基于染色体单片段代换系的水稻孕穗期耐冷性关键基因克隆及遗传机制研究

Rice is a cold-sensitive plant that has its origin in tropical or sub-tropical areas. Spikelet fertility of rice decrease when rice are exposed to low temperature, especially at the booting stage, due to the failure of microspore development under low temperature conditions. Hence, cold tolerance at the booting stage (CTB) is a critical trait, and now been considered as one of the major determinants for a stable yield of rice (Oryza sativa L.). In recent years, to overcome the problem of damage caused by low temperature, rice breeders and genetists have been making efforts to reveal the genetic mechanism of CTB in rice, and a large amount of quantitative trait loci (QTL) have been identified. However, due to the difficulty in precisely evaluating CTB and the limitations of genetic population employed in QTL analysis, most of the QTL identified cannot be repeatedly detected in the difference crosses, even though in the same crosses by different researches. Therefore, few of QTL identified previously has been cloned except CTB1 so far, and the genetic mechanism of CTB in rice remains unclear yet. In our previous study, two QTL, qPF10.1 and qPF10.2, associated with CTB were successfully identified in the chromosomal segment substitution line population, and their interaction was also found to be responsible for CTB. In the project, we aim (1) to clone qPF10.1 and qPF10.2 by using the advanced genetic population together with the cold tolerance evaluation in the artificial climatic chamber; (2) to develop the near-isogenic lines for qPF10.1 and qPF10.2, respectively, consequently, subject to analyzing their expression pattern of the candidates, and to further dissect the mechanism of interaction between qPF10.1 and qPF10.2 through molecular biology methods ; and (3) to design the functional molecular markers on the qPF10.1 and qPF10.2 loci based on the genomic sequence diversity among the indica/japonica germplasm, which will be used as the selection markers in the rice CTB improvement program in rice. We believe that the results obtained from the project will highlight the genetic mechanism of CTB in rice and will be helpful for rice CTB improvement program.

水稻孕穗期耐冷性是一个十分重要的性状，已成为制约水稻安全生产的重要因素。近年来科学家们围绕水稻孕穗期的耐冷性开展了许多研究，定位了大量的QTL。然而，由于性状鉴定困难和使用遗传群体的局限性，鲜有相关QTL被成功克隆。我们在前期研究中，利用染色体单片段代换系群体鉴定了两个孕穗期耐冷性的QTL qPF10.1和qPF10.2，并且它们之间存在互作。本研究拟在此基础上：（1）通过构建高级遗传分析群体，结合人工气候室鉴定，分别克隆qPF10.1和qPF10.2；（2）通过分析qPF10.1和qPF10.2近等基因系中候选基因的表达，结合分子生物学方法，解析qPF10.1和qPF10.2互作的分子机制；（3）根据qPF10.1和qPF10.2基因的序列变异，设计相应功能标记，用于水稻孕穗期耐冷性的遗传改良。本项目的开展将揭开水稻耐冷性的遗传机制，对开展水稻孕穗期耐冷性遗传改良具有重要意义。

水稻孕穗期耐冷性是一个十分重要的性状，已成为制约水稻安全生产的重要因素。近年来科学家们围绕水稻孕穗期的耐冷性开展了许多研究，定位了大量的QTL。然而，由于性状鉴定困难和使用遗传群体的局限性，鲜有相关QTL被成功克隆。我们在前期研究中，利用染色体单片段代换系群体鉴定了两个孕穗期耐冷性的QTL qPF10.1和qPF10.2，并且它们之间存在互作。本研究在此基础上：（1）通过人工气候室和海南自然低温鉴定，我们重复验证了qPF10.1和qPF10.2位点。两个位点互作明显的小穗育性降低，小穗育性降低是由花粉败育引起，表明低温引起雄配子的部分败育。（2）在进行qPF10.1和qPF10.2位点精细定位过程中，我们发现了一个新的影响孕穗期耐冷性的QTL位点qPF10.3，并构建了三个位点之间的互作模型。只导入qPF10.1或qPF10.2时，植株对低温敏感败育；同时导入qPF10.1和qPF10.3或 qPF10.2和qPF10.3时，qPF10.3能够与qPF10.1或qPF10.2互作，恢复正常表型；同时导入qPF10.1和qPF10.2时，qPF10.1和qPF10.2会发生互作，恢复正常表型；只导入qPF10.3时，植株表型正常。（3）通过构建目标区段的染色体片段叠代系，将qPF10.1精细定位在215kb范围内，共23个候选基因；将qPF10.2精细定位在74kb范围内，共10个候选基因；将qPF10.3精细定位在191kb范围内，共31个候选基因。. 本研究定位了三个孕穗期耐冷性相关的QTL qPF10.1、qPF10.2和qPF10.3，并获得了与之连锁的分子标记。阐述了影响孕穗期耐冷性状的三位点互作模式，低温敏感败育位点的遗传机制。为育种家培育和选择耐冷水稻，提高水稻稳产能力提供了参考价值。