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Comparative transcript profiling of fertile and sterile flower buds from multiple-allele-inherited male sterility in Chinese Cabbage (Brassica campestris L. ssp. pekinensis)

ABSTRACT: We studied the causes of sterility underlying multiple-allele-inherited male sterility in Chinese cabbage by identifying differentially expressed genes (DEGs) related to pollen sterility between fertile and sterile flower buds. In this work,we performed transcriptome analysis of mRNA isolated from fertile and sterile buds using Illumina HiSeq 2000 platform sequencing. Approximately 80% of ~229 million high quality paired-end reads were uniquely mapped to the reference genome. In sterile buds, 699 genes were significantly up-regulated and 4,096 genes were down-regulated. Among the DEGs, 28 pollen cell wall-related genes, 53 transcription factor genes, 45 phytohormone-related genes, 20 anther and pollen-related genes, 212 specifically expressed transcripts (SETs), and 417 DEGs located in linkage group R07 were identified. Six transcription factor genes BrAMS, BrMS1, BrbHLH089, BrbHLH091, BrAtMYB103, and BrANAC025, were identified as putative sterility-related genes. The weak auxin signal that is regulated by BrABP1 may be one of the key factors causing pollen sterility observed here. Moreover, several significantly enriched GO terms “cell wall organization or biogenesis” (GO:0071554), “intrinsic to membrane” (GO:0031224), “integral to membrane” (GO:0016021), “hydrolase activity, acting on ester bonds” (GO:0016788) and one significantly enriched pathway “starch and sucrose metabolism” (ath00500) were obtained in this work. qRT-PCR and in situ hybridization validated that our RNA-seq transcriptome analysis was accurate and reliable. This study will lay the foundation for elucidating the molecular mechanism(s) underlying sterility, and provide valuable information for studying multiple-allele-inherited male sterility in Chinese cabbage line ‘AB01’.

ORGANISM(S): Brassica rapa subsp. pekinensis

PROVIDER: GSE95242 | GEO | 2017/02/23

SECONDARY ACCESSION(S): PRJNA376499

REPOSITORIES: GEO

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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.

Project description:Background: The fertile and sterile plants are derived from the self-pollinated offspring of the F1 hybrid between novel restorer line NR1 and Nsa CMS line in Brassica napus, which possess the identical cytoplasmic genetic material arising from Nsa CMS line. As far as the nuclear genetic background is concerned, both fertile and sterile plants have the complete set of chromosomes from Brassica napus, except one or two members of the added Sinapis arvensis chromosome pair in the fertile plant. To elucidate gene expression and regulation caused by the A and C subgenomes, the alien chromosome and cytoplasm from S. arvensis during the development of young floral buds, we performed genome-widely high-throughput transcriptomic sequencing between young floral buds of sterile and fertile plants. Results: In this study, equal amount of RNA taken from young floral buds of sterile and fertile plants were sequenced using Illumina/Solexa platform. A total of 4,415,866 and 4,244,140 raw tags were obtained in sterile plant (Ste) and fertile plant (Fer) libraries, respectively. After filtering out low quality data, a total of 2,760,574 and 2,714,441 clean tags remained from the two libraries, from which 242,163 (Ste) and 253,507 (Fer) distinct tags were obtained. To identify the genes corresponding to the distinct tags in each library, all distinct sequencing tags were annotated using all possible CATG+17-nt sequences of the genome and transcriptome of Brassica rapa and those of Brassica oleracea as the reference sequences, respectively. Many genes showed substantial differences in expression between the two libraries. In total, there were 3231 genes of B. rapa and 3371 genes of B. oleracea which were detected with significant differential expression levels. GO and pathway-based analyses were performed to determine and further understand the biological functions of differentially expressed genes (DEGs). In addition, there were 1089 specially expressed unknown tags in Fer, which were neither mapped to B. oleracea nor mapped to B. rapa, and these unique tags were presumed to arise basically from the added alien chromosome of S. arvensis. Fifteen genes were randomly selected and confirmed their expression levels by quantitative RT-PCR, fourteen of the fifteen genes showed expression patterns consistent with the digital gene expression (DGE) data. Conclusions: A number of genes were differentially expressed between the young floral buds of sterile and fertile plants. Some of these genes may be candidates for future research on CMS in Nsa line, fertility restoration and improved agronomic traits in NR1 line. Further study of the unknown tags which were specially expressed in Fer will help to dig those genes with desirable agronomic traits from wild species. mRNA profiles of fertile buds (Fer) and sterile buds (Ste) were generated by deep sequencing.

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Comparative transcript profiling of fertile and sterile flower buds from multiple-allele-inherited male sterility in Chinese Cabbage (Brassica campestris L. ssp. pekinensis)

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