Construction Of EMS Mutant Library Of Flax And Identification And Analysis Of Als-resistant Herbicides And High Oleic Acid Mutants

Flax（Linum usitatissimum L.,2n=30）is one of the important oil crops in the world,with oil content up to 35-45%and rich in unsaturated fatty acids,which have important edible and industrial values.China is the second largest producer of flaxseed,increasing the content of oleic acid or linolenic acid is the main goal for flax breeding.In addition,there is no safe and effective broad-leaved herbicides in flax production,so breeding herbicide-resistant flax has important application value.In this study,a Flax mutant library was constructed by EMS（Ethyl methyl sulfone）chemical mutagenesis with an elite cultivar Longya10,as the receptor.Several mutants with important agronomic trait variation were screened out,and the resistance mechanism of herbicide-resistant mutant was analyzed.Moreover,the mutant gene associated with high oleic acid was predicted by multi-omics,it provides basic materials and theoretical basis for breeding new varieties and mining functional genes of flax.The main results are as follows:1.About 100,000 individuals of M₁ population were induced by EMS.Various types of variation in plant type,flower type,leaf type and flowering period were observed in the M₂ population（about 4,400 lines）.Through three consecutive generations of screening（1185 in M₃generation,367 in M₄ generation and 108 in M₅ generation）,32 fatty acid extreme value mutants with stable inheritance were obtained.The results of field experiments in the main flax producing area（Lanzhou,Gansu Province）showed that 9 of the mutants had breeding value,two mutants with high oil content（increased by 2.95-3.33%）,two mutants with high oleic acid content（increased by 4.24-7.45%）,and five mutants with high linolenic acid content（increased by 5.09-7.51%）.2.An ALS-resistant herbicide mutant R10 was screened from M₂population.The results of resistance test showed that R10 was able to tolerate 593.92 g ai.ha^-1 TBM,showing high resistance to TBM（SU）.Field experiment showed that R10 grew well under 18.56 g ai.ha^-1 TBM treatment and was effective in removing broadleaf weeds.The Lu ALS gene functional mutation site identification of R10 showed that the Lu ALS1 gene underwent a base mutation C556T,resulting in a proline substitution for serine at position 186（At ALS-Pro197）in Lu ALS1,which was first identified in flax.By analyzing the docking model,it is found that reduced hydrogen bonding and weakened hydrophobic force between Lu ALS1-R10 and TBM.T₂ generation transgenic Arabidopsis thaliana overexpressing 35S:Lu ALS1-R10 was able to survive with 0.145g ai.ha^-1 TBM treatment,verifying the resistance function of the Pro197Ser mutant site.3.Two CAPS markers were developed based on the mutation sites of Lu ALS1 gene,which can accurately detect the genotyping of Lu ALS1and are applicable to several main cultivars in China.4.The mutant M45 with high oleic acid was screened from the mutant library.The results of fatty acid composition showed that the oleic acid content of M45 continued to increase,while Longya10 showed a decreasing trend from 10 to 20 d.The oleic acid content of M45 at 40 d was 43.22%,which was 21.23%higher than that of Longya10.5.180 genes with SNPs and 42 genes with Indel variant loci associated with high oleic acid traits were screened by BSA-Seq.By analyzing the transcriptome sequencing data,it was found that two SAD homologs（L.us.o.g.scaffold194.104,L.us.o.g.scaffold205.47）and two FAD2 homologs（novel.1295,novel.1294）in the differentially expressed genes between M45 and Longya10 were in accordance with the high oleic acid phenotype.It was hypothesized that these four genes may be the main cause of high oleic acid in M45.The lipid metabolome results showed opposite trends in the accumulation of free oleic acid between M45 and Longya10 at 10-20 d,indicating that the difference of oleic acid content was mainly reflected in free fatty acids.Based on the combined analysis of BSA,transcriptome and metabolome,it was found that there was a negative correlation between the expression level of L.us.o.g.scaffold122.86 and the accumulation of oleic acid,and there were three variant sites in the 3’UTR.So presumably,L.us.o.g.scaffold122.86 may be a candidate gene for high oleic acid content in M45.