PCR Marker-based Analysis of Wild and Cultivated Yams (<Emphasis Type="Italic">Dioscorea</Emphasis> spp.) in Nigeria: Genetic Relationships and Implications for <Emphasis Type="Italic">ex situ</Emphasis> Conservation

Reliable characterization of the variation among wild and cultivated yams in Nigeria is essential for improved management and efficient utilization of yam genetic resources. RAPD and double stringency PCR (DS-PCR) analyses were used to investigate genetic relationships and the extent of redundancy among 30 accessions of two cultivated, and 35 accessions of four wild yam species collected from Nigeria. Twenty-five selected random decamer and two microsatellite primers were used individually and in combination to generate DNA profiles for each accession of the six Dioscorea species. The number of amplified fragments varied from 7 to 18 fragments per primer/primer combination. Different levels of intraspecific genetic diversity were found, with Dioscorea rotundata Poir. being the most variable. Based on identical profiles for the RAPD and DS-PCR primers, 12 duplication groups consisting of a total number of 37 accessions were observed in the present study. An UPGMA analysis grouped the majority of plants according to the species. Cultivated yams belonging to the D. cayenensis–rotundata species complex, which were classified into seven morphotypes/varietal groups, could be clearly separated into two major groups corresponding to D. rotundata Poir. and D. cayenensis Lam. D. cayenensis cultivars exhibited a low level of intraspecific variation and were genetically close to the wild species Dioscorea burkilliana J. Miège. D. rotundata cultivars classified into six varietal groups showed a high degree of DNA polymorphism and were separated into two major groups that appeared most closely related to Dioscorea praehensilis Benth. and Dioscorea liebrechtsiana de Wild. We propose, based on these results, that cultivars classified into D. cayenensis should be considered as a taxon separate from D. rotundata. The implications of intraspecific variability for the ex situ conservation of wild and cultivated yam germplasm in Nigeria are discussed.     

Variation and Intraspecific Relationships in Indian Wild Musa balbisiana (BB) Population as Evidenced by Random Amplified Polymorphic DNA

Sixteen collections of the wild Musa species, Musa balbisiana Colla collected from different regions of India were studied for their intraspecific relationships using random amplified polymorphic DNA (RAPD) markers. Out of 80 primers screened, 34 primers produced reproducible bands and four primers among them showing polymorphic bands were used. In all, 43 DNA fragments were amplified averaging 10.75 per primer. Of these, 31 amplified fragments showed polymorphism (averaging of 7.75 per primer). The extent of polymorphism (74.6%) has indicated the existence of considerable variation at the DNA level within the species. The 16 accessions were clustered into four as against seven clusters obtained through morphotaxonomic characterization. The inter relationships based on geographical origin in comparison with molecular characterization have been discussed.     

AFLP Analysis of the Phenetic Organization and Genetic Diversity in the Sugarcane Complex, Saccharum and Erianthus

Amplified fragment length polymorphism (AFLP) markers were evaluated for determining the phylogenetic relationships, and the diversity in the Saccharum complex using 30 clones belonging to S. officinarum, S. robustum, S. spontaneum, S. barberi, S. sinense and the related genus Erianthus. The phenetic tree of the species clones based on AFLP data was consistent with the known taxonomical relationships. AFLP gave higher resolution of closely related species into discrete groups than that by RAPD and RFLP markers, reported earlier. The levels of diversity within the various Saccharum species were also found to be higher than those obtained previously with the same set of clones using RAPD markers. The intraspecies similarity in S. barberi and S. sinense was much higher than interspecies similarity suggesting a clear separation of the two, which are considered ‘horticultural species’. The genetic similarity matrix derived from a single primer combination highly correlated (r = 0.980) with that obtained from all the 12 primer combination used in the study, thus highlighting the efficiency of a single primer combination in delineating species relationships. All the primer combinations could identify markers that are specific to each of the species and the genus Erianthus. Among the species, specific markers were highest in S. spontaneum followed by S. robustum, S. barberi, S. officinarum and S. sinense. Erianthus had a distinct profile with 30% of the total amplified fragments being specific to it. This offers great scope for identifying intergeneric hybrids, which has been very difficult using morphological traits and RAPD markers. High degree of correspondence between the results from the cluster analysis based on Jaccard's similarity index, Neighbour Joining tree based on Sokal and Michener distance matrix and AFTD (Analyses Factorielle on Table of Distances) analysis clearly demonstrated that AFLP markers would be an appropriate tool in providing better information about the relationships among the species, estimation of diversity, and in revealing species and genus specific markers that could be directly applied in sugarcane breeding programmes.     

