Intra-specific DNA polymorphism in pineapple (<Emphasis Type="Italic">Ananas comosus</Emphasis> (L.) Merr.) assessed by AFLP markers

Pineapple (Ananas comosus (L.) Merr.) cultivars, often derived from somatic mutations, are propagated vegetatively. It has been suggested by isozyme data that there is little genetic variation among Smooth Cayenne cultivars. A thorough investigation of the genetic variation within the cultivated speciesAnanas comosus, particularly among commercial cultivars, will provide critical information needed for crop improvement and cultivar protection. One-hundred and forty-eight accessions ofA. comosus and 14 accessions of related species were evaluated with AFLP markers. The average genetic similarity ofA. comosus was 0.735 ranging from 0.549 to 0.972, suggesting a high degree of genetic variation within this species. With AFLP markers, discrete DNA fingerprints were detected for each commercial cultivar, breeding line, and intra-specific hybrid. Self-incompatibility, high levels of somatic mutation, and intraspecific hybridization may account for this high degree of variation. However, major cultivar groups of pineapple, such as Cayenne, Spanish, and Queen, could not be distinctively separated. These cultivar groups are based on morphological similarity, and the similar appearance can be caused by a few mutations that occurred on different genetic background. Our results suggest that there is abundant genetic variation within existing pineapple germplasm for selection, and discrete DNA fingerprinting patterns for commercial cultivars can be detected for cultivar protection. The genetic diversity and relationships of fourAnanas species are also discussed.     

Collection of Lupinus angustifolius L. Germplasm and Characterisation of Morphological and Molecular Diversity

Lupinus angustifolius L. is a Mediterranean species, domesticated in the 20th century, representing an important grain legume crop in Australia and other countries. This work is focused on the collection of wild germplasm and on the characterisation of morphological and molecular diversity of germplasm accessions. It reports the collection of 81 wild L. angustifolius accessions from the South and Centre of Portugal, available at the ‘Instituto Superior de Agronomia Gene Bank’, with subsequent morphological and molecular characterisation of a selection of these and other accessions. A multivariate analysis of morphological traits on 88 L. angustifolius accessions (including 59 wild Portuguese accessions, 15 cultivars and 14 breeding lines) showed a cline of variation on wild germplasm, with plants from Southern Portugal characterised by earlier flowering, higher vegetative development and larger seeds. AFLP and ISSR molecular markers grouped modern cultivars as sub-clusters within the wider diversity of wild germplasm, revealing the narrow pool of genetic diversity on which domesticated accessions are based. The importance of preserving, characterising and using wild genetic resources for L. angustifolius crop improvement is outlined by the results obtained.     

Genetic variation within and among species of five sections of the genus Arachis L. (Leguminosae) using RAPDs

Some Arachis species are widely used as commercial plants, e.g. the groundnut A. hypogaea, an important source of good quality protein and oil, and A. pintoi and A. glabrata, that are utilized as forage species. Germplasm of most Arachis species is available in germplasm banks. However, little it is known about the genetic attributes of this germplasm, and mainly about its genetic variability, which is very important for its maintenance. In the present study RAPDs were used to assay the genetic variation within and among 48 accessions of five sections of the genus Arachis and to establish the genetic relationships among these accessions. Ten of 34 primers tested were selected for DNA amplification reactions since they yielded the largest numbers of polymorphic loci. A dendrogram was constructed based on data from the 10 primers selected. Eighty RAPD polymorphic bands were analyzed among the accessions studied. The relationships among species based on RAPDs were similar to those previously reported based on morphological, cytological and crossability data; demonstrating that RAPDs can be used to determine the genetic relationships among species of the different sections of the genus Arachis. In general, wide variation was found among accessions and low variation was found within the accessions that had two or more plants analyzed. However, higher polymorphism was found in the section Trierectoides and in one accession of A. major, indicating that generalizations should be avoided and each species should be analyzed in order to establish collection and maintenance strategies.     

