印迹基因对人工合成甘蓝型油菜杂种败育的影响

The rich resources of Brassica rapa and B. oleracea can be used to resynthesize B.napus and to create a new germplasm of rapeseed. However, a crossing barrier exists between B. rapa and B. oleracea, and hybrid abortion preferably occurs when B. oleracea is used as the female parent. The mechanism underlying this phenomenon needs to be elucidated. This research aims to compare the compatibility of different parents and reciprocal cross, and observe the development of embryo and endosperm in hybrids. Five cultivars of B. rapa and five cultivars of B. oleracea will be adopted as parents for complete diallel crossing. With RNA-seq analysis, the imprinting genes from female or male parents will be screened, specific imprinting genes of B. rapa and B. oleracea will be identified, and the endosperm imprinting genes' tissue expression pattern and distribution on A and C genomes will be determined. Functional analysis of imprinting genes that influence endosperm development and are specific to the maternal parent is also performed. In this study, methylation changes in the promoters of these imprinting genes in the embryo and endosperm will be identified using bisulfite sequencing. These results can help revealing the regulatory effects of DNA methylation on endosperm imprinting gene expression and elucidate the cytological basis and molecular mechanism of interspecific hybridization barrier and hybrid abortion. This study will provide a new strategy to improve the hybridization between B. rapa and B. oleracea, and be valuable for the germplasm innovation and utilization of B. napus.

利用资源丰富的白菜和甘蓝人工合成甘蓝型油菜是创制油菜新种质的重要途径之一，但白菜和甘蓝种间杂交存在障碍，尤其是甘蓝作母本更易导致杂种的败育，这一现象发生的机制尚不清楚。本研究选用5种甘蓝和5种白菜作双亲进行双列杂交，比较白菜和甘蓝不同品种间杂交以及正反交亲和性的差异，并观察杂种胚和胚乳的发育过程；通过转录组分析筛选母源和父源印迹基因以及白菜和甘蓝特异的印迹基因，明确胚乳印迹基因的组织表达特性及在A/C基因组上的分布特征；对影响杂种胚乳发育的印迹基因以及甘蓝特异的母源印迹基因进行功能验证，利用亚硫酸氢钠测序法分析杂种胚和胚乳中这些印迹基因启动子区域的甲基化变化，分析DNA甲基化对胚乳印迹基因表达调控的影响，最终阐明甘蓝和白菜种间杂交障碍以及甘蓝作母本易败育的细胞学基础和分子机制。本研究能为进一步提高白菜和甘蓝的可交配性提供新思路，对甘蓝型油菜种质资源创新和利用具有重要的理论意义和应用价值。

本项目利用不同白菜型油菜和甘蓝品种，通过种间杂交和胚胎挽救创建了表型变异丰富的人工合成甘蓝型油菜，并分析了不同杂交组合的杂种获得效率。通过不同甘蓝型油菜品种（扬油6号和中双11）的正反交杂种和亲本胚乳的RNA-seq分析，鉴定获得甘蓝型油菜的297个候选印迹基因，包括283个母本表达的印迹基因（MEG）和14个父本的印迹表达基因（PEG），并对其与其他植物物种印迹基因的保守性进行了分析，发现不同物种中的印迹基因并不保守。通过甘蓝型油菜印迹基因的注释和表达分析，发现杂种胚乳中AGLs（AGAMOUS-LIKE MADS-box genes）和果胶相关基因的表达水平较亲本发生了显著差异，这些印迹基因的表达变化可能与杂种胚乳细胞和胚的发育相关。此外，本项目通过二倍体白菜型油菜（特矮青）、甘蓝（中花芥兰）及其正反交胚乳的高通量测序，鉴定获得人工合成甘蓝型油菜的267个候选印迹基因。本研究创建了候选印迹基因的突变体材料，为甘蓝型油菜印迹基因调控种子发育的分子机制奠定了工作基础。