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Identification of mitochondrial genome rearrangements unique to novel cytoplasmic male sterility in radish (Raphanus sativus L.)
Abstract
A novel cytoplasmic male-sterility (CMS) radish (Raphanus sativus L.) and its associated mitotype (DCGMS) were previously identified; however, no mtDNA fragments flanking the atp6 gene were found in the DCGMS mitotype. Unlike three other mitotypes in this study, a unique mtDNA organization, atp6-nad3-rps12, was found to be the major mtDNA structure associated with this mitotype. This organization may have arisen from short repeat sequence-mediated recombination events. The short repeat clusters involved in the mtDNA rearrangement around the atp6 gene also exist as repetitive sequences in the complete mitochondrial genomes of other members of the Brassicaceae family, including rapeseed and Arabidopsis. These sequences do not exist as repetitive elements in the mtDNA of tobacco, sugar beet, or rice. While studying the regions flanking atp6, we identified a truncated atp6 mtDNA fragment which consists of the 5' part of the atp6 gene linked to an unidentified sequence. This mtDNA structure was present in all mitotypes; however, a single nucleotide insertion mutation leading to a frame-shift was identified only in the DCGMS mitotype. Although this truncated atp6 organization was transcribed, there was no significantly different expression between male-sterile and fertile segregating individuals from the BC(1)F(1) population originating from a cross between male-sterile and restorer parents. Comprehensive survey of the single base-pair insertion showed that it was maternally inherited and unique to the DCGMS mitotype. Therefore, this single nucleotide polymorphism (SNP) in the coding sequence of the mtDNA will be a useful molecular marker for the detection of the DCGMS mitotype.
... Furthermore, Bang et al. (2011) developed the novel CMS lines in Radish (R. sativus) carrying cytoplasm of B. maurorum by intergeneric hybridization followed by embryo rescue. In recent decades, a novel CMS type designated as DCGMS (Dongbu cytoplasmic and genic male sterility) was discovered in Radish (R. sativus L.) (Lee et al. 2008(Lee et al. , 2009). The fertility restoration of DCGMS is controlled by single nuclear locus, Rfd1, and there is no linkage of this locus with Ogura-CMS Rf locus 'Rfo' (Cho et al. 2012). ...
... accession from Uzbekistan orf463 cox1 Raphanus sativus Lee et al. (2008Lee et al. ( , 2009) and Park et al. (2013) mur CMS Protoplast fusion Interspecific hybridization Yarrow et al. (1990), Wang et al. (1995, Singh and Brown (1993), Ke and Song (1989) and Ke et al. (1992) nap CMS Landgren et al. (1996) Tournefortii-Stiewe CMS Brassica tournefortii (somatic cell fusion) orf193 atp9-2 ...
Key message
Overview of the current status of GMS and CMS systems available in Brassica vegetables, their molecular mechanism, wild sources of sterile cytoplasm and exploitation of male sterility in hybrid breeding.
Abstract
The predominantly herbaceous family Brassicaceae (crucifers or mustard family) encompasses over 3700 species, and many of them are scientifically and economically important. The genus Brassica is an economically important genus within the tribe Brassicaceae that comprises important vegetable, oilseed and fodder crops. Brassica vegetables display strong hybrid vigor, and heterosis breeding is the integral part in their improvement. Commercial production of F1 hybrid seeds in Brassica vegetables requires an effective male sterility system. Among the available male sterility systems, cytoplasmic male sterility (CMS) is the most widely exploited in Brassica vegetables. This system is maternally inherited and studied intensively. A limited number of reports about the genic male sterility (GMS) are available in Brassica vegetables. The GMS system is reported to be dominant, recessive and trirecessive in nature in different species. In this review, we discuss the available male sterility systems in Brassica vegetables and their potential use in hybrid breeding. The molecular mechanism of mt-CMS and causal mitochondrial genes of CMS has been discussed in detail. Finally, the exploitation of male sterility system in heterosis breeding of Brassica vegetables, future prospects and need for further understanding of these systems are highlighted.
... In radish CMS-Rf system, the mitochondrial gene orf138/orf125 coincides with Ogura/Kosena CMS plants in which anthers are completely devoid of pollen grains (Bonhomme et al. 1992;Krishnasamy and Makaroff 1993;Iwabuchi et al. 1999). Recently, two novel radish CMS types, NWB and DCGMS, have been reported (Nahm et al. 2005;Lee et al. 2008Lee et al. , 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. 2008Lee et al. , 2009. ...
... Recently, two novel radish CMS types, NWB and DCGMS, have been reported (Nahm et al. 2005;Lee et al. 2008Lee et al. , 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. 2008Lee et al. , 2009. Distinct genetic controls of radish CMS restoration have been proposed using different populations (Nieuwhof 1990;Koizuka et al. 2000;Bett and Lydiate 2004). ...
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.
... Two CMS-specific SCAR markers were developed to distinguish N-cytoplasm from S-cytoplasm and AFLP markers linked to the fertility restorer gene Rf [260]. The CAPS marker (E-AGC/M-GCA122) linked to the Pr locus that is related to the partial restoration of fertility in CMS was also reported in chili pepper (Capsicum annuum L.) [261]. The SCAR marker, CRF3S1S, was highly efficient (100%) at differentiating restorers from maintainers. ...
To address the complex challenges faced by our planet such as rapidly changing climate patterns, food and nutritional insecurities, and the escalating world population, the development of hybrid vegetable crops is imperative. Vegetable hybrids could effectively mitigate the above-mentioned fundamental challenges in numerous countries. Utilizing genetic mechanisms to create hybrids not only reduces costs but also holds significant practical implications, particularly in streamlining hybrid seed production. These mechanisms encompass self-incompatibility (SI), male sterility, and gynoecism. The present comprehensive review is primarily focused on the elucidation of fundamental processes associated with floral characteristics, the genetic regulation of floral traits, pollen biology, and development. Specific attention is given to the mechanisms for masculinizing and feminizing cucurbits to facilitate hybrid seed production as well as the hybridization approaches used in the biofortification of vegetable crops. Furthermore, this review provides valuable insights into recent biotechnological advancements and their future utilization for developing the genetic systems of major vegetable crops.
... there were repeat sequences in the region of nad1-trnV and the repeat sequences were detected by the website https://www.novoprolabs.com/tools/repeats-sequences-finder, accessed on 22 April 2022) [37][38][39]. As more and more unprecedented Pleosporales species and new lineages are continuously emerging, analyses regarding gene rearrangement and homologous blocks are still worthy of detecting to find out recombinational hotspots and rearrangement patterns a step further. ...
The Pleosporales is the most predominant order in the Dothideomycetes class, which contains over 4700 species that function in a variety of ways. The material used in this research was previously isolated from the Chinese white wax scale insect, and it was determined to be a Paraconiothyrium genus species that belonged to the Pleosporales order. For further molecular analysis, we assembled the complete mitochondrial genome of Paraconiothyrium sp. based on short reads of BGISEQ sequencing and subreads from Pacbio sequencing. The results showed that it was 42,734 bp in length and contained 8 open reading frames, 12 protein-coding genes and 31 non-coding genes. Phylogenetic analysis showed it was affiliated to the Pleosporales order and formed a sister relationship with Pithomyces chartarum. Compared to the seven other species in the Pleosporales order, Paraconiothyrium sp. has generally conserved gene content and structure, while the homologous blocks and gene order were shown to be significantly rearranged, in accordance with the species diversity in the Pleosporales order. In this study, we presented the first mitochondrial genome of Paraconiothyrium fungi to be reported, and we also showed gene order diversity in the Pleosporales order. These findings will lay the foundation for further species studies regarding molecular diversity and our understanding of species characteristics in the Paraconiothyrium genus.
... Many studies have shown that amplification, rearrangement, and recombination of mitochondrial genes in higher plants leads to formation of new ORFs and CMS. The first CMS-related gene identified in higher plants was urf13 in the mitochondrial genome of maize; specific genes associated with CMS were subsequently identified in the mitochondrial genomes of rice, sunflower, Brassica, radish, and pepper (Dewey et al., 1986;Singh and Brown, 1993;Dieterich et al., 2003;Kim and Kim, 2006;Wang et al., 2006;Kim et al., 2007;Ashutosh et al., 2008;Lee et al., 2009;Nizampatnam et al., 2009;Das et al., 2010;Jing et al., 2011). ...
Cytoplasmic male sterility (CMS) is a maternally inherited trait that derives from the inability to produce functional pollen in higher plants. CMS results from recombination of the mitochondrial genome. However, understanding of the molecular mechanism of CMS in pepper is limited. In this study, comparative transcriptomic analyses were performed using a near-isogenic CMS line 14A (CMS-14A) and a maintainer line 14B (ML-14B) as experimental materials. A total of 17,349 differentially expressed genes were detected between CMS-14A and ML-14B at the PMC meiosis stage. Among them, six unigenes associated with CMS and 108 unigenes involved in energy metabolism were identified. The gene orf165 was found in CMS-14A. When orf165 was introduced into ML-14B, almost 30% of transgenic plants were CMS. In addition, orf165 expression in transgenic CMS plants resulted in abnormal function of some genes involved in energy metabolism. When orf165 in transgenic CMS plant was silenced, the resulted orf165-silenced plant was male fertile and the expression patterns of some genes associated with energy metabolism were similar to ML-14B. Thus, we confirmed that orf165 influenced CMS in pepper.
... The fertile or sterile phenotype differed depending on the flowers in one plant (Table 3, Fig. 5). Such an unstable phenotype was not described in any of previous studies on DCGMS male sterile cytoplasm (Lee et al. 2008(Lee et al. , 2009Kim et al. 2010;Cho et al. 2012;Park et al. 2013). This observation suggested that the function of orf463 or that of fertility restoring gene(s) against orf463 was not stable in the radishes of the 'Niger' group. ...
