Assessment of gene copy number in the homosporous fernsCeratopteris thalictroides andC. richardii (Parkeriaceae) by restriction fragment length polymorphisms

Homosporous ferns are generally considered polyploid due to high chromosome numbers, but genetically diploid since the expression of isozymes is generally controlled by a single locus. Gene silencing over evolutionary time is one means by which this apparent contradiction can be explained. A prediction of this hypothesis is that silenced gene sequences still reside in the genomes of homosporous ferns. We examined the genomes ofCeratopteris richardii andC. thalictroides for sequences which are similar to expressed gene sequences. Genomic DNA blots hybridized withC. richardii cDNA clones showed that the majority of these clones detected multiple fragments, suggesting that most gene-like sequences are duplicated inCeratopteris. Hybridization signal intensity often varied between fragments of the same size between accessions, sometimes dramatically, which indicates that not all sequences are equivalent, and may represent the products of silenced genes. Observed reciprocal differences in intensity could be due to reciprocally silenced genes. In addition, an unusual segregation pattern for one locus followed by one probe may indicate homeologous chromosome pairing and segregation.     

Comparison of RFLP and RAPD markers to estimating genetic relationships within and among cruciferous species

Restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers are being used widely for evaluating genetic relationships of crop germplasm. Differences in the properties of these two markers could result in different estimates of genetic relationships among some accessions. Nuclear RFLP markers detected by genomic DNA and cDNA clones and RAPD markers were compared for evaluating genetic relationships among 18 accessions from six cultivated Brassica species and one accession from Raphanus sativus. Based on comparisons of genetic-similarity matrices and cophenetic values, RAPD markers were very similar to RFLP markers for estimating intraspecific genetic relationships; however, the two marker types gave different results for interspecific genetic relationships. The presence of amplified mitochondrial and chloroplast DNA fragments in the RAPD data set did not appear to account for differences in RAPD- and RFLP-based dendrograms. However, hybridization tests of RAPD fragments with similar molecular weights demonstrated that some fragments, scored as identical, were not homologous. In all these cases, the differences occurred at the interspecific level. Our results suggest that RAPD data may be less reliable than RFLP data when estimating genetic relationships of accessions from more than one species.     

Distribution of sequences related to the Bg transposable element of maize in Zea and related genera

Thirty-four accessions from Zea and 10 accessions from related genera were assayed for the presence of Bg, a transposable element originally found in maize (Zea mays ssp. mays). Bg-like sequences, identified as hybridizing bands on Southern blots, were visualized in all Zea accessions and were present in approximately equal numbers in teosinte and maize. With the exception of Tripsacum dactyloides, all accessions from related genera failed to hybridize with the Bg probes, even at reduced stringency. A comparison of the restriction patterns of related inbred lines revealed numerous common hybridizing fragments. An index of molecular similarity (MS) was used to determine the degree of similarity between pairs of inbred lines. Computed MS values endorse an inbred relationship and are in good agreement with published results of cluster analysis on these inbred lines.     

Interspecific hybridization in natural populations ofCyrtandra (Gesneriaceae) on the Hawaiian Islands: Evidence from RAPD markers

Interspecific hybridization among Hawaiian species ofCyrtandra (Gesneriaceae) was investigated using randomly amplified polymorphic DNA (RAPD) markers. Thirty-three different primers were used to investigate interspecific hybridization for 17 different putative hybrids based on morphological intermediacy and sympatry with putative parental species. RAPD data provided evidence for the hybrid origin of all putative hybrid taxa examined in this analysis. However, the patterns in the hybrid taxa were not found to be completely additive of the patterns found in the parental species. Markers missing in the hybrid taxa can be attributed to polymorphism in the populations of the parental species and the dominant nature of inheritance for RAPD markers. Unique markers found within hybrid taxa require further explanation but do not necessarily indicate that the taxa are not of hybrid origin. The implications suggest that these interspecific hybridization events had, and continue to have, an effect on the adaptive radiation and conservation biology ofCyrtandra.     

