ALLOZYME COMPARISONS AMONG SPECIES OF THE BULINUS FORSKALII GROUP (GASTROPODA: PLANORBIDAE) IN CAMEROON

Allozyme comparisons were made for adult Bulinus forskalii,B. camerunensis, and B. senegalensis from populations in Cameroonusing starch gel electrophoresis. Fifteen loci were examinedbut no differences were found between B. forskalii and B. camerunensis,bringing into question the validity of B. camerunensis as aseparate species. Bulinus senegalensis differed from B. forskaliiand B. camerunensis in allozymes for acid phosphatase, alpha-glycero-phosphatedehydxogenase, hydroxybutyrate dehy-drogenase, and phosphoglucomutase.No differences were found, as reported elsewhere, between B.forskalii and B. senegalensis for aspartate amino-transferase,isocitrate debydrogenase, phosphogluco-isomerase, and xanthineoxidise. No polymorphism was seen in B. camerunensis or variousB. senegalensis populations examined. Three alleles for isocitratedehydrogenase were observed for B. forskalii. Mixed populationsof B. forskalii and B. senegalensis were found at four sitesbut no evidence of hybridization between these species was found. (Received 9 May 1988; accepted 30 August 1988)     

Potential taxonomic use of random amplified polymorphic DNA (RAPD): a case study in Brassica

Summary The potential use of RAPDs for taxonomic studies were investigated using Brassica, Sinapis and Raphanus taxa. Principal coordinate analysis of 284 RAPD bands revealed the classical U triangle relationship between diploid and amphidiploid Brassica taxa. Raphanus sativus and S. alba were distinct from the Brassica taxa. It appears that at least ten primers with approximately 100 total bands are needed to adequately portray these relationships. Cultivars of cabbage and cauliflower were separated by RAPDs. Analysis of RAPDs from individual plants of B. carinata cv. dodola resulted in 69 RAPDs, with 91.7% monomorphic and 8.3% polymorphic bands. RAPDs appear to be useful for taxonomic studies at levels ranging from populations to species and perhaps genera.     

Rapid identification of white-Engelmann spruce species by RAPD markers

Fragments of random amplified polymorphic DNA (RAPDs) were used as markers to distinguish Picea glauca (Moench) Voss (white spruce) and Picea engelmannii Parry (Engelmann spruce). These species and their putative hybrids are difficult to differentiate morphologically and are collectively known as interior spruce. Four oligodeoxynucleotide decamer primers showed species-specific amplification products between white spruce and Engelmann spruce. These fragments are highly conserved among seed lots and individual trees of each species from diverse geographic origins. The consistency and reproducibility of these species-specific amplification products were tested in more than two amplification reactions. Therefore, RAPD markers can provide genetic markers for easy and rapid identification of the specific genetic entry of these spruce species and their reported putative hybrids. According to the frequencies of the species-specific RAPD markers, it is possible to estimate the hybrid fraction, indicative of true introgression between the two species. These results are useful for quick identification of both species and their hybrid swarms at any stage in the sporophyte phase of the life cycle, for determining the occurrence and the magnitude of introgressive hybridization in an overlap zone between the two species, and for certification purposes in operational re-forestation and tree-improvement programs.     

Qualitative and quantitative characterization of RAPD variation among snap bean (Phaseolus vulgaris) genotypes

