Developmental Application For Rice Mutant Screening And Molecular-assisted Breeding Based On CRISPR/Cas9 System

Mutant screening plays an important role in plant functional genomics research,crop improvement and breeding.Targeted genome editing tools,ZFN,TALEN and CRISPR/Cas9 system,provides a novel method for mutant screening,which was different from random inactivation of natural variation,physical,chemical,T-DNA insertional mutagenesis.Among them,CRISPR/Cas9 system has been widely used for genome modification in various species with inexpensive cost,easy operation,multiplex editing and high-efficiency.Rice not only belongs to the three major grain crops,which supports food source for people worldwild,but also is used as model plant to study growing development for monocotyledons and evolution of agronomic traits in cereal crop.Therefore,mutant screening of rice important agronomic trait genes plays a vital role in yield increase,and also provides critical materials for gene function analysis.In our work,35 rice genes were performed to targeted genome editing through CRISPR/Cas9technology,and mutant events for 7 inflorescence related genes,1brassinolide related gene and 2 agricultural production genes,CSA and SD1,were generated.Given the phenotype characteristic of mutant events induced by CRISPR/Cas9 and the analysis for editing efficiency of CRISPR/Cas9 system,the conclusions mainly included as follows.1.CRISPR/Cas9 system was used for target editing of the photo-sensitive genic male sterile gene,CSA,and dwarf gene,SD1,separately.The results showed that the fertility convertion of 9522^csa lines and JY5B^csa lines was observed in different day length,and the feritily of KY131^csa lines may associated with both photoperiod and temperature.And the dwarfing of plant height caused by modifying SD1 gene using CRISPR/Cas9 was showed in diverse rice varieties,inculding NG9015,JY5B,JD138,C15,9815B and P405.These results together indicate that CRISPR/Cas9 system has the great applicable prospect for molecular-assisted breeding in rice.2.The targeted editing lines of OsMYC2 utilizing CRISPR/Cas9system exhibited short in plant height compared with wild type,indicating OsMYC2 may be involved in cotrolling plant height.Meanwhile,mutant events in LOC_Os01g51260,LOC_Os06g46560 and LOC_Os04g56150based on CRISPR/Cas9 system displayed a significant change in spikelets number per panicle,secondary branch number,spikelet length and seed setting rate,suggesting that the genes may participate in regulating panicle development.3.By characteristics analysis of target sequence and rice genome,refering to genome sequence extending from the middle position of target sequence to the flanking sequence separately in rice,in 23 rice candidate genes,we found that the target sequence with some rules may be easier to modify through CRISPR/Cas9 system.Firstly,two PAM sequences exists in both ends flanked by target sequence,respectively;Sceondly,when the GC content of target sequence is in 50⁷0%,the GC content in rice genome ranged from 20 bp to 80 bp or 80¹00 bp increases or decreases with extended nucleotide lengh separately,and decrease for within 80¹00 bp and increase for within 20⁸0 bp.Then,when the rate of GC in target sequence reaches 75%,the GC content of rice genome within the extent of20¹00 bp is 75±3%.Finally,when the GC content arrives 80%in target sequence,the GC content of rice genome with a range of 20¹00 bp is fluctuant with a range of 72%to 88%.