Development Of New Rice Germplasms With Improved Heading Date Via CRISPR/Cas9 Technology

Heading date is one of the important agronomic traits of rice,which determines the regional adaptability and seasonal adaptability of rice varieties.The regulation and improvement of rice heading date can shorten the growing period of current rice varieties and expand the planting area of high-quality rice varieties and increase the yield.In recent years,the research of rice heading date regulatory network has made some progress,and a large number of heading date related genes have been cloned.Recently,gene editing technology represented by CRISPR/Cas9 has gradually developed into an important tool for the research of heading date related genes,and its wide application in rice breeding and variety improvement has also greatly accelerated the process of rice breeding.In this study,we used CRISPR/Cas9 to edit the promoter of RFT1 and the coding region of Hd16 which are rice heading date related genes under the background of Huaidao5 and Nanjing5055.We also analyzed the rice quality of the new PHYC knockout germplasm under the background of Nanjing46.The main results are as follows:1.Edit the promoter region of RFT1 to create mutants with variation in heading dateHuidao5 and Nanjing5055 belong to late-maturing medium japonica and early-maturing late japonica respectively,and are suitable for planting in central and southern Jiangsu.In order to improve their heading date,we used the CRISPR/Cas9 technology to edit the promoter region of the RFT1 which is the rice florigen gene.Firstly,the transgenic positive plants obtained by genetic transformation were sequenced and analyzed,and secondly,the exogenous genes were eliminated.After the addition of generation homozygous and exogenous gene deletion,17 homozygous mutants with different genotypes were obtained under the background of Huaidao5,and 2 homozygous mutants with different genotypes were obtained under the background of Nanjing5055.The investigation results of heading date of mutant lines under natural long-day conditions in Nanjing showed that most of the mutant lines in the RFT1 promoter region under the background of Huidao5 was later than that of the wild type.Among them,the heading date of rft1-10,rft1-11 and rft1-12 were about 10 days later than the wild type.Notably,the heading date of rft1-1,rft1-9 and rft1-15 were earlier than that of wild type.There was no significant chang of the heading date of the mutant lines in the RFT1 promoter region under the background of Nanjing5055.The agronomic traits of three mutant lines rft1-10,rft1-11 and rft1-12 with late heading stage under the background of Huaidao 5 showed that except for the increase of plant height of rft1-12,there was no significant difference between wild type and other agronomic traits of mutant lines.In yield-related traits,the ear length and grain number per ear of the three mutant lines decreased significantly compared with the wild type,while the1000-grain weight did not increase significantly,which led to a significant decline in yield.These changes suggested the yield of mutants decreased.2.Edit Hd16 coding region to create early-mutants with variation in heading dateIn this study,the coding region of the flowering inhibitor Hd16 in the photoperiod flowering regulation pathway of rice under long-day light was functionally knocked out in Huidao5 and Nanjing5055.Four homozygous mutants hd16-1,hd16-2,hd16-3 and hd16-4 with different genotypes were finally obtained after the addition of the generations to produce homozygous and the deletion of foreign genes.Among the two cultivars,the heading date of the mutant with hd16-4 genotype was significantly earlier,which was consistent with the expected effect.The heading date of mutants with hd16-1,hd16-2 and hd16-3 genotypes unexpected did not change significantly in the background of Huaidao5.The results of the survey of agronomic traits showed that there was no significant difference between wild type and mutant in tiller number and ear length under two different background materials.The plant height of mutants with hd16-4 genotype in both backgrounds was significantly lower than that of the wild type,which was related to the shortening of the vegetative growth period.It is worth noting that in the background of Huidao5 the number of grains per panicle of the mutant with hd16-4 genotype was significantly increased compared with the wild type;while the hd16-4 gene in the background of Nanjing5055.However,the number of grains per panicle decreased significantly in the mutants,which may be related to the effect of genes at heading stage affected by genetic background and environmental conditions.3.Rice quality analysis of PHYC knock-out mutantsThe heading date of the new germplasm with PHYC knockout under the background of Nanjing46 was about 1 week earlier than that of the wild type,which could be inherited stably.The rice quality of the new germplasm planted in Nanjing area was analyzed,and the results showed that the chalky grain rate and chalkiness degree of PHYC knockout new germplasm increased significantly,the transparency of milled rice decreased,and the appearance quality was lower than that of wild type.The measurement results of the eating quality index of the PHYC knockout new germplasm showed that its amylose content decreased significantly which made it softer than Nanjing 46 after cooking,while the gel consistency did not change significantly.We suggested this germplasm is considered suitable for planting in northern Jiangsu where light and temperature conditions are more suitable.In conclusion,this study used CRISPR/cas9 gene editing technology edited the heading date genes in Jiangsu high-quality japonica rice varieties,and obtained new rice breeding germplasm with heading date improved.Then the agronomic traits and rice quality of the new germplasm were analyzed and identified.This study lays a foundation for cultivating new varieties with improved heading date.