Fine Mapping And Genetic Analysis Of A Brittle And Susceptible Mutant Bc-sub1 In Rice

The mechanical strength of rice stem is an important agronomic trait in rice breeding.The mechanical strength of plant stem depends on the structure and composition of cell walls,which play critical roles in plant nutrient transportation,signal transmission and defense response.Plant cell wall mutants showing deficient in mechanical strength are important genetic resources for deciphering the molecular regulation of cell wall structure maintenance.In addition,these mutants are useful for investigating the molecular mechanism of cell wall as a natural barrier in immune response against pathogens invasion.We identified a brittle and susceptible mutant bc-sub1 from Ethylmethylsμlfone-induced mutation population of cultured rice Subl.We analyzed the agronomic traits,mutation sites and blast resistance of mutant bc-sub1.The main results are summarized as following:1.The brittle phenotype of the mutant started to appear at seedling stage,and was evident throughout the life cycle of rice.2.The bc-subl mutant showed brittleness on leaf,stem,glume and other tissues and organs.However,the bc-sub1 mutant was similar to wild type Subl in other agronomic traits including plant height,seed setting rate,1000-grain weight and panicles per plant.3.Genetic analysis indicated that a single recessive gene is responsible for the reduced mechanical strength of mutant bc-subl.Map-based cloning showed that the gene is primary located on chromosome 10 between molecular marker RM258 and RM25486.By enlarging the F2 segregating populations,we finally identified the candidate gene that was located between the molecular markers,Inde13 and Inde122,within a 66.82 kb region.In this 66.82 kb region,there are 10 candidate genes out of which six have functional annotation.The six annotated genes respectively encode OsCesA7-synthase(LOC_Osl0g32980),putative receptor like protein kinase 2 precursor(LOC—Os 10g32990),putative unclassified retrotransposon proteins(LOC_Osl0g33000)and(LOC_Osl0g33010),putative receptor like protein kinase precursor(LOC_Os10g33040),and putative hcr2-5D proteins(LOC_Os10g33060).4.In order to identify the genes containing recessive mutations,we amplified and sequenced the candidate gene LOC_Os10g32980 encoding OsCesa7-synthase from seven independent F2 lines that show brittleness as mutant bc-subl.We found that the OsCesA7 gene in all the seven lines contains a nucleotide substation at position 3661 which was changed from C to T.Consequently,the 847th amino acid in OsCesa7-synthase is changed from leucine(Leu)to phenylalanine(Phe).These results showed that the gene LOC_OslOg32980 very likely determines the brittleness phenotype of the bc-sub1.5.By assaying the susceptibility of rice against rice blast pathogen Magnaporthe oryzae,we found that mutant bc-sub1 was more susceptible than the wild type.These results indicated that OsCesA7 gene could affect the resistance of rice against Magnaporthe oryzae.6.We further employed RT-PCR to determine the expression of the pathogenesis related gene(PR gene)in bc-sub1 mutant and wild type.The results showed that the expression levels of pathogenesis related(PR)gene,Os04gl0010 and OsKS4 were significantly decreased in bc-sub1 plants compared to wild type.The expression of PR gene can be induced in the wild type when treated with flg22 and chitin.But the induction of gene expression was severely compromised in mutant bc-sub1.Collectively,these results suggest that OsCesA7 gene plays an important role in the immune response to disease resistance in rice.