Cytoplasmic Male Sterility In Cotton Research Articles

Integrated Methylome and Transcriptome Analysis Widen the Knowledge of Cytoplasmic Male Sterility in Cotton (Gossypium barbadense L.).

DNA methylation is defined as a conserved epigenetic modification mechanism that plays a key role in maintaining normal gene expression without altering the DNA sequence. Several studies have reported that altered methylation patterns were associated with male sterility in some plants such as rice and wheat, but global methylation profiles and their possible roles in cytoplasmic male sterility (CMS), especially in cotton near-isogenic lines, remain unclear. In this study, bisulfite sequencing technology and RNA-Seq were used to investigate CMS line 07-113A and its near-isogenic line 07-113B. Using integrated methylome and transcriptome analyses, we found that the number of hypermethylated genes in the differentially methylated regions, whether in the promoter region or in the gene region, was more in 07-113A than the number in 07-113B. The data indicated that 07-113A was more susceptible to methylation. In order to further analyze the regulatory network of male sterility, transcriptome sequencing and DNA methylation group data were used to compare the characteristics of near-isogenic lines 07-113A and 07-113B in cotton during the abortion stage. Combined methylation and transcriptome analysis showed that differentially expressed methylated genes were mainly concentrated in vital metabolic pathways including the starch and sucrose metabolism pathways and galactose metabolism. And there was a negative correlation between gene methylation and gene expression. In addition, five key genes that may be associated with CMS in cotton were identified. These data will support further understanding of the effect of DNA methylation on gene expression and their potential roles in cotton CMS.

The cotton mitochondrial chimeric gene orf610a causes male sterility by disturbing the dynamic balance of ATP synthesis and ROS burst

Plant cytoplasmic male sterility (CMS) is maternally inherited and often manifested as aborted pollen development, but the molecular basis of abortion remains to be identified. To facilitate an investigation of CMS in cotton, the complete sequence of cotton mitochondrial (mt) genome for CMS-D2 line ZBA was determined. The mt genome was assembled as a single circular molecule with 634,036 bp in length. A total of 194 ORFs, 36 protein‐coding genes, six rRNAs, and 24 tRNAs were identified. Several chimeric genes encoding hypothetical proteins with transmembrane domains were identified. Among them, a previously unknown chimeric gene, orf610a, which is composed of atp1 and a 485-bp downstream sequence of unknown nature, was identified. RT-PCR and qRT-PCR validation indicated that orf610a was expressed specifically in a sterile line. Ectopic expression of orf610a in yeast resulted in excessive accumulation of reactive oxygen species and reduction in ATP content, in addition to inhibition of cellular growth. Transgenic A. thaliana overexpressing orf610a fused with a mitochondrial targeting peptide displayed partial male sterility. Interaction between ORF610a and the nuclear-encoded protein RD22 indicated an association between ORF610a and pollen abortion. Positive feedback during transcriptional regulation between nuclear regulatory factors and the mt CMS gene may account for the male sterility of ZBA.

Mitochondrial gene expression analysis reveals aberrant transcription of cox3 in Gossypium barbadense CMS line H276A.

Cotton cytoplasmic male sterility (CMS) has been extensively studied; however, information regarding its molecular mechanisms has not yet been disclosed. Therefore, to explore the molecular mechanism of pollen abortion of cotton CMS line H276A, transcript profiles of 30 mitochondrial protein-encoding genes at tetrad stage were conducted with northern blot and a differential expression gene cox3 was identified. Quantitative reverse-transcribed PCR (qRT-PCR) analysis indicated that the expression level of cox3 in the CMS line H276A was 0.39-fold compared to its maintainer line H276B. In addition, the immunoblot analysis revealed that the amount of COX3 protein was decreased to 59.38% in CMS line H276A. The 5` and 3` terminals of the transcript of cox3 in two materials were determined simultaneously with circularized RNA reverse-transcribed PCR (CR-RT-PCR). The data indicated that seven 5` end of transcript of cox3 in H276A (-451/-464/-465/-467/-471/-472/-508 respect to ATG) were identified which were different from that of H276B (-411/-412). A total of 15 single nucleotide polymorphisms (SNPs) was detected by clone sequencing analysis of upstream of cox3. To our knowledge, we are the first to comprehensively analyze the transcripts of the mitochondrial genome in the cotton CMS line and to identify the 5` and 3` terminals of the transcript of cox3 in cotton. Our data will provide a framework for a better understanding of molecular mechanisms of CMS and mitochondrial gene expression in cotton.

