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Exploring the genes associated with male sterility mutant (msm) in Chinese cabbage (Brassica campestris ssp. pekinensis) using RNA-Seq

ABSTRACT: The hybrid seed production was performed using the male sterility line, which is an important way of heterosis utilization in Chinese cabbage. A stably inherited male sterile mutant msm was obtained from a Chinese cabbage DH line ‘FT’ using the isolated microspore culture combined with 60Co γ-rays mutagenesis. Compared to the wild type ‘FT’, the msm exhibited completely degenerated stamens and no pollen phenotype, and other characters had no significant difference except for stamen. The genetic analysis indicated that the msm mutant phenotype was controlled by a single recessive nuclear gene. Cytological observation showed that the stamen abortion of msm began at the tetrad period, and tapetum cells were abnormally expanded and highly vacuolated, leading to microspore abortion. Comparative transcriptome analysis on the flower buds of ‘FT’ and msm using RNA-Seq technology revealed a total of 1,653 differentially expressed genes (DEGs). Among which, a large number of genes associated with male sterility were found, including 64 pollen development and pollen tube growth-related genes, 94 pollen wall development-related genes, 11 phytohormone-related genes and 16 transcription factor-related genes, and the overwhelming majority of these genes were down-regulated in the msm vs. ‘FT’ comparison. Furthermore, KEGG pathway analysis indicated that a variety of carbohydrate metabolic and lipid metabolic pathways were significantly enriched, which may be related to pollen abortion. The expression patterns of 24 male sterility-related genes were analyzed using qRT-PCR. In addition, a total of 24,476 single nucleotide polymorphisms and 413,073 insertion-deletion events were specifically detected in msm. These results facilitate to elucidate the regulatory mechanisms of male sterility in Chinese cabbage.

ORGANISM(S): Brassica rapa subsp. pekinensis

PROVIDER: GSE125485 | GEO | 2019/01/23

REPOSITORIES: GEO

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Project description:The leaf of Chinese cabbage is the major place of photosynthesis, the mutation of leaf may directly affect the rate of plant growth and development and the formation of leafy head, and ultimately influence the yield and quality of Chinese cabbage. We identified a developmentally retarded mutant (drm) exhibiting stable inheritance, which was derived from Chinese cabbage DH line ‘FT’ using a combination of isolated microspore culture and radiation treatment (60Co γ-rays). The drm exhibited slow growth and development at the seedling and heading stages, leading to the production of a tiny, leafy head, as well as chlorophyll-deficient leaves, especially in seedlings. Genetic analysis indicated that the phenotype of drm was controlled by a single recessive nuclear gene. Compared with wild-type line ‘FT’, the drm’s chlorophyll content was significantly reduced and its chloroplast structure was abnormal. Moreover, the photosynthetic efficiency and chlorophyll fluorescence parameters were significantly decreased. The changes in leaf color, combined with these altered physiological characters may influence the growth and development of plant, ultimately resulting in the developmentally retarded phenotype of drm. To further understand the molecular regulatory mechanisms of phenotypic differences between ‘FT’ and drm, comparative transcriptome analysis were performed using RNA-Seq, a total of 338 differentially expressed genes (DEGs) were detected between ‘FT’ and drm. According to GO and KEGG pathway analysis, a number of DEGs which involved in the chlorophyll degradation and photosynthesis were identified, such as chlorophyllase and ribulose-1,5-bisphosphate carboxylase/oxygenase. In addition, the expression patterns of 12 DEGs, including three chlorophyll degradation- and photosynthesis-related genes and nine randomly selected genes, were confirmed by qRT-PCR. Numerous single nucleotide polymorphisms were also identified, providing a valuable resource for research and molecular marker-assistant breeding in Chinese cabbage. These results contribute to our understanding of the molecular regulatory mechanisms underlying growth and development and lay the foundation for future genetic and functional genomics studies in Chinese cabbage.

Project description:Background: Pepper (Capsicum annuum L.) is a major cash crop throughout the world. Male sterility is an important characteristic in crop species that leads to a failure to produce functional pollen, and it has crucial roles in agricultural breeding and the utilization of heterosis. Objectives: In this study, we identified many crucial factors and important components in metabolic pathways in anther and pollen development, and elucidated the molecular mechanism related to pollen abortion in pepper. Methods: Pepper pollen was observed at different stages to detect the characteristics associated with male sterility and fertility. The phytohormone and oxidoreductase activities were detected in spectrophotometric and redox reaction assays, respectively. Proteins were extracted from male sterile and fertile pepper lines, and identified by TMT/iTRAQ (Tandem mass tags/isobaric tags for relative and absolute quantitation) and LC-MS/MS (liquid chromatograph-mass spectrometer) analysis. Differentially abundant proteins (DAPs) were analyzed based on Gene Ontology annotations and the Kyoto Encyclopedia of Genes and Genomes database according to |fold change)| > 1.3 and P value < 0.05. DAPs were quantified in the meiosis, tetrad, and binucleate stages by parallel reaction monitoring (PRM). Results: In this study, we screened and identified one male sterile pepper line with abnormal cytological characteristics in terms of pollen development. The peroxidase and catalase enzyme activities were significantly reduced and increased, respectively, in the male sterile line compared with the male fertile line. Phytohormone analysis demonstrated that the gibberellin, jasmonic acid, and auxin contents changed by different extents in the male sterile pepper line. Proteome analysis screened 1645 DAPs in six clusters, which were mainly associated with the chloroplast and cytoplasm based on their similar expression levels. According to proteome analysis, 45 DAPs were quantitatively identified in the meiosis, tetrad, and binucleate stages by PRM, which were related to monoterpenoid biosynthesis, and starch and sucrose metabolism pathways. Conclusions: We screened 1645 DAPs by proteomic analysis and 45 DAPs were related to anther and pollen development in a male sterile pepper line. In addition, the activities of peroxidase and catalase as well as the abundances of phytohormones such as gibberellin, jasmonic acid, and auxin were related to male sterility. The results obtained in this study provide insights into the molecular mechanism responsible for male sterility and fertility in pepper.

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Exploring the genes associated with male sterility mutant (msm) in Chinese cabbage (Brassica campestris ssp. pekinensis) using RNA-Seq

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