Physical map of chloroplast DNA of aerial yam,Dioscorea bulbifera L.

Summary A physical map of chloroplast DNA (ctDNA) of aerial yam, Dioscorea bulbifera L. was constructed using three restriction endonucleases, PstI, SalI, and SmaI. In addition, a clone bank of the BamHI-digested fragments were generated, and the locations of most BamHI fragments on the map were also determined. The ctDNA of D. bulbifera was found to be a circular molecule with a total size of ca. 152 kb involving two inverted repeats of ca. 25.5 kb, and small and large single copy regions of ca. 18.5 and 83.4 kb, respectively. The genes for the large subunit of the ribulose 1,5-bisphosphate carboxylase (rbcL) and the ATP-synthase subunits and (atpB/atpE) were mapped.Contribution from the Plant Germ-plasm Institute and the Laboratory of Genetics (No. 504), Faculty of Agriculture, Kyoto University, Japan. The work was supported in part by a Grant-in-Aid (No. 60400005) from the Ministry of Education, Science and Culture, Japan     

An elicitor-induced cDNA from aerial yam (Dioscorea bulbifera L.) encodes a pathogenesis-related type 4 protein

We established Dioscorea bulbifera (aerial yam) cell suspension cultures to study the expression of defense-related genes upon elicitation with the yam pathogenic ascomycete Colletotrichum gloeosporioides. The induction of phenylalanine-ammonia lyase (PAL) mRNA, coding for a key enzyme involved in phytoalexin biosynthesis, was observed upon elicitation. Using RT-PCR, we isolated an elicited cDNA clone with an open reading frame of 453 nucleotides which showed high homology to cDNA sequences of pathogenesis-related proteins belonging to the PRP-4 group. Yam PRP-4 expression is increased in elicited cell cultures as well as in elicited leaves, and is encoded by a small multigene family. This is the first example for the cloning of a cDNA that might be involved in defense reactions of yam plants. Received: December 1997 / Revision received: June 1998 / Accepted: November 1998     

Intraspecific variation of chloroplast DNA in Dioscorea bulbifera L.

Summary Restriction fragment length polymorphism (RFLP) analysis of chloroplast (ct) DNAs from 15 accessions of Dioscorea bulbifera collected from Africa and Asia was carried out using the Southern hybridization technique. Eight cloned ctDNA fragments of D. bulbifera and D. opposita, which cover 80% of the total chloroplast genome, were used as the probes to detect variation in ctDNA digested with nine restriction endonucleases. Ten variable sites, located in the large and small single-copy regions, were disclosed among the 15 accessions, of which six showed base substitution and four carried length mutation. Positions of the latter mutations were determined on the physical map of ctDNA. Based on these results, chloroplast genomes of the 15 accessions could be classified into nine types. Their phylogenetic relationships are assumed to be as follows: (1) African and Asian chloroplast genomes diverged from each other at the earliest point in time; (2) E-type chloroplast genome, occurring in the south-east edge of the Asian continent, appears to be the most ancient among all the Asian chloroplast genomes; and (3) four chloroplast genomes, found in Asian insular regions, are probably derived independently from the E-type genome. The discrepancy between the taxonomic relationship and the proposed chloroplast genome phylogeny of the present materials is noted.     

Genetic relationships and variation among ecotypes of seashore paspalum (Paspalum vaginatum) determined by random amplified polymorphic DNA markers.

Random amplified polymorphic DNA (RAPD) markers were used to assess genetic relationships and variation among ecotypes of the turfgrass seashore paspalum (Paspalum vaginatum Swartz). Vegetative tissues or seeds of 46 seashore paspalum ecotypes were obtained from various locations in the United States, Argentina, and South Africa. Leaf DNA extracts were screened for RAPD markers using 34 10-mer random primers. A total of 195 reproducible RAPD fragments were observed, with an average of six fragments per primer. One hundred and sixty-nine fragments (87% of the total observed) were polymorphic, among which 27 fragments (16%) were present in three or less ecotypes, indicating the occurrence of a high level of genetic variation among the examined accessions of this species. Cluster analysis (UPGMA) and principal coordinates analysis were performed on the RAPD data set. The results illustrate genetic relationships among the 46 ecotypes, and between ecotypes and their geographical origins. Ecotypes from southern Africa could be differentiated from the U.S. and most of the Argentinean ecotypes. With a few exceptions, ecotypes collected from Argentina, Hawaii, Florida, and Texas were separated into distinct clusters.     

Direct comparison of levels of genetic variation in tomato detected by a GACA-containing microsatellite probe and by random amplified polymorphic DNA.