Ten snap bean (Phaseolus vulgaris) genotypes were screened for polymorphism with 400 RAPD (random amplified polymorphic DNA) primers. Polymorphic RAPDs were scored and classified into three categories based on ethidium bromide staining intensity. An average of 5.19 RAPD bands were scored per primer for the 364 primers that gave scorable amplification products. An average of 2.15 polymorphic RAPDs were detected per primer. The results show that primer screening may reduce the number of RAPD reactions required for the analysis of genetic relationships among snap-bean genotypes by over 60%. Based on the analysis of the distribution of RAPD amplification, the same number of polymorphic RAPDs were amplified from different genotypes for all RAPD band intensity levels. A comparison of RAPD band amplification frequency among genotypes for the three categories of bands classified by amplification strength revealed a measurable difference in the frequencies of RAPDs classified as faint (weakly amplifying) compared to RAPD bands classified as bold (strongly amplifying) indicating a possible scoring error due to the underscoring of faint bands. Correlation analysis showed that RAPD bands amplified by the same primer are not more closely correlated then RAPD bands amplified by different primers but are more highly correlated then expected by chance. Pairwise comparisons of RAPD bands indicate that the distribution of RAPD amplification among genotypes will be a useful criterion for establishing RAPD band identity. For the average pairwise comparison of genotypes, 50% of primers tested and 15.8% of all scored RAPDs detected polymorphism. Based on RAPD data Nei's average gene diversity at a locus was 0.158 based on all scorable RAPD bands and 0.388 if only polymorphic RAPD loci were considered. RAPD-derived 1 relationships among genotypes are reported for the ten genotypes included in this study. The data presented here demonstrate that many informative, polymorphic RAPDs can be found among snap bean cultivars. These RAPDs may be useful for the unique identification of bean varieties, the organization of bean germplasm, and applications of molecular markers to bean breeding.     

Hybridization in staminate and pistillate Salix alba and S. fragilis (Salicaceae): morphology versus RAPDs

 The polyploid Salix alba–Salix fragilis hybrid complex is rather difficult to study when using only morphological characters. Most of the characters have a low diagnostic value for unambiguously identifying the hybrids, introgression patterns and population structures. Morphology and molecular variation determined with random amplified polymorphic DNAs (RAPDs) were investigated in a set of staminate and pistillate willows from Belgium. A thorough screening of possible RAPD markers was done to select homologous amplification products. The selected amplified products proved to be useful in a principal coordinate analysis for the identification of individuals from a morphological continuum comprising presumed pure species and introgressants. The RAPD based identity of the individuals or clones was checked against those based on morphological characters. A correspondence analysis indicated that all pubescence related characters were associated but separated from the size related characters. The RAPDs also revealed that the S. fragilis genotypes mainly consisted of staminate individuals whereas most of the pistillate trees belonged to the S. alba genotype cluster. It was suggested that both species have kept their gene pools well separated and that morphologically intermediate plants are not necessarily genetically intermediate. Received August 31, 1999 Accepted December 12, 2000     

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.     

AN EVALUATION OF RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) FOR THE IDENTIFICATION AND PHYLOGENY OF FRESHWATER SNAILS OF THE GENUS BULINUS (GASTROPODA: PLANORBIDAE)

The Random Amplified Polymorphic DNA (RAPD) assay was used tostudy genetic variation within and between 9 species of thegenus Bulinus and to determine whether RAPD profiles could beused as markers for identification purposes. RAPDs were generatedwith 8 primers of two different sizes (l0mers & 15mers)and were visualised using both polyacrylamide gel electrophoresis(PAGE) with silver staining and agarose gel electrophoresiswith ethidium bromide staining. The species groups of Bulinushad few similarities in their RAPD profiles and there was interspecificvariation within groups. Intrapopulation variation was observed,with all primers, for B globosus collected from a single sitein Zimbabwe PAGE/silver staining methods visualised a greaternumber of RAPDs in comparison with agarose/ethidium bromidemethods. Phenetic analysis indicated that distance estimatesbetween taxa were sometimes non-additive and the phylo-geneticanalysis of such non-metnc data is discussed. The resultantphenograms, constructed using a least squares method, were constrainedalmost into a polytomy with topologies often differing betweendata sets. It was concluded that this phenomenon was most likelyattributable to large nucleotide divergences between the speciesgroups which go beyond the phylogenetic scope of RAPD analysis.RAPD profiles, when used in conjunction with other taxonomicmethods, may contribute to the identification of species ofBulnus on a regional basis, but the observed variability ina natural population suggests that a diagnostic RAPD profilefor each species throughout its geographic range is unlikely. (Received 19 April 1995; accepted 1 September 1995)     

