The Functional Analysis Of Rice CCT Family Genes And The Recloning Of Hd1

The heading date is a crucial trait for rice diversification and domestication,greatly determines the regional adaptability and the yield of rice.And it is controlled by multiple quantitative trait loci(QTLs)involved many CCT-domain containing genes.There are 41 members in the CCT domain-containing gene family in rice,which are divided into 3subfamilies: COL,CMF and PRR.The first flowering gene to be isolated by map-based cloning,Heading date 1(Hd1),which is the orthologue of CO in rice,belongs to COL.The central regulator of plant development,Ghd7,belongs to CMF.The major role in controlling rice distribution to high latitudes,Ghd7.1/PRR37,belongs to PRR.Both of Hd1,Ghd7 and Ghd7.1 simultaneously control grain number,plant height and the heading date.To date,13 CCT family genes from these three subfamilies have been shown to regulate flowering.Some of them have pleiotropic effects on grain yield,plant height,and abiotic stresses,and others function as circadian oscillators.CCT family genes are rich in natural variation because rice cultivars have been subjected to natural and artificial selection for different day lengths in the process of domestication and improvement.Alleles of several crucial CCT family genes such as Hd1,Ghd7 and Ghd7.1 exhibit geographic distribution patterns and are highly associated with yield potentials.In addition,CCT family genes are probably involved in the responses to abiotic stress,which should be emphasized in future.In general,CCT family genes play important roles in regulating flowering,plant growth and grain yield.The functional identification and elucidation of the molecular mechanisms of CCT family genes would help construct a flowering regulatory network and maximize their contribution to rice production.To efficiently identify more heading date-related genes from the CCT family.In this study,we use CRISPR/Cas9 technology to generate targeted mutagenesis in CCT family genes in rice.The average frequencies of T0 mutant lines were close to 60%,including10% homozygous or bi-allelic mutant lines.Only 4 of 13 reported genes showed similar phenotypes to those described in previous reports,Which ghd7 and dth2 mutants affect the heading date and plant height at the same time,but hd1 and oscol10 mutants onlychanged the plant height.In addition,we idengtified 4 novel CCT family genes that regulated heading date,Os CCT3,Os CCT22,Os CCT38 and Os CCT41.While Os CCT41,Os CCT5 and Os CCT25 regulate the plant height of rice.We defined eight expression patterns of CCT family genes by q RT-PCR and the expression pattern may relate to functional redundancy.Multiple CCT family genes may shared functional redundancy in floral regulation since 14 groups of double CCT-domain gene mutants showed no obvious change of phenotype.The functional identification of CCT family genes through CRISPR/Cas9-mediated editing would help us effective construct a flowering regulatory network and maximize their contribution to rice production.we observed extremely late flowering phenotype from the pyramid of Ghd7 and Ghd8 in Zhenshan97 background(referred as Zhenshan-Ghd7/8).However,93-11,an indica variety,which carries the same functional Ghd7 and Ghd8 alleles,flowered over35 days earlier than Zhenshan-Ghd7/8.An F2 population was produced by crossing93-11 with Zhenshan-Ghd7/8.Via Map-based cloning approach,Hd1,the previously characterized major flowering time regulator was fished out to be the regulator,which caused extremely late heading of Zhenshan-Ghd7/8.Ghd7,Ghd8 and Hd1 are the major QTLs for heading date in F2 population from the cross between Zhenshan 97 and 93-11,and significant three-way interaction and digenic interactions were observed in the F2 population.Then Ghd7,Ghd8 and Hd1 were re-sequenced in cultivated rice(Oryza sativa)and wild rice(Oryza rufipogon)in order to reveal the polymorphism of nucleotide and evolutionary.Besides,we associated the SNPs with the heading date and the yield,only one site of Ghd7 associated with yield.Strong LD(linkage disequilibrium)was observed among Ghd7,Ghd8,and Hd1 in indica group,while low LD were detected in japonica group except strong LD between Ghd7 and Ghd8.