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The genetic classification of cytoplasmic male sterility systems in Brassica napus L
... G. L. Sun Department of Biology, Saint Mary's University, Halifax, NS B3H 3C3Canada because rapeseed is biennial. Restriction fragment length polymorphism analysis of mtDNA is a fast method to distinguish cytoplasm types (Yang et al. 1998; Wei et al. 2005), but not suitable in high-throughput genotyping due to the requirement of large amounts of mtDNA, appropriate restriction endonucleases and suitable probes in southern hybridization. PCR-based markers developed from speciWc gene sequences are eYcient and high-throughput tool for determining cytoplasm type. ...
... To date, the methods developed for identiWcation of cytoplasm type in rapeseed include classical test-cross (Shiga et al. 1976Shiga et al. , 1978), RFLP analysis of mtDNA (Yang et al. 1998) and PCR marker developed from gene sequence of mtDNA (Wei et al. 2005). Among these methods, test-cross is the most common but time-consuming, because winter rapeseed is biennial. ...
Cytoplasmic male sterility (CMS) has widely been used as an efficient pollination control system in rapeseed hybrid production. Identification of cytoplasm type of rapeseed accessions is becoming the most important basic work for hybrid-rapeseed breeding. In this study, we report a simple multiplex PCR method to distinguish the existing common cytoplasm resources, Pol, Nap, Cam, Ogu and Ogu-NWSUAF cytoplasm, in rapeseed. Cytoplasm type of 35 F(1) hybrids and 140 rapeseed open pollinated varieties or breeding lines in our rapeseed breeding programme were tested by this method. The results indicated that 10 of 35 F(1) hybrids are the Nap, and 25 the Pol cytoplasm type, which is consistent with the information provided by the breeders. Out of 140 accessions tested, 100 (71.4%), 21 (15%) and 19 (13.6%) accessions possess Nap, Cam and Pol cytoplasm, respectively. All 19 accessions with Pol cytoplasm are from China. Pedigree analysis indicated that these accessions with Pol cytoplasm were either restorers for Pol CMS, including Shaan 2C, Huiyehui, 220, etc. or derived from hybrids with Pol CMS as female parent. Our molecular results are consistent with those of the classical testcross, suggesting the reliability of this method. The multiplex PCR assay method can be applied to CMS "three-line" breeding, selection and validation of hybrid rapeseed.
... Recently, great progress had been made in the identification and application of different CMS systems to cruciferous vegetables. Restorer and maintainer relationships combined 60 Theor Appl Genet (2018) 131:59-66 1 3 with RFLP analysis have been used to classify different CMS types in Brassica napus (Yang 1998). Moreover, test crosses and molecular genetics methods have been used to show that hau CMS cytoplasm is fundamentally different from pol, nap, ogu, and tour CMS cytoplasm (Wan et al. 2008). ...
Key message
oxa CMS is a new cytoplasmic male sterility type in Brassica juncea.
Abstract
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 no negative effect on 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.
... These results suggest that 681A and pol-CMS have the same restoring-maintaining relationship and belong to the same type of CMS on the basis of restoring-maintaining relationship. ÔShaan 2AÕ has proved to have the same restoring-maintaining relationship as pol-CMS (Yang et al. 1998). A chimeric open reading frame orf224 is shown to be unique to the pol-CMS mitochondrial genome (L'Homme and Brown 1993). ...
... Ninty individuals of 0912A were selected for fertility identification during the whole flowering period, and the sterile plant ratio was calculated after exact survey. The pollen fertility was classified according to method of Yang et al. (1998). All the 90 plants were artificially bagged for self-pollination, and their seed settings were counted for statistical analysis after harvested. ...
