Genetic erosion and in situ conservation of Lima bean (Phaseolus lunatus L.) landraces in its Mesoamerican diversity center

Lima bean (Phaseolus lunatus L.) is an important crop in traditional Mayan agriculture of the Yucatan Peninsula, Mexico, its Mesoamerican center of diversity. Genetic erosion in this species is currently a threat in this region out of 3 of 21 landraces dominate 71.24% of the cultivated area, and 12 are rare landraces grown only in 6.29%. Using 90 ISSR loci, we estimated the diversity and genetic relationships for 21 landraces to analyzing their risk of genetic erosion, and generate data for their in situ conservation. Total genetic diversity was high (h = 0.29), however it was lower than wild gene pool reported (h = 0.69). The abundant landraces had genetic diversity values lower (h = 0.13, I = 0.17) than the common (h = 0.26, I = 0.33) and rare landraces (h = 0.24, I = 0.27). However, the rare landraces are in a higher risk of genetic erosion due to local extinction. The cluster analysis showed no groups corresponding to morpho-phenological characteristics, geographic origin or traditional classification, which resulted from high inter-landraces gene flow levels. The molecular data confirmed that the domesticated Lima bean pool of the Yucatan Peninsula has a high risk of genetic erosion. If current tendencies in landrace cultivation continue, many will no longer be planted within two to three generations, with a consequent loss of their alleles. Programs urgently need to be established for in situ conservation of Lima bean landraces in this region.     

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.     

Geographical barriers and ecological gradients: Geographical distribution of the subspecies and morphotypes of Deutonura deficiens Deharveng, 1979 (Collembola: Neanuridae)

This work explores the links between ecogeographical barriers and clines, and the boundaries of infraspecific biological entities. The distribution of the three subspecies and eight morphotypes of the Neanurinae Deutonura deficiens (Collembola) are mapped and commented based on 570 records. The species inhabits a wide range of forest habitats from the north of Portugal to southeastern France. The subspecies and morphotypes constitute homogeneous geographic sets arranged as a mosaic of closely related forms. A few overlapping zones between subspecies or morphotypes have been detected, some of them having the character of hybrid zones; on the whole however, overlap is small or rare and allopatry or parapatry is the general pattern. Current range boundaries of subspecies and morphotypes were found to be usually not determined by major hydrographical and topographical barriers, with the noticeable exception of the Rhône River. At subspecies level, the morphological cline of increasing tuberculization correlated to the bioclimatic gradient from Mediterranean to Atlantic climates, already recorded by Deharveng (1984. Polymorphisme et polytypisme morphologiques chez quelques Neanurinae européens. Rev. Ecol. Biol. Sol. 21, 533–562), is confirmed on the present data set. Conversely, ecological boundaries, known to constrain species distribution in many living groups of the region, do not fit the contact zones between the different morphotypes of the three subspecies of D. deficiens. The eight recognized forms appear to be at different degrees of reproductive isolation, illustrating active speciation process possibly inherited from recent ecogeographical events. A more detailed sampling on contact zones and a robust phylogenetic hypothesis are needed at this stage for progressing in our understanding of this complex pattern.     

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.     

Oligonucleotide DNA fingerprinting revealing polymorphism in Phaseolus lunatus L.

Digoxigenated synthetic oligonucleotides complementary tosimple repetitive DNA sequences were used to detect polymorphisms inPhaseolus lunatus L. (Lima bean)genotypes. Twenty two accessions, including wild and cultivatedrepresentatives of the Mesoamerican and Andean gene pools andintermediate forms were included in this study. It could be shownthat five repetitive motifs, namely (AAT)₅,(ATG)₅,(GACA)₄,(GATA)₄,(GGAT)₄, were able to give clear-cutbands when used as probes and to reveal high levels of variation. Thehighest resolving power was revealed by the(AAT)₅ probe, which was able to detect eveninter-individual polymorphisms. Therefore, oligonucleotidefingerprinting has proved to be a useful tool to analyse geneticdiversity in Lima bean. Phylogenetic reconstruction based onJACCARD's similarity index confirmed the existence of two majorgene pools and the presence of a minor group of genetically separatedintermediate genotypes.     

