| Rice heading date (HD) refers to the time from sowing to heading (flowering). HD is one of the most important factors determining rice distribution and grain yield. Heilongjiang province constitutes the largest rice cultivation region and the most important elite rice production and exporting base in China. Heilongjiang province located at the northeast of China, and local rice cultivation condition is lower temperature and longer day length. With the northward of rice growth region, accumulated temperature is decreasing and varieties have to flower early to fulfill their life cycle. So the growth of elite varieties is restricted to specific local region.Heading date is complex quantitative traits, and controlled by multiple genes and diverse environmental factors. To identify major and minor HD genes, and the reveal their effect in determining heading date of Heilongjiang rice cultivars, we comprehensive combined linkage analysis and association analysis. Main results are as follows:1. F7 RIL (recombination inbred lines) population between KY131 and DN422, which are major cultivars in the second and third temperature zone respectively, was used for analysis of HD QTL, and it was revealed that Hd2 is a major gene. The single amino acid variation L710P between KY131 and DN422 contributed to early flowering of KY131. Quantitative real-time PCR (QRT-PCR) analysis show that expression levels of Hd3a, RFT1 and Ehdl were significantly up-regulated in the extremely early flowering parent KY131 compared to DN422 under LD conditions, suggesting that Hd2 regulate flowering through Ehd1 dependent pathway. Moreover, association analysis of 395 rice collections of Heilongjiang province also shows that Hd2 is the main HD gene, which supports our QTL analysis results.2. Biochemical characteristics analysis shows that Hd2 is a nucleus localized protein and can form homo-dimers through its CCT domain. Interestingly, we identified LTG1 and DELLA as HD2 interaction protein by yeast two hybrid assay, and confirm their interaction via in vitro pull-down, Bimolecular fluorescence complementation (BiFC) and LUC complementation assays. The biological significance of these interactions will be elaborated in future work.3. We surveyed the heading date phenotype of 395 collected cultivars and analyze the haplotype of HD related genes among these cultivars. Association analysis show that Hdl, Hd2/OsPRR37, Hd4/Ghd7, Hd5/DTH8/Ghd8 are major genes and Hd6, Hd16, Hd17 are minor genes in determining rice heading date in Heilongjiang province. Further, we reveal that diverse combination of these HD genes control the regional distribution of rice cultivars.4. Based on identified HD genes above mentioned, we performed molecular design breeding strategy to more quickly breed elite rice material that fit to local growth circumstance. To this end, two approaches were adopted. One is to develop the molecular markers for these HD genes, and use these markers to assist selection in multiple backcross generation. Another is to employ the CRISPR/CAS9-mediate genome editing techniques to knock out specific HD gene. We expect to develop a series of materials which maintain excellent characterization of common elite cultivar, but show diverse heading date resulting from utilizing different haplotypes of HD genes. Thus, this study can contribute to expand growth areas of elite cultivar and breed extremely early cultivar. |
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