Genetic Relationships and Variation among Biotypes of Dallisgrass (Paspalum dilatatum Poir.) and Related Species Using Random Amplified Polymorphic DNA Markers

The genus Paspalum L. consists of more than 400 species. Around twenty-five informal groups of species are recognized in Paspalum and the Dilatata group is of special interest because its members are excellent potential forage grasses. Seventy-five germplasm accessions, representing 15 taxa, were analyzed using randomly amplified polymorphic DNA (RAPD). Polymorphisms were observed with twenty-two primers in the Dilatata group and 16 of those were analyzed. Four hundred and four different RAPD fragments were generated, resulting in an average of 25.2 bands per primer. Among the 404 markers analyzed, 48 (11.88%) were exclusive for the P. dilatatum Poir. biotypes, 31 (7.67%) were exclusive to taxa belonging to other groups included in this study, 28 markers (6.93%) were diagnosed for other species of the Dilatata group and 16 (3.96%), for natural hybrids. Extensive RAPD variation was found among the species studied. Inter- and intra-taxonomic polymorphisms were detected. A dendrogram based on the RAPD data shows some clusters corresponding to the same taxa. However, the biotypes of P. dilatatum do not form a cluster. The present work confirms that the RAPD technique can be used to determine genetic relationships between the taxa belonging to the Dilatata group.     

Characterization of genetic variation and relationships among Choix germplasm accessions using RAPD markers

Choix, a plant in the tribe Maydeae of the grass family, has been cultivated in Asia for several thousand years. It is a potential gene resource for improvement of other cereal crops because of its nutritional value and tolerance to stress. Genetic variation and relationships among 21 Choix lachryma-jobi L. accessions were characterized by random amplified polymorphic DNA (RAPD) markers. A total of 205 DNA fragments across all materials were amplified with 31 random primers, averaging 6.61 per primer. Among amplified fragments, 115 showed polymorphism averaging 3.71 per primer. Of amplified markers, 56.1% were polymorphic, indicating considerable variation at the DNA level among these accessions. Some fragments were accession-specific. Pair-wise genetic similarity (GS) among 21 accessions ranged from 0.809 to 0.301. The 21 accessions clustered into two major groups. Three exotic Choix accessions clustered together. Three other Choix accessions, collected from Guangxi, China, clustered into a cohesive subgroup. Four wild types of Choix clustered into the same subgroup. These results indicated that the classification by RAPD data reflected the differences in geographic origins and evolution in Choix.     

Relationships among species of Hystrix Moench and Elymus L. assessed by RAPDs

To assess the generic delimitation and the interspecific relationships between Hystrix and Elymus, three Hystrix and 10 Elymus species were used for random amplified polymorphic DNA(RAPD) assay. Of the 54 primers tested, 26 (48%) produced polymorphic products. A total of 167 products amplified from 16 primers were selected for RAPD analysis, among which 156 (93.4%) amplified products were found to be polymorphic among the 13 species. The polymorphism produced by each primer ranged from 4 to 13, with an average of 9.8. Data were used to generate Jaccard's similarity coefficients and to construct a dendrogram using UPGMA in the NTSYS computer programs. It is concluded from this study that: (1) there were clear differences between Hystrix and Elymus, which possibly suggest that Hystrix is a valid genus; (2) great diversity existed among the species of Hystrix and Elymus; (3) the species similar to each other in morphological characters were grouped together; (4) the species from neighboring geographical regions were clustered; (5) the species with the same genomes and polyploidy level were clustered together; (6) RAPD results are comparable with those obtained from studies on morphology and cytology. It is a useful additional method for assessing the relationships among genera and species in Triticeae.     

