Isozyme variation in cultivated and wild pineapple

Summary Isozyme variation was studied in 161 accessions of pineapple including four species of Ananas and one of Pseudananas. Six enzyme systems (ADH, GPI, PGM, SKDH, TPI, UGPP) involving seven putative loci revealed 35 electromorphs. Considerable variation exists within and between species of Ananas. Sixty-six distinct zymotypes were identified. Multivariate analyses of isozyme variation indicated that A. comosus contains five genetically diverse groups that do not match perfectly with the traditional varietal groups. Isozyme evidence also suggests that A. erectifolius is a conspecific variant of A. comosus, and that among other wild species, A. ananassoides is more closely related to A. comosus than A. bracteatus. Pseudananas is genetically distinct from all species of Ananas. It is evident from our study that differentiation among the species of Ananas may be due to ecological isolation rather than genetic divergence with breeding barriers and therefore may represent a species complex.This is journal series No. 3956 of the Hawaii Institute of Tropical Agriculture and Human Resources.     

Phylogenetic relationships as in Ocimum revealed by RAPD markers

Genetic relationships were examined among thirty germplasm accessions belonging to five Ocimum species using RAPD markers. A very high degree of polymorphism (98.20%) was observed. UPGMA cluster analysis of genetic similarity indices grouped all the accessions into two major clusters corresponding to previously reported botanical sections. Intra-clustering within the two clusters precisely grouped the accessions belonging to one species in one sub-cluster as expected from their genetic background. Our results show that RAPD technique is a sensitive, precise and efficient tool for genomic analysis in Ocimum species, that may be useful in future studies, by assigning new unclassified germplasm accessions to specific taxonomic groups and reclassifying previously classified accessions of other Ocimum species by traditional criteria on a more objective basis. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetic variability measures among Bromus catharticus Vahl. populations and cultivars with RAPD and AFLP markers

The objectives of this study were to optimize RAPD and AFLP techniques in B. catharticus, and to determine the genetic variability of populations and commercial prairie grass cultivars with the aforementioned molecular markers. Two populations with contrasting morphological characteristics were evaluated from individual and bulked DNA samples using RAPD markers. Both analyses showed a similar information about inter population variability. Each accession was sampled by a single leaf bulk of 10 plants. Accession similarities were established with 276 RAPD and 714 AFLP bands using Jaccard similarity coefficient. The dendrogram of the accessions using RAPD markers showed that they shared high similarity values (>94%). A similar result was obtained with AFLP markers (similarity values >98%), revealing a narrow genetic basis in the analyzed accessions. Consequently, molecular characterization of germplasm should be considered in addition to morphological criteria, to choose the parental genotypes for breeding programs of this forage crop. The AFLP technique was more efficient to detect DNA polymorphism in our experiments and unique fingerprints were detected for all the accessions. RAPD is a simple and non expensive technique, suitable to estimate genetic similarity. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetic relationship of Mediterranean mandarins (Citrus deliciosa Tenore) using RAPD markers

Summary Random amplified polymorphic DNA (RAPD) analysis was carried out to evaluate polymorphism and genetic similarity between 39 Mediterranean mandarin genotypes. One hundred eleven amplification products were identified using 21 random primers. An average of 2.2 RAPD markers was obtained for each primer, corresponding to 42% of the amplification products. Genotype-specific RAPD markers were also found, mainly in known hybrids. UPGMA cluster analysis revealed the low level of genetic variation between accessions of Mediterranean mandarins, whereas their hybrids with other Citrus species showed greater genetic dissimilarity. Twenty accessions yielded very similar patterns, suggesting either that they could be a single clone, or that the technique was not able to detect genomic variation. However, for the other specimens genetic polymorphism can easily be detected by RAPD, although the genetic variation between accessions was quite low. The large number of hybrids and the low polymorphism between accessions support the hypothesis that Mediterranean mandarins are all true hybrid of Common mandarins (Citrus reticulata Blanco).     