In this study, a direct comparison was made of the ability of four selected random amplified polymorphic DNA (RAPD) primers and a GACA-containing microsatellite probe to detect genetic variation in Lycopersicon. Of the 89 RAPD primers initially tested, 85 showed differences between a representative of Lycopersicon pennellii and L. esculentum, but only 4 distinguished among three L. esculentum cultivars. These four primers were subsequently tested on representatives of six Lycopersicon species. In pairwise comparisons of species, all or 14 of the 15 combinations could be distinguished by single primers. When the primers were tested on 15 L. esculentum cultivars, 90 of the 105 combinations could be distinguished by the four primers together. Finally, none of 118 tested primers showed reproducible differences among calli or progeny of régénérants from tissue culture, although some of the plants had inherited morphological mutations. The probe pWVA16, which detects GACA-containing microsatellites, could distinguish in TaqI-digested DNA the representatives of Lycopersicon species as well as all the L. esculentum cultivars tested. The probe was unable to detect polymorphisms among calli and the progeny of regenerants from tissue culture. An analysis of the results showed that the four selected RAPD primers were able to detect polymorphic bands among species at a frequency of 80%, and among cultivars at a frequency of 44%. In contrast, the microsatellite probe detected polymorphic bands at a frequency of 100 and 95%, respectively. The GACA-containing probe did not detect any common bands among the representatives of the six species, while band sharing with RAPDs was 48%. These results indicate that the two methods detect two types of DNA that differ in their degree of variability.     

Genomic diversity within Taxus cuspidata var. nana revealed by random amplified polymorphic DNA markers

Taxus cuspidata var. nana is a cultivated variety of Taxus cuspidata and contains taxol, a valuable secondary metabolite of medical importance, both in their stems and leaves. In this paper, random amplified polymorphic DNA (RAPD) markers were used to assess the genomic diversity of individual plants within T. cuspidata population. Seventy-four randomly selected plants were analyzed by 29 selected primers among which 25 primers produced polymorphic banding patterns. The coefficient of similarity among the plants ranged from 0.30 to 1.00 with a mean of 0.605. Our results showed that a surprisingly high level of genomic diversity existed within T. cuspidata, and RAPD markers were effective in revealing the diversity. Cluster analysis divided the plants into two groups. This data, when taken together with earlier findings showing variation in taxol content within a natural population of T. cuspidata, suggests a tantalizing possibility for selecting genomically homogeneous T. cuspidata plant lines with elevated and stable taxol content. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 5, pp. 771–776. The text was submitted by the authors in English.     

红豆杉科植物RAPD分析及其系统学意义

利用随机扩增多态性ＤＮＡ（ＲＡＰＤ）技术分析了红豆杉科（Ｔａｘａｃｅａｅ）红豆杉属（Ｔａｘｕｓ）、白豆杉属（Ｐｓｅｕｄｏｔａｘｕｓ）、穗花杉属（Ａｍｅｎｔｏｔａｘｕｓ）和榧树属（Ｔｏｒｒｅｙａ）的６种植物：红豆杉（Ｔａｘｕｓ ｃｈｉｎｅｎｓｉｓ （Ｐｉｌｇｅｒ）Ｒｅｈｄ）、南方红豆杉（Ｔａｘｕｓ ｃｈｉｎｅｎｓｉｓ ｖａｒ．ｍａｉｒｅｉ（Ｌｅｍｅｅ ｅｔＬｅｖｌ．）Ｃｈｅｎｇ ｅｔＭａｉｒｅＹｅｗ     

Genetic variation in wild sorghum (Sorghum bicolor ssp. verticilliflorum (L.) Moench) germplasm from Ethiopia assessed by random amplified polymorphic DNA (RAPD)

The extent and distribution of genetic variation in wild sorghum (Sorghum bicolor ssp. verticilliflorum (L.) Moench) collected from five different geographical regions in Ethiopia were analyzed using random amplified polymorphic DNA (RAPD) markers for 93 individuals representing 11 populations. Nine decamer primers generated a total of 83 polymorphic bands with 8-12 bands per primer and a mean of 9 bands across the 93 individuals. The amount of genetic variation among the populations (H = 0.37) and among the geographical region (H = 0.44) was low to moderate, despite the high degree of polymorphic bands per primer. Similarly, the mean genetic distance (0.08) among populations as well as among regions of origin (0.04) of the population was found to be low. The low genetic variation may be due to the reduced population size of the wild sorghum in Ethiopia because of habitat change. Partitioning of the genetic variation into between and within the population as well as between and within the regions of origin revealed that 75% and 88% of the variation was found within the populations and within the regions, respectively. Cluster analysis of genetic distance estimates further confirmed low level of differentiation of wild sorghum populations both on population and regional bases. The implications of the results for genetic conservation purposes are discussed.     

