RAPD Bands Research Articles

How selfing and intra- and interspecific crossing influence seed set, morphology and ploidy level in Euphrasia: An experimental study of species occurring in the Alps of Switzerland

Annual alpine species rely on selfing rather than on cross-pollination for successful reproduction. However, insect visits may occasionally cause cross-pollination not only within but also between closely related species. The aim of this study was to investigate four species of Euphrasia for their efficiency in spontaneous selfing and their success in intra- and interspecific crossing. We used the seed sets that followed spontaneous selfing and artificial cross- and selfpollination to measure the breeding success. We compared the morphological characters of species and hybrids and determined their ploidy level using flow cytometry. We verified the hybridogenous origin of plants resulting from interspecific crosses using RAPD banding patterns. While spontaneous seed set was high in the two small-flowered species, seed set in the large-flowered species was small and affected by external circumstances. We obtained F1 and F2 hybrids from interspecific crosses of two diploid species and detected polyploid individuals in both generations.

Molecular Mapping of Rice Blast Resistance Gene Pi-kh in the Rice Variety Tetep

We have used rice line Tetep as a resistant donor with the aim of mapping a durable blast resistance gene Pi-kh using RAPD and AFLP techniques in conjunction with bulk segregant analysis. An F2 mapping population consisting of 205 plants was generated by crossing Tetep with HP2216, a highly susceptible cultivar. Inoculation with specific isolate (PLP-1) of Magnaporthe grisea at seeding stage showed that the Pi-kh gene inherited as a single dominant gene in F2 population. RAPD analysis was performed with 240 primers to detect polymorphism between resistant and susceptible parents. Of these, 48 primers produced polymorphic banding pattern between resistant and susceptible parents. Bulk segregant analysis was performed with 48 primers of which 5 showed polymorphism between resistant and susceptible bulks. A 700 bp DNA band was obtained in resistant F2 plants with primer 5-129 indicating its linkage to the resistance gene. Out of 64 AFLP primer combinations used for polymorphism survey between HP 2216 and Tetep, 11 AFLP primer combinations were able to distinguish the resistant and susceptible bulks. An AFLP band of 75 bp obtained with primer combination, E-TAlM-CTC co-segregated with the resistance gene. The RAPD marker 5-129700 and AFLP75 were placed on the linkage map at a distance of 2.1 eM and 15.1 eM flanking to Pi-khgene, respectively. The RAPD band closely linked to Pi-kh gene was sequenced and used for the development of CAPs markers which also co-segregated with resistant phenotype in the mapping population. On sequence analysis and homology search of RAPD fragment with whole rice genome sequence database and the information available on physical, genetic and sequence maps of rice, the co-segregating CAPs marker was placed at long arm of rice chromosome 11. CAPs marker developed in this study showed polymorphism in different rice cultivars grown in North-Western Himalayan region and is being used for the pyramiding of Pi-kh gene along with other blast resistance genes using marker-assisted selection.

Molecular Polymorphism of Strains within Paramecium septaurelia (Ciliophora, Oligohymenophorea)

RAPD-PCR fingerprinting and ARDRA riboprinting revealed polymorphism within P. septaurelia strains from Russia (4 strains from Lower Volga Basin), and one strain from USA, Florida. However, the first method showed the existence of four RAPD genotypes while the second revealed only two groups of strains with different band patterns. All studied strains had a high percentage of surviving hybrid clones in the inter-strain crosses, with little differentiation of strains within species. Intra-species differentiation of strains in RAPD band patterns may be connected with the degree of inbreeding for the studied species. Species of the P. aurelia complex can be arranged according to the degree of inbreeding characteristic for each, which is correlated with the degree of DNA polymorphism revealed by the RAPD method from extreme inbreeders (e.g. P. sexaurelia), moderate inbreeders (e.g. P. triaurelia) to weak inbreeders (e.g. P. pentaurelia). P. septaurelia of the P. aurelia complex should be included in the group of extreme inbreeder.

