Pol Cytoplasmic Male Sterility Research Articles

Genetic dissection of thousand-seed weight and fine mapping of cqSW.A03-2 via linkage and association analysis in rapeseed (Brassica napus L.).

KEY MESSAGE: cqSW.A03-2, one of the six identified quantitative trait loci associated with thousand-seed weight in rapeseed, is mapped to a 61.6-kb region on chromosome A03 and corresponds to the candidate gene BnaA03G37960D. Seed weight is an important factor that determines the seed yield of oilseed rape (Brassica napus L.). To elucidate the genetic mechanism of thousand-seed weight (TSW), quantitative trait locus (QTL) mapping was conducted using a double haploid population derived from the cross between an elite line ZY50 and a pol cytoplasmic male sterility restorer line 7-5. The genetic basis of TSW was dissected into six major QTLs. One major QTL denoted as cqSW.A03-2, which explained 8.46-13.70% of the phenotypic variation, was detected across multiple environments. To uncover the genetic basis of cqSW.A03-2, a set of near-isogenic lines were developed. Based on the test of self-pollinated progenies, cqSW.A03-2 was identified as a single Mendelian factor and the ZY50 allele at cqSW.A03-2 showed a positive effect on TSW. Fine mapping delimited the cqSW.A03-2 locus into a 61.6-kb region, and 18 genes within this region were predicted. Candidate gene association analysis and expression analysis indicated that a histidine kinase gene (BnaA03G37960D) is likely to be the candidate gene for the cqSW.A03-2 locus. Our results may contribute to a better understanding of the molecular mechanism of seed weight regulation and promote the breeding program for yield improvement in rapeseed.

Morphological and genetic characterization of a new cytoplasmic male sterility system (oxa CMS) in stem mustard (Brassica juncea).

KEY MESSAGE: oxa CMS is a new cytoplasmic male sterility type in Brassica juncea. oxa CMS is a cytoplasmic male sterility (CMS) line that has been widely used in the production and cultivation of stem mustard in the southwestern China. In this study, different CMS-type specific mitochondrial markers were used to confirm that oxa CMS is distinct from the pol CMS, ogu CMS, nap CMS, hau CMS, tour CMS, Moricandia arvensis CMS, orf220-type CMS, etc., that have been previously reported in Brassica crops. Pollen grains of the oxa CMS line are sterile with a self-fertility rate of almost 0% and the sterility strain rate and sterility degree of oxa CMS is 100% due to a specific flower structure and flowering habit. Scanning electron microscopy revealed that most pollen grains in mature anthers of the oxa CMS line are empty, flat and deflated. Semi-thin section further showed that the abortive stage of anther development in oxa CMS is initiated at the late uninucleate stage. Abnormally vacuolated microspores caused male sterility in the oxa CMS line. This cytological study combined with marker-assisted selection showed that oxa CMS is a novel CMS type in stem mustard (Brassica juncea). Interestingly, the abortive stage of oxa CMS is later than those in other CMS types reported in Brassica crops, and there is nonegative effecton the oxa CMS line growth period. This study demonstrated that this novel oxa CMS has a unique flower structure with sterile pollen grains at the late uninucleate stage. Our results may help to uncover the mechanism of oxa CMS in Brassica juncea.

Identification of different cytoplasms based on newly developed mitotype-specific markers for marker-assisted selection breeding in Brassica napus L.

Different mitotype-specific markers were developed to distinguish different cytoplasms in Brassica napus L. Mitotype-specific markers have been developed to distinguish different mitotypes in plant. And use of molecular markers to identify different mitotypes in Brassica napus would enhance breeding efficiency. Here, we comparatively analyzed six sequenced mitochondrial genomes in Brassica napus and identified collinear block sequences and mitotype-specific sequences (MSSs) of these mitochondrial genomes. The collinear block sequences between mitochondrial genomes of nap, cam, and pol cytoplasmic male sterility (CMS) lines were higher than those of other lines. After comparative analysis of the six sequenced mitochondrial genomes (cam, nap, ole, pol CMS, ogu CMS, and hau CMS), 90 MSSs with sizes ranging from 101 to 9981bp and a total length of 103,756bp (accounting for 6.77% of the mitochondrial genome sequences) were identified. Additionally, 12 mitotype-specific markers were developed based on the mitochondrial genome-specific sequences in order to distinguish among these different mitotypes. Cytoplasms of 570 different inbred lines collected across scientific research institutes in China were identified using the MSS markers developed in our study. In addition to confirming the accuracy of the cytoplasmic identification, we also identified mitotypes that have not been reported in Brassica napus. Our study may provide guidance for the classification of different mitotypes in B. napus breeding.

