In this study, we have investigated the cytoplasmic male sterility (CMS) of a novel male sterile radish line, designated NWB CMS. The NWB CMS was crossed with 16 fertile breeding lines, and all the progenies were completely male sterile. The degree of male sterility exhibited by NWB CMS is more than Ogura CMS from the Cruciferae family. The NWB CMS was found to induce 100% male sterility when crossed with all the tested breeding lines, whereas the Ogura CMS did not induce male sterility with any of the breeding lines. PCR analysis revealed that the molecular factor that influenced Ogura CMS, the orf138 gene, was absent in the NWB CMS line, and that the orf138 gene was not also expressed in this CMS line. In order to identify the cytoplasmic factors that confer male sterility in the NWB CMS line, we carried out RFLP analyses with 32 mitochondrial genes, all of which were used as probes. Fourteen genes exhibited polymorphisms between the NWB CMS line and other radish cultivars. Based on these RFLP data, intergenic primers were developed in order to amplify the intergenic regions between the polymorphic genes. Among these, a primer pair at the 3' region of the atp6 gene (5'-cgcttggactatgctatgtatga-3') and the 5' region of the nad3 gene (5'-tcatagagaaatccaatcgtcaa-3') produced a 2 kbp DNA fragment as a result of PCR. This DNA fragment was found to be specific to NWB CMS and was not present in other CMS types. It appears that this fragment could be used as a DNA marker to select NWB CMS line in a radish-breeding program.
"However, several important pieces of the puzzle are still missing, including the impact of cytoplasmic genes. A number of studies have been carried out for characterization and identification of CMS systems in many crops, including rice, wheat, sugar beet, sunflower , common bean, onion, Arabidopsis, pearl millet and radish, based on mtDNA analyses (Delorme et al. 1997; Schnable and Wise 1994; Horn and Fried 1999; Nahm et al. 2005). CMS is an important approach to exploit heterosis (Fu et al. 1995). "
[Show abstract][Hide abstract] ABSTRACT: We analysed DNA and complementary DNA (cDNA) from two inbred lines (CMS-ARIT121 and CMS-ARIT2) along with their respective maintainer lines with eight known genes (atpa, atp6, atp9, coxI, coxII, orfB, orf222 and orf224) reported in other cytoplasmic male sterility (CMS) systems. DNA analyses indicated that only orf224 was different in the maintainer and CMS in ARIT121. Sequencing analysis indicated that CMS-ARIT121 has a similar sequence to that of polima CMS. However, CMS-ARIT2 was quite unique, with no sequence difference at DNA or cDNA level in all eight candidate genes, with the exception of a two-base substitution in the atp6 gene between the CMS and maintainer lines. The similar amplification pattern with all primers, especially orf224, indicates that CMS-ARIT2 is unique and does not correspond to polima, ogura or nap. However, further research is required to establish the causes of sterility in this cms, since we could not find any significant difference at transcript level between CMS-ARIT2 and its corresponding maintainer line. However, the same restorer PR121 as for CMS-ARIT121 was able to restore CMS-ARIT2, and as such can be used for hybrid seed production in Brassica napus L. Overall, this study provides important information for breeders selecting new cytoplasmic male sterility (CMS) in B. napus L. and use of molecular markers to identify CMS lines at early seedling stage that will enhance the B. napus L. breeding program.
