The Variations Of High-Molecular-Weight Glutenin Subunits In EMS-Treated Tetraploid Wheat

High-molecular-weight glutenin subunits（HMW-GS）are one of the major storage proteins in wheat kernels.Although HMW-GS is only about 10%of wheat endosperm,it plays a key role in affecting the processing quality of wheat flour.The HMW-GS encoding genes have a number of allelic variations in wheat and its wild relatives.In order to explore the impact of different subunits on the quality of wheat processing and make a comprehensive evaluation to different HMW-GS,it is necessary to produce the wheat lines harboring different subunit composition.In this study,the tetraploid wheat Jianyangailanmai（AS2283）was treated by ethyl methanesulfonate（EMS）to obtain different types of HMW-GS deletion mutants.The main results are listed as the following:1.The seeds of tetraploid wheat Jianyangailanmai were treated with three different concentrations of EMS 0.2%（V/V）,0.3%（V/V）and 0.4%（V/V）in 2015.One thousand seeds were used in for each treatment with different concentration.Finally,a M₂ mutation population of 1157 lines were constructed including 733 lines from EMS 0.2%（V/V）treatment,367 lines from EMS 0.3%（V/V）treatment,and 57 lines from EMS 0.4%（V/V）treatment,and with the rates of 73.3%,36.7%and 0.57%,respectively,in 2016.2.Three seeds were selected from each M₂ line of the M₂ population for sodium dodecylsulfate-polyacrylamide gel（SDS-PAGE）analysis.The results showed that four lines lacked 1Ax subunits and one line lacked 1Bx subunit,and four lines deleted 1By.There are three mutant lines,6 lines,and 0 lines for EMS concentration 0.41%,1.63%,0%.The remaining M₂ generation seeds were grown in the field in 2016,.a total of 7565 lines of M₃ generation were harvested in 2017.HMW-GS profiles indicated that two more 1By deletion mutants were characterized from one M₂ line.3.The mutation mechanism of the identified mutants was revealed by gene cloning.The results indicated that due to the single base substitution has resulted in the premature stop codons,is the reason of HMW-GS gene silencing.For example,in the 1Ax subunit-deleted line No.162-3,HMW-GS gene has a single base substitution at position380 in the coding region,leading to the change from cytosine（C）to thymine（T）,resulting in 127 serine（S）turns to phenylalanine（F）,and the single base substitution occurs at the2142 position in the coding region,eventually causing the glutamine（Q）codon change from CAA to the stop codon TAA,leads to silencing of the 1Ax gene,resulting in deletion of the protein band.For the line No.1039-3,single base substitution from cytosine（C）to thymine（T）at the coding regions 1540 and 2209has led to phenylalanine（F）becoming serine（S）and stop codon appear early,resulting in the lack of 1Bx subunit.A single-nucleotide mutation from cytosine（C）to thymine（T）occurs in position 339 of the line No.107-2 change the codon from glutamine（Q）codon CAA to stop codon TAA,resulted the deletion of 1By subunit.