Transcriptome and MiRNAomics Analyses Identify Genes Associated with Cytoplasmic Male Sterility in Cotton (Gossypium hirsutum L.).

Cytoplasmic male sterility (CMS) is important for large-scale hybrid seed production. Rearrangements in the mitochondrial DNA (mtDNA) for the cotton (Gossypium hirsutum L.) CMS line J4A were responsible for pollen abortion. However, the expression patterns of nuclear genes associated with pollen abortion and the molecular basis of CMS for J4A are unknown, and were the objectives of this study by comparing J4A with the J4B maintainer line. Cytological evaluation of J4A anthers showed that microspore abortion occurs during meiosis preventing pollen development. Changes in enzyme activity of mitochondrial respiratory chain complex IV and mitochondrial respiratory chain complex V and the content of ribosomal protein and ATP during anther abortion were observed for J4A suggesting insufficient synthesis of ATP hindered pollen production. Additionally, levels of sucrose, starch, soluble sugar, and fructose were significantly altered in J4A during the meiosis stage, suggesting reduced sugar metabolism contributed to sterility. Transcriptome and miRNAomics analyses identified 4461 differentially expressed mRNAs (DEGs) and 26 differentially expressed microRNAs (DEMIs). Pathway enrichment analysis indicated that the DEMIs were associated with starch and sugar metabolism. Six deduced target gene regulatory pairs that may participate in CMS were identified, ghi-MIR7484-10/mitogen-activated protein kinase kinase 6 (MAPKK6), ghi-undef-156/agamous-like MADS-box protein AGL19 (AGL19), ghi-MIR171-1-22/SNF1-related protein kinase regulatory subunit gamma-1 and protein trichome birefringence-like 38, and ghi-MIR156-(8/36)/WRKY transcription factor 28 (WRKY28). Overall, a putative CMS mechanism involving mitochondrial dysfunction, the ghi-MIR7484-10/MAPKK6 network, and reduced glucose metabolism was suggested, and ghi-MIR7484-10/MAPKK6 may be related to abnormal microspore meiosis and induction of excessive sucrose accumulation in anthers.

Deviant DNA methylation play a key role in the pollen abortion of Gossypium barbadense L. cytoplasmic male sterility

DNA methylation is a conserved epigenetic modification mechanism in higher plants that affects several biological processes by regulating gene expression without alteration in DNA sequence. Although, gene expression of cotton cytoplasmic male sterility (CMS) line H276A has been conducted but its regulation mechanism of gene expression is unknown. Therefore, a bisulfite sequencing (BS-Seq) technology was utilized to evaluate genome wide relative study of DNA methylation of the cotton CMS line H276A and its maintainer line H276B. Data showed that 1498 differentially methylated genes (DMGs) and 5762 differentially methylated regions (DMRs) were observed between two samples, respectively. The KEGG pathway analysis exhibited that 139 DMGs were highly enriched in eight pathways, i.e. starch and sucrose metabolism, ABC transporters and oxidative phosphorylation. Moreover, 74 DMGs were identified as encoding transcription factors (TFs). Eight DMGs were randomly selected to confirm the correlation between DNA methylation and gene expression using qRT-PCR. Results indicated that 6 of 8 DMGs showed a negative relationship between gene expression and DNA methylation. Twelve key DMGs, which involved in carbohydrate and energy metabolism, TFs and male gametophyte development, potentially related to cotton CMS were also identified. These data will provide better understanding of DNA methylation alteration and their potential roles in cotton CMS.

Integrated Methylome and Transcriptome Analysis between the CMS-D2 Line ZBA and Its Maintainer Line ZB in Upland Cotton.