Detection of the Diversity of Cytoplasmic Male Sterility Sources in Broccoli (Brassica Oleracea var. Italica) Using Mitochondrial Markers.

Broccoli (Brassica oleracea var. italica) is an important commercial vegetable crop. As part of an efficient pollination system, cytoplasmic male sterility (CMS) has been widely used for broccoli hybrid production. Identifying the original sources of CMS in broccoli accessions has become an important part of broccoli breeding. In this study, the diversity of the CMS sources of 39 broccoli accessions, including 19 CMS lines and 20 hybrids, were analyzed using mitochondrial markers. All CMS accessions contained the ogu orf138-related DNA fragment and the key genes of nap CMS, pol CMS, and tour CMS were not detected. The 39 CMS accessions were divided into five groups using six orf138-related and two simple sequence repeat markers. We observed that ogu CMS R3 constituted 79.49% of the CMS sources. CMS6 and CMS26 were differentiated from the other accessions using a specific primer. CMS32 was distinguished from the other accessions based on a 78-nucleotide deletion at the same locus as the orf138-related sequence. When the coefficient was about 0.90, five CMS accessions (13CMS6, 13CMS23, 13CMS24, 13CMS37, and 13CMS39) exhibiting abnormal floral organs with poor seed setting were grouped together. The polymerase chain reaction amplification profiles for these five accessions differed from those of the other accessions. We identified eight useful molecular markers that can be used to detect CMS types during broccoli breeding. Our data also provide important information relevant to future studies on the possible origins and molecular mechanisms of CMS in broccoli.

Basic helix-loop-helix transcription factor BcbHLHpol functions as a positive regulator of pollen development in non-heading Chinese cabbage.

Cytoplasmic male sterility (CMS) is a common trait in higher plants, and several transcription factors regulate pollen development. Previously, we obtained a basic helix-loop-helix transcription factor, BcbHLHpol, via suppression subtractive hybridization in non-heading Chinese cabbage. However, the regulatory function of BcbHLHpol during anther and pollen development remains unclear. In this study, BcbHLHpol was cloned, and its tissue-specific expression profile was analyzed. The results of real-time polymerase chain reaction showed that BcbHLHpol was highly expressed in maintainer buds and that the transcripts of BcbHLHpol significantly decreased in the buds of pol CMS. A virus-induced gene silencing vector that targets BcbHLHpol was constructed and transformed into Brassica campestris plants to further explore the function of BcbHLHpol. Male sterility and short stature were observed in BcbHLHpol-silenced plants. The degradation of tapetal cells was inhibited in BcbHLHpol-silenced plants, and nutrients were insufficiently supplied to the microspore. These phenomena resulted in pollen abortion. This result indicates that BcbHLHpol functions as a positive regulator in pollen development. Yeast two-hybrid and bimolecular fluorescence complementation assays revealed that BcbHLHpol interacted with BcSKP1 in the nucleus. This finding suggests that BcbHLHpol and BcSKP1 are positive coordinating regulators of pollen development. Quantitative real-time PCR indicated that BcbHLHpol and BcSKP1 can be induced at low temperatures. Thus, we propose that BcbHLHpol is necessary for meiosis. This study provides insights into the regulatory functions of the BcbHLHpol network during anther development.

Abortive Process of a Novel Rapeseed Cytoplasmic Male Sterility Line Derived from Somatic Hybrids Between Brassica napus and Sinapis alba

Somatic hybridization is performed to obtain significant cytoplasmic male sterility (CMS) lines, whose CMS genes are derived either from the transfer of sterile genes from the mitochondrial genome of donor parent to the counterpart of receptor or production of new sterile genes caused by mitochondrial genome recombination of the biparent during protoplast fusion. In this study, a novel male sterile line, SaNa-1A, was obtained from the somatic hybridization between Brassica napus and Sinapis alba. The normal anther development of the maintainer line, SaNa-1B, and the abortive process of SaNa-1A were described through phenotypic observations and microtome sections. The floral organ of the sterile line SaNa-1A was sterile with a shortened filament and deflated anther. No detectable pollen grains were found on the surface of the sterile anthers. Semi-thin sections indicated that SaNa-1A aborted in the pollen mother cell (PMC) stage when vacuolization of the tapetum and PMCs began. The tapetum radically elongated and became highly vacuolated, occupying the entire locule together with the vacuolated microspores. Therefore, SaNa-1A is different from other CMS lines, such as ogu CMS, pol CMS and nap CMS as shown by the abortive process of the anther.

