Studies On Cytology And Molecular Biology Of Male Sterility Line H276A In Gossypium Barbadense

Cytoplasmic male sterility(CMS)is the basis of plant heterosis utilization and has been widely used in rice,maize and other crops.Cotton(Gossypium barbade,nse L.)is an important economic crop and also has significant heterosis.However,due to few types of cotton CMS,its heteriosis utilization has been limited.Therefore,it is significantly to study the molecular mechanism of cotton CMS in order to get better utilization of cotton heterosis.The gossypium barbadense H276A is a new cotton CMS line,which was modified genetically by Liu Dongmei who transferred the kenaf gene(HcPDIL5-2a)in H276.As the CMS line H276A is derived from its maintainer line H276B,so they are iso-genic lines at mitochondrial genome levels.In the study of the mechanism of cotton CMS,the interference of non-CMS related redundant genetic information can be excluded.They provides valuable materials for the study of molecular mechanism of cotton CMS.In this study,we investigated the abortion mechanism of CMS H276A from cytology,mitochondrial genome and transcriptome and obtained the main conclusions as follows:1.The comparative analysis of anther development between CMS line and its maintainer line was performed by using paraffin section method.The abortion stage of cotton CMS line was confirmed at tetrad stage and its main abortion features were cell nuclear vacuole of tetrad,tapetum integrated and didn't degrade during the anther development.The ultrastructure observation of anther cells showed that the mitochondria of the tapetum cells in the male sterile line appeared to be degraded at tetrad stage.2.Comparative RFLP analysis of mitochondrial gennome between CMS and its maintainer line was carried out using EcoR I and Hind? restriction endonuclease.The results showed that atp1,nad4,nad7,,nad9,ccmb showed RFLP polymorphisms between sterile and maintainer line with EcoR I,and atp4,nad5,nad9,sdh4,cox3 showed RFLP polymorphisms between sterile and maintainer line with Hind? and other genes exhibited no polymorphism.Our data indicated that DNA mutated at coding region or near the coding region of above genes,and inferred that these mutations may be related to occurrence of cotton CMS.3.RNA blot was performed with 35 mitochondrial gene probe.The results showed that there was no transcript length polymorphism between CMS line and its maintainer line.However,it was found that the transcript abundance of cox3 gene was significantly decreased(0.3-0.4)in CMS line.The subsequent quantitative analysis of cox3 indicated that the relative expression level of the gene was 0.39 times in H276A than that of in H276B.We inferred that the abnormal expression of cox3 may be related to the lack of energy during the formation of CMS.4.The 3'and 5'transcript terminal of cox3 were obtained with circularized RNA reverse transcribed PCR(CR-RT-PCR).The results showed that the full-length transcript of cox3 was 1515bp in H276B,the transcription start site and termination site were located at-412bp,1103bp of the initiation codon(ATG),in respectively.H276A has the same transcription terminal site as that of in H276B.But at transcription initiation site,it has three sites(-451,-466 and-473 respect to ATG),which are different from that of H276B.Seven SNPs were identified by homologous clonning which were located around original site of transcript of cox3 in H276A.We inferred that the seven SNPs influenced the transcription recognition site of cox3 in H276A,which leaded to significantly reduced expression of cox3.5.In the study of the expression analysis of ATP synthase genes,we found that except atp4(0.94),the relative expression levels of atp 1(0.1),atp6(0.64),atp8(0.59)and atp9(0.61)decreased significantly in H276A compared with that of in H276B.On the other hand,the expression levels of atp1(1.25),atp4(2.06),atp6(1.64),atp8(1.55)and atp9(2.21)in fertility F1 were significantly higher than that of H276B.The results indicated that the introduction of the restore gene increased the expression of ATP synthase gene,and further confirmed that the occurrence of cotton CMS may be related to the energy deficiency.6.The features and the editing frequencies of each editing sites of ATP synthase genes were detected by cloning sequence.A total of 41 editing sites have been identified,among them 27 were full edited and 14 were partialy edited.As a result of RNA editing,there was increase in hydrophobic amino acids content in the protein polypeptide chain leading to increased protein stability.In addition,two new stop codon created by RNA editing were detected,one was at 787th of atp6 and the other was at 223th of atp9.However,the RNA editing frequency in three materials indicated that there was no correlation between RNA editing of ATP synthase genes and cotton CMS.7.Based on the 6bp deletion of upstream of atp8 in CMS line H276A,a molecular marker was developed to identify male sterile cytoplasm(MSC).Validation of 41 known fertility germplasm resources of cotton,the results showed that the molecular marker was stable and reliable.It provides a more convenient tool for molecular marker assisted selection breeding.8.The transcriptome comparative analysis was conducted with Illumina Hiseq4000.A total of 64,675genes were detected,3603(5.57%)genes differentially expressed(1363 up-regulation,2240 down-regulation).Bioinformatic analysis on differentially expressed genes showed that 76 of them participated in TCA,oxidative phosphorylation,glycolysis and the other energy metabolism and most of them were down regulation genes.At the same time,some differentially expressed genes associated with PPR protein,MYB transcription factor and anther specific expression were identified.To evaluate the accuracy 15 DEGs were selected randomly and detected by RT-PCR and the results indicated that the transcriptome sequencing were reliable.Further studies on the gene regulatory networks of these differentially expressed genes may help us to elucidate the mechanism of cotton cytoplasmic male sterility.