Wheat phylogeny determined by RFLP analysis of nuclear DNA. 2. Wild tetraploid wheats

Intra- and inter-specific variations in the nuclear DNA of Triticum dicoccoides Körn. (2n = 28, genome constitution AABB) and T. araraticum Jakubz. (2n = 28, AAGG), wild species, respectively, of the Emmer and Timopheevi group, were studied by restriction fragment length polymorphism (RFLP) analysis. Total DNAs of 32 T. dicoccoides and 24 T. araraticum accessions, collected from throughout the distribution areas of these species, were treated with two 6-bp cutters and hybridized with 30 nuclear DNA clones as probes to detect RFLPs. A total of 167 hybrid bands were observed per accession. All the enzyme-probe combinations showed RFLPs between accessions. The average genetic distance between the T. dicoccoides accessions was 0.0135 ± 0.0031 and that between the T. araraticum accessions 0.0036 ± 0.0015, indicative of about a four-fold intraspecific variation in T. dicoccoides as compared to T. araraticum in terms of genetic distance. No significant genetic differentiation was found for the geographical populations of these species, the genetic distance between the two species being 0.0482 ± 0.0022. The interspecific divergence corrected for intraspecific divergence was 0.0395, about three times that for T. dicoccoides and 11 times that for T. araraticum. The results show that in the wild state the Emmer and Timopheevi groups are clearly differentiated and that T. dicoccoides has much greater variation than T. araraticum, suggesting a relatively recent origin for the latter and therefore a diphyletic origin for these species.Contribution from the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, Japan, No. 535     

A new synthetic species of Cucumis (Cucurbitaceae) from interspecific hybridization and chromosome doubling

A new synthetic species,Cucumis hytivus J.-F. Chen & J. H. Kirkbr. (2n=38), obtained by doubling the number of chromosomes in the sterile interspecific F₁ hybrid (2n=19) betweenC. sativus L. (2n=14) andC. hystrix Chakr. (2n=24), is described.     

Genetic relationships between peanut and wild species ofArachis sect.Arachis (Fabaceae): Evidence from RAPDs

Twenty-six accessions of wildArachis species and domesticated peanuts,A. hypogaea, introduced from South America were analyzed for random amplified polymorphic DNA (RAPD). The objective of the study was to investigate inter- and intraspecific variation and affinities among species of sect.Arachis which have been proposed as possible progenitors for the domesticated peanut. Ten primers resolved 132 DNA bands which were useful for separating species and accessions. The most variation was observed among accessions ofA. cardenasii andA. glandulifera whereas the least amount of variation was observed inA. hypogaea andA. monticola. The two tetraploid species could not be separated by using RAPDs.Arachis duranensis was most closely related to the domesticated peanut and is believed to be the donor of the A genome. The data indicated thatA. batizocoi, a species previously hypothesized to contribute the B genome toA. hypogaea, was not involved in its evolution. The investigation showed that RAPDs can be used to analyze both inter- and intraspecific variation in peanut species. Southern hybridization of RAPD probes to blots containing RAPD of theArachis species provided information on genomic relationships and revealed the repetitive nature of the amplified DNA.     

Independent origins of tetraploid cryptic species in the fern <Emphasis Type="Italic">Ceratopteris thalictroides</Emphasis>

Ceratopteris thalictroides (L.) Brongn is a tetraploid fern species that contains at least three cryptic species, the south, the north and the third type. In this study we combined data from both chloroplast DNA (cpDNA) and nuclear DNA sequences of three diploid species and three cryptic species of C. thalictroides to unravel the origin of the cryptic species, particularly of the reticulate relationships among the diploid and tetraploid taxa in the genus Ceratopteris. Of the three diploid species examined, C. cornuta had cpDNA identical to that of the tetraploid third type plants, and this diploid species is a possible maternal ancestor of the tetraploid third type. Analysis of the homologue of the Arabidopsis thaliana LEAFY gene (CLFY1) identified ten alleles in the genus Ceratopteris, with six alleles found in C. thalictroides. The unrooted tree of the CLFY1 gene revealed four clusters. Each cryptic species showed fixed heterozygosity at the CLFY1 locus and had two alleles from different clusters of the CLFY1 tree. Consideration of the cpDNA sequences, CLFY1 genotypes of the cryptic species and CLFY1 gene tree in concert suggested that the cryptic species of C. thalictroides had originated through independent allopolyploidization events involving C. cornuta and two unknown hypothetical diploid species.     