Genetic Characterization of Brazilian Annual <Emphasis Type="Italic">Arachis</Emphasis> Species from Sections <Emphasis Type="Italic">Arachis</Emphasis> and <Emphasis Type="Italic">Heteranthae</Emphasis> using RAPD Markers

The genus Arachis is divided into nine taxonomic sections. Section Arachis is composed of annual and perennial species, while section Heteranthae has only annual species. The objective of this study was to investigate the genetic relationships among 15 Brazilian annual accessions from Arachis and Heteranthae using RAPD markers. Twenty-seven primers were tested, of which nine produced unique fingerprintings for all the accessions studied. A total of 88 polymorphic fragments were scored and the number of fragments per primer varied from 6 to 17 with a mean of 9.8. Two specific markers were identified for species with 2n = 18 chromosomes. The phenogram derived from the RAPD data corroborated the morphological classification. The bootstrap analysis divided the genotypes into two significant clusters. The first cluster contained all the section Arachis species, and the accessions within it were grouped based upon the presence or absence of the ‘A’ pair and the number of chromosomes. The second cluster grouped all accessions belonging to section Heteranthae.     

AFLP Fingerprinting in Pigeonpea (Cajanus cajan (L.) Millsp.) and its Wild Relatives

Detection of DNA polymorphism in cultivated pigeonpea (Cajanus cajan) and two of its wild relatives Cajanus volubilis and Rhynchosia bracteata is reported here for the first time using amplified fragment length polymorphism (AFLP) fingerprinting. For this purpose, two EcoRI (three selective nucleotides) and 14 MseI (three selective nucleotides) primers were used. The two wild species shared only 7.15% bands with the pigeonpea cultivars, whereas 86.71% common bands were seen among cultivars. Similarly, 62.08% bands were polymorphic between C. volubilis and pigeonpea cultivars in comparison to 63.33% polymorphic bands between R. bracteata and pigeonpea cultivars, and 13.28% polymorphic bands among pigeonpea cultivars. The cluster analysis revealed low polymorphism among pigeonpea cultivars and very high polymorphism between cultivated pigeonpea and its wild relatives. The AFLP analysis also indicated that only one primer combination (EcoRI + ACT and MseI + CTG), at the most any four primer pair combinations, are sufficient for obtaining reliable estimation of genetic diversity in closely related cultivars like pigeonpea material analyzed herein. AFLP analysis may prove to be a useful tool for molecular characterization of pigeonpea cultivars and its wild relatives and for possible use in genome mapping.     

Assessment of genetic relationships among Pyrus species and cultivars using AFLP and RAPD markers

Twenty-five Pyrus communis L. cultivars including eight traditional Portuguese pears, and four commercial Pyrus pyrifolia (Burm.) Nak. (Japanese pear or `nashi') cultivars were analysed by RAPD and AFLP techniques focusing on their molecular discrimination and the assessment of their genetic relatedness. Twenty-five primers generated 324 RAPD markers, among which 271 (84%) were polymorphic. The AFLP technique, using seven primer combinations, revealed a similar level of molecular polymorphisms (87%), representing 418 polymorphic bands among a total of 478 scored in autoradiographs. The high reproducibility of RAPD and AFLP techniques was confirmed comparing DNA samples from different extractions and different digestions of DNA from the same plant. Three genetic similarity matrices and respective dendrograms were elaborated on using RAPD, AFLP or joint RAPD and AFLP data. Both molecular marker techniques proved their reliability to assess genetic relationships among pear cultivars. P. pyrifolia cultivars exhibit a closer genetic relatedness, clustering apart from P. communis cultivars. Within P. communis, `William's', as well as `Doyenne du Comice', cluster close to their hybrids. Most of the Portuguese cultivars tend to cluster together, indicating to constitute a relatively independent genetic pool, which can be of interest in pear breeding programs.     

