Heilongjiang2. Heilongjiang Provincial Key Laboratory of Resistance Gene Engineering and Preservation of Biodiversity in Cold Areas, Qiqihar 161006, Heilongjiang3. Qiqihar Agricultural Technology Extension Center, Qiqihar 161006, Heilongjiang4. College of Horticulture and Landscape Architecture, Northeast Agricultural University, Harbin 150030 |
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| Abstract: | 【Objective】 Based on the result of fruit shape gene preliminary mapping by genotyping-by-sequencing (GBS) high-density genetic map, the fine mapping of the fruit shape gene was conducted by using map-based cloning method, and the functional molecular markers were developed in watermelon. The present study could facilitate comprehensive study on the function of fruit shape gene and molecular marker-assisted selection breeding. 【Method】GWAS (Genome-wide association study) analysis was performed on 114 watermelon inbred lines by using whole-genome re-sequencing data and their fruit shape index phenotypes. The candidate regions of fruit shape gene ClFSI were confirmed by combing the results of GWAS with fruit shape gene preliminary mapping. The F2 segregating population were derived from a cross between two inbred lines K2 (oval, FSI=1.54±0.13) and L1 (round, FSI=1.11±0.07), which were purified from commercial small watermelon varieties. Fine-mapping of the fruit shape gene ClFSI was conducted by developing molecular markers. Candidate gene was identified by gene annotation of the candidate region in watermelon reference genome ‘97103’ v1 and validated by using real-time quantitative PCR (qRT-PCR). 【Result】 The 1 152 F2 plants were used for fine mapping and the ClFSI gene was finally mapped into 63 kb region on chromosome 3 between FMFSI-1 and FMFSI-2, containing a total of 5 annotated genes. The Cla011257 gene belonged to the SUN gene family that has been reported to control the fruit shape. There were two SNPs identified in the genomic region of third exon of ClFSI gene in the watermelon oval line K2. One SNP was a mutation from G to A at Chr3: 26846636 (‘97103’ v1), which resulted in a mutation from asparagine acid (Asn) to aspartic acid (Asp). Another SNP was a mutation from G to A at Chr3: 26846636 (‘97103’ v1), which resulted in another mutation from glutamic acid (Glu) to lysine acid (Lys). The FSICAPS-2 functional molecular marker was developed based on the Chr3: 26847041 SNP site. qRT-PCR expression analysis showed that candidate gene expression level was not significantly difference in K2 (oval) and Charleston Gray (elongated), but both were significantly higher than that in L1 (round). 【Conclusion】In this study, the ClFSI gene was mapped into a 63 kb candidate region on chromosome 3, and Cla011257 was the good candidate gene. Chr3: 26847041 and Chr3: 26846636 mutations were important loci that caused elongation of different degrees in fruit shape. A functional marker FSICAPS-2 was developed, which could identify multiple mutation types of Cla011257 simultaneously. |
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| Keywords: | watermelon fruit shape gene map-based cloning molecular marker |
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