Variations in morphological and agronomic traits among Jerusalem artichoke (Helianthus tuberosus L.) accessions

Jerusalem artichoke is a diversely-utilized crop. Selection for high yield, inulin content and other economically important traits are useful for improving this crop. The objectives of the present study were to evaluate genetic variability for qualitative and quantitative traits among Jerusalem artichoke accessions and to identify different groups of accessions using morphological and agronomic traits. Seventy-nine accessions were evaluated in a randomized complete block design with two replications in the late rainy season 2008, the early rainy season 2009 and the late rainy season 2009 at Khon Kaen University agronomy farm, Thailand. Morphological and agronomic characteristics were evaluated for genetic variations. High variations were found among Jerusalem artichoke accessions for qualitative and quantitative characters, and selection for these characters is possible. High variations were observed for tuber width, number of tubers/plant, biomass, fresh tuber yield and tuber size. Correlation coefficient between fresh tuber yield and tuber size was positive and significant (0.58, P ≤ 0.01). Improvement of tuber size is a means to improve yield and tuber quality. Based on morphological and agronomic characteristics, Jerusalem artichoke accessions were clustered into four distinct groups (R² = 0.88). These groups may be used as parental material to generate progenies for further improvement of this crop. This information will enable breeders to make informed decisions about possible heterotic groups for their breeding programs and germplasm conservation.     

Morphological and genetic diversity of Momordica cochinchinenesis (Cucurbitaceae) in Vietnam and Thailand

Momordica cochinchinensis (Cucurbitaceae) is native to South East Asia and is important for its nutritional and medicinal properties. There is little information available on morphological and genetic diversity, crop selection and strategic germplasm collection for M. cochinchinensis. In this study, 42 accessions of M. cochinchinensis collected from Vietnam, Thailand and Australia were analysed for diversity study both at a morphological and molecular level. Both morphological and molecular analyses were in agreement and clustered the accessions based on the country of origin. The markers investigated in this study could be used for future applications with samples from unknown origins or for gene tracking for important cultivars. The Momordica accessions were highly polymorphic based on the markers suggesting high genetic diversity. Therefore the results from this study will be important in future strategic germplasm collection, conservation, horticultural development and commercial propagation of M. cochinchinensis.     

Germplasm characterization of Sesbania accessions based on multivariate analyses

Nineteen Sesbania accessions were investigated, in a total of six species, to characterize the accessions based on morphological and agronomic data using multivariate methods, to identify a core set of morphological and agronomic attributes to be used in germplasm characterization of Sesbania spp., and to evaluate the forage potential of these accessions. Plots, in randomized block design, consisted of single 16 m-long rows of eight plants, spaced at 2 m between plants and 3 m between rows. Morphological data were obtained from the first four plants in each plot. The four remaining plants, submitted to four cuts at a height of 1 m, provided agronomic data. Principal component analysis indicated that variance accumulated by the first two components for morphological and agronomic data was 74.4% and 77.0%, respectively. Two large groups of accessions were formed in the cluster analysis for morphological data, one consisting of S. virgata, subgenus Daubentonia and the second group comprising the other five species. Cluster analysis using agronomic data classified higher yielding perennial species in three groups, and the annual species, which stood out principally in relation to seed production, in three other groups. Accession n° 8 of S. sesbanwas classified in an isolated group due to its forage superiority. A character discard resulted in the selection of eight characters: floral initiation, pod and leaf length, number of leaflet pairs, plant regrowth height, leaf dry matter yield, seed production and vigour, indicated as a core set of attributes for Sesbania germplasm characterization.     