Intraspecific genetic diversity of Pyrenophora tritici-repentis (Died.) Drechs. (Drechslera tritici-repentis[Died.] Shoem.) detected by random amplified polymorphic DNA assays

Abstract

Random amplified polymorphic DNAs (RAPDs) were used to study the genetic variation of Pyrenophora tritici-repentis isolates causing wheat tan spot. Two independent experiments were conducted in 2002 – 2003. In 2002, 40 isolates collected in Russia (Krasnodar region, Bashkiria), Germany, and the Czech Republic were studied and 35 unique RAPD genotypes were identified. Most of the genetic variation (72%) was observed within populations and 28% between them. In 2003, 69 new isolates from Russia (Dagestan, North Osetia, Bashkiria), Germany, and the Czech Republic were studied and 47 unique RAPD genotypes were identified. As in 2002, most of the genetic variation (75%) was observed within populations and 25% between them. Total gene diversity in each group ranged from 0.67 – 1.00 for 2002 and was 1.00 for 2003. The average gene diversity was estimated between 0.13 and 0.20 in 2002 and between 0.07 and 0.18 in 2003. A dendrogramme based on genetic distances between isolates illustrates that the variation is distributed on a small scale (0.3 – 4.0%). Estimated F_ST values and clustering of isolates on dendrogrammes suggest that groups of isolates from Bashkiria and groups of isolates from Dagestan and North Osetia are separated from others and may be considered as different geographical populations. No clear differentiation between isolates from other sites was revealed.     

Genetic diversity and relationships in olive ( Olea europaea L.) germplasm collections as determined by randomly amplified polymorphic DNA

Genetic diversity studies using the RAPD technique were carried out in a set of 103 olive cultivars from the World Germplasm Bank of the Centro de Investigación y Formación Agraria (CIFA) "Alameda del Obispo" in Cordoba (Spain). A total of 126 polymorphisms (6.0 polymorphic markers per primer) out of 135 reproducible products (6.4 fragments per primer) were obtained from the 21 primers used. The number of bands per primer ranged from 4 to 11, whereas the number of polymorphic bands ranged from 3 to 10, corresponding to 83% of the amplification products. The dendrogram based on unweighted pair-group cluster analysis using Jaccard's index includes three major groups according to their origin: (1) cultivars from the Eastern and Central Mediterranean areas, (2) some Italian and Spanish cultivars, and (3) cultivars from the Western Mediterranean zone. The pattern of genetic variation among olive cultivars from three different Mediterranean zones (West, Centre and East) was analysed by means of the analysis of molecular variance (AMOVA). Although most of the genetic diversity was attributable to differences of cultivars within Mediterranean zones (96.86%) significant phi-values among zones (phi(st) = 0.031; p < 0.001) suggested the existence of phenotypic differentiation. Furthermore, the AMOVA analysis was used to partition the phenotypic variation of Spain, Italy (Western region), Greece and Turkey (Eastern region) into four categories: among regions, among countries (within regions), within countries, and among and within countries of each region. Most of the genetic diversity was attributable to differences among genotypes within a country. These results are consistent with the predominantly allogamous nature of Olea europaea L. species. This paper indicates the importance of the study of the amount and distribution of genetic diversity for a better exploration of olive genetic resources and the design of plant breeding programmes.     

Characterization of genetic identities and relationships of Brassica oleracea L. via a random amplified polymorphic DNA assay

Summary Effective conservation and the use of plant genetic resources are essential for future agricultural progress. Critical to this conservation effort is the development of genetic markers which not only distinguish individuals and accessions but also reflect the inherent variation and genetic relationships among collection holdings. We have examined the applicability of the random amplified polymorphic DNA (RAPD) assay for quick, cost-effective, and reliable use in addressing these needs in relation to collection organization and management. Twenty-five decamer oligonucleotide primers were screened individually with a test array composed of individuals representing a range of genetic relationships in Brassica oleracea L. (vegetable and forage cole crops). Over 140 reproducible, polymorphic fragments were generated for study. Each individual of the test array exhibited a unique molecular genotype and composites specific for accessions and botanical varieties could be established. An analysis of similarity based on amplified DNA fragments reflected the known genetic relationships among the selected entries. These results demonstrated that RAPD markers can be of great value in gene bank management for purposes of identification, measurement of variation, and establishment of genetic similarity at the intraspecific level.     