DNA methylation is an important epigenetic modification involved in multiple biological processes. Altered methylation patterns have been reported to be associated with male sterility in some plants, but their role in cotton cytoplasmic male sterility (CMS) remains unclear. Here, integrated methylome and transcriptome analyses were conducted between the CMS-D2 line ZBA and its near-isogenic maintainer line ZB in upland cotton. More methylated cytosine sites (mCs) and higher methylation levels (MLs) were found among the three sequence contexts in ZB compared to ZBA. A total of 4568 differentially methylated regions (DMRs) and 2096 differentially methylated genes (DMGs) were identified. Among the differentially expressed genes (DEGs) associated with DMRs (DMEGs), 396 genes were upregulated and 281 genes were downregulated. A bioinformatics analysis of these DMEGs showed that hyper-DEGs were significantly enriched in the “oxidative phosphorylation” pathway. Further qRT-PCR validation indicated that these hypermethylated genes (encoding the subunits of mitochondrial electron transport chain (ETC) complexes I and V) were all significantly upregulated in ZB. Our biochemical data revealed a higher extent of H2O2 production but a lower level of adenosine triphosphate (ATP) synthesis in CMS-D2 line ZBA. On the basis of the above results, we propose that disrupted DNA methylation in ZBA may disrupt the homeostasis of reactive oxygen species (ROS) production and ATP synthesis in mitochondria, triggering a burst of ROS that is transferred to the nucleus to initiate programmed cell death (PCD) prematurely, ultimately leading to microspore abortion. This study illustrates the important role of DNA methylation in cotton CMS.

Using Transcriptome Analysis to Screen for Key Genes and Pathways Related to Cytoplasmic Male Sterility in Cotton (Gossypium hirsutum L.).

Cotton (Gossypium hirsutum L.) is one of the most important cash crops worldwide. Cytoplasmic male sterility (CMS) is an excellent breeding system for exploitation of heterosis, which has great potential to increase crop yields. To understand the molecular mechanism of CMS in cotton, we compared transcriptome, cytomorphological, physiological and bioinformatics data between the CMS line C2P5A and its maintainer line C2P5B. By using high-throughput sequencing technology, 178,166 transcripts were assembled and 2013 differentially expression genes (DEGs) were identified at three different stages of C2P5A anther development. In this study, we identified DEGs associated with reactive oxygen species (ROS), peroxisomes, aldehyde dehydrogenases (ALDH), cytochrome oxidase subunit VI, and cytochrome P450, and DEGs associated with tapetum development, Jojoba acyl-CoA reductase-related male sterility protein, basic helix-loop-helix (bHLH) and MYB transcription factors. The abnormal expression of one of these genes may be responsible for the CMS C2P5A line. In gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, DEGs were mainly related to carbohydrate metabolism, amino acid metabolism, transport and catabolism, and signal transduction. Carbohydrate metabolism provides energy for anther development, starch and sucrose metabolism, fatty acid biosynthesis and metabolism and ascorbate and aldarate metabolism. These results showed that numerous genes and multiple complex metabolic pathways regulate cotton anther development. Weighted correlation network analysis (WGCNA) indicated that three modules, ‘turquoise,’ ‘blue,’ and ‘green,’ were specific for the CMS C2P5A line. The ‘turquoise’ and ‘blue’ modules were mainly related to carbohydrate metabolism, amino acid metabolism, energy metabolism, peroxisomes, pyruvate metabolism as well as fatty acid degradation. The ‘green’ module was mainly related to energy metabolism, carbon metabolism, translation, and lipid metabolism. RNA-sequencing and WGCNA polymerization modules were screened for key genes and pathways related to CMS in cotton. This study presents a new perspective for further research into the metabolic pathways of pollen abortion in the CMS C2P5A line and also provides a theoretical basis for its breeding and production.

RNA editing analysis of ATP synthase genes in the cotton cytoplasmic male sterile line H276A

BackgroundPollen development is an energy-consuming process that particularly occurs during meiosis. Low levels of adenosine triphosphate (ATP) may cause cell death, resulting in CMS (cytoplasmic male sterility). DNA sequence differences in ATP synthase genes have been revealed between the N- and S-cytoplasms in the cotton CMS system. However, very few data are available at the RNA level. In this study, we compared five ATP synthase genes in the H276A, H276B and fertile F1 (H276A/H268) lines using RNA editing, RNA blotting and quantitative real time-PCR (qRT-PCR) to explore their contribution to CMS. A molecular marker for identifying male sterile cytoplasm (MSC) was also developed.ResultsRNA blotting revealed the absence of any novel orf for the ATP synthase gene sequence in the three lines. Forty-one RNA editing sites were identified in the coding sequences. RNA editing showed that proteins had 32.43% higher hydrophobicity and that 39.02% of RNA editing sites had proline converted to leucine. Two new stop codons were detected in atp6 and atp9 by RNA editing. Real-time qRT-PCR data showed that the atp1, atp6, atp8, and atp9 genes had substantially lower expression levels in H276A compared with those in H276B. By contrast, the expression levels of all five genes were increased in F1 (H276A/H268). Moreover, a molecular marker based on a 6-bp deletion upstream of atp8 in H276A was developed to identify male sterile cytoplasm (MSC) in cotton.ConclusionsOur data substantially contributes to the understanding of the function of ATP synthase genes in cotton CMS. Therefore, we suggest that ATP synthase genes might be an indirect cause of cotton CMS. Further research is needed to investigate the relationship among ATP synthase genes in cotton CMS.

