Complete mitochondrial genome sequencing and identification of candidate genes responsible for C5-type cytoplasmic male sterility in cabbage (B. oleracea var. capitata).

Cytoplasmic male sterility (CMS) is widely used in cruciferous vegetables hybrid breeding. The C5-type CMS cabbage line exhibits stable male sterility and offers great value for cabbage breeding. However, the underlying CMS mechanism remains unclear. Here, the complete mitochondrial genome was sequenced and assembled for this line. The genome size was 221,862 bp. Mitochondrial genome comparison showed that the mitochondrial genome was likely generated by recombination with a nap-type CMS B. napus strain. Sixty-seven unknown-function open reading frames (ORFs) were identified. Seven orfs, orf114a, orf123a, orf188a, orf222a, orf261a, orf286a, and orf322a, were specifically identified in this genome. The presence of these candidate CMS genes decreased ATPase activity and ATP content by affecting the transcript levels of energy metabolism-related genes and F1F0-ATP synthase assembly. Among them, orf188a, orf222a, orf261a, orf286a, and orf322a possessed a transmembrane structure, and orf188a was cotranscribed with rps7 and trnfM. orf222a was partially homologous to atp8 and coexpressed with nad5. orf261a and orf322a were cotranscribed with cox1 and atp9, respectively. Additionally, orf114a was cotranscribed with atp8. Yeast two-hybrid assays showed that the ORF222a protein interacts with a B. oleracea ATP17 homolog (Bo7g114140) during F0-type ATP synthase assembly, reducing the quantity and activity of assembled F1F0-ATP synthase. Cytological sections showed that premature separation of the tapetum from the connective tissue and delayed tapetal programmed cell death (PCD) might be the immediate causes of CMS in C5-type CMS cabbage lines. Our results may help uncover the molecular mechanism of C5-type CMS in B. oleracea from the perspectives of the whole mitochondrial genome and cytology of anther development.

Comparative Genomic Analysis of Xanthomonas campestris pv. campestris Isolates BJSJQ20200612 and GSXT20191014 Provides Novel Insights Into Their Genetic Variability and Virulence.

Black rot is a disease that has a severe impact on cabbage yield and quality in China. Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot of Brassicaceae crops. So far, the whole genomic sequences of more than 30 Xcc isolates have been sequenced; however, little information about genomic variability and virulence has been reported. In this study, 12 Xcc isolates were isolated from diseased cabbage leaves in seven Chinese provinces and two municipalities from July 2019 to November 2020. Pathogenicity analysis showed that isolate GSXT20191014 was more aggressive than BJSJQ20200612 and HRIW 3811 on cabbage inbred line 1371. Both BJSJQ20200612 and GSXT20191014 were sequenced and comparatively analyzed. The results showed that BJSJQ20200612 and GSXT20191014 have a single circular chromosome comprising 5,115,975 and 4,975,682 bp, respectively. Compared to the other six sequenced strains, 26 and 47 variable genomic regions were found in BJSJQ2020061 and GSXT20191014 genomic sequences, respectively. The variable genomic regions could be responsible for the genetic variation in Xcc strains and have led to the differences in type III secreted effector repertoires, virulence factors and secreted proteins between these two strains. Among the identified secreted proteins, two copies of peptidase S8/S53 were found in GSXT20191014-specific chromosomal segments. The common effectors xopR, xopH, avrBs1, and xopAH are found in most Xcc genomes, but they are absent in the GSXT20191014 genome. Variations in the composition of exopolysaccharides (EPS) and lipopolysaccharides (LPS) may aid GSXT20191014 isolate infections to evade recognition by the host immune system. Our results revealed a direct correlation between genomic variability and Xcc virulence. We also developed several markers for detecting BJSJQ20200612 and GSXT20191014 isolates and further tested the rest of our other 10 isolates. Finally, the isolated Xcc strains were classified into three genetic subgroups by specific molecular markers and multilocus sequence typing (MLST) approach. BJSJQ20200612 and GSXT20191014 isolates were also classified into two subgroups of Xcc according to the core-genome-based phylogenetic tree. This study extended our understanding of Xcc genomic features and provided the foundation to further characterize the mechanisms for Xcc virulence and a clue for black rot resistance breeding.