"Molecular biological analysis in BC 4 plants derived from BC 3 plants carrying the cytoplasm and chromosomes of M. arvensis Total DNA of BC 4 plants and their parent plants was extracted according to the CTAB method (Doyle and Doyle 1987). In order to clarify the organelle genome, we performed PCR analysis using a primer pair specific to mitochondrial 32 genes, described in Nahm et al. (2005), and to the chloroplast intergenic region (forward primer; 5′GAA CGACGGGAATTGAACC3′, reverse primer; 5′TGCTGT TGAGGCTCCATCTA3′), which was designed based on the sequence of trnH and psbA in Arabidopsis thaliana (accession number AP000423). The 10 μl reaction mixtures contained 100 ng genomic DNA, 0.5 μM primers, 0.2 mM dNTPs (Fermentas, Vilnius, Lithuania), 1 × DreamTaq buffer and 0.5 unit of DreamTaq DNA polymerase (Fermentas) in 0.2 ml tubes, and PCR was carried out using a Program Temp Control system PC-320 (ASTEC Co., Fukuoka, Japan). "
[Show abstract][Hide abstract] ABSTRACT: Intergeneric hybridization was performed between Moricandia arvensis and four inbred lines of Brassica rapa following embryo rescue. Three F(1) hybrid plants were developed from three cross combinations of M. arvensis × B. rapa, and amphidiploids were synthesized by colchicine treatment. Six BC(1) plants were generated from a single cross combination of amphidipolid × B. rapa 'Ko1-303' through embryo rescue. One BC(2) and three BC(3) plants were obtained from successive backcrossing with B. rapa 'Ko1-303' employing embryo rescue. Alloplasmic and monosomic addition lines of B. rapa (Allo-MALs, 2n = 21) were obtained from backcrossed progeny of three BC(3) plants (2n = 21, 22 and 23) without embryo rescue. An alloplasmic line of B. rapa (2n = 20) degenerated before floliation on 1/2 MS medium due to severe chlorosis. Allo-MALs of B. rapa (2n = 21) showed stable male sterility without any abnormal traits in vegetative growth and female fertility. Molecular analyses revealed that the same chromosome and cytoplasm of M. arvensis had been added to each Allo-MAL of B. rapa. This Allo-MAL of B. rapa may be useful material for producing cytoplasmic male sterile lines of B. rapa.
"Recently, two novel radish CMS types, NWB and DCGMS, have been reported (Nahm et al. 2005; Lee et al. 2008, 2009). A small number of aggregated pollen grains are observed in both of the DCGMS and NWB male-sterile lines, but the mitotypes are different from each other (Nahm et al. 2005; Lee et al. 2008, 2009). Distinct genetic controls of radish CMS restoration have been proposed using different populations (Nieuwhof 1990; Koizuka et al. 2000; Bett and Lydiate 2004). "
[Show abstract][Hide abstract] ABSTRACT: In this work, we have identified a chimeric pentatricopeptide repeat (PPR)-encoding gene cosegregating with the fertility
restorer phenotype for cytoplasmic male sterility (CMS) in radish. We have constructed a CMS-Rf system consisting of sterile line ‘9802A2’, maintainer line ‘9802B2’ and restorer line ‘2007H’. F2 segregating population analysis indicated that male fertility is restored by a single dominant gene in the CMS-Rf system described above. A PPR gene named Rfoc was found in the restorer line ‘2007H’. It cosegregated with the fertility restorer in the F2 segregating population which is composed of 613 fertile plants and 187 sterile plants. The Rfoc gene encodes a predicted protein 687 amino acids in length, comprising 16 PPR domains and with a putative mitochondrial targeting
signal. Sequence alignment showed that recombination between the 5′ region of Rfob (EU163282) and the 3′ region of PPR24 (AY285675) resulted in Rfoc, indicating a recent unequal crossing-over event between Rfo and PPR24 loci at a distance of 5.5kb. The sterile line ‘9802A2’ contains the rfob gene. In the F2 population, Rfoc and rfob were observed to fit a segregation ratio 1:2:1 showing that Rfoc was allelic to Rfo. Previously we have reported that a fertile line ‘2006H’, which carries the recessive rfob gene, is able to restore the male fertility of CMS line ‘9802A1’ (Wang et al. in Theor Appl Genet 117:313–320, 2008). However, here when conducting a cross between the fertile line ‘2006H’ and CMS line ‘9802A2, the resulting plants were
male sterile, which shows that sterile line ‘9802A2’ possesses a different nuclear background compared to ‘9802A1’. Based
on these results, the genetic model of fertility restoration for radish CMS is also discussed.
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