In addition to Ogura cytoplasmic male sterility (CMS), which is used worldwide in radish and Brassica crops, another type of CMS was found and named DCGMS. A mitochondrial candidate gene (orf463) of DCGMS was identified, but the distribution of this gene in radish species has not been clarified to date. We found that orf463 is distributed specifically in black radish cultivars belonging to the ‘Niger’ group and a line of wild species, Raphanus raphanistrum. The progeny tests using the cultivars having orf463 as a female parent demonstrated that the cytoplasm induces male sterility in radishes. Although the DNA sequences of orf463 possessed by the ‘Niger’ group cultivars are identical to those of DCGMS, the orf463 sequence of the wild radish line contained nine nucleotide substitutions, seven of which were nonsynonymous. All radishes with orf463 had both DCGMS-type and ‘Black radish’- type mitochondrial structures according to multiplex PCR. Based on these results, the mechanism of rearrangement between the two mitochondrial types was studied.
... The atp6 gene is one of the most frequently rearranged mitochondrial genes in plants [12,47,48]. Correlation between the chimeric atp6 gene and CMS phenotypes have been found in several species, including pepper [48], maize [49], radish [50], monkeyflower [51], etc. It is interesting to note, that in sugar beet the chimeric ORF presented by the atp6 5′ elongated transcript has been translated in vivo and caused CMS phenotype [47]. ...
Background
More than 70 cytoplasmic male sterility (CMS) types have been identified in Helianthus, but only for less than half of them, research of mitochondrial organization has been conducted. Moreover, complete mitochondrion sequences have only been published for two CMS sources – PET1 and PET2. It has been demonstrated that other sunflower CMS sources like MAX1, significantly differ from the PET1 and PET2 types. However, possible molecular causes for the CMS induction by MAX1 have not yet been proposed. In the present study, we have investigated structural changes in the mitochondrial genome of HA89 (MAX1) CMS sunflower line in comparison to the fertile mitochondrial genome.
Results
Eight significant major reorganization events have been determined in HA89 (MAX1) mtDNA: one 110 kb inverted region, four deletions of 439 bp, 978 bp, 3183 bp and 14,296 bp, respectively, and three insertions of 1999 bp, 5272 bp and 6583 bp. The rearrangements have led to functional changes in the mitochondrial genome of HA89 (MAX1) resulting in the complete elimination of orf777 and the appearance of new ORFs - orf306, orf480, orf645 and orf1287. Aligning the mtDNA of the CMS sources PET1 and PET2 with MAX1 we found some common reorganization features in their mitochondrial genome sequences.
Conclusion
The new open reading frame orf1287, representing a chimeric atp6 gene, may play a key role in MAX1 CMS phenotype formation in sunflower, while the contribution of other mitochondrial reorganizations seems to appear negligible for the CMS development.
Electronic supplementary material
The online version of this article (10.1186/s12870-019-1637-x) contains supplementary material, which is available to authorized users.
... Among the markers mentioned above, SCAR markers were more specific and reproducible. And SCAR markers were also used in other crops such as rice (Yashitola et al. 2004), pepper (Kim and Kim 2005) and radish (Lee et al. 2009). Recently, mitochondrial SSR markers were also used to identify cytoplasm types (Rajendrakumar et al. 2007). ...
With 8 figures and 3 tables
Cytoplasmic male sterility (CMS) line P30A of Upland cotton has been used in commercial production of F1 seeds. However, cytoplasm-specific molecular markers in this ‘three-line’ (CMS line P30A, maintainer line P30B and restorer line Y18R) system are mostly unknown. Twenty mitochondrial gene probes were used to identify the differences between P30B fertile (N-) and P30A sterile (S-) cytoplasm. Among six genes that revealed restriction fragment length polymorphisms (RFLPs), the gene for α-subunit of F1ATPase (atpA) revealed significant differences between N- and S-cytoplasm. All of the EcoRI restriction bands of atpA were amplified by inverse PCR (iPCR) technique, indicating that both N- and S-cytoplasm contained an intact and a 3′ truncated atpA copy, but the truncating site (breakpoint) was different. According to the chimerical sequences following the breakpoints, three cytoplasm-specific sequence characterized amplified region (SCAR) markers were developed. In addition, a simple sequence repeat (SSR) locus in the 3′ flanking sequences of the intact atpA gene was found between N- and S-cytoplasm. The four PCR markers were used to distinguish CMS lines from maintainer lines.
... Classifications of radish mitotypes and certain genes in radish mitochondria have been reported (Sakai and Imamura, 1992;Albaum et al., 1995;Kim et al., 2007). The involvement of short repeat sequences in dynamic mtDNA rearrangements has also been studied in the radish (Lee et al., 2009). ...
To explore the mitochondrial genes of the Cruciferae family, the mitochondrial genome of Raphanus sativus (sat) was sequenced and annotated. The circular mitochondrial genome of sat is 239,723 bp and includes 33 protein-coding genes, three rRNA genes and 17 tRNA genes. The mitochondrial genome also contains a pair of large repeat sequences 5.9 kb in length, which may mediate genome reorganization into two sub-genomic circles, with predicted sizes of 124.8 kb and 115.0 kb, respectively. Furthermore, gene evolution of mitochondrial genomes within the Cruciferae family was analyzed using sat mitochondrial type (mitotype), together with six other reported mitotypes. The cruciferous mitochondrial genomes have maintained almost the same set of functional genes. Compared with Cycas taitungensis (a representative gymnosperm), the mitochondrial genomes of the Cruciferae have lost nine protein-coding genes and seven mitochondrial-like tRNA genes, but acquired six chloroplast-like tRNAs. Among the Cruciferae, to maintain the same set of genes that are necessary for mitochondrial function, the exons of the genes have changed at the lowest rates, as indicated by the numbers of single nucleotide polymorphisms. The open reading frames (ORFs) of unknown function in the cruciferous genomes are not conserved. Evolutionary events, such as mutations, genome reorganizations and sequence insertions or deletions (indels), have resulted in the non-conserved ORFs in the cruciferous mitochondrial genomes, which is becoming significantly different among mitotypes. This work represents the first phylogenic explanation of the evolution of genes of known function in the Cruciferae family. It revealed significant variation in ORFs and the causes of such variation.
... Up to now, there have been a number of genes associated with the CMS phenomena identified in the mitochondrial genome of various plant species (Schnable and Wise 1998). For example, specific CMS associated genes have been reported in maize (Dewey et al. 1986), petunia (Young and Hanson 1987), bean (Johns et al. 1992), Brassica (Bonhomme et al. 1992; Grelon et al. 1994), radish (Makaroff et al. 1990; Lee et al. 2008), sunflower (Moneger et al. 1994), rice (Akagi et al. 1994), carrot (Kanzaki et al. 1991), sorghum (Tang et al. 1996), pepper (Kim et al. 2007), among others. It is commonly believed that CMS is frequently associated with the dysfunction of mitochondria during male meiosis and pollen development , which could be caused by post-transcriptional regulations of CMS-causing chimeric open reading frames (ORF) and/or expression of specific CMSinducing proteins. ...
Cytoplasmic male sterility (CMS) is a maternally inherited trait that fails to produce functional pollen grains. The CMS system
is widely employed to facilitate the utilization of heterosis in major crops. However, little is known about the CMS associated
genes in Upland cotton (Gossypium hirsutum). The objective of this study was to compare CMS cotton (CMS-D2) with the cytoplasm from G. harknessii and its isogenic maintainer line with the normal fertile Upland cotton cytoplasm to identify CMS-D2 specific gene(s) and
to develop CMS-specific sequence characterized amplified region (SCAR) markers. Based on Southern blot analysis using 10 mitochondrial
gene-specific probes (cob, cox2, atp6, atp9, nad3, cox3, atpA, cox1, nad6 and nad9), three probes (cox3, atpA, and nad6) revealed restriction fragment length polymorphisms (RFLP) between the CMS-D2 and its isogenic maintainer line. RT-PCR confirmed
that the three genes were differentially expressed between the two lines. These results indicated that there existed structural
and expression variations in the three genes when the mitochondrial D2 genome was transferred into Upland cotton. Genome walking
and rapid amplification of cDNA ends (RACE) were further performed to analyze the sequences of these genes and their flanking
regions. For cox3 and nad6, there was only one different nucleotide each in the gene regions between the two lines. Also some nucleotides upstream of
the ATG codon were different. For atpA, the sequences downstream the atpA were significantly different between the two cytoplasmic lines. Furthermore, two nucleotides at the -4 and -5 position from
ATG codon were also changed between the two cytoplasms (i.e., CG→AA), and this mutation also exists in RNA sequences. Interestingly,
nine nucleotides (ATGCAACTA) were also inserted at the location of 899bp upstream of ATG codon in the CMS line. The results
suggest that the abnormal sequence and expression of atpA gene is associated with CMS expression in Upland cotton. According to the significant different sequences downstream the
atpA gene, a CMS-D2 specific SCAR marker was developed. The CMS-specific PCR bands were verified for 10 cultivars containing either
normal- or CMS-D2cytoplasm. This will allow quick and reliable identification of the cytoplasmic types of individual plants
at the seedling stage, and assessment of the purity of F1 seed lots.
KeywordsUpland cotton–CMS–
atpA
–ATG codon–SCAR
... orf108, which is co-transcibed with the atpA gene, was found to be associated with Moricandia arvensis CMS, and the long atpA transcript is spliced within orf108 in fertility-restored lines (Ashutosh et al., 2008). A single nucleotide insertion mutation that results in the truncation of atp6 may be associated with DCGMS CMS in radish (Raphanus sativus L.) (Lee et al., 2009). However, these male sterility-associated ORFs are rarely transformed into fertile plants, which further confirm their functions. ...
