Cytoplasmic male sterility and nuclear restorer genes in a natural population of Beta maritima: genetical and molecular aspects

Summary One natural population (F₀ generation) of Beta maritima situated on the French Atlantic coast has been analysed. It was composed of 62% female, 30% hermaphrodite and 8% intermediate plants. The analysis of half-sib progeny (F₁ generation) obtained from in situ open pollination demonstrates the cytoplasmic determination of male sterility in Beta maritima and the restoration of fertility by nuclear genes. The mitochondrial DNA (mtDNA) and the chloroplast DNA (ctDNA) of sixteen F₁ plants, extracted from offspring of the three sexual phenotypes, were analysed using the restriction enzymes Sal I and Bam HI, respectively. Two cytoplasmic lines with their own peculiar genetic characteristics were distinguished using the restriction enzyme patterns of mtDNA: (i) the S cytoplasmic line was found in segregating progeny of two F₀ plants; all three phenotypes were produced (that is, progeny including hermaphrodite, female and intermediate plants); (ii) the N cytoplasmic line was found in the progeny of one F₀ hermaphrodite plant; this produced only hermaphrodites. Thus, segregating and non-segregating hermaphrodite F₀ plants can be distinguished. The nuclear genes maintaining sterility or restoring fertility are expressed in line S. At the same time the analysis of Beta vulgaris material has been carried out at the molecular level: N cytoplasmic lines of B. vulgaris and B. maritima differed only by 3 fragments of mtDNA; but the S cytoplasmic line of B. maritima was very different from Owen's cytoplasmic male sterile line of B. vulgaris. No variation in the ctDNA pattern was detected within and between the two taxa.     

A new cytoplasmic male sterile genotype in the sugar beet Beta vulgaris L.: a molecular analysis

Summary Mitochondrial DNA (mtDNA) from fertile (N) and possibly new cytoplasmic male sterile (CMS) genotypes was studied in the sugar beet Beta vulgaris L. It was found by restriction endonuclease analysis that BMC-CMS, a cytoplasm that was derived from the wild beet Beta maritima, contained a unique type of mtDNA which is distinguishable from both the N and S-CMS, the only other CMS genotype that is currently availabe in B. vulgaris L. The organization of three genes: coxI, coxII and cob, was analyzed by hybridization with heterologous probes from maize. These genes have a similar structure in N and BMC-CMS that is different from S-CMS. It is concluded that BMC-CMS is a novel CMS genotype in the sugar beet.     

Two new types of cytoplasmic male sterility found in wild Beta beets

Summary Mitochondrial (mt) and chloroplast (ct) DNAs from sugar beet carrying normal fertile and different cytoplasmic male sterile (cms) cytoplasms were compared by restriction analysis and for the occurrence of minicircles. One of the cms materials had the Owen cms cytoplasm currently used for hybrid production in sugar beet; the other three cms materials were derived from wild Beta beets. The mtDNAs from two of the latter cms types (C 7051, C 8640) differed from both the Owen and the fertile cytoplasms in fragment patterns seen after restriction enzyme analysis and in minicircle composition. The third cms type (C 8684) differed from the Owen cytoplasm in mini-circle composition, but restriction enzyme analysis revealed no differences. The presence of the different minicircles was confirmed by Southern hybridization using minicircle-specific clones. All bands hybridized as predicted by gel electrophoresis except a band in the cms type C 8640, which migrated in a similar manner as the c.c.c. form of the a minicircle. This band hybridized only faintly to a minicircle a-specific probe and could be removed by treatment with nuclease S1. In contrast to the large mtDNA variation, restriction analysis of ctDNA detected little variation between cytoplasms. The molecular characterization of the new sources of cms supports the results of previous crossings. Two of the cytoplasms are not only of independent origin, but are also most likely functionally different and thus may be of value in future production of hybrid sugar beet varieties.     

