Analysis Of Allelic Variations Of Wheat Plant Height Regulation Genes Rht-1 And GID1 And Their Interaction Mechanism

Wheat is a major source of human food.There are 43 countries in the world that use wheat as a staple food,which accounts for 35% of the world's total population.The ultimate objective of grain production is high yield and high quality to meet people's needs.As an important agronomic trait,Plant height has an important influence on crop yield and lodging resistance.Proper dwarfing can increase the harvest index.Rht-1 is the main object of wheat dwarf breeding,through the regulation of plant height to improve harvest index and lodging resistance.Wheat plant height regulation gene Rht-1 is associated with GA signaling pathway.Thus,to explore new allelic variations of wheat Rht-1 gene promoter and the regulatory mechanism of Rht-1 coding proteins,and further analyzing the mechanism of Rht-1 gene regulating plant height,has important theoretical and practical significance for wheat genetic improvement and breeding.Therefore,this study analyzed the allelic variations of Rht-1 and the GA receptor gene GID1,and their interactions between the new allelic variants derived from Rht-1 and GID1.The following results were obtained: 1.Discovery of the wheat dwarf gene Rht-1 allelic variationsThe cloned sequences of Rht-1 gene promoter region in Chinese micro-core collections were spliced with the previously cloned Rht-1 coding region sequences of Aixia Li.The results showed that except for the wild type?China spring type?,we detected a total of 74 allelic variations in the A,B and D genomes.Among them,there are 29 in A genomes,21 in B genomes,and 24 in D genomes.There are 52 varieties of Rht-A1₂1 type,accounting for 19.8%,Rht-A1₂ has 17 varieties,accounting for 6.5%,14 varieties of Rht-A1₂8,accounting for 5.3%,Rht-A1₂4 and Rht-A1₂9 each have 8 varieties,each with 3.1%.Rht-B1₃ type has 7 varieties,accounting for 2.7%,Rht-B1₁2 has 9 varieties,accounting for 3.4%,and Rht-B1₁4 has 10 varieties,accounting for 3.8%.Rht-D1₁8 type has 23 varieties,accounting for 8.8%,Rht-D1₉ has 7 varieties,accounting for 2.7%,and Rht-B1₂1 has 12 varieties,each accounting for 4.6%.Interestingly,we found new insertion types of 191 bp,197bp,and 217 bp in the wheat A and B subgenomes,cis-acting elements of these three types of insertions were predicted,in addition to the common cis-acting elements in the promoter region,they also include some elements related to environmental response,hormonal response,and tissue-specific expression.These results indicate that the adjustment network of these three allelic variations of Rht-1 are more complicated,especially in the 217 bp insertion,which has a CCGTCC-box cis-acting element that is associated with meristem expression,which may change the plant height of the allelic variant.The co-dominant markers PA-191MF1/R1,PB-197MF1/R1,and PA-217MF1/R1 were developed to detect 191 bp,197bp,and 217 bp allelic variants.At the same time,the three allelic variants of Arabidopsis thaliana GUS expression vectors were constructed and transgenic Arabidopsis thaliana plants with T1 generation were obtained.2.Discovery and functional analysis of new allelic variations in wheat gibberellin receptor gene GID1The GID1 genes of subgenomic groups A,B,and D from 262 Chinese wheat micro core collections were obtained by homologous cloning,the three groups of GID1 genes consist of two exons and one intron,except wild type?China spring type?,a total of 31 new allelic variations were discovered?7 GID1-A genotypes,12 GID1-B genotypes,and 12 GID1-D genotypes?.Among them,GID1-B₈ mutations accounted for the largest proportion,32 species belong to this mutation type,the frequency is 12.21%.GID1-B₅ and GID1-B₉ both have 9 varieties with a frequency of 3.44%.GID1-D₂,GID1-D₁0,GID1-B₃ and GID1-D₅ contained 6,5,4 and 3 varieties,the frequency are 2.29%,1.91%,1.53% and 1.15% respectively.The GID1-A₃,GID1-A₆,GID1-B₄,and GID1-D₇ variants all contain 2 varieties with a frequency of 0.76%.The remaining variants contain only one species and the frequency is 0.38% for each of them.In terms of plant height traits,there are GID1-A₃?127cm?,GID1-A₇?125cm?,GID1-D₃?124cm?,GID1-D₈?126cm?,and GID1-D₉?122cm?,which are more than 10 cm higher than the wild-type Chinese Spring?CS,111cm?.GID1-D₉?122cm?,GID1-D₁1?127cm?,GID1-B₄?91cm?,GID1-B₇?53cm?,GID1-B₁0?81cm?,GID1-D₁?86cm?,GID1-D₄?92cm?and GID1-D₅?99cm?,which are more than 10 cm shorter than the wild type CS.Due to the conservation of the coding region of GID1,there are 11 allelic variants in the exonic region and only 6 sense mutations,the intron region includes 20 allelic variants.We performed a yeast two-hybrid experiment with the two frameshift mutations?GID1-A₆,GID1-B₁1?and one missense mutation?GID1-D₁0?in the exon region and the DELLA protein.The results showed that GID1-A₆ and GID1-B₁1 frame-shift mutations did not interact with DELLA protein under the condition of GA,GID1-D₁0 missense mutations and DELLA protein had GA-dependent interactions.However,there is no significant difference between the plant height of the corresponding material and the wild type.It may be the reason for the functional redundancy of the GID1 genes in the A,B and D subgenomes of wheat.