Project description:we performed transcript profiling of male sterile and fertile buds from a multiple-allele inherited male sterile AB line using the Illumina high-throughput sequencing platform and analyzed differential gene expression at the transcriptional level. Examination of mRNA levels in sterile and fertile buds of chinese cabbage Please note that the 'Table_S*.xls' files contain the further-processed supplementary data. The data processing details are provided in the readme.xlsx.
Project description: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’.
Project description:we performed transcript profiling of male sterile and fertile buds from a multiple-allele inherited male sterile AB line using the Illumina high-throughput sequencing platform and analyzed differential gene expression at the transcriptional level.
Project description:To identify genes associated with genic male sterility (GMS) that could be useful for hybrid breeding in Chinese cabbage (Brassica rapa ssp. pekinensis), floral bud transcriptome analysis was carried out using a B. rapa microarray with 300,000 probes (Br300K). Among 47,548 clones deposited on a Br300K microarray with seven probes of 60 nt length within the 3' 150 bp region, a total of 10,622 genes were differentially expressed between fertile and sterile floral buds; 4,774 and 5,848 genes were up-regulated over 2-fold in fertile and sterile buds, respectively. However, the expression of 1,413 and 199 genes showed fertile and sterile bud-specific features, respectively. Genes expressed specifically in fertile buds, possibly GMS-related genes, included homologs of several Arabidopsis male sterility-related genes, genes associated with the cell wall and synthesis of its surface proteins, pollen wall and coat components, signaling components, and nutrient supplies. However, most early genes for pollen development, genes for primexine and callose formation, and genes for pollen maturation and anther dehiscence showed no difference in expression between fertile and sterile buds. Some of the known genes associated with Arabidopsis pollen development showed similar expression patterns to those seen in this study, while others did not. BrbHLH89 and BrMYP99 are putative GMS genes. Additionally, 17 novel genes identified only in B. rapa were specifically and highly expressed only in fertile buds, implying the possible involvement in male fertility. All data suggest that Chinese cabbage GMS might be controlled by genes acting in post-meiotic tapetal development that are different from those known to be associated with Arabidopsis male sterility. A total of 14 chips were used for the microarray experiment. Experiments were performed with two biological replicates.
Project description:To identify genes associated with genic male sterility (GMS) that could be useful for hybrid breeding in Chinese cabbage (Brassica rapa ssp. pekinensis), floral bud transcriptome analysis was carried out using a B. rapa microarray with 300,000 probes (Br300K). Among 47,548 clones deposited on a Br300K microarray with seven probes of 60 nt length within the 3' 150 bp region, a total of 10,622 genes were differentially expressed between fertile and sterile floral buds; 4,774 and 5,848 genes were up-regulated over 2-fold in fertile and sterile buds, respectively. However, the expression of 1,413 and 199 genes showed fertile and sterile bud-specific features, respectively. Genes expressed specifically in fertile buds, possibly GMS-related genes, included homologs of several Arabidopsis male sterility-related genes, genes associated with the cell wall and synthesis of its surface proteins, pollen wall and coat components, signaling components, and nutrient supplies. However, most early genes for pollen development, genes for primexine and callose formation, and genes for pollen maturation and anther dehiscence showed no difference in expression between fertile and sterile buds. Some of the known genes associated with Arabidopsis pollen development showed similar expression patterns to those seen in this study, while others did not. BrbHLH89 and BrMYP99 are putative GMS genes. Additionally, 17 novel genes identified only in B. rapa were specifically and highly expressed only in fertile buds, implying the possible involvement in male fertility. All data suggest that Chinese cabbage GMS might be controlled by genes acting in post-meiotic tapetal development that are different from those known to be associated with Arabidopsis male sterility.
Project description:Comparative transcript profiling of fertile and sterile flower buds from multiple-allele-inherited male sterility in Chinese Cabbage (Brassica campestris L. ssp. pekinensis)
Project description: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.
Project description:Pistil development is a complicated process in plants, and female sterile mutant is an ideal material for screening and cloning the pistil development-related genes. In our previous study, a female sterile mutant fsm (namely fsm1 here) was obtained from a Chinese cabbage DH line ‘FT’ using a combination of isolated microspore culture and ethyl methanesulfonate (EMS) mutagenesis. BraA04g009730.3C was predicted as the candidate gene for mutant fsm1. BraA04g009730.3C encoded STERILE APETALA (SAP), a transcriptional regulator, which played a role in regulating floral organ development. In this study, another female sterile mutant (namely fsm2) was derived from a population combining EMS mutagenesis and germinating seeds of ‘FT’. The phenotype of mutant fsm2 was consistent with that of fsm1, exhibiting pistil abortion, and smaller floral organs. Genetic analysis indicated that the phenotype of mutant fsm2 was controlled by a single recessive nuclear gene. Allelism testing showed that the mutant genes of fsm1 and fsm2 were allelic, named as Brfsm. A single-nucleotide mutation (G-to-A) in the first exon of BraA04g009730.3C caused a missense mutation from GAA (glutamic acid) to GGA (glycine) in mutant fsm2. Comparative transcriptome analysis on the pistils of wild-type ‘FT’ and mutant fsm1 revealed that a total of 3,855 differentially expressed genes (DEGs) were obtained, among which 29 genes related to ovule development and 16 genes related to organ size were identified. Based on the validation of qRT-PCR, we proposed the possible regulatory pathways whereby SAP may mediate pistil development in the fsm mutant. The mutation of BraA04g009730.3C in fsm plants was involved in the pistil abortion and smaller floral organ in Chinese cabbage. These results lay a solid foundation for elucidating the molecular mechanism of pistil development in Chinese cabbage.
Project description:Using 300K′-high density microarray covering the chinese cabbage whole genome, genome-wide expression analyses of cold stress conditions.
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 âFTâ 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 âFTâ, the drmâs 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 âFTâ and drm, comparative transcriptome analysis were performed using RNA-Seq, a total of 338 differentially expressed genes (DEGs) were detected between âFTâ 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. The RNA from the third true leaves (day 15 to day 24 after the appearance of the third true leaves) of a developmentally retarded mutant (drm) and its wild type âFTâ in Chinese cabbage were sequenced by RNA-Seq, in triplicate.