Case studies on breeding systems and its consequences for germplasm conservation

Summary This study gives the results of allozyme diversity within and among 20 wild Lima bean populations uniformly distributed throughout the Central Valley of Costa Rica. The electrophoretic analysis of seven enzyme systems show five monomorphic loci and a relatively low level of polymorphism for the other loci. This moderate level of polymorphism is unexpected for a species for which a fair amount of allogamy rates has been reported, at least among the cultivated forms. The genetic parameters (mainly percentage of polymorphic loci, mean genetic diversity, percentage of heterozygotes and fixation index) indicate a tendency for a predominantly selfing breeding system in the wild Lima beans although some values range between selfing and mixed-animal breeding systems. Very low within-population diversity is observed while a good diversity is found among populations. Results also show a departure from Hardy-Weinberg equilibrium on most analyzed populations in the target site This might be due to populations divided into subpopulations among which no natural crosses occur randomly, to weeding practices or to overlap of generations within some populations. All the results obtained in this work are discussed in view of further studies for the planning of in situ conservation in a quickly evolving tropical environment.     

Diversity of native rice (Oryza Poaceae:) species of Costa Rica

We found several populations of wild Oryza species in the lowlands of Costa Rica. The plants showed extensive morphological variation, suggesting the presence of several species. In the morphologic study, 33 traits were scored for plants of all the species. A principal component analysis revealed the significant morphological separation of the different species. The analyses indicated that there are three species, O. grandi­glumis, O. latifolia and O. glumaepatula. Two putative hybrid types were found, both significantly differing in their morphology from the known species and intermediate at several traits. O. grandi­glumis is a new record for Costa Rican flora. Its main population is located in Caño Negro Wildlife Refuge, Los Chiles, Alajuela. O. latifolia is distributed throughout the lowlands of the country and the plants of the Atlantic slope are significantly bigger in general habit than those of the Guanacaste area. During this study a population of O. glumaepatula of hundreds of thousands of plants was discovered in the Medio Queso River wetland, Los Chiles, Alajuela. This population is the most important source of genes for cultivar's improvement from the primary gene pool of rice in Costa Rica. The small ligule and the wide flag leaf characteristic of the two CCDD species separated them from the AA diploid O. glumaepatula. Seed size, ligule size, number of branches in the panicle, plant height and sterile lemma length are all bigger in O. grandi­glumis, and influenced the second factor that separated the CCDD species in two discrete clusters. The species found offer great possibilities for the improvement of rice cultivars and they should be thoroughly studied and appropriately protected.     

Morphologic and Agronomic Diversity of Wild Genetic Resources of Medicago sativa L. Collected in Spain

One hundred and three natural populations of Medicago L. were collected in Spain, mainly from roadsides, non-irrigated or grazed lands. This germplasm was evaluated at Montpellier (France) with control cultivars and Spanish landraces under completely random block design, replicated four times, and observed for 64 qualitative or quantitative characters. Differences between natural populations and cultivated controls are highly significant, but gene flow occurs between wild and cultivated compartment and hybrid populations were identified. On the basis of multivariable analysis, the accessions were grouped into four clusters depending on their proximity with the cultivated pool to facilitate their management and ex situ conservation. The relationship between environment of the site of collection and phenotypic characteristics of the natural populations was also discussed. Different policies of conservation of these genetic resources are suggested to avoid their disappearance even when they disappeared from the other regions of the western Mediterranean. Spanish wild pool of alfalfa, also called ‘Mielga’, appears of great interest for the breeding of alfalfa because it contains a large diversity of characteristics (prostrate habit, rhizomes) linked to tolerance to grazing or drought environment. With the need for more sustainable systems in agriculture, the erosion of natural habitats and the necessity for rehabilitation of degraded ecosystems, the importance of this wild pool is really inestimable.     

Conservation strategies and management guidelines for wild Prunus genetic resources in Andalusia, Spain

Six species of the genus Prunus occur inAndalusia. Matters regarding their ecology, ethnobotanical aspectsand conservation are discussed, as well as knowledge about thegermplasm of those species with greater economic importance and theirdegree of conservation, namely: P.avium, P.mahaleb and P.insititia. Various conservation measures areproposed for wild germplasm and for the germplasm of local varieties:a) conservation efforts should target the best populations,except in the case of P.avium and P.insititia, where every individual should beaddressed, including both wild and local cultivars; b) theDehesa del Camarate (Sierra Nevada) is proposed as an areafor in situ germplasm conservation; c) theawareness of the importance of Prunus germplasmshould be increased among forest workers, environmental managers andthe public; d) some silvicultural techniques are proposed, suchas mixed Prunus patches, hedge treatment,Z-tree selection at the early stages and the protection ofroot suckers; e) improved, selected P.avium and P.mahaleb germplasm should be used at highlyproductive sites since these two species can be cultivated as highvalue timber trees; f) some Prunus speciescan also provide profitable NTFPs in the region. Details regardingdifferent collection areas are covered. Finally, some topics arerecommended for scientific research. Three tables and a distributionmap of Prunus species in Andalusia areincluded.     