Genetic diversity in tomato (Solanum lycopersicum L.) germplasm using fruit variation implemented by tomato analyzer software based on high throughput phenotyping

The decrease of genetic variation in crops like tomato (Solanum lycopersicum L.) due to domestication, breeding, and population bottle necks for fresh market consumptions has increased the need for this cash crop’s conservation and characterizations. To study the genetic variation of this valuable genetic resource in the National Plant Gene Bank of Iran, a collection of 589 tomato accessions from different regions was characterized using semi-automatic and high-throughput techniques. Based on the fruit shape, the studied accessions were classified into ten groups and significant differences for all the fruit characteristics studied indicated an abundant diversity within the germplasm collected from different regions. Studied tomato germplasm was grouped into nine main clusters based on hierarchical cluster analysis. More than 83% of data variation was explained by seven components in which the first two components explained for 50% of the variation where fruit shape index and proximal/distal fruit end shape showed a high contribution in the variation of the first component. This unique genetically divergent germplasm could be utilized to select tomato breeding lines of interest. Also, accessions belonged to the heterotic clusters could be selected for hybridization and conservation purpose as well as enrichment of pre-breeding programs of the tomato germplasm.

     

Realizing value of genetic resources of <Emphasis Type="Italic">Allium</Emphasis> in India

The genetic resources of Allium in India are potential source of genes for widening the crop genetic base. Despite their high economic value, limited number of germplasm accessions of wild species have been collected and conserved mainly due to difficult access to areas of occurrence. The present work mainly highlights information on species diversity distribution and utilization of wild Allium species to assess the value of Indian germplasm and prioritization for future collection and conservation programmes.     

Genetic Diversity in Accessions of Wild Rice Oryza granulata from South and Southeast Asia

Oryza granulata, an upland wild rice species, represents an unique germplasm for possessing abilities of tolerance to shade and drought, immune to bacterial blight and resistance to brown planthopper. Although low degree of genetic variability has been revealed within its populations, little genetic information at the species level is available in determining rational conservation strategies. Here we used dominant DNA marker random amplified polymorphism DNA (RAPD) to assess the genetic variability among 23 accessions of O. granulata that collected from main distribution areas worldwide. Twenty decamer primers generated a total of 243 bands, with 83.5% of them (203 bands) being polymorphic. Calculation of Shannon index of diversity revealed an average value of 0.42 ± 0.25, indicating that O. granulata maintains a relatively high degree of genetic diversity on the species level. Analysis of genetic dissimilarity (GD) showed that genetic differentiation occurred among studied accessions, which supports the feasibility of current ex situ conservation strategies. We also suggested that information based on population studies, which could be achieved by international co-operation, is needed to conserve this widespread germplasm more effectively.     

Morphological and molecular diversity of an underutilized fruit crop - <Emphasis Type="Italic">Cordia myxa</Emphasis> L. germplasm from the arid region of Rajasthan,India

Twenty two germplasm accessions of Cordia myxa were collected from Rajasthan and established at the field gene bank for conservation and evaluation. Morphological characterization of 10 year-old trees for 17 traits indicated wide variations among the accessions tested. Higher number of flowers per cyme was found in accession ACHM11 and higher pulp:stone ratio in AHCM25. Overall, AHCM22 was found to be a superior germplasm line for most of the horticulturally useful traits among the accessions tested as it had higher percent of fruit set, pulp:stone ratio and fruit weight. High significant positive correlation was obtained between leaf, fruit characters and pulp:stone ratio. However, these characters were found to be negatively correlated with number of flowers per cyme. Out of 50 random decamer primers used for random amplification (RAPD), 25 were polymorphic. Average polymorphism resolved by these markers among these accessions was 69.8% with an average polymorphic information content of 0.43. Genetic diversity revealed by Jaccard’s co-efficient was between 0.44 and 0.94, and three major clusters were identified among these accessions by phylogenetic analysis using NTSYSpc-2.02e software. RAPD markers associated with leaf size and pulp:stone ratio were also identified. This study shows the existence of high genetic diversity among these accessions.     

