Molecular Mechanism Of Male Sterility Research Articles

Integrated cytological, physiological, and comparative transcriptome profiling reveals the regulatory network for male sterility in Welsh onion (Allium fistulosum L.)

Welsh onion (Allium fistulosum L.) is a specialty crop with value as a vegetable, condiment, and medicine. However, the molecular mechanisms related to pollen abortion and cytoplasmic male sterility are poorly understood, limiting the heterosis utilization and hybrid production of A. fistulosum. This study investigated the dynamic differences between male-sterile (MS) plants and male-fertile (MF) plants at different anther developmental stages based on integrated cytological, physiological, and transcriptomic analyses. Cytological observations revealed that pollen abortion in the MS plants began during the tetrad stage and was caused by vacuolated microspores, abnormal degradation of the tapetum, and inadequate nutrient supply. The physiological results showed that MS plants exhibited low total soluble sugar and starch contents and decreased antioxidant enzyme activities (peroxidase and catalase). Comparative transcriptome analysis identified 7005 differentially expressed genes (DEGs) and functional enrichment analysis indicated that significant transcriptomic differences were related to oxidative phosphorylation, starch and sucrose metabolism, and cutin, suberine, and wax biosynthesis. The relative expression levels of key DEGs in these three important metabolic pathways were determined using quantitative real-time polymerase chain reaction. On this basis, a core transcriptome-mediated MS network was proposed for MS plants with four functional components: inhibited sucrose synthesis and transport; blocked cutin, suberine, and wax synthesis; accelerated reactive oxygen species generation; and abnormal tapetal programmed cell death. These results provide theoretical guidance for further exploring the molecular mechanisms of male sterility in A. fistulosum.

Molecular Mechanism of Male Sterility Induced by 60Co γ-Rays on Plutella xylostella (Linnaeus).

Plutella xylostella (Linnaeus) is one of the notorious pests causing substantial loses to numerous cruciferous vegetables across many nations. The sterile insect technique (SIT) is a safe and effective pest control method, which does not pollute the environment and does not produce drug resistance. We used proteomics technology and bioinformatics analysis to investigate the molecular mechanisms responsible for the effects of different doses of radiation treatment on the reproductive ability of male P. xylostella. A total of 606 differentially expressed proteins (DEPs) were identified in the 200 Gy/CK group, 1843 DEPs were identified in the 400 Gy/CK group, and 2057 DEPs were identified in the 400 Gy/200 Gy group. The results showed that after 200 Gy irradiation, the testes resisted radiation damage by increasing energy supply, amino acid metabolism and transport, and protein synthesis, while transcription-related pathways were inhibited. After 400 Gy irradiation, the mitochondria and DNA in the testis tissue of P. xylostella were damaged, which caused cell autophagy and apoptosis, affected the normal life activities of sperm cells, and greatly weakened sperm motility and insemination ability. Meanwhile, Western blotting showed that irradiation affects tyrosine phosphorylation levels, which gradually decrease with increasing irradiation dose.

High-Quality Genome Assembly and Genome-Wide Association Study of Male Sterility Provide Resources for Flax Improvement.

Flax is an economic crop with a long history. It is grown worldwide and is mainly used for edible oil, industry, and textiles. Here, we reported a high-quality genome assembly for "Neiya No. 9", a popular variety widely grown in China. Combining PacBio long reads, Hi-C sequencing, and a genetic map reported previously, a genome assembly of 473.55 Mb was constructed, which covers ~94.7% of the flax genome. These sequences were anchored onto 15 chromosomes. The N50 lengths of the contig and scaffold were 0.91 Mb and 31.72 Mb, respectively. A total of 32,786 protein-coding genes were annotated, and 95.9% of complete BUSCOs were found. Through morphological and cytological observation, the male sterility of flax was considered dominant nuclear sterility. Through GWAS analysis, the gene LUSG00017705 (cysteine synthase gene) was found to be closest to the most significant SNP, and the expression level of this gene was significantly lower in male sterile plants than in fertile plants. Among the significant SNPs identified in the GWAS analysis, only two were located in the coding region, and these two SNPs caused changes in the protein encoded by LUSG00017565 (cysteine protease gene). It was speculated that these two genes may be related to male sterility in flax. This is the first time the molecular mechanism of male sterility in flax has been reported. The high-quality genome assembly and the male sterility genes revealed, provided a solid foundation for flax breeding.

