| Rice(Oryza sativa L.) is one of the most important crop which plays an important role in the food security. Tillering and flowering are two of the most important agronomic traits of rice. Study on tillering and flowering could provide theoretical and material foundation for high-yield and wide adaptability rice breeding. Related studies showed that the process of tillering and flowering formation in rice are affected by various environmental and endogenous genetic network, but their accurate molecular regulation mechanism are still need to elucidated. In our study, by screening of ethyl methyl sulfone(EMS) mutagenesis pool, two tillering mutants (sc64&sc312) and an early flowering mutant (s61) were identified respectively. Firstly, we surveyed and compared the phenotype and major agronomic traits of the mutants and their wild-types. Then, genetic analysis and genes mapping were carry out. Finally, we successfully cloned the candidate genes of the mutants. Our study will be helpful to further reveal the molecular mechanisim of tillering and flowering formation in rice. The main results were described as follows.1)The tillering mutant sc64:Phenotypic analysis showed that, compared with the wild-type, sc64 exhibited increased tillering and reduced height, while its flag length, grain number per panicle and seed-setting rate were decreased. Compared the length of each internode between sc64 and wild-type showed that the dwarf phenotype of sc64 was caused by average decrease of each internode. Genetic analysis illustrated that high tillering of sc64 were controlled by one pair of recessive nuclear gene. Map-based cloning, showed that the mutant gene was located in 326 kb region between two InDel markers S1 and S5 on chromosome 4, and the genetic distances were 0.81 cM and 0.23 cM, respectively. Sequence alignment and analysis revealed that there was a single nucleotide substitution from thymine (T) to cytosine (C) at 385 bp of the CDS in LOC_Os04g46470 which has been named HTD1(High Tillering Dwarf 1)in sc64 mutant, and led to a single amino acid substitution from serine (S) to prolin (P) at 129th amino acids. Therefore, we surmised that HTD1 was identified as the candidate of the SC64 gene, and the abnormal phenotypes of sc64 mutant is probably caused by one amino acid change in HTD1.2) The tillering mutant sc312:The major agronomic traits investigate of the mutant sc312 showed that, sc312 are high tillering and dwarfed compared with wild-type plants. Moreover, the panicle length and seed-setting rate of the mutant sc312 were less than wild-type. Compared the length of each internode indicated that the third and fourth internode of sc312 were decreased most seriously. Genetic analysis showed that the phenotypes of the sc312 mutant were controlled by a pair of recessive nuclear genes. By map-based cloning, the target gene was narrowed in 106 kb region between two InDel markers W4 and W5 on chromosome 6, with 1.00 cM and 1.12 cM genetic distances, respectively. Analysis and alignment of sequence of the candidate gene between sc312 and wild-type revealed that, there was a single nucleotide substitution in the sc312 from guanine (G) to adenine (A) at 1557 bp of CDS of LOC_Os06g06050 which has been named D3(Dwarf-3), which further resulted in a single amino acid substitution from tryptophan to an early stop codon at 519th amino acids. Therefore, we concluded that D3 was identified as the candidate gene of SC312, and the loss of function of the D3 gene was very likely responsible for the abnormal phenotypes of sc312.3) The flowering mutant s61:We characterized an early flowering time mutant s61 from EMS mutagenesis pool of a late flowering time mutant lvpl, s61 exhibits an early flowering time compared with wild-type lvpl. Genetic analysis indicated that the early flowering phenotype of s61 was controlled by a pair of recessive nuclear genes. By MutMap cloning, the target gene was finally mapped on chromosome 3. Sequence analysis and gene functional annotation showed that LOC_Os03g05680-LOC_Os03g05690 is probably the candidate gene of S61 gene. SNP analysis of the candidate region by SNP index exhibited LOC_Os03g05680-LOC_Os03g05690 changed a single base site from guanine (G) to adenine(A) at the splice-recognition site between 5th intron and 6th exon, causing one nucleotide deletion in the sixth exon and lesion of its protein. Therefore, we concluded that the LOC_Os03g05680-LOC_Os03g05690 gene was responsible for the early flowering mutant phenotype which was probably allelic to Se14. Besides, compared with wild-type plants, s61 exhibits less tillering, shorter height, panicle and flag leaf, suggest that Se14 not only mediates flowering pathway, but also plays an important role in other growth and development pathways in rice, which will help to further understand the function of the histone deacetylases Se14 in future. |
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