The cytoplasmic male-sterile type and normal type mitochondrial genomes of sugar beet share the same complement of genes of known function but differ in the content of expressed ORFs

The complete nucleotide sequence (501,020 bp) of the mitochondrial genome from cytoplasmic male-sterile (CMS) sugar beet was determined. This enabled us to compare the sequence with that previously published for the mitochondrial genome of normal, male-fertile sugar beet. The comparison revealed that the two genomes have the same complement of genes of known function. The rRNA and tRNA genes encoded in the CMS mitochondrial genome share 100% sequence identity with their respective counterparts in the normal genome. We found a total of 24 single nucleotide substitutions in 11 protein genes encoded by the CMS mitochondrial genome. However, none of these seems to be responsible for male sterility. In addition, several other ORFs were found to be actively transcribed in sugar beet mitochondria. Among these, Norf246 was observed to be present in the normal mitochondrial genome but absent from the CMS genome. However, it seems unlikely that the loss of Norf246 is causally related to the expression of CMS, because previous studies on mitochondrial translation products failed to detect the product of this ORF. Conversely, the CMS genome contains four transcribed ORFs (Satp6presequence, Scox2-2 , Sorf324 and Sorf119) which are missing from the normal genome. These ORFs, which are potential candidates for CMS genes, were shown to be generated by mitochondrial genome rearrangements.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by R. Hagemann     

Sugar beet BAC library construction and assembly of a contig spanning <Emphasis Type="Italic">Rf1</Emphasis>, a restorer-of-fertility gene for Owen cytoplasmic male sterility

Rf1 is a nuclear gene that controls fertility restoration in cases of cytoplasmic male sterility caused by the Owen cytoplasm in sugar beet. In order to isolate the gene by positional cloning, a BAC library was constructed from a restorer line, NK198, with the genotype Rf1Rf1. The library contained 32,180 clones with an average insert size of 97.8 kb, providing 3.4 genome equivalents. Five AFLP markers closely linked to Rf1 were used to screen the library. As a result, we identified eight different BAC clones that were clustered into two contigs. The gap between the two contigs was filled by chromosome walking. To map the Rf1 region in more detail, we developed five cleaved amplified polymorphic sequence (CAPS) markers from the BAC DNAs identified, and carried out genotyping of 509 plants in the mapping population with the Rf1-flanking AFLP and CAPS markers. Thirteen plants in which recombination events had occurred in the vicinity of the Rf1 locus were identified and used to map the molecular markers relative to each other and to Rf1. In this way, we were able to restrict the possible location of the Rf1 gene to a minimum of six BAC clones spanning an interval of approximately 250 kb. The first two authors contributed equally to this work.     

Minicircle variation in Beta mitochondrial DNA

Summary Mitochondrial DNAs from nine male fertile and eight cytoplasmic male sterile (cms) accessions of wild and cultivated Beta beets were investigated for the presence of low molecular weight DNA molecules. Five different supercoiled DNA molecules were detected, varying in size from 1.33 to 1.63 kb. Southern hybridizations revealed multimeric forms and sequence homologies between the minicircles. The occurrence of the different minicircles among the 17 accessions was investigated by agarose gel electrophoresis and Southern hybridization using minicircle specific probes. The 1.33 and 1.63 kb minicircles were found in most accessions, the other three minicircles were found in one or two of the wild Beta beet accessions. The presence of a low number of small, more or less homologous, minicircles in all investigated plants makes these molecules a general characteristic of Beta mtDNA. No association is found between the presence or absence of specific minicircles and the expression of male sterility. Neither does the distribution of the different minicircles in Beta beets indicate any essential biological role of these minicircles.     

Cytoplasmic male sterility in hybrids of sterile wild beet (Beta vulgaris ssp. maritima) and O-type fertile sugar beet (Beta vulgaris L.): molecular analysis of mitochondrial and nuclear genomes