Nature of taro (Colocasia esculenta (L.) Schott) genetic diversity prevalent in a Pacific Ocean island, Vanua Lava, Vanuatu

Taro (Colocasia esculenta (L.) Schott), cultivated in Vêtuboso, a village of northern Vanuatu, Melanesia, was surveyed to: (1) assess the extent of morphological and molecular variation being maintained by growers at the village level and, (2) compare this diversity with the diversity found in the crops in Vanuatu. Ethnobotanical data were combined with AFLP analysis to elucidate possible sources of variation. Folk assessment of variation is based on: (a) morphological characteristics (11 characters), (b) names and (c) classification according to habitat, uses, origin and agronomic adaptation. This 3-fold approach allowed growers to differentiate 96 morphotypes, all of which are given distinct vernacular names. AFLP fingerprints successfully differentiated all these 96 morphotypes which do not present a significant intra-clonal variation. But genetic results showed no clear groupings according to geographic origin or habitat of morphotypes and stated that the diversity found within the village was comparable with the overall diversity found in Vanuatu. Local nomenclature and stories associated with each cultivar suggested three sources of diversity: introductions (38%), somatic mutations (15%) and sexual recombinations (48%). AFLP results confirm folk beliefs about origin at least for three pairs of mutants. The 11 so-called wild forms analysed by AFLP were suggested to be feral, escapes from domestication. A dynamic in situ conservation strategy (DISC), favouring a broadening of the national genetic base, was discussed for taro.     

Phaseolin diversity in wild Lima bean (Phaseolus lunatus L.) in American centres of origin

Summary Phaseolin variability was analyzed in 86 wild Phaseolus lunatus accessions, representing a wide geographical distribution from Mexico to Argentina, to determine geographical patterns of variability. The collection can be divided into two major groups. One group consists of accessions from Mexico, Central America, Cuba, Columbia, Argentina showing two Mesoamerican (M) phaseolin pattern types, already described in cultivated materials, as well as a variety of additional banding patterns. This suggests that at least two genotypes in the Mesoamerican gene pool were involved in a domestication process and spread throughout the world. The other group, consisting of wild Andean (A) forms from Northern Peru and belonging to the Andean gene pool, showed, among others, two A pattern types comparable to those found in cultivated large-seeded accessions.     

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.     

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.     

Identification of duplicates and fingerprinting of primary and secondary wild annual <Emphasis Type="Italic">Cicer</Emphasis> gene pools using AFLP markers

Wild annual Cicer gene pools contain valuable germplasm for chickpea improvement programs. Previous research showed that duplication might exist in accessions collected from these gene pools, which would hinder chickpea breeding and related research. AFLP (amplified fragment length polymorphism) markers were used to fingerprint the world collections of the primary and secondary gene pools including C. reticulatum Lad., C. bijugum K.H. Rech., C. judaicum Boiss. and C. pinnatifidum Jaub. et Sp. Duplicates were detected in a total of 24 accessions in both the gene pools, highlighting the necessity to fingerprint the germplasm. Genotypic difference was detected as gene pool specific, species specific and accession specific AFLP markers. These were developed into fingerprinting keys for accession identification between and within species and gene pools. Use of AFLP markers to detect duplicates and to identify accessions is a reliable method which will assist in the characterisation and use of wild annual Cicer germplasm in chickpea improvement programs. We recommend the procedure presented in this paper as a standard approach for the precise genetic identification and characterisation of future world collections of wild Cicer, to keep germplasm integrity and to benefit chickpea breeding and related research programs.     

Systematics and taxonomic delimitation of vegetable,grain and weed amaranths: a morphological and biochemical approach

Amaranthus is a cosmopolitan genus comprising large number of species with many morphotypes cultivars/accessions having diversified morphological features. The species, morphotypes within Amaranthus are very closely related requiring microclassification even revision in present taxonomic status. Most of the works have been done employing grain Amaranthus and their putative ancestors applying morphological and molecular data. In present study along with morphological features, phenolic compounds (secondary metabolites), isozyme profile of acid phosphatase were applied to get comprehensive reflection of interrelationship and relative closeness among the species, morphotypes/accessions of few vegetable, grain and weed Amaranthus. Both the vegetable and grain Amaranthus are supposed to have originated from respective weed progenitor through occasional outcrossing and gradual domestication. The study strongly vouched the previous interpretations regarding interrelationship of grain Amaranthus and their derivation from their putative progenitor also showed clear separate clustering of vegetable and grain Amaranthus with their close relatives. A sharp congruence in cluster pattern was achieved between morphological dendrogram and cumulative dendrogram computed on morphological and biochemical parameters, which is a rare event in systematics.     

Biochemical and cytological analyses in natural populations ofVicia benghalensis L.

