美洲南瓜矮生基因的精细定位与功能分析

In the simplified cultivation and production of Cucurbita pepo, dwarf type of cultivars is important, but the dwarfing mechanism is not clear at present. In preliminary studies, we found that the dwarf trait was controlled with single dominant gene according to the genetic analysis of a F2 segregated population derived from two inbred lines DW4-5 (dwarf type) and V6-1 (vine type). For further research, we mapped the dwarf gene into a 103 kb interval on chromosome 10 of C. pepo with the strategy of BSA-Seq. Based on these research foundations, we will screen recombinant plants using an enlarged F2 population to fine map the dwarf related gene, which candidate gene will be identified by gene function prediction. Gene function analysis will be carried out with gene expression analysis, genetic transformation and other biological experiments. Transcriptome sequencing, hormone sensitivity test and cytological microscopic observation at different developmental stages will also be used for elucidating the mechanism of dwarf formation. Meanwhile, with 100 varieties of C. pepo germplasm resources with different vine patterns, the polymorphism of gene sequence and structure of target gene will be analyzed, which can be used to develop specific markers for marker-assisted selection of dwarf trait. Gene expression analysis will be performed to identify the correlation between the phenotype and the mutant sites. This study will provide theoretical basis for the mechanism of dwarfism in C. pepo and provide supportive information for marker-assisted selection for dwarf pumpkin accurate breeding.

矮生类型是美洲南瓜简约化生产中的重要栽培类型，但其矮化机制目前尚不明确。在前期工作中，我们以矮生自交系DW4-5和蔓生自交系V6-1为亲本构建分离群体，发现美洲南瓜矮生性状受单显性基因控制，并进一步利用BSA-Seq技术将其定位至10号染色体103kb区间内。在此基础上，本项目拟扩大F2分离群体筛选重组单株，精细定位美洲南瓜矮生基因，通过功能预测等方法确定候选基因，利用基因表达分析、遗传转化等生物学手段验证其基因功能；并结合节间发育不同时期转录组测序、激素敏感性测定和细胞学显微观察初步阐明美洲南瓜矮生性状的形成机制。同时，通过100份不同蔓生美洲南瓜种质资源，比较该矮生基因在序列和结构上的差异，结合基因表达分析，获得矮生基因突变位点与表型的相关性，开发特异性分子标记。项目研究结果将为解析美洲南瓜矮生性状形成的调控机制提供新的基因资源和理论依据，为株型精准育种提供技术支持。

矮化株型是美洲南瓜重要的育种目标，但其矮化机制不明确。本项目以美洲南瓜矮生自交系X10（DW4-5）和蔓生自交系JIN234（V6-1）为试材，通过表型、细胞学观察、外源激素处理和内源激素测定等生理试验明确了矮生性状的生物学特性；利用BSA-seq、KASP和CAPS技术对美洲南瓜矮生基因进行了精细定位，通过功能预测、基因表达分析等方法确定了候选基因，通过对候选基因进行超表达转基因研究，结合表型和分子生物学手段，初步确定了候选基因的功能。对近等基因系材料节间生长发育不同时期的转录组进行差异分析，挖掘出与美洲南瓜矮生性状形成相关的基因及调控网络，为进一步探明美洲南瓜短蔓形成的分子机理以及南瓜的株型育种提供理论依据。