Anther Ultrastructure Observation And Mitochondrial Genome Analysis Of K-type Male Sterile Wheat

The utilization of wheat heterosis is of great significance to improve yield,quality and adaptability.Male sterility is one of the important ways of heterosis utilization.But the mechanism of male sterility in wheat still has no clear system explanation.In this study,the relationship between male sterility of K type wheat and microstructure of anther cells and the difference of mitochondrial genome sequence were discussed.The objective of this study was to gain an insight into the abortion mechanism of K-type cytoplastic male sterile(CMS)line of wheat.The microstructure and ultrastructure of anthers of the K-CMS line,K519 A,and its maintainer line 519 B were observed with semithin and ultrathin sections.The expression patterns of tapetal-related genes,RAFTIN1 and APs,during microspore development were also analyzed.Using high-throughput sequencing technology and bioinformatics methods,the mitochondrial genome of the K-CMS line and its maintainer were assembled and compared.The main findings are as follows:1.The tapetum of K519 A degenerated at tetrad stage and earlier than that of 519 B.Partial tapetal cells entered the anther chamber,and only cell contour and a small amount of ubisch bodies were observed at binuclear and trinuclear stages.The cell wall of microspore of K519 A were thicker than that of 519 B at mononuclear(2.35 folds over that of 519B)and binuclear stage,but became thinner than that of 519 B at trinuclear stage(0.89 fold over that of 519B).Besides,abnormal pollen grains and plasmolysis were observed in K519 A at trinuclear stage.These results indicate that sterility of K519 A probably occurs at tetrad and binuclear stage.The abortion might be related to the abnormal of tapetal structure,resulting that partial tapetal cells intrude into the chamber and occupy the room for the normal development of microspore at tetrad stage.The early degradation of tapetal cells causes thickening of cell wall at mononuclear stage by consuming energies and less synthesis of sporopollenin at binuclear and later stages due to energy deficiency,as a result,the microspore cell walls becomes thinner in the sterile line than in the maintainer line at trinuclear stage and pollen abortion eventually.2.From microspore mother cell to mononuclear stage,the relative expression levels of RAFTIN1 and APs were significantly higher in K519 A than in 519 B,but significantly lower in K519 A at binuclear stage.This hypothesis can be validated by the expression analysis of RAFTIN1 and APs genes,and the two genes are considered to be involved in the sterile process of K519 A.3.The complete mtDNA sequence of a wheat K-type(with cytoplasm of Aegilops kotschyi)CMS line,K519 A,was assembled into a master circle molecule of 420543 bp,AT-content of 55.86%,including 34 protein coding genes,23 tRNA genes and 3 rRNA gene,as well as 138 open reading frames(ORFs;greater than 300 bp in length).The maintainer line contains 433560 bp(AT-content of 55.86%),including 35 known protein-coding genes,three rRNA and 24 tRNA genes,as well as 148 open reading frames(ORFs;greater than 300 bp in length).4.Comparison and analysis of genetic sequences of the K-type male sterile line,K519 A,and its maintainer line 519 B.Most of the protein-coding genes are highly conserved,except nad1,atp4,atp8 and rpl16.We also defined 32 single nucleotide polymorphisms(SNPs)in 10 protein-coding,fourteen of them were non-synonymous mutations and seven were synonymous mutations.In the sterile line,K-atpA-like and K-ndhB-like two wheat chloroplast homologous genes were found.But in the mitochondrial genome of the sterile line,there was no annotation to atp6,ccmB and rps13 genes.In addition,21 specific ORFs were selected.In summary,sterility of K519 A probably occurs at tetrad and binuclear stage,the abortion might be related to the abnormal of tapetal structure.RAFTIN1 and APs genes,the two genes are considered to be involved in the sterile process of K519 A.There were many differences in the mitochondrial genome of the sterile line K519 A and its maintainer line 519 B,and these differences may be the key factor for anther abortion.