The organization of the mitochondrial genome of B3, B4 and B5generations of hybrids created by backcrossing sterile wild beet Betamaritima with a fertile O-type sugar beet line was studied usingrestriction fragment length polymorphism (RFLP) analysis. Random amplifiedpolymorphic DNA (RAPD) analysis was used to study restoration of the fertile(O-type) sugar beet genotype in hybrids after multiple backcrossings.Restriction of mtDNAs from the cytoplasm of B. maritimaandhybrids revealed BamHI, EcoRI andXhoI restriction patterns different from those for sterileand fertile sugar beet lines. The most conspicuous feature of our accession ofsterile wild beet mtDNA was the absence of the 10.7-kbEcoRI fragment detected in the cytoplasm of S-type sterileB. maritima and sugar beet. The hybridization of digestedmtDNAs with coxII, atpA andatp6 homologous probes revealed alterations within thesegene loci that distinguished wild beet and hybrids from sugar beets.Characteristic hybridization profiles for the wild beet and B3, B4 and B5hybrids were observed for all probes regardless of the restrictase used todigest mtDNA. Notable changes in atpA andatp6 genes resulted when probes that comprised the5flanking sequences of these genes and a small part of the coding sequences wereused. RFLP analysis of the sterile B. maritimamitochondrial genome further supported the unique character of this source ofwild beet sterility. The genotypic differences between hybrids and parentalaccessions were determined by scoring PCR-RAPD reaction products for nineselected primers. The diversity of the B. maritimagenotyperesulted in a lower genetic similarity index in comparison with hybrids,sterileand fertile lines of sugar beet. The dendrogram obtained after cluster analysisdistinguished hybrids as a group that differed from wild beet and themaintainersugar beet line used for backcrossing. These results may indicate incompleterestoration of the fertile sugar beet genotype in hybrids.     

Comparison of restriction endonucleases and sources of probes for their efficiency in detecting restriction fragment length polymorphisms in lettuce (Lactuca sativa L.)

Summary Five accessions of members of the C group of male sterile maize cytoplasms (BB, C, ES, PR, and RB) in two nuclear backgrounds (A619 and A632) were examined to elucidate the nature of mitochondrial genome diversity within a related group of cytoplasms. Cosmid and plasmid clones carrying single copy and recombinationally active sequences from N and S cytoplasms of maize were used as probes. Although restriction patterns are quite similar, each of the five could be discriminated by evidence of sequence duplication and recombination, deletion of recombinationally active sequences of N, normal cytoplasm, population of mini-circular DNAs, and by restriction patterns. Each member of the group carried a 1,913 bp minicircular mtDNA, while all entries but RB carried a 1,445 bp minicircular mtDNA. Members of the C group clearly are not molecularly identical; evolution of the group included principal genome reorganization involving sequence duplication/deletion events, apparently independent of the cms trait.Cooperative Investigations of Agricultural Research Service, U.S. Department of Agriculture, and Institute of Food and Agricultural Sciences, University of FloridaMention of trademark, proprietary product, or vendor does not constitute a guarantee of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable     

Cytoplasmic male sterility inBeta is associated with structural rearrangements of the mitochondrial DNA and is not due to interspecific organelle transfer

Summary Chloroplast (ct) and mitochondrial (mt) DNAs from four cytoplasmic male sterile (cms) and 22 normal fertile sugar beet lines and accessions of wild beets from the genusBeta have been compared with restriction analyses and Southern hybridizations. We have used restriction analyses of ctDNA as a phylogenetic marker to confirm the taxonomic relationships between the different cytoplasms. According to the ctDNA data, all four cms cytoplasms belong to the same taxonomic section,Beta. Restriction patterns of ct and mtDNA from fertile accessions produced analogous trees of similarity and showed a close correlation between the organellar DNA diversity and the accepted taxonomic classification of the species studied. However, the mtDNA restriction profiles of the four cms types differed dramatically from each other and from those of all fertile accessions from the genus. No indication of cytoplasmic introgression was found in any of the four investigated cms types. Southern hybridization to mtDNA revealed variant genomic arrangements in the different fertile and cms cytoplasms, indicating that rearrangement of the mitochondrial genome is a common denominator to the different cms systems inBeta. It may, indeed, be a common property to spontaneously occurring cms in all or most species.     

Differences between,and possible origins of,the cytoplasms found in fertile and male-sterile onions (Allium cepa L.)

Summary The DNA of the organellar genomes of Allium cepa has been examined to detect restriction fragment length polymorphisms. Differences can be shown between both the chloroplastal and mitochondrial genomes of the N and cms-S cytoplasms in their restriction fragment profiles. Southern blot analysis of the mtDNA profiles using probes containing defined mitochondrial genes also detected polymorphisms. No differences can be shown between the organellar genomes of the N and cms-T onions by either of these techniques. These data indicate different origins for the two sterility-conferring cytoplasms, suggesting autoplasmic and alloplasmic origins for the cms-T and cms-S cytoplasms, respectively. No evidence of the presence of virus-like particles was found in any of the cytoplasms.     

