Molecular phylogeny and DNA amplification fingerprinting of Petunia taxa

The relationship of five species of Petunia and ten cultivars of the cultivated petunia, Petunia x hybrida, were investigated using DNA-amplification fingerprinting (DAF). Reproducible banding profiles were obtained from P. parodii and P. axillaris DNA from different seed sources. In contrast, other petunias such as P. inflata, P. violacea and P. integrifolia produced variable fingerprints when different plants were examined. However, representative profiles of the variable Petunia taxa were obtained by bulking the leaf tissue from ten different individual plants. Each of ten octamer primers revealed polymorphic loci between taxa. Among a total of 201 bands produced, 146 (73%) loci were polymorphic and distinguished all species and cultivars. Phenetic and cluster analysis using DAF markers separated P. axillaris from P. parodii and distinguished between the violet-flowered species, P. inflata, P. violacea, and P. integrifolia. P. parodii grouped together with the monophyletic set of the ten cultivars of P. x hybrida examined, indicating that it had made a major contribution to the development of these cultivars. Cultivars were distributed within the dendograms by flower color. The results demonstrated the utility of DAF in establishing relationships among closely related species and cultivars of Petunia.     

DNA amplification variation within cultivars of turf-type Couch grasses (Cynodon spp.)

Arbitrary primed polymorphic DNA was employed to investigate relationships among 18 Cynodon cultivars available in Australia. Thirteen out of the 20 random primers screened gave reproducible banding patterns for all samples. The cultivars showed a high level of polymorphism. Each cultivar was readily distinguishable with a combination of primers. One primer was able to discriminate between all the cultivars except Tifdwarf and its `off-type' sample. The Cynodon grasses used in this study separated into two distinct groups based on a distance matrix calculated from the DNA amplification data. The results clearly demonstrate a methodology based on arbitrary primed DNA amplification can be used to identify and fingerprint Cynodon cultivars. Received: 22 October 1996 / Revision received: 11 March 1997 / Accepted: 4 April 1997     

DNA amplification fingerprinting: A strategy for genome analysis

A novel strategy to detect genetic differences among organisms, DNA amplification fingerprinting (DAF), uses a thermostable DNA polymerase directed by usually one short (≥5 bp) oligonucleotide primer of arbitrary sequence to amplify short segments of genomic DNA and generate a range of DNA extension products. These products can be analyzed by polyacrylamide gel electrophoresis and silver staining. DAF is rapid and sensitive and is independent of cloning and prior genetic characterization. Here we describe this new methodology, its application to plant genotyping, and its perspectives in DNA fingerprinting and genome mapping.     

Enhanced detection of polymorphic DNA by multiple arbitrary amplicon profiling of endonuclease-digested DNA: identification of markers tightly linked to the supernodulation locus in soybean

Multiple endonuclease digestion of template DNA or amplification products can increase significantly the detection of polymorphic DNA in fingerprints generated by multiple arbitrary amplicon profiling (MAAP). This coupling of endonuclease cleavage and amplification of arbitrary stretches of DNA, directed by short oligonucleotide primers, readily allowed distinction of closely related fungal and bacterial isolates and plant cultivars. MAAP analysis of cleaved template DNA enabled the identification of molecular markers linked to a developmental locus of soybean (Glycine max L. Merrill). Ethyl methane sulfonate (EMS)-induced supernodulating, near-isogenic lines altered in the nts locus, which controls nodule formation, could be distinguished from each other and from the parent cultivar by amplification of template pre-digested with 2–3 restriction enzymes. A total of 42 DNA polymorphisms were detected using only 19 octamer primers. In the absence of digestion, 25 primers failed to differentiate these soybean genotypes. Several polymorphic products co-segregated tightly with the nts locus in F₂ families from crosses between the allelic mutants nts382 and nts1007 and the ancestral G. soja Sieb. & Succ. PI468.397. Our results suggest that EMS is capable of inducing extensive DNA alterations, probably around discrete mutational hot-spots. EMS-induced DNA polymorphisms may constitute sequence-tagged markers diagnostic of specific genomic regions.     

