Sequence analysis of a chloroplast intergenic spacer for phylogenetic estimates in Allium section Cepa and a PCR-based polymorphism detecting mixtures of male-fertile and male-sterile cytoplasmic onion

Restriction-enzyme analysis of the chloroplast (cp) DNA yielded maternal phylogenies supporting a close phylogenetic relationship among normal (N) male-fertile and male-sterile (S) cytoplasmic bulb onion (Allium cepa), Allium altaicum, Allium fistulosum, Allium galanthum, Allium roylei, and Allium vavilovii. The S cytoplasm of onion is most likely an alien cytoplasm introduced in antiquity into onion populations. We previously showed that size differences in an intergenic spacer in the cp DNA distinguish N and S cytoplasms of onion. We cloned and sequenced this intergenic spacer from the N and S cytoplasms of onion, A. altaicum, A. fistulosum, A. galanthum, Allium pskemense, Allium oschaninii, A. roylei, and Allium ampeloprasm (outgroup) to identify the nature of previously described RFLPs and to develop a PCR-based marker revealing N-cytoplasmic contamination of S-cytoplasmic hybrid seed lots. Phylogenies based on restriction-enzyme analysis of the entire cp DNA were similar, but not identical, to those based on sequence divergence in this intergenic region. Received: 29 November 1999 / Accepted: 28 April 2000     

Fertility restoration and mitochondrial nucleic acids distinguish at least five subgroups among cms-S cytoplasms of maize (Zea mays L.)

Summary Differences in fertility restoration and mitochondrial nucleic acids permitted division of 25 accessions of S-type male sterile cytoplasm (cms-S) of maize into five subgroups: B/D, CA, LBN, ME, and S(USDA). S cytoplasm itself (USDA cytoplasm) was surprisingly not representative of cms-S, since only two other accessions, TC and I, matched its mitochondrial DNA pattern. CA was the predominant subgroup, containing 18 of the 25 accessions. The B/D and ME subgroups were the most fertile and LBN the most sterile. The exceptional sterility of LBN cytoplasm makes it the most promising of the 25 cms-S accessions for the production of hybrid seed. The most efficient means of quantifying the fertility of the subgroups was analysis of pollen morphology in plants having cms-S cytoplasm and simultaneously being heterozygous for nuclear restorer-of-fertility (Rf) genes. This method took advantage of the gametophytic nature of cms-S restoration. The inbred NY821LERf was found to contain at least two restorer genes for cms-S. Fertility differences were correlated with mitochondrial nucleic acid variation in the LBN, ME, and S (USDA) subgroups.Paper No. 9498 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC     

A PCR-marker for the CMS(1) inducing cytoplasm in chives derived from recombination events affecting the mitochondrial gene atp9

The complete coding and 3′-flanking region of the mitochondrial gene atp9 of chives (Allium schoenoprasum L.) was determined in order to develop primers that allow the identification of atp9-related sequences in subsequent PCR-amplifications. One of these sequences is of a chimerical nature, consisting of atp9-homologous regions on its end, interrupted by an insertion that is composed of one atp6-homologous part and one part of unknown origin. This PCR-fragment is 762 bp in size and exclusively amplified in the sterility inducing cytoplasm of CMS₁. Thus it can be used as a PCR-marker in order to distinguish this cytoplasm type from the remaining cytoplasm types of chives. The chimerical marker sequence forms a putative open reading frame (orfA501), from which CMS₁ might originate. Received: 6 June 2001 / Accepted: 3 August 2001     

Identification of cytoplasms using the polymerase chain reaction to aid in the extraction of maintainer lines from open-pollinated populations of onion

S-cytoplasm is the most common source of cytoplasmic-genic male sterility (CMS) used to produce hybrid-onion seed. Identification of the cytoplasm of a single plant takes from 4 to 8 years and is complicated by the segregation of a nuclear gene that restores fertility. Although CMS in onion may be due to an incompatibility between the mitochondrial and nuclear genomes, Southern analyses of DNA from individual plants from crosses of S- and N-cytoplasmic plants supported maternal inheritance of the chloroplast and mitochondrial DNA and, therefore, polymorphisms in the chloroplast DNA may be used to classify cytoplasms. Amplification by the polymerase chain reaction of a fragment that carries an autapomorphic 100-bp insertion in the chloroplast DNA of N-cytoplasm offers a significantly quicker and cheaper alternative to crossing or Southern analysis. Molecular characterization of N- and S-cytoplasms and frequencies of the nuclear non-restoring allele allow onion breeders to determine the proportion of plants in open-pollinated populations that maintain CMS and can significantly reduce the investment required to identify individual maintainer plants.     

