Breeding Of Selectable Maker-free High Quality Rice

Rice is a staple food for nearly 60 percent of the Chinese population.Increasing rice yields has become a priority for breeders and quality improvement was neglected for a long period in the past years.Therefore,rice quality in our country can’t meet people’s demands.Rice quality traits are controlled by multiple genes with complex genetic basis.Therefore,it is not easy to further improve rice quality characters only by conventional breeding methods.For improving the appearance quality,eating and cooking quality(ECQ)of rice,the CRISPR/CAS9 technology was employed to edit Wx,GS9,BADH2 and SSSII-2 genes of 2 rice varieties previously in our lab,which are suitable for cultivated in Jiangsu province.In the present study,new selectable-marker free lines with mutation in both GS9 and BADH2 genes were obtained.For selection of lines with better comprehensive characters,the ECQ and agronomic traits of these lines were further evaluated.The main results are as follows:1.Two selectable-marker free homozygous with mutation in both GS9 and BADH2 genes were obtained from the Japonica line ZZ-16.The genotype of the hygromycin resistance(HPT)gene and the target genes,Wx,GS9,BADH2 and SSSII-2 in the 12 lines obtained previously was confirmed.2.Real-time PCR analysis showed that the expressions of the target genes in most of the lines were reduced accompanying with mutation in the target genes(Wx,GS9,BADH2 and SSSII-2)compared to the wild type(WT).3.The amylose content(AC)of the two lines,HDW-1 and HDW-2 derived from the Japonica variety Huaidao 5(HD5H)with shift mutation in Wx gene was reduced significantly compared to HD5H and close to that of glutinous rice(AC<2%).The gel consistency(GC)of these 2 lines was increased significantly compared to HD5H.Their gelatinization temperature(GT)was reduced significantly.These indicated that they are softer than the WT.Most RVA(Rapid visco-analyser)values of these lines were reduced significantly and taste values were increased significantly.The AC,GC,GT,RVA values and taste values of the three lines,HDGBW-1-3 derived from HD5H with mutation in Wx,GS9 and BADH2 genes were similar to HDW-land HDW-2.And their seeds were longer with fragrance.4.The grain length of HDGBW-4,HDGBW-5 and ZGBW-1,ZGBW-2 derived from HD5H and ZZ-16 respectively with mutation in GS9 and BADH2 genes was increased significantly compared to the corresponding WT.These four lines have fragrance.And the taste values of HDGBW-4 and HDGBW-5 was increased significantly compared to HD5H.5.The AC of the three fragrant lines,KBS-1-3 derived from the Japonica rice Kangtiaowuyunjing 8(K8)with mutation in BADH2 and SSSII-2 genes was reduced significantly compared to K8.And it is higher than that of soft rice(AC 5%～12%).The higher GC and lower GT values indicated softer characters of KBS-1～3 compared to K8.Some RVA values of these three lines were lower than those of K8,including the peak viscosity(PKV),hot paste viscosity(HPV),cool paste viscosity(CPV),and setback viscosity(SBV).Breakdown viscosity(BDV)of KBS-1～3 was increased significantly compared to K8.The taste values of these three lines were higher than that of K8 and their endosperm retained transparency,similar to K8.6.Most agronomic traits of all the lines obtained in present study had no significant change compared to the WT,except for some lines with slight differences in 1000-grain weight,grain number per spike and seed setting rate.Finally,four lines with improved quality and good agronomic traits,ZGBW-1,HDW-1,HDGBW-1,and KBS-2,were selected from the 14 selectable marker-free lines.