The comparison of four mitochondrial genomes reveals cytoplasmic male sterility candidate genes in cotton

BackgroundThe mitochondrial genomes of higher plants vary remarkably in size, structure and sequence content, as demonstrated by the accumulation and activity of repetitive DNA sequences. Incompatibility between mitochondrial genome and nuclear genome leads to non-functional male reproductive organs and results in cytoplasmic male sterility (CMS). CMS has been used to produce F1 hybrid seeds in a variety of plant species.ResultsHere we compared the mitochondrial genomes (mitogenomes) of Gossypium hirsutum sterile male lines CMS-2074A and CMS-2074S, as well as their restorer and maintainer lines. First, we noticed the mitogenome organization and sequences were conserved in these lines. Second, we discovered the mitogenomes of 2074A and 2074S underwent large-scale substitutions and rearrangements. Actually, there were five and six unique chimeric open reading frames (ORFs) in 2074A and 2074S, respectively, which were derived from the recombination between unique repetitive sequences and nearby functional genes. Third, we found out four chimeric ORFs that were differentially transcribed in sterile line (2074A) and fertile-restored line.ConclusionsThese four novel and recombinant ORFs are potential candidates that confer CMS character in 2074A. In addition, our observations suggest that CMS in cotton is associated with the accelerated rates of rearrangement, and that novel expression products are derived from recombinant ORFs.

Transcriptome, cytological and biochemical analysis of cytoplasmic male sterility and maintainer line in CMS-D8 cotton.

Key message This research based on RNA-seq, biochemical, and cytological analyses sheds that ROS may serve as important signaling molecules of cytoplasmic male sterility in CMS-D8 cotton. To understand the mechanism of cytoplasmic male sterility in cotton (Gossypium hirsutum), transcriptomic, cytological, and biochemical analysis were performed between the cytoplasmic male sterility CMS-D8 line, Zhong41A, and its maintainer line Zhong41B. A total of 2335 differentially expressed genes (DEGs) were identified in the CMS line at three different stages of anther development. Bioinformatics analysis of these DEGs indicated their relationship to reactive oxygen species (ROS) homeostasis, including reduction-oxidation reactions and the metabolism of glutathione and ascorbate. At the same time, DEGs associated with tapetum development, especially the transition to secretory tapetum, were down-regulated in the CMS line. Biochemical analysis indicated that the ability of the CMS line to eliminate ROS was decreased, which led to the rapid release of H2O2. Cytological analysis revealed that the most crucial defect in the CMS line was the abnormal tapetum. All these results are consistent with the RNA sequencing data. On the basis of our findings, we propose that ROS act as signal molecules, which are released from mitochondria and transferred to the nucleus, triggering the formation of abnormal tapetum.

Physical mapping and candidate gene prediction of fertility restorer gene of cytoplasmic male sterility in cotton

BackgroundCytoplasmic male sterility (CMS) is a maternally inherited trait failing to produce functional pollen. It plays a pivotal role in the exploitation of crop heterosis. The specific locus amplified fragment sequencing (SLAF-seq) as a high-resolution strategy for the identification of new SNPs on a large-scale is gradually applied for functional gene mining. The current study combined the bulked segregant analysis (BSA) with SLAF-seq to identify the candidate genes associated with fertility restorer gene (Rf) in CMS cotton.MethodsIllumina sequencing systematically investigated the parents. A segregating population comprising of 30 + 30 F2 individuals was developed using 3096A (female parent) as sterile and 866R (male parent) as a restorer. The original data obtained by dual-index sequencing were analyzed to obtain the reads of each sample that were compared to the reference genome in order to identify the SLAF tag with a polymorphism in parent lines and the SNP with read-associated coverage. Based on SLAF tags, SNP-index analysis, Euclidean distance (ED) correlation analysis, and whole genome resequencing, the hot regions were annotated.ResultsA total of 165,007 high-quality SLAF tags, with an average depth of 47.90× in the parents and 50.78× in F2 individuals, were sequenced. In addition, a total of 137,741 SNPs were detected: 113,311 and 98,861 SNPs in the male and female parent, respectively. A correlation analysis by SNP-index and ED initially located the candidate gene on 1.35 Mb of chrD05, and 20 candidate genes were identified. These genes were involved in genetic variations, single base mutations, insertions, and deletions. Moreover, 42 InDel markers of the whole genome resequencing were also detected.ConclusionsIn this study, associated markers identified by super-BSA could accelerate the study of CMS in cotton, and as well as in other crops. Some of the 20 genes’ preliminary characteristics provided useful information for further studies on CMS crops.