Genetic diversity in tef [Eragrostis tef (Zucc) Trotter] and its relatives as revealed by Random Amplified Polymorphic DNAs

Tef is one of the staple cereal crops in Ethiopia. To evaluate genetic diversity of tef and its relatives, 47 accessions of tef, three accessions of E. pilosa, and six accessions of E. curvulawere analyzed using random amplified polymorphic DNA (RAPD) markers. The level of polymorphism among the wild species was extremely high, while low polymorphism was detected among tef accessions. All cultivars and wild species under study could be distinguished with the help of different primers, thereby indicating the potential of RAPD in the genetic fingerprinting of tef. Accessions from E. curvula and E. pilosa can be differentiated by a single selected primer. In spite of low polymorphism within tef, accessions under study could be distinguished by a combination of selected primers. Cluster analysis indicated that tef is a very closely related species to E. pilosa with 45%similarity, supporting the hypothesis that tef originated from E. pilosa based on morphological data. Given that RAPD are relatively quick, simple to use, and are not subjected to environmental influences, they provide a valuable new approach for the genetic fingerprinting and study of genetic diversity in tef. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular marker diversity and bacterial wilt resistance in wild <Emphasis Type="Italic">Solanum commersonii</Emphasis> accessions from Uruguay

Solanum commersonii is a wild tuber-bearing species native to Uruguay with high potential for use in potato breeding programs. Little is known about the genetic diversity within this wild species and the relationship with the resistance to the bacterial pathogen Ralstonia solanacearum. We studied 30 S. commersonii clonal accessions, 20 of which were collected from geographically different areas across the country, while the other ten were grown from seeds from a single plant. Resistance against R. solanacearum was tested and different levels of resistance were found, ranging from delayed wilting to asymptomatic reactions. The genetic variation and the relationships among individuals in this germplasm collection were studied by different molecular markers: Random Amplified Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP) and Microsatellites or Simple Sequence Repeats (SSR). AFLP markers generated the largest number of total and polymorphic fragments per assay unit while SSR revealed the highest frequency of polymorphic bands (100%), followed by AFLP (96.2%) and RAPD (89.4%). In contrast, when comparing the number of different genetic profiles generated, the SSR markers exhibited the lowest discriminatory power. The clustering pattern obtained with the three marker systems showed a similar distribution of the S. commersonii germplasm revealing a high correlation between the three methods employed. All three dendrograms grouped most of the accessions into two main clusters, containing the same accessions regardless of the marker type. Bacterial wilt resistant accessions were present in both clusters. Accessions originated from different seeds of the same plant were grouped within one of the major clusters, and differed in the response to R. solanacearum revealing segregation of resistance. Furthermore, the distribution in two main clusters showed high correspondence with the geographical origin of the accessions, from the north and south of the country, and with the subspecies malmeanum and commersonii morphologically identified.     

The taxonomic characterisation of annual Beta germplasm in a genetic resources collection using RAPD markers

Summary Annual beets in the genus Beta section Beta represent an important genetic resource. Representative accessions of annual beets from a beet germplasm collection were analysed using RAPD to assess the patterns of variation and relationships among them. Using arbitrary primers, markers showing variation across accessions were identified. A dendrogram of similarity was produced using these molecular markers. All the accessions analysed were classified into three major groups corresponding to species or subspecies macrocarpa, adanensis and maritima. Macrocarpa was shown to be the most divergent group in this section. Using RAPD molecular markers, it was possible to ascribe an accession to one of three taxonomic groups and overcome much of the confusion encountered when morphological traits are used for identification. The group of maritima was found to be more polymorphic than either the group of macrocarpa or adanensis at both accession and subspecies levels.     