Cytoplasmic male sterility (CMS) is a widespread phenomenon in higher plants, and several studies have established that this
maternally inherited defect is often associated with a mitochondrial mutant. Approximately 10 chimeric genes have been identified
as being associated with corresponding CMS systems in the family Brassicaceae, but there is little direct evidence that these
genes cause male sterility. In this study, a novel chimeric gene (named orf288) was found to be located downstream of the atp6 gene and co-transcribed with this gene in the hau CMS sterile line. Western blotting analysis showed that this predicted open reading frame (ORF) was translated in the mitochondria
of male-sterile plants. Furthermore, the growth of Escherichia coli was significantly repressed in the presence of ORF288, which indicated that this protein is toxic to the E. coli host cells. To confirm further the function of orf288 in male sterility, the gene was fused to a mitochondrial-targeting pre-sequence under the control of the Arabidopsis APETALA3 promoter and introduced into Arabidopsis thaliana. Almost 80% of transgenic plants with orf288 failed to develop anthers. It was also found that the independent expression of orf288 caused male sterility in transgenic plants, even without the transit pre-sequence. Furthermore, transient expression of orf288 and green fluorescent protein (GFP) as a fused protein in A. thaliana protoplasts showed that ORF288 was able to anchor to mitochondria even without the external mitochondrial-targeting peptide.
These observations provide important evidence that orf288 is responsible for the male sterility of hau CMS in Brassica juncea.
... Several mitochondrial genes responsible for male sterility have been cloned in some plant species and most of them have been revealed as chimeric genes created through mtDNA rearrangement (Hanson and Bentolila 2004). The information of these chimeric genes has been used to design PCR primers to identify diVerent cytoplasms in B. napus (Wei et al. 2005), onion Engelke et al. 2003), radish (Kim et al. 2007;Lee et al. 2009) and other plant species (Engelke and Tatlioglu 2002;Cheng et al. 2009). ...
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.
Vegetables are the integral part of our daily diet as they are loaded with various phytonutrients and considered as an important component for nutritional security and alleviating malnutrition. There is a huge demand of vegetables to fulfil the requirement of ever-growing population. This can be achieved only through vertical expansion by enhancing productivity. In this respect exploitation of heterosis and hybrid breeding will be a feasible and sustainable intervention. Vegetable crops exhibit robust and durable hybrid vigour, and exploitation of heterosis is the important way forward in their genetic improvement. Production of hybrid seeds in a commercial scale and cost-effective way, pollination control mechanism like male sterility system is considered as robust and proven arsenal in vegetables. Among the various types of male sterility mechanism available in plant species, cytoplasmic male sterility is the most extensively utilized in different vegetable crops. In this chapter, we thoroughly documented the various available male sterility mechanisms; their potential utilization in F1 hybrid seed production; sterilizing cytoplasm, their sources and diversification; fertility restoration mechanism, inheritance, molecular mapping and cloning of Rf elements; recent advances in deciphering male sterility determinants; role of marker-assisted selection in hybrid breeding utilizing CMS system; and adoption of modern omics technology to elucidate the CMS systems in vegetables. Moreover, this chapter also summarizes the current status and future strategies of utilization and improvement of the available CMS systems in various vegetables.KeywordsVegetablesHybrid seed productionCMS systemFertility restoration
Male sterility in plant is a reproductive physio-morphological inability to articulate incompetent or inoperative male reproductive parts in flowers deterring sexual reproduction. The phenomenon of male sterility in plants is governed by both genetic and/or environmental conditions, which results in plant reproductive biology abnormalities ranging from hampered microsporogenesis, non-functional stamens to production/release of viable pollens. Existence of male sterility has been reported in many cereals, pulses, oilseeds and horticultural crops. Nevertheless, male sterility is a constraint for natural gene flow in plants. But the phenomenon of natural male reproductive inability of the plants is a gift to the breeders to harness the heterosis through hybrid technology. Heterosis is the manifestation of higher vigour in the offspring pertinent to the yield and its attributing traits, resistance to biotic and abiotic stresses and enhanced nutritional quality traits. Several reports are available in cereals, pulses, oilseeds and in horticultural crops, stating the yield plateau due to repeated cultivation of inbreed varieties in spite of being cultivated using the best management practices. In this regard, hybrid technology is the way ahead to break the yield plateau and achieve sustainable productivity increase and resilience in different agricultural crops.KeywordsBreedingFertility restorationHeterosisHybridMale sterility
Hybrid production using lines with cytoplasmic male sterility (CMS) has become an important way to utilize heterosis in vegetables. Ogura CMS, with the advantages of complete pollen abortion, ease of transfer and a progeny sterility rate reaching 100%, is widely used in cruciferous crop breeding. The mapping, cloning, mechanism and application of Ogura CMS and fertility restorer genes in Brassica napus, Brassica rapa, Brassica oleracea and other cruciferous crops are reviewed herein, and the existing problems and future research directions in the application of Ogura CMS are discussed.
Radish is an important vegetable crop that is widely distributed throughout the world. Because of the long cultivation history, radish is morphologically diverse and a large number of cultivars with widely varying morphology, especially in root shapes, sizes, skin, and flesh colors, are produced. According to the morphology of its edible root and its intended uses, cultivated radishes are classified into five main varieties. Radish has a wide range of genetic diversity which provides a tremendous scope for genetic improvement of economic traits. Studies on DNA polymorphisms and genetic diversity of radish help us to determine the genetic relationship between radish cultivars and to understand the origin and evolution of this species. In the past two decades, various molecular tools provide easy, less laborious means for identifying the DNA polymorphisms and genetic diversity of radish. Here, we review recent research on the DNA polymorphisms, including cytoplasmic and nuclear genomes, summarize the genetic relationship between wild and cultivated radishes, and discuss the origin and evolution of Raphanus according to their genetic diversity.
Key message
We utilized a combination of BSA and RNA-Seq to identify SNPs linked to the
Rfd1
locus, a restorer-of-fertility gene in radish. A high-density linkage map was constructed using this approach.
Abstract
Male fertility of cytoplasmic male sterility conditioned by the Dongbu cytoplasmic and genic male-sterility cytoplasm can be restored by a restorer-of-fertility locus, Rfd1, in radish. To construct a high-density linkage map and to identify a candidate gene for the Rfd1 locus, bulked segregant analysis and RNA-seq approaches were combined. A total of 26 and 28 million reads produced from male-fertile and male-sterile bulked RNA were mapped to the radish reference unigenes. After stringent screening of SNPs, 327 reliable SNPs of 109 unigenes were selected. Arabidopsis homologs for 101 of the 109 genes were clustered around the 4,000 kb region of Arabidopsis chromosome 3, which was syntenic to the Rfd1 flanking region. Since the reference unigene set was incomplete, the contigs were de novo assembled to identify 134 contigs harboring SNPs. Most of SNP-containing contigs were also clustered on the same syntenic region in Arabidopsis chromosome. A total of 21 molecular markers positioned within a 2.1 cM interval including the Rfd1 locus were developed, based on the selected unigenes and contigs. A segregating population consisting of 10,459 individuals was analyzed to identify recombinants containing crossovers within this interval. A total of 284 identified recombinants were then used to construct a high-density map, which delimited the Rfd1 locus into an 83-kb syntenic interval of Arabidopsis chromosome 3. Since no candidate gene, such as a pentatricopeptide repeat (PPR)-coding gene, was found in this interval, 231 unigenes and 491 contigs containing putative PPR motifs were analyzed further, but no PPR gene in linkage disequilibrium with the Rfd1 locus could be found.
Brassicaceae crops display strong hybrid vigor, and have long been subject to F1 hybrid breeding. Because the most reliable system of F1 seed production is based on cytoplasmic male sterility (CMS), various types of CMS have been developed and adopted in practice to breed Brassicaceae oil seed and vegetable crops. CMS is a maternally inherited trait encoded in the mitochondrial genome, and the male sterile phenotype arises as a result of interaction of a mitochondrial CMS gene and a nuclear fertility restoring (Rf) gene. Therefore, CMS has been intensively investigated for gaining basic insights into molecular aspects of nuclear-mitochondrial genome interactions and for practical applications in plant breeding. Several CMS genes have been identified by molecular genetic studies, including Ogura CMS from Japanese radish, which is the most extensively studied and most widely used. In this review, we discuss Ogura CMS, and other CMS systems, and the causal mitochondrial genes for CMS. Studies on nuclear Rf genes and the cytoplasmic effects of alien cytoplasm on general crop performance are also reviewed. Finally, some of the unresolved questions about CMS are highlighted.
A novel cytoplasmic male sterility (CMS) conferred by Dongbu cytoplasmic and genic male-sterility (DCGMS) cytoplasm and its restorer-of-fertility gene (Rfd1) was previously reported in radish (Raphanus sativus L.). Its inheritance of fertility restoration and profiles of mitochondrial DNA (mtDNA)-based molecular markers were reported to be different from those of Ogura CMS, the first reported CMS in radish. The complete mitochondrial genome sequence (239,186 bp; GenBank accession No. KC193578) of DCGMS mitotype is reported in this study. Thirty-four protein-coding genes and three ribosomal RNA genes were identified. Comparative analysis of a mitochondrial genome sequence of DCGMS and previously reported complete sequences of normal and Ogura CMS mitotypes revealed various recombined structures of seventeen syntenic sequence blocks. Short-repeat sequences were identified in almost all junctions between syntenic sequence blocks. Phylogenetic analysis of three radish mitotypes showed that DCGMS was more closely related to the normal mitotype than to the Ogura mitotype. A single 1,551-bp unique region was identified in DCGMS mtDNA sequences and a novel chimeric gene, designated orf463, consisting of 128-bp partial sequences of cox1 gene and 1,261-bp unidentified sequences were found in the unique region. No other genes with a chimeric structure, a major feature of most characterized CMS-associated genes in other plant species, were found in rearranged junctions of syntenic sequence blocks. Like other known CMS-associated mitochondrial genes, the predicted gene product of orf463 contained 12 transmembrane domains. Thus, this gene product might be integrated into the mitochondrial membrane. In total, the results indicate that orf463 is likely to be a casual factor for CMS induction in radish containing the DCGMS cytoplasm.