Expression profiles of a cytoplasmic male sterile line of Gossypium harknessii and its fertility restorer and maintainer lines revealed by RNA-Seq

The Gossypium harknessii background cytoplasmic male sterility (CMS) system has been used in cotton hybrid breeding in China. However, the mechanism underlying pollen abortion and fertility restoration in CMS remains to be determined. In this study, we used RNA-seq to identify critical genes and pathways associated with CMS in G. harknessii based CMS lines (588A), the near isogenic restorer lines (588R), and maintainer lines (588B). We performed an assembly of 80,811,676 raw reads into 89,939 high-quality unigenes with an average length of 698 bp. Among these, 72.62% unigenes were annotated in public protein databases and were classified into functional clusters. In addition, we investigated the changes in expression of genes between 588A and 588B (588R); the RNA-seq data showed 742 differentially expressed genes (DEGs) between 588A and 588B and 748 DEGs between 588A and 588R. They were mainly down-regulated in 588A and most of them distributed in metabolic and biosynthesis of secondary metabolites pathways. Further analysis revealed 23 pollen development related genes were differentially expressed between 588A and 588B. Numerous genes associated with tapetum development were down-regulated in 588A, implicating tapetum dysplasia may be a key reason for pollen abortion in CMS lines. Also, among DEGs between 588A and 588R, we identified two PPR genes which were highly up-regulated in restorer line. This study may provide assistance for detailed molecular analysis and a better understanding of harknessii based CMS in cotton.

Transcriptome sequencing and de novo analysis of cytoplasmic male sterility and maintenance in JA-CMS cotton.

Cytoplasmic male sterility (CMS) is the failure to produce functional pollen, which is inherited maternally. And it is known that anther development is modulated through complicated interactions between nuclear and mitochondrial genes in sporophytic and gametophytic tissues. However, an unbiased transcriptome sequencing analysis of CMS in cotton is currently lacking in the literature. This study compared differentially expressed (DE) genes of floral buds at the sporogenous cells stage (SS) and microsporocyte stage (MS) (the two most important stages for pollen abortion in JA-CMS) between JA-CMS and its fertile maintainer line JB cotton plants, using the Illumina HiSeq 2000 sequencing platform. A total of 709 (1.8%) DE genes including 293 up-regulated and 416 down-regulated genes were identified in JA-CMS line comparing with its maintainer line at the SS stage, and 644 (1.6%) DE genes with 263 up-regulated and 381 down-regulated genes were detected at the MS stage. By comparing the two stages in the same material, there were 8 up-regulated and 9 down-regulated DE genes in JA-CMS line and 29 up-regulated and 9 down-regulated DE genes in JB maintainer line at the MS stage. Quantitative RT-PCR was used to validate 7 randomly selected DE genes. Bioinformatics analysis revealed that genes involved in reduction-oxidation reactions and alpha-linolenic acid metabolism were down-regulated, while genes pertaining to photosynthesis and flavonoid biosynthesis were up-regulated in JA-CMS floral buds compared with their JB counterparts at the SS and/or MS stages. All these four biological processes play important roles in reactive oxygen species (ROS) homeostasis, which may be an important factor contributing to the sterile trait of JA-CMS. Further experiments are warranted to elucidate molecular mechanisms of these genes that lead to CMS.