Random amplified polymorphic DNA (RAPD) variation in Fagopyrum tataricum Gaertn. accessions from China and the Himalayan region

The diversity among 52 landraces and cultivars of tartary buckwheat (Fagopyrum tataricum Gaertn.) and one accession of its wild ancestor, F. tataricum ssp. potanini Batalin, from diverse geographic origins was examined using random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) markers. Eighteen primers produced a total of 240 fragments, of which 153 (63.75%) were monomorphic and 87 (36.25%) polymorphic bands. UPGMA-based pairwise Jaccard’s coefficient of similarity was used to deduce the relationships among 53 genetically diverse accessions. The similarity between cultivated tartary buckwheat accessions ranged from 0.61 to 1.00. Four distinct clusters were formed which corresponded well with the geographic distribution of the tartary buckwheat. Nepalese accessions showed maximum diversity followed by Chinese accessions. Tartary buckwheat accessions from the Himalayan region of northwestern India revealed a narrow gene pool. The wild buckwheat accession did not group with any of the three cultivated tartary buckwheat groups, and formed its own single-entry group. Genetic similarity (0.59) of Chinese buckwheat accessions with the wild ancestor reaffirmed that cultivated tartary buckwheat originated in the Yunnan province of northwestern China. Consistent with some earlier reports, our study demonstrated the usefulness of the RAPD technique for the characterization of plant genetic resources and assessment of diversity between species. This revised version was published online in August 2006 with corrections to the Cover Date.     

Species relationships in the subgenus Ceratotropis (genus Vigna) as revealed by RAPD analysis

Summary The genetic variation among 23 accessions of 5 species in the subgenus Ceratotropis, genus Vigna, were investigated by random amplified polymorphic DNA (RAPD) analysis. A total of 404 fragments amplified with 24 primers were scored and analyzed by cluster analysis. The accessions used were separated into two main groups with an average of 70% differences. Within the main groups, five subgroups were recognized, which are in complete agreement with taxonomic species. Wild forms were always grouped with their most closely related cultivated forms and they showed variation in each species. The largest intraspecific variation was found in V. radiata (mungbean), in which wild forms (V. radiata var. sublobata) were highly different from each other and from cultivated forms. V. angularis (adzuki bean) showed the least variation and thus, was probably differentiated in relatively recent times.     

Molecular Marker based Genetic Diversity Analysis in Rice (Oryza sativa L.) using RAPD and SSR markers

The availability of an array of molecular marker systems allowed comparing the efficiency of two of these marker systems to estimate the relationships among various taxa. The objective of this study was to assess the genetic diversity among 40 cultivated varieties and five wild relatives of rice, Oryza sativa L. involving simple sequence repeat (SSR) randomly amplified polymorphic DNA (RAPD) markers. The accessions were evaluated for polymorphisms after amplification with 36 decamer primers and 38 SSR primer pairs. A total of 499 RAPD markers were produced among the 40 cultivated varieties and five wild relatives with a polymorphism percentage of 90.0. Out of 38 SSR primer pairs used, only one locus viz., RM115 was monomorphic. The average Polymorphism Information Content (PIC) value was 0.578 and it ranged from a low of zero (RM 115) to a high of 0.890 (RM 202). The Mantel matrix correspondence test was used to compare the similarity matrices and the correlation coefficient was 0. 582. The test indicated that clusters produced based on RAPD and SSR markers were not conserved since matrix correlation value was 0.582 as against the minimum required value of 0.800. The two marker systems contrasted most notably in pair-by-pair comparisons of relationships. SSR analysis resulted in a more definitive separation of clusters of genotypes indicating a higher level of efficiency of SSR markers for the accurate determination of relationships between accessions that are too close to be accurately differentiated by RAPD markers. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic diversity of wild coffee (Coffea arabica L.) using molecular markers

