Ogura CMS Research Articles

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)

BackgroundCytoplasmic 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.ResultsIn 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.ConclusionsThe 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.

Characterization of Ogura CMS fertility-restored interspecific hybrids and backcross progenies from crosses between broccoli and rapeseed

Interspecific F1 hybrids were obtained from crosses between two Ogura cytoplasmic male sterility (Ogura CMS) broccoli lines (B14 and 137) and a rapeseed restorer line to introgress the Ogura CMS’s fertility-restoration gene Rfo from rapeseed into broccoli. Ten Rfo-positive interspecific, triploid plant progenies, phenotypically between rapeseed and broccoli, were obtained but still contained multiple rapeseed genes, with reduced fertility and seed setting potential under natural pollination. For fertility and seed setting improvement, successive backcrosses with broccoli were carried out to yield BC2 progenies. Screening revealed six plants with Rfo among 25 BC2 progenies, which were investigated for agronomic traits, ploidy determination, pollen viability and SSR (simple sequence repeat) markers to evaluate for ideal individuals closer to broccoli. Pollen viability tests were carried out at different stages for various hybrid parents. The results were as follows: 137-F1 (68.54%) > B14-F1 (65.32%) > 137-BC1 (48.15%) > B14-BC1 (32.41%). Although there were significant differences among the BC1 and BC2 individuals, the genetic background of BC2 was closer to the parent broccoli compared with that of BC1 plants. The existence of the Rfo gene in broccoli not only facilitates broccoli germplasm innovation, combination of interspecific hybridization and backcrossing, but may also shed light on trait segregation in different generations.

Genetic characterization of a new radish introgression line carrying the restorer gene for Ogura CMS in Brassica napus.

Creating a homologous restorer line for Ogura cytoplasmic male sterility (Ogu-CMS) in Brassica napus is meaningful for the wider application of Ogu-CMS system in rapeseed production. Previously, an independent development of a new Ogu-CMS restorer line (CLR650) was reported locally from crossing between Raphanobrassica (AACCRR, 2n = 56) and B. napus and a new version of Ogu CMS lines CLR6430 derived from CLR650 was characterized in this study. The results showed that the fertility restoration gene in CLR6430 presented a distorted segregation in different segregating populations. However, the majority of somatic cells from roots had a regular chromosome number (2n = 38) and no radish signal covered a whole chromosome was detected using GISH. Thirty-two specific markers derived from the introgressed radish fragments were developed based on the re-sequencing results. Unique radish insertions and differences between CLR6430 and R2000 were also identified through both radish-derived markers and PCR product sequences. Further investigations on the genetic behaviors, interactions between the fertility restoration and other traits and specific molecular markers to the introgression in CLR6430 were also conducted in this study. These results should provide the evidence of nucleotide differences between CLR6430 and R2000, and the specific markers will be helpful for breeding new Ogura restore lines in future.

BREEDING OF WINTER RAPESEED INBRED LINES FOR THE DEVELOPMENT OF PARENT FORMS FOR HYBRIDS

Introduction. The V.S. Pustovoit All-Russian Research Institute of Oil Crops (VNIIMK) performs the breeding of interline rapeseed hybrids using the Ogura CMS. The program of interline winter rapeseed hybrid development was aimed at studying the efficiency of self-pollinated line breeding from population cultivars, assessing the impact of inbreeding depression on economically important traits, analyzing the changes in the lines’ traits after their transition to the sterile basis, and obtaining fertility restorer lines with an acceptable level of glucosinolate content in seeds.Materials and methods. Intraspecific hybrids, cultivars developed at the Pustovoit Institute and those of foreign breeding were employed as parent material for the development of self-pollinated lines. Self-pollination of rapeseed plants was performed using spunbond isolators. Over 1000 plants were self-pollinated every year. Generations of self-pollinated lines S1 –S4 were planted on 2.25 m2 plots, and during the growth season their visual screening was undertaken to assess their morphobiological traits. Starting with S5, the lines were evaluated for their yield on 7.5 m2 plots.Results. The inbreeding depression was found to not exceed 3–15% of the seed yield. The transition of the lines to sterile cytoplasm did not have any negative consequences. A series of experimental hybrids were developed, exceeding the reference cultivar ‘Loris’ in seed yield by 23–48%. New hybrid combinations also exceeded the reference in seed yield by 21–54%.

