Ogura Cytoplasmic Male Sterility Line Research Articles

Comparative Transcriptome Analysis Reveals a Potential Regulatory Network for Ogura Cytoplasmic Male Sterility in Cabbage (Brassica oleracea L.)

Ogura cytoplasmic male sterility (CMS) lines are widely used breeding materials in cruciferous crops and play important roles in heterosis utilization; however, the sterility mechanism remains unclear. To investigate the microspore development process and gene expression changes after the introduction of orf138 and Rfo, cytological observation and transcriptome analysis were performed using a maintainer line, an Ogura CMS line, and a restorer line. Semithin sections of microspores at different developmental stages showed that the degradation of tapetal cells began at the tetrad stage in the Ogura CMS line, while it occurred at the bicellular microspore stage to the tricellular microspore stage in the maintainer and restorer lines. Therefore, early degradation of tapetal cells may be the cause of pollen abortion. Transcriptome analysis results showed that a total of 1287 DEGs had consistent expression trends in the maintainer line and restorer line, but were significantly up- or down-regulated in the Ogura CMS line, indicating that they may be closely related to pollen abortion. Functional annotation showed that the 1287 core DEGs included a large number of genes related to pollen development, oxidative phosphorylation, carbohydrate, lipid, and protein metabolism. In addition, further verification elucidated that down-regulated expression of genes related to energy metabolism led to decreased ATP content and excessive ROS accumulation in the anthers of Ogura CMS. Based on these results, we propose a transcriptome-mediated induction and regulatory network for cabbage Ogura CMS. Our research provides new insights into the mechanism of pollen abortion and fertility restoration in Ogura CMS.

Morphological characterization, combining ability and heterosis for important horticultural traits in snowball cauliflower (B. oleracea var. botrytis L.)

AbstractCauliflower is an important vegetable crop grown worldwide. Development and characterization of suitable cytoplasmic male sterile (CMS) lines and male fertile inbreds is instrumental in developing heterotic hybrids. A study was undertaken to estimate heterosis and combining ability in snowball cauliflower lines through line × tester analysis involving five Ogura CMS lines and seven male fertile testers. The assessment of F1s along with their parental lines for different horticultural traits revealed a wide range of heterosis. Ogu-13-01 was the best general combiner for leaf length, leaf width and plant height, Ogu-13-85 for number of leaves/plant, Ogu-101 for days to 50% curd initiation, days to 50% curd maturity and net curd weight and Ogu-119 for harvest index. The lines with better general combining ability (GCA) were involved in majority of the heterotic hybrids. The tester Kt-18 was the best general combiner for leaf width, DB-1305 for number of leaves/plant and DB-187 for leaf length, plant height, gross plant weight and days to 50% curd initiation. However, GCA alone was not sufficient to determine and identify the potential parental lines. Hybrids Ogu-101 × DB-1305 and Ogu-119 × Suprimax Late were the best specific combinations for marketable curd weight, marketable curd yield, net curd weight and net curd yield. The hybrid Ogu-119 × Suprimax Late was the best heterotic combination for marketable curd weight, marketable curd yield, net curd weight and net curd yield, followed by Ogu-101 × DB-1305, Ogu-119 × Sel-26 and Ogu-101 × DB-187. Our findings will be instrumental to identify suitable parental lines and developing heterotic F1 hybrids in cauliflower.

Development of Ogura CMS restorers in Brassica oleracea subspecies via direct RfoB gene transformation.

The Ogura CMS RfoB restorer developing via RfoB gene transformation was utilized to produce specific morphological Ogura CMS restorers and clubroot resistance lines in Brassica oleracea subspecies. Brassica oleracea vegetables including cabbage, cauliflower, kohlrabi, Brussels sprouts and Chinese kale are morphologically very different despite being members of the same species. The Ogura cytoplasmic male sterility (CMS) system is the most stable strategy for the hybrid breeding of these species. However, this limits the utilization of some excellent genes due to the lack of fertile restorer genes in the system. Herein, to efficaciously use Ogura CMS, the Ogura CMS RfoB restorer was produced by transforming the modified RfoB restorer gene into the Ogura CMS line 'CMS2016' of B. oleracea var. capitata. This gene was shown to recover fertility of natural Ogura CMS lines in B. oleracea subspecies and create transient Ogura CMS RfoB restorers such as the clubroot resistance Ogura CMS RfoB restorer. Interestingly, clubroot resistant individuals without transgenic elements were screened in the progenies of hybrids between B. oleracea inbred lines and the clubroot resistance Ogura CMS RfoB restorer. In addition, 18 different morphological Ogura CMS restorers were developed to specifically recover fertile of Ogura CMS cultivars in B. oleracea subspecies.