Integrated analysis of transcriptome and proteome changes related to the male-sterile mutant MS7-2 in wucai (Brassica Campestris L.)

Male sterility is a complex developmental process involving the differentiation of different tissues and the coordinated expression of genes. It is not only used to produce hybrids with effective utilization of heterosis but also an ideal tool to study plant development networks. Here, we carried out morphological and cytological observations on the flower buds of the previously studied male-sterile mutant MS7-2 and its wild-type fertile plant MF7-2 at different developmental stages and performed transcriptomic and proteomic analyses. Compared with the anthers of MF7-2, the anthers of MS7-2 were obviously atrophied, the anther surface was concave, the tapetum was abnormally expanded at the mononuclear microspore stage and normal pollen grains could not be produced. Proteomic analysis showed that the differentially expressed proteins (DEPs) were mainly involved in lipid transport, lipid binding and lipase activity and were mainly concentrated in carbohydrate metabolism, biosynthesis of other secondary metabolites and lipid metabolism. Transcriptomic results showed that the differentially expressed genes (DEGs) were mainly related to cell membrane components and were significantly enriched in carbohydrate metabolism, lipid metabolism, membrane transport and other pathways. In addition, transcription factors involved in anther and pollen development were also found to be significantly down-regulated in MS7-2, such as AMS, bHLH66, bHLH69 and WRKY 34. Weighted gene coexpression network analysis (WGCNA) screened two key modules of anther abortion in wucai. The DEGs in the two modules were mainly enriched in the “terpenoid backbone biosynthesis,” “pentose and glucuronate interconversions” and “cutin, suberine and wax biosynthesis” pathways, and three hub genes were screened as BraA02g028550.3C (LACS5), BraA01g016670.3C (VHA-G3) and BraA03g043400. 3C (AMS). Through transcriptomic and proteomic analyses, a regulatory network of MS7-2 male sterility of was constructed. It is speculated that the down-regulation of these genes and proteins in MS7-2 anthers inhibits the synthesis of some lipids and endogenous metabolites and accumulates a large amount of reactive oxygen species (ROS) in the tapetum, leading to the failure of normal PCD and the abortion of MS7-2. This study provides new insights into the molecular mechanism of male sterility in wucai.

Cloning and Expression Analysis of HAT1 and HDAC1 in the Testes of Mature Yaks and Their Sterile Hybrids.

Simple SummaryCattle-yak is the hybrid between male cattle (Bos taurus) and female yak (Bos grunniens). Male cattle-yak can not produce normal sperm. The mechanisms that underlie cattle-yak male sterility have not been elucidated. Histone acetylation is a common regulation mode that plays an important role in the development of gametes. The objective of this study was to explore the molecular mechanism of male sterility in yak hybrids based on histone acetyltransferase 1 (HAT1) and histone deacetylase 1 (HDAC1), two enzymes that regulate histone acetylation. The mRNA and protein expression levels of HAT1 in the testes of adult cattle-yaks were significantly lower than in adult yaks, and the protein expression levels of HDAC1 were significantly higher than in yaks. In addition, H3K9 acetylation levels in cattle-yak testes were significantly lower than in yaks. These results suggest that male sterility in cattle-yaks might be associated with decreased histone acetylation levels in the testes.The objective of this study was to explore the molecular mechanism of male sterility in yak hybrids based on HAT1 and HDAC1. Total RNA was extracted from the testes of adult yaks (n = 11) and sterile cattle-yaks (n = 11) followed by reverse transcription. The coding sequence (CDS) of yak HAT1 and HDAC1 were obtained by conventional polymerase chain reaction (PCR) and gene cloning. The testicular mRNA and protein levels of HAT1 and HDAC1 in yaks and cattle-yaks were detected by quantitative PCR (qPCR) and Western blotting, respectively, and the histone H3 lysine 9 (H3K9) histone acetylation level in the testes of yaks and cattle-yaks was assayed using enzyme linked immunosorbent assay (ELISA). The results showed that the CDS of HAT1 and HDAC1 were 1242 bp and 1449 bp in length, encoding 413 and 482 amino acids, respectively; yaks had a similar mRNA sequence as cattle in both genes. The testicular mRNA and protein levels of HAT1 of cattle-yaks were significantly lower than those of yaks, and the protein level of HDAC1 was significantly higher than that of yaks. ELISA showed that the acetylation level of testicular H3K9 was significantly lower in yak hybrids than that of yaks. The present results suggest that the decreased level of HAT1 and increased level of HDAC1 may result in the decreased H3K9 acetylation in cattle-yaks and might be associated with their sterility.