품종 특이성을 이용한 제주마 판별 표지인자 재발

This study was conducted to analyze genetic characteristics and to develop the specific marker for Cheju native horse (Coo) at the level of sequence characterized amplified regions (SCARs). We collected blood samples from Cheju native horse and Thoroughbred horse (Th) and obtained genomic DNA from the blood of 50 individuals randomly selected within the breeds. Seven hundred primers were chosen randomly and were used to examin the polymorphism and 40 kinds of primers showed polymorphic RAPD band patterns between two breeds. Thirty primers of them showed horse specific bands. With the primer MG 30, amplified band of 2.0 kb showed the specificity to Cheju native horse (Cnh). Additionally MG 53 detected the thoroughbred horse (Th) specific markers at size of 2.3 kb. As the next, 2.3 kb band from MG 53 was checked with the all individuals from all the breeds of this study, and it maintained the reproducible breed specificity to thoroughbred horse (Th). With this results, 2.3 kb band was cloned into plasmid vector and sequenced bidirectionally from both ends of the cloned fragment. With the obtained sequences 10 nucleotide extended primers including the original arbitray primer were designed as a SCARs primer. Finally, the primer with extended sequence showed the reproducible breed differentiation pattern and it was possible to identify Cheju native horse (Cnh) from other breeds. The SCARs marker 2.3 kb from MG 53 could be used to identify Cheju native horse (Cnh) for not only registration but also horse breeding programe.

PCR-BASED MARKERS AND CUT FLOWER LONGEVITY IN CARNATION

In carnation, the identification of molecular markers linked to flower vase life character could be an important tool to improve the efficiency of breeding programs, considering that this is one of the most important traits selected by breeders. Longevity is probably a complex quantitative trait, involving several genes showing predominantly additive effects. A previous study carried out on cv. Roland, cv. Milady, and their progenies showed that some RAPD bands significantly discriminated a population with longer vase life. With the aim to verify the general use of these markers for assisted selection, 12 commercial varieties of carnation were collected and analyzed with the RAPD technique. The 23 fragments produced with ten decamers were not able to discriminate the genotypes with greater vase life. In order to identify more effective markers, preliminary analyses were also conducted on four genotypes, using 30 primer sets designed to amplify internal sequences from ethylene biosynthesis and response pathway genesPCR products were obtained with 22 primer pairs, and some polymorphic fragments were observed even in the agarose gels.

Genetic variation among isolates of Alternaria spp. from select Egyptian crops

Nine different species of Alternaria represented by 27 isolates were collected from several Egyptian crops and analyzed by RAPD-PCR for genetic diversity using four different RAPD primers. Most isolates produced a distinct pattern of DNA fragments which was used as a measure of the degree of similarity among isolates. Genetic similarity between isolates of A. alternata species-group ranged from 14.8 to 100%, whereas the range was from 14.81 to 96.30% between the other species used in this study. The results indicated considerable genetic variability among Alternaria isolates, even within the same species-group. The phylogenetically related outgroup genus Stemphylium could not be clearly separated using RAPD banding criteria. Of the isolates studied, 62% were pathogenic on tomato fruits. There was no correlation between RAPD cluster analysis and pathogenic abilities of the isolates tested.

Simple Identification of Some Bacillus Species and Related Genera in Food by RAPD Analysis Combined with Morphological Observation

Template DNA from Brevibaciiius brevis, Geobaciiius stearothermophilus, Paenibaciiius macerans, P. polymyxa and 10 species of Bacillus was used with the commercially available RAPD Analysis Primers to generate DNA fragments by PCR. Different RAPD band patterns were generated from the DNA of 14 different Bacillus species and related genera with Primer 1 or 4. The result suggested that the single use of Primer 1 or 4 is effective for the simple identification of these strains of Bacillus and related genera by the RAPD analysis. By the RAPD analyses of the template DNA prepared from bacterial colonies formed on nutrient agar plates after cultivation at 35°C for 24 to 48 h, B. brevis, B. circulans, B. firmus, B. iicheniformis, P. macerans, P. polymyxa, B. pumilus, B. sphaericus, B. subtilis and B. thuringiensis were identified by the combination of the present RAPD analysis using Primer 4 and morphological observations of colonies, vegetative cells and spores. For identification of B. coagulans and G. stearothermophilus, the RAPD analyses of the template DNA prepared from colonies formed on standard plate agar after cultivation at 55°C for 24 to 48 h might be effective though further improvements of the assay are required for identification of these Bacillus species. For simple identification of B. cereus and B. megaterium by the PCR-based method, the use of other RAPD primers or a specific PCR primer set should be developed in addition to morphological observations of colonies, vegetative cells and spores.

