Identification Of Fertility-conversion Genes And Molecular Mechanism For TaGAMYB To Regulate Fertility In Thermo-sensitive Male Sterile Wheat

As an important food in China,wheat plays a vital role in food safety and economic stability.The utilization of wheat heterosis is an important way to improve crop yield,and male sterility is an effective way to utilize heterosis.Therefore,the discovery of fertility regulation genes and the study of their molecular mechanisms have far-reaching significance for the research and utilization of wheat breeding.The materials used in this experiment are a thermo-sensitive genic male sterile wheat line,Yan Zhan 4110S,and its near-isogenic line,Yan Zhan 4110.To determine the basic characteristics of abortion under different temperature treatment conditions,phenotypic and cytological observation for Yan Zhan 4110S was carried out,mainly including the external morphology of the plant,the characteristics of flower organs,the development process of tapetum,microspore and pollen wall.To further explore the molecular mechanism on male sterility,transcriptome sequencing and differential expression analysis of candidate genes,gene silencing mediated by barley stripe mosaic virus,overexpression and CRISPR/Cas9-knockout test in Arabidopsis thaliana were used to verified the relationship between candidate genes and male sterility,and finally established a work model to regulate fertility.The main results obtained in this study were as follows:1.According to the phenotypic and cytological observation results,the pistil of the flower organ of Yanzhan 4110S developed normally,but it has the following characteristics of fertility conversion:the florets were more stretched,and the period with significant difference in anthers is the trinucleate stage,which is pale yellow,thin and does not crack.The outer epidermal cells were arranged irregularly,and the fiber cells were disorderly and cross arranged.The pollen grains were shrunk and deformed,and the germination pores collapse.I₂-KI solution staining is not enough,and the type of pollen abortion is dye abortion.TUNEL,tissue section and ultrastructure observation showed that the degradation of tapetum cells was delayed,the development process from microspore to mature pollen grains was abnormal,and finally the tectum of pollen outer wall was bend and deformed.Therefore,we speculated that the reason for fertility conversion of Yanzhan 4110S under high temperature may be the delayed degradation of tapetum,which can not provide the nutrients needed in the process of microspore development in a timely manner,resulting in abnormal microspore development and finally the defective of exine.2.The transcriptome sequencing results of Yanzhan 4110S and Yanzhan 4110 at the later uninucleate stage anthers under different temperatures showed that 1422 differentially expressed genes（DEGs）were unique DEGs of Yanzhan 4110S.The core gene set determined by WGCNA has a total of 1340 genes.According to comparative analysis,some genes in the core gene set are consistent with the unique DEGs of Yan Zhan 4110S.To find the gene set closely related to male sterility,we conducted the intersection analysis of the above two gene sets,and look for the genes that specifically expressed in the anther.Three genes TaLIM2,TaGELP073,and TaGAMYB,related to the development of pollen,exine,and tapetum,separately,were finally identified for the verification of gene function.3.There were 29 and 509 members were identified from the genome-wide analysis of LIM and GELP gene families in wheat,which were unevenly distributed in the wheat chromosomes.TaLIMs gene were divided into two types:DA1&DAR subfamily and CPR-like subfamily;The TaGELPs can be classified into 8 subfamilies,and the number of TaGELPs members in different branches is significantly different.The analysis of gene structure and conserved motif showed that the member evolution of each small branch was relatively conservative,and its cis-acting elements were involved in hormones,plant development,biological and abiotic stress response,etc.The results of expression analysis in sterile and fertile Yan Zhan 4110S anthers showed that these family members might be important genes related to pollen development during fertility conversion.In order to confirm this conjecture,transient gene silencing and overexpression analysis were performed on the DEGs of TaLIM2 and TaGELP073 obtained from the transcriptome database.The anthers of TaLIM2 and TaGELP073 silenced plants showed development defects in microspore and exine.The plants bolting time of TaLIM2 overexpression lines were earlier than that of wild type.The pollen vitality of TaLIM2-g overexpression line was reduced,the pollen tube hardly germinated and the pods were not full.The mutant gelp5050 gelp1890 of the homologous gene At GELP5050 and At GELP1890 of TaGELP073 was constructed by gene knockout.It was found that the pods of the plant became shorter and the fertility decreased.It showed that the genes TaLIM2 and TaGELP073 are closely related to the fertility of Yanzhan 4110S.4.Observation of anther ultrastructure of silenced plants showed that silencing TaGAMYB resulted in defects in tapetum and exine development.To fully explore the function of the gene,TaGAMYB coding sequences（TaGAMYB-d and TaGAMYB-g）were cloned in fertile and sterile Yan Zhan 4110S,respectively.Sequence analysis indicated that there were three nucleotide differences.They belonged to the same branch as At MYB97,At MYB101 and At MYB120.The mutants of myb97 myb101 myb120 in Arabidopsis was constructed by CRISPR/Cas9 gene editing technology,and it was found that their pollen germination rates and fertility decreased.After constructing the overexpression lines in Arabidopsis,we found that the accumulation of TaGAMYB expression led to the early flowering of TaGAMYB-d overexpression line,while the fertility was normal.The reduction of pollen vitality and germination rates of TaGAMYB-g overexpression line finally led to the decline of fertility.The results indicated that TaGAMYB is also closely related to the fertility of Yanzhan 4110S,and high temperature can cause fertility decline.5.After treatment of fertile Yan Zhan 4110S with gibberellin,the anthers were thin and small,the pollen grains were neithor stained by I₂-KI nor Alexander with a shrunken shape,the pollen tubes did not grow,and the seed setting rates decreased significantly.The content of GA3 in anthers treated at binuclear stage was higher than that of the control.q RT-PCR showed that GA3 treatment reduced the expression level of TaGAMYB,and was more significant at binuclear stage and trinuclear stage,so TaGAMYB was regulated by GA3.In the yeast single hybrid and dual luciferase activity assay showed that TaWRKY2 can bind to the promoter of TaGAMYB and regulate its gene expression.The expression of TaWRKY2 in the anthers treated with GA3 was upregulate at each stage,therefore,GA3 may inhibit the expression of TaGAMYB through TaWRKY2,and then affected the expression of PAL and FLS related to pollen wall formation.In summary,the fertility of TGMS wheat Yan Zhan 4110S was regulated by TaGAMYB through gibberellin signal.