Using isozyme polymorphism to assess genetic variation within cultivated yams (Dioscorea cayenensis/Dioscorea rotundata complex) of the Republic of Benin

Four hundred and sixty-seven accessions of cultivated Guinea yam (Dioscorea cayenensis/Dioscorea rotundatacomplex) collected from different localities of Benin Republic were analysed to study isoenzymatic variability in seven enzyme systems: aspartate aminotransferase (AAT), esterase (EST), glucose-6-phosphate dehydrogenase (G6PDH), isocitrate dehydrogenase (IDH), phosphoglucomutase (PGM), phosphoglucoisomerase (PGI), and shikimate dehydrogenase (SKDH) using starch gel electrophoresis. Polymorphism was observed in all of the enzyme systems and a total of 62 electromorphs of different frequency and variability patterns were recorded. Different combinations of banding patterns of these systems led to identification of 227 different cultivars within the 467 accessions analysed. For an old and vegetatively propagated crop (with a considerable number of vernacular names) such as yam, and for which a high rate of duplication is expected, the 227 cultivars were found to be good enough to be considered as the adequate number of accessions representing the diversity in the germplasm analysed. Cluster analysis (UPGMA) produced a most likely division of the 467 accessions into two groups corresponding to D. rotundata Poir. and D. cayenensis Lam., supporting the concept that the two forms of guinea yam represent different genetic entities. The different clusters formed within the white yams (D. rotundata) did not exactly conform to the known cultivar groups. Additional polymorphic enzymes are needed for an accurate isozyme-based genetic discrimination of most of the cultivar groups.     

Genetic Diversity of the Greater Yam (<Emphasis Type="Italic">Dioscorea alata</Emphasis> L.) and Relatedness to <Emphasis Type="Italic">D. nummularia</Emphasis> Lam. and <Emphasis Type="Italic">D. transversa</Emphasis> Br. as Revealed with AFLP Markers

Amplified fragment length polymorphism markers were used to assess the genetic relatedness between Dioscorea alata and nine other edible Dioscorea. These species include D. abyssinica Hoch., D. bulbifera L., D. cayenensis-rotundata Lamk. et Poir., D. esculenta Burk., D. nummularia Lam., D. pentaphylla L., D. persimilis Prain. et Burk., D. transversa Br. and D. trifida L. Four successive studies were conducted with emphasis on the genetic relationship within D. alata and among species of the Enantiophyllum section from Vanuatu. Study 1 was carried out to select a set of polymorphic primer pairs using 11 combinations and eight species belonging to five distinct sections. The four most polymorphic primer pairs were used in study 2 among six species of the Enantiophyllum section. Study 3 focussed mainly on the genetic relationship among 83 accessions of D. alata, mostly from Vanuatu (78 acc.) but also from Benin, Guadeloupe, New Caledonia and Vietnam. The ploidy level of 53 accessions was determined and results indicated the presence of tetraploid, hexaploid and octoploid cultivars. Study 4, included 35 accessions of D. alata, D. nummularia and D. transversa and was conducted using two primer pairs to verify the taxonomical identity of the cultivars `langlang', `maro' and `netsar' from Vanuatu. The overall results indicated that each accession can be fingerprinted uniquely with AFLP. D. alata is an heterogeneous species which shares a common genetic background with D. nummularia and `langlang', `maro' and `netsar'. UPGMA cluster analysis revealed the existence of three major groups of genotypes within D. alata, each assembling accessions from distant geographical origins and different ploidy levels. The analysis also revealed that `langlang', `maro' and `netsar' clustered together with the cultivar `wael' (D. transversa) from New Caledonia. Results are discussed in the paper.     