Isolation of an ascorbate peroxidase in Brassica napus and analysis of its specific interaction with ATP6

In Brassica napus , ATP6 is related to Pol cytoplasmic male sterility. To understand the mechanism of Pol CMS, proteins which interact with ATP6 were screened in a yeast two-hybrid system. A partial sequence of a putative and ascorbate peroxidase ( Bn-APX ) was isolated from Brassica napus . By use of rapid amplification of cDNA ends method, the full length of Bn-APX coding sequence was cloned. The deduced amino acid sequence contained 438 amino acid residues with a conserved ascorbate peroxidase domain and shared 77% identity with that of APX from Arabidopsis thaliana . Further analyses revealed that the region of Bn-APX interacting with ATP6 was at its c-terminal. It was also observed that the expressions of ATP6 and Bn-APX were strongly similar in the floral tissue of CMS line and the restoring line. Furthermore, in restoring line, the expression of Bn-APX is higher in the flower than that in other tissues.

Towards Positional Cloning in Brassica napus: Generation and Analysis of Doubled Haploid B. rapa Possessing the B. napus pol CMS and Rfp Nuclear Restorer Gene

The Polima (pol) system of cytoplasmic male sterility (CMS) and its fertility restorer gene Rfp are used in hybrid rapeseed production in Brassica napus. To facilitate map-based cloning of the Rfp gene, we have successfully transferred the pol cytoplasm and Rfp from the amphidiploid B. napus to the diploid species B. rapa and generated a doubled haploid pol cytoplasm B. rapa population that segregates for the Rfp gene. This was achieved through interspecific crosses, in vitro rescue of hybrid embryos, backcrosses, and microspore culture. Male fertility conditioned by Rfp was shown to co-segregate in this population with Rfp-specific mitochondrial transcript modifications and with DNA markers previously shown to be linked to Rfp in B. napus. The selfed-progeny of one doubled haploid plant were confirmed to be characteristic B. rapa diploids by cytogenetic analysis. Clones recovered from a genomic library derived from this plant line using the RFLP probe cRF1 fell into several distinct physical contigs, one of which contained Rfp-linked polymorphic restriction fragments detected by this probe. This indicates that chromosomal DNA segments anchored in the Rfp region can be recovered from this library and that the library may therefore prove to be a useful resource for the eventual isolation of the Rfp gene.

Post-transcriptional and developmental regulation of a CMS-associated mitochondrial gene region by a nuclear restorer gene.

Transcripts of the mitochondrial gene region orf224/atp6, which is associated with the Polima or pol cytoplasmic male sterility (CMS) of Brassica napus, differ among fertile, sterile and nuclear-restored plants. We show here that the effects of the restorer gene Rfp on orf224/atp6 transcripts varies among different floral organs. Relative to monocistronic atp6 transcripts, levels of the dicistronic transcripts spanning orf224 and atp6 are dramatically reduced in petals, stamens and carpels, but not sepals, of restored flowers. In pol CMS plants, the relative levels of different orf224/atp6 transcripts are similar among the floral organs. Analysis of guanylyltransferase-labeled mtRNA indicates that only the dicistronic 2.2 and 1.9 kb orf224/atp6 transcripts carry an initiator 5' terminus; hence the 1.4 and 1.3 kb transcripts of restored plants, as well as the 1.1 kb atp6 transcript common to all genotypes, are generated by RNA processing and not de novo initiation. Although steady-state levels of dicistronic transcripts in flower buds are lower in restored than in sterile plants, run-on transcription experiments show that these transcripts are synthesized at the same rate in both types of flowers. These findings imply that the restorer gene acts by conditioning the removal of sequences from the 5' end of dicistronic transcripts in a developmentally regulated manner. Run-on transcription experiments indicate that the single 1.1 kb atp6 transcript of nap cytoplasm is also generated by removal of sequences from the 5' end of a precursor. We suggest that specific endonucleolytic cleavage of a precursor RNA, followed by non-specific 3' to 5' exonuclease action, may represent a common mechanism for tailoring transcripts in plant mitochondria.