CRISPR/Cas9-Induced Mutagenesis of TMS5 Confers Thermosensitive Genic Male Sterility by Influencing Protein Expression in Rice (Oryza sativa L.)

The development of thermosensitive genic male sterile (TGMS) lines is the key to breeding two-line hybrid rice, which has been widely applied in China to increase grain yield. CRISPR/Cas9 has been widely used in genome editing to create novel mutants in rice. In the present study, a super grain quality line, GXU 47, was used to generate a new TGMS line with specific mutations in a major TGMS gene tms5 generated with CRISPR/Cas9-mediated genome editing in order to improve the rice quality of two-line hybrids. A mutagenesis efficiency level of 75% was achieved, and three homozygous T-DNA-free mutant lines were screened out. The mutants exhibited excellent thermosensitive male fertility transformation characteristics with complete male sterility at ≥24 °C and desirable male fertility at around 21 °C. Proteomic analysis based on isobaric tags for relative and absolute quantification (iTRAQ) was performed to unveil the subsequent proteomic changes. A total of 192 differentially expressed proteins (DEPs), including 35 upregulated and 157 downregulated, were found. Gene ontology (GO) analysis revealed that the DEPs were involved in a single-organism biosynthetic process, a single-organism metabolic process, oxidoreductase activity, and catalytic activity. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the DEPs were involved in ubiquinone and other terpenoid quinone biosynthesis, the biosynthesis of secondary metabolites, metabolic pathways, and phenylpropanoid biosynthesis. Our study shows that high mutation efficiency was achieved in both target sites, and T-DNA-free mutant lines were obtained in the T1 generation. The present study results prove that it is feasible and efficient to generate an excellent mutant line with CRISPR/Cas9, which provides a novel molecular mechanism of male sterility caused by the mutation of tms5.

Conjunctive Analyses of BSA-Seq and BSR-Seq Unveil the Msβ-GAL and MsJMT as Key Candidate Genes for Cytoplasmic Male Sterility in Alfalfa (Medicago sativa L.).

Knowing the molecular mechanism of male sterility in alfalfa is important to utilize the heterosis more effectively. However, the molecular mechanisms of male sterility in alfalfa are still unclear. In this study, the bulked segregant analysis (BSA) and bulked segregant RNA-seq (BSR) were performed with F2 separation progeny to study the molecular mechanism of male sterility in alfalfa. The BSA-seq analysis was located in a candidate region on chromosome 5 containing 626 candidate genes which were associated with male sterility in alfalfa, while the BSR-seq analysis filtered seven candidate DEGs related to male sterility, and these candidate genes including EF-Tu, β-GAL, CESA, PHGDH, and JMT. The conjunctive analyses of BSR and BSA methods revealed that the genes of Msβ-GAL and MsJMT are the common detected candidate genes involved in male sterility in alfalfa. Our research provides a theory basis for further study of the molecular mechanism of male sterility in alfalfa and significant information for the genetic breeding of Medicago sativa.

The chimeric gene atp6c confers cytoplasmic male sterility in maize by impairing the assembly of the mitochondrial ATP synthase complex

Cytoplasmic male sterility (CMS) is a powerful tool for the exploitation of hybrid heterosis and the study of signaling and interactions between the nucleus and the cytoplasm. C-type CMS (CMS-C) in maize has long been used in hybrid seed production, but the underlying sterility factor and its mechanism of action remain unclear. In this study, we demonstrate that the mitochondrial gene atp6c confers male sterility in CMS-C maize. The ATP6C protein shows stronger interactions with ATP8 and ATP9 than ATP6 during the assembly of F1Fo-ATP synthase (F-type ATP synthase, ATPase), thereby reducing the quantity and activity of assembled F1Fo-ATP synthase. By contrast, the quantity and activity of the F1' component are increased in CMS-C lines. Reduced F1Fo-ATP synthase activity causes accumulation of excess protons in the inner membrane space of the mitochondria, triggering a burst of reactive oxygen species (ROS), premature programmed cell death of the tapetal cells, and pollen abortion. Collectively, our study identifies a chimeric mitochondrial gene (ATP6C) that causes CMS in maize and documents the contribution of ATP6C to F1Fo-ATP synthase assembly, thereby providing novel insights into the molecular mechanisms of male sterility in plants.