Genetic diversity was studied using RAPD markers among119 coffee (Coffea arabica L.) individuals representing 88 accessions derived from spontaneous and subspontaneous trees in Ethiopia, the primary centre of species diversity, six cultivars grown locally in Ethiopia, and two accessions derived from the genetic populations Typica and Bourbon, spread in the 18^th century, which gave rise to the most currently grown cultivars. Twenty-nine polymorphic fragments were used to calculate a similarity index and construct dendrograms. The Ethiopian material was separated from the Typica- and Bourbon-derived accessions and classified in four groups: one with most of the collected material from southwestern Ethiopia and three from southern and southeastern Ethiopia. Almost all detected diversity was found in the southwestern group while the southern and southeastern groups presented only 59% of identified markers. The genetic distances were low between the southwestern group and the southern and southeastern groups, and between the southwestern group and the Typica- and Bourbon-derived accessions. The cultivated coffee derived from the genetic populations Typica and Bourbon appeared little differentiated from wild coffee growing in the southwest. The results supported the hypothesis that southwestern Ethiopian coffee trees could have been introduced recently in the south and southeast. A separate analysis of the 80accessions classified in the southwestern group allowed identifying particular spontaneous- and subspontaneous-derived accessions and redundancies in the collected material from southwestern Ethiopia. RAPD markers did not detect any within-collection polymorphism except for two trees that were identified as off-types in the CATIE field genebank. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inter and intra-population variability of <Emphasis Type="Italic">Jatropha curcas</Emphasis> (L.) characterized by RAPD and ISSR markers and development of population-specific SCAR markers

Jatropha curcas (Euphorbiaceae) is an oil-bearing species with multiple uses and considerable potential as a bioenergy crop. The present investigation has been undertaken to assess the extent of genetic diversity in a representative set of 42 accessions of J. curcas encompassing different crop growing regions in India along with a non-toxic genotype from Mexico as a prelude for utilization of promising and genetically divergent materials in the breeding programmes. Molecular polymorphism was 42.0% with 400 RAPD primers and 33.5% with 100 ISSR primers between accessions indicating modest levels of genetic variation in the Indian germplasm. The within-population variation based on RAPD polymorphism was 64.0% and was on par with the inter-population variation. Polymorphic ISSR markers have been identified that could differentiate the Indian accessions from the Mexican genotype and two of them were converted to SCAR markers. The SCAR primer pair ISPJ1 amplified a 543 bp fragment in all the Indian populations, while ISPJ2 with a specific amplicon of 1,096 bp was specific to the Mexican genotype. Population-specific bands have been identified for the accession from Kerala (2 RAPD markers), Neemuch-1 from Rajasthan (1 each of RAPD and ISSR markers) and the non-toxic genotype from Mexico (17 RAPD and 4 ISSR markers), which serve as diagnostic markers in genotyping. The study indicates an immediate need for widening the genetic base of J. curcas germplasm through introduction of accessions with broader geographical background.     

Genetic diversity of Cannabis sativa germplasm based on RAPD markers

Random amplified polymorphic DN A (RAPD) markers were generated from 13 cultivars and accessions of Cannabis sativa L. Approximately 200 fragments generated by 10 primers of arbitrary sequence were used to assess the level of DNA variation. Statistical analysis was performed using the Dice coefficient of similarity and principal coordinate analysis. The grouping of the accessions according to the cluster analysis was in good agreement with their origin and lines with common ancestors were grouped together. Principal coordinates 1 and 2 revealed a clear separation of Italian and Hungarian germplasm and a third group, including a mixture of genotypes coming from different places; the third coordinate separated the Korean group which is probably the most divergent germplasm. Variability within the two cultivars ‘Carmagnola’ and ‘Fibranova1’ was also shown, suggesting good possibilities for long–term selection work. RAPD markers provide a powerful tool for the investigation of genetic variation in cultivars/accessions of hemp.     

Selection of wild Cicer accessions for the generation of mapping populations segregating for resistance to ascochyta blight

In order to determine genetically diverse parents for the generation of mapping populations segregating for resistance to ascochyta blight in wild Cicer species, the genetic diversity between a selection of resistant and susceptible accessions was assessed using molecular markers. Twenty Cicer accessions — comprising eight C. reticulatum accessions, six C. echinospermum accessions, five C. bijugum accessions, and one C. arietinum accession — were compared using a combination of seven RAPD primers and seven ISSR primers. A total of 231 polymorphic bands were scored and used to determine the genetic distances between accessions using Jaccard similarity coefficients. The most genetically diverse parents for the generation of intraspecific and interspecific populations segregating for resistance to ascochyta blight are reported. This revised version was published online in July 2006 with corrections to the Cover Date.     

