Development Of New Rice Germplasms With Improved Fertility,Starch Quality And Heading Date Using CRISPR/Cas9 Technology

In recent years,CRISPR/Cas9,one of the gene editing technologies,has been widely used in rice genomics research and germplasm development due to its high efficiency,ease of use,and accuracy.In this study,we used the CRISPR/Cas9 system to edit several genes responsible for rice fertility,starch quality and heading date.Multiple lines with improved target traits were obtained.The main results are as follows.1.CRISPR/Cas9-mediated gene editing of TMS5.TMS5,a key gene for thermo-sensitive genic male sterility,is widely used in two-line hybrid rice.In order to accelerate the breeding process of thermo-sensitive sterility lines,CRISPR/Cas9 technology was employed to edit TMS5 in the Wuyunjing 7(a high-yield japonica rice)background,and several homozyougou mutation lines were developed.Cytological observation and artificial pollination investigated that the pollen of tms5 mutant was aborted,but the stigma receptivity was normal.These results indicated that thermo-sensitive male sterile lines could be rapidly obtained by knocking out TMS5 in a japonica rice.2.CRISPR/Cas9-mediated gene editing of FLO2.FL02,encoding a tetratricopeptide repeat domain(TPR)-containing protein,regulates starch amylose content in rice.In order to obtain new germplasms with low amylose content,the FLO2 gene in the Suken118 background was edited by CRISPR/Cas9 technology and two homozygous mutants were obtained.The amylose contents of these two mutants were decreased by 9.2%and 8.8%,respectively.However,the 1000-grain weight of these two mutants significantly decreased.3.CRISPR/Cas9-mediated gene editing of PHYC.The PHYC gene plays important roles in plant photomorphogenesis,photosynthesis and heading date.Nanjing46 is an excellent but late-maturing variety.The PHYC gene in the Nanjing46 background was edited and two homozygous mutants were obtained.The heading dates of these two mutant were 8.2d and 8.1d,earlier,respectively,than Nanjing46.4.CRISPR/Cas9-mediated multiplex gene editing of OsDof12 and DTH2.Under long-day conditions,the OsDof12 and DTH2 genes promote flowering in rice.The OsDof12 and DTH2 were simultaneously edited in the Zhendao 99 background and two homozygous double mutants were obtained.Under natural long-day conditions in Yangzhou,the heading dates of double mutants were 13.0d and 7.8d later than Zhendao 99.In this study,we developed several new germplasms by CRISPR/Cas9-mediated editing genes responsible for thermo-sensitive genic male sterility,amylose content and heading date,providing new genetic resources in molecular breeding in rice.