Restorer genes for different forms of Brassica cytoplasmic male sterility map to a single nuclear locus that modifies transcripts of several mitochondrial genes.

The oilseed rape plant, Brassica napus, possesses two endogenous male sterile cytoplasms, nap and pol. Previous studies have shown that nuclear restoration of pol cytoplasmic male sterility (CMS) is conditioned by a gene, Rfp, that is also involved in modifying transcripts of the pol CMS-associated orf224/atp6 mtDNA region. We now find that the nap nuclear restorer gene Rfn apparently is identical to Mmt, a gene that conditions the modification of transcripts from several different mtDNA regions, including one that is associated with nap CMS and contains orf222, a chimeric gene related to orf224. Mmt, in turn, is found to be allelic to Rfp, suggesting that restorer genes for the two cytoplasms represent different alleles or haplotypes of a single nuclear locus. This view is supported by restriction fragment length polymorphism mapping studies that indicate that Rfn and Rfp map to the same chromosomal position. Thus, in contrast to CMS in other species, different forms of Brassica CMS are restored by alleles of a single nuclear locus, and the restoration properties of these alleles reflect their involvement in the modification of transcripts of corresponding CMS-associated mtDNA regions. A survey of 51 varieties from 8 Brassica and Sinapis species failed to find evidence of Rfn(Mmt) in other than fertility-restored, nap cytoplasm B. napus. This suggests that Rfn(Mmt) arose in Brassica with nap cytoplasm and that the necessity for fertility restoration may have provided the selective pressure for its origin and maintenance.

Genetic mapping of nuclear fertility restorer genes for the ‘Polima’ cytoplasmic male sterility in canola (Brassica napus L.) using DNA markers

Co-segregation of male fertility with DNA markers selected by targeted mapping approaches as being potentially linked to the Rfp1 restorer gene for the pol cytoplasmic male sterility (CMS) was analyzed using two canola (Brassica napus L.) backcross populations. Eleven DNA markers (10 RFLP markers and one RAPD marker) directly linked to the Rfp1 locus were identified. The linkage group containing the Rfp1 locus was found to correspond to B. napus linkage group 18 of Landry et al. (1991). A similar pattern of co-segregation between DNA markers and male fertility was observed in a backcross population segregating for the pol restorer gene Rfp2 from line ‘UM2383’; one RFLP marker, cRF1b, showed perfect linkage with both Rfp1 and Rfp2 and detected identical polymorphic fragments in both the Rfp1 and Rfp2 restorer lines. Our findings indicate that restoration of pol CMS is controlled by a single nuclear genetic locus on linkage group 18 and that Rfp1 and Rfp2 are likely allelic.

Genetic correlation of the orf224/atp6 gene region with Polima CMS in Brassica somatic hybrids.

To identify regions of the mitochondrial genome from the polima or pol male-sterile cytoplasm of Brassica napus that are genetically correlated with cytoplasmic male sterility (CMS) we analyzed mtDNAs of three male-sterile somatic hybrids formed by the fusion of broccoli (B. oleracea L. var. italica) and pol CMS B. napus protoplasts. Fragments characteristic of a 4.5 kb DNA segment that comprises the single organizational difference between sterile pol and fertile cam Brassica mitochondrial genomes were found in all three sterile somatic hybrids. One of these hybrids possessed a mitochondrial genome that was, apart from a limited region around this 4.5 kb CMS-associated segment, collinear with B. oleracea mtDNA. Previous studies have indicated that expression of transcripts spanning the atp6 gene and a chimeric gene, orf24, located on this 4.5 kb DNA segment, is associated with male sterility. The present results indicate that the orf224/atp6 gene region is genetically correlated with male sterility and provide significant additional support for the view that this gene region may be involved in specifying the CMS trait.

RNA editing of transcripts of a chimeric mitochondrial gene associated with cytoplasmic male-sterility in Brassica.

