Random amplified polymorphic DNA and genetic diversity in IndianMusa germplasm

Summary Fifty-seven accessions ofMusa including cultivated clones of 6 genomic groups (AA, AB, AAA, AAB, ABB, ABBB),M. balbisiana Colla (BB),M. acuminata Colla ssp.banksii F. Muell. (AA),M. acuminata Colla ssp.malaccensis Ridl. (AA) andM. velutina Wendl. & Drude were examined for random amplified polymorphic DNA (RAPD) genetic markers using PCR with sixty 10-mer random primers. Forty-nine of 60 tested primers gave reproducible DNA amplification patterns. The number of bands resolved per amplification was primer dependent and varied from 1 to a maximum of 24. The size range of the amplification products also differed with the selected primer sequence/genotype and ranged from 0.29 to 3.0 kb. RAPD data were used to generate Jaccard's similarity coefficients which were analyzed phenetically. Phenetic analysis separated clones into distinct groupings that were in agreement with clusterings revealed when data were subsequently analyzed by principal coordinate analysis (PCO). In both the phenetic and the PCO analyses, previously unclassified cultivars grouped with cultivars previously classified for their genomic group based on morphological keys. The implications of RAPD analysis forMusa germplasm classification, clonal identification, and management are discussed.     

Genetic relationships among Arachis hypogaea L. (AABB) and diploid Arachis species with AA and BB genomes

The cultivated peanut (Arachis hypogaea L.) is an allotetraploid, with two types of genomes, classified as AA and BB, according to cytogenetic characters. Similar genomes to those of A. hypogaea are found in the wild diploid species of section Arachis, which is one of the nine Arachis sections. The wild species have resistances to pests and diseases that affect the cultivated peanut and are a potential source of genes to increase the resistance levels in peanut. The aim of this study was to analyze the genetic variability within AA and BB genome species and to evaluate how they are related to each other and to A. hypogaea, using RAPD markers. Eighty-seven polymorphic bands amplified by ten 10-mer primers were analyzed. The species were divided into two major groups, and the AA and the BB genome species were, in general, separated from each other. The results showed that high variation is available within species that have genomes similar to the AA and the BB genomes of A. hypogaea.     

The genetic characterization of Turkish watermelon (<Emphasis Type="Italic">Citrullus lanatus</Emphasis>) accessions using RAPD markers

Genetic diversity of the Turkish watermelon genetic resources was evaluated using different Citrullus species, wild relatives, foreign landraces, open pollinated (OP) and commercial hybrid cultivars by RAPD markers. The germplasm was consisted of 303 accessions collected from various geographical regions. Twenty-two of 35 RAPD primers generated a total of 241 reproducible bands, 146 (60.6%) of which were polymorphic. Based on the RAPD data the genetic similarity coefficients were calculated and the dendrogram was constructed using UPGMA (Unweighted pair-group method with arithmetic average). Cluster analysis of the 303 accessions employing RAPD data resulted in a multi-branched dendrogram indicating that most of the Turkish accessions belonging to var. lanatus of Citrullus lanatus (Thunb.) Matsum et Nakai were grouped together. Accessions of different Citrullus species and Praecitrullus fistulosus (Stocks) Pangalo formed distant clusters from C. lanatus var. lanatus. Among 303 accessions, a subset of 56 accessions was selected representing different groups and a second dendrogram was constructed. The genetic similarity coefficients (GS) within the Turkish accessions were ranged from 0.76 to 1.00 with 0.94 average indicating that they are closely related. Taken together, our results indicated that low genetic variability exist among the watermelon genetic resources collected from Turkey contrary to their remarkable phenotypic diversity.     

Studying the extent of genetic diversity among Gossypium arboreum L. genotypes/cultivars using DNA fingerprinting

Genetic diversity is an area of concern for sustaining crop yield. Information on genetic relatedness/diversity among Gossypium arboreum L. cultivars/genotypes is scanty. We have used random amplified polymorphic DNA (RAPD) analysis to assess the genetic divergence/relationship among 30 genotypes/cultivars of G. arboreum. Of 45 primers surveyed, 63% were polymorphic. Out of the total number of loci amplified, 36% were polymorphic. The calculated genetic similarity between the cultivars/genotypes was in the range of 47.05–98.73%. Two genotypes, HK-244 and Entry-17, were the most distantly related. The average genetic relatedness among all the genotypes was 80.46%. However, most of the cultivated varieties showed a close genetic relationship, indicating a narrow genetic base in comparison to the non-cultivated germplasm. The calculated coefficients were used to construct a dendrogram using the unweighted pair group of arithmetic means (UPGMA) algorithm, which grouped the genotypes/cultivars into two major and three smaller clusters. The study is the first comprehensive analysis of the genetic diversity of G. arboreum germplasm and identifies cultivars that will be useful in extending the genetic diversity of cultivated varieties and future genome mapping projects.     

