| Originating in China,Soybean has been cultivated for more than 5,000 years.Its seeds are full of protein and fat.It is also one of the major sources of edible oil and seed proteins in the world.Soybean fats are full of unsaturated fatty acids,accounting for 80-90%of total fatty acids and saturated fatty acids accounting for 6-20%.The composition of fatty acids is linoleic acid 41-62%,oleic acid 12.4-39%,palmitic acid 10-14%,linolenic acid 5-14.7%,and stearic acid 1.63-5.43%.Soybean fatty acid composition and ratio has directly effects on the nutritional value,storage,transportation and processing of soybean oil,and is the most important factor to determine the quality of soybean oil.Therefore,The increase of fat content is an important content of soybean quality breeding.In this study,a number of soybean germplasm resources with high protein,high fat,high oleic acid,linoleic acid,and low linolenic acid were successfully created by using EMS mutagenesis techniques.On the one hand,the quality of soybeans in Northeast China and even in China was improved.Germplasm resources,on the other hand,lay the foundation for better research on soybean-related functional genes.At the same time,based on the phenotype information of the mutants,the GmFAD2-1A/2A/1B gene was successfully cloned from soybean high and low linoleic acid mutants and wild-type parents,and the SNP was developed for the mutation site of the GmFAD2-2A gene.Mark,preliminary evaluation of its accuracy.The main findings are as follows:1.The EMS optimum mutagenesis conditions were determined.The study used soybean“JiNong 18”as the material,set 12 treatments with different EMS concentrations?0.3%,0.5%,0.7%,0.9%?and different treatment times?2h,4h,6h?and control,and took the median lethal dose As the sensitivity index,the best mutagenesis condition was determined as EMS concentration 0.5%and treatment time 6h.2.Genetic analysis of main quality traits in the M2 generation of soybean EMS mutagenesis population.From the analysis of the variable coefficient,the variable coefficient of oleic acid,linolenic acid and stearic acid in the M2 generation was relatively large;the generalized genetic forces of the M2 generation were relatively high in linoleic acid,linolenic acid and plmitic,but fat and stearic acid were very low.Correlation analysis results showed that the main quality traits of oleic acid and palmitic acid had significantly positive correlation,oleic acid and linoleic acid,linolenic acid had significantly negative correlation,linolenic acid and linoleic acid had significantly positive correlation.3.A batch of soybean fatty acid mutant resources was obtained.A total of 561soybean M3 fatty acid mutants were obtained,including 110 high oil mutants,150high linoleic acid mutants,55 low linoleic acid mutants,35 high linolenic acid mutants,and low flaxseed.There were 55 acid mutants,34 high-stearate mutants,21low-stearate mutants,36 high-palmitic acid mutants,and 65 low-palmitic acid mutants.4.Three FAD2 gene mutation sites were initially identified.PCR product sequencing results DNAMAN6.0 software was used to compare the sequencing results of the high linoleic acid mutants,the low linoleic acid mutants and their wild-type parent GmFAD2-1A/2A/1B genes.The GmFAD2-1A gene isolated from the oleic acid mutant?2017MT52?has a base mutation?G?A?at+928 bp and+1037 bp after the start codon;the isolated GmFAD2-2A The gene has a mutation at one base site?G?A?at+521 bp after its start codon,resulting in a stop codon that prematurely terminates translation;the isolated GmFAD2-1B gene,at its initiation codon There is a base mutation?G?A?at+247bp and+1978bp,respectively.The GmFAD2-1A gene isolated from soybean high linoleic acid mutant?2017MT71?has a base mutation?G?A?at+574 bp after the start codon;the isolated GmFAD2-2A gene There is a base site mutation?G?A?at+407 bp after its start codon;the isolated GmFAD2-1B gene has a base at+318 bp and+507 bp after the start codon,respectively.At the site mutation?T?C?,there was a base mutation?G?A?at+1096bp and+1635 bp.5.A PCR-based SNP marker was developed.Specific primers for the allele PCR reaction were designed by using the genomes of mutant plants and wild-type plants as templates to obtain a set of specific primers with ideal amplification effect.The mismatch bases of a set of specific primers were introduced at the 3?end.At the 3position,base pairing follows the strong and weak collocation principle.The size of the PCR product is 756 bp and the allele of the SNP locus can be detected by 1%agarose gel electrophoresis.6.The accuracy of SNP markers was evaluated.For the SNP-specific primers screened for the GmFAD2-2A gene,the SNP locus genotype of the mutant soybean material was detected by direct PCR product sequencing.The results obtained by the sequencing method were compared with the results identified by the SNP marker.The results showed that the detection results of the specific primer FAD2-2A-R2 were consistent with the sequencing results,indicating that the SNP marker we developed was accurate. |
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