The orf224 gene is a chimeric open reading frame associated with the Polima or pol cytoplasmic male sterility of Brassica napus. The first 58 codons and 5' upstream region of orf224 are derived from a conventional mitochondrial gene, orfB, while the origin of the remaining portion of the gene is unknown. Transcripts of the orf224 gene were found to be edited at a single site in the region of the gene that does not correspond to a known sequence. Oligonucleotides corresponding to the edited and unedited forms were shown to hybridize specifically to respective in vitro orf224 transcripts. Analysis of floral mtRNA by this method indicated that virtually all orf224 transcripts of both sterile and fertile, nuclear-restored pol cytoplasm plants are edited. Our results indicate that transcripts of novel, CMS-associated genes may be edited, but that, at least in this case, the degree of editing does not appear to be directly related to the male-sterile phenotype.

SEED PRODUCTION OF HYBRID SUMMER RAPE IN THE FIELD USING THE POL CYTOPLASMIC MALE STERILITY SYSTEM: A FIRST ATTEMPT

Hybrid summer rape (Brassica napus L.) seed production blocks were established at two locations in Manitoba in 1986 and 1987 to examine the effect of distance from the pollen source on seed yield, (both total and hybrid) and percent hybridity of seed produced on rows of a male sterile line of the open pollinated population B. napus cultivar, Marnoo, possessing the pol cytoplasmic male sterility inducing cytoplasm. A 10:1 ratio of male sterile line (A-line) to pollen parent was employed. Leaf cutter bees (Megachile rotundata F.) were used as the pollen vector between the two parents. In 1986, no significant differences in total seed yield were found among A-line rows, while in 1987, significant differences in total seed yields of A-line rows were found. Differences in hybrid seed yields among A-line rows were significant for all locations and years. Leaf cutter bees were found to be effective pollinators of the A-line plants. Less than half and less than a third of the seed produced on the A-line rows in 1986 and 1987, respectively, was hybrid seed. The high percentage of non-hybrid seed present in the seed lot may have been due to incomplete male sterility of the Marnoo A-line population. Total seed yields, hybrid seed yields and percent hybridity all declined linearly as distance from the pollen source increased. Improvement in the degree of male sterility of the Marnoo A-line population and/or a reduction in the 10:1 ratio of parents, and subsequent maximum A-line row to R-line row distance, will be necessary if hybrid summer rape seed production using this pol CMS A-line is to be commercially viable.Key words: Brassica napus L., CMS, hybrid, hybridity

Inheritance of male fertility restoration and allelism of restorer genes for the Polima cytoplasmic male sterility system in oilseed rape

The inheritance of male fertility restoration for the pol cytoplasmic male sterility (CMS) system in summer rape (Brassica napus L.) was determined. Male fertility:sterility segregation ratios observed in F2 and backcross generations derived from crosses and backcrosses between two pol CMS A lines and the male fertility restorer gene(s) sources Italy and UM2353 were used in this study. Italy and UM2353 were found to possess a single Mendelian dominant gene with high male fertility restoration capabilities for the pol CMS system. Tests for allelism of the restorer genes were also conducted using male fertility:sterility segregation ratios observed in F3 families derived from crosses between F1 plants containing genes for male fertility restoration from the Italy and UM2353 restorer gene sources. The male fertility restorer gene from Italy (designated Rfp1) was found to be different (i.e., nonallelic) from the restorer gene possessed by UM2353 (designated Rfp2).Key words: Brassica napus L., oilseed rape, male fertility restoration, pol cytoplasmic male sterility.

Morphologic and fundoscopic investigations into drug-induced and age-dependent retinal pigmentation in the dog

Somatic hybridization is performed to obtain significant cytoplasmic male sterility (CMS) lines, whose CMS genes are derived either from the transfer of sterile genes from the mitochondrial genome of donor parent to the counterpart of receptor or production of new sterile genes caused by mitochondrial genome recombination of the biparent during protoplast fusion. In this study, a novel male sterile line, SaNa-1A, was obtained from the somatic hybridization between Brassica napus and Sinapis alba. The normal anther development of the maintainer line, SaNa-1B, and the abortive process of SaNa-1A were described through phenotypic observations and microtome sections. The floral organ of the sterile line SaNa-1A was sterile with a shortened filament and deflated anther. No detectable pollen grains were found on the surface of the sterile anthers. Semi-thin sections indicated that SaNa-1A aborted in the pollen mother cell (PMC) stage when vacuolization of the tapetum and PMCs began. The tapetum radically elongated and became highly vacuolated, occupying the entire locule together with the vacuolated microspores. Therefore, SaNa-1A is different from other CMS lines, such as ogu CMS, pol CMS and nap CMS as shown by the abortive process of the anther.