Previously, novel cytoplasmic male-sterility (CMS) caused by DCGMS cytoplasm was discovered in radish (Raphanus sativus L.) introduced from Uzbekistan. We performed extensive progeny tests and identified two fertility restorer lines (‘R171’
and ‘R121’) for this new CMS. Two F1 hybrid populations were self-pollinated and backcrossed to produce F2 and BC populations. Inheritance patterns of male-sterility in segregating populations varied depending on paternal lines.
Segregation of male-sterility in F2 populations originating from the cross between MS19 and R121 showed that a single locus was involved in fertility restoration.
However, populations originating from the cross between MS15 and R171 showed the involvement of more than one restorer-of-fertility
genes. The single fertility restorer locus identified in the cross between MS19 and R121 was designated Rfd1 locus. Bulked segregant analysis was performed using RAPD and AFLP, which identified one marker each. Both RAPD and AFLP
markers were converted into simple PCR-based co-dominant markers after their isolated flanking sequences were analyzed. Indels
773-bp and 67-bp in length were identified between two Rfd1 allele-linked flanking sequences of the RAPD and AFLP fragments, respectively, then utilized to develop simple PCR markers.
In addition, we prove that the newly identified Rfd1 locus is independent of the Rfo locus, another radish fertility restorer for CMS caused by Ogura cytoplasm.
KeywordsRadish (Raphanus sativus L.)-Cytoplasmic male-sterility-Restorer-of-fertility genes-Molecular markers-DCGMS cytotype
Cytoplasmic male sterility caused by Dongbu cytoplasmic and genic male-sterility (DCGMS) cytoplasm and its nuclear restorer-of-fertility locus (Rfd1) with a linked molecular marker (A137) have been reported in radish (Raphanus sativus L.). To construct a linkage map of the Rfd1 locus, linked amplified fragment length polymorphism (AFLP) markers were screened using bulked segregant analysis. A 220-bp linked AFLP fragment sequence from radish showed homology with an Arabidopsis coding sequence. Using this Arabidopsis gene sequence, a simple PCR marker (A220) was developed. The A137 and A220 markers flanked the Rfd1 locus. Two homologous Arabidopsis genes with both marker sequences were positioned on Arabidopsis chromosome-3 with an interval of 2.4 Mb. To integrate the Rfd1 locus into a previously reported expressed sequence tag (EST)-simple sequence repeat (SSR) linkage map, the radish EST sequences located in three syntenic blocks within the 2.4-Mb interval were used to develop single nucleotide polymorphism (SNP) markers for tagging each block. The SNP marker in linkage group-2 co-segregated with male fertility in an F(2) population. Using radish ESTs positioned in linkage group-2, five intron length polymorphism (ILP) markers and one cleaved amplified polymorphic sequence (CAPS) marker were developed and used to construct a linkage map of the Rfd1 locus. Two closely linked markers delimited the Rfd1 locus within a 985-kb interval of Arabidopsis chromosome-3. Synteny between the radish and Arabidopsis genomes in the 985-kb interval were used to develop three ILP and three CAPS markers. Two ILP markers further delimited the Rfd1 locus to a 220-kb interval of Arabidopsis chromosome-3.
To gain further insight into the abortive stages and ultrastructural changes leading to pollen degeneration of a novel cytoplasmic male sterile radish 805A, we compared differences of cellular and subcellular structure of sterile anther with fertile anther by light and electron microscopy analysis. Two types of locule degeneration in sterile anther were detected, of which the time of degeneration occurred and completed was different. In type I, abnormality of pollen mother cells (PMCs) and tapetal cells, including condensation of cytoplasm and large vacuoles within tapetal cells, was shown at PMC stage. In type II, meiosis and early tetrad stage progressed normally except for large vacuoles that appeared in tapetal cells. Ultrastructural alterations of the cellular organization were observed in the type II locules, such as chromatin condensation at the periphery of the nucleus and degeneration of the karyotheca, compared with normal pollen development. The results suggested that the cytoplasmic male sterility anther degeneration was probably caused by dysfunctions of tapetum and vacuolation of tapetum, PMCs, and microspores. Thus, the identical factors, which induced CMS in the same cytoplasmic and nuclear genetic background, might affect development of tapetum and microspore at different stages during the cytoplasmic male sterile 805A anther development.
Spontaneous reversion to fertility was studied in the progeny of a cytoplasmic male-sterile (CMS) Brassica napus cybrid containing recombinant B. napus/Ogura radish mitochondrial genomes. This reversion is concomitant with the disappearance of a 2.5 kb NcoI fragment present in the mitochondrial DNA of Ogura radish, and of CMS cybrids derived from plants carrying Ogura cytoplasm, and absent in the mitochondrial genome of normal Brassicas and fertile cybrids. This specific fragment hybridizes to a 1.4 kb transcript found only in male-sterile plants bearing an Ogura derived cytoplasm.
Several descriptive models have been proposed to explain the occurrence of deletion-duplication events in the plant mitochondrial genome. In order to investigate the dynamics of these events, we have simulated them using a computer model. The simulation shows that whatever the recombination rates between repeats, if a mitochondrial sequence becomes unnecessary for the proper function of mitochondria, this sequence can be deleted and another sequence can be duplicated in consequence. Furthermore, the model shows that the organization of the sequences with respect to the origin of replication has a great influence over the dynamics of the deletion-duplication events.
A PCR analysis of mitochondrial (mt) genomes of cybrid rapeseed plants revealed substoichiometric concentrations of molecules bearing different configurations of the gene (orf138) responsible for Ogura cytoplasmic male sterility (CMS). These substoichiometric molecules are also present in plants bearing the unmodified Ogura cytoplasm. In one cybrid family, which shows reversion of the male sterile phenotype, we observed changes in the respective proportions of these molecules. The phenotypic (sterility-fertility) reversion occurs as a result of a modification of the equilibrium state between the different forms of the orf138 gene and is very probably determined by the level of expression of this gene. Stable situations are always characterized by one predominant form; the others, when present, exist in substoichiometric amounts. We report results indicating that the different forms of the orf138 gene are continuously interconverted by recombination and that an active mechanism is involved in the maintenance of some substoichiometric molecules.
The plant mitochondrial genome is characterized by a complex, multipartite structure. In cytoplasmic male-sterile (CMS) common bean, the sterility-inducing mitochondrial configuration maps as three autonomous DNA molecules, one containing the sterility-associated sequence pvs-or f 239. We constructed a physical map of the mitochondrial genome from the direct progenitors to the CMS cytoplasm and have shown that it maps as a single, circular master configuration. With long-exposure autoradiography of DNA gel blots and polymerase chain reaction analysis, we demonstrate that the three-molecule CMS-associated configuration was present at unusually low copy number within the progenitor genome and that the progenitor form was present substoichiometrically within the genome of the CMS line. Furthermore, upon spontaneous reversion to fertility, the progenitor genomic configuration as well as the molecule containing the pvs-or f 239 sterility-associated sequence were both maintained at substoichiometric levels within the revertant genome. In vitro mitochondrial incubation results demonstrated that the genomic shift of the pvs-or f 239-containing molecule to substoichiometric levels upon spontaneous reversion was a reversible phenomenon. Moreover, we demonstrate that substoichiometric forms, apparently silent with regard to gene expression, are transcriptionally and translationally active once amplified. Thus, copy number suppression may serve as an effective means of regulating gene expression in plant mitochondria.
The recombination and copy number shifting activities of the plant mitochondrial genome are widely documented across plant genera, but these genome processes have not been as well examined with regard to their roles in plant evolution. Because of the extensive plant collections of Phaseolus spp and the degree to which cytoplasmic male sterility (cms) has been characterized in the common bean, this system would be valuable for investigating mitochondrial genome dynamics in natural populations. We have used the cms-associated sequence pvs-orf239 as a mitochondrial genetic marker for these studies and have demonstrated its universal presence throughout a diversity of undomesticated Phaseolus lines. Within these populations, the pvs-orf239 sequence is present in high copy number in approximately 10% of the lines, but substoichiometric in all others. This mitochondrial sequence, derived apparently by at least two recombination events, is well conserved with two point mutations identified that are both apparently silent with regard to the sterility phenotype. A putative progenitor sequence was identified in Phaseolus glabelus in substoichiometric levels, suggesting that the present-day pvs-orf239 sequence was likely introduced substoichiometrically. Copy number shifting within the mitochondrial genome results in a 1000- to 2000-fold change, so that substoichiometric forms are estimated at less than one copy per every 100 cells. On the basis of PCR analysis of root tips, we postulate that a mitochondrial "transmitted form" resides within the meristem to assure transmission of a complete genetic complement to progeny.
