Control Of Thousand-grain Weight And Heading Date By OsMADS56 In Rice

Thousand-grain weight（TGW）is one of the three components of yield traits which is mainly determined by grain size（GS）.Most of the cloned QTL for grain weight are major QTL,but minor QTL are less favored due to its small effect,difficulty in mapping and functional verification lead to slow progress in gene cloning.In the previous study,a minor QTL qTGW10-20.8 controlling TGW was mapped in a 70.7 kb region on chromosome 10using a population derived from an indica rice cross Teqing/IRBB52.OsMADS56 was the most likely candidate gene based on nucleotide polymorphism and expression analysis between the parental alleles.In this study,the positive regulation of OsMADS56 on grain weight and grain size in rice was determined using near-isogenic line（NIL）populations combined with gene knockout and overexpression assay.Besides,the role of OsMADS56was analyzed in indica rice cultivars.The function of OsMADS56 was initially explored.Main results are as follows:1.OsMADS56 is the causal gene for qTGW10-20.8According to the previous fine mapping results,three residual heterozygous plants were selected from one segregating population of Teqing/IRBB52 and three NIL populations were constructed.The segregating region of TE1 covers the entire interval of qTGW10-20.8.The segregating region of TE2 only covers OsMADS56.The segregating region of TE3 is adjacent to the downstream boundary of qTGW10-20.8 and does not contain OsMADS56.The three populations were tested for two years in the same field and examined TGW,grain length（GL）,grain width（GW）and heading date（HD）.Significant differences were detected for all traits in TE1 and TE2 but not in TE3,suggesting that OsMADS56 is the causative gene responsible for qTGW10-20.8.The function of OsMADS56 was further verified by gene knockout and overexpression analysis.While knockout of OsMADS56 resulted a reduced GS and TGW.Overexpression of OsMADS56showed an increased GS and TGW.2.The expression level of OsMADS56 affects TGW,GS and HDThe expression level of OsMADS56 was detected using young panicles in TE2 NIL population,knockout population and overexpression population.The relative expression of OsMADS56^IRBB52was significantly lower compared to OsMADS56^TQ,which was associated with the decreased TGW,GS and longer HD in OsMADS56^IRBB52.Knockout mutants showed a significant decrease in expression of OsMADS56 compared to wild-type,the degree of reduction in premature termination mutant was higher than the mutant with a small amount of amino acid deletion,so as the TGW and GS,due to the weak effect of OsMADS56 on HD,knockout mutants showed different variations on HD may influenced by background genes.Scanning electron microscopy of spikelet hull from the knockout mutant showed that OsMADS56 increased the length and width of spikelet hull by promoting cell divisions,and finally increased GS and TGW.Compared with the wild-type,the expression level of the overexpressed plant was significantly increased,and the TGW and GS were also significantly increased.The expression level of OsMADS56 was positively correlated with GL,in the segregating population,the HD of positive plant was significantly shortened.It indicated that OsMADS56 could positively regulate TGW and GS,and promote heading.Sequencing of the parental OsMADS56 allele promoter revealed that-320 bp（A/G）and-793 bp（T/C）resulted in OsMADS56^TQ had two additional TATA-boxes related to transcription initiation and one CAAT-box related to transcription frequency than OsMADS56^IRBB52,OsMADS56^TQ may had higher expression efficiency.3.OsMADS56 mainly regulates GW and HD in indica rice cultivarsThe haplotypes of OsMADS56 were analyzed by using 294 indica rice cultivars based on that polymorphism of OsMADS56 coding region and four traits including TGW,GL,GW and HD were examined.OsMADS56^TQ（Hap1）and OsMADS56^IRBB52（Hap5）are the main haplotypes among five haplotypes.Differences in GW and HD were observed between Hap5 and other three haplotypes.Similar results were founded in subpopulation after classifying GS3 and GSE5 according to their function,suggesting that 9 bp deletion in Hap5 presumably associated with GW and HD.4.Possible function mode of OsMADS56Research showed that OsMADS56 interacted with OsMADS6,OsMADS14,OsMADS15 and OsMADS57,respectively.Os RDCP1 and Os WRKY68 were downstream target genes of OsMADS56.OsMADS56 may performing function on TGW,GS and HD through the above interaction.In conclusion,we verified that OsMADS56 is the casual gene of qTGW10-20.8,which positively regulates TGW and GS,also promotes heading.This study provides an additional gene resource for rice molecular breeding,and contributes a theoretical basis for the improvement of rice TGW and GS.