甜瓜黄/绿柱头颜色主效基因精细定位与功能分析

Melon stigma color was only found two color categories，green and yellow. Different stigma color effect to insect attraction of flowers, while in the artificial pollination practice, we found green stigma melon materials with higher artificial pollination efficiency. Study in this proposal focuses on melon stigma color. Based on the results of pre-research foundation of mapping the major melon stigma decision genes with the GWAS (Genome wide association study) strategy analysis of a natural population containing 380 melon line with different stigma color. Two sets of F2 genetic populations were constructed from the bi-parents with different stigma color to fine map the key stigma color decision genes; finally use the map-based cloning combined with candidate gene identification to clone the melon stigma color decision genes. By gene sequence analysis, gene over-expression, CRISPR/CAS9 based gene editing, other biological experimental methods, and combining with different stigma development transcriptome sequencing analysis, gene function and melon stigma color formation mechanism will be preliminarily described. At the same time, a melon yellow/green stigma marker-assisted selection system will be established for the creation of specific melon germplasm resources, breeding of green stigma elite lines and cultivars. The results of this study will be used to improve the efficiency of seed production and provide the important gene resources.

甜瓜柱头颜色有绿色和黄色两种，不同柱头颜色影响花粉对昆虫的吸引力，绿色柱头甜瓜材料具有较高的人工授粉效率。本研究以甜瓜柱头颜色为研究对象，在利用GWAS策略对380份甜瓜材料的自然群体开展研究，初步定位甜瓜柱头颜色决定基因的研究基础之上，采用不同柱头颜色亲本构建两套分离群体，精细定位柱头颜色决定基因；最后利用图位克隆手段结合候选基因鉴定，获得甜瓜柱头颜色决定基因。通过基因序列分析、基因超量表达、基因编辑以及其它生物学实验手段进行基因功能分析；结合不同柱头发育阶段转录组测序分析，初步阐明甜瓜柱头颜色形成机理。同时建立甜瓜黄/绿色柱头分子标记辅助选择技术体系，为创制甜瓜特异种质资源，选育绿色柱头优异品系及品种，提高制种效率，提供重要基因资源。

本研究对甜瓜黄/绿柱头的色素进行定性定量分析，发现绿色柱头叶绿素含量与类胡萝卜素含量均高于黄色柱头。对甜瓜黄绿柱头不同发育时期进行显微观察发现不同颜色柱头在各时期叶绿体发育均有较大差异。选用甜瓜绿色柱头材料MR-1×黄色柱头材料M1-32杂交获得的F2作图群体进行初步定位，通过QTL-seq结合CAPS标记获得2个QTL位点；为了获得稳定准确的QTL位点继续选用甜瓜绿色柱头材料MR-1×黄色柱头材料M4-7杂交并自交获得F2及F2：3家系作图群体，通过CAPS标记共获得3个QTL位点。进一步扩大两套材料的作图群体，通过加密QTL置信区间标记数量，最后锁定8号染色体SC8.1（GC8.1）为甜瓜柱头颜色主效QTL位点。对黄绿柱头不同发育时期进行RNA测序，通过GO term分析，KEGG分析和WGCNA分析等手段，进一步明确光系统I、光系统II、叶绿体、叶绿体类囊体、光合作用、光反应、叶绿素结合、类胡萝卜素合成与甜瓜黄/绿柱头形成相关；结合基因精细定位结果，锁定MELO03C003165为调控甜瓜柱头颜色的主效基因。为验证目的基因，利用VIGS技术沉默绿色柱头材料MR-1中MELO03C003165，与对照植株相比，沉默植株出现褪绿现象；进一步RT-PCR验证该基因在沉默植株中表达量显著降低。本研究阐明甜瓜柱头颜色形成机理。构建了甜瓜绿色柱头紧密连锁的分子标记1套，为创制甜瓜特异种质资源，选育绿色柱头优异品系及品种，提高制种效率，提供重要基因资源。