Allozyme genetic distances and evolutionary relationships in species of akodontine rodents (Cricetidae: Sigmodontinae)

Allozyme genetic distances were estimated for ten species of akodontine rodents, as compared with the Oryzomyini Oligoryzomys longicaudatus , which was used as an outgroup to assess plesiomorphic character-states. Twenty-six loci were analysed. Distribution patterns of allele frequencies were determined by both phenetic (UPGMA) and cladistic (PAUP') techniques. Allozyme analysis confirmed monophyly for the Akodontini, and among them, the distinctiveness of the genus Oxymycterus. Genetic divergence among the eight species of Akodon was small compared to most known species of rodents. Phenrtic and phylogenetic analysis between Bolonys obscurus and species of Akodon was m agreement with previous chromosomal work but in disagreement with the indications of morphology. The general lack of allozymic differentiation among members of the Akodontini suggests that in this group molecular divergence is unrelated to speciation. Molecular clock estimation calibrated by fossils showed that generic divergence within Akodontini started at least in the late Miocene and that divergence of Akodintini from Orizomyini occurred within the Miocene.     

Low-Cot DNA sequences for fingerprinting analysis of germplasm diversity and relationships in Amaranthus

We examined genetic diversity and relationships among 24 cultivated and wild Amaranthus accessions using the total low-Cot DNA and five individual repetitive sequences as probes. These low-Cot DNA probes were obtained by the isolation of various classes of repetitive-DNA sequences, including satellites, minisatellites, microsatellites, rDNA, retrotransposon-like sequences, and other unidentified novel repetitive sequences. DNA fingerprints generated by different types of repetitive-DNA probes revealed different levels of polymorphism in the Amaranthus genomes. A repetitive sequence containing microsatellites was found to be a suitable probe for characterizing intraspecific accessions, whereas more conservative sequences (e.g. rDNA) were informative for resolving phylogenetic relationships among distantly related species.Genetic diversity, measured as restriction fragment length polymorphism (RFLP) and the similarity index at the low-Cot DNA level, was equally high among intraspecific accessions between the two species groups: grain amaranths (A. caudatus, A. cruentus, and A. hypochondriacus) and their putative wild progenitors (A. hybridus, A. powellii, and A. quitensis). At the interspecific level, however, the grain amaranth species are less divergent from each other than their wild progenitors. With the rare exceptions of certain A. caudatus accessions, grain amaranths were found to be closely related to A. hybridus. The results based on low-Cot DNA were comparable with previous RAPD and isozyme studies of the same set of species/accessions of Amaranthus, indicating that low-Cot DNA sequences are suitable probes for a fingerprinting analysis of plant germplasm diversity and for determining phylogenetic relationships. Received: 19 October 1998 / Accepted: 8 January 1999     

Random amplified polymorphic DNA (RAPD) analysis reveals genetic relationships among the annual Cicer species

 Random amplified polymorphic DNA markers were used to distinguish between nine different Cicer taxa representing the cultivated chickpea and eight other related annual wild species. Of the 75 random10-mer primers tested, only 8 amplified genomic DNA across all the species. A total of 115 reproducibly scorable RAPD markers were generated, all except 1 polymorphic, and these were utilized to deduce genetic relationships among the annual Cicer species. Four distinct clusters were observed and represented C. arietinum, C. reticulatum and C. echinospermum in first cluster followed by C. chorassanicum and C. yamashitae in the second cluster, while C. pinnatifidum, C. judaicum and C. bijugum formed the third cluster. Cicer cuneatum did not cluster with any of the species and was most distantly placed from the cultivated species. Except for the placement of C. chorassanicum and C. yamashitae, deduced species’ relationships agreed with previous studies. In addition, species-diagnostic amplification products specific to all the nine species were identified. The results clearly demonstrate a methodology based on random-primed DNA amplification that can be used for studying Cicer phylogeny and chickpea improvement. Received: 27 July 1998 / Accepted: 5 August 1998     

