The Function Verification Of The Rice CCT Gene Family, Mutation Mechanism Research Of The New Allele OsMADS3

1. Plant flowering is a complex process regulated by the external environment and internal genes, which involved in many gene families. CCT domain-containing genes generally regulate flowering in plants. Currently, only six of the 41 CCT family genes have been confirmed to control flowering in rice. To efficiently identify more heading date-related genes from the CCT family, we identified functional genes through combination of candidate gene-based association mapping with linkage analysis and verified the gene function with the transformation in this project. We also study the function of the one CCT gene OsCCT01. Our results are performed as follow: 1.1 The candidate gene association analysis and functional verificationA total of 59 heading date QTLs were merged into 12 chromosomes by integrating closely linked QTLs, then we located 41 CCT family genes and 59 integrated QTLs on 12 chromosomes according to their physical positions. 25 CCT family genes were located in the QTL regions. Association mapping showed that a total of 19 CCT family genes were associated with the heading date. We chose a total of 18 genes to test their functions on heading by transformation. The results show that five of the eight associated genes within QTL regions and two of four associated genes outside of the QTL regions were confirmed to regulate heading date by transformation. None of the six non-associated genes outside of the QTL regions regulates heading date. 1.2 The expression profilings and nucleotide diversity of the CCT genesThe CCT family was clearly resolved to three subfamilies corresponding to CMF, COL and PRR. We extracted expression data on 35 CCT family genes generated from the japonica rice Nipponbare and investigated them in leaves and stems from five different stages. The results show that the the three subfamily genes expressed rich in leaves; some genes also have higher expression in stem. A total of 4,002 SNPs were identified in 41 CCT family genes from 529 cultivars and 107 wild rice accessions. The average nucleotide diversity of CCT family genes is that the π = 2.7 × 10-3. The pairwise nucleotide diversity ranged from one to eight SNPs per kilobase for most CCT genes. 1.3 The function study of the OsCCT01OsCCT01 belong to the CMF subfamily, the overexpression of OsCCT01 greatly delayed heading by more than 25 days in LDs and 15 days in SDs, while the suppression of Os CCT01 did not observe the heading date phenotype. We compared the expression of OsCCT01 and other key flowering regulators, such as OsCCT21(Hd1), Hd3 a, RFT1 and Ehd1, in young leaves between wild-type Zhonghua 11 and OsCCT01-overexpressing plants using qRT-PCR. The overexpression of Os CCT01 delayed flowering through suppressing the expression of Ehd1, Hd3 a and RFT1 under both long day and short day conditions. OsCCT01-overexpressing plants also showed the decreased plant height, tillers and yield. OsCCT01 was responsible significantly to the BR treatment. We detected the expression of the BR related genes OsCCT01-overexpressing plants and supposed that the OsCCT01 regulated the BR signaling pathway. We also detected the cell cycle regulation genes and found that the expression of the three G2/M phase genes were largely downregulated, this result suggested that OsCCT01 functions putativley as a negative modulator downstream of cell cycle genes to regulate cell division.2. Natural mutation is the source of natural variation, which is the fundamental basis for the genetic improvement of crops. During the process of developing a recombinant inbred line(RI), a spontaneous mutagenesis in RI127 led to the production of the recessive male-sterile line RI127 S. Via a map-based cloning approach, the gene controlling the male sterility was identified as OsMADS3, which was previously reported to be associated with floral organ development and male sterility. Thermal asymmetric interlaced PCR isolated one 1633-bp insertion in OsMADS3 in R127 S, which damaged its function due to failed transcription. The 1633-bp insertion was derived from a fragment flanked by retrotransposon genes on chromosome 5. Seven haplotypes of OsMADS3 were observed among 529 cultivars and 107 wild rice accessions, and 98% of the investigated genotypes carried the same H2 haplotype, indicating that OsMADS3 is highly conserved housekeeping gene. RI127 S has the combined genome constitution of its parents, indica rice Teqing and japonica 02428, and carries the widely compatible S5 gene donated by 02428. RI127 exhibits good performance in regard to its agronomic traits and has a wide compatibility. Therefore, RI127 S would be an elite mediator for recurrent breeding in cases requiring a tedious hand-crossing-based inter-crossing phase. RI127 S can be crossed not only with indica rice but also with japonica rice, thus providing breeders with flexible arrangements in recurrent breeding programs.