Genetic diversity of wild banana (<Emphasis Type="Italic">Musa balbisiana</Emphasis> Colla) in China as revealed by AFLP markers

Wild banana Musa balbisiana Colla is one of the progenitors of cultivated bananas and plantains. It is native to Southeast Asia and the western Pacific. South China represents the northern limit of its distribution range. The genetic diversity of Musa balbisiana was assessed by the amplified fragment length polymorphism (AFLP) fingerprinting in 15 populations of China. Four primer pairs produced 199 discernible loci. High levels of genetic diversity were detected, with P = 78.5%, H _E = 0.241, and H _pop = 0.3684 at population level, and P = 100%, H _T = 0.3362 and H _sp = 0.5048 at species level. Significant genetic differentiation among populations was detected based on Hickory’s analysis (27.6%), Shannon’s diversity index (27.0%) and AMOVA (27.1%). The AFLP results are discussed and compared with data obtained by microsatellites method. The estimates of genetic diversity and differentiation between each pair of populations computed with microsatellites and AFLP markers were not significantly correlated. Conservation strategies for Musa balbisiana in China are proposed.     

Development,characterization, and variability analysis of microsatellites from a commercial cultivar of <Emphasis Type="Italic">Musa acuminata</Emphasis>

We report the sequence and variability parameters of 23 microsatellite primers obtained from a commercial cultivar Gongjiao (Musa acuminata) using selectively amplified microsatellite (SAM) analysis. Polymorphisms were evaluated in a collection of 26 banana cultivars and 11 related species/subspecies. The mean number of alleles amplified per primer was 4.55 (range, 2–9), with a total of 100 alleles identified. The mean PIC value was 0.48 (range, 0.10–0.74). In addition, 22 markers also showed robust cross-species/genera amplification across 11 related species/subspecies, with the exception of ‘Xiangtuijiao’ (Ensete glaucum). Unweighted pair-grouping method with arithmetic averages (UPGMA) cluster analysis divided all the banana accessions into three main groups. The results demonstrate the usefulness of microsatellites for identification, similarity studies, and germplasm conservation in banana and related species.     

Analysis of genetic diversity in Piper nigrum L. using RAPD markers

RAPD analysis was conducted in 22 cultivars of P. nigrum(black pepper) from South India and one accession each of P. longum and P. colubrinum. Twenty-four primers generated 372 RAPD markers of which 367 were polymorphic. Jaccard's similarity between pairs of accessions ranged between 0.11 and 0.66 with a mean of 0.38. Among P. nigrum cultivars, the similarity ranged between 0.20 and 0.66 and the mean was 0.42. The existence of wide genetic diversity as revealed in the present study is supported by earlier reports of extensive inter- and intrapopulation morphological variability in pepper cultivars from South India. UPGMA dendrogram and PCO plot revealed P. colubrinum to be most distant of the three species. Genetic proximity among P. nigrum cultivars could be related to their phenotypic similarities or geographical distribution. Greater divergence was observed among landraces than among advanced cultivars. Landraces grown in southern parts of coastal India and those grown in more northern parts were grouped in separate clusters of the dendrogram.     

Identification and evaluation of <Emphasis Type="Italic">Forsythia</Emphasis> germplasm using molecular markers

This study identifies Forsythia germplasm and evaluated the genetic relationships of F. ×intermedia hybrids, cultivars and their putative parental species. Leaf samples of F. ×intermedia cultivars and species, such as F. koreana and F. suspensa, were collected in the Netherlands, Korea, and USA. Total genomic DNA was extracted and evaluated by randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) analyses. Dendrograms were constructed by using the neighbor-joining (NJ) clustering algorithm applying the interior branch (IB) test or analyzed by STRUCTURE. In the dendrogram generated by RAPD markers, two major clusters were observed. One cluster (CL-I) contained most of the F. ×intermedia cultivars, F. suspensa, and F. koreana. The other cluster (CL-II) included F. europaea, F. ovata, F. densiflora, F. mandshurica, F. japonica, F. viridissima, and cultivars derived from F. ovata. In the AFLP dendrogram, the placement of F. ×intermedia cultivars with F. suspensa was similar, forming cluster CL-A I. The RAPD and AFLP results clearly separated most F. ×intermedia cultivars from F. ovata derived cultivars. The full range of genetic diversity of F. suspensa and F. viridissima should be investigated to verify whether these two species are truly parental taxa for F. ×intermedia. Placement of F. viridissima, F. ovata, and F. japonica in different sub-clusters requires further investigation regarding genetic diversity in the species, and their close relationship with F. koreana, F. mandshurica, and F. saxatalis.     