Allozyme variation within and among populations of onion (Allium cepa L.) from West Africa

Local germplasm of onion (Allium cepa L.) in West Africa is threatened by extinction. Sixteen populations of onion collected in five countries in West Africa were investigated for isozyme polymorphism using four polymorphic enzyme systems (ADH, MDH, 6-PGDH and PGI) among nine enzyme systems assayed. This is the first report on the genetic diversity of local landraces of onion. The inheritance of two dimeric enzyme systems PGI and MDH was demonstrated using F₂ progeny arrays. The PGI system revealed a single locus with three alleles, and the MDH system revealed three loci with four alleles. Four polymorphic systems revealed nine alleles (adh-a1 and a2, mdh-c1 and c2, 6-pgdh-a1 and a2, and pgi-a1, a2 and a3) in the 16 local populations observed. The mean number of alleles per polymorphic locus was 2.25, and 67% of the alleles were present in all populations. Allele 6-pgdh-a2 was present in only two landraces (from Niger and Nigeria); it is considered to be a rare allele (frequency approximately 2%). Among the 16 populations, within-population diversity was greater (90%) than between-population diversity (10%). Genetic distance analyses showed an aggregate of all populations except for two, which originated from Nigeria, an English-speaking country. Received: 24 August 1995 / Accepted: 26 February 2001     

A putative donor of S-cytoplasm and its distribution among open-pollinated populations of onion

Summary Cytoplasmic-genic male-sterility systems are used to economically produce hybrid onion seed. Previous studies have indicated that the source of cytoplasmic male sterility discovered in 1925 by Jones (S-cytoplasm) may be an alien cytoplasm. Restriction enzyme analysis of the chloroplast DNA (cpDNA) revealed five polymorphisms between S and normal (N) fertile cytoplasms. S-cytoplasm was different from the Allium species closely related to the bulb onion, and cladistic estimates of phylogenies supported introduction from an unknown species. S-cytoplasm was identical for all polymorphisms in the cpDNA to Pran, a triploid viviparous onion. Pran shares morphological characteristics with Italian Red 13–53, the single plant source of S-cytoplasm. Densiometric scans of autoradiograms revealed that 12 of 31 open-pollinated populations of onion possessed S-cytoplasm and that introgression may have occurred since the discovery of S-cytoplasm.     

DNA polymorphism in Allium cepa cytoplasms and its implications concerning the origin of onions

Summary Mitochondrial and chloroplast DNA was isolated from fertile and cytoplasmic male sterile cultivars of cultivated onions. Restriction fragment length polymorphism led to the distinction between cytoplasms S and M. Mitochondrial DNA patterns from S cytoplasms appeared dentical and characterized mostly male sterile lines. An open-pollinated variety was found to bear this cytoplasm and thought to be the origin of S types. Mitochondrial DNA patterns from M cytoplasms were subdivided into four types, M₁ and M₂ corresponding to normal N cytoplasm, M₃ and M₄ probably corresponding to T cytoplasms. S and M cytoplasms were also distinguished by chloroplast DNA restriction patterns. Our results confirm previous genetic distinction between S, N and T cytoplasms.     

Agrobacterium tumefaciens-mediated transformation and transgenic-plant regeneration of onion (Allium cepa L.)

 An Agrobacterium tumefaciens-mediated transformation method has been developed for onions (Allium cepa L.) using immature embryos as the explant source. Transgenic plants were recovered from the open-pollinated onion cultivar Canterbury Longkeeper at a maximum transformation frequency from immature embryos of 2.7%. The method takes between 3–5 months from explant to primary regenerant entering the glasshouse. Multiple-shoot formation from primary transgenic material made possible the clonal multiplication of transformants. The binary vector used carried the nptII antibiotic resistance gene and the m-gfp5-ER reporter gene. Transgenic cultures were initially screened for their ability to fluoresce and to grow in the presence of geneticin (5–25 mg/l). The transgenic nature of individual plants was confirmed by Southern blot analysis. Received: 12 October 1998 / Revision received: 17 May 1999 Accepted: 14 June 1999     

Phosphatase and ATPase activities in isonuclear lines of cytoplasmic male-sterile and male-fertile petunia