Comparative transcriptome analysis reveals differential gene expression in sterile and fertile rubber tree varieties during flower bud differentiation

Plant male sterility (MS) is an important agronomic trait that provides an efficient tool for hybridization and heterosis utilization of crops. Based on phenotypic and cytological observations, our study performed a multi-comparison transcriptome analysis strategy on multiple sterile and fertile rubber tree varieties using RNA-seq. Compared with the male-fertile varieties, a total of 1590 differentially expressed genes (DEGs) were detected in male-sterile varieties, including 970 up-regulated and 620 down-regulated transcripts in sterile varieties. Key DEGs were further assessed focusing on anther development, microsporogenesis and plant hormone metabolism. Twenty DEGs were selected randomly to validate transcriptome data using quantitative real-time PCR (qRT-PCR). Eleven key genes were subjected to expression pattern analysis using qRT-PCR and fluorescence in situ hybridization. Among them, nine genes, i.e., A6, GAI1, ACA7, TKPR1, CYP704B1, XTH26, MS1, MS35 and MYB33, that regulate callose metabolism, pollen wall formation, tapetum and microspores development were identified as candidate male-sterile genes. These findings provide insights into the molecular mechanism of male sterility in rubber tree.

Integrative analysis of transcriptomic and proteomic changes related to male sterility in Tagetes erecta.

Male sterile and male fertile two-type lines are important in heterosis utilization and breeding in Tagetes erecta, but the genes and pathways involved in male sterility are poorly understood. To explore these topics, transcriptome data (by RNA-seq) and proteome data (by iTRAQ) were gathered from flower buds of the male sterile line 'MS2-2' and male fertile line 'MF2-2' and integrated for a better understanding of the underlying molecular mechanisms of male sterility in T. erecta. The RNA-seq procedure generated 285,139,740 clean reads and 63359 unigenes and 6640 differentially expressed genes (DEGs) were identified, of which 4136 were downregulated and 2504 were upregulated in 'MS2-2'. DEGs related to flower development, pollen development, pollen wall assembly, endogenous hormones and transcription factors were identified. The iTRAQ analysis identified 3950 proteins in total; 789 were differentially expressed proteins (381 upregulated, 408 downregulated), which were mainly annotated to the Ribosome, Carbon metabolism and Biosynthesis of amino acids pathways. An association analysis revealed strong correlation (r Pearson = 0.6019) between the transcriptomic and proteomic data, and 256 and 34 proteins showed the same and opposite expression patterns with regard to their transcripts, respectively. Pathways such as photosynthesis, fatty acid biosynthesis and phenylpropanoid biosynthesis which influence tapetum and pollen development in male sterile plants, were significantly enriched at the transcript and protein levels. Most genes involved in these pathways were downregulated in 'MS2-2'. The low expression of these genes or functional loss of proteins could be associated with flower development, pollen development and related to changes in fertility in T. erecta. This study provided transcriptomic and proteomic information for T. erecta that could illuminate the mechanism of male sterility.

Male Sterility is linked to the Flavonoid Biosynthesis Pathways in Prunus mira.

Sterility plays an important role in plant adaptation and evolution and has contributed to the development of high yielding crop hybrids. We used the widely targeted metabolomics profiling to survey the metabolites and biological pathways associated with male sterility in Prunus mira by comparing flowers from fertile and sterile trees. Male sterile flowers displayed abnormal stamen, uncolored anthers, and distorted and shrunken pollen grains with an apparent lack of turgidity. We report 566 metabolites in six flower samples and 140 differentially accumulated metabolites (DAMs) between both flower types. Most of the DAMs belong to the phenyl propanoid biosynthesis pathway, particularly flavonoid, flavone and flavonol biosynthesis pathways, implying that alterations in these key pathways link to male sterility in P. mira. The known link between low levels of flavonoid metabolites, weak expression levels of several structural genes from the phenyl propanoid biosynthesis pathway and hyper accumulation of reactive oxygen species were highlighted for understanding the underlying mechanism leading to the abnormal or aborted pollen grains observed in the sterile flowers. Data on the molecular mechanism of male sterility in Prunus mira will facilitate further in-depth investigations on this important agronomic and ecological trait.