Molecular evidence for hybridization ofIlex x wandoensis (Aquifoliaceae) by RAPD analysis

Based on the presence of intermediate morphological characters, such as serrated leaf margins and flower structures,Ilex x wandoensis was initially described as a putative natural hybrid betweenI. cornuta andI. Integra, and was formally described as a new hybrid species,I. x wandoensis C. F. Mill., and M. Kim. However, using molecular markers generated via random amplified polymorphic DNA (RAPD), we have now discovered hybridization in populations of theI. x wandoensis complex collected from Wando and Jeju Islands, Korea. Marker bands of the putative parent taxa also were found in some populations ofI. x wandoensis, confirming its hybrid origin. Morphological variability within and among those populations was confirmed by model-based clustering methods, using multilocus genotype data. Phenograms generated from RAPD bands indicated that some accessions ofI. x wandoensis clustered with one of the parental species. This implied the occurrence of hybridization and recurring backcrosses of the hybrid to both parents, resulting in various hybrid derivatives because of the segregation and recombination of traits.Ilex x wandoensis was more closely related toI. cornuta than toI. integra suggesting that it backcrossed more with the former than with the latter.     

Electrophoretic seed albumin patterns inVicia species of sectt.Hypechusa andPeregrinae (Fabaceae)

This work is a continuation of electrophoretic investigations aimed at revealing a wild relative ofVicia faba. Electrophoretic analysis (PAGE) of seed albumins covered 52 accessions representing eightVicia species of sect.Hypechusa and two species of sect.Peregrinae. Most of the examined species showed an intraspecific variation due to differences between accessions and/or individual variation within accessions. In spite of the intraspecific variation, marked interspecific differences were recorded. However, none of the investigated species displayed electrophoretic seed albumin patterns similar to those reported earlier forV. faba. Contribution of the obtained results to characterization of the examined taxa is discussed.     

The discovery and phylogenetic implications of a novel 41 bp plastid DNA deletion in wild potatoes

Insertions and deletions (indels) are common in intergenic spacer regions of plastid DNA and can provide important phylogenetic characters for closely related species. For example, a 241-bp plastid DNA deletion in the trnV-UAC/ndhC intergenic spacer region has been shown to have major phylogenetic importance in determining the origin of the cultivated potato. As part of a phylogenetic study of the wild potato Solanum series Piurana group we screened 199 accessions of 38 wild potato species in nine of the 19 tuber-bearing (Solanum section Petota) series that have not been examined before for indels in the trnV-UAC/ndhC intergenic spacer region. A novel 41 bp deletion (but no 241 bp deletion) was discovered for 30 accessions of three species: S. chiquidenum (5 of 10 accessions), S. chomatophilum (19 of 28), and S. jalcae (6 of 6). Accessions with and without this deletion are found throughout much of the north-south range of all three species in northern and central Peru, but not east of the Marañón River. Multivariate morphological analyses of these 44 accessions showed no morphological associations to the deletion. The results suggest extensive interspecific gene flow among these three species, or a common evolutionary history among species that have never been suggested to be interrelated.     

A second spontaneous hybrid in the genusLeucaena (Leguminosae, Mimosoideae)

The detection of hybridity inLeucaena is discussed in relation to: (i) traditional criteria, (ii) molecular criteria and (iii) models to predict hybrid leaf morphology. Morphological, geographical and molecular evidence for the occurrence of interspecific hybrids betweenL. leucocephala andL. diversifolia in south-central Mexico, northern Guatemala, Jamaica, Dominican Republic, the Philippines and Papua New Guinea is presented. Predicted mean hybrid leaf trait values calculated from parent material are compared with data from putative hybrids and shown to be similar. The origin of these hybrids is discussed and shown to be the result of artificial sympatry resulting from indigenous, and recent exotic, domestication of the parent species. The hybrid is described asL. ×spontanea.     

High frequency somatic embryogenesis and plant regeneration from zygotic embryo-derived callus cultures of three Allium species

The plant regeneration ability of zygotic embryo-derived callus cultures was studied for 12 A. cepa varieties and accessions, two A. fistulosum varieties, one A. fistulosum x A. cepa interspecific hybrid and two A. porrum varieties. Compact embryogenic callus was induced on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid. The embryogenic calluses of all three Allium species were similar in appearance. For all accessions tested plants could be regenerated at a high frequency from this compact callus through somatic embryogenesis, when using kinetin supplemented MS medium (regeneration medium). Addition of abscisic acid to the regeneration medium stimulated the formation of both somatic embryos and shoots for a number of varieties. Concerning shoot regeneration from callus cultures, significant differences existed between genotypes of all accessions except one.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - VDH Van Der Have Seed company     