Inheritance of polymorphic markers generated by DNA amplification fingerprinting and their use as genetic markers in soybean

DNA amplification fingerprinting (DAF) using a high primer-to-template ratio and single, very short arbitrary primers, was used to generate amplified fragment length polymorphic markers (AFLP) in soybean (Glycine max (L.) Merr.). The inheritance of AFLPs was studied using a cross between the ancestral Glycine soja PI468.397 and Glycine max (L.) Merr. line nts382, F1 and F2 progeny. The amplification reaction was carried out with soybean genomic DNA and 8 base long oligounucleotide primers. Silver-stained 5% polyacrylamide gels containing 7 M urea detected from 11 to 28 DAF products with primers of varying GC content (ranging from 50 to 100% GC). Depending on their intensity, AFLPs were classified into three classes. DAF profiles were reproducible for different DNA extractions and gels. Forty AFLPs were detected by 26 primers when comparing G. soja and G. max. Most AFLPs were inherited as dominant Mendelian markers in F1 and F2 populations. However, abnormal inheritance occured with about 25% of polymorphisms. One marker was inherited as a maternal marker, presumably originating from organelle DNA while another showed apparent paternal inheritance. To confirm the nuclear origin and utility of dominant Mendelian markers, three DAF polymorphisms were mapped using a F11 mapping population of recombinant inbred lines from soybean cultivars Minsoy × Noir 1. The study showed that DAF-generated polymorphic markers occur frequently and reliably, that they are inherited as Mendelian dominant loci and that they can be used in genome mapping.     

Phylogenetic relationships in some diploid species of Triticineae: cytogenetic analysis of interspecific hybrids

Summary Ten diploid species from genera Triticum, Aegilops, Haynaldia and Secale were included in a diallel crossing program. Forty-one different interspecific hybrids were obtained. The number of associations between chromosome arms at metaphase I of meiosis in pollen mother cells from the hybrids was taken as an indication of the degree of homology between parental genomes. Genome relationships were defined and indicated a possible pattern of differentiation from a common ancestor. Breeding strategies based on this information are proposed.     

Multilocus DNA fingerprinting and RAPD reveal similar genetic relationships between strains of Oreochromis niloticus (Pisces: Cichlidae)

Two molecular techniques which reveal highly variable DNA polymorphisms, RAPD and multilocus DNA fingerprinting, were used to evaluate genetic diversity between six aquacultural strains of Oreochromis niloticus (tilapia) from the Philippines. The results using both techniques were in close agreement Within-strain heterozygosity values were similar and were correlated between the two data sets, but statistical errors associated with the RAPD data set were lower. Although genetic distances between strains were greater using DNA fingerprinting, the distances measured using both methods were significantly correlated. Both methods were useful in estimating variation between strains, but they offered different advantages. RAPD was technically easier to perform and produced results with low statistical error, whereas DNA fingerprinting detected greater genetic differentiation between strains. The theoretical basis for using RAPD and multilocus minisatellite markers for population studies is discussed.     

Evaluation of genetic relationships in Dalbergia species using RAPD markers

Conservation of identified germplasm is an important component forefficient and effective management of plant genetic resources. Traditionally,species identification has relied on morphological characters like growth habit,floral morphology like flower colour, and agronomic characteristics of the plant.Dalbergia species are important wind-dispersed tropicaltimber trees which exhibit high intrafruit seed abortion because of intensesibling competition for maternal resources. Studies were undertaken foridentification and genetic relationships in five species ofDalbergia and to evaluate genetic diversity withinpopulations of Dalbergia sisso, D.latifolia, D. paniculata, D.assamica and D. spinosa by using randomamplified polymorphic DNAs (RAPD) markers. Analysis was started by using 30decamer primers that allowed to distinguish five species and to select a reducedset of primers. The selected primers were used for identification and forestablishing a profiling system to estimate genetic relationships and toevaluate the genetic variability among the individuals in a population ofDalbergia species. A total of 120 distinct DNA fragments(bands), ranging from 0.3 to 4.0 kb, were amplified byusing nine selected random decamer primers. The genetic similarity was evaluated onthe basis of presence or absence of bands, which revealed a wide range ofvariability within the species. The cluster analysis indicated that five speciesof Dalbergia formed two major clusters. The first clusterconsisted of D. spinosa, D. latifolia and D.sisso. The second cluster was represented by two species, i.e.D. paniculata and D. assamica.A maximum similarity of 60% was observed in D. paniculata andD. assamica and they formed a minor cluster.Dalbergia latifolia and D. sissoformed another minor cluster with more than 50% similarity. Dalbergiaspinosa shared up to 40% similarity with D.latifolia and D. sisso. All the species sharemore than 20% similarity among themselves. The closest genetic distance existedwithin populations of different Dalbergia species. Thus,these RAPD markers have the potential for conservation of identified clones andcharacterization of genetic relatedness among the species. This is also helpful intree breeding programs and provides an important input into conservation biology.     