Comparison of the genetic maps of Brassica napus and Brassica oleracea

 The genus Brassica consists of several hundreds of diploid and amphidiploid species. Most of the diploid species have eight, nine or ten pairs of chromosomes, known respectively as the B, C, and A genomes. Genetic maps were constructed for both B. napus and B. oleracea using mostly RFLP and RAPD markers. For the B. napus linkage map, 274 RFLPs, 66 RAPDs, and two STS loci were arranged in 19 major linkage groups and ten smaller unassigned segments, covering a genetic distance of 2125 cM. A genetic map of B. oleracea was constructed using the same set of RFLP probes and RAPD primers. The B. oleracea map consisted of 270 RFLPs, 31 RAPDs, one STS, three SCARs, one phenotypic and four isozyme marker loci, arranged into nine major linkage groups and four smaller unassigned segments, covering a genetic distance of 1606 cM. Comparison of the B. napus and B. oleracea linkage maps showed that eight out of nine B. oleracea linkage groups were conserved in the B. napus map. There were also regions in the B. oleracea map showing homoeologies with more than one linkage group in the B. napus map. These results provided molecular evidence for B. oleracea, or a closely related 2n=18 Brassica species, as the C-genome progenitor, and also reflected on the homoeology between the A and C genomes in B. napus. Received: 14 June 1996 / Accepted: 11 October 1996     

Polymorphic microsatellites in the African freshwater snail,Bulinus forskalii (Gastropoda,Pulmonata)

Eleven microsatellites were isolated in the freshwater snail Bulinus forskalii, intermediate host for the medically important trematode Schistosoma intercalatum. Characterization in 60 snails from three populations of B. forskalii from Cameroon revealed 4 to 18 alleles per locus. Low observed heterozygosity but higher expected heterozygosity, high F_IS estimates, significant departures from Hardy–Weinberg equilibrium and genotypic linkage disequilibria all indicate that B. forskalii is a preferential selfer. High F_ST estimates suggest that effective dispersal is limited and genetic drift is an important determinant of genetic structure. The potential utility of the microsatellite primers in other closely related Bulinus species was explored.     

Genome analysis of amaranths: Determination of inter- and intra-species variations

Amaranths are an important group of plants and include grain, vegetable and ornamental types. Despite the economic importance of the amaranths, there is very little information available about the extent and nature of genetic diversity present in the genus Amaranthus at molecular level. We now report the randomly amplified polymorphic DNA (RAPD) profiles of different species of Amaranthus as well as different accessions of the species. These RAPD analyses have been carried out using 65 arbitrary sequence decamer primers. From the RAPD data, an UPGMA dendrogram illustrating the inter-as well as intra-species relationships has been computed. The putative hybrid origin of A.dubious from A. hybridus and A. spinosus is also ruled out by the RAPD data. The trends of species relationships amongst the amaranths determined by RAPDs is consistent with their cytogenetic and evolutionary relationships that have already been determined. NBRI Communication No:464 (N.S.).     

DNA polymorphisms in grain sorghum (Sorghum bicolor (L.) Moench)

Molecular markers [random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP)] were used to determine the frequency of DNA polymorphism in grain sorghum (Sorghum bicolor (L.) Moench). Twenty-nine oligonucleotide primers were employed for RAPDs, generating a total of 262 DNA fragments, of which 145 were polymorphic in at least one pairwise comparison between 36 genotypes. Individual primers differed significantly in their ability to detect genetic polymorphism in the species. The overall frequency of polymorphisms was low with a mean frequency of 0.117 polymorphisms per RAPD band being obtained from all pairwise comparisons between genotypes, with maximum and minimum values of 0.212 and 0.039, respectively. Results from phenetic analysis of bandsharing data were consistent with current sub-specific groupings of the species, with clusters of Durra, Zerazera, Caud-Nig, Caud-Kaura and Caffrorum being discernible. The results also indicated that individuals of a similar taxonomic grouping but different geographic origin may be genetically less identical than previously considered. Similar frequencies of polymorphism to that obtained with RAPDs were obtained with RFLPs. Results from these experiments indicated that a high level of genetic uniformity exists within S. bicolor.     

Segregating random amplified polymorphic DNAs (RAPDs) in Betula alleghaniensis

Summary Molecular markers are currently being developed for Betula alleghaniensis Britton using random amplified polymorphic DNA (RAPD). Arbitrarily designed 11-mer primers were tested on three intraspecific controlled crosses for which more than 15 full-sibs were available. Using two of these primers, we were able to genetically characterize a total of nine polymorphic RAPD markers. Segregation of these markers was consistent with a biparental diploid mode of inheritance, and all appeared dominant. RAPDs were valuable in detecting contaminants and, therefore, in assessing the validity of controlled crosses. Limitations of the technique are discussed in relation to the determination of parental genotypes and construction of linkage maps for hardwood species.     