Comparison of chloroplast and mitochondrial DNA from five morphologically distinct Beta vulgaris cultivars: sugar beet,fodder beet,beet root,foliage beet,and Swiss chard

Summary Two cytoplasms, N and S, are used in the breeding of sugar beet, Beta vulgaris var. altissima. These cytoplasms can be distinguished by their mitochondrial DNA. In an attempt to detect new cytoplasms, we compared the restriction profiles of chloroplast and mitochondrial DNA from five different cultivars of Beta vulgaris. All restriction patterns of chloroplast DNA were identical. With the exception of sugar beet with S-cytoplasm, all cultivars studied showed the same restriction profile of mitochondrial DNA, indicating that these cultivars all contain the N-cytoplasm. These results are discussed with regard to the large morphological differences of the cultivars and the cytoplasmic variability found in natural populations of the wild beet, Beta maritima.     

The use of constrained selection indexes in breeding for economic merit

Summary Various methods exist for the derivation of restricted and/or desired gains selection indexes, and their use in applied breeding has been advocated. It is shown that there exists a set of implied linear economic weights for all constrained indexes and their derivation is given. Where economic weights are linear and known, a standard selection index is, by definition, optimal and thus a constrained index will usually be suboptimal. It is argued that economic weights can always be estimated and that the effects of uncertain weights can be examined by sensitivity analysis. If economic weights are nonlinear, use of the first order (linear) economic weights or a derived linear index, using previously described methods, will give very close to optimum economic selection responses. Examples from the literature indicate that severe losses of potential economic gain can possibly occur through use of a constrained index. It is concluded that constrained indexes should be avoided for economic genetic selection.     

红麻不育系和保持系中cob基因的克隆与分析

开展红麻的CMS机理的研究有利于更好地利用其杂种优势。分别提取红麻CMS系和保持系线粒体DNA,Southern blot分析表明,cob基因的组织形式存在差异。根据不同物种cob基因的保守序列设计引物,在红麻CMS系和保持系中克隆到了cob基因,GenBank序列号分别为HM535786和HM535787。基因长度均为1179bp,编码392个氨基酸残基,分子量约为43kD。序列比较发现cob基因在CMS系和保持系同源性达99.8%,和其他物种中cob基因的同源性大于92.3%。研究结果为下一步克隆cob基因的侧翼序列,进而揭示红麻的CMS机理提供了很好的研究基础。     

Low-copy-number molecules are produced by recombination, actively maintained and can be amplified in the mitochondrial genome of Brassicaceae: relationship to reversion of the male sterile phenotype in some cybrids

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 sub-stoichiometric 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. Received: 14 July 1997 / Accepted: 16 September 1997     

A new source of cytoplasmic male sterility in maize induced by the nuclear gene,iojap

Summary Cytoplasmic male sterility (cms) was found in plants derived from the F₂ progeny of fertile, normal cytoplasm plants of the inbred R181 pollinated with a genetic stock carrying the recessive nuclear gene, iojap. The male sterile plants were maintained by back-crossing with the inbred W182BN which maintains all known sources of cytoplasmic male sterility. The new male sterile progeny were found to exhibit stable male sterility under field conditions in two environments. However, they were partially fertile in the hot, dry summer of 1983 at Aurora, NY. It was found that these lines were restored by lines that characteristically restore cms S group cytoplasms. Pollen phenotype studies indicated that the restoration was gametophytic in nature, also characteristic of the cms S group. Agarose gel electrophoresis of undigested mitochondrial DNA (mtDNA) from these steriles indicated that these lines have the S-1 and S-2 episomes characteristic of the cms S group. Restriction endonuclease digest patterns of mtDNA from these sterile lines digested with BamH I indicated that these steriles fit into the CA subgroup of the cms S group. The new source of cms has been designated cms Ij-1.     

Genetic analyses supported by molecular methods provide evidence of a new genic (st1) and a new cytoplasmic (st2) male sterility in Allium schoenoprasum L.