Yam (Dioscorea ssp.) domestication by the Nago and Fon ethnic groups in Benin

Guinea yams (Dioscorea cayenensis-rotundata complex; D. rotundata Poir. and D. cayenensis Lam.) have been described as resulting from a process of domestication of wild yams of the section Enantiophyllum by African farmers. Although currently practised by few farmers, the process of yam domestication is still on-going in Benin. In order to document the practices used and the indigenous knowledge maintained by Nago and Fon farmers, 27 villages were surveyed. In total, 57 farmers domesticating yam were identified, and 68 yams newly domesticated (or in domestication) were collected. Fon and Nago farmers domesticate yam mainly to widen the genetic basis of the existing diversity or for simple curiosity. Among the three wild yams species (D. abyssinica Hochst. ex Kunth, D. praehensilis Benth. and D. burkilliana J. Miège) used, D. praehensilis is the most important and the most exploited. Tuber of the wild yams are collected either in the bush (most often near the village) or in the forests (far from the village) during hunting. The domestication process consists of bringing into cultivation selected individuals which go through intense vegetative multiplication and selection procedures (over a lengthy but variable period of time) that induce morphological and biochemical changes in the plant mainly at the tuber level. Individuals resulting from these manipulations were found to be, either similar or identical to known landraces or completely new based on both morphological and isozyme analysis. Because it leads to some new cultivars, this process of domestication has potential in yam breeding and appears to be a strategy that could be useful to breeders, while developing a methodology for participatory breeding of yam.     

Diversification of Multipurpose Plant, <Emphasis Type="Italic">Perilla Frutescens</Emphasis>

The traditional Asian crop, Perilla frutescens has multiple uses. There are specialized cultivars for seed oil and for medicinal use, as well as wild/weedy forms growing in various habitats. Based on selective characteristics of leaf odor, anthocyanin pigmentation, seed hardness and seed diameter, the diversity of this species was investigated to clarify the intraspecific differentiation. P. frutescens was divided into five groups by the combination of three qualitative characteristics: leaf odor, anthocyanin pigmentation and seed hardness. Most of the plants cultivated for oil belonged to one group, while medicinal plants belonged to three other groups. Wild/weedy forms were in the last group. The five groups could not be distinguished by seed diameter. Though the plants cultivated for oil tended to have larger seeds than the medicinal and wild/weedy plants, there was no boundary either between the two crops, or among various phenotypes of P. frutescens.     

Genetic Nature of Yams (Dioscorea sp.) Domesticated by Farmers in Benin (West Africa)

‘Domestication’ is a traditional farmers’ practice reported for yams (Dioscorea sp.) in Benin (West Africa). It involves introducing ‘spontaneous’ (naturally occurring) yams, supposedly wild (D. abyssinica and D. praehensilis), in varieties of the D. cayenensis–D. rotundata cultivated species complex. In this study, we established the genetic nature of ‘predomesticated’ yam plants using the amplified fragment length polymorphism (AFLP) technique. A total of 213 accessions, consisting of 32 predomesticated yams, 70 D. cayenensis–D. rotundata, 86 D. abyssinica and 25 D. praehensilis yams were analysed. Using 91 AFLP markers, three groups of accessions were distinguished, broadly corresponding to the above botanical species. Of the 32 predomesticated accessions, 16% were clustered with D. praehensilis, 37% with D. abyssinica and the remaining 47% with D. cayenensis–D. rotundata. These results demonstrated the use of wild plants by farmers in their domestication process, and suggested that plants derived from intervarietal and interspecific hybridisation may also be subject to this process. This study has shown that through domestication farmers influence and increase the genetic diversity in yam by using sexual reproduction of wild and possibly cultivated yams.     

Cultivation of lotus (<Emphasis Type="Italic">Nelumbo nucifera</Emphasis> Gaertn. ssp. <Emphasis Type="Italic">nucifera</Emphasis>) and its utilization in China

Lotus (Nelumbo nucifera Gaertn. ssp. nucifera), one of 12 aquatic species used as vegetable, has been cultivated for more than 2,000 years, and now has been widely cultivated in almost all provinces in China. The largest area under cultivation of lotus is located in the regions surrounding mid-down Yangtse River, including Hubei, Jiangsu, Zhejiang, Anhui, Jiangxi and Hunan provinces. According to different purposes or morphological differences, the Chinese lotus (N. nucifera ssp. nucifera) is usually classified into three types: rhizome lotus, seed lotus and flower lotus. Rhizome lotus is mainly cultivated in Hubei, Jiangsu, Anhui and Zhejiang provinces; Seed lotus in Jiangxi, Fujian and Hunan, and flower lotus in Wuhan, Hubei province, and Beijing. Up to the year 2002, a total of 572 lotus accessions (including landraces, cultivars and breeding lines) were conserved in National Garden of Aquatic Vegetable, Wuhan, Hubei province, including those collections from 153 counties in 18 provinces, and lines bred by breeders. Out of these accessions, 310 were rhizome lotus which contains 201 landraces and 109 breeding lines; 229 were flower lotus including 172 cultivars and 57 breeding lines; and the rest 33 were seed lotus with 18 cultivars.     

