Map-Based Cloning And Functional Analysis Of The Dth3Gene Controlling Head Date In Rice (ORYZA Sativa L.)

Rice (Oryza sativa L.) is not only an important cereal crop and model species monocot, but also a short day model plant. Heading date which affects cultivation areas and seasonal adaptability of different rice cultivars is one of the important agronomic traits of rice and plays an important role in rice breeding. Therefore, it has broad application prospects to investigate the genetic law, fine mapping and clone genes of heading date for improving the rice growth period traits in molecular level. Meanwhile, studying the regulation network of flowering gene in rice will be helpful for disclosing the molecular mechanism of flowering regulation pathway and provide guidance for rice breeding. In our study, we developed an NIL (nearly isogenic line) via consecutive back-crosses and molecular marker-assisted selection (MAS), where the japonica variety Dianjingyou1(DJY1) was used as the background parent, and the African rice variety as the donor parent. Both in long day and short day condition, the NIL flowered later than DJY1, we fine mapping this heading date gene (DTH3, Days To Head On Chromosome3) and establish its candidate gene a MADS-box familiy gene. We cloned this gene and domestication the function of DTH3. This gene may be associated with evolution. Taken together, the main founding are as follows:1. QTL analysis of heading date in rice and fine mapping DTH3The japonica variety Dianjingyou1(DJY1) is from Yunan Province, China, and IRGC102203is an O. glaberrima accession. We developed a NIL (a near-isogenic line) by backcrossinges and molecular marker-assisted selection (MAS) using DJY1as the recurrent parent, and IRGC102203as the donor. To assess the purity of the DJY1background of NIL, we surveyed it using192SSR markers that were evenly distributed across the12chromosomes of rice. Only one foreign chromosomal fragment about2BAC was identified on chromosome3. We named DTH3that control early flower from DJY1, and dth3for NIL. DJY1×NIL F2population (n=18,000) was used for fine mapping DTH3under natural long-day in Nanjing, the early flowering gene DTH3was located between marker g71and RM523and the physical distance between the two markers was64kb.10,000of F2population plants were grown under natural short days in Hainan, the result was the same as in natural long day condition. We get a MADS-box family gene in mapped region, the sequence was different between DJY1and NIL. These results were basis for map-based cloning of the DTH3.2. Cloning and functional analysis of the DTH3geneBy sequencing DTH3which encodes a MADS-box gene, there were some differences between DJYl and NIL in the coding sequence. Subcellular localization experiments show that the gene was localized in the nucleus, and overexpression DTH3lead to flower in rice callus. Quantitative PCR analysis of DTH3and dth3, expression of dih3was lower than DTH3both in long day and short day condition, and our results show that DTH3positive regulates Ehdl and RFT1to promote flowering. The expression of DTH3/dth3was the highest expression at dawn, and the lowest and at the evening, and the expression level dth3was always lower than DTH3. Yeast two-hybrid experiments showed that DTH3interaction with OsMADS56, and dth3can not interaction with OsMADS56. By analyzing DTH3in cultivars, we found dth3only exist in the African cultivated rice.