Assessment of genetic diversity in cultivars and wild accessions of Luohanguo (<Emphasis Type="Italic">Siraitia grosvenorii</Emphasis> [Swingle] A. M. Lu et Z. Y. Zhang), a species with edible and medicinal sweet fruits endemic to southern China,using RAPD and AFLP markers

Genetic variation of wild populations and cultivars of Luohanguo (Siraitia grosvenorii), a plant species endemic to southern China, was assessed using random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers. Based on the results for 130 individuals from seven populations, a high level of genetic diversity of Luohanguo was observed at the species level. The percentage of polymorphic loci (P) was 89.4%, Nei’s gene diversity (H _e) was 0.239, and Shannon’s information index (H _o) was 0.373 based on the combined AFLP and RAPD data. There was a high degree of genetic differentiation, with 45.1% of the genetic variation attributed to differences between the populations. The genetic diversity of the Luohanguo cultivars is much lower than that of wild populations (P = 41.8%, H _e = 0.141, H _o = 0.211), and a distinct genetic differentiation is observed between the cultivars and wild accessions. The pool of genetic variation in the wild populations provides an excellent gene resource for Luohanguo breeding.     

Inter-Simple Sequence Repeat fingerprints to assess genetic diversity in Tunisian fig (Ficus carica L.) germplasm

The genetic diversity of 18 Tunisian fig cultivars was investigated at the DNA level using the Inter Simple Sequence Repeat (ISSR) associated with the Polymerase Chain Reaction (PCR). Using a set of primers, the most informative ones were selected that were characterized by an important Resolving power value of 29.6. A total of 47 discernible fragments were scored from samples, with a mean of 11.7 fragments per primer. The 90.4% of sample that were polymorphic were scored as molecular markers to examine the Tunisian fig germplasm polymorphism at DNA level. A large genetic diversity as related to ISSR patterns was found within the local Tunisian fig germplasm. An UPGMA dendrogram exhibits the unstructured variability in this crop. Moreover, the principal component analysis shows that the observed diversity was typically continuous. Our data provide a large number of ISSR markers that are useful in the fingerprinting of Ficus carica L. cultivars, and in the understanding of the genetic relationships among these accessions.     

Genetic relationships and diversity among Brazilian cultivars and landraces of common beans (Phaseolus vulgaris L.) revealed by AFLP markers

The genetic variation and relationships among 31 accessions of Phaseolus vulgaris L., and two representatives of Vigna unguiculata L., were evaluated by AFLP analysis. A total of 263 DNA fragments across all materials were scored using nine primer combinations, averaging 32 per primer. More than 95% of the amplification products showed polymorphism, indicating high variation at the DNA level among these accessions. Pair-wise genetic similarity (Jaccard's coefficient) ranged from 0.553 to 0.840, with a mean of 0.765. Twenty-three accessions (70%) clustered into three groups. A majority of the commercial cultivars (91%) clustered within a single group, whereas the landraces were distributed along all the variation. An apparent correlation with phaseolin types was detected. Results of this study suggest that Brazilian landraces truly represent the overall genetic variability of Phaseolus vulgaris, confirming the multiple origins of these materials, and their potential as a source of variation for breeding programs.     

Nuclear DNA content estimations in wild olive ( Olea europaea L. ssp. europaea var. sylvestris Brot.) and Portuguese cultivars of O. europaea using flow cytometry

Olive tree (Olea europaea L.) is an economically important woody fruit crop widely distributed in the Mediterranean regions. In this work the genome size of six Portuguese cultivars of olive (O. europaea ssp. europaea var. europaea) and wild olive (O. europaea spp. europaea var. sylvestris) was estimated for the first time. The nuclear DNA content of O. europaea cultivars ranged between 2.90 ± 0.020 pg/2C and 3.07 ± 0.018 pg/2C and the genome size of wild olive was estimated as 3.19 ± 0.047 pg/2C DNA. These results suggest a low intraspecific variation at least among the studied cultivars and between them and wild olive. This is not in accordance with previous results in some Italian cultivars where high genome size heterogeneity was found. The methodology presented here seems appropriate for genome size estimations within this genus and opens good perspectives for a large screening of estimation of nuclear DNA content among O. europaea cultivars and Olea species that could clarify this issue.     

