Establishment Of Mutant Library By EMS Treatment And Screening Of ALS-inhibitor Herbicide-resistant Mutants In Brassica Napus L.

Oilseed rape(Brassica napus L.)is the most important oil crops in our country.Weed control is a bottleneck to realize rapeseed industry mechanization in our country.It is the most economic approach to control weeds by using herbicide-resistant varieties.To enhance the genetic resource and creat mutants with resistance to herbicide tribenuron-methyl,the dry seeds of Zhongshuang No.9(B.napus),a widely used conventional cultivar in our country,was treated by different concentrations of EMS.The results obtained are as follows.1.EMS treatment depressed the germination percentage of M1 population,but did not impact the germination percentage of M2.With EMS concentration increasing,the depressing effect increased.When EMS concentration increase to a threshold,the germination percentage of M1 population dropped sharply.There was a nolinear relationship between EMS concentration and germination percentage of M1 population.EMS treatment prolonged emerging time of M1 and M2 populations.So,EMS mutagenesis not only mangled M1 population but also restrained M2 population remarkably.2.Different kind of mutants,including those with flowers more than 4 petals,no pistil,flowers without blossoming,white new leaves,were detected in the mutant library,ranging from mutants of cotyledons,leaves,flowers,plant-types to siliques.All these mutants will become the basis materials for studying gene functions and improving varieties with new traits in B.napus.3.By counting the mutunts of cotyledons,leaves,flowers,plant-types and siliques in M2 population which included 16,176 plants in total,the phenotype variations in four M2 populations(0.4%0.8%,1.0%and 1.2%EMS solution treatment)were 11.26%,14.82%,27.19%and 12.38%,respectively.So,1.0%of EMS solution treatment was optional for mutagenesis of Zhongshuang No.9 dry seeds.4.The effects of different mutagen concentrations varied among different organs.In our experiment,the mutant rate was highest in the population treated by 1.0%EMS solution,however,this concentration EMS treatment also induced siliques becoming shorter.The mutants with some new traits,including mutants with almost all flowers more than 4 petals,or with flowers without blossoming,were discovered in the population treated by 0.4%EMS solution.Higher concentration(1.2%EMS)has positive effects on the root.So,treatment with lower concentration of EMS solution is benefit to reproductive organs,and that higher concentration of EMS treatment is benefit to improve the root trait.5.Three mutants with resistance to tribenuron-methyl were screened out,recorded as Rl,R4 and R5,and the mutant rate is about 1.0*10-6.By cloning and sequencing of the ALS1,ALS2 and ALS3 genes of Zhongshuang No.9,R1,R4 and R5,we discovered that ? ALS1 of R5 has a nonsynonymous mutation,+544(C?T),resulting in P197 to S(C544CT?TCT),the amino acid number is based on Arabidopsis thaliana ALS gene(the same below),which maybe the main reason to the resistance;?of R1 and R4 has a nonsynonymous mutation,+535(C?T),resulting in P197 to S(C535CT?TCT),which maybe the main reason of R1 and R4 to the resistance;the resistant level of R5 is higher than R4's;? Compared with ALS3(Z11526.1 in Genbank),ALS3 of Zhongshuang No.9 has five SNPs,including four synonymous mutations +135(A?T),+312(C?T),+660(A?T)and +816(C?T),and one nonsynonymous mutation,+975(G?C),which results in the coding amino acid E to D(GAG975?GAC).