Microsatellite (SSR) variation in a collection of Malus (apple) species and hybrids

A collection of 142 accessions of 23 Malus species, derived hybrids and cultivar accessions from the USDA-ARS Plant Genetic Resources Unit's core collection, which represents an extensive range of Malus species, was screened with a set of previously described SSR (simple sequence repeat) markers. The markers were used to determine genetic identities, estimate genetic diversity, identify genetic relationships among the accessions, and determine the utility of SSR primers developed from Malus ×domestica for making genetic assessments across the whole Malus genus. All eight primer pairs amplified multiple fragments when used in polymerase chain reactions with DNA from these accessions. High levels of variation were detected with a mean of 26.4 alleles per locus and a mean direct count heterozygosity across all eight loci equal to 0.623. The eight primer pairs used in this study unambiguously differentiated all but five pairs of accessions in this collection of 142 accessions of 23 Malus species, derived hybrids and cultivars. These SSR data were not useful in identifying genetic relationships among this diverse collection of accessions, with the majority of the accessions not clustering in ways concordant with taxonomic information and/or geographic origin. The resulting phenogram resolved only two meaningful clusters, for the taxonomically isolated Section Chloromeles and for M. fusca accessions, reflecting genetic relationships arising from geographic origin. The detection of identical accessions in the collection, which were previously considered to be unique, highlights the critical need to further bolster collections of certain Malus species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Penetrance of creeping-rootedness in clonal progenies of lucerne and observations on underground morphology of plants differing for this character

Elaeis oleifera or ‘caiaué’, a close relative of oil palm (E. guineensis), has some agronomic traits of great interest for the oil palm genetic breeding such as slow growth, oil quality (mostly unsaturated) and disease resistance. An analysis of a Brazilian oil palm germplasm collection was carried out using RAPD (Random Amplified Polymorphic DNA) markers with the objective of understanding the genetic variation of ‘caiaué’ accessions collected in the Amazon Forest in the last two decades. A sample of 175 accessions obtained along the Amazon River Basin was analyzed and compared to 17 accessions of oil palm from Africa. Ninety-six RAPD markers were used in the analysis, of which fourteen were shown to be specific to oil palm, while twelve were specific to ‘caiaué’. Results showed that the Brazilian ‘caiaué’ accessions studied have moderate levels of genetic diversity as compared to oil palm accessions. The data allowed the establishment of similarity groups for ‘caiaué’ accessions, which is useful for selecting parental plants for population breeding. Cluster analysis showed that, in general, genetic similarities are not correlated to geographical distances, but consistent with geographical dispersal along the Amazon River network. AMOVA showed that most of the genetic variation is found within populations, as expected for anallogamous and long-lived perennial species. The study provides important information to define strategies for future collection expeditions, for germplasm conservation and for the use of E. oleifera in breeding programs. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetic diversity for RAPD markers between cultivated and wild accessions of Coffea arabica

Summary Random amplified polymorphic DNA (RAPD) markers have been successfully employed to analyse the genetic diversity among cultivated and subspontaneous accessions of Coffea arabica. The narrow genetic base of commercial cultivars was confirmed. On the other hand, a relatively large genetic diversity was observed within the germplasm collection demonstrating the importance of collecting missions. Results suggested an East-West differentiation in Ethiopia, the primary centre of diversification of C. arabica. The large heterosis effect reported in intergroup hybrids could be related to such genetic differentiation. RAPD method appeared to be effective in resolving genetic variations and in grouping germplasm in C. arabica.     