Restoration of pollen fertility to cytoplasmic male-sterile common bean by nuclear gene Fr is accompanied by mitochondrial (mt) DNA rearrangements within restored plants. These rearrangements are also observed upon spontaneous cytoplasmic reversion to fertility. An mtDNA fragment of at least 25 kilobases was lost from the genome upon restoration or reversion. This fragment contained DNA segments that were not repeated elsewhere in the genome and, therefore, were not detected within the genome upon fertility restoration. This result suggested that the particular mtDNA configuration absent from restored plants could not be maintained by a constant process of recombination but rather by autonomous replication. No evidence of excision of this region from the mt genome, in the form of a junction fragment associating flanking DNA regions, was detected in fertile restored plants. DNA gel blot hybridization of this mtDNA region, compared with hybridization to related regions of the mitochondrial genome that shared sequence homology, indicated that the mtDNA region associated with sterility was present in lower copy number. These observations, as well as the occurrence of similar or identical rearrangements upon spontaneous cytoplasmic reversion, indicate that the restoration of pollen fertility may be accompanied by loss of an independently replicating subgenomic DNA molecule from the mitochondrial genome.
The entire mitochondrial genome of rice (Oryza sativa L.), a monocot plant, has been sequenced. It was found to comprise 490,520 bp, with an average G+C content of 43.8%. Three rRNA genes, 17 tRNA genes and five pseudo tRNA sequences were identified. In addition, eleven ribosomal protein genes and two pseudo ribosomal protein genes were found, which are homologous to 13 of the 16 genes for ribosomal proteins in the mitochondrial genome of the liverwort (Marchantia polymorpha). A greater degree of variation in terms of presence/absence and integrity of genes was observed among the ribosomal protein genes and tRNA genes of rice, Arabidopsis and sugar beet. Transcription and post-transcriptional modification (RNA editing) in the rice mitochondrial sequence were also examined. In all, 491 Cs in the genomic DNA were converted to Ts in cDNA. The frequency of RNA editing differed markedly depending upon the ORF considered. Sequences derived from plastid and nuclear genomes make up 6.3% and 13.4% of the mitochondrial genome, respectively. The degree of conservation of plastid sequences in the mitochondrial genome ranged from 61% to 100%, suggesting that sequence migration has occurred very frequently. Three plastid DNA fragments that were incorporated into the mitochondrial genome were subsequently transferred to the nuclear genome. Nineteen fragments that were similar to transposon or retrotransposon sequences, but different from those found in the mitochondrial genomes of dicots, were identified. The results indicate frequent and independent DNA sequence flow to and from the mitochondrial genome during the evolution of flowering plants, and this may account for the range of genetic variation observed between the mitochondrial genomes of higher plants.
Cytoplasmic male sterility (CMS) in plants is a classical example of genomic conflict, opposing maternally-inherited cytoplasmic genes (mitochondrial genes in most cases), which induce male sterility, and nuclear genes, which restore male fertility. In natural populations, this type of sex control leads to gynodioecy, that is, the co-occurrence of female and hermaphroditic individuals within a population. According to theoretical models, two conditions may maintain male sterility in a natural population: (1) female advantage (female plants are reproductively more successful than hermaphrodites on account of their global seed production); (2) the counter-selection of nuclear fertility restorers when the corresponding male-sterility-inducing cytoplasm is lacking. In this review, we re-examine the model of nuclear-mitochondrial conflict in the light of recent experimental results from naturally occurring CMS, alloplasmic CMS (appearing after interspecific crosses resulting from the association of nuclear and cytoplasmic genomes from different species), and CMS plants obtained in the laboratory and carrying mitochondrial mutations. We raise new hypotheses and discuss experimental models that would take physiological interactions between cytoplasmic and nuclear genomes into account.
The plant mitochondrial genome is retained in a multipartite structure that arises by a process of repeat-mediated homologous recombination. Low-frequency ectopic recombination also occurs, often producing sequence chimeras, aberrant ORFs, and novel subgenomic DNA molecules. This genomic plasticity may distinguish the plant mitochondrion from mammalian and fungal types. In plants, relative copy number of recombination-derived subgenomic DNA molecules within mitochondria is controlled by nuclear genes, and a genomic shifting process can result in their differential copy number suppression to nearly undetectable levels. We have cloned a nuclear gene that regulates mitochondrial substoichiometric shifting in Arabidopsis. The CHM gene was shown to encode a protein related to the MutS protein of Escherichia coli that is involved in mismatch repair and DNA recombination. We postulate that the process of substoichiometric shifting in plants may be a consequence of ectopic recombination suppression or replication stalling at ectopic recombination sites to effect molecule-specific copy number modulation.
Ogura cytoplasmic male sterility (CMS) in radish (Raphanus sativus) is caused by an aberrant mitochondrial gene, Orf138, that prevents the production of functional pollen without affecting female fertility. Rfo, a nuclear gene that restores male fertility, alters the expression of Orf138 at the post-transcriptional level. The Ogura CMS/Rfo two-component system is a useful model for investigating nuclear-cytoplasmic interactions, as well as the physiological basis of fertility restoration. Using a combination of positional cloning and microsynteny analysis of Arabidopsis thaliana and radish, we genetically and physically delimited the Rfo locus to a 15-kb DNA segment. Analysis of this segment shows that Rfo is a member of the pentatricopeptide repeat (PPR) family. In Arabidopsis, this family contains more than 450 members of unknown function, although most of them are predicted to be targeted to mitochondria and chloroplasts and are thought to have roles in organellar gene expression.
Apart from their agronomic importance in hybrid seed production, mutations that encode cytoplasmic male sterility (CMS) provide a means to probe the role of the mitochondrion in reproductive development. Fertility restorers are examples of nuclear genes that affect cytoplasmic gene expression, and
Strong evidence exists for polyploidy having occurred during the evolution of the tribe Brassiceae. We show evidence for the dynamic and ongoing diploidization process by comparative analysis of the sequences of four paralogous Brassica rapa BAC clones and the homologous 124-kb segment of Arabidopsis thaliana chromosome 5. We estimated the times since divergence of the paralogous and homologous lineages. The three paralogous subgenomes of B. rapa triplicated 13 to 17 million years ago (MYA), very soon after the Arabidopsis and Brassica divergence occurred at 17 to 18 MYA. In addition, a pair of BACs represents a more recent segmental duplication, which occurred approximately 0.8 MYA, and provides an exception to the general expectation of three paralogous segments within the B. rapa genome. The Brassica genome segments show extensive interspersed gene loss relative to the inferred structure of the ancestral genome, whereas the Arabidopsis genome segment appears little changed. Representatives of all 32 genes in the Arabidopsis genome segment are represented in Brassica, but the hexaploid complement of 96 has been reduced to 54 in the three subgenomes, with compression of the genomic region lengths they occupy to between 52 and 110 kb. The gene content of the recently duplicated B. rapa genome segments is identical, but intergenic sequences differ.
X-irradiated protoplasts of a Brassica napus line carrying the Ogura Raphanus sativus male sterile cytoplasm were fused to protoplasts of male fertile B. napus cv. Olga. Plants were regenerated from six out of 34 randomly selected clones. In one clone, Bn(RS)26, a plant with male sterile flowers was obtained. Mitochondria of this plant are non-parental as revealed by DNA-DNA hybridization using a species specific probe. Its chloroplasts, however, derive from the fertile parent which results in loss of the sensitivity to low temperatures associated with R. sativus plastids in the male sterile parent. The novel cytoplasm of the Bn(RS)26 cybrid was transmitted through seed.
Cytoplasmic differences between male-fertile and male-sterile Brassica napus as well as Raphanus sativus were investigated. Plastids of the male-fertile B. napus were found to differ from those of male-sterile B. napus and R. sativus with respect to DNA restriction enzyme patterns. Differences between male-fertile and male-sterile B. napus mitochondria were detected not only in the restriction fragment patterns of their DNA, but also at the level of expression by in organello translation of mitochondrial polypeptides.
The chlorophyll deficiency obtained upon transferral of the male-sterility-conferring radish cytoplasm to a winter variety of B. napus had been corrected earlier through protoplast fusion. The cytoplasmic composition of the corrected lines was analysed using DNA restriction analysis and in organello translation. The stability of the recombined cytoplasm in the corrected lines was confirmed by analysis of the subsequent seed-derived generation.
To establish a cytoplasmic male-sterile/restored fertility (cms-Rf) system for F1 seed production in Brassica napus, we transferred a gene from fertillity restored radish to B. napus by protoplast fusion. X-irradiated protoplasts, isolated from shoots of Raphanus sativus cv Kosena (Rf line), were fused with iodoacetamide-treated protoplasts of a B. napus cms cybrid. Among 300 regenerated plants, six were male-fertile. The fertile plants were characterized for petal color, chromosome number and the percentage of viable pollen grains. Three fertile plants had aneuploid chromosome numbers and white or cream petals, which is a dominant marker in radish. Of these three plants, one which had 2n = 47 chromosomes and white petals was used for further backcrosses. After two backcrosses, chromosome number and petal color became identical to that of B. napus. No female sterility was observed in the BC3 generations.
'BLAST 2 SEQUENCES', a new BLAST-based tool for aligning two protein or nucleotide sequences, is described. While the standard BLAST program is widely used to search for homologous sequences in nucleotide and protein databases, one often needs to compare only two sequences that are already known to be homologous, coming from related species or, e.g. different isolates of the same virus. In such cases searching the entire database would be unnecessarily time-consuming. 'BLAST 2 SEQUENCES' utilizes the BLAST algorithm for pairwise DNA-DNA or protein-protein sequence comparison. A World Wide Web version of the program can be used interactively at the NCBI WWW site (http://www.ncbi.nlm.nih.gov/gorf/bl2.html). The resulting alignments are presented in both graphical and text form. The variants of the program for PC (Windows), Mac and several UNIX-based platforms can be downloaded from the NCBI FTP site (ftp://ncbi.nlm.nih.gov).