Genetic diversity and species delimitation in the cultivated and wild Guinea yams (Dioscorea spp.) from Southwest Ethiopia as determined by AFLP (amplified fragment length polymorphism) markers

Yams (Dioscorea spp.) rank as the fourth most important root and tuber crop after potatoes, cassava and sweet potatoes. They are an economic crop in most of the tropics especially in West Africa, which produces over 95 % of the world output. Despite their cultural and economic importance there is taxonomic confusion regarding Guinea yams. The currently used classification scheme, which relies on vegetative and inflorescence characters, does not consistently delimit species boundaries between members of Guinea yams (D. cayenensis Lam.–D. rotundata Poir. complex), their wild relatives (D. abyssinica Kunth and D. praehensilis Benth.,) and D. sagittifolia Pax. Establishing the taxonomic identity of the germplasm and understanding the systematic relationships among crops is vital to the management of genetic resources and the utilization of accessions. In this study, amplified fragment length polymorphism (AFLP) genetic fingerprinting was used to evaluate and characterize 43 individual plants, belonging to different populations of wild and cultivated Guinea yams. The three primer combinations used in the AFLP analyses generated 158 scorable bands, with an overall polymorphism of 78 %. Ordination and cluster analyses of AFLP data failed to produce any clear species boundary between either the Guinea yam accessions or between them and their wild relatives. The average genetic similarity between the study individuals of Guinea yams and their wild relatives ranged from 60 to 100 %. The first, second and third principal coordinates axes cumulatively account for 77.45 % of the total variation. AFLP analyses also revealed a higher genetic divergence among cultivated Guinea yam accessions of the Sheko cultivars. Ordination and cluster analysis using UPGMA revealed no clear species boundaries between members of the complex. Thus, the taxonomy of these “species” needs to be revisited using other markers.     

Processes affecting genetic structure and conservation: a case study of wild and cultivated <Emphasis Type="Italic">Brassica rapa</Emphasis>

When planning optimal conservation strategies for wild and cultivated types of a plant species, a number of influencing biological and environmental factors should be considered from the outset. In the present study Brassica rapa was used to illustrate this: to develop Scandinavian conservation strategies for wild and cultivated B. rapa, DNA-marker analysis was performed on 15 cultivated and 17 wild accessions of B. rapa plus 8 accessions of the cross compatible B. napus. The B. rapa cultivars were bred in Sweden and Finland in 1944–1997 and the wild B. rapa material was collected from Denmark, Sweden and United Kingdom. The B. napus accessions were bred within the last 20 years in the Scandinavian countries. Results were based on scoring of 131 polymorphic ISSR markers in the total plant material. A Bayesian Markov chain Monte Carlo (MCMC) approach implemented in NewHybrids demonstrated a clear distinction of B. rapa and B. napus individuals except for three individuals that seemed to be backcrosses. The backcrossed hybrids descended from two Swedish populations, one wild and one escaped. The overall pattern of genetic variation and structure in B. rapa showed that cultivated and wild B. rapa accessions formed two almost separated clusters. Geographical origin and breeding history of cultivars were reflected in these genetic relationships. In addition, wild populations from Denmark and Sweden seemed to be closely related, except for a Swedish population, which seemingly was an escaped cultivar. The study point to that many processes, e.g. spontaneous introgression, naturalisation, breeding and agricultural practise affected the genetic structure of wild and cultivated B. rapa populations.     