Six cytoplasmic male sterile (CMS) lines of Brassica vegetable Cai-xin (Brassica rapa L. ssp. chinensis var. utilis Tsen et Lee) were newly developed by backcrossing of six local inbred lines with a new eru CMS line of the vegetable purple cai-tai (B. rapa L. var. purpurea Bailey) from South China. The sterility of these Cai-xin eru CMS lines was stable and complete. The line 4A represented typical sterility and produced six off-white and shriveled stamens without pollen grains in the anthers. Paraffin sectioning comparison of the anthers between the CMS line 4A and its maintainer line 4B showed that their developmental differentiations started at the early to middle mononucleate microspore stage. The tapetum in the CMS line 4A was vacuolized highly and expanded radically, which extruded the developing microspores and caused their adhesion with each other and failure to develope mature pollen. With six CMS lines as female parents and 6 fertile lines as pollen parents, 36 hybrids were produced and their potentials of heterosis were analyzed. In general, mid-parent heterosis was detected for most of the 14 agronomic, yield and quality traits, such as petiole length (19.01%), weight of main flower stalk (35.96%), and soluble protein content (1.20%). These results indicated that the six newly derived eru CMS lines of Cai-xin provided a novel CMS system for its breeding and heterosis utilization.
... So far, nine CMS lines in B. napus have been characterized into four types (pol, ogu, nap and tour) based on analysis of restorer-maintainer relationships and mtDNA restriction fragment length polymorphisms (RFLPs) (Yang et al. 1998). Another CMS system named ''126-1'' was identified in a population of doubled haploids of synthetic B. napus ISN 706 and subsequently transferred to B. juncea (Sodhi et al. 2006). ...
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.
... Ninty individuals of 0912A were selected for fertility identification during the whole flowering period, and the sterile plant ratio was calculated after exact survey. The pollen fertility was classified according to method of Yang et al. (1998). All the 90 plants were artificially bagged for self-pollination, and their seed settings were counted for statistical analysis after harvested. ...
... The pol CMS is sensitive to environment in certain nuclear backgrounds leading to breakdown of sterility, which reduces hybridity levels in F 1 hybrids. So far, nine CMS lines in B. napus have been characterized into four types: pol, ogu, nap, tour (Yang et al. 1998 ). The recent reports of CMS in Brassica include CMS 681A of spontaneous origin in a mutant line of a B. napus cv. ...
Use of male sterile plants has become an important technique in heterosis breeding,which simplify and reduce the cost of hybrid seed production. Cruciferous vegetables are very important crops in the world, and two types of male sterility have been mainly explored in cruciferous vegetables (1) nuclear male sterility, this kind of male sterility is controlled by the dominant or recessive nuclear genes, and its sterility is easy to be restored, but diffi cult to maintained; (2) cytoplasmic male sterility, in which male sterility is controlled by a particular cytoplasmic male sterile gene (S). Cytoplasmic male sterility is easy to be maintained but complicated to be recovered. Male sterility can be produced by different ways; natural mutation, wide hybridization, and protoplast fusion. Ogu CMS and Polma CMS were found in rad-ish and B. napus respectively by natural mutations. Among the male sterile materi-als, most of them were obtained by the wide hybridization among varieties, species and genera. CMS cabbage was produced by the fusion of leaf protoplasts from fer-tile cabbage and CMS Ogura broccoli lines. The Pol CMS had been transferred from CMS B. napus to Chinese cabbage. Chinese breeders produced many cabbage vari-eties after introduced male sterility from other materials. A new cabbage hybrid varieties Zhonggan no.16, 17 and 18 were produced by hybridization of dominant genic male sterility line and inbred line in China. A stable CMS line of tuber mustard was developed by distant crosses and subsequent backcrosses and induced 100 % male sterility. To date, extensive efforts have been made on identifi cation of male sterility systems and the possibilities of development of hybrids in application.
... Genetic experiments and restriction mapping have shown that the CMS lines can be classified as two cytoplasmic groups, one including T-CMS and S-CMS [22][23][24], which is commonly referred to as the ''nap'' cytoplasm present in normal fertile accessions, and the other group as ''pol'' cytoplasm including pol-CMS, MI CMS, and shaan 2A CMS [25][26][27], which has been the most widely applied line in the world. During the past few decades, the fact that the grouped CMS accessions from multiple independent findings have the same genetic regulation has been a perplexing issue for researchers [28]. In order to identify the mechanism for natural CMS in B. napus, the main mitochondrial genome of pol-CMS was sequenced and compared to the sequence of Westar (nap cytoplasm), uncovering structural and evolutionary differences. ...