The study of Brassica napus L. plants carrying restorer genes introgressed from radish (Raphanus sativus L.) showed that these genes ensured restoration of male fertility in rapeseed for all the male sterility-inducing cytoplasm studied, i.e., "Ogura"-type cytoplasm and that of four cybrids obtained by protoplast fusion. Plants with high levels of restored male fertility were obtained. However, the introduction of restorer genes was accompanied by a large decrease in seed set. Observations of embryo sacs inside the ovules and correlation between number of seeds per pod and percentage of octonucleate embryo sacs indicated that low seed set could be attributed to a high rate of embryo sac abortion, mainly at the uninucleate stage. Introduction of too much radish genetic information was assumed to be the cause of this low female fertility. Female fertility must be improved before the restored material can be used for F1 hybrid rapeseed production.Key words: Brassica napus, Raphanus sativus, cytoplasmic male sterility, restorer, cybrid, female fertility, embryo sac.
Rapeseed plants have been regenerated after fusion between protoplasts bearing cytoplasms of different genera. Cybrids combine, in a first experiment Brassica napus chloroplasts and a cytoplasmic male sterility (cms) trait coming from Raphanus sativus, in a second experiment chloroplasts of a triazine resistant Brassica campestris and cms trait from Raphanus sativus. Transfer of chloroplasts has been confirmed by restriction cp DNA analysis and two dimensional thylakod protein electrophoresis. These plants may be very useful for Brassica hybrid seed production.
In order to further investigate sequences that are responsible for low-frequency recombination in plant mitochondrial DNAs
and RNA editing in radish mitochondria, thenad3/rps12 locus has been isolated and characterized from a normal cultivar of radish and the male-sterile Ogura cytoplasm. A repeated
sequence that has been implicated in other radish mitochondrial DNA rearrangements was identified at the breakpoint between
the two loci indicating that it was also involved in thenad3/rps12 rearrangement. Similar to some other radish mitochondrial genes,nad3/rps12 genomic sequences already contain several, but not all, of the bases that are typically edited in plant mitochondrialnad3 andrps12 genes. Analysis ofnad3/rps12 cDNAs indicated that the mRNAs are not edited. One partially edited transcript was identified out of the twenty two that
were examined. This finding, along with the observation thatnad3/rps12 RNAs are present at very low levels, raises the possibility that radish mitochondria may not encode functional copies of
these genes. Consistent with this hypothesis, DNA-blot analysis detectsnad3/rps12 sequences in the nucleus.
Alterations to mitochondrial genomes containing atpA in Brassica napus cybrids and their descendants were previously reported. We now have examined the possible causes of the alteration of the mt genomes in cybrid progeny. Alterations in the atpA fragments were transmitted to the BC3 generation of a B. napus cybrid. A comparison of the restriction maps of the BamHI fragments indicated that the alterations to the genomes were caused by stoichiometrical changes in the existing molecules rather than by recombinations. To confirm the supposed low copy number of the molecules, we used DNA-DNA hybridization to investigate the mitochondrial DNA of BC2 and BC3 plants of a B. napus cybrid. atpA did not hybridize to the radish-specific 4.0 kb BamHI fragment in the cybrid or it progeny. Both plants also underwent the polymerase chain reaction (PCR) with radish-specific oligonucleotides as the primers. Although no hybridization signal was found for radish atpA, an expected 700 bp fragment was amplified by the radish-specific primer, evidence that both plants have a subgenome with the radish atpA region which is below the limits of Southern hybridization detection.
The structure of mitochondrial DNA (mtDNA) from cultured cells of the liverwort, Marchantia polymorpha, was analyzed by pulsed-field gel electrophoresis (PFGE) and moving pictures of the fluorescently labeled molecules. Previous electron microscopic analysis with this liverwort revealed a unique property among land plants: mtDNA circles of only one size, that of the 186 kb genome, with no subgenomic circles. Most of the mtDNA was immobile in PFGE and contained complex structures, larger than the genome size with a bright fluorescent node and multiple attached fibers. The mobile mtDNA was mostly linear molecules in monomeric to pentameric lengths of the unit genome that increased following mung bean nuclease digestion, with a corresponding decrease in the immobile fraction. From 0 to 5% of the mtDNA was found as circular molecules the size of the genome and its oligomers; no subgenome-sized circles were present. Radiolabeling revealed that mtDNA synthesis began soon after transfer of cells to fresh medium and most newly replicated mtDNA was immobile; the circular form of the genome was not rapidly labeled.
A characteristic of CMS mutations in plants, in contrast to the single base changes in human mitochondrial mutants (49, 140), is the presence of chimeric genes or chimeric loci; different open reading frames are joined together, or placed in proximal locations and cotranscribed with standard mitochondrial genes. Despite much progress, and the identification of several mitochondrial loci that specify CMS, the molecular basis of this defect is not understood in any plant species. Observations of altered electron transport in Petunia and toxin-mediated membrane disruption in maize plants, bacteria, and yeast expressing the maize urf13 gene product, provide clues to possible mechanisms for disruption of pollen development. Whether disruption in a particular mitochondrial function is at the root of CMS in all species, or whether defects in numerous mitochondrial activities can produce sterility, will only be revealed by further probing of physiological and biochemical defects present in CMS genotypes.
The mitochondrial genome (mtDNA) organization from a fertile revertant line (V3) derived from the maize cytoplasmic male sterile type T (cmsT) callus tissue culture has been determined. We report that the sequence complexity can be mapped on to a circular "master chromosome" of 705 kb which includes a duplication of 165 kb of DNA when compared to its male sterile progenitor. Associated with this event is also a 0.423-kb deletion, which removed the cmsT-associated urf13 gene. As found for the maize normal type (N) and cmsT mitochondrial genomes, the V3 master chromosome also exists as a multipartite structure generated by recombination through repeated sequences.
Comparison of the modern fertile maize mitochondrial genome (N) with an ancestral maize mitochondrial genome (RU) reveals a 12 kb duplication (containing the atpA gene) in the modern genome that is absent from the ancestor. Cloning, mapping, and sequencing of the relevant portions of the ancestral genome shows that this duplication probably arose via a three-stage recombination process involving substoichiometric intermediates. Comparison with analogous observations on yeast mitochondrial genomes suggests that this three-stage model of genome reorganization can be generally applied to plant mitochondrial genomes to explain both deletions and the creation of novel repeats, common features of plant mitochondrial genome evolution.
Intraspecific variation was examined among 25 mitochondrial DNAs (mtDNAs), representing between two and five lines of eight agriculturally important Brassica species. Each of the approximately 140 restriction sites surveyed was invariant within each species. Only two length polymorphisms, deletions of 700 bp and 100 bp in a Brassica nigra line, were detected. A single inversion polymorphism was found; this distinguished two different mtDNA populations within a single line of Brassica hirta. Approximately 60% of the mtDNA molecules in this line and in two other B. hirta lines were identical, whereas the other 40% of the molecules in the first line differed by a 62-kb inversion. Levels of within-species variability in mtDNA appear to be lower in Brassica than in other groups of plants. These mtDNA comparisons are in agreement with cpDNA studies regarding the maternal ancestry of three amphidiploid Brassica species. This agreement and others imply that the two cytoplasmic genomes must have shared a common, maternal mode of transmission throughout the history of the genus. Finally, analysis of a supercoiled fraction of mtDNA from cauliflower (Brassica oleracea) provides the strongest evidence yet in support of the multicircular model for plant mtDNAs.
Restriction mapping studies reveal that the mitochondrial genome of white mustard (Brassica hirta) exists in the form of a single circular 208 kb chromosome. The B. hirta genome has only one copy of the two sequences which, in several related Brassica species, are duplicated and undergo intramolecular recombination. This first report of a plant mitochondrial DNA that does not exist in a multipartite structure indicates that high frequency intramolecular recombination is not an obligatory feature of plant mitochondrial genomes. Heterologous filter hybridizations reveal that the mitochondrial genomes of B. hirta and B. campestris have diverged radically in sequence arrangement, as the result of approximately 10 large inversions. At the same time, however, the two genomes are similar in size, sequence content, and primary sequence.
The genome sizes of mitochondrial DNA from darkgrown (etiolated) shoots of several higher plants were determined by reassociation kinetics and restriction analysis. Kinetic complexities obtained from reassociation kinetics measured spectrophotometrically indicate a mitochondrial genome size of 1600 Md for muskmelon, 1000 Md for cucumber, 560 Md for zucchini squash and 220 Md for watermelon (four species in the cucurbit family), as well as 240 Md for pea and 320 Md for corn. The kinetic curves also reveal the presence (except in corn) of sequences of a few magadaltons of complexity, reiterated about 10-50 times and representing 5%- 10% of the DNA in each mitochondrial genome. Molecular weight summation of fragments resulting from digestion with restriction endonucleases Sal I and Kpn I give genome size estimates similar to those obtained from reassociation kinetics, except for muskmelon and cucumber, for which the large number of fragments of similar size limits our estimate to at least 500 Md. The number of mitochondrial genomes per diploid cell is estimated to be about 110 to 140 for muskmelon, zucchini and watermelon. We consider the possible evolutionary mechanisms by which the mitochondrial genome has grown within the cucurbit family and the possible reasons for the existance of a seven to eight-fold range in mitochondrial genome size among such closely related species.
Transcription of a putative mitochondrial gene (orf138) has previously been correlated with Ogura cytoplasmic male-sterility (CMS) in rapeseed cybrids. In this paper, studies performed on a Brassica cybrid with a different organization of the orf138 locus confirm this association. We also show that mitochondria isolated from male-sterile rapeseed plants synthesize a polypeptide of 19 kDa, which is absent in fertile revertants. Antibodies against a glutathione S-transferase-ORF138 fusion protein were raised to establish that this 19 kDa polypeptide is the product of orf138. The anti-ORF138 serum was used to demonstrate that the orf138 translation product occurs only in sterile cybrids and co-purifies with the mitochondrial membrane fraction.