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.     

A phenetic analysis of morphological variation in Citrullus lanatus in Namibia

Morphological data recorded from field trials using Citrullus lanatus germplasm collected in Namibia were used to analyse and compare the various morphotypes of this species. The experiment comprised wild types and local landraces as well as commercial cultivars. Cluster analysis supported the indigenous classification system used in Namibia, in which Citrullus types are distinguished based on gross morphology, ecology and usage and grouped into seed, cooking and fresh-eating (watermelon) types. Commercial watermelon cultivars formed a distinct cluster. Wide variation was found within the local types whereas the genetic basis of the commercial type appears to be narrow. The commercial cultivars were most closely related to local watermelon types and more distantly related to the wild types, whereas the cooking melons form an intermediate group.     

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.     

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 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.     

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.     

Genetic Diversity Among Brazilian Cultivars and Landraces of Tomato Lycopersicon Esculentum Mill. Revealed by RAPD Markers

Random amplified polymorphic DNA markers (RAPD) were used to estimate the variability of 35 tomato accessions (Lycopersicon esculentum Mill.). A total of 257 reproducibly scorable bands were obtained from 20 primers, 78.6% of which were polymorphic. The percentage distribution of RAPD markers shows a bimodal distribution, and the frequency of rare alleles is similar in commercial and landrace accessions. Genetic distances among accessions were calculated and a dendrogram showing the genetic relationships among them was constructed allowing for the separation of four groups. Twenty out of 23 Brazilian landraces fell within one group, whereas commercial cultivars were distributed in the four groups. AMOVA analysis of RAPD data showed that, despite the high within Brazilian landraces and commercial cultivars variation, these two groups are significantly different, indicating that landraces can be a source of variation for breeding programs.     

Molecular Markers for the Classification of Switchgrass (Panicum virgatum L.) Germplasm and to Assess Genetic Diversity in Three Synthetic Switchgrass Populations

Information regarding the amount of genetic diversity is necessary to enhance the effectiveness of breeding programs and germplasm conservation efforts. Genetic variation between 21 switchgrass genotypes randomly selected from two lowland (‘Alamo’ and ‘Kanlow’) and one upland (‘Summer’) synthetic cultivars were estimated using restriction fragment length polymorphism (RFLP) markers. Comparison of 85 RFLP loci revealed 92% polymorphism between at least two genotypes from the upland and lowland ecotypes. Within ecotypes, the upland genotypes showed higher polymorphism than lowland genotypes (64% vs. 56%). ‘Kanlow’ had a lower percent of polymorphic loci than ‘Alamo’ (52% vs. 60%). Jaccard distances revealed higher genetic diversity between upland and lowland ecotypes than between genotypes within each ecotype. Hierarchical cluster analysis using Ward's minimum variance grouped the genotypes into two major clusters, one representing the upland group and the other the lowland group. Phylogenetic analysis of chloroplast non-coding region trnL (UAA) intron sequences from 34 switchgrass accessions (6 upland cultivars, 2 lowland cultivars, and 26 accessions of unknown affiliation) produced a neighbor-joining dendrogram comprised of two major clusters with 99% bootstrap support. All accessions grouped in the same cluster with the lowland cultivars (‘Alamo’ and ‘Kanlow’) had a deletion of 49 nucleotides. Phenotypic identification of greenhouse-grown plants showed that all accessions with the deletion are of the lowland type. The deletion in trnL (UAA) sequences appears to be specific to lowland accessions and should be useful as a DNA marker for the classification of upland and lowland germplasm.     

RAPD markers efficiently distinguish heterogenous populations of wild potato (Solanum)

Genetic characterization of germplasm is important for setting objective guidelines for conservation. One common problem found in genebanks is determining the value of populations with insufficient or unreliable data regarding their geographic origin. In this study, a genetic analysis based on RAPD markers was conducted to characterize a `mystery' population of Solanum sucrense, a polysomic tetraploid potato (2n=4x=48), for which adequate documentation was lacking. The comparative analysis of genetic similarities between this mystery population and each one of 30 other S. sucrense populations in the genebank revealed that all populations within this species, including the mystery population, are significantly different from being duplicates, and are therefore worthy of separate conservation. RAPD markers also distinguished the mystery population from closely related tetraploid species S. oplocense, S. gourlayi and S. tuberosum ssp. andigena, suggesting that it is also not a duplicate of a population of these species. If RAPDs can clearly differentiate populations within highly heterogeneous tetraploids like S. sucrense, they should be generally useful for determining germplasm organization within potato species.     

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.