RNA editing events in mitochondrial genes by ultra-deep sequencing methods: a comparison of cytoplasmic male sterile, fertile and restored genotypes in cotton

Cytoplasmic male sterility (CMS) is a maternally inherited trait resulting in failure to produce functional pollen and is widely used in the production of hybrid seed. Improper RNA editing is implicated as the molecular basis for some CMS systems. However, the mechanism of CMS in cotton is unknown. This study compared RNA editing events in eight mitochondrial genes (atp1, 4, 6, 8, 9, and cox1, 2, 3) among three lines (maintainer B, CMS A, and restorer R). These events were quantified by ultra-deep sequencing of mitochondrial transcripts and sequencing of cloned versions of these genes as cDNAs. A comparison of genomic PCR and RT-PCR products detected 72 editing sites in coding sequences in the eight genes and four partial editing sites in the 3'-untranslated region of atp6. The most frequent alteration (61.4 %) resulted in changes of hydrophilic amino acids to hydrophobic amino acids and the most common alteration was proline (P) to leucine (L) (26.7 %). In atp6, RNA editing created a stop codon from a glutamine in the genomic sequence. Statistical analysis of the frequencies of RNA editing events detected differences between mtDNA genes, but no differences between cotton cytoplasms that could account for the CMS phenotype or restoration. This study represents the first work to use next-generation sequencing to identify RNA editing positions and efficiency, and possible association with CMS and restoration in plants.

Transcriptome analysis of cytoplasmic male sterility and restoration in CMS-D8 cotton

A global view of differential expression of genes in CMS-D8 of cotton was presented in this study which will facilitate the understanding of cytoplasmic male sterility in cotton. Cytoplasmic male sterility (CMS) is a maternally inherited trait in higher plants which is incapable of producing functional pollen. However, the male fertility can be restored by one or more nuclear-encoded restorer genes. A genome-wide transcriptome analysis of CMS and restoration in cotton is currently lacking. In this study, Affymetrix GeneChips© Cotton Genome Array containing 24,132 transcripts was used to compare differentially expressed (DE) genes of flower buds at the meiosis stage between CMS and its restorer cotton plants conditioned by the D8 cytoplasm. A total of 458 (1.9%) of DE genes including 127 up-regulated and 331 down-regulated ones were identified in the CMS-D8 line. Quantitative RT-PCR was used to validate 10 DE genes selected from seven functional categories. The most frequent DE gene group was found to encode putative proteins involved in cell wall expansion, such as pectinesterase, pectate lyase, pectin methylesterase, glyoxal oxidase, polygalacturonase, indole-3-acetic acid-amino synthetase, and xyloglucan endo-transglycosylase. Genes in cytoskeleton category including actin, which plays a key role in cell wall expansion, cell elongation and cell division, were also highly differentially expressed between the fertile and CMS plants. This work represents the first study in utilizing microarray to identify CMS-related genes by comparing overall DE genes between fertile and CMS plants in cotton. The results provide evidence that many CMS-associated genes are mainly involved in cell wall expansion. Further analysis will be required to elucidate the molecular mechanisms of male sterility which will facilitate the development of new hybrid cultivars in cotton.

RFLP analysis of mitochondrial DNA in two cytoplasmic male sterility systems (CMS-D2 and CMS-D8) of cotton

Cytoplasmic male sterility (CMS) in higher plants is a maternally inherited trait and CMS-associated genes are known to be located in the mitochondrial genome. However, CMS-inducing genes in CMS-D2 and CMS-D8 of Upland cotton (Gossypium hirsutum L., AD1) are currently unknown. The objective of this study was to identify potential candidate DNA or gene sequences for CMS-D2 and CMS-D8 through restriction fragment length polymorphism (RFLP) analysis. Seven mtDNA gene probes and five restriction enzymes were first used to compare D2 (from G. harknessii Brandegee) and AD1 cytoplasms. With cox1, cox2, and atp1 as probes, RFLP polymorphisms were detected with one or more restriction enzyme digestions. The most notable difference was an additional fragment in the normal AD1 cytoplasm detected by cox2 in digests of three enzymes, and by cox1 and atp1 in digests with PstI. The RFLP analysis was then conducted among CMS-D2, CMS-D8 (from G. trilobum (DC.) Skovst.), and AD1 cytoplasms. Two probes from maize, atp1 and atp6, detected polymorphism among the different cytoplasmic lines. However, no difference in RFLP patterns was noted between male sterile (A) and restorer (R) lines with the D2 or D8 cytoplasm, indicating that the presence of the D2 or D8 restorer gene does not affect mtDNA organization in Upland cotton. The results demonstrate that RFLP using atp1 and atp6 as probes can distinguish the three cytoplasms. The atp1 and atp6 in CMS-D8 and these two genes together with cox1 and cox2 in CMS-D2 could be the candidates of CMS-associated genes in the mitochondrial genome, providing information for further molecular studies and developing PCR-based markers for the CMS cytoplasms in breeding. This research represents the first work using RFLP to analyze the genetic basis of CMS in cotton.