Relationships betweenNor-loci from differentTriticeae species

TheNor-loci of polyploid wheats and their putative diploid progenitor species were assayed by probing isolated nuclear DNA with ribosomal DNA spacer sequences (spacer rDNA sequences, isolated by cloning), from theNor-loci of genomes B (Triticum aestivum), G (T. timopheevi), B (syn. S,T. speltoides), A (T. monococcum) and V (Dasypyrum villosum). DNA samples for analysis were digested with the restriction endonuclease Taq 1 and assayed by DNA-DNA hybridization under standard (37°C) and high stringency (64°C) conditions. The assay procedure emphasized differences between the divergent spacer sequences of the polyploid species and allowed relative homologies to the respective sequences in diploid species to be established. — The studies indicated thatT. timopheevi andT. speltoides contain different sets of spacer rDNA sequences which were readily distinguishable and, in the case ofT. timopheevi, assigned toNor-loci on different chromosomes. This contrast with the spacer rDNA sequences of the majorNor-loci on chromosomes 1 B and 6 B inT. aestivum, which were difficult to distinguish and were deduced to contain very similar sequences. Among the diploid progenitor species only the spacer rDNA fromT. speltoides shared close homology with polyploid wheat species. OneNor-locus inT. timopheevi (on chromosome 6 G) did not show close homology with any of the rDNA spacer probes available. — The data suggestsT. speltoides was the origin of someNor-loci for both theT. timopheevi andT. turgidum lines of tetraploid wheats. The possibility that the 6GNor-locus inT. timopheevi may have derived from an unknown diploid species by introgressive hybridization is discussed. The spacer rDNA sequence probe fromT. monococcum shared good homology with some accessions ofD. villosum and a line ofT. dicoccoides; the implications of this finding for evolution of present-day wheats are discussed.     

Assessment of the degree of restriction fragment length polymorphism in Brassica

Summary The feasibility of creating a restriction fragment length polymorphism (RFLP) linkage map in Brassica species was assessed by screening EcoRI-, HindIII-, or EcoRV-digested total genomic DNA from several accessions of B. campestris, B. oleracea, and B. napus using random genomic DNA clones from three Brassica libraries as hybridization probes. Differences in restriction fragment hybridization patterns occurred at frequencies of 95% for comparisons of accessions among species, 79% for comparisons of accessions among subspecies within species, and 70% for comparisons among accessions within subspecies. In addition, species differences in the level of hybridization were noted for some clones. The high degree of polymorphism found even among closely related Brassica accessions indicates that RFLP analysis will be a very useful tool in genetic, taxonomic, and evolutionary studies of the Brassica genus. Development of RFLP linkage maps is now in progress.     

Variability of chloroplast DNA and nuclear ribosomal DNA in cassava (Manihot esculenta Crantz) and its wild relatives

Chloroplast DNA (cp) and nuclear ribosomal DNA (rDNA) variation was investigated in 45 accessions of cultivated and wild Manihot species. Ten independent mutations, 8 point mutations and 2 length mutations were identified, using eight restriction enzymes and 12 heterologous cpDNA probes from mungbean. Restriction fragment length polymorphism analysis defined nine distinct chloroplast types, three of which were found among the cultivated accessions and six among the wild species. Cladistic analysis of the cpDNA data using parsimony yielded a hypothetical phylogeny of lineages among the cpDNAs of cassava and its wild relatives that is congruent with morphological evolutionary differentiation in the genus. The results of our survey of cpDNA, together with rDNA restriction site change at the intergenic spacer region and rDNA repeat unit length variation (using rDNA cloned fragments from taro as probe), suggest that cassava might have arisen from the domestication of wild tuberous accessions of some Manihot species, followed by intensive selection. M. esculenta subspp flabellifolia is probably a wild progenitor. Introgressive hybridization with wild forms and pressures to adapt to the widely varying climates and topography in which cassava is found might have enhanced the crop's present day variability.     

Identification of homoeologous chromosomes in hexaploid oat (A. byzantina cv Kanota) using monosomics and RFLP analysis

The use of RFLP markers, together with a partial set of monosomics available in Avena byzantina cv Kanota, has enabled us to identify putative homoeologous chromosome sets in hexaploid Avena species (2n = 6x = 42, AACCDD). We first identified probes producing distinct three-band patterns on Southern blots that possibly reflect orthologous loci of the three genomes present in the hexaploid. Using monosomic analysis, 51 different restriction fragments that hybridized to 26 probes were localized to 12 different chromosomes for which monosomic stocks were available. These DNA restriction fragments were localized to specific monosomics using image analysis to quantify band intensity relative to other bands in the same lane. From these data, we have tentatively identified two complete homoeologous sets of three chromosomes each and two partial sets of two of the three chromosomes. The results indicate that RFLP dosage analysis is useful in the characterization of homoeologous chromosomes in hexaploid oat where nullisomics for many of the chromosomes are not available.Mention of a trademark or proprietary product does not constitute a guarantee or warranty by the USDA-ARS or the University of Minnesota and does not imply approval over other products that also may be suitableJoint contribution of the Minnesota Agricultural Experiment Station and USDA-ARS. Scientific Journal Series Paper no. 20 650 of the Minnesota Agricultural Experiment Station     