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.

Development of a novel allele-specific Rfo marker and creation of Ogura CMS fertility-restored interspecific hybrids in Brassica oleracea.

A novel allele-specific Rfo marker was developed and proved to be effective for MAS of Rfo gene in B. oleracea background and six Ogu-CMS fertility-restored interspecific hybrids were created for the first time. Ogura cytoplasmic male sterility (Ogu-CMS) has been extensively used for Brassica oleracea hybrid production. However, because of maternal inheritance, all the hybrids produced by CMS lines are male sterile and cannot be self-pollinated, which prohibits germplasm maintenance and innovation. This problem can be overcome by using the Ogu-CMS restorer line, but restorer material is absent in B. oleracea crops. Here, Rfo, a fertility-restored gene of Ogu-CMS, was transferred from rapeseed restorer lines into a Chinese kale Ogu-CMS line using interspecific hybridization combined with embryo rescue. Nine interspecific, triploid plant progenies were identified at morphological and ploidy level, with phenotypes intermediate between those of rapeseed and Chinese kale. Because the Rfo marker (Hu et al., Mol Breeding 22:663-674, 2008) cannot distinguish the Rfo and its homologies under a B. oleracea background, a novel allele-specific Rfo marker was developed based on the BLAST analysis of highly homologous Rfo sequences in B. oleracea. Screening using the novel Rfo marker found that six interspecific hybrids carrying Rfo were also fertile, although fertility varied during different flowering periods. Furthermore, BC1 offsprings with the Rfo gene were selected with the allele-specific Rfo marker and showed restored fertility. These results indicated that the novel allele-specific marker could be used for the MAS of Rfo gene in B. oleracea, and this study lays the foundation for the development of Ogu-CMS restorer material in cabbage and its related other subspecies.

Differential gene expression profiling of Ogura CMS line and its maintainer in radish (Raphanus sativus L.)

Cytoplasmic male sterility (CMS) is a maternally inherited trait, which becomes an important way in heterosis breeding to simplify the seed production procedures and reduce the production cost. Cytological observation of pollen development stages showed a clear difference between the newly developed CMS line and its maintainer; the profiling comparisons between floral buds of CMS line and its maintainer were conducted using differential display reverse transcription PCR (DDRT-PCR) technology. Thirty genes were up-regulated and sixty genes were down-regulated in newly developed CMS line when compared with its maintainer. These genes were involved in cell wall biosynthesis and regulation, transporter and ion channel, flower development and protein metabolism, etc. Expression patterns of six genes encoding RsCAM6, RsGPI, RsPMEI, RsRac, RsCHS, and RsSTP9 were verified by RT-PCR and qRT-PCR in different development stage of floral buds and different organs of CMS line and its maintainer. The expression level of RsRac was higher in stamens and microspores of CMS line than in maintainer. In comparison, the expression levels of the other five genes in CMS line were lower compared with maintainer. This expression profile suggests that these genes played important roles in the development of the pollen and may be closely related to male sterility. The results observed in this study will contribute to understanding the mechanism of pollen abortion during CMS in radish.

