CRISPR/Cas9-mediated Editing Of GS3 To Improve Flowering Time In Japonica Rice

Rice is one of the main cereal crops in the world.The planting area of rice is about 3067hm² in China,of which japonica rice accounts for about 21.7%.However,the planting area of hybrid japonica rice barely accounts for 5 percent of Japonica rice.Compared with hybrid indica rice,the development of hybrid japonica is slower relatively and its application is limited.One of the utmost factors,which affects the development of hybrid japonica rice,is low seed production.Additionally,the poor flowering habits of japonica rice are the main factor restricting the yield of seed production.The poor flowering habits mainly reflect in the lack of concentration and dispersion of flowering,and the peak period is not obvious.Over the past years,the breeding workers did many researches on the flowering habits and the results indicated that the same subspecies?indica or japonica rice?have different flowering habits.The longer the grain,the earlier the flowering,the rounder the grains and the later the flowering,which is particularly prominent in the japonica rice varieties.Because the grain length and width ratio have the stable genetic traits,which could provide references to select the sterile lines in early flowering,and it hopefully regulate the flower opening time by changing the length and length-width ratios.In this study,we used CRISPR/Cas9-mediated method to edit GS3 in 13 japonica varities and got13 pairs of near-isogenic lines.The main results were shown as follows:1.Construction of target pointsThe GS3 gene is located on the chromosome 3 in rice and contains five exons.It encodes a transmembrane protein involving 232 amino acids,which may function as a negative regulator for grain size.According to the CRISPR/Cas9 system to identify the specificity of Protospacer adjacent motif,we design the gRNA target point at the upstream and downstream of the ATG on the first exon.2.Agrobacterium transformation and the mutation validation of transgenic T₀ plantsWe transform the target PC1300-Cas9-GS3 vector into Agrobacterium EHA105 by heat shock,and then respectively transformed it into pro-embryogenic callus of rice varieties changbai25,jijing102,kongyu131,kenjiandao6hao,zhejing22,zhejing88,wuyunjing27,yangjing4227,nanjing9108,J5933,J6167,J5938,and J42.The genomic DNA of them of T0 plants was extracted by the CTAB method,then it was processed by PCR-RE and sequencing analysis.3.A study on agronomic traitsThe agronomic traits including the days from sowing to heading,plant height,grain length,grain width,and length-width ratio of homozygous mutant were investigated.There was no significant difference between transgenic lines and wild lines in sowing to heading and grain width.The grain length and length-width ratio of transgenic lines were longer compared to the wild type,however the plant height is decreased in most of the transgenic lines.4.Difference analysis of flowering timeFlowering time analysis showed that the homozygous mutants were earlier than those of wild type,especially the flowering time of the gs3 mutants of Jijing102,Wuyunjing27,Kongyu131,Zhejing88 and Yangjing4227 were significantly earlier than those of the wild type.The others were also earlier,but there was no significant difference.CRISPR/Cas9-mediated editing of GS3 is to improve flowering time in japonica rice,which could provide references to the study of grain-type breeding of japonica rice,and it would accelerate the breeding process of long-grain japonica parents and promote the development of hybrid japonica rice.