Cytoplasmic male sterility (CMS) has been identified in numerous plant species. Brassica napus CMS plants, such as Polima (pol), MI, and Shaan 2A, have been identified independently by different researchers with different materials in conventional breeding processes. How this kind of CMS emerges is unclear. Here, we report the mitochondrial genome sequence of the prevalent mitotype in the most widely used pol-CMS line, which has a length of 223,412 bp and encodes 34 proteins, 3 ribosomal RNAs, and 18 tRNAs, including two near identical copies of trnH. Of these 55 genes, 48 were found to be identical to their equivalents in the "nap" cytoplasm. The nap mitotype carries only one copy of trnH, and the sequences of five of the six remaining genes are highly similar to their equivalents in the pol mitotype. Forty-four open reading frames (ORFs) with unknown function were detected, including two unique to the pol mitotype (orf122 and orf132). At least five rearrangement events are required to account for the structural differences between the pol and nap sequences. The CMS-related orf224 neighboring region (∼5 kb) rearranged twice. PCR profiling based on mitotype-specific primer pairs showed that both mitotypes are present in B. napus cultivars. Quantitative PCR showed that the pol cytoplasm consists mainly of the pol mitotype, and the nap mitotype is the main genome of nap cytoplasm. Large variation in the copy number ratio of mitotypes was found, even among cultivars sharing the same cytoplasm. The coexistence of mitochondrial mitotypes and substoichiometric shifting can explain the emergence of CMS in B. napus.
... At present, Polima and Shaan 2A CMS are the only two CMS widely applied in heterosis utilization in China. Although Shaan 2A CMS and Polima CMS have different origin, they are sometimes assumed to be the same type of CMS, because they share the same restorer lines and maintainer lines (Yang and Fu, 1991), and have the same cytoplasm as revealed by restriction fragment length polymorphism (RFLP) and mtDNA fragment polymorphism analyses (Yang et al., 1998). ...
Polima and Shaan 2A are the two most widely used forms of cytoplasmic male sterility (CMS) in the utilization of heterosis of rapeseed (Brassica napus) in China. A previous study indicated that the mitochondrial gene, orf224, was the only gene with a differential expression pattern among the normal, sterile and fertility-restored lines in rapeseed. DNA sequences of orf224, including coding sequences from Shaan 2A and Polima CMS, were then amplified and analyzed. DNA sequence alignment indicated both the coding sequences were 675 bp in length and had 99.9 and 99% homology in nucleotides and amino acids, respectively, and shared certain similarity to homologues from other Brassica spp. and Arabidopsis thaliana. The probable promoter regions of orf224 were conserved between B. napus and A. thaliana, but the upstream regions of probable promoter regions were completely divergent from each other. Additionally, analysis of the primary and secondary structure of the proteins encoded by orf224 from the two lines predicted that the proteins contain a a-helix, extended strand, and random coil. After cloning a in vitro experiment showed that these two proteins could be expressed in Escherichia coli BL21.
A single cytoplasmic source of foxtail millet male sterile lines has become a major limiting factor for wide utilization of heterosis in foxtail millet. To explore genetic diversity of Chinese foxtail millet cytoplasm, 23 pairs of mitochondrial DNA (mtDNA) primers that showed a high level of polymorphism among selected 14 genetically diverse foxtail millet accessions were identified after screening 34 pairs of consensus mtDNA primers that were derived from various plant species. The 23 pairs of primers were used to analyze genetic diversity of cytoplasm in 111 Chinese elite foxtail millet accessions. Genetic similarity coefficients for differentiation of cytoplasmic types were calculated based on the phylogenetic tree and female-parent-derived pedigree graph of all the tested accessions. The results show that the genetic diversity of foxtail millet cytoplasm is low, which makes Chinese foxtail millet germplasm vulnerable to the infection by cytoplasm-related diseases. The 111 tested accessions can be classified into eight cytoplasmic types: Qinyuanmujizui, Huangruangu, Riben60ri, Dahuanggu, Yingsuigu, Heizhigu, MissingI, and MissingII. The eight cytoplasmic types matched with the geographic and ecological distribution pattern of the most tested accessions. The results provided a scientific foundation for rationally utilizing these germplasm lines, diversifying cytoplasmic types in male sterile lines, and further improving hybrid foxtail millet cultivars.