Summary 27 accessions ofVicia benghalensis from different geographical origins constitute the pool on which the present study was performed. Genetic variation among the samples was biochemically and cytologically evaluated: seed storage protein profiles and C-banded karyotypes were analysed from single individuals of each accession. SDS-PAGE has shown the possibility to divide the samples into two groups, each characterized by specific protein profiles. The two patterns were indicated as A and B. From individual seed electrophoresis it was ascertained that samples possessing the pattern A showed a low level of individual variation, while those possessing the pattern B were highly polymorphic, thus suggesting differences in the allogamic rate. The cytological analysis demonstrated the presence of two groups of accessions, one being much richer in heterochromatin as evidenced by C-banding (H⁺) than the other (H^-). The analysis of biochemical and karyological data showed a constant association between pattern A and karyotype H⁻ and between pattern B and karyotype H⁺. On the basis of these results it is proposed to considerV. benghalensis as a highly heteromorphic species, in which two groups may be identified.     

Genetic evidence for speciation in <Emphasis Type="Italic">Cannabis</Emphasis> (Cannabaceae)

Sample populations of 157 Cannabis accessions of diverse geographic origin were surveyed for allozyme variation at 17 gene loci. The frequencies of 52 alleles were subjected to principal components analysis. A scatter plot revealed two major groups of accessions. The sativa gene pool includes fiber/seed landraces from Europe, Asia Minor, and Central Asia, and ruderal populations from Eastern Europe. The indica gene pool includes fiber/seed landraces from eastern Asia, narrow-leafleted drug strains from southern Asia, Africa, and Latin America, wide-leafleted drug strains from Afghanistan and Pakistan, and feral populations from India and Nepal. A third putative gene pool includes ruderal populations from Central Asia. None of the previous taxonomic concepts that were tested adequately circumscribe the sativa and indica gene pools. A polytypic concept of Cannabis is proposed, which recognizes three species, C. sativa, C. indica and C. ruderalis, and seven putative taxa.     

Variation and genomic polymorphism of lectin-related proteins in Lima bean (Phaseolus lunatus L.) seeds

Variation of the lectin and the two lectin-related proteins, AIL (-amylase inhibitor-like) and ARL (arcelin-like) was examined in wild and cultivated accessions of Lima bean (Phaseolus lunatus L.) using electrophoresis of total seed proteins, immunoblot and RFLP analysis of lectin-related genes. Results confirm that divergence of the two major Lima bean gene pools, Andean and Mesoamerican, also apply to this protein family. All three members of the family are present in both gene pools, with differences in size, abundance and composition between gene pools, giving the possibility to distinguish Andean from Mesoamerican lectin pattern types. Both patterns show some variants, such as lack of lectin or its presence as an abundant protein. The observed variation reflects, at least in part, into genomic polymorphism. The presence of arcelin- and -amylase-related proteins in Lima bean could represent a tool to increase our knowledge in the evolution of the lectin family in Phaseolus species.     

Analysis of genetic diversity in Indian taro [Colocasia esculenta (L.) Schott] using random amplified polymorphic DNA (RAPD) markers

Taro [Colocasia esculenta (L.) Schott] germplasm accessions collected from different parts of India were subjected to RAPD (Random Amplified Polymorphic DNA) analysis to assess the genetic diversity prevalent and also to test the genetic basis of morphotypic classification. Thirteen random decamer primers out of the 22 tested were used to analyse 32 taro accessions belonging to 28 morphotypes. Three out of these thirteen primers analysed showed 100 per cent polymorphism. Per cent polymorphism varied from 60 to 100 among the polymorphic primers. High genetic diversity was revealed as the similarity coefficient values ranged from 0.50 to 0.98. No two accessions analysed in the present study showed a similarity coefficient value of one thereby indicating their distinctness and presence of high genetic diversity in Indian taro germplasm. Dendrogram obtained from UPGMA analysis grouped 32 accessions in four clusters and three accessions were placed as outliers. Clustering pattern did not show any strict relationship with geographical distribution, morphotype classification and genotypic diversity. Further, accessions classified, as belonging to the same morphotypic group did not always cluster together. Presence of a very close genepool of the wild, weedy and cultivated forms with extreme levels of phenotypic and genotypic variation is suggested as the reason for high genetic diversity reported. Usefulness of DNA markers such as RAPD in characterising and assessing the genetic diversity in Indian taro germplasm is hereby demonstrated.     