Natural hybrids between cultivated and wild sunflowers (<Emphasis Type="Italic">Helianthus</Emphasis> spp.) in Argentina

Two introduced wild species Helianthus annuus L. and H. petiolaris Nutt. have become widespread in central Argentina and overlap the sunflower crop region. Intermediate off-type plants between the wild and cultivated species are often found, which is of concern because of the recent release of imidazolinone resistant varieties and the likely use of genetically modified sunflower cultivars. The progeny of 33 off-type plants obtained from 14 representative sites of the diffusion area were studied to confirm hybrid origin. Germination, survival, morphological traits and days to flowering confirmed hybridization between crop and both wild species, when compared to eight accessions of typical wild plants. Some progenies were presumably crop–wild H. annuus hybrids, some originated from the cross of cultivated plants and H. petiolaris, and two were the advanced generation of a cultivated hybrid. Hence, morphological traits are a good clue for the identification of spontaneous hybrid plants at field. The results indicate that crop–wild hybridization and introgression occur at various places in central Argentina. This fact may represent a way to herbicide resistance escape and future transgene escape if GM sunflower cultivars are released for commercial use.     

Evaluation and utilization of <Emphasis Type="Italic">Aegilops</Emphasis> and wild <Emphasis Type="Italic">Triticum</Emphasis> species for enhancing iron and zinc content in wheat

Grains of 80 accessions of nine species of wild Triticum and Aegilops along with 15 semi-dwarf cultivars of bread and durum wheat grown over 2 years at Indian Institute of Technology, Roorkee, were analyzed for grain iron and zinc content. The bread and durum cultivars had very low content and little variability for both of these micronutrients. The related non-progenitor wild species with S, U and M genomes showed up to 3–4 folds higher iron and zinc content in their grains as compared to bread and durum wheat. For confirmation, two Ae. kotschyi Boiss. accessions were analyzed after ashing and were found to have more than 30% higher grain ash content than the wheat cultivars containing more than 75% higher iron and 60% higher zinc than that of wheat. There were highly significant differences for iron and zinc contents among various cultivars and wild relatives over both the years with very high broad sense heritability. There was a significantly high positive correlation between flag leaf iron and grain iron (r = 0.82) and flag leaf zinc and grain zinc (r = 0.92) content of the selected donors suggesting that the leaf analysis could be used for early selection for high iron and zinc content. ‘Chinese Spring’ (Ph ^I ) was used for inducing homoeologous chromosome pairing between Aegilops and wheat genomes and transferring these useful traits from the wild species to the elite wheat cultivars. A majority of the interspecific hybrids had higher leaf iron and zinc content than their wheat parents and equivalent or higher content than their Aegilops parents suggesting that the parental Aegilops donors possess a more efficient system for uptake and translocation of the micronutrients which could ultimately be utilized for wheat grain biofortification. Partially fertile to sterile BC₁ derivatives with variable chromosomes of Aegilops species had also higher leaf iron and zinc content confirming the possibility of transfer of required variability. Some of the fertile BC₁F₃ and BC₂F₂ derivatives had as high grain ash and grain ash iron and zinc content as that of the donor Aegilops parent. Further work on backcrossing, selfing, selection of fertile derivatives, leaf and grain analyses for iron and zinc for developing biofortified bread and durum wheat cultivars is in progress. Nidhi Rawat, Vijay K. Tiwari, and Neelam Singh have contributed equally to the work.     

Polymorphisms Among Chloroplast and Mitochondrial Genomes of <Emphasis Type="Italic">Citrullus</Emphasis> Species and Subspecies

Twelve and six DNA clones representing various parts of chloroplast and mitochondrial genomes, respectively, were used to detect polymorphism among five watermelon cultivars and 21 U.S. Plant Introductions (PIs) collected from diverse geographical locations and representing major groups of Citrullus species. Cluster analysis based on 20 chloroplast DNA (cpDNA) and 10 mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP) markers differentiated the accessions into three major phenetic groups: PIs and watermelon cultivars of Citrullus lanatus subsp. vulgaris (Schrad. ex Eckl. et Zeyh.) Fursa (also designated as C. lanatus var. lanatus) (group I), PIs of C. lanatus var. citroides (of C. lanatus subsp. lanatus Schrad. ex Eckl. et Zeyh.)(group II), and C. colocynthis (L.) Schrad. PIs (group III). The chloroplast and mitochondrial genomes of watermelon cultivars are distinct, but closely related to those of the C. lanatus var. lanatus PIs. On the other hand, the chloroplast and mitochondrial genomes of the wild species C. colocynthis are more similar to those of C. lanatus var. citroides. Polymorphic cpDNA and mtDNA markers identified in this study can complement isozyme and nuclear DNA data used in earlier phylogenetic and phenetic classifications of Citrullus PIs. These cpDNA and mtDNA markers are being used in experiments designed to enhance watermelon cultivars by replacing the chloroplast and mitochondrial genome of cultivated watermelon with those of the wild species C. colocynthis.     