DNA fingerprinting: a tool for determining genetic distances between strains of poultry

Summary DNA fingerprinting, a technique based on the detection of hypervariable minisatellite regions in DNA restriction fragments, was tested for its applicability to conduct population genetics in poultry. Using MspI digestion and phage M13 DNA as a probe, between 25 and 35 minisatellite-containing DNA fragments were observed per bird. Comparison of the banding pattern of offspring with their parents revealed that the bands were inherited as stable genetic traits. The variability of the DNA fingerprinting pattern was reduced in inbred strains. DNA fingerprints of chickens from five well-defined populations of known genetic relationships were analyzed and indices of genetic distances were computed. They correctly reflected the history of these strains, indicating that DNA fingerprinting may be a powerful tool to characterize genetic relationships between different breeding populations of the same species.     

Studies on genetic diversity and phylogenetic relationships of limpograss (Hemarthria altissima) and related species based on combined chloroplast DNA intergenic spacer data

Hemarthria R. Br. is a genus which includes important forage grasses. However, there is currently a lack of data analysis on the chloroplast DNA (cpDNA) of Hemarthria species. This study is to use three cpDNA intergenic spacers (trnL-F, trnC-ycf6 and psbC-trnS) to obtain phylogenetic information in 36 Hemarthria samples including four Hemarthria species: Hemarthria altissima (Poir.) Stapf et C. E. Hubb., Hemarthria compressa (L. f.) R. Br., Hemarthria uncinata R. Br., and Hemarthria japonica (Hack.) Roshev. Data analysis revealed that non-significant genetic diversity existed in our samples, which was implied by nucleotide sequences information and the results of haplotypic and nucleotide diversity. The results of phylogenetic trees based on maximum likelihood (ML) and Bayesian inference (BI) revealed that H. altissima and H. compressa samples were not entirely distinct, suggesting that the two species share an intimate genetic relationship. A haplotype median-joining (MJ) network revealed broadly similar results to those derived from the ML and BI trees and implied that haplotype H3 may represent an ancient haplotype. Analysis of the population statistic F_ST revealed little genetic differentiation among the seven populations of H. altissima in Africa.     

Parametric relationships between genotype x environment interaction and genetic correlation when two environments are involved

Summary Parametric relationships between the genotype x environment interaction and the genetic correlation of the same attribute measured in two different environments are derived. It is shown that the criticism by Fernando et al. (1984) of Yamada's method (1962) in the case of unbalanced data is irrelevant.     

Use of microsatellites to evaluate genetic diversity and species relationships in the genus Lycopersicon

In order to determine how informative a set of microsatellites from tomato is across the genus Lycopersicon, 17 microsatellite loci, derived from regions in and around genes, were tested on 31 accessions comprising the nine species of the genus. The microsatellite polymorphisms were used to estimate the distribution of diversity throughout the genus and to evaluate the efficacy of microsatellites for establishing species relationships in comparison with existing phylogeny reconstructions. Gene diversity and genetic distances were calculated. A high level of polymorphism was found, as well as a large number of alleles unique for species. The level of polymorphism detected with the microsatellite loci within and among species was highly correlated with the respective mating systems, cross-pollinating species having a significantly higher gene diversity compared to self-pollinating species. In general, microsatellite-based trees were consistent with a published RFLP-based dendrogram as well as with a published classification based on morphology and the mating system. A tree constructed with low-polymorphic loci (gene diversity <0.245) was shown to represent a more-reliable topology than a tree constructed with more-highly polymorphic loci. Received: 19 February 2001 / Accepted: 26 March 2001     