Random-amplified-polymorphic DNA markers in sorghum

Conditions have been identified that allow reproducible amplification of RAPD markers in sorghum. High resolution of RAPD markers was accomplished by radiolabeling PCR-amplified DNAs followed by separation on denaturing 5% polyacrylamide gels. Reaction parameters including MgCl₂ concentration and temperature significantly influenced yield and the type of amplification products synthesized. Unexplained amplified DNAs increased when more than 35 cycles of PCR amplification were used. Under standard conditions, approximately 80% of the primers tested amplified DNA, and most revealed 1–5 polymorphisms between BTx 623 and IS 3620C. Primers were used to amplify RAPDs in 32 genotypes of sorghum. In addition, 8 primers detected RAPDs in a population previously used to create an RFLP map for sorghum. These RAPDs were mapped successfully using a population of 50 F₂ plants.     

Variation among and within Capsicum species revealed by RAPD markers

 Germplasm characterization is an important link between the conservation and utilization of plant genetic resources. A total of 134 accessions from six Capsicumspecies maintained at the Asian Vegetable Research and Development Center were characterized using 110 randomly amplified polymorphic DNA (RAPD) markers. Ten pairs of potentially duplicated accessions were identified. Multidimensional scaling analysis of the genetic distances among accessions resulted in clustering corresponding to a previous species assignment except for six accessions. Diagnostic RAPDs were identified which discriminate among the Capsicumspecies. The diagnostic markers were employed for improved taxonomic identification of accessions since many morphological traits used in the identification of Capsicumare difficult to score. Three Capsicumaccessions, misclassified based on morphological traits, were reassigned species status based on diagnostic RAPDs. Three accessions, not previously classified, were assigned to a species based on diagnostic RAPDs. Definitive conclusions about the species assignment of three other accessions were not possible. The level of diversity between Capsicum annuumaccessions from the genebank and the breeding program were compared and no differences were observed either for RAPD variation or diversity. The utilization of genetic resources as a source of variance for useful traits in the breeding program may be the reason for the similarity of these two groups. Received: 1 September 1998 / Accepted: 28 December 1998     

Template mixing: a method of enhancing detection and interpretation of codominant RAPD markers

Ten codominant RAPD markers, ranging in size from about 300 to about 1350 bp, were identified in mapping populations of chickpea (Cicer arietinum L.) and diploid strawberry (Fragaria vesca L.). A distinguishing feature of all ten markers, and perhaps of codominant RAPD markers in general, was the presence in heterozygous individuals of a non-parental, heteroduplex band migrating more slowly than either of the respective parental bands. This non-parental band could also be generated by mixing parental DNAs before PCR (template mixing). As a means of identifying primers likely to detect codominant RAPD markers, parental and mixed-template (parent-parent) PCR-product gel lanes were compared for 20 previously untested RAPD primers (10-base oligomers). Four primers that produced a total of five non-parental, heteroduplex bands in mixed-template reactions were selected, and then used to detect a total of five segregating, codominant markers and nine dominant markers in the respective F₂ mapping population, a codominant marker frequency of 35.7%. When closely migrating fast and slow bands of codominant RAPDs were difficult to differentiate, parent-progeny template mixing was used to deliberately generate heteroduplex bands in fast- or slow-band F₂ homozygotes, respectively, allowing confirmation of marker phenotype.     

Construction of a genetic linkage map in <Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis> (Osbeck) using RAPD and SSR markers

The primary genetic linkage maps of Fenneropenaeus chinensis (Osbeck) were constructed by using the “two-way pseudo-testcross” strategy with RAPD and SSR markers. Parents and F1 progeny were used as segregating populations. Sixty-one RAPD primers and 20 pairs of SSR primers were screened from 460 RAPD primers and 44 pairs of SSR primers. These primers were used to analyze the parents and 82 progeny of the mapping family. About 146 primers (128 RAPDs, 18 microsatellites) in the female and 127 primers (109 RAPDs, 18 microsatellites) in the male were segregating markers. The female linkage map included eight linkage groups, nine triplets and 14 doublets, spanning 1,173 cM with the average marker density of 11.28 cM, and the observed coverage was 59.36%. The male linkage map included 10 linkage groups, 12 triplets and seven doublets, spanning 1,144.6 cM with the average marker density of 12.05 cM, and the observed coverage was 62.01%. The construction of the F. chinensis genetic linkage maps here opened a new prospect for marker-assisted selection program, comparative genomics and quantitative trait loci (QTL) gene location and cloning.     