The RAPD analysis of several cultivars of grapevine (Vitis viniferaL.) and their clones

Nine identification RAPD markers (Moravcová et al. 2003) were used to distinguish 24 clones and grapevine cultivars. No polymorphism was detected among all the tested clones of Chardonnay, Pinot gris and Zweigeltrebe from Polešovice. Pinot noir, Pinot gris, Pinot blanc and Pinot Meunier were indistinguishable within clones, they also showed the identical RAPD profile within cultivars (except discussed sample No. 26). On the other hand, Auxerrois as a relative to cultivars of Pinot group showed unique patterns and may be classified as a different cultivar. Some irregularities within the cultivars of Pinot family from Oblekovice were also found, several of them gave different results from those expected: Pinot blanc sample 26 has the RAPD profile typical of Chardonnay. A new abnormal RAPD pattern as a marker of typical Chardonnay and Pinot profiles was observed in two cases. While RAPD banding patterns could not distinguish between the known clones, they were useful for distinguishing between phenotypically similar cultivars and for assessing the origins of cultivars thought to have originated as sports.    

Molecular Identification of the Entomorpathogenic fungus, Metarhizium anisopliae Isolate Released for Biological Control of the Legume Flower Thrips, Megalurothrips sjostedti (Trybom)

The control of insect pests with entomopathogens requires the ability to distinguish released pathogen from locally endemic isolates of the same species. In this study, random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) technique was carried out on Metarhizium anisopilae (Sorok isolate ICIPE 69 (isolated from a soil sample) released for biologist control of Megalurothrips sjostedti (Trybom) on cowpea in Kenya, the recovered isolate and five other isolates from different host and geographic locations. RAPD bands for the recovered isolate, ICIPE 69, was identical to those of the released isolate but different from other five isolates recovered from Maruca vitrata Fab. and Clavigraila spp. collected from the same treated plot and other isolates obtained from the germplasm. This relatively easy, fast and efficient technique has proved useful in identification of entomopathogenic hyphomycete used for thrips control and may also be valuable in modelling epizootiology of the released pathogen.

Genetic diversity and its implications in the conservation of endangeredZostera japonica in Korea

As part of the on-going effort to conserve endangeredZostera japonica Ascher. & Graebn. in Korea, we have used RAPD band patterns to analyze its genetic structure and diversity. Out of 50 primers tested, 45 formed amplified bands with its genome, including 814 polymorphic and 28 monomorphic bands. The highest number (120) was found in the population of Geoje-do; the smallest (58), in Anmyeon-do. An examination of its genetic structure with AMOVA revealed that about 50% of all variations could equally be assigned to within and between populations. The statistical value Gst (index of genetic differences) was 0.49, and the average number of individuals exchanged between populations per generation (Nem) was calculated as 0.26. Although the habitats ofZ. japonica in Korea are disappearing at an alarming rate, significant levels of genetic variation still exist, especially in the Geoje-do population. Therefore, any recovery strategy for this endangered species should be planned on the basis of this genetic diversity among populations.

Genomic Relations among Two Non-mangrove and Nine Mangrove Species of Indian Rhizophoraceae

Random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers were used to study the genomic relationship among 11 members of Indian Rhizophoraceae represented by nine true mangroves and two non-mangrove species. The AFLP and RAPD bands were scored and analyzed for genetic similarities and cluster analysis was done which separated the 11 species studied into two main groups, the true mangroves and the non-mangroves. The polymorphism observed for these markers showed a high degree of genetic diversity among the constituent taxa of the family. The phylogenetic relationship inferred from molecular marker systems supported the traditional taxonomic classification of the family Rhizophoraceae based on morphological characters at the levels of tribe, phylogeny and delimitation of genera and species, except the intra-generic classification of the genus Bruguiera and the placement of Rhizophora in the family Rhizophoraceae.

Assessing genetic diversity of Ginkgo biloba l. (Ginkgoaceae) populations from China by RAPD markers.

Genetic diversity and differentiation of nine populations of Ginkgo biloba L. (Ginkgoaceae)from China were evaluated using RAPD. Of 47 clear and repeatable RAPD bands, 46 were polymorphic (overall polymorphism = 97.9%). A ranged from 1.57 to 1.83 with a mean of 1.75. Mean He was 0.3159 (0.2429-0.3603). The Shannon index ranged from 0.3432 to 0.5119 with a mean of 0.4489. The GST was 0.1609 and AMOVA analysis indicated 89% of the variation within populations. UPGMA clustered the 9 populations into two groups: one containing only population JF and the other including 8 populations. Genetic diversity and differentiation of Chinese populations were higher than those of Korean and North American populations, which are ultimately descended from China, as reported previously.

