Identification And Gene Cloning Of Rice Tillering Mutant 5575

Rice is an important food crop,with more than half of the world’s population relying on rice as a staple food.High and stable rice yields are crucial to protecting China’s food security.Growth and development factors such as rice plant height,tillering number and panicle stage have an important impact on rice yield.We found a mutant material with abnormal growth and development in the rice T-DNA insertion mutant library with the background of Zhonghua 11,named 5575.The mutant showed a phenotype such as few tillers,light green leaf color at seedling stage,delayed spike stage,and low setting rate.In this study,the mutant 5575 was phenotypically identified,the main agronomic traits were investigated,and the photosynthetic pigment content was statistically and determined,and the candidate gene was further cloned by BSA sequencing.The main results are as follows1.The 5575 mutant has slow growth and development at the seedling stage,with few tillers and light green leaf color;The panicle stage is later than that of the wild type;Compared with the wild type,the plant height,solid grain number per panicle number,setting rate and tillering number of mutants were significantly or very significantly reduced.2.At the seedling stage,the analysis results of photosynthetic pigment content showed that the chlorophyll a(Chla),chlorophyll b(Chlb)and total chlorophyll(Total Chl)of the mutant 5575 leaves were significantly lower than those of the wild type.It is speculated that this may be due to obstruction of chlorophyll synthesis.3.The wild type was crossed with the mutant 5575,the F1 representative type was normal,and the F2 generation was separated by traits.Among them,there were 138 wild type phenotypes and 62 mutant phenotypes,and chi-square detection showed that it conformed to the 3:1 Mendelian genetic law.This indicates that the phenotype of mutant 5575 is controlled by a pair of recessive nuclear genes.4.Using BSA mixed-pool sequencing,the candidate gene was mapped between chromosome 3 1920k-2795 k.Comparing wild-type and 5575 sequences in this interval shows that four SNP sites within this interval in which the PLS-type protein(LOC＿Os03g04390)gene encoding the PPR family undergoes a deletion of one base C on its exon,resulting in a frameshift of the protein sequence.The 5575 gene encodes a PPR protein family containing781 amino acids,belongs to the PLS family,contains a DYW domain at its N-terminus and 13 PPR motifs at its C-terminus,and PPR protein family is involved in the editing,splicing,maturation and translation of RNA in plant plastids.5.In order to verify the candidate gene,the gene was knocked out in Zhonghua 11 by gene editing technology to obtain Cas9-5575,and it was found that the phenotype of Cas9-5575 and 5575 was similar,indicating that LOC＿Os03g04390 was indeed a candidate gene of5575.6.Compared with the wild type,the splicing efficiency of chloroplast transcripts atpF,rpl16,ndhB-2,trnV,rps16 in 5575 was significantly reduced,while the splicing efficiency of trnA and ndhA-1 in 5575 mutant was higher than that of wild type.