Genetic variation in two sympatric European populations of Bosmina spp. (Cladocera) tested with RAPD markers

We used RAPDs (Random Amplified Polymorphic DNA) to test genetic divergence between two populations of Bosmina spp. in Lake Östersjön, Sweden. Previous taxonomic studies on European species within the genus Bosmina have been based on morphological characters alone. RAPD markers distinguished the two populations and supported the specific status of B. coregoni and B. longispina based on morphological characters. Furthermore, juveniles with a long antennule and a mucro were classified as B. coregoni. RAPDs also revealed genetic differences among the tested individuals, suggesting several clones within each species.     

RAPD analysis: a method to investigate aspects of the reproductive biology of Hypericum perforatum L.

Recent interest in breeding strategies for Hypericum perforatum L. requires a better understanding of the floral biology of this medicinal plant. The aim of the present study was to check, whether RAPD fingerprinting may be a useful tool for research on the mode of reproduction of this species. Progenies from three defined single plants of two accessions, as well as progenies from a random sample of seeds of a wild population, of H. perforatum were characterized by RAPD analyses using six primers. The results obtained by DNA fingerprints indicate the predominance of an identical mode of reproduction for this species, obviously due to apomixis. Nevertheless, non-identical reproduction was evident as a minor effect in H. perforatum, as could be demonstrated by significant deviations in the RAPD fingerprints of progenies from one single plant. It is concluded that RAPD fingerprint analysis is a suitable technique to discover identity or non-identity in H. perforatum populations. Therefore, RAPDs may be used in addition to cytological studies to confirm the mode of reproduction by apomixis versus self-pollination, haploid parthenogenesis or cross-fertilization. Received: 12. August 1999 / Accepted: 27 August 1999     

DNA fingerprinting of Peronospora parasitica,a biotrophic fungal pathogen of crucifers

The fungus Peronospora parasitica (Pers. ex Fr.) Fr. is an obligate biotroph infecting a wide range of host species in the family Cruciferae. Isolates from different hosts are morphologically similar, and pathotypes are usually distinguished on the basis of host range. Random Amplified Polymorphic DNA (RAPD) fingerprints were generated from a range of P. parasitica isolates from different Brassica species. Reaction conditions, in particular DNA template, primer and Mg²⁺ concentrations, were optimized to ensure that amplifications were reproducible. Possible artefacts arising through host plant DNA were assessed by including such DNA in control reactions. Confirmation that diagnostic RAPD bands were generated from fungal DNA was also obtained by Southern hybridization of a RAPD band to genomic fungal DNA. By screening 20 decamer primers, 2 were found to detect sufficient genetic variation to allow complete differentiation between pathotypes. These results illustrate the potential value of RAPDs for detecting polymorphisms between isolates of a non-culturable plant pathogenic fungus.     

Development of cost-effective Hordeum chilense DNA markers: molecular aids for marker-assisted cereal breeding

Hordeum chilense is a potential source of useful genes for wheat breeding. The use of this wild species to increase genetic variation in wheat will be greatly facilitated by marker-assisted introgression. In recent years, the search for the most suitable DNA marker system for tagging H. chilense genomic regions in a wheat background has lead to the development of RAPD and SCAR markers for this species. RAPDs represent an easy way of quickly generating suitable introgression markers, but their use is limited in heterogeneous wheat genetic backgrounds. SCARs are more specific assays, suitable for automatation or multiplexing. Direct sequencing of RAPD products is a cost-effective approach that reduces labour and costs for SCAR development. The use of SSR and STS primers originally developed for wheat and barley are additional sources of genetic markers. Practical applications of the different marker approaches for obtaining derived introgression products are described.