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.     

The Weedy Rice Complex in Costa Rica. I. Morphological Study of Relationships Between Commercial Rice Varieties,Wild <Emphasis Type="Italic">Oryza</Emphasis> Relatives and Weedy Types

Weedy rice is a complex of Oryza morphotypes widely distributed in commercial rice fields, which interfere with rice cultivation, seed production, industrial processing and commercialization of this crop in several countries. The objective of this study was to characterize the weedy rice complex of Costa Rica by comparing it with the cultivated and wild rice species found in the country. A collection of weedy rice accessions, representative of the morphotypes found in the country, was established and characterized. Their morphometric relationships were established by comparing 27 morphological traits with commercial rice cultivars, landraces and wild Oryza species and by performing a multivariate analysis. Twenty-one weedy rice morphotypes were identified among 735 weedy accessions by using a three-digit code based on seed characters. Three principal components (PCs) explained 66.25% of the variation observed. The first PC accounted for 36.21% of the variation and separated CCDD genome type Oryza latifolia and O. grandiglumis from AA genome species O. sativa, O. glumaepatula, O. rufipogon and O. glaberrima. The second (18.9%) and third (11.14%) PCs separated the weedy morphotype groups from the AA genome species O.sativa, O. glaberrima and O. rufipogon. The weedy morphotypes were scattered between the indica commercial rice varieties, the cluster landraces–glaberrima and O. rufipogon. Additionally, a group of morphotypes showed intermediate characteristics between O. sativa and O. rufipogon, suggesting that hybridization could have taken place in the past between these species. None of the morphotypes collected in Costa Rica clustered with the allotetraploids CCDD species or O. glumaepatula.     

Use of GIS for Optimizing a Collecting Mission for a Rare Wild Pepper (<Emphasis Type="Italic">Capsicum flexuosum</Emphasis> Sendtn.) in Paraguay

This paper presents an effective method for prioritizing areas within a country for acquisition of germplasm of a crop gene pool for ex situ conservation. The method was applied to the rare wild pepper species, Capsicum flexuosum Sendtn., in south-east Paraguay. A model to prioritize areas for collecting germplasm was constructed by combining (1) a prediction of the species' geographic distribution based on the climate at previous collection points, (2) the distribution of forest margins (the species' natural habitat) and (3) areas accessible by road. The model was then tested in the field by visiting 20 sites having both high and low predicted probability of occurrence of C. flexuosum. Six new populations were found, representing a significant improvement over two previous collecting missions for the species in the same region, undertaken without the use of GIS targeting. Using the most optimistic analysis of model performance, C. flexuosum was found at five out of seven points predicted to harbour the species and not found at four of five points predicted not to harbour the species. The model was then improved by the use of higher resolution climate surfaces. It is recommended that future explorers use more recent and higher resolution satellite images to locate suitable habitats. The method is replicable for different species in different geographic regions and is offered as a means of optimizing efficiency in financially constrained, national plant genetic resources programs.     

The common bean populations from Basilicata (Southern Italy). An evaluation of their variation

The variation within a collection constituted by 36 populations of common bean (Phaseolus vulgarisL.) recently collected in Basilicata region (Southern Italy) was studied. These populations are cultivated in marginal areas of the region mainly for farms' self-consumption. An appreciable variation of seed shape, colour and type of pattern was observed; 34 populations are of climbing growth habit. The frequency of the phaseolin types within the collection was investigated. C, T and S phaseolin types were observed, the type C being predominant (18 of 36 landraces); intra-population variation was detected only for two landraces. Local populations were compared with 28 commercial cultivars widely grown in the Basilicata region and accounting for 80–90% of common bean production. Significant divergences in growth habit and frequency of phaseolin types were observed: 22 cultivars had bush growth habit and 20 showed T phaseolin type. These results stress the urgency of actions devoted to the safeguard of this local germplasm. In fact, the diffusion of alloctonous and not typical germplasm is the first step towards the erosion of useful or valuable genes present in those established local populations.     

Genetic Diversity among Wild Common Beans from Northwestern Argentina Based on Morpho-agronomic and RAPD Data

The genetic diversity among 10 wild populations of common bean Phaseolus vulgaris var. aborigineus was analyzed by means of RAPD markers and morpho-agronomic data. The study was performed on populations collected from different sites located in the provinces of Jujuy, Salta and Tucumán in northwestern Argentina. Ten quantitative traits and 33 random primers were scored. Clustering based on morpho-agronomic traits and RAPD markers generated similar phenograms that grouped bean populations based on their site of collection. The levels of diversity observed among populations were low suggesting they have a common ancestor. The levels of diversity shown by morpho-agronomic traits were higher compared to those of molecular markers, most probably due to the effect of the environment. Furthermore, a 480-bp DNA band identified a group of wild populations collected from similar sites. Breeding strategies need to exploit this diversity to broaden the genetic base of commercial beans to develop high yield cultivars.     