Genetic diversity of <Emphasis Type="Italic">Nelumbo</Emphasis> accessions revealed by RAPD

Total 65 lotus accessions in genus Nelumbo mainly collected from China, were subjected to random amplified polymorphic DNA (RAPD) markers to estimate the genetic diversity and to test the genetic basis of the relationships between morphotypes and molecular markers. Seventeen primers generated a total of 195 highly reproducible and discernible loci, among which 173 were polymorphic. Percent polymorphism varied from 66.7 to 100 with an average of 88.72, and five primers out of them, OPC05, OPG10, OPN20, OPP09 and OPS17, showed 100% polymorphism. A relatively high genetic diversity was detected among all the samples with the similarity coefficient values ranging from 0.45 to 0.85, and Nei’s gene diversity (h) 0.30, and Shannon index (I) 0.46. The UPGMA dendrogram clustered 65 accessions in four clusters and the clustering pattern showed two groups, N. nucifera ssp. nucifera and those accessions related to the American lotus, and some special cultivars, landraces, hybrids and the American lotus. Principal Coordinate Analysis (PCA) further indicated that the genetic diversity of Nelumbo accessions was not evenly distributed, instead, was presented by a clustered distribution pattern. Similar to the results revealed by the dendrogram, two main groups representing the two subspecies of N. nucifera, as well as some special landraces, cultivars of Chinese lotus, the Japanese lotus and hybrids out of the two groups were obtained. Neither the UPGMA dendrogram nor the PCA analysis exhibited strict relationship with geographic distribution and morphotypes among the accessions.     

Study of clonally propagated cassumunar ginger (<Emphasis Type="Italic">Zingiber montanum</Emphasis> (Koenig) Link ex Dietr.) and its relation of wild <Emphasis Type="Italic">Zingiber</Emphasis> species from Thailand revealed by RAPD markers

The genetic relatedness among 51 accessions, 14 species of the genus Zingiber and genetic variability of a clonally propagated species, Zingiber montanum (Koenig) Link ex Dietr., from Thailand were studied using random amplified polymorphic DNA (RAPD) profiles. Twenty-nine random primers gave reproducible amplification banding patterns of 607 polymorphic bands out of 611 scored bands accounting for 99.40% polymorphism across the genotypes. Jaccard’s coefficient of similarity varied from 0.119 to 0.970, indicative of distant genetic relatedness among the genotype studied. UPGMA clustering indicated eight distinct clusters of Zingiber, with a high cophenetic correlation (r = 1.00) value. Genetic variability in Z. montanum was exhibited by the collections from six regions of Thailand. High molecular variance (87%) within collection regions of Z. montanum accessions was displayed by AMOVA and also explained the significant divergence among the sample from six collection regions. Our results indicate that RAPD technique is useful for detecting the genetic relatedness within and among species of Zingiber and that high diversity exists in the clonally propagated species, Z. montanum.     

Application of random amplified polymorphic DNA to study genetic diversity in Paspalum scrobiculatum L. (Kodo millet, Poaceae)

Summary Genetic diversity and patterns of geographic variation among collections of Paspalum scrobiculatum (kodo millet) and P. polystachyum were studied using molecular markers generated through the random amplified polymorphic DNA (RAPD) method. A high level of polymorphism in RAPD markers was observed among the individual accessions, demonstrating the high genetic diversity of the crop. The markers obtained from the RAPD method were analyzed with the cluster analysis, principal coordinates and minimum spanning tree methods. Three major groups were resolved, one representing the African accessions, and two for the Indian accessions. The accessions of the north African kodo millet and P. polystachyum (considered conspecific with P. scrobiculatum) were quite distinct. The Australian kodo millet showed higher affinity to the African types. The study demonstrated that the RAPD technique can be applied to resolving degrees and patterns of genetic variation at the population and species levels, identifying cultivars, and defining gene pools of this crop.     