Characterisation of germplasm accessions of Napier grass (pennisetum purpureum and P. purpureum×P. glaucum Hybrids) and comparison with farm clones using RAPD

Fifty six germplasm accessions of the important East African fodder crop Napier grass, Pennisetum purpureum, and its hybrids with P. glaucum, were characterised using 67 random amplified polymorphic DNA (RAPD) fragments. No or very low intra-accession variation was found for 49 of the accessions examined, confirming field observations that this species is predominantly clonally propagated. Comparison of intra and inter-accession variation identified several groups of identical/similar accessions that could be targeted if the collection is to be rationalised, and also highlighted two misplantings of germplasm material during transfer to a field trial site. A neighbour joining dendrogram of Jaccard's similarity estimates, clearly separated 50 accessions of P. purpureum from three P. glaucum individuals, and placed six hybrid accessions in an intermediate position. These groupings were well supported by a nested AMOVA (P<0.001; 29.5% of total variance due to taxonomic delineation). The main group of P. purpureum individuals could be further differentiated into five sub-groups (designated East Africa, Southern Africa, USA1, USA2 and Miscellaneous, to reflect the majority membership of sub-groups) and examination of the within P. purpureum component of the nested AMOVA, found them to be significantly different (P<0.001; 18.8% of variance). Genetic diversity across all accessions was found to be fairly high (Shannon's diversity index 0.306) and thus the collection probably represents a wide genetic base for this species. In addition to germplasm accessions, 25 Kenyan farm clones were also analysed. A principal coordinate analysis found that all but one of the clones clustered with the main P. purpureum group of accessions, indicating that the majority are probably not interspecific hybrids. The origin and pedigree of clones is discussed based on genetic similarity amongst clones and to germplasm accessions.     

Multivariate analysis of phenotypic diversity of Coffea arabica

Summary Eighteen different morphological and agronomical traits observed in a field collection of 148 accessions of Coffea arabica were analyzed using multivariate analysis. For the first time, a clear structure is observed within this species with the identification of two main groups. Accessions of group 1 have a more erect branching habit, narrower leaves and are on average more resistant to coffee leaf rust and coffee berry disease than accessions of group 2. Group 1 mostly contains Ethiopian accessions collected west of the Great Rift Valley, whereas group 2 mostly contain commonly cultivated varieties throughout the world and Ethiopian accessions collected east of the Great Rift Valley in Ethiopia. This phenotypic structure together with historical evidence suggest that group 1 has not been involved in the domestication of C. arabica. The present results confirm that sub-spontaneous genotypes west of the Rift Valley are highly valuable for enriching the genetic basis of cultivated C. arabica germplasm. The potential value of this material in breeding of C. arabica is discussed.     

A wide range of genetic diversity in pear (<Emphasis Type="Italic">Pyrus ussuriensis</Emphasis> var. <Emphasis Type="Italic">aromatica</Emphasis>) genetic resources from Iwate,Japan revealed by SSR and chloroplast DNA markers

Iwateyamanashi (Pyrus ussuriensis var. aromatica) is one of the Pyrus species which grows wild in Japan. The number of Iwateyamanashi trees has been decreasing, so conservation and evaluation is urgently needed. Over 500 accessions of Pyrus species collected from Iwate in northern Tohoku region are maintained at Kobe University as an Iwateyamanashi germplasm collection. In order to investigate the genetic diversity, five SSR (simple sequence repeat) markers, developed from Japanese and European pear were examined for 86 Pyrus individuals including 58 accessions from Iwate. These SSR loci could discriminate between all the Iwate accessions except for 10 that bear seedless fruit, as well as determine the genetic diversity in Iwateyamanashi germplasms. High levels of variation were detected in 41 alleles and the mean observed heterozygosity across 5 loci was 0.50 for the Iwate accessions. Seedless accessions sharing identical SSR genotype with the local pear variety “Iwatetanenashi” were supposed to have been propagated vegetatively via grafting. In an UPGMA phenogram, Japanese pear varieties (P. pyrifolia) were clustered into two groups with some Iwate accessions including seedless ones. Another 38 Iwate accessions were not clustered clearly, and there was no clear relationship between these accessions and geographical distribution or morphological characters. Allele frequency revealed that the Iwate accessions were genetically more divergent than the Japanese pear varieties. Most Japanese pears possessed a 219 bp deletion at a spacer region between the accD and psaI genes in the chloroplast DNA (cpDNA), but other Pyrus species and two Iwateyamanashi trees did not. In the Iwate accessions, 79.3% had a deletion type cpDNA and others had a standard type cpDNA without deletion. These results are indicative of the wide range of genetic diversity in the Iwate accessions which include Japanese pear varieties. A combination of SSR and cpDNA analyses revealed high heterogeneity in Iwateyamanashi and coexistence of Iwateyamanashi and hybrid progeny with P. pyrifolia. These could be reasons for the wide range of continuous morphological variation described previously.     