ITRAQ-based proteomic analysis reveals several key metabolic pathways associated with male sterility in Salvia miltiorrhiza.

Male sterility is a common phenomenon in flowering plants, and it has been widely used in hybrid seed production in a number of economically important crops. In 2002, our team discovered a natural male sterile mutant of Salvia miltiorrhiza. It provided us with the possibility of obtaining stable and controllable quality. To study the molecular mechanism of male sterility in S. miltiorrhiza, we generated proteomic profiles comparing the male sterile mutant type (MT) and wild type (WT) using iTRAQ sequencing. We found a total of 639 differential abundant proteins (DAPs) between MT and WT buds. The DAPs associated with male sterility were mainly involved in (1) carbohydrate and energy metabolism, and (2) protein synthesis and degradation. Based on a comparison between the protein expression profiles of MT and WT, we elucidated a potential protein interaction network involved in male sterility. These results provide new potential biomarkers and insights into the molecular mechanism of male sterility in S. miltiorrhiza.

Gene characterization and molecular pathway analysis of reverse thermosensitive genic male sterility in eggplant (Solanum melongena L.)

The naturally occurring mutant eggplant line 05ms was identified with reverse thermosensitive genic male sterility (rTGMS), but its temperature-responsive fertility mechanisms remain largely unknown. Here, we studied the flower morphology, anther cellular structure, and genome-wide gene expression of this rTGMS line. Candidate genes for thermosensitive male sterility during the microspore development of 05ms and the temperature-insensitive line S63 under low-temperature (LT) and high-temperature (HT) conditions were identified. Under LT, tapetum cells were vacuolated and had delayed disintegration in 05ms. RNA-seq analysis indicated that DEGs were enriched in the KEGG pathways ‘plant hormone signal transduction’, ‘starch and sucrose metabolism’, and ‘phenylpropanoid biosynthesis’. We identified two genes, 4CLL1 (Sme2.5_00368.1_g00010.1) and CKI1 (Sme2.5_10056.1_g00002.1), which could potentially regulate eggplant anther development and may be candidate genes for rTGMS. Finally, we propose a working model of anther abortion for rTGMS in eggplant. CKI1 responds to LT stress and causes expression changes in genes related to anther development, such as 4CLL1, and the cellular structure of the tapetum becomes abnormal, causing male sterility. The findings of this study explain the underlying molecular mechanisms of male sterility in eggplant rTGMS lines.

Identification and Mapping of a New Soybean Male-Sterile Gene, mst-M.

The use of sterility is common in plants and multiple loci for hybrid sterility have been identified in crops such as rice. In soybean, fine-mapping and research on the molecular mechanism of male sterility is limited. Here, we identified a male-sterile soybean line, which produces larger, abnormal pollen grains that stain poorly with I2-KI. In an inheritance test, all F1 plants were fertile and the F2 and F2:3 populations conformed with the expected segregation ratio of 3:1 (fertility:sterility) (p = 0.82) and showed a 1:2:0 ratio of homozygous fertile: heterozygous fertile: homozygous sterile genotypes (p = 0.73), suggesting that the sterility was controlled by a single recessive gene (designated “mst-M”). Bulked segregant analysis showed that almost all single-nucleotide polymorphisms (SNPs; 95.92%) were distributed on chromosome 13 and 868 SNPs (95.81%) were distributed in the physical region of Chromosome 13.21877872 to Chromosome 13.22862641. Genetic mapping revealed that mst-M was flanked by W1 and dCAPS-1 with genetic distances of 0.6 and 1.8 cM, respectively. The order of the consensus markers and known sterility genes was: Satt146 – (5.0 cM) – st5 – (2.5 cM) – Satt030 – (15.3 cM) – ms6 – (5.0 cM) – Satt149 – (39.5 cM) – W1 – (0.6 cM) – mst-M – (14.1 cM) – Satt516 (7.5 cM) – ms1 – (16.3 cM) – Satt595. These results suggest that mst-M is a newly identified male-sterility gene, which represents an alternative genetic resource for developing a hybrid seed production system for soybean.

QTL mapping of male sterility and transmission pattern in progeny of Satsuma mandarin.