Summary Soluble and membrane-bound fractions of plant leaves, cell suspension cultures and seedlings of petunia were examined for phosphohydrolase activity on p-nitrophenyl phosphate (pNPPase) and adenosine triphosphate (ATPase). One cytoplasmic male-sterile (CMS) and one fertile (F) line was examined for each tissue. Both pNPPase and ATPase exhibited a broad optimal activity between pH 5.5–7.0 for the membrane-bound fraction and between 4.5–7.0 for the soluble fractions. The activity of both were inhibited by divalent ions including Mg²⁺. At pH 7.2, the activities on various triphosphonucleotides were similar and they were hydrolyzed by a rate of 20–50% of that of ATP. Significant differences between CMS and F extracts were: (a) higher activities in CMS membranes; (b) lower Ea (energy of activation) values for activities in CMS membrane functions; (c) seedling and cell-culture CMS extracts exhibited a higher sensitivity to high temperature denaturation; (d) the hydrolase activity on monoand triphospho-cytosine compounds was significantly higher in CMS than in F membranes.Contribution from the Agricultural Research Organization, The Volcani Center, Bet-Dagan, Israel No. 355-E, 1992 series     

Phylogenetic relationships among fertile and petaloid male-sterile accessions of carrot, Daucus carota L.

 Mitochondrial (mt) DNA variation for six petaloid cytoplasmic male-sterile (CMS) and three fertile maintainer lines of carrot was assessed to establish genetic relationships. Total DNA was digested with restriction enzymes and probed with six homologous mtDNA cosmid probes. The six CMS accessions derived from wild carrot, four from Guelph, Ontario, one from Orleans, Massachusetts, and one from Madison, Wisconsin, were more closely related with each other (F=0.91) than with fertile maintainer lines derived from cultivated germplasm (F=0.62). The fertile maintainer lines were likewise found to be more similar to each other (F=0.78) than to the sterile lines. Three sterile lines, originating from wild carrot populations within 1 km of each other in Guelph, Ontario, were most closely related (F=0.96). The high degree of similarity among the six petaloid CMS lines which originated from individual wild carrot plants, some from geographically diverse regions, suggests that the cytoplasm responsible for this trait was imported to, or else evolved, only once in North America. Received: 1 December 1997 / Accepted: 12 December 1997     

Rapid hybridization-based assays for identification by DNA probes of male-sterile and male-fertile cytoplasms of the sugar beet Beta vulgaris L.

Summary Methods are described whereby hybridization of mitochondrial (mt) DNA with different DNA probes can definitely distinguish male-fertile and and male-sterile (cms) cytoplasms of sugar beet Beta vulgaris L. We have developed two types of miniassays. (1) Comparative methods requiring the isolation and restriction of total cellular DNA, hybridization with cloned mtDNA fragments from either fertile or male-sterile cytoplasms, and comparison of the hybridization patterns to the fertile-and sterile-specific patterns of mtDNA of sugar beet for the given mtDNA probe. For these analyses, we routinely used 1 g of plant material to determine the type of cytoplasm. (2) Noncomparative (plus-minus) methods requiring neither the isolation of pure DNA nor restriction, electrophoresis, or Southern blotting. Instead, alkaline-SDS plant extracts from as little as 50 mg of plant material were dot-blotted and hybridized with fertile-specific (mitochondrial minicircular DNA) and/or cms-specific probes (consisting of a 2.3-kb mtDNA sequence exclusively occurring in the cms cytoplasm). The assays are simple to perform, give definitive results, are nonde-structive to the plants, and may be used in mass screening of sugar beet populations for hybrid production or in in vitro culture processes.     

Molecular diversity of male sterility inducing and male-fertile cytoplasms in the genus Helianthus

The organisation of mtDNA was investigated for 28 sources of cytoplasmic male sterility (CMS) and a fertile line (normal cytoplasm) of Helianthus annuus by Southern hybridisation. In addition to nine known mitochondrial genes (atp6, atp9, cob, coxI, coxII, coxIII, 18S, 5S and nd5) three probes for the open reading frames in the rearranged area of PET1, orfH522, orfH708 and orfH873, were used. Genetic similarities of the investigat-ed cytoplasms varied between 0.3 and 1. Cluster analyses using the UPGMA method allowed the distinction of ten mitochondrial (mt) types between the 29 investigated cytoplasms. Most mitochondrial types comprise two or more CMS sources, which could not be further separated, like the PET1-like CMS sources (with the exception of ANO1 and PRR1), or ANN1/ANN2/ANN3, ANN4/ ANN5, ARG3/RIG1, BOL1/EXI1/PEF1/PEP1 and GIG1/ PET2. ANL1, ANL2 and the fertile cytoplasms are also regarded as one mitochondrial type. Unique banding patterns were only observed for ANT1 (atp6), MAX1 (atp6, orfH522 and orfH708) and PRR1 (coxII). However, four of the mitochondrial types showed unique hybridisation signals: ANN4/ANN5 had characteristic bands for atp6 and orfH708, PEF1/PEP1/EXI1/BOL1 for atp6 and coxII, and PET2/GIG1 for atp9. The PET1-like cytoplasms all shared the same patterns for orfH522, orfH708 and cob (except ANO1). It could be demonstrated that CMS sources, like, e.g., PET2 and PEF1, are different from PET1 in mtDNA organisation and the CMS mechanism. Therefore, these CMS sources represent interesting candidates for the development of new hybrid breeding systems based on new CMS mechanisms. Received: 20 April 2001 / Accepted: 3 August 2001     