Genetic Diversity Among Traditional Ethiopian Highland Maize Accessions Assessed by Simple Sequence Repeat (SSR) Markers

Over the past three centuries, maize has become adapted to complex environmental conditions in the highlands of Ethiopia. We analyzed 62 traditional Ethiopian highland maize accessions, using 20 simple sequence repeat (SSR) markers and 15 morphological traits, to assess genetic diversity and relationships among these accessions and to assess the level of correlation between phenotypic and genetic distances. The accessions varied significantly for all of the measured morphological traits. The average number of alleles per locus was 4.9. Pair-wise genetic dissimilarity coefficients ranged from 0.27 to 0.63 with a mean of 0.49. Ward minimum variance cluster analysis showed that accessions collected from the Northern agroecology were distinct from the Western and Southern agroecologies. However, there was no differentiation between the Western and Southern accessions. This suggested gene flow between these regions. The relationship between morphological and SSR-based distances was significant and positive (r = 0.43, p = 0.001). The high genetic diversity observed among these set of accessions, suggests ample opportunity for the development of improved varieties for different agroecologies of Ethiopia. From conservation perspective, sampling many accessions from all agroecologies would be an effective way of capturing genetic variation for future collections and conservation.     

Variation in melon (<Emphasis Type="Italic">Cucumis melo</Emphasis>) landraces adapted to the humid tropics of southern India

We present here the first comprehensive genetic characterization of melon landraces from the humid tropics of southern India. The genetic diversity among 50 melon landraces collected from 3 agro-ecological regions of southern India (6 agro-ecological sub-regions) was assessed by variation at 17 SSR loci, morphological traits of plant habit and fruit, 2 yield-associated traits, pest and disease resistance, biochemical composition (ascorbic acid, carotenoids, titrable acidity) and mineral content (P, K, Fe, Zn). Differences among accessions were observed in plant and fruit traits. Melon germplasm with high titrable acidity, higher than average amounts of mineral content and resistance to Cucumber mosaic virus, Zucchini yellow mosaic virus, powdery mildew (races 1, 2, 3, 5), Fusarium wilt (races 1, 2), Aphis gossypii and leafminer was recorded in the collection. A high level of genetic variability in melon germplasm was suggested by the SSR analysis. Comparative analysis using SSRs of the genetic variability between Indian melons from north, south, and east regions and reference accessions of melon from Spain, France, Japan, Korea, Iraq, Zambia showed regional differentiation between Indian melon accessions and that Indian germplasm was weakly related to the melon accessions from other parts of the world, suggesting that an important portion of the genetic variability found within this melon collection has not been used yet for the development of new cultivars. Additional collections of acidulus melon germplasm should be made in southern India and adequate management of this important genetic resource is clearly a necessity.     

Morphological and chemical variation in 25 collections of the Indian medicinal plant, <Emphasis Type="Italic">Withania somnifera</Emphasis> (L.) Dunal (Solanaceae)

Six phenotypic characters and three withanolide markers were assessed in 25 accessions of Withania somnifera collected from different states of India for studying genetic variability. The variability ranges observed at phenotypic and chemotypic levels were polymorphic. Based on D² values and PCA (Principal Component Analysis) of phenotypic traits like plant height, no. of branches/plant, no. of seeds/berry, root length, root diameter and root yield, these 25 accessions were grouped in five clusters. The relative contribution of each character towards genetic divergence was worked out. Five accessions–AGB002 (Rajasthan), AGB003 (J&K), AGB004 (Madhya Pradesh), AGB006 (J&K) and AGB009 (Punjab) representing clusters 2 and 4 exhibited maximum intra and inter-cluster divergence. Cluster 5 representing accession AGB053 (Andhra Pradesh) was having mixed traits. Chemically most of the accessions in cluster 3 showed uniformity in presence of three marker withanolides Withaferin A, Withanone and Withanolide A in the leaves.     