Spontaneous mutations leading to male sterility have been described for many different crops and are of great importance to hybrid breeding, provided that their inheritance is resolved. This paper describes an efficient method to characterise male sterilities with respect to cytoplasmic factors that might be causally related to them. The differentiation of cytoplasmic (CMS) and genic (GMS) male sterility is achieved by a specific transfer of nuclear male sterility factors to different cytoplasm types which have previously been distinguished by means of RFLP analyses using mitochondrial gene probes. The nuclear sterility factors of Allium schoenoprasum used, st1 and st2, showed a monogenic recessive inheritance in their original cytoplasms. While st1 was expressed in four different cytoplasm types, st2 did not show itself in a cytoplasm type differing from the original. Therefore, the st1-sterility is a GMS, while a cytoplasmic factor is necessary for the occurrence of st2-sterility. This cytoplasmic factor was verified by a reciprocal cross, and the CMS system was completed by the selection of maintainer genotypes. Neither of these new sterilities were influenced by high temperatures or tetracycline. The benefits of a new CMS system to practical breeding and the advantages and disadvantages of the environmental influences on the expression of male sterility are discussed. Received: 24 November 1999 / Accepted: 3 December 1999     

Cloning and Sequence Analysis of a Specific Mitochondrial DNA Fragment Related to Wild Abortive Type Cytoplasmic Male Sterility in Rice

A specific amplified mtDNA fragment R2-630 WA was obtained from wild abortive type (WA) male-sterile cytoplasm by AP-PCR technique. Polymorphisms between mtDNAs of male-sterile and normal cytoplasms were detected by Southern hybridization with the probe of R2-630 WA fragment. This fragment was sequenced by the didoxy chain-termination method. It is 629 bp in length. Its base components of A+T is 54. I%. A comparison of R2-630 WA fragmerft and 1236 plant genes (including sixteen mitochondrial genes of rice) from Gene Bank reveals low homology (<50%). The nucleotide sequence of R2-630 WA contains a small inverted-repeated sequence 5′ -ACCATATGGT-3′, which is located at the positions from 262 to 272. And it has a coding region of 20 amino acid residues, which is located at the positions from 379 to 439. The results imply that the specific fragment R2-630 WA may associate with WA type cytoplasmic male sterility in rice. The small inverted-repeated sequence probably play a role in the generation of male sterility.     

Comparative restriction endonuclease analysis and molecular cloning of plastid DNAs from wild species and cultivated varieties of the genus Beta (L.)

Summary A phyletic tree of the genus Beta has been constructed based on EcoRI and PstI plastid DNA restriction patterns of eight species from three sections of the genus. In contrast to the remarkable morphological variability of the varieties of B. vulgaris the restriction patterns of the plastid DNA of this species were found to be almost identical. The comparison of plastic DNAs of B. vulgaris crassa fertile and sterile lines with 13 different restriction enzymes revealed only a single fragment polymorphism in the HindIII patterns. Hybridization analyses in the plastidal rDNA region revealed an interesting loss of an EcoRI restriction site in all cultivated B. vulgaris varieties in contrast to wild species. The results of the construction of clone banks for SalI and BamHI fragments of plastid DNA from fertile B. vulgaris crassa are reported and difficulties in the cloning of specific fragments are discussed.     

Plastid DNA diversity in natural populations of Beta maritima showing additional variation in sexual phenotype and mitochondrial DNA

Summary Plants of two natural populations of Beta maritima, characterized by high percentages of male-sterile plants, have been investigated for organelle DNA polymorphism. We confirm the two classes of mitochondrial DNA variation previously described: (i) mitochondrial DNA (mtDNA) type N is associated with male fertility, whereas mtDNA type S can cause cytoplasmic male sterility (CMS); (ii) the 10.4-kb linear plasmid is observed in both types of mitochondria and is not correlated with the cytoplasmic male sterility occurring in this plant material. A third polymorphism is now described for chloroplast DNA (ctDNA). This polymorphism occurs within single populations of Beta maritima. Three different ctDNA types have been identified by HindIII restriction analysis. Among the plants studied, ctDNA type 1 is associated with N mitochondria and type 2 with S mitochondria. Chloroplast DNA type 3 has been found both in a fertile N plant and in a sterile S plant. This finding suggests that the chloroplast DNA polymorphism reported is not involved in the expression of male sterility. A comparison with Beta vulgaris indicates that ctDNA type 3 of Beta maritima corresponds to the ctDNA of fertile sugar beet maintainer lines. The three types of Beta maritima ctDNA described in this study differ from the ctDNA of male-sterile sugar beet.