A novel Pol CMS named Polima temperature-sensitive cytoplasmic male sterile (Pol TCMS) is sterile at high temperatures and partially fertile at low temperatures. Its temperature sensitivity is controlled by several minor genes, and probably influenced by the genetic background, making it quite difficult to breed a stable Pol TCMS line through the conventional breeding methods. The present study aimed to obtain stable homozygous Pol TCMS lines by microspore culture. The highest frequency of embryogenesis was induced from cultures of buds sampled at 4–7 days before first blossom. The second method of microspore cultures mentioned in this study could significantly increase the production of embryos. Analysis of fatty acid composition, glucosinolate content and genetic diversity by simple sequence repeat (SSR) markers indicated that microspore culture was effective for selecting Pol TCMS lines.
A novel cytoplasmic male sterility-fertility restoration system has been developed in rapeseed (Brassica napus). The cytoplasmic male sterile line 681A was derived from a spontaneous male sterile mutant in a newly released double-low rapeseed cultivar ‘Xiangyou 13′. The restorer line 714R was identified in the interspecific progeny from a B. napus×B. juncea-cross. Genetic analysis showed that fertility restoration for 681A cytoplasmic male sterility was controlled by a single dominant nuclear gene which might originate from B. juncea. The RAPD marker S1039-520 was found to be linked to the restorer gene in F2 progeny of 681A × 714R with a recombination frequency of 5.45%.
The Polima cytoplasmic male sterility (CMS) system has been successfully used in three/two-line hybrid production in rapeseed
(Brassica napus L.). However, the sterility of the Polima (pol) CMS lines is sensitive to temperature fluctuations. Also, traces of pollen
can cause self-pollination within the CMS lines, which results in reduced levels of F1 hybrid seed purity and leads to a significant yield loss. Self-incompatibility (SI) is another important approach for hybrid
seed production in rapeseed. Despite having a wide range of restorers and being easily selected in a breeding program, SI
system has some drawbacks. In this study, SI genes from a self-incompatible line of Brassica napus were transferred to a pol CMS line and S372A, a novel line of combined cytoplasmic male sterility with self-incompatibility
was bred. Due to the SI genes, this line produced very few seeds when it was selfed at low temperature and no seeds at high
temperature. This suggested that the line with CMS+SI had combined the advantages and overcome the disadvantages of both
the pol CMS and SI systems. Furthermore, our results showed that most of the maintainers and all the restorers of the pol
CMS system were also maintainers and restorers of the CMS+SI line, respectively. This indicates that the CMS+SI system
can be easily used to establish three-line hybrids of rapeseed, and we believe this novel system could be extended to other
species of Brassica.
A novel cytoplasmic male sterility (CMS) was identified in Brassica juncea, named as hau CMS (00-6-102A). Subsequently, the male sterility was transferred to B. napus by interspecific hybridization. The hau CMS has stable male sterility. Flowers on the A line are absolutely male sterile, and seeds harvested from the line following pollinations with the maintainer gave rise to 100% sterile progeny. The anthers in CMS plants are replaced by thickened petal-like structures and pollen grains were not detected. In contrast, in other CMS systems viz. pol, nap, tour, and ogu, anthers are formed but do not produce viable pollen. The sterility of hau CMS initiates at the stage of stamen primordium polarization, which is much earlier compared with the other four CMS systems. We have successfully transferred hau CMS from B. juncea to B. napus. Restorer lines for pol, ogu, nap, and tour CMS systems were found to be ineffective to restore fertility in hau CMS. Sixteen out of 40 combinations of mitochondrial probe/enzyme used for RFLP analysis distinguished the hau CMS system from the other four systems. Among these sixteen combinations, five ones alone could distinguish the five CMS systems from each other. The evidence from genetic, morphological, cytological and molecular studies confirmed that the hau CMS system is a novel CMS system.
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