Diversity among landraces of Indian snapmelon (<Emphasis Type="Italic">Cucumis melo</Emphasis> var. <Emphasis Type="Italic">momordica</Emphasis>)

Diversity among 36 snapmelon landraces, collected from 2 agro-ecological regions of India (9 agro-climatic sub-regions), was assayed using RAPD primers, morphological traits of plant habit and fruit, 2 yield-associated traits, pest and disease resistance and biochemical composition (TSS, ascorbic acid, titrable acidity). Typical differences among accessions were observed in plant and fruit characteristics and snapmelon germplasm with high titrable acidity and possessing resistance to downy mildew, Cucumber mosaic virus, Zucchini yellow mosaic virus, Papaya ringspot virus, Aphis gossypii and Meloidogyne incognita was noticed in the collection. RAPD based grouping analysis revealed that Indian snapmelon was rich in genetic variation and region and sub-region approach should be followed across India for acquisition of additional melon landraces. Accessions of var. agrestis and var. momordica clustered together and there was a separate cluster of the accessions of var. reticulatus. Comparative analysis of the genetic variability among Indian snapmelons and an array of previously characterized reference accessions of melon from Spain, Israel, Korea, Japan, Maldives, Iraq, Pakistan and India using SSRs showed that Indian snapmelon germplasm contained a high degree of unique genetic variability which was needed to be preserved to broaden the genetic base of melon germplasm available with the scientific community. N. P. S. Dhillon and Ranjana contributed equally to this work and are considered the first authors.     

Delimitation of Amaranthus cruentus L. and Amaranthus caudatus L. using micromorphology and AFLP analysis: an application in germplasm identification

The ‘Morelos’ accessions of Amaranthus from Mexico demonstrate taxonomic ambiguity at the basic morphologic level. The main cause is the enormous morphological and genetic variation exhibited by the species in the genus. Although basic morphological criteria can be applied to herbarium specimens or germplasm collections for quick taxonomic identification, the morphological data alone can be misleading. To ascertain the taxonomic identity of the ‘Morelos’ accessions and their hypothesized species affiliation to Amaranthus caudatus or Amaranthus cruentus, we conducted a comparative analysis of phylogenetic relationships among these taxa/accessions using amplified fragment length polymorphism (AFLP) and micromorphology methods. Based on AFLP data, all the controversial ‘Morelos’ accessions can be consistently placed into a single A. cruentus species clade, which is clearly separated from the A. caudatus species clade. The AFLP-based phylogenetic relationship of ‘Morelos’ and delimitation of A. cruentus and A. caudatus are further supported by micromorphology, showing that the combination of these techniques can provide more reliable data for germplasm identification than each method used alone.     

Genetic identity of three mint accessions stored by different conservation procedures: field collection, in vitro and cryopreservation

At the genebank of IPK (Leibniz Institute of Plant Genetics and Crop Plant Research) in Gatersleben, Germany, three long-term conservation methods (field genebank, in vitro slow-growth and cryopreservation) are used for mint germplasm. The plant material of the field genebank was the source to establish the slow-growth in vitro culture collection, from which the cryopreserved collection was set up, using a droplet-vitrification protocol. The genetic identity of 161 samples of three mint accessions (MEN 198, MEN 166 and MEN 186), stored for several years using those three methods, was studied using RAPD markers. Accession ‘MEN 198’ was the only one with a unique RAPD fragment pattern for all its samples, independently of the conservation procedure employed. The field collections of accessions ‘MEN 166’ and ‘MEN 186’ were made up of different genotypes. None of the genotypes detected in the plots of these accessions was represented in the in vitro and cryopreserved samples analyzed, which showed a unique genotype for each accession. From this work we conclude that, for an appropriate management of a germplasm collection of vegetatively propagated species, the determination of the genetic composition of the donor material and a periodical assessment of the preserved material should be carried out.