Chromosomal relationships among cultivars of Citrus reticulata Blanco, its hybrids and related species

Karyotypes of 93 individuals belonging to 18 accessions of mandarins, mandarin hybrids and two related species were analysed with the fluorochromes CMA and DAPI, to identify marker chromosomes. The karyotypes revealed highly differentiated banding patterns and could be classified in four groups (I–IV) according to the presence/absence of chromosomes with three bands (type A) or with two bands (one proximal and one terminal, type B, or both terminal, type C). The accessions of group I exhibited the simplest and homozygous karyotypes (lacking chromosome types A, B and C), represented by `Sunki' and `Cleopatra'. Group II (lacking chromosome types A and B) included three accessions of Mediterranean mandarins and `Cravo' mandarin, all of them with very similar and almost completely homozygous karyotypes. All other karyotypes of groups II and III (lacking chromosome type A) were heterozygous for one or more chromosome pairs and most of them seemed to be hybrid derivatives from non-mandarin accessions. Group IV (with chromosome types A and B) was represented only by two heterozygous hybrids (`Murcott' and `King'). The karyotype of most hybrids agrees with one of the possible combinations resulting from chromosome types segregation from their putative ancestor karyotypes, but at least `Orlando' seemed to be a more complex hybrid. Comparing with banding patterns of other Citrus species, those of group I and the Mediterranean mandarins (group II) are the best candidates to represent C. reticulata (sensu Swingle) as a true species.     

Canine Dimorphism and Interspecific Canine Form in Cebus

I investigated canine dimorphism and interspecific canine form in adult specimens from 4 capuchin species (Cebus albifrons, C. apella, C. capucinus, and C. olivaceus). I used various univariate and multivariate statistics, which are based on 6 variables, to test several hypotheses that are based upon the finding that canine dimorphism is strongly associated with intermale competition in platyrrhines (Kay et al., 1988), Greenfield (1992a), Plavcan and van Schaik (1992, 1994). Results from the indices of canine dimorphism and the unpaired t-tests corroborate the prediction that males of each species possess significantly larger canines than those of females. Large male canines are especially prominent in 2 variables, maxillary and mandibular canine height. Greene's t-test (1989) does not support the prediction that Cebus apella and C. olivaceus possess a larger degree of canine dimorphism relative to C. albifrons and C. capucinus. No interspecific differences in degree of canine dimorphism are indicated by this test. Results of the discriminant function analyses (DFA) do not find that Cebus apella and C. olivaceus possess different canine form relative to C. albifrons and C. capucinus as predicted. However, Cebus apella is differentiated from the untufted capuchins (C. albifrons, C. capucinus, and C. olivaceus) by the DFA. I suggest that intermale competition is the primary selective force underlying the observed morphological patterns; however, it does not explain all the variation associated with canine dimorphism in Cebus.     

Stylosanthes sp. aff.S. scabra: a putative diploid progenitor ofStylosanthes scabra (Fabaceae)

Stylosanthes sp. aff.S. scabra is an undescribed taxon showing affinities with the allotetraploid speciesS. scabra, but distinct in a number of attibutes. Several collections show potential as forage for clay soils in northern Australia. Twelve accessions have been analysed using STS (sequence-tagged-sites) as genetic markers, and they all displayed STS phenotypes of typical diploid species. Taking into account their morphological similarities, the STS analysis provides strong evidence thatStylosanthes sp. aff.S. scabra might be a diploid progenitor of the allotetraploidS. scabra. This speculation was supported by cytological examinations. Somatic chromosome numbers of two of these accessions were counted and both were found to be diploid (2n = 20). The level of polymorphism among the 12Stylosanthes sp. aff.S. scabra accessions, estimated using randomly amplified polymorphic DNA (RAPD) as markers, was 7.8%, and the dissimilarity value betweenStylosanthes sp. aff.S. scabra andS. viscosa (the other putative progenitor ofS. scabra) was 89%.