<Emphasis Type="Italic">Rhizobium</Emphasis>,<Emphasis Type="Italic"> Bradyrhizobium</Emphasis> and<Emphasis Type="Italic"> Agrobacterium</Emphasis> strains isolated from cultivated legumes

The present study was conducted to isolate and characterize rhizobial strains from root nodules of cultivated legumes, i.e. chickpea, mungbean, pea and siratro. Preliminary characterization of these isolates was done on the basis of plant infectivity test, acetylene reduction assay, C-source utilization, phosphate solubilization, phytohormones and polysaccharide production. The plant infectivity test and acetylene reduction assay showed effective root nodule formation by all the isolates on their respective hosts, except for chickpea isolate Ca-18 that failed to infect its original host. All strains showed homology to a typical Rhizobium strain on the basis of growth pattern, C-source utilization and polysaccharide production. The strain Ca-18 was characterized by its phosphate solubilization and indole acetic acid (IAA) production. The genetic relationship of the six rhizobial strains was carried out by random amplified polymorphic DNA (RAPD) including a reference strain of Bradyrhizobium japonicum TAL-102. Analysis conducted with 60 primers discriminated between the strains of Rhizobium and Bradyrhizobium in two different clusters. One of the primers, OPB-5, yielded a unique RAPD pattern for the six strains and well discriminated the non-nodulating chickpea isolate Ca-18 from all the other nodulating rhizobial strains. Isolate Ca-18 showed the least homology of 15% and 18% with Rhizobium and Bradyrhizobium, respectively, and was probably not a (Brady)rhizobium strain. Partial 16S rRNA gene sequence analysis for MN-S, TAL-102 and Ca-18 strains showed 97% homology between MN-S and TAL-102 strains, supporting the view that they were strains of B. japonicum species. The non-infective isolate Ca-18 was 67% different from the other two strains and probably was an Agrobacterium strain.     

Diversity of the ectomycorrhiza community at a uranium mining heap

Ectomycorrhiza (EM) community structure was analyzed at one bare heap site (BHS), one heap site with organic cover (HS-OH) and one reference site (RS) in the former uranium mining area near Ronneburg (Thuringia, Germany). Twenty-three EM morphotypes were distinguished, and 14 of them were additionally characterized by polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) and internal transcribed spacer (ITS) sequence analysis. Colonization of birch by the different morphotypes was quantified, and the EM diversity at the different sites was investigated. Compared to RS, total EM colonization was reduced by 6% (P=0.851) at HS-OH and by 58% (P<0.001) at BHS. Likewise, EM diversity was reduced by 16% (P=0.229) at HS-OH and 52% (P<0.001) at BHS. The Sørensen similarity between EM samples from RS was nearly independent from the sampling date, whereas at HS-OH and especially BHS, the Sørensen similarity decreased with increasing time between the samplings. All EM fungal species dominating at the two heap sites were also present at RS. Thus, fungi with high tolerance against uranium and other stress factors at the heap sites (e.g. heavy metals, nutrient limitation, drought) were selected among all EM fungi of the area. Highly adapted fungi with a distribution restricted to the contaminated soils were not detected.Electronic Supplementary Material Supplementary material is available for this article at .     

Biodiversity in the barnyard millet (<Emphasis Type="Italic">Echinochloa frumentacea</Emphasis> Link,Poaceae) germplasm in India

One hundred ninety four accessions of barnyard millet collected from different eco-geographical regions of India were evaluated for 14 quantitative traits during kharif 2003 and 2005. These accessions were grouped into 5 groups on the basis of place of collection. These groups differed significantly in their mean values for quantitative traits and magnitude of correlation among traits indicates origin based association. The group ‘C’ (source of origin is unknown) was found most diverse group (mean coefficient of variation 17.67%), while rest of the groups recorded mean coefficient of variation between 12 and 13%. Correlation studies indicate that flag leaf width, number of racemes along with internode length should be considered while performing selection in segregating generations. The plotting of first and second principal component axes scores suggests that presence of three distinct morphotypes in the present study. These morphotypes are quite similar to three botanical varieties viz., var. robusta, var. intermedia and var. stolonifera in their morphological traits, while botanical variety laxa (endemic to Sikkim area of India) was completely absent from the present study.     

Phyletic relationships within the genus Hordeum using PCR-based markers

Fifteen Hordeum species and subspeciesbelonging to groups with different genomes were studied usingPCR-based markers to establish phylogenetic relationshipswithin the genus. Two hundred and seventeen RAPD and STS markers wereused to calculate genetic distances and construct phylogenetic trees.The phenetic analysis clearly separated the primary gene pool,represented by H. vulgaressp. vulgare, H.vulgare ssp. vulgare convar. vulgare f. agriochriton andH. vulgare ssp.spontaneum, from the secondary gene pool,represented by H. bulbosumand the tertiary gene pool, represented by American wild barleys andH. bogdanii. Data obtainedfrom PCO analysis are in complete agreement with taxonomicclassifications proposed previously, which were comparisons ofnumerous morphological, cytological and reproductivecharacters.