| Heading date,also known as flowering time in rice,is one of the essential agronomic traits,which largely determines the seasonal and regional adaptability of the varieties.Under limited natural light and temperature conditions and land resources,the high and stable yield breeding goals can be achieved by adjusting the heading date of rice.The heading date is regulated by a complex molecular network,which requires the integration of exogenous environmental signals and endogenous signals.The mining of early maturing genes and the study of molecular mechanisms can provide genetic resources and theoretical basis for manipulating heading date.This research focuses on the mining of rice early maturity genes,the creation of early maturity resources,and the development and utilization of related technologies,and can be divided into the following two parts.1.In this study,F1plants were obtained by crossing an early maturing indica cultivar MH1602（’Minghui 1602’）with a late maturing indica cultivar MNH1（’Mingnuohui 1’）.The heading date of reciprocal F1 plants were similar to that of MH1602,indicating that the early maturing gene in MH1602 was dominant.The heading date of this F2population was continuously distributed and had the phenomenon of transgressive segregation in LDs,which indicated that the early flowering phenotype of MH1602 was controlled by multiple genes.SNP,In Del and SSR markers were used for QTL analysis.Two QTLs for heading date were detected on the third and seventh chromosomes,named q Hd3 and q Hd7,respectively.q Hd3 locus accounted for 30.55%of the phenotypic variation,with LOD value of 17.93.The additive effect of q Hd3 came from the locus of MH1602.The contribution rate of q Hd7 was 7.67%,the LOD value was 6.64,and the additive effect value was 1.80.By using MNH1 as recurrent parent to construct BC3F2 population,the two QTLs,q Hd3 and q Hd7 locus were repeatly detected,this verified the reliability of QTL mapping results.In the background of MH1602,the coding region of DTH7 has an 8 bp deletion of’GAACGTTG’,which resulted in the deletion of CCT domain,suggesting that q Hd7 was the allele of DTH7.Under LDs,the non-functional DTH7 gene promoted the heading date of MH1602.There were two reported genes near q Hd3,including DTH3 and Ef-cd.Sanger sequencing reveals no difference in the coding region of DTH3 between these two parents,which ruled out the possibility that q Hd3 was an allele of DTH3.There were only two SNPs in the genomic sequence of Ef-cd between these two parents,and both locate in the intron.Further analysis using the rice 3K data showed that MH1602 and MNH1 could be divided into two different haplotypes,but the difference of heading date between the two haplotypes was only 1.3 days,and the difference was not statically significant.Therefore,q Hd3 is likely to be a new QTL for early maturity in rice.By constructing NILs with different QTLs,it was found that NIL（q Hd3）flowered 11days earlier than the recurrent parent MNH1,and there was no significant difference in yield related agronomic traits,indicating that q Hd3 locus has certain breeding value.The q Hd3 locus can be transferred into late maturing rice cultivar by backcross method.2.The development of CRISPR/Cas9 gene editing technology has accelerated the process of rice breeding.Ningjing7 and Ningjing8 are two elite rice cultivars bred by our laboratory,and they belong to late-maturing medium japonica and early-maturing late japonica respectively.They are suitable for planting in central and southern Jiang Su province.In order to further expand the suitable planting area of these two varieties,we first used the CRISPR/Cas9 system to knock out the coding regions of the main LD flowering suppressor genes Hd1,Ghd7,DTH7,DTH8,Os HAPL1,Hd16,respectively.As expected,the heading dates of the two varieties were significantly earlier after knocking out of Hd1,Ghd7 or DTH8.Surprisingly,the heading dates of the two cultivars were significantly delayed after knocking out of DTH7,while editing of Os HAPL1 and Hd16 had no effect on the heading date.This may be due to the genetic background and environmental conditions effects of the heading date genes.To overcome the large effect of direct gene knockout that might cause negative effects on yield,this study designed random targets to edit the promoters of key heading date genes to fine-tune the expression of these genes,and to obtain genetic resources with continuous variation.In order to construct a technical system for promoter editing,this study constructed a p CRISPR-zero vector suitable for efficient multi-target editing by modifying the TKC vector.Eight targets were designed to edit the promoter regions of Hd1,Ghd7,DTH7,and DTH8.Sequencing of the obtained T0 generation transgenic seedlings showed that various types of mutations appeared in the designed targets,which verified the high efficiency of using p CRISPR-zero vector for multi-target editing of the promoter.According to the obtained mutant group of multi-targets editing in the promoter region,improved varieties with suitable heading date can be obtained from it,which is conducive to the introduction of Ningjing 7 and Ningjing 8 to broader regions,and further increases their value. |