Genetic relationships among NE Turkish Lilium L. (Liliaceae) species based on a random amplified polymorphic DNA analysis

Random amplified polymorphic DNA (RAPD) fingerprinting was used to study species boundaries in six closely related NE Turkish Lilium (Liliaceae) taxa of the section Liriotypus. The investigated taxa were L. ciliatum, L. akkusianum, L. ponticum, L. kesselringianum, L. armenum, and L. szovitsianum. Of the 108 primers screened, 11 provided polymorphic and reproducible bands. A total of 93 polymorphic bands were scored for 122 individuals from 18 populations of the six Lilium taxa and principle coordinate analysis and neighbour-joining cluster analysis based on these RAPD profiles were performed. The results demonstrate a clear distinction between the two species L. ciliatum and L. akkusianum, and the other four species. While populations of the two species groups are found to be allopatrically distributed, the two species groups overlap in their geographical ranges. Analysis of molecular variance (AMOVA) indicated that nearly half of the total molecular variance is found within the individual populations and that the molecular variance among species is as high as the variance within the individual species, indicating that genetic differentiation of the species is rather weak.     

Genomic fingerprinting of Clostridium difficile isolates by using a random amplified polymorphic DNA (RAPD) assay

Abstract This study describes the use of a new and easy method called random amplfied polymorphic DNA (RAPD) assay to distinguish strains of C. difficile . We used two single short primers (AP4 and AP5) with arbitrary nucleotide sequences in a polymerase chain reaction to amplify genomic DNA. The profiles observed after electrophoretic separation were able to distinguish 20 reference C. difficile strains previously serotyped by Delmées method. The fingerprints of 11 epidemiologically unrelated C. diffiile strains clearly yielded a DNA polymorphism between all the strains. Latterly, RAPD profiles of 11 C. difficile strains isolated from 2 independant suspected outbreaks showed, in each case, a predominant banding pattern correponding to an epidemic strain. These results suggest that RAPD assay could be a valuable tool for epidemiological studies.     

Genome relationships among Lotus species based on random amplified polymorphic DNA (RAPD)

The ability of random amplified polymorphic DNA (RAPD) to distinguish among different taxa of Lotus was evaluated for several geographically dispersed accessions of four diploid Lotus species, L. tennis Waldst. et Kit, L. alpinus Schleich., L. japonicus (Regel) Larsen, and L. uliginosus Schkuhr and for the tetraploid L. corniculatus L., in order to ascertain whether RAPD data could offer additional evidence concerning the origin of the tetraploid L. corniculatus. Clear bands and several polymorphisms were obtained for 20 primers used for each species/accession. The evolutionary pathways among the species/accessions presented in a cladogram were expressed in terms of treelengths giving the most parsimonious reconstructions. Accessions within the same species grouped closely together. It is considered that L. uliginosus which is most distantly related to L. corniculatus, may be excluded as a direct progenitor of L. corniculatus, confirming previous results from isoenzyme studies. Lotus alpinus is grouped with accessions of L. corniculatus, which differs from previous studies. With this exception, these findings are in agreement with previous experimental studies in the L. corniculatus group. The value of the RAPD data to theories on the origin of L. corniculatus is discussed.     

Phylogenetic relationships among ten Elymus species based on random amplified polymorphic DNA

Ten species of Elymus (Poaceae: Triticeae) were analyzed using random amplilied polymorphic DNA (RAPD) markers. Thirty-four decamer oligonucleotide random primers from Opron Technologies were used for polymorphic selection. 25 (73.53%) produced polymorphic products. A total of 136 bands amplified from 16 primers were selected for RAPD analyses. The data were used to generate Nei's similarity coefficients and to construct a dendrogram using UPGMA in NTSYS programs. The result showed that: (1) Three tetraploid species, E. sibiricus L., E. caninus (L.) L. and E. lanceolatus (Scribner et Smith) Gould, were clustered in one group, while seven hexaploid species, E. nutans Griseb., E. dahuricusTurcz., E. brachyaristatus A Löve, E. submuticus (Keng) Keng f., E. tangutorum (Nevski)Hand.-Mazz., E. excelsus Turcz. and E. cylindricus (Franch) Honda, were clustered in another group. 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DNA fragments of organellar origin in random amplified polymorphic DNA (RAPD) patterns of sugar beet (Beta vulgaris L.)

The technique of random amplified polymorphic DNA (RAPD) offers a broad range of applications in the investigation of plant genomes. A promising prospect is the use of RAPD products as genetic markers. We have investigated a possible organellar source of fragments in RAPD patterns of total DNA. Two nearly-isogenic lines of cytoplasmic male-sterile and male-fertile sugar beet (Beta vulgaris L.) were subjected to RAPD analysis with six different primers. Total, nuclear, mitochondrial (mt), and chloroplast (cp), DNA from each line were investigated. Reproducible DNA fingerprints could be obtained from both organellar DNAs. Differences in band patterns of mtDNA between cytoplasmic male-sterile and -fertile lines were observed with five out of six primers, whereas different cpDNA patterns were generated by one of the primers. Consequently, the RAPD technique can be used to discriminate between different cytoplasms. Clear evidence is provided for the organellar origin of fragments in genomic (total DNA) RAPD patterns. The consequences of these results for the interpretation of RAPD analyses are discussed.