Size and location of radish chromosome regions carrying the fertility restorer Rfk1 gene in spring turnip rape

In spring turnip rape (Brassica rapa L. spp. oleifera), the most promising F1 hybrid system would be the Ogu-INRA CMS/Rf system. A Kosena fertility restorer gene Rfk1, homolog of the Ogura restorer gene Rfo, was successfully transferred from oilseed rape into turnip rape and that restored the fertility in female lines carrying Ogura cms. The trait was, however, unstable in subsequent generations. The physical localization of the radish chromosomal region carrying the Rfk1 gene was investigated using genomic in situ hybridization (GISH) and bacterial artificial chromosome–fluorescence in situ hybridization (BAC–FISH) methods. The metaphase chromosomes were hybridized using radish DNA as the genomic probe and BAC64 probe, which is linked with Rfo gene. Both probes showed a signal in the chromosome spreads of the restorer line 4021–2 Rfk of turnip rape but not in the negative control line 4021B. The GISH analyses clearly showed that the turnip rape restorer plants were either monosomic (2n = 2x = 20 + 1R) or disomic (2n = 2x = 20 + 2R) addition lines with one or two copies of a single alien chromosome region originating from radish. In the BAC–FISH analysis, double dot signals were detected in subterminal parts of the radish chromosome arms showing that the fertility restorer gene Rfk1 was located in this additional radish chromosome. Detected disomic addition lines were found to be unstable for turnip rape hybrid production. Using the BAC–FISH analysis, weak signals were sometimes visible in two chromosomes of turnip rape and a homologous region of Rfk1 in chromosome 9 of the B. rapa A genome was verified with BLAST analysis. In the future, this homologous area in A genome could be substituted with radish chromosome area carrying the Rfk1 gene.

Transfer of the Kosena Rfk1 gene, required in hybrid seed production, from oilseed rape to turnip rape

The Ogura cytoplasmic male sterility and fertility restoring (cms/Rf) system is one of the most promising hybrid systems in Brassica species. The Ogura cms has been introduced earlier into spring turnip rape (Brassica rapa L.) and the sterility has been stable. To enable the production of F1 hybrids in turnip rape, the Kosena fertility restoring gene (Rfk1), a homologue of the Ogura fertility restoring gene (Rfo), was transferred from spring oilseed rape (Brassica napus L.) into spring turnip rape by the traditional backcross method. The male fertility, measured here as a pollen production, of the F1 hybrids between turnip rape and oilseed rape was good (83%). The transmission rate of the Rfk1 gene during backcrossing was not essentially different between the pollen (35%) and egg (33%) cells. For the selection of homozygous (Rfk1/Rfk1) turnip rape plants, both testcrosses to male sterile (Ogura cms) turnip rape line and TaqMan based real-time qPCR method were used simultaneously. The TaqMan qPCR detection system was good when used for selecting homozygous plants out of heterozygous ones before flowering. This enabled interpollination instead of inbreeding, which is a benefit with cross-pollinating crop species like turnip rape. According to testcross results of the offspring between male sterile line and homozygous (Rfk1/Rfk1) turnip rape plants, 100% fertility was reached. It was a good indication, that turnip rape with Ogura cms can be restored with the Kosena Rfk1 gene. However, during the subsequent selection of homozygous (Rfk1/Rfk1) plants, it was observed that the Rfk1 gene was still unstable in the genome of turnip rape, and thus the Rfk1 gene needs to be stabilized before its full exploitation in commercial hybrid production is possible.