Identification of <Emphasis Type="Italic">SUB1A</Emphasis> alleles from wild rice <Emphasis Type="Italic">Oryza rufipogon</Emphasis> Griff.

Submergence stress is a major constraint to rice production in South and Southeast Asia. Most rice (Oryza sativa L.) cultivars die within a week of complete submergence, while a small number of accessions are submergence-tolerant for up to 2 weeks or more. These cultivars have the tolerant allele of the SUB1A gene, one of three ERF genes at this locus on rice chromosome 9. In all O. sativa varieties studied, the SUB1A gene is limited to a subset of indica accessions of O. sativa. Thus far, there has been no published report of the SUB1A gene in wild rice species. Here we report evidence of the SUB1A gene found in wild species of O. rufipogon Griff. accessions by the use of degenerate primers corresponding to the most highly conserved regions of the SUB1 locus. The results indicated that two SUB1A-like alleles, e.g. OrSub1A-1 and OrSub1A-2, were identified from two O. rufipogon accessions. Submergence treatment shows that both of the accessions with SUB1A-like genes were submergence-intolerant. This preliminary study provides insight into the origin and allelic variation of SUB1A, an agronomically important gene that is rapidly being introduced into widely-grown rice cultivars.     

Genetic structure and differentiation among grapevines (<Emphasis Type="Italic">Vitis vinifera</Emphasis>) accessions from Maghreb region

Three gene pools representative of Vitis vinifera L. subsp. vinifera (=subsp. sativa Beger) growing in the Maghreb regions (North Africa) from Tunisia (44), Algeria (31) and Morocco (18) and 16 wild grape accessions (Vitis vinifera L. subsp. sylvestris (Gmelin) Beger) from Tunisia were analysed for genetic diversity and differentiation at twenty nuclear microsatellites markers distributed throughout the 19 grape chromosomes. 203 alleles with a mean number of 10.15 alleles per locus were observed in a total of 109 accessions. Genetic diversities were high in all populations with values ranging from 0.6775 (Moroccan cultivars) to 0.7254 (Tunisian cultivars). F _st pairwise values between cultivated grapevine populations were low but found to be significantly different from zero. High F _st pairwise values were shown between wild and cultivated compartments. Two parent offspring relationships, two synonyms and two clones of the same cultivar were detected. The rate of gene flow caused by vegetative dissemination of cultivated grapevine plants was not sufficient to genetically homogenise the pools of cultivars grown in different regions. The Neighbour Joining cluster analysis showed a clear separation according to geographical origins for the cultivated grapevines gene pools and revealed a high dissimilarity between cultivated and wild grapevine. However, three cultivars (Plant d’Ouchtata 1, Plant de Tabarka 3 and Plant d’Ouchtata 3) are very close to wild accessions and may result from a hybridisation between cultivated and wild accessions. The high level of differentiation between cultivated and wild accessions indicates that the cultivated accessions do not derive directly from local wild populations but could mostly correspond to imported materials introduced from others regions during historical times or derived from crossing between them.     

Diversity analysis of Central Asia and Caucasian lentil (<Emphasis Type="Italic">Lens</Emphasis><Emphasis Type="Italic">culinaris</Emphasis> Medik.) germplasm using SSR fingerprinting

Diversity analysis was performed among 39 cultivated lentil (Lens culinaris Medik.) accessions of Central Asia and Caucasian origin using five highly polymorphic microsatellite markers. A total of 33 alleles determined ranging from 3 to 8 per locus. Estimated gene diversity value for 33 loci was 0.66. Genetic similarity indices among 39 accessions ranged from 0.24 to 1.0. Cluster analysis using the unweighted pair group method with arithmetic mean method classified accessions into six major groups at 0.5 similarity coefficient. More than half accessions from Tajikistan formed large cluster. On the other hand, a few accessions from each country showed unique genotypes. Overall, most of the accessions, except ones with closely related origin, were distinguished by the present high quality DNA fingerprinting. This molecular diversity information gives important basis for conservation strategy in gene bank and exotic germplasm introduction in breeding programs in Central Asia and Caucasian countries.     