Seedlessness is one of the important traits in citrus breeding. Male sterility derived from Satsuma mandarin (Citrus unshiu) has been used in Japanese citrus breeding programs to obtain seedless cultivars. The efficiency of seedless cultivar breeding would be improved by developing a selection marker linked to seedlessness. In this study, we performed QTL mapping in ‘Okitsu No. 46’ × ‘Okitsu No. 56’ (O46-O56) crosses for the number of pollen grains per anther (NPG) and apparent pollen fertility (APF), two traits used as an index of male sterility, and detected a candidate QTL for NPG (MS-P1) on linkage group 8 with a significant LOD score (7.31) and 47% of variance explained. The QTL for APF (MS-F1) was detected on linkage group 6 with a significant LOD score (5.71) and 63.6% of variance explained. The role of both MS-P1 in reducing NPG and MS-F1 in decreasing APF were confirmed with the ‘Okitsu No.46’ × ‘Kara’ (O46-K) cross. Pedigree analysis inferred that both MS-P1 and MS-F1 in ‘Okitsu No. 46’ were derived from kunenbo (Citrus nobilis) through hassaku (C. hassaku) and ‘Sweet Spring’. Cytoplasm analysis revealed that both male-sterile ‘Sweet Spring’ and ‘Okitsu No. 46’ have cytoplasm derived from Kishu (C. kinokuni hort. ex Tanaka), but the cytoplasm of male-sterile kunenbo and hassaku were derived from other varieties rather than Kishu. These results suggest that MS-P1 and MS-F1 primarily reduce the NPG and decrease APF, but their expression requires a cytoplasm derived from Kishu. These findings will improve our understanding of the molecular mechanism of male sterility in citrus and help to develop a DNA marker for seedless breeding in citrus.

Comparative phenotype and microRNAome in developing anthers of wild-type and male-sterile Lycium barbarum L.

Lycium barbarum L. (L. barbarum) is an economically important plant, as its fruit is highly marketable for its healthy nutrient content. In this study, we characterized the anther development of a major cultivar (Ningqi No. 1) and a male-sterile mutant (Ningqi No. 5) of L. barbarum. We initially investigated the phenotypes of Ningqi No. 1 and Ningqi No. 5 using microscopy and chemical staining, which showed that Ningqi No. 5 failed in the degradation of anther callose, leading to an absence of mature pollen grains and thus to male sterility. Then, to understand the dynamic profile of miRNA expression during the development of the anthers, we collected anther samples from both Ningqi No. 1 and Ningqi No. 5 throughout anther development, and we further identified 137 novel miRNAs from these anther samples by using next-generation deep sequencing technology. Of these 137 novel miRNAs, 96 miRNAs were conserved miRNAs classified into 65 miRNA families, including a few well-known miRNA families related to anther development, such as miR156, miR159 and miR172. In addition, the remaining 41 miRNAs were considered lineage-specific miRNAs, which had no orthologues in other species. The expression data showed that 45 of the 137 miRNAs were differentially expressed in the different samples, including 4 Ningqi No. 5-specific miRNAs and 15 stage-specific miRNAs. The expression patterns of six miRNAs and their predicted targets were verified by Q-PCR, and one of miRNAs and its target were chosen for transient co-expression in Nicotiana benthamiana leaves to verify the correlations between the miRNA and its predicted target. Overall, the identification of the miRNAs in the anther development of Ningqi No. 1 and Ningqi No. 5 provides a valuable resource for understanding the molecular mechanisms of male sterility in L. barbarum.

BrEXL6, a GDSL lipase gene of Brassica rapa, functions in pollen development

Multiple allele-inherited male sterility has been widely used by breeders of Brassica rapa L. ssp. pekinensis, but the molecular mechanisms of male sterility are not yet clear. In this study, we isolated the full-length cDNA of a new gene (not included in the Brassica database). This gene, comprising 1 054 bp, encodes a 39.99 kDa protein with a Gly-Asp- Ser-Leu (GDSL)-lipase domain that is a member of the lipolytic protein GDSL family. The sequence of candidate gene is the most similar to extracellular lipase 6 (EXL6) of Arabidopsis and was therefore designated BrEXL6 and submitted to NCBI (accession No. JX131630.1). Reverse transcription semi-quantitative PCR and Western blot analysis showed that BrEXL6 and its encoded protein were significantly more expressed in fertile buds than in sterile buds. Quantitative PCR and in situ hybridization showed that BrEXL6 was highly expressed in the anthers of fertile buds, especially anthers at the pollen-development stages, but only weakly expressed in other tissues and floral organs of fertile plants and whole sterile plants. These results suggest that BrEXL6 is a pollen development-related gene. The results of this study provide clues for understanding the mechanisms underlying multiple allele-inherited male sterility.