RAPD and ISSR fingerprinting in cultivated chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) and its wild progenitor <Emphasis Type="Italic">Cicer reticulatum</Emphasis> Ladizinsky

Detection of genetic relationships between 19 chickpea cultivars and five accessions of its wild progenitor Cicer reticulatum Ladizinsky were investigated by using RAPD and ISSR markers. On an average, six bands per primer were observed in RAPD analysis and 11 bands per primer in ISSR analysis. In RAPD, the wild accessions shared 77.8% polymorphic bands with chickpea cultivars, whereas they shared 79.6% polymorphic bands in ISSR analysis. In RAPD analysis 51.7% and 50.5% polymorphic bands were observed among wild accessions and chickpea cultivars, respectively. Similarly, 65.63% and 56.25% polymorphic bands were found in ISSR analysis. The dendrogram developed by pooling the data of RAPD and ISSR analysis revealed that the wild accessions and the ICCV lines showed similar pattern with the dendrogram of RAPD analysis. The ISSR analysis clearly indicated that even with six polymorphic primers, reliable estimation of genetic diversity could be obtained, while nearly 30 primers are required for RAPD. Moreover, RAPD can cause genotyping errors due to competition in the amplification of all RAPD fragments. The markers generated by ISSR and RAPD assays can provide practical information for the management of genetic resources. For the selection of good parental material in breeding programs the genetic data produced through ISSR can be used to correlate with the relationship measures based on pedigree data and morphological traits to minimize the individual inaccuracies in chickpea.     

Use of random amplified DNA markers to analyse genetic variability and relationships of Coffea species

Summary The use of random amplified DNA fragments as genetic markers in Coffea was investigated. Arbitrary oligonucleotides were used as primers to amplify genomic DNA of different coffee accessions representing major Coffea species by polymerase chain reaction. Intraspecific variation was easily detected in C. canephora and C. liberica whereas the primers assayed failed to reveal polymorphism between C. arabica accessions. Extensive interspecific variation was observed. Genetic relationships between Coffea species are deduced from the degrees of similarity in amplified product profiles. Random amplified DNA markers appeared to be of high value for characterization, analysis and utilization of coffee genetic resources.     

Identification of some Benin Republic's Guinea yam (Dioscorea cayenensis/Dioscorea rotundata complex) cultivars using Randomly Amplified Polymorphic DNA

DNA from twenty-three late maturing cultivars of Guinea yams (D. cayenensis/D. rotundata complex) from the Benin Republic that could not be separated using isozyme markers, were examined using randomly amplified polymorphic DNA (RAPD) markers with decamer primers of arbitrary sequence. All the twelve primers tested were informative and yielded 63 amplified DNA bands from which 47 (75%) were polymorphic. Although no single primer produced polymorphic bands in all cultivars, the great majority of the cultivars were separated with the combinations of polymorphic bands generated by various primers. Putative duplicates and cultivar misclassifications were identified. Many morphologically distinct cultivars were close. The dwarf cultivar Tam-Sam considered as derived from Tabane, appeared more distant from the latter than was believed. RAPD analysis was found as a practical tool for the identification of duplicates toward establishment of an accurate core collection of Guinea yams in Benin Republic and in the other countries of the African yam belt.     

Different methods for the evaluation of species of the genus Mesaphorura (Collembola, Tullbergiinae)

Field studies and laboratory investigations based upon cultures, isoenzyme electrophoresis and RAPD-PCR were carried out to get a better understanding of the value of morphospecies within the genus Mesaphorura.Culturing of seven Mesaphorura species under constant as well as variable conditions provided a basis for evaluation of morphological characters, but did not yield any statistically supportable evidence that intraspecific variability reached interspecific delimitations.In enzyme tests, esterases gave the best results. The search for species-specific band patterns is, however, time and material consuming. DNA investigations using RAPD-PCR resulted in band patterns that appeared to be species specific. Nevertheless, these require further investigations.None of the studies carried out gave any conclusive evidence for invalidation of the morphologically defined species of the genus Mesaphorura.