Metabolism of reactive oxygen species in cotton cytoplasmic male sterility and its restoration

To elucidate reactive oxygen species (ROS) metabolism of cotton cytoplasmic male sterility and the effects of restorer gene on the metabolism of ROS, the metabolism changes in the production and scavenging of ROS and gene expression related to ROS-scavenging enzymes were investigated in the anther mitochondria of CMS line, maintainer line and hybrid F(1). During the abortion preliminary stage (sporogenous cell division stage), anthers of CMS line had a little higher superoxide (O(2)(-)) production rate and hydrogen peroxide (H(2)O(2)) and malondialdehyde (MDA) contents than those of maintainer or hybrid F(1. )Simultaneously, a little higher ROS contents might serve as a signal to increase the activity of superoxide dismutase (SOD) in anthers of CMS line to reduce the ROS damage to the anther development. But at the abortion peak (pollen mother cell meiosis stage), anthers of CMS line had extraordinarily higher ROS contents and lower ROS-scavenging enzymic activities compared with the hybrid F(1), during which the ROS contents and ROS-scavenging enzymic activities in hybrid F(1) were approximate to those of maintainer line. The expression of Mn-sod and apx mRNA in anther of CMS line was obviously inhibited when ROS produced with a great deal during anther abortion, however the gene expression in hybrid F(1) kept normal with the maintainer. Excessive accumulation of O(2)(*-) , H(2)O(2 )and MDA, significant reduction of ROS-scavenging enzymic activities and lower gene expression level of ROS-scavenging enzyme were coinstantaneous with male cells death in anthers of CMS line. But when the restorer gene was transferred into CMS line, excessive production of ROS could be eliminated in the anthers of hybrid F(1). The restorer gene likely plays an important role in keeping the dynamic balance between the production and elimination of ROS.

Inheritance and Genetic Relationships of the D8 and D2‐2 Restorer Genes for Cotton Cytoplasmic Male Sterility

In cotton, cytoplasmic male sterility conditioned by the D8 alloplasm (CMS‐D8) is independently restored to fertility by its specific D8 restorer (D8R) and by the D2 restorer (D2R) that was developed for the D2 cytoplasmic male sterile alloplasm (CMS‐D2). However, knowledge of the relationship between the two restorer genes is lacking. The objectives of these studies were to determine the inheritance and genetic relationship between D2R and D8R. A series of intercrosses and test crosses were used to determine the inheritance and relationships. CMS‐D2 lines crossed with D8R resulted in all sterile F1 plants, confirming that the D8 restorer gene does not suppress D2 cytoplasmic dysfunction. A segregation ratio of one fertile to one sterile in three testcross combinations revealed that D2R contained one restorer gene, Rf, for both CMS‐D8 and CMS‐D2. The Rf gene functioned sporophytically, whereas the D8R gene is known to function gametophytically. However, Rf pollen with the D8 or D2 cytoplasm was preferentially effective in fertilization based on the low number of sterile plants obtained when alloplasmic F1 plants were self‐pollinated or used as the pollen‐parent in crosses with CMS‐D8. In tests for allelism only 8 sterile plants out of 1921 were obtained in two testcrosses of (D8R × D2R)F1 × normal cotton. Thus, the two restorer loci are not allelic but are tightly linked with an average genetic distance of 0.93 cM. The D2 gene Rf is redesignated Rf1, and Rf2 is assigned to the D8 restorer gene.

Effects of temperature and day length on cytoplasmic male sterility in cotton (Gossypium)

Temperature and day length were shown to affect, to a major and minor degree respectively, the expression of male sterility in G. hirsutum stocks carrying G. anomalum or G. arboreum cytoplasms. Generally, sterility increased with increasing temperature and day length. Day temperatures above 33°C were required for the consistent expression of male sterility in the sterile A lines tested, while the maintainer, or B, lines became completely sterile at day temperatures above 36�. It was concluded that while the production of hybrid cotton seed by means of the sterile A lines currently available may be feasible in some tropical areas of Australia, it would be desirable to develop genotypes in which the cytoplasmic male sterile character is stable under a much wider set of temperature regimes.