The use of mitochondrial DNA polymorphism in the classification of individual onion plants by cytoplasmic genotypes

Summary Individual plants of a Japanese onion variety Sapporo-ki, which is characterized by the occasional occurrence of male-sterile plants, have been investigated for mitochondrial (mt) DNA polymorphism. Male-fertile and the Jones' cytoplasmic male-sterile (CMS) onions were also included for comparison. Southern blot hybridization with rrn26, cox-I, cox-II, cob, atpA and atp9 genes as probes revealed the two classes of mtDNA variation within a population of Sapporo-ki: Out of the 41 plants examined 19 contained mtDNA typical of malefertile plants, and 22 individuals contained mtDNA typical of the Jones' CMS genotype. Our results thus indcate that the use of the mitochondrial gene probes may greatly facilitate the classification of individual plants by cytoplasmic genotypes.     

RFLP mapping of the maize gametophytic restorer-of-fertility locus (rf3) and aberrant pollen transmission of the nonrestoring rf3 allele

 Cytoplasmic male sterility (CMS) is the maternally inherited inability to produce functional pollen. The Rf3 allele of the nuclear gene rf3 gametophytically restores male fertility to maize plants with the S-type of CMS. The rf3 locus is on the long arm of maize chromosome two (2L). Using 2L RFLPs and three-point mapping analysis we showed that the rf3 locus is located an estimated 4.3 cM distal to the whp locus and 6.4 cM proximal to the bnl17.14 locus. This information was used in combination with RFLPs on two additional maize chromosomes to show that Rf3/rf3 CMS-S plants may aberrantly transmit the nonrestoring allele, rf3, through the male gametophyte. Received: 30 September 1996/Accepted: 21 March 1997     

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.     

Effect of root exudate fractions from P-deficient and P-sufficient onion plants on root colonisation by the arbuscular mycorrhizal fungus Gigaspora margarita

 The effect of root exudates from P-deficient onion on root colonisation by an arbuscular mycorrhizal fungus was examined. Onions (Allium cepa L.) were grown in solution culture at phosphorus concentrations of 0 (P0) and 2 (P2) mg P l^–1. Root exudates were collected and fractionated with Amberlite XAD-4 resin to give EtOH and water soluble fractions. Onions inoculated with the arbuscular mycorrhizal fungus Gigaspora margarita Becker & Hall were grown with or without (control) root exudates and exudate fractions in a growth chamber. After 24 days, arbuscular mycorrhiza levels and appressoria formation had increased in plants treated with P0-root exudate or the P0-EtOH fraction when compared to corresponding P2 treatments or control plants. P0 and P2 water-soluble fractions did not significantly affect either aspect of fungal development. These results suggest that hydrophobic compounds found in root exudates from P-deficient onion increase appressorium formation and, therefore, enhance mycorrhiza development. Accepted: 2 June 1998     

Effect of onion (Allium cepa) root exudates on the hyphal growth of Gigaspora margarita

 The effect of root exudates from onions differing in P status on spore germination and hyphal growth of arbuscular mycorrhizal fungi was investigated. Onion (Allium cepa) was grown in solution culture at different phosphorus concentrations (0, 0.1, 1.0, 8.0 and 24.0 mg P l^–1) and root exudates were collected. When spores of the arbuscular mycorrhizal fungus, Gigaspora margarita were incubated with these root exudates, spore germination was only slightly affected but hyphal growth was greatly affected, particularly with exudates from P-deficient plants. This suggests that the P nutrition of host plants influences the composition of root exudates and thereby the hyphal growth of arbuscular mycorrhizal fungi. Accepted: 25 June 1995