Production Of Waxy Tetraploid Wheat(Triticum Turgidum)by EMS Mutagenesis

Starch is the main component of wheat grain and it?s relatives,accounting for 50-70﹪of its dry weight.The waxy gene（Wx-1）encodes the granule-bound starch synthase I（GBSSI）,which is the sole key enzyme that catalyzes amylose synthesis in wheat.Because the amylose contents largely affect the flour quality,production of wheat lines with varied amylose content would be valuable in wheat quality breeding.In this study,we detected the Wx null mutant in the tetraploid wheat mutant library which was developed in our previous work.We further explore the mechanism of gene inaction,and further generated Waxy tetraploid wheat lines by crossing between single Wx null muntant,and investigated the effect of the loss of Wx protein on granules structure and starch content and composition of.The main results obtained are as follows:1.In this study,we characterized the Wx null mutants from ethyl methanesulfonate（EMS）mutagenized tetraploid wheat（Triticum turgidum）M2 populations,which is composed of3474 plants and was devoloped in our previous work.The Wx-A1 or Wx-B1 null mutants were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE）,and named as M2-219 and M2-504,respectively.The results of gene cloning showed that there was a single nucleotide polymorphism（SNP）of G1086A happened at position of1086bp from the start codon in Wx-A1 of line M2-219,mutation of codon from TGG to stop codon TGA,thereby resulting in premature termination of the Wx-A1 protein.For Wx-B1 of line M2-504,two SNP mutations from G to A at the start codons 207 and 1122,named G207A and G1122A,were detected in Wx-B1 ORF;the former resulted in the amino acid substitution from Serine to Asparagine,while G1122A occurred in the fifth exon,which mutated the codon from the codon of Tryptophan TGG to stop codon TGA and thus result in the lack of Wx-B1 protein.2.These M2 mutants of null Wx-A1 or Wx-B1 were self-crossed to M3,SDS-PAGE analysis showed that all M3 progenies of the three M2 mutants are homozygous of the Wx-1loci,with the genotype of Wx-A1 null or Wx-B1 null.Hybridization was conducted by crossing the Wx-A1 null mutant with the Wx-B1 null mutant,producing Waxy tetraploid durum wheat.The Wx-A1 and Wx-B1 subunits in the identified F1 were not deleted,and the hybridization was successful.After the identified F1 seeds were selfed,four types of lines with different Wx protein compositions were identified:Wx-A1+Wx-B1,Wx-A1null+Wx-B1,Wx-A1+Wx-B1 null,Wx null（Wx-A1 null+Wx-B1 null）.3.The starch properties of four different types Waxy tetraploid wheat lines were determined.In terms of GBSSI activity,all four Wx types of materials all showed the law of increasing first and then decreasing,and the greatest activity on the 10^th day,the GBSSI activity followed WT>Wx-A1 null>Wx-B1 null>Wx null at 10d,15d and 20d.The results showed that all lines except Wx-A1 null mutant lines had no significant change;As for the amylose content,the amylose content of the Wx null line is the lowest,ranging from 0.379﹪to 4.132﹪,the amylose content of the Wx-B1 null line is 8.590﹪,and the Wx-A1null line is 17.744﹪,and the lack of Wx-B1 results in an greater decrease in amylose content than loss of Wx-A1;Through the observation of SEM,the starch granules in the Wx null lines showed high incompleteness and edge damage,the gap between the starch granules was significantly reduced,and the A-type starch granules showed a lot of damage B-type starch granules appear shedding and the lowest amylose content and peak gelatinization temperature,highest enthalpy.