水稻籼粳交F2代杂合株致死的分子机理

Reproductive isolation in indica-japonica subspecies of rice was important for the maintenance and development of species, but it hampered the exploitation of the great heterosis and limited the communication of the favorable genes. Genetic mechanism of it was very complex, and it was controlled by multiple genes in rice. Among them, three ORFs in S5 loci were the important genes to control reproductive isolation in indica-japonica subspecies. Our team found a new problem in hybrid offspring from indica crossed with japonica. It was the premature death of some heterozygous plants in F2 segregation population in bud or seedling stage, limiting the restructuring of favorable genes from parents in F2 population. Preliminary studies suggested that ORF3, ORF4, ORF5 these three genes at S5 loci could regulate the death of heterozygous genotype plants in F2 populations from the crosses between indica and japonica rice, and its genetic effect were easily affected by the external environment. In this project, Huajingxian74 single segment substitution lines were used as the major study objects to acquire F2 segregation population from the combination of different genotypes in S5 loci. Then, we explore the molecular mechanism of the death of heterozygous genotype plants in F2 populations from indica-japonica hybridization, through adversity treatment, gene knockout, gene expression and transgenic technology. Our project reveals the complexity of reproductive isolation between subspecies, and it would offer some valuable guidances for the research of genetic differentiation in subspecies and the exploitation of the application of favorable genes from indica and japonica rice.

水稻籼粳亚种间的生殖隔离有利于物种的维持和发展，但是阻碍了亚种间强大的杂种优势的利用，也限制了亚种间优异基因的交流。其遗传机制非常复杂，是由多个基因共同控制的，其中S5是控制籼粳亚种间生殖隔离的重要基因。本研究团队发现籼稻和粳稻杂交后代存在一种新的问题，即F2代分离群体中部分杂合株在芽期或幼苗期提前死亡，阻碍了亲本优异基因在F2代的重组。初步研究表明S5位点的ORF3、ORF4、ORF5三个基因可能是参与调控籼粳交F2代分离群体中杂合株致死的重要基因，并且其遗传效应容易受外界环境的影响。本项目以遗传背景完全一致的华粳籼74单片段代换系为主要的研究材料，将S5位点不同基因型的材料杂交配组获得F2分离群体，利用逆境处理、基因敲除、基因超表达以及转基因等技术探索籼粳交F2代杂合株致死的分子机理。本项目揭示了亚种间生殖隔离复杂性，将为研究水稻的遗传分化以及籼稻与粳稻间优异基因的利用提供参考和借鉴。

水稻籼粳亚种间具有强大的杂种优势，长期以来被认为是进一步提高水稻产量潜力的一种有效方法。但是籼粳亚种的生殖隔离严重阻碍了亚种间杂种优势的利用，也限制了亚种间优异基因的交流。S5位点ORF3、ORF4和ORF5基因是调控籼粳亚种间生殖隔离的重要基因。本项目以华粳籼74单片段代换系为主要研究材料，并应用CRISPR/Cas9基因编辑技术对ORF3和ORF4基因进行定点编辑，创制出5个不同的等位基因，用于分析籼粳交F2代杂合株致死的分子机理。结果表明不同等位基因配子相结合产生的杂种F1表现出不同程度的败育，而F2分离群体中杂合株的比值也具有显著差异。转录组分析表明ORF3+ORF4-ORF5+/ORF3-ORF4+ORF5-等位基因互作将导致与碳代谢、MAPK信号转导、苯丙素的生物合成等代谢途径相关基因在F2分离群体中杂合株的表达量显著下调，从而致使部分杂合株中抗氧化酶的活性降低，抗逆性降低，从而无法正常生长。但是敲除粳型配子中的ORF4+基因，与籼型配子ORF3+ORF4-ORF5+相结合能产生正常胚囊育性的杂种F1，并且其F2代分离群体中不存在杂合株死亡。本项目初步分析水稻籼粳亚种间F2代杂合株致死的分子机理，并提出利用广亲和基因ORF5n 或敲除粳型配子的ORF4基因一定程度上可以打破籼粳亚种间生殖隔离，该项结果将为籼稻与粳稻间优异基因的聚合及其在水稻育种上的应用提供参考和借鉴。