Phytomonas: analysis of polymorphism and genetic relatedness between isolates from plants and phytophagous insects from different geographic regions by RAPD fingerprints and synapomorphic markers

The random amplification of polymorphic DNA was used for easy, quick and sensitive assessment of genetic polymorphism within Phytomonas to discriminate isolates and determine genetic relationships within the genus. We examined 48 Phytomonas spp., 31 isolates from plants and 17 from insects, from different geographic regions. Topology of the dendrogram based on randomly amplified polymorphic DNA fingerprints segregated the Phytomonas spp. into 5 main clusters, despite the high genetic variability within this genus. Similar clustering could also be obtained by both visual and cross-hybridization analysis of randomly amplified synapomorphic DNA fragments. There was some concordance between the genetic relationship of isolates and their plant tissue tropism. Moreover, Phytomonas spp. from plants and insects were grouped according to geographic origin, thus revealing a complex structure of this taxon comprising several clusters of very closely related organisms.     

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.     

Allozyme variation and genetic inter-relationships between seven flatfish species (Pleuronectiformes)

A total of 11 enzymes coded by 14 loci were assayed for each of 17 populations (from the north east Atlantic and Mediterranean) of seven flatfish species, including representatives of three of the four European families of the order Pleuronectiformes. Diagnostic alleles were observed for each species and there were fixed differences between the species at many loci. Thus all species were genetically distinct, although there were some common alleles. H_obs was higher than the average of a range of fish species and was also higher than that of vertebrate species as a whole. It seems that flatfish as a group may show higher levels of genetic variation than other fish. Values of genetic identity for all pairwise comparisons fell comfortably within the ranges expected. The data support the grouping of plaice and flounder into a single genus, Pleuronectes , but it is concluded that the retention of the dab in a separate genus, Limanda , is justified. A high level of genetic divergence was found between Dover and thickback soles. Genetic divergence data support the hypothesis that Pleuronectes flesus luscus in the Aegean Sea is a distinct subspecies of Pleuronectes flesus (flounder). The data show a clear separation of the Mediterranean Dover sole population from those in the Atlantic. Low genetic divergence was observed between the Aegean Sea and Atlantic brill populations. It is speculated that about 5 Mya the families Pleuronectidae and later Soleidae evolved from the ancestral Scophthalmidae.     

Development of highly regenerable callus lines and biolistic transformation of turf-type common bermudagrass [<Emphasis Type="Italic">Cynodon dactylon</Emphasis> (L.) Pers.]

Common bermudagrass, Cynodon dactylon, is a widely used warm-season turf and forage species in the temperate and tropical regions of the world. Improvement of bermudagrass via biotechnology depends on improved tissue culture responses, especially in plant regeneration, and a successful scheme to introduce useful transgenes. When the concentration of 6-benzylaminopurine was adjusted in the culture medium, yellowish, compact calluses were observed from young inflorescence tissue culture of var. J1224. Nine long-term, highly regenerable callus lines (including a suspension-cultured line) were subsequently established, of which six were used for biolistic transformation. Five independent transgenic events, with four producing green plants, were obtained following hygromycin B selection from one callus line. Three transgenic events displayed resistance to the herbicide glufosinate, and one of these showed -glucuronidase activity since the co-transformation vector used in the experiments contained both the gusA and bar genes.Abbreviations ABA Abscisic acid - BAP 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA ₃ Gibberellic acid - GUS -Glucuronidase - hph Hygromycin phosphate transferase - hyg B Hygromycin B - NAA -Naphthaleneacetic acid - SEC Somatic embryo cluster Communicated by P. Ozias-Akins