Inter and intra-species Inter Simple Sequence Repeat (ISSR) variations in the genus Cicer

Intra and inter-species ISSR variation and use of ISSR markers in determination of genetic relationship were investigated in an accession collection representing twoperennial and six annual Cicerspecies. Screening of Ciceraccessions with SSR primers revealed highly reproducible amplicon profiles with relatively high multiplex ratios. Many of the primers generated amplicon profiles with which not only the differences among species can readily be identified, but also polymorphisms within species could be detected more efficiently. PCR products at 150 gel positions detected using six SSR primers in Cicer accessions were treated as dominant DNA markers and utilized to compute the distances among accessions and species. Cluster analysis of accessions and species revealed groupings that corroborate our previous studies of relationships based on allozyme and AFLP analysis. Consistent with the AFLP analysis carried out in the same accession collection, ISSR-based groupings indicated that perennial C. incisumis genetically close to the annuals of the second crossability group (C. pinnatifidum,C. bijugum, C. judaicum) while C. reticulatum is the closest wild species to the cultivated chickpea. ISSR-based variation estimates were relatively higher when compared to previous estimates computed from RAPD and AFLP data. Technically, ISSR analysis combines the PCR-based targeting of microsatellite-associated polymorphisms with no prior sequence requirement and stringent PCR conditions. Similarly, when compared to AFLP analysis, it is less technically demanding allowing to survey polymorphic loci in the genome. Thus, ISSR-PCR technology is a reliable, fast, and cost-effective marker system that can be used to study genetic variation and genetic relationships in the genusCicer. This revised version was published online in July 2006 with corrections to the Cover Date.     

Assessment of genetic relationships in Mentha species

A set of 60 random primers was used to analyse 11accessions from six taxa of Mentha developed byCIMAP. These accessions were maintained in the nationalgene bank for medicinal and aromatic plants at CIMAP.A total of 630 bands could be detected as amplifiedproducts upon PCR amplification, out of which 589 werepolymorphic (93.5%). Further analysis of these RAPDprofiles for band similarity indices clearlydifferentiated five of the Mentha arvensis L.accessions from the rest. Among two accessions of Mentha spicata L. CIMAP/C33 could bedistinguished from CIMAP/C32. Mentha × gracilis Sole cv. cardiaca showed a muchhigher similarity with Mentha spicata L. as wellas Mentha arvensis L. which amongst themselvesshowed rather a greater distance indicating that Mentha × gracilis Sole cv. cardiaca might have evolved as a natural hybridbetween M arvensis L. and M. spicataL. In terms of uniqueness of amplified bands fordeveloping RAPD markers, it was observed that at taxalevel 298 bands were unique to one of the six taxa,singly amounting to 47.3% of total amplifiedfragments. Primers MAP 10 and 17 produced polymorphismonly in case of M. spicata L. and Menthaspicata L. cv. viridis while MAP 08 producedpolymorphic bands in all 4 other species than thesetwo. Similarly unique patterns were observeddifferentiating all six species and could be used asRAPD markers for differentiating Mentha species.     

Assessment of genetic variability in grapefruits (Citrus paradisi Macf.) and pummelos (C. maxima (Burm.) Merr.) using RAPD and SSR markers

The genetic variability of 38 grapefruit (Citrus paradisi Macf.) and three pummelos (C. maxima (Burm.) Merr..) accessions was evaluated using RAPD, and single sequence repeat (SSR) analyses. Approximately49% of the 198 RAPD were polymorphic, and 4.6 alleles per SSR loci were identified. PIC values changed from 0.093 to 0.450. A UPGMA phenetic tree was constructed and two main grapefruit groups were identified. The grapefruit accessions `do Cabo' and `Siamesa-Filipinas'clustered very close to the pummelos in Group A. The Group B consisted of three sub-groups, which comprised all of the other grapefruit accessions. The majority of grapefruit accessions showed a narrow genetic base suggesting that the observed morphological polymorphism within the group must be associated with somatic mutations, which were not detected by these molecular markers. This revised version was published online in August 2006 with corrections to the Cover Date.