Comparative analysis of the complete mitochondrial genome sequences and anther development cytology between maintainer and Ogura-type cytoplasm male-sterile cabbage (B. oleracea Var. capitata).

BACKGROUND: Cytoplasmic male sterility (CMS) has been widely used for commercial F1 hybrid seeds production. CMS is primarily caused by chimeric genes in mitochondrial genomes. However, which specific stages of anther development in cabbage are affected by the chimeric genes remain unclear. RESULTS: In the present study, the complete mitochondrial genomes were sequenced and assembled for the maintainer and Ogura CMS cabbage lines. The genome size of the maintainer and Ogura CMS cabbage are 219,962 bp and 236,648 bp, respectively. There are 67 and 69 unknown function ORFs identified in the maintainer and Ogura CMS cabbage mitochondrial genomes, respectively. Four orfs, orf102a, orf122b, orf138a and orf154a were specifically identified in the Ogura CMS mitochondrial genome, which were likely generated by recombination with Ogura type radish during breeding process. Among them, ORF138a and ORF154a possessed a transmembrane structure, and orf138a was co-transcribed with the atp8 and trnfM genes. orf154a is partially homologous to the ATP synthase subunit 1 (atpA) gene. Both these genes were likely responsible for the CMS phenotype. In addition, cytological sections showed that the abnormal proliferation of tapetal cells might be the immediate cause of cytoplasmic male-sterility in Ogura CMS cabbage lines. RNA-seq results showed that orf138a and orf154a in Ogura CMS might influence transcript levels of genes in energy metabolic pathways. CONCLUSIONS: The presence of orf138a and orf154a lead to increased of ATPase activity and ATP content by affecting the transcript levels of genes in energy metabolic pathways, which could provide more energy for the abnormal proliferation of tapetal cells. Our data provides new insights into cytoplasmic male-sterility from whole mitochondrial genomes, cytology of anther development and transcriptome data.

Identification and expression profiling analysis of NBS-LRR genes involved in Fusarium oxysporum f.sp. conglutinans resistance in cabbage.

As one of the most important resistance (R) gene families in plants, the NBS-LRR genes, encoding proteins with nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains, play significant roles in resisting pathogens. The published genomic data for cabbage (Brassica oleracea L.) provide valuable data to identify and characterize the genomic organization of cabbage NBS-LRR genes. Ultimately, we identified 105 TIR (N-terminal Toll/interleukin-1 receptor)-NBS-LRR (TNL) genes and 33 CC (coiled-coil)-NBS-LRR (CNL) genes. Further research indicated that 50.7% of the 138 NBS-LRR genes exist in 27 clusters and there are large differences among the gene structures and protein characteristics. Conserved motif and phylogenetic analysis showed that the structures of TNLs and CNLs were similar, with some differences. These NBS-LRRs are evolved under negative selection and mostly arose from whole-genome duplication events during evolution. Tissue-expression profiling of NBS-LRR genes revealed that 37.1% of the TNL genes are highly or specifically expressed in roots, especially the genes on chromosome 7 (76.5%). Digital gene expression and reverse transcription PCR analyses revealed the expression patterns of the NBS-LRR genes upon challenge by Fusarium oxysporum f.sp. conglutinans: nine genes were upregulated, and five were downregulated. The major resistance gene Foc1 probably works together with the other four genes in the same cluster to resist F. oxysporum infection.

Integrated analysis of transcriptome and proteome changes related to the Ogura cytoplasmic male sterility in cabbage.