Can Phaseolin Patterns Help Resolve the Phaseolus–Vigna Complex?

Although common bean (Phaseolus vulgaris L.) is cultivated throughout India, the Himalayas hold largest diversity of bean germplasm. No studies on characterization of phaseolin types on this germplasm have been conducted earlier. In order to determine whether the common bean cultivars collected from various areas in the Northern Himalayas represent introductions from the Central American and Andean domestication centers or are local domesticates, we have analyzed the electrophoretic variation (SDS-PAGE) of phaseolin types in several bean accessions. A few species of Vigna were also included in this study to determine whether phaseolin (vignin in Vigna) patterns can be used to resolve the Phaseolus–Vigna complex. The present investigation on phaseolin (globulin) patterns of Phaseolus vulgaris and Vigna spp. clearly shows much variability in globulin patterns. Three new types of phaseolin patterns were recorded. An attempt to resolve phylogenetic problems in this complex was made using the phaseolin data.     

Collection and Characterization of Citrus indica Tanaka and C. macroptera Montr.: Wild Endangered Species of Northeastern India

Citrus indica and C. macroptera are the wild endangered species of Citrus occurring in northeastern India. Surveys were undertaken in this region for ascertaining distribution, studying variability and for collection of germplasm of these two species. C. indica, an endemic species of this region, was collected from the Citrus Gene Sanctuary located in buffer zone of Nokrek Biosphere Reserve in the Garo hills of Meghalaya. In addition, a putative natural hybrid of C. indica and C. limon was collected for the first time from the south Garo hills. C. macroptera had much wider distribution and was collected from Mizoram and Meghalaya states. In Jantia hills of Meghalaya, natural populations of this species are in a highly threatened state. The two species were unevenly distributed all over the explored territory. Morphological characterization of leaves, fruits and seeds indicated the presence of sizable variability within collected accessions of these two Citrus species. Indigenous technical knowledge gathered on the use and socio-economic importance indicated commercial potential for these two species in northeastern India. However, lack of cultivation of these species and clearing of forest cover at an alarming rate has led to an urgent need to adopt complementary conservation strategies to safeguard these species and to ensure their availability for future utilization. A major emphasis on developing methods for their propagation, multiplication and regeneration in in situ and ex situ conditions is required.     

Isozyme diversity and analysis of the mating system of the wild rice Oryza latifolia Desv. in Costa Rica

Oryza latifolia is the most abundant and widespread wild relative of rice in the lowlands of Costa Rica. It also has the most diverse morphology. However, little is known about the genetic diversity and mating system of this allotetraploid species. Genetic analyses of nine populations from different life zones from the Pacific and Atlantic slopes were performed using six isozyme loci. Differences in genetic variability were observed among populations, of which Palo Verde and Santa Rosa were the most diverse. Most Atlantic populations clustered together, and a similar result was obtained with the Pacific populations. High levels of interpopulation diversity were observed while most populations were monomorphic for at least one genotype, suggesting little genetic flow within populations. Even in polymorphic populations no variation was observed within progeny groups, combined with Hardy-Weinberg disequilibria in most populations, commonly observed in autogamous species or in species with clonal reproduction. However, the high frequency of heterozygous-like patterns may suggest that the reproductive system of O. latifolia might be more complex. Future research may explain the genetic patterns as well as the reproductive biology of this species. The knowledge of the genetic diversity and mating system of O. latifolia could contribute to the implementation of genetic resource conservation strategies and gene flow analyses, as well as of breeding programs for rice improvement.     

Selecting in situ conservation sites for grape genetic resources in the USA

Previously, there has not been any in situ conservation sites for crop germplasm within the United States Department of Agriculture (USDA), Agricultural Research Service (ARS), National Plant Germplasm System (NPGS). Using morphologic and molecular (SSR markers) techniques, we assessed the genetic variation present in populations of rock grape (Vitis rupestris Scheele), a native American grape species, throughout its range. We identified seven in situ conservation sites for rock grape using a strategy based on morphologic and molecular data, taxonomic information, population size and integrity, and landholder commitment. In collaboration with federal and state landholding agencies, we have established the first NPGS in situ conservation sites for American wild relatives of a crop.     

