Geeneration Of High-quality Japonica Rice Lines Using Gene Editing Technology

Rice is one of the major food crops in the world.It is the staple food of more than half of the world’s population,especially in China.Rice production also plays a very important role in the national economy.However,rice quality improvement started later in China.The produced rice so far can not fully meet the needs of different people populations,and its competitiveness in the international market is not strong,such as long-grain fragrant rice.Therefore,generating of rice varieties with excellent taste and appearance quality has become an important objective of rice breeding in China.The traditional method of rice variety improvement based on hybridization and backcross is low efficiency and taking far too long.With the wide application of gene editing technology in crop breeding,it is feasible to combine several excellent quality traits to improve rice quality.For improvement of the appearance and taste quality of rice,the CRISPR/CAS9 constructs for Wx,GS9,BADH2 and SSSII-2 genes were introduced into different rice varieties previously.In the present study,selectable-marker free homozygous mutants for the target genes were screened from the offspring of these transforms.And the grain shape,appearance and taste quality were evaluated.The main results are as follows:1.Real-time PCR analysis showed that the mutations in Wx,BADH2 and SSSII-2 genes resulted lower expression levels of the target genes compared to those of the recipient parents in most lines,and the expression of GS9 gene was very low in both mutant and wild type(WT)endosperm.2.Mutation in BADH2 gene leads to fragrance in both leaves and endosperm of the transforms.3.The amylose content of the transgenic lines with mutation in BADH2 and SSSII-2 genes was significantly lower than that of the WT,Kangtiaowuyunjing 8.And more soft gel consistency presented for these lines.The amylose content of the lines,HW-1,HW-3 and HW-4 with mutation in Wx gene derived from Huaidao 5,decreased significantly,and the gel consistency of these lines increased significantly compared with the WT.The amylose content of the lines,HGBW-1 and HGBW-2 with mutation in Wx,BADH2 and GS9 genes derived from Huaidao 5,decreased significantly,and the gel consistency of these lines increased significantly compared with the WT.4.The results of starch viscosity spectrum analysis showed that RVA pastes viscosity val ue and RVA profile indices of the lines derived from Kangtiaowuyunjing8 with mutation in BADH2 and SSSII-2 genes,including lower setback values(SBV)and consistence value(CSV),and increased breakdown viscosity(BDV).Lower CSV was observed for most transgenic lines derived from Huaidao 5.5.The results of starch thermodynamic analysis showed that gelatinization temperature(GT)of the lines with mutated BADH2 and SSSII-2 genes decreased.And the GT of the lines with mutated Wx gene had no regularly.6.Rice taste was evaluated using rice taste meter.The results showed that the appearance,viscosity and taste value of the most rice lines with edited target genes were improved,which indicated that the cooking taste quality of these lines was improved.7.Compared with the WT,the grain length and length-width ratio increased significantly,the grain width decreased significantly in the lines with edited GS9 gene.There were no significant difference in most agronomic traits of the other transgenic lines,except for 1000-grain weight and seed setting rate in some lines.