Cabbage (Brassica oleracea L. var. capitata), an important vegetable crop in the Brassicaceae family, is economically important worldwide. In the process of hybrid seed production, Ogura cytoplasmic male sterility (OguCMS), controlled by the mitochondrial gene orf138, has been extensively used for cabbage hybrid production with complete and stable male sterility. To identify the critical genes and pathways involved in the sterility and to better understand the underlying molecular mechanisms, the anther of OguCMS line R2P2CMS and the fertile line R2P2 were used for RNA-seq and iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) proteome analysis. RNA-seq analysis generated 13,037,109 to 13,066,594 SE50-clean reads, from the sterile and fertile lines, which were assembled into 36,890 unigenes. Among them, 1,323 differentially expressed genes (DEGs) were identified, consisting of 307 up- and 1016 down-regulated genes. For ITRAQ analysis, a total of 7,147 unique proteins were identified, and 833 were differentially expressed including 538 up- and 295 down-regulated proteins. These were mainly annotated to the ribosome, spliceosome and mRNA surveillance pathways. Combined transcriptomic and proteomic analyses identified 22 and 70 genes with the same and opposite expression profiles, respectively. Using KEGG analysis of DEGs, gibberellin mediated signaling pathways regulating tapetum programmed cell death and four different pathways involved in sporopollenin synthesis were identified. Secretion and translocation of the sporopollenin precursors were identified, and the key genes participating in these pathways were all significantly down-regulated in R2P2CMS. Light and transmission electron (TE) microscopy revealed fat abnormal tapetum rather than vacuolization and degradation at the tetrad and microspore stages of the OguCMS line. This resulted in the failed deposition of sporopollenin on the pollen resulting in sterility. This study provides a comprehensive understanding of the mechanism underlying OguCMS in cabbage.

Transcriptome Analysis of Pepper (Capsicum annuum) Revealed a Role of 24-Epibrassinolide in Response to Chilling.

Brassinosteroids (BRs) have positive effects on many processes during plant growth, development, and various abiotic stress responses. However, little information is available regarding the global gene expression of BRs in response to chilling stress in pepper. In this study, we used RNA sequencing to determine the molecular roles of 24-epibrassinolide (EBR) during a chilling stress response. There were 39,829 transcripts, and, among them, 656 were differently-expressed genes (DEGs) following EBR treatment (Chill+EBR) compared with the control (Chill only), including 335 up-regulated and 321 down-regulated DEGs. We selected 20 genes out of the 656 DEGs for RT-qPCR analysis to confirm the RNA-Seq. Based on GO enrich and KEGG pathway analysis, we found that photosynthesis was significantly up-enriched in biological processes, accompanied by significant increases in the net photosynthetic rate (Pn), Fv/Fm, and chlorophyll content. Furthermore, the results indicate that EBR enhanced endogenous levels of salicylic acid (SA) and jasmonic acid (JA) while suppressing the ethylene (ETH) biosynthesis pathway, suggesting that BRs function via a synergistic cross-talk with SA, JA, and ETH signaling pathways in response to chilling stress. In addition, EBR induced cellulose synthase-like protein and UDP-glycosyltransferase, suggesting a contribution to the formation of cell wall and hormone metabolism. EBR also triggered the calcium signaling transduction in cytoplasm, and activated the expression of cellular redox homeostasis related genes, such as GSTX1, PER72, and CAT2. This work, therefor, identified the specific genes showed different expression patterns in EBR-treated pepper and associated with the processes of hormone metabolism, redox, signaling, transcription, and defense. Our study provides the first evidence of the potent roles of BRs, at the transcription level, to induce the tolerance to chilling stress in pepper as a function of the combination of the transcriptional activities, signaling transduction, and metabolic homeostasis.

Transcriptome Profiling of Resistance to Fusarium oxysporum f. sp. conglutinans in Cabbage (Brassica oleracea) Roots.

Fusarium wilt caused by Fusarium oxysporum f. sp. conglutinans (FOC) is a destructive disease of Brassica crops, which results in severe yield losses. There is little information available about the mechanism of disease resistance. To obtain an overview of the transcriptome profiles in roots of R4P1, a Brassica oleracea variety that is highly resistant to fusarium wilt, we compared the transcriptomes of samples inoculated with FOC and samples inoculated with distilled water. RNA-seq analysis generated more than 136 million 100-bp clean reads, which were assembled into 62,506 unigenes (mean size = 741 bp). Among them, 49,959 (79.92%) genes were identified based on sequence similarity searches, including SwissProt (29,050, 46.47%), Gene Ontology (GO) (33,767, 54.02%), Clusters of Orthologous Groups (KOG) (14,721, 23.55%) and Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) (12,974, 20.76%) searches; digital gene expression analysis revealed 885 differentially expressed genes (DEGs) between infected and control samples at 4, 12, 24 and 48 hours after inoculation. The DEGs were assigned to 31 KEGG pathways. Early defense systems, including the MAPK signaling pathway, calcium signaling and salicylic acid-mediated hypersensitive response (SA-mediated HR) were activated after pathogen infection. SA-dependent systemic acquired resistance (SAR), ethylene (ET)- and jasmonic (JA)-mediated pathways and the lignin biosynthesis pathway play important roles in plant resistance. We also analyzed the expression of defense-related genes, such as genes encoding pathogenesis-related (PR) proteins, UDP-glycosyltransferase (UDPG), pleiotropic drug resistance, ATP-binding cassette transporters (PDR-ABC transporters), myrosinase, transcription factors and kinases, which were differentially expressed. The results of this study may contribute to efforts to identify and clone candidate genes associated with disease resistance and to uncover the molecular mechanism underlying FOC resistance in cabbage.