Analyses of mitochondrial transcription and in organello translation were performed with the Brassica tournefortii cytoplasm. This cytoplasm causes alloplasmic male sterility when combined with the nuclear genomes of B. napus and B. juncea. Mitochondrial RNA and protein banding patterns were compared between the fertile wild species B. tournefortii, an alloplasmic male-sterile B. juncea line, an alloplasmic male-sterile B. napus line and an alloplasmic B. napus line with restored fertility. The analyses were carried out to identify differences in gene expression and to investigate whether alterations in gene expression accompanied male sterility. A difference in transcription patterns between the fertile B. tournefortii and the alloplasmic lines was found for the atp6 gene. The atp6 region was investigated further, since a similar alteration in atp6 transcription has been observed in two other Brassica cytoplasms which are associated with cytoplasmic male sterility (CMS). The additional longer atp6 transcript detected in the alloplasmic lines in the present study was found to contain an open reading frame (ORF) located downstream of the atp6 gene. DNA sequencing revealed that the ORF, orf263, could encode a protein with a predicted molecular weight of about 29 kDa. In organello analysis detected two proteins of 29 and 32 kDa respectively, which were found only in the alloplasmic lines. Furthermore, the 32 kDa protein accompanied male sterility since it was absent in alloplasmic plants restored to fertility. The protein analysis might indicate that orf263 is translated and causes CMS.
We have determined the complete sequence of the mitochondrial DNA in the model plant species Arabidopsis thaliana, affording access to the first of its three genomes. The 366,924 nucleotides code for 57 identified genes, which cover only 10% of the genome. Introns in these genes add about 8%, open reading frames larger than 100 amino acids represent 10% of the genome, duplications account for 7%, remnants of retrotransposons of nuclear origin contribute 4% and integrated plastid sequences amount to 1%-leaving 60% of the genome unaccounted for. With the significant contribution of duplications, imported foreign DNA and the extensive background of apparently functionless sequences, the mosaic structure of the Arabidopsis thaliana mitochondrial genome features many aspects of size-relaxed nuclear genomes.
'BLAST 2 Sequences', a new BLAST-based tool for aligning two protein or nucleotide sequences, is described. While the standard BLAST program is widely used to search for homologous sequences in nucleotide and protein databases, one often needs to compare only two sequences that are already known to be homologous, coming from related species or, e.g. different isolates of the same virus. In such cases searching the entire database would be unnecessarily time-consuming. 'BLAST 2 Sequences' utilizes the BLAST algorithm for pairwise DNA-DNA or protein-protein sequence comparison. A World Wide Web version of the program can be used interactively at the NCBI WWW site (http://www.ncbi.nlm.nih.gov/gorf/bl2.++ +html). The resulting alignments are presented in both graphical and text form. The variants of the program for PC (Windows), Mac and several UNIX-based platforms can be downloaded from the NCBI FTP site (ftp://ncbi.nlm.nih.gov).
We determined the complete nucleotide sequence of the mitochondrial genome of an angiosperm, sugar beet (Beta vulgaris cv TK81-O). The 368 799 bp genome contains 29 protein, five rRNA and 25 tRNA genes, most of which are also shared by the
mitochondrial genome of Arabidopsis thaliana, the only other completely sequenced angiosperm mitochondrial genome. However, four genes identified here (namely rps13, trnF-GAA, ccb577 and trnC2-GCA) are missing in Arabidopsis mitochondria. In addition, four genes found in Arabidopsis (ccb228, rpl2, rpl16 and trnY2-GUA) are entirely absent in sugar beet or present only in severely truncated form. Introns, duplicated sequences, additional
reading frames and inserted foreign sequences (chloroplast, nuclear and plasmid DNA sequences) contribute significantly to
the overall size of the sugar beet mitochondrial genome. Nevertheless, 55.6% of the genome has no obvious features of information.
We identified a novel tRNACys gene (trnC2-GCA) which shows no sequence homology with any tRNACys genes reported so far in higher plants. Intriguingly, this tRNA gene is actually transcribed into a mature tRNA, whereas
the native tRNACys gene (trnC1-GCA) is most likely a pseudogene.
Mapping predicts that the mitochondrial genome of the liverwort Marchantia polymorpha exists as a circular molecule, although nearly all the mitochondrial DNA (mtDNA) is found as genome-sized and multigenomic molecules in linear and branched form. We used restriction enzymes with one recognition site per genome, end-specific exonucleases and pulsed-field gel electrophoresis (PFGE) to analyze the arrangement of genomic units and the terminal structure of the molecules. We find a head-to-tail arrangement in the concatemers and circular permutation in both the monomeric and multigenomic molecules. The termini contain covalently bound protein at the 5' end and an open (unblocked) 3' end. We find that the standard in-gel procedure used to prepare large DNA molecules for PFGE may introduce extraction artifacts leading to erroneous conclusions about the termini. These artifacts can be reduced by omitting high salt (high EDTA) and protease during mitochondrial lysis. Our results suggest that the mtDNA may use a T4 phage-like mechanism of replication and that the linear molecules may be due to strand breaks mediated by type II topoisomerase.
Conservation of gene order in vertebrates is evident after hundreds of millions of years of divergence, but comparisons of the Arabidopsis thaliana sequence to partial gene orders of other angiosperms (flowering plants) sharing common ancestry approximately 170-235 million years ago yield conflicting results. This difference may be largely due to the propensity of angiosperms to undergo chromosomal duplication ('polyploidization') and subsequent gene loss ('diploidization'); these evolutionary mechanisms have profound consequences for comparative biology. Here we integrate a phylogenetic approach (relating chromosomal duplications to the tree of life) with a genomic approach (mitigating information lost to diploidization) to show that a genome-wide duplication post-dates the divergence of Arabidopsis from most dicots. We also show that an inferred ancestral gene order for Arabidopsis reveals more synteny with other dicots (exemplified by cotton), and that additional, more ancient duplication events affect more distant taxonomic comparisons. By using partial sequence data for many diverse taxa to better relate the evolutionary history of completely sequenced genomes to the tree of life, we foster comparative approaches to the study of genome organization, consequences of polyploidy, and the molecular basis of quantitative traits.
Cytoplasmic male sterility (CMS) in plants is a maternally inherited inability to produce functional pollen, and is often associated with mitochondrial DNA abnormalities. Specific nuclear loci that suppress CMS, termed as restorers of fertility (Rf), have been identified. Previously, we identified an Rf for the CMS Kosena radish and used genetic analysis to identify the locus and create a contig covering the critical interval. To identify the Rf gene, we introduced each of the lambda and cosmid clones into the CMS Brassica napus and scored for fertility restoration. Fertility restoration was observed when one of the lambda clones was introduced into the CMS B. napus. Furthermore, introduction of a 4.7-kb BamHI/HpaI fragment of the lambda clone is enough to restore male fertility. A cDNA strand isolated from a positive fragment contained a predicted protein (ORF687) of 687 amino acids comprising 16 repeats of the 35-amino acid pentatricopeptide repeat (PPR) motif. Kosena CMS radish plants were found to express an allele of this gene possessing four substituted amino acids in the second and third repeats of the PPR suggesting that the domains formed by these repeats in ORF687 are essential for fertility restoration. Protein levels of the Kosena CMS-associated mitochondrial protein ORF125 were considerably reduced in plants in which fertility was restored, although mRNA expression was normal. Regarding the possible role for PPR-containing proteins in the regulation of the mitochondrial gene, we propose that ORF687 functions either directly or indirectly to lower the levels of ORF125, resulting in the restoration of fertility in CMS plants.
A single radish nuclear gene, Rfo, restores Ogura (ogu) cytoplasmic male sterility (CMS) in Brassica napus. A map-based cloning approach relying on synteny between radish and Arabidopsis was used to clone Rfo. A radish gene encoding a 687-amino-acid protein with a predicted mitochondrial targeting pre-sequence was found to confer male fertility upon transformation into ogu CMS B. napus. This gene, like the recently described Petunia Rf gene, codes for a pentatricopeptide repeat (PPR)-containing protein with multiple, in this case 16, PPR domains. Two similar genes that do not appear to function as Rfo flank this gene. Comparison of the Rfo region with the syntenic Arabidopsis region indicates that a PPR gene is not present at the Rfo-equivalent site in Arabidopsis, although a smaller and related PPR gene is found about 40 kb from this site. The implications of these findings for the evolution of restorer genes and other PPR encoding genes are discussed.
The entire mitochondrial genome of rapeseed (Brassica napus L.) was sequenced and compared with that of Arabidopsis thaliana. The 221 853 bp genome contains 34 protein‐coding genes, three rRNA genes and 17 tRNA genes. This gene content is almost
identical to that of Arabidopsis. However the rps14 gene, which is a pseudo‐gene in Arabidopsis, is intact in rapeseed. On the other hand, five tRNA genes are missing in rapeseed compared to Arabidopsis, although the set of mitochondrially encoded tRNA species is identical in the two Cruciferae. RNA editing events were systematically
investigated on the basis of the sequence of the rapeseed mitochondrial genome. A total of 427 C to U conversions were identified
in ORFs, which is nearly identical to the number in Arabidopsis (441 sites). The gene sequences and intron structures are mostly conserved (more than 99% similarity for protein‐coding regions);
however, only 358 editing sites (83% of total editings) are shared by rapeseed and Arabidopsis. Non‐coding regions are mostly divergent between the two plants. One‐third (about 78.7 kb) and two‐thirds (about 223.8 kb)
of the rapeseed and Arabidopsis mitochondrial genomes, respectively, cannot be aligned with each other and most of these regions do not show any homology
to sequences registered in the DNA databases. The results of the comparative analysis between the rapeseed and Arabidopsis mitochondrial genomes suggest that higher plant mitochondria are extremely conservative with respect to coding sequences
and somewhat conservative with respect to RNA editing, but that non‐coding parts of plant mitochondrial DNA are extraordinarily
dynamic with respect to structural changes, sequence acquisition and/or sequence loss.