Genetic diversity in cowpea [Vigna unguiculata (L.) Walp.] as revealed by RAPD markers

The present study, using RAPD analysis, was undertaken to characterize genetic variation in domesticated cowpea and its wild progenitor, as well as their relationships. The materials used consisted of 26 domesticated accessions, including accessions from each of the five cultivar-group, and 30 wild/weedy accessions, including accessions from West, East and southern Africa. A total of 28 primers generated 202 RAPD bands. One hundred and eight bands were polymorphic among the domesticated compared to 181 among wild/weedy cowpea accessions. Wild accessions were more diverse in East Africa, which is the likely area of origin of V. unguiculata var. spontanea. Var. spontanea is supposed to have spread westward and southward, with a loss of variability, loss counterbalanceed in southern Africa by introgressions with local perennial subspecies. Although the variabilty of domesticated cowpea was the highest ever recorded, cultivar-groups were poorly resolved, and several results obtained with isozyme data were not confirmed here. However primitive cultivars were more diverse than evolved cultivars, which still suggests two consecutive bottlenecks within domesticated cowpea evolution. As isozymes and AFLP markers, although with a larger number of markers, RAPD data confirmed the single domestication hypothesis, the gap between wild and domesticated cowpea, and the widespread introgression phenomena between wild and domesticated cowpea.

Characterization of genotypes and genetic relationships of cili (Rosa roxburghii) and its relatives using RAPD markers

AbstractCili (Rosa roxburghii Tratt), characterized by containing the highest vitamin C content among fruits and showing attractive senescence-retarding and cancer-preventing effects, has gained widespread interest. RAPD markers were applied to identify the seven genotypes of cili and to evaluate the genetic relationships within cili, as well as among its relatives. Sixteen arbitrary primers screened from 154 were adopted to analyse polymorphism in RAPD profiles for the 15 samples. A total of 137 RAPD bands ranging in size from 480 bp to 3.3 kb were obtained, among which 95 were polymorphic, covering 69.3% of the total bands obtained; and an average of 8.6 bands/primer was scored. The genotypes of cili and seedless cili could be identified efficiently by 14 genotype-specific bands, which were obtained from the polymorphic primers OPB-11, OPAF-16 and OPW-02. Additionally, using the unweighted pair group method with arithmetic mean, a dendrogram showing genetic relationships among the 15 samples was constructed based on cluster analysis of genetic distance. The possible origin of seedless cili and multiple-corolla cili is also discussed.

Detection and genetic distance of resistant populations of Pseudosuccinea columella (Mollusca: Lymnaeidae) to Fasciola hepatica (Trematoda: Digenea) using RAPD markers

Twelve natural populations of Pseudosuccinea columella snails, sampled in the western and central regions of Cuba, were analyzed using the RAPD-PCR technique to screen for resistance to Fasciola hepatica. Ten OPA primers previously shown to produce marker bands for resistance and susceptibility were tested. A new population of P. columella (El Azufre, Pinar del Rı́o) exhibited the amplification patterns of resistant snails, and its resistant status was confirmed after experimental exposure to miracidia . No genetic variability was detected across or within the susceptible isolates. Similarly, the novel resistant isolate displayed an RAPD profile identical to the profile of two other isolates previously identified as resistant to F. hepatica. However, clear differences in RAPD banding patterns and genetic distance were observed between resistant and susceptible isolates.

USE OF RAPD MARKERS FOR THE GENETIC CHARACTERIZATION OF LIMONIUM SPECIES

The genus Limonium (fam. Plumbaginaceae) consists of about 300 species of mostly herbaceous perennials, some low shrubs, and annuals. Most botanical species are endemics in the Mediterranean region, but many species have their centre of origin in Caucaso, Turkestan, Caspian Sea, Russia, Iran, China, and South Africa. Limonium is grown in several regions of the world for use as a cut flower for both fresh and dry-flower arrangements.In this work, RAPD analyses were used for the study of genetic relationships in Limonium. Thirteen wild species were tested with 10 primers. A total of 244 bands were scored and used for the analysis of genetic distances. The dendrogram obtained from cluster analysis showed high similarity among three species that some authors report as synonymous and that appeared very similar from our previous phenotypic observations (L. caspia, L. bellidifolium and L. otolepis). In order to clarify the genetic relationships, further analyses were carried out on several genotypes belonging to these species. The new dendrogram, obtained scoring 151 RAPD bands, showed that the genotypes did not group in clear clusters. Analysis of molecular variance (AMOVA) confirmed this trend: the highest genetic variation resulted among genotypes and only 6,58 % of the total variation resulted among the species. These results suggest that the species can be considered synonymous. The use of RAPD markers in our case was thus useful for clarifying the highly probable identity of the three Limonium species, in a plant genus that is notably of difficult interpretation.