Identification of tapetum-specific genes by comparing global gene expression of four different male sterile lines in Brassica oleracea.

The tapetum plays an important role in anther development by providing necessary enzymes and nutrients for pollen development. However, it is difficult to identify tapetum-specific genes on a large-scale because of the difficulty of separating tapetum cells from other anther tissues. Here, we reported the identification of tapetum-specific genes by comparing the gene expression patterns of four male sterile (MS) lines of Brassica oleracea. The abortive phenotypes of the four MS lines revealed different defects in tapetum and pollen development but normal anther wall development when observed by transmission electron microscopy. These tapetum displayed continuous defective characteristics throughout the anther developmental stages. The transcriptome from flower buds, covering all anther developmental stages, was analyzed and bioinformatics analyses exploring tapetum development-related genes were performed. We identified 1,005 genes differentially expressed in at least one of the MS lines and 104 were non-pollen expressed genes (NPGs). Most of the identified NPGs were tapetum-specific genes considering that anther walls were normally developed in all four MS lines. Among the 104 NPGs, 22 genes were previously reported as being involved in tapetum development. We further separated the expressed NPGs into different developmental stages based on the MS defects. The data obtained in this study are not only informative for research on tapetum development in B. oleracea, but are also useful for genetic pathway research in other related species.

Mapping and analysis of a novel candidate Fusarium wilt resistance gene FOC1 in Brassica oleracea.

BACKGROUND: Cabbage Fusarium wilt is a major disease worldwide that can cause severe yield loss in cabbage (Brassica olerecea). Although markers linked to the resistance gene FOC1 have been identified, no candidate gene for it has been determined so far. In this study, we report the fine mapping and analysis of a candidate gene for FOC1 using a double haploid (DH) population with 160 lines and a F2 population of 4000 individuals derived from the same parental lines. RESULTS: We confirmed that the resistance to Fusarium wilt was controlled by a single dominant gene based on the resistance segregation ratio of the two populations. Using InDel primers designed from whole-genome re-sequencing data for the two parental lines (the resistant inbred-line 99-77 and the highly susceptible line 99-91) and the DH population, we mapped the resistance gene to a 382-kb genomic region on chromosome C06. Using the F2 population, we narrowed the region to an 84-kb interval that harbored ten genes, including four probable resistance genes (R genes): Bol037156, Bol037157, Bol037158 and Bol037161 according to the gene annotations from BRAD, the genomic database for B. oleracea. After correcting the model of the these genes, we re-predicted two R genes in the target region: re-Bol037156 and re-Bol0371578. The latter was excluded after we compared the two genes' sequences between ten resistant materials and ten susceptible materials. For re-Bol037156, we found high identity among the sequences of the resistant lines, while among the susceptible lines, there were two types of InDels (a 1-bp insertion and a 10-bp deletion), each of which caused a frameshift and terminating mutation in the cDNA sequences. Further sequence analysis of the two InDel loci from 80 lines (40 resistant and 40 susceptible) also showed that all 40 R lines had no InDel mutation while 39 out of 40 S lines matched the two types of loci. Thus re-Bol037156 was identified as a likely candidate gene for FOC1 in cabbage. CONCLUSIONS: This work may lay the foundation for marker-assisted selection as well as for further function analysis of the FOC1 gene.