Land plants exhibit a significant evolutionary plasticity in their mitochondrial DNA (mtDNA), which contrasts with the more conservative evolution of their chloroplast genomes. Frequent genomic rearrangements, the incorporation of foreign DNA from the nuclear and chloroplast genomes, an ongoing transfer of genes to the nucleus in recent evolutionary times and the disruption of gene continuity in introns or exons are the hallmarks of plant mtDNA, at least in flowering plants. Peculiarities of gene expression, most notably RNA editing and trans-splicing, are significantly more pronounced in land plant mitochondria than in chloroplasts. At the same time, mtDNA is generally the most slowly evolving of the three plant cell genomes on the sequence level, with unique exceptions in only some plant lineages. The slow sequence evolution and a variable occurrence of introns in plant mtDNA provide an attractive reservoir of phylogenetic information to trace the phylogeny of older land plant clades, which is as yet not fully resolved. This review attempts to summarize the unique aspects of land plant mitochondrial evolution from a phylogenetic perspective.
Tobacco is a valuable model system for investigating the origin of mitochondrial DNA (mtDNA) in amphidiploid plants and studying the genetic interaction between mitochondria and chloroplasts in the various functions of the plant cell. As a first step, we have determined the complete mtDNA sequence of Nicotiana tabacum. The mtDNA of N. tabacum can be assumed to be a master circle (MC) of 430,597 bp. Sequence comparison of a large number of clones revealed that there are four classes of boundaries derived from homologous recombination, which leads to a multipartite organization with two MCs and six subgenomic circles. The mtDNA of N. tabacum contains 36 protein-coding genes, three ribosomal RNA genes and 21 tRNA genes. Among the first class, we identified the genes rps1 and psirps14, which had previously been thought to be absent in tobacco mtDNA on the basis of Southern analysis. Tobacco mtDNA was compared with those of Arabidopsis thaliana, Beta vulgaris, Oryza sativa and Brassica napus. Since repeated sequences show no homology to each other among the five angiosperms, it can be supposed that these were independently acquired by each species during the evolution of angiosperms. The gene order and the sequences of intergenic spacers in mtDNA also differ widely among the five angiosperms, indicating multiple reorganizations of genome structure during the evolution of higher plants. Among the conserved genes, the same potential conserved nonanucleotide-motif-type promoter could only be postulated for rrn18-rrn5 in four of the dicotyledonous plants, suggesting that a coding sequence does not necessarily move with the promoter upon reorganization of the mitochondrial genome.
A major objective of breeders using the Ogu-INRA cytoplasmic male sterility (cms) system in rapeseed (Brassica napus L.) is to obtain double low restorer lines with a shorter introgression and a good agronomic value. The development of low glucosinolate content (low GC) restorer lines often occurs through the deletion of a part of the introgression. One of these lines has lost the radish Pgi-2 allele expression, without recovering that of the rapeseed Pgi-2 allele. This line shows a defect in the meiotic transmission of the restorer gene Rfo and a very poor agronomic value. We initiated a programme to force non-spontaneous recombination between this Rfo-carrying introgression and the rapeseed homologous chromosome from a low GC B. napus line. Gamma ray irradiation was used to induce chromosome breakage just prior meiosis aiming at just such a recombination. Low GC cms plants were crossed with the pollen of irradiated plants that were heterozygous for this introgression. The F(2) families were scored for their vigour, transmission rate of Rfo and female fertility. One family of plants, R2000, showed an improved behaviour for these three traits. This family presented a unique combination of molecular markers when compared to other rapeseed restorers analysed, which suggests that the recombination event allowed the recovery of B. oleracea genetic information that was originally replaced by the radish introgression in the original restorers. This resulted in a duplicated region (originating from radish and B. oleracea) on the chromosome carrying the introgression in the R2000 family.
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.
Plant mitochondrial genomes have complex configurations resulting from the multipartite structures and highly rearranged substoichiometric molecules created by repetitive sequences. To expedite the reliable classification of the diverse radish (Raphanus sativus L.) cytoplasmic types, we have developed consistent molecular markers within their complex mitochondrial genomes. orf138, a gene responsible for Ogura male-sterility, was detected in normal cultivars in the form of low-copy-number substoichiometric molecules. In addition to the dominant orf138-atp8 Ogura mitochondrial DNA (mtDNA) organization, three novel substoichiometric organizations linked to the atp8 gene were identified in this study. PCR amplification profiles of seven atp8- and atp6-linked sequences were divided into three groups. Interestingly, the normal cytoplasm type, which had previously been considered a single group, showed two patterns by PCR amplification. The most prominent difference between the two normal mtDNAs was size variation within four short-repeat sequences linked to the atp6 gene. This variation appeared to be the result of a double crossover, mediated by these homologous, short-repeat sequences. Specific PCR amplification profiles reflecting the stoichiometry of different mtDNA fragments were conserved within cultivars and across generations. Therefore, the specific sequences detected in these profiles were used as molecular markers for the classification of diverse radish germplasm. Using this classification system, a total of 90 radish cultivars, or accessions, were successfully assigned to three different mitotypes.
A male-sterile (MS) radish (Raphanus sativus L.) was found in an accession collected from Uzbekistan. Unlike Ogura MS radishes in which no pollen grain is typically visible during anthesis, a small number of pollen grains stuck together in the dehiscing anthers was observed in the newly identified MS radish. Fluorescein diacetate tests and scanning electron micrographs showed that pollen grains in the new MS radish were severely deformed and non-viable. Cytological examination of pollen development stages showed a clear difference in the defective stage from that seen in Ogura male-sterility. Reciprocal cross-pollination with diverse male-fertile lines indicated that pollen grains of the new MS radish were completely sterile, and the female organs were fully fertile. When the new MS radish and Ogura MS lines were cross-pollinated with a set of eight breeding lines, all F1 progeny originating from crosses with the new MS radish were male-sterile. In contrast, most of the F1 progeny resulting from crosses with Ogura MS lines were male-fertile. These results demonstrated that factors associated with induction of the newly identified male-sterility are different from those of Ogura male-sterility. The lack of restorer lines for the newly identified male-sterility led us to predict that it might be a complete cytoplasmic male-sterility without restorer-of-fertility genes in nuclear genomes. However, cross-pollination with more diverse radish germplasm identified one accession introduced from Russia that could completely restore fertility, proving the existence of restorer-of-fertility gene(s) for the new male-sterility. Meanwhile, the PCR amplification profile of molecular markers for the classification of radish mitochondrial genome types revealed that the new MS radish contained a novel mitotype.
The structure of mitochondrial DNA from the Liverwort, Marchatia polymorpha
- Dj
- Bendich
- Aj
DJ, Bendich AJ (1998) The structure of mitochondrial DNA from the Liverwort, Marchatia polymorpha. J Mol Biol 276:745–758
Intergeneric cytoplasmic hybridization in Crucife-rae by protoplast fusion A new combined double low restorer line for the Ogu-INRA cms in rapeseed
- G C Primard
- F Vedel
- Remy P R Chetrit
- Rousselle P C Poupard Jp
- Horvais R F Eber
- Renard G M Pelletier
- Delourme
G, Primard C, Vedel F, Chetrit P, Remy R, Rousselle P, Re-nard M (1983) Intergeneric cytoplasmic hybridization in Crucife-rae by protoplast fusion. Mol Gen Genet 191:244–250 Primard-Brisset C, Poupard JP, Horvais R, Eber F, Pelletier G, Renard M, Delourme R (2005) A new combined double low restorer line for the Ogu-INRA cms in rapeseed (Brassica napus L.). Theor Appl Genet 111:736–746
Mitochondrial DNA from the Liv-erwort Marchantia polymorpha: Circularly permuted linear mol-ecules, head-to-tail concatemers, and a 5Ј protein IntraspeciWc variation and multicircularity in Bras-sica mitochondrial DNAs
- Dj
- Bendich
- Aj
DJ, Bendich AJ (2001) Mitochondrial DNA from the Liv-erwort Marchantia polymorpha: Circularly permuted linear mol-ecules, head-to-tail concatemers, and a 5Ј protein. J Mol Biol 310:549–562 Palmer JD (1988) IntraspeciWc variation and multicircularity in Bras-sica mitochondrial DNAs. Genetics 118:341–351
Discovery of a novel cytoplasmic male-sterility and its re-storer lines in radish (Raphanus sativus L.) Fertility restoration is associated with loss of a portion of the mitochondrial genome in cytoplasmic male-sterile common bean
- Y S Park
- Kim C H Lim
- H Lim
- Y Ahn
- S Sung
- M Yoon
- Kim
Y, Park S, Lim C, Kim H, Lim H, Ahn Y, Sung S, Yoon M, Kim S (2008) Discovery of a novel cytoplasmic male-sterility and its re-storer lines in radish (Raphanus sativus L.). Theor Appl Genet 117:905–913 Mackenzie SA, Chase CD (1990) Fertility restoration is associated with loss of a portion of the mitochondrial genome in cytoplasmic male-sterile common bean. Plant Cell 2:905–912
Substoichiometric shifting in the plant mitochondrial genome is influenced by a gene homologous to MutS
- R V Abdelnoor
- Yule R Elo
- A Christensen
- A C Meyer-Gauen
- G Mackenzie
- RV Abdelnoor