Apomixis and Sexuality in Hexaploid Potentilla Argentea

Hexaploid Potentilla argentea L. (s.l.) plants from twelve populations in southern Sweden were investigated with RAPD to determine the pattern of genetic variability. The ploidy level of the plants was ascertained by flow cytometry. Thirteen different RAPD band patterns (genotypes) were found among 64 plants investigated. Nine of the populations had unique band patterns, two populations shared the same, and in one population three different patterns were found among three plants. Also a small isozyme investigation detected genetic variability, again distributed primarily between populations. Analyses with RAPD of offspring derived from bagged flower-stalks and from artificial crosses between different hexaploid genotypes, showed that seeds are normally produced by apomixis. However, one hybrid offspring with a nonoploid (9x) ploidy level was found, proving that pollen produced by hexaploid P. argentea is capable of fertilization, and that sexually derived seeds can develop and mature on hexaploid plants. The broad genetic variability found in the RAPD and isozyme investigations indicates that gamete production after reduction division may also occur on the female side, though at a low frequency. Judging from its isozyme patterns, hexaploid P. argentea is an allopolyploid having diploid P. argentea as one of its parents.

A microsatellite-based linkage map of the honeybee, Apis mellifera L.

A linkage map for the honeybee (Apis mellifera) was constructed mainly from the progeny of two hybrid queens (A. m. ligustica x A. m. mellifera). A total of 541 loci were mapped; 474 were microsatellite loci; a few were additional bands produced during PCRs, one of the two rDNA loci (using ITS), the MDH locus, and three sex-linked markers (Q and FB loci and one RAPD band). Twenty-four linkage groups were estimated of which 5 were minute (between 7.1 and 22.8 cM) and 19 were major groups (>76.5 cM). The number of major linkage groups exceeded by three the number of chromosomes of the complement (n = 16). The sum of the lengths of all linkage groups amounts to 4061 cM to which must be added at least 320 cM to link groups in excess, making a total of at least 4381 cM. The length of the largest linkage group I was 630 cM. The average density of markers was 7.5 cM and the average resolution was about one marker every 300 kb. For most of the large groups, the centromeric region was determined genetically, as described in (accompanying article in this issue), using half-tetrad analysis of thelytokous parthenogens in which diploid restoration occurs through central fusion. Several cases of segregation distortion that appreared to result from deleterious recessives were discovered. A low positive interference was also detected.

Inheritance of random amplified polymorphic DNA (RAPD-PCR) markers and their use in population genetic studies of Calomys musculinus (Rodentia, Muridae), the reservoir of Argentine hemorrhagic fever.

In order to assess the reliability of RAPD markers in the estimation of the genetic structure of natural populations of the murid rodent Calomys musculinus (reservoir of Junin virus, ethiological agent of Argentine Hemorrhagic Fever), we have analyzed the heritability of RAPD bands in 10 parents and their offspring (33 individuals). Fourteen out of a total of 119 bands obtained were absent in the parental patterns, but consistently amplified in offspring from some families. These bands can be eliminated from analyses. Overall degree of band sharing between individuals, including non-parental bands, correctly grouped members of a family in the same cluster in a UPGMA tree, with a high bootstrap percentage. Results support the usefulness of RAPDs as hereditable markers. One hundred polymorphic RAPD loci were identified in three natural populations of C. musculinus. Mean expected heterozygosity in three natural populations ranged from 0.206 to 0.220. Allele frequency based and phenotype based measures of genetic differentiation among natural populations of C. musculinus gave similar results (Weir and Cockerham's theta = 0.133; Excoffier et al.'s phi = 0.127). These values were considerably higher than those found previously using allozymes as genetic markers, and are compatible with moderate to low levels of gene flow among populations.

Genetic diversity of cacao accessions selected for resistance to witches’ broom based on RAPD markers

To increase the genetic diversity sources of resistance to witches' broom disease, 59 cacao trees of commercial plantations of the State of Bahia, Brazil, were selected and cloned. The diversity of these clones was evaluated with RAPD markers. Their pedigree was investigated by comparison to traditional resistance sources used as parents for most hybrid mixtures cultivated in the State. One hundred and six RAPD bands were obtained, 85.8% of which were polymorphic. Genetic distances among the clones varied from 0.04 to 0.41. The MDS plot showed large genetic variability among clones. Analyses based on rare markers inherent to Scavina-6 and Scavina-12 clones, which represent important resistance sources, showed a close relationship of these sources to most accessions. However, some accessions did not present any of the rare Scavina markers, suggesting potentially different alleles, which could contribute to broaden the gene pool of breeding programs for resistance to witches' broom.