Upregulation of the AT-hook DNA binding gene BoMF2 in OguCMS anthers of Brassica oleracea suggests that it encodes a transcriptional regulatory factor for anther development.

Ogura cytoplasmic male sterility (OguCMS) is the most important CMS system used for F1 hybrid cabbage production worldwide. The anther abortion and defective pollen development exhibited in OguCMS are coordinately regulated by the mitochondrial male sterile gene orf138 and many nuclear transcriptional regulatory factors. AT-hook DNA binding proteins regulate cell-specific gene expression. In this study, we cloned the gene encoding the AT-hook DNA binding protein BoMF2 using the cDNA-AFLP TDF sequence, which was upregulated in OguCMS cabbage flower buds, as a querying probe. BoMF2 contains a 783-nt continuous complete open reading frame encoding a 260 amino-acid polypeptide. In vivo transient expression assays using GFP fusions showed that BoMF2 protein was located in the nucleus. BoMF2 was preferentially expressed in cabbage stamens, with a short expression window at anther development stage 7-8. However, in OguCMS flowers, BoMF2 expression continued into the mature pollen stage and was concomitant with the continued proliferation of tapetum cells exhibited in this mutant. Arabidopsis plants overexpressing BoMF2 showed significantly shorter siliques than the wild type, as well as decrease of pollen viability. These results suggest that BoMF2, a transcriptional regulatory factor, might regulate tapetum proliferation during anther development.

Global analysis of gene expression in flower buds of Ms-cd1 Brassica oleracea conferring male sterility by using an Arabidopsis microarray.

The dominant male sterility gene Ms-cd1 is identified in Brassica oleracea. Electron microscopical observations revealed that abortion of pollen development starts after tetrad formation. This important male sterility phenotype is characterized by lack of degradation of the primary pollen mother cell (PMC) wall and delayed degradation of callose surrounding the tetrads and thus arrest of microspore release. Gene expression of the male sterile and fertile buds was analyzed by heterologous hybridization of Brassica oleracea cRNA onto an Arabidopsis whole genome oligonucleotide microarray. A total of 277 suppressed genes including 40 kinase-, 32 cell wall modification and 29 transport related genes were found to be significantly down regulated >3-fold in the male sterile mutant. The vast majority of the differentially expressed transcripts are found to present late pollen stage specific genes. Kinase genes, cell wall modification genes and ion transport genes were greatly over-represented when compared to their percentage of all flower bud expressed genes and represent 36.5% of the genes suppressed by Ms-cd1. Our results also suggest that Ms-cd1 may blocks an anther developmental pathway with a small number of genes suppressed in tapetum cells which prevent the degradation of callose and PMC wall, which further leads to the suppression of a large number of genes involved in signaling pathways, cell wall modification and ion transport in pollen grains.

[Gene cloning and expression analysis of a male sterility related gene BoDHAR from broccoli].

A differentially expressed cDNA fragment obtained from a cDNA-AFLP analysis, which performed on floral buds of male sterile and fertile lines of cabbage, was used as a querying probe to blast the Genbank and Arabidopsis databases. Based on the assembled homologous cDNA sequences, a full-length cDNA of 633 bp for BoDHAR was cloned by RT-PCR. Furthermore, we have experimentally cloned and sequenced the 5' flanking sequence of gene BoDHAR by genomic walking method based on ligation-mediated PCR. The full length DNA sequence with 1486bp, containing two introns, was achieved. Homologous analysis shows that gene has 82.3% identity at nucleotide level, and 79.6% identity at amino acid level with Arabidopsis dehydroascorbate reductase (DHAR) gene AT1 G19570.1. Structurally, BoDHAR encodes a polypeptide of 210 amino acids, which contains a GST-c-DHAR domain highly conserved among other members of the DHAR superfamily and has multiple phosphorylation sites. Promoter predictions software indicated that the 5' upstream region contained putative transcription signals and conserved sequences, one CAAT-box, one G-box and four TGAC-like motifs. To advance our understanding of gene BoDHAR, tissue expression pattern were analyzed by semi-quantitative RT-PCR. The results indicate that expression level of gene BoDHAR is higher in fertile buds than that in sterile buds, and expressed intensively in the anther.