Genetic Diversity of the Vigna Germplasm from Thailand and Neighboring Regions Revealed by AFLP Analysis

Thailand is a center of diversity for section Angulares of the Asian Vigna (genus Vigna subgenus Ceratotropis) and 4 Vigna species are cultivated in Thailand. Using newly collected wild and cultivated germplasm of Vigna from Thailand and outgroup accessions AFLP analysis was conducted to clarify genetic diversity and relationships. The results suggest that cultivated V. umbellata and V. mungo evolved from wild relatives in a single domestication event. Vigna umbellata is poorly differentiated from its wild and weedy relatives compared to V. mungo. Results suggest northern Thailand and the neighboring Shan state, Myanmar, is the probable center of domestication for V. umbellata as wild accessions from this area and cultivated rice bean from a wide area in Asia are not greatly diverged. Vigna minima, V. tenuicaulis and V. exilis accessions in Thailand are well differentiated with considerable intra-specific variation. Vigna hirtella consists of two well differentiated subgroups, suggesting taxonomic revision may be necessary. Close genetic relationships between V. radiata and V. grandiflora, and between V. mungo and V. trinervia are confirmed. Naturally growing V. mungo populations in northern Thailand appear to be true wild species as they are well differentiated from Indian wild and Thai cultivated populations. The origin of naturally growing cowpea in Thailand needs to be further studied using a more comprehensive set of materials. This study clarifies inter and intra-specific genetic diversity and inter species relationships of Thai Vigna species.     

Germplasm Sources for Improvement of Forage Quality in Lotus corniculatus L. and L. uliginosus Schkuhr (Fabaceae)

Extensive germplasm resources are present in collections of Lotus corniculatus and L. uliginosus, two forage species that are valuable in situations where other legumes are not persistent. Naturalised populations in many regions are the primary gene pool for genetic improvement of these species and interspecific hybrids are an important source of variation for agronomic and forage quality traits. Although previous research has provided evidence that selection for optimal levels of condensed tannins and herbage productivity are feasible breeding objectives, less is known about correlations of condensed tannin with other parameters of nutritive value. A germplasm collection of 38 accessions in both species from 9 geographic regions was used as a data base to determine these correlations. Forage of each accession was sampled in two successive years from a field site in south-eastern Australia and analysed for condensed tannin, in vitro digestible dry matter, nitrogen, acid detergent fibre and neutral detergent fibre. In both species condensed tannin was negatively correlated with in vitro digestible dry matter and nitrogen. This result helped to identify potentially valuable accessions with low condensed tannin (<4% of dry weight) and high in vitro digestible dry matter (>70%). One of these was an interspecific hybrid of L. uliginosus and L. corniculatus, confirming the view that interspecific hybridization would be a valuable avenue for improvement of agronomic and forage quality characters in Lotus species.     

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.     

Diversity of common bean (Phaseolus vulgaris L.) germplasm from Portugal

Common bean (Phaseolus vulgarisL.) is a traditional crop in Portugal, where farmers growvarieties selected and maintained by themselves. A collection of 88landraces of common bean was evaluated for 17 quantitative andqualitative traits and the biochemical marker phaseolin to displaythe degree of variation of this germplasm. Agronomic data weresubjected to cluster analysis and several groups were identified,with three groups clustering most of the landraces. Regardingphaseolin variation the C and T banding patterns are the mostfrequent ones, so the origin of the Portuguese beans is thus probablythe Andean region of South America. These results give informationabout the origin, diversity and breeding value of the Portuguesegermplasm, that could be useful to widen the genetic base ofcurrently cultivated bean varieties in Europe.     

Evidence for two gene pools of the Lima bean,Phaseolus lunatus L., in the Americas

Summary The lima bean,Phaseolus lunatus L., is a bean species with a broad distribution in the Americas that rivals that of common bean (P. vulgaris). In order to better understand the organization of genetic diversity and the pattern of domestication in lima bean, a review was conducted of the available information on the geographic distribution of wild and cultivated forms of this species. In addition, one-dimensional SDS polyacrylamide gel electrophoresis of seed proteins was also conducted on a sample of 84 wild, 6 weedy, and 426 cultivated forms. Results show that wild forms can be divided into two groups, one with smaller seeds and a very extensive distribution that includes Mexico, Central America, and the eastern slope of the Andes, and the other with a more circumscribed distribution on the western slope of the Andes in Ecuador and northern Peru. Electrophoretic analyses of seed proteins confirmed this subdivision and, additionally, showed that the large-seeded cultivars had been domesticated from the large-seeded wild lima beans in western South America. For the small-seeded lima bean cultivars, it was not possible to determine a domestication center as the most abundant protein pattern in the cultivars also had a widespread distribution in the small-seeded wild progenitor. Electrophoretic analyses showed, however, that domestication led to a reduction of genetic diversity in the small-seeded, Mesoamerican group, but not in the large-seeded group. The latter may be due to insufficient sampling of the larger-seeded, wild 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.