Differential gene expression analysis of a new Ogura CMS line and its maintainer in non-heading Chinese cabbage by cDNA-AFLP

CMS is induced by the coordinated expression of certain mitochondrial and nuclear genes in flower development. Mitochondrial genes regulate manifestation of CMS, whereas nuclear genes regulate fertility phenotype and thus affect negatively. In this article, the buds of newly bred Ogura CMS of non-heading Chinese cabbage and its maintainer line as plant materials, genes differentially expressed transcripts were analyzed by cDNA-AFLP. Seventeen differently expressed genes were found in new Ogura CMS and nine genes in maintainer line. These genes were involved in energy metabolism, signal transduction, flower development, stress-related metabolism, transcription, etc. Expression patterns of three genes encoding BrCAM6, BrANK, BrTUB3 were verified by qRT-PCR in different organs and various stage flower buds of CMS and its maintainer line. The results revealed that two genes related to signal transduction, BrCAM6 and BrANK, were highly expressed in stamens and microspores of CMS than in maintainer line. As believed, these two genes involved in signal transduction of male sterile in CMS line. In comparison, BrTUB3 gene was accumulated in stamens and was expressed in significantly lower level in CMS line than in maintainer line. It expressed significantly lower in CMS than maintainer line after tetrad stage. This expression profile suggests that BrTUB3 played an important role in the development of the pollen, and may be closely related to male sterility.

Nectar secretion in winter oilseed rape, Brassica napus— quantitative and qualitative variability among 71 genotypes

AbstractThe genotypic variability in nectar secretion among 71 genotypes of winter oilseed rapes was tested for floral nectar volume, sugar composition and concentration. Differences were found between seed quality genotypes: double low [99] oilseed rape varieties tended to secrete much more nectar than single low [0 +] and double high [++] varieties. Male‐sterile lines (Ogura CMS) secreted one‐half to one‐third the amount of their isogenic male‐fertile counterparts. Restored lines behaved as conventional rape varieties. On average, amounts of nectar were 2 μl/flower and ranged from 0.7 to 6 μl/flower. Among the genotypes under test, the male‐sterile lines were not the least productive. The analysis of (Samourai) winter oilseed rape showed that total sugar concentrations in nectar were highest at the beginning of the flowering period (30g/100 ml) and lowest towards the end (10g/100 ml). At a given date, total sugar contents in nectar of [0 +] varieties were significantly higher than those of [++] varieties and were due to higher fructose contents. Differences between genotypes within the same type also occurred, depending on the day of sampling. Oilseed rape nectar was confirmed to be predominantly composed of hexose (glucose and fructose). From the results of these investigations, it is concluded that the opportunity exists to select oilseed rape genotypes, including male‐sterile lines, that secrete sufficient amounts of nectar to attract honey‐bees to ensure pollination of male‐sterile lines for hybrid seed production.

Synthesis of Brassica oleracea/Brassica napus somatic hybrid plants with novel organeile DNA compositions

Broccoli (Brassica oleracea L. italica) hypocotyl protoplasts were fused with mesophyll protoplasts of two B. napus lines, one carrying the Ogura (ogu) cms cytoplasm, and the other carrying a hybrid cytoplasm consisting of ogu mitochondria combined with triazine-tolerance-conferring chloroplasts from ctr cytoplasm. Two male-sterile somatic hybrids were recovered from the fusion of broccoli protoplasts with those of ogu/ctr cybrid B. napus. The ogu mtDNAs and ctr cpDNAs were not altered in these hybrids. Four male-sterile plants were recovered from the somatic hybridization of broccoli with ogu cms B. napus. Three of these possessed mitochondrial genomes that appeared to have resulted from recombination between the ogu and normal B. oleracea (ole) mtDNAs, while the fourth possessed an unrearranged ogu mtDNA. All four of these plants had B. oleracea cpDNA, and none displayed the seedling chlorosis associated with ogu chloroplasts. Most of the plants recovered from these fusions had the chromosome number expected of B. oleracea + B. napus hybrids (2n = 56). The novel cytoplasms may prove to be useful for the molecular analysis of Brassica cms and for the production of hybrid Brassica.