The influence of European and American wild germplasm in hop (<Emphasis Type="Italic">Humulus lupulus</Emphasis> L.) cultivars

Microsatellite variation at the nuclear and chloroplast genomes was evaluated for wild European and wild American hops, in order to assess the genetic diversity and origin of cultivated hops. Seven nuclear loci and 32 chloroplast loci were used in the analysis of 182 hop accessions including wild European (68), wild American (48), and cultivars (66). A total of 116 alleles were identified using 7 nuclear microsatellites showing different averages of polymorphism and distribution in the wild American and European accessions and cultivars. Two main groups were established as revealed by several statistical analyses; one including European wild accessions and cultivars and a second group consisting of American wild accessions. Three polymorphic chloroplast microsatellite loci were detected, six alleles were scored which defined a total of five haplotypes that were exclusive or presented different distribution between American and European wild accessions. A major influence of the wild European haplotypes was detected among hop cultivars. To the best of our knowledge, this is the first work reporting the use of chloroplast microsatellites in hops.     

The Pacific yam (<Emphasis Type="Italic">Dioscorea nummularia</Emphasis> Lam.), an under-exploited tuber crop from Melanesia

This study aims to explore the potential of Dioscorea nummularia Lam. and to gather information as a prelude to its improvement by plant breeding. After a taxonomic clarification and a review of the botanical data, its geographical areas of origin and distribution are determined. A germplasm survey conducted on nine islands of Vanuatu enabled the collection of 110 accessions corresponding to 84 cultivars. These were grouped into: (1) wild forms under domestication, (2) annual types, (3) perennial-type cultivars of D. nummularia and (4) natural interspecific hybrids with D. alata L. Within each group, remarkable morphological variation exists for stem spininess, leaf shape, emerging leaf colour, tuber shape and tuber flesh colour. Selected accessions examined for ploidy had chromosome numbers ranging from 2n = 3x = 60 to 2n = 6x = 120. Chloroplast counts conducted on stomatal guard cells confirmed the variation in ploidy levels. Eight different male cultivars were studied for pollen fertility, but <5% of the pollen grains appeared to be fertile when stained with acetocarmine. However, profuse flowering and synchrony favour pollination and result in fruit set. All accessions are resistant to anthracnose. Viruses were detected but are morphologically not discernible, with no incidence on yield. Physicochemical analyses of tubers from 16 accessions revealed that D. nummularia and interspecific hybrids have high percentages of dry matter (mean of 33.11%) and starch (82.81%). The Pacific yam could be improved through conventional hybridisation or used for interspecific crosses with related economically important species. Recommendations are made for further research.     

Genetic Characterization of Brazilian Annual <Emphasis Type="Italic">Arachis</Emphasis> Species from Sections <Emphasis Type="Italic">Arachis</Emphasis> and <Emphasis Type="Italic">Heteranthae</Emphasis> using RAPD Markers

The genus Arachis is divided into nine taxonomic sections. Section Arachis is composed of annual and perennial species, while section Heteranthae has only annual species. The objective of this study was to investigate the genetic relationships among 15 Brazilian annual accessions from Arachis and Heteranthae using RAPD markers. Twenty-seven primers were tested, of which nine produced unique fingerprintings for all the accessions studied. A total of 88 polymorphic fragments were scored and the number of fragments per primer varied from 6 to 17 with a mean of 9.8. Two specific markers were identified for species with 2n = 18 chromosomes. The phenogram derived from the RAPD data corroborated the morphological classification. The bootstrap analysis divided the genotypes into two significant clusters. The first cluster contained all the section Arachis species, and the accessions within it were grouped based upon the presence or absence of the ‘A’ pair and the number of chromosomes. The second cluster grouped all accessions belonging to section Heteranthae.