Identification and Functional Analysis of microRNAs Involved in the Anther Development in Cotton Genic Male Sterile Line Yu98-8A.

Hybrid vigor contributes in a large way to the yield and quality of cotton (Gossypium hirsutum) fiber. Although microRNAs play essential regulatory roles in flower induction and development, it is still unclear if microRNAs are involved in male sterility, as the regulatory molecular mechanisms of male sterility in cotton need to be better defined. In this study, two independent small RNA libraries were constructed and sequenced from the young buds collected from the sporogenous cell formation to the meiosis stage of the male sterile line Yu98-8A and the near-isogenic line. Sequencing revealed 1588 and 1536 known microRNAs and 347 and 351 novel miRNAs from male sterile and male fertile libraries, respectively. MicroRNA expression profiles revealed that 49 conserved and 51 novel miRNAs were differentially expressed. Bioinformatic and degradome analysis indicated the regulatory complexity of microRNAs during flower induction and development. Further RT-qPCR and physiological analysis indicated that, among the different Kyoto Encyclopedia Gene and Genomes pathways, indole-3-acetic acid and gibberellic acid signaling transduction pathways may play pivotal regulatory functions in male sterility.

Differential proteomics analysis to identify proteins and pathways associated with male sterility of soybean using iTRAQ-based strategy

To further elucidate the molecular mechanism of cytoplasmic male sterility (CMS) in soybean, a differential proteomic analysis was completed between the CMS line NJCMS1A and its maintainer NJCMS1B using iTRAQ-based strategy. As a result, 180 differential abundance proteins (DAPs) were identified, of which, 60 were down-regulated and 120 were up-regulated in NJCMS1A compared with NJCMS1B. Bioinformatic analysis showed that 167 DAPs were annotated in 41 Gene Ontology functional groups, 106 DAPs were classified into 20 clusters of orthologous groups of protein categories, and 128 DAPs were enrichment in 53 KEGG pathways. Fifteen differential level proteins/genes with the same expression pattern were identified in the further conjoint analysis of DAPs and the previously reported differential expression genes. Moreover, multiple reaction monitoring test, qRT-PCR analysis and enzyme activity assay validated that the iTRAQ results were reliable. Based on functional analysis of DAPs, we concluded that male sterility in NJCMS1A might be related to insufficiencies in energy supply, unbalance of protein synthesis and degradation, disruption of flavonoid synthesis, programmed cell death, abnormalities of substance metabolism, etc. These results might facilitate our understanding of the molecular mechanisms behind CMS in soybean. Biological significanceSoybean is an important global crop that provides protein and oil. Heterosis is a significantly potential approach to increase the yield of soybean. Cytoplasmic male sterility (CMS) plays a vital role in the production of hybrid seeds. However, the genetic and molecular mechanisms of male sterility in soybean still need to be further elucidated. In the present paper, a differential proteomic analysis was carried out and the results showed that several key proteins involved in key pathways were associated with male sterility in soybean. This work provides a new insight to understand the genetic and molecular mechanisms underlying CMS in soybean.

Comparative sequence and expression analysis of tapetum specific male sterility related genes in Medicago truncatula.

Heterosis, or enhancement through outbreeding, is one of the most promising approaches for increasing crop yield. Male sterility (MS), which promotes heterosis, has been widely applied in hybrid crop production. Medicago truncatula is a model legume species and is closely related to M. sativa, an important legume forage plant. Although the molecular mechanisms of MS in M. truncatula and M. sativa remain unclear, several studies of MS have been conducted in Arabidopsis thaliana. Previous research has shown that MS is associated with the destruction of tapetal cell layers. Disruption of tapetum developmental processes may result in pollen abortion. In an effort to identify genes useful for breeding in M. sativa, we identified MS related genes in M. truncatula using BLAST and homology to A. thaliana genes. In this study, we identified 63 tapetum specific male sterility (TSMS) related genes. The length of TSMS genes varied from 225 to 3747 bp. We identified 15 conserved domains and 8 cis-elements associated with TSMS related genes. Analysis of the phylogenetic relationships among these genes allowed them to be classified into three groups, MtTsms A, MtTsms B, and MtTsms C. Expression analyses revealed that these genes may be involved in developmental processes and response to abiotic stress.