Isolation and characterization of a male sterility gene homolog BcMS2 from Chinese cabbagge-pak-choi that expressing in an anther-specific manner

A male sterility gene homolog, designated BcMS2, was isolated from flower buds using gene-specific primer pairs and was submitted to GenBank under accession number EF093533. Comparison of BcMS2 gene with MS2 from Arabidopsis thaliana and MS2Bnap from Brassica napus revealed some differences in gene structure and evolution. The full genomic DNA sequence of BcMS2 was 2,576 bp in length containing 8 exons and 7 introns, more than those of MS2Bnap but less than MS2. RT-PCR showed that BcMS2 gene expressed only in stage III flower buds of male fertile Chinese cabbage-pak-choi 'ZUBajh97-01B' and there were no detection in all organs of Polima cytoplasmic male sterility (CMS) line 'Bpol97-05A' and Ogura CMS line 'Bogu97-06A'. Furthermore, RT-PCR revealed that BcMS2 expressed only in anthers of male fertile material and there were no expression in sepals, petals, filaments and pistils. These results suggested that BcMS2 was an anther-specific gene and might be essential for the fertility of Chinese cabbage-pak-choi.

Molecular cloning and characterization of BcMYBogu, a novel member of the MYB family involved in OguCMS in Brassica campestris ssp. chinensis

In the attempt to elucidate the molecular mechanism of CMS. Ogura cytoplasmic male sterile (OguCMS) lines were obtained in Chinese cabbage after interspecific hybridization between Brassica. napus L. OguCMS and B. campestris ssp. chinensis followed by recurrent backcross with B. campestris ssp. chinensis as the pollen donor. The CMS lines were significantly characterized by the whitish anther and indehiscence of anther. The tapetal hypertrophy with excess vacuola-tion was the first observed defective soon after the tetrad stage, subsequently the microspores defected in pollen wall forma-tion, and later the cytoplasm detached from the exine wall and underwent degeneration. With aid of cDNA-AFLP and RACE approaches, we cloned the BcMYBogu(GenBank accession No: EF127861) in Chinese cabbage, which is premature expressed in early and middle stage floral buds of OguCMS lines, and predicted to encode a novel protein with a DNA binding domain: SH[AL]QKY[RF] motif at the N-terminus. Phylogenetic comparison revealed that the BcMYBogu was clustered with AtMYB32, AtMYB26 and AtMYB4, which were indicated to be involved in male sterility in Arabidopsis thaliana. The BcMYBogu transcript was detected in rosette leaves, floral buds and stems by RT-PCR analysis. Compared with the maintainer, the expression level of BcMYBogu was increased in these organs, especially in floral buds of OguCMS lines. Our investigation suggests that BcMYBogu is a new member of the MYB family involved in male sterility in Chinese cabbage.

Endogenous Hormones in Seeds, Germination Behaviour and Early Seedling Characteristics in a Normal andoguraCytoplasmic Male Sterile Line of Rapeseed (Brassica napusL.)

Endogenous hormones, namely cytokinins (CKs), indole-3-acetic acid (IAA) and abscisic acid (ABA) were quantified by specific enzyme-linked immunosorbent assay (ELISA) in the mature seed of normal (cv. Westar) and ogura (ogu) cytoplasmic male sterile (CMS) lines of rapeseed (Brassica napus L.). Dihydrozeatin (DZ) and dihydrozeatin riboside (DZR) were the major CK base and riboside, respectively, in seeds of both the normal and ogu CMS lines. The normal seed had more than 4-fold DZ levels in comparison to that of ogu CMS. On the other hand, the ogu CMS seed had higher levels of CK o-glucosides and CK. nucleotides than normal seed. Seeds of the normal line contained 5-fold more IAA but had one-quarter the level of ABA in comparison to those of the ogu CMS line. The normal line also had greater seed diameter and weight than the ogu CMS line and the normal seed germinated earlier than the male sterile seed. DZ (10−6 M) promoted the germination of ogu CMS seeds, but it was not comparable to that of the normal line. ABA (10−6 M) inhibited seed germination of ogu CMS but had little effect on the normal line. The normal seedlings had shorter primary roots, more lateral roots, longer hypocotyls, greater cotyledon fresh weight and higher chlorophyll levels in comparison to ogu CMS seedlings. Exogenously supplied DZ, IAA and ABA affected the various parameters of both the normal and ogu CMS seedlings, but in most cases did not fully restore the differences in the two lines. The results presented show that in the ogura cytoplasmic male sterile line of B. napus (1) a number of seed and seedling characteristics are affected, and (2) the altered seed morphology is accompanied by changes in the levels of various hormones.