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.     

Exploitation of <Emphasis Type="Italic">Malus</Emphasis> EST-SSRs and the utility in evaluation of genetic diversity in <Emphasis Type="Italic">Malus</Emphasis> and <Emphasis Type="Italic">Pyrus</Emphasis>

A total of 8117 suitable SSR-contaning ESTs were acquired by screening from a Malus EST database, among which dinudeotide SSRs were the most abundant repeat motif, within which, CT/TC followed by AG/GA were predominant. Based on the suitable sequences, we developed 147 SSR primer pairs, of which 94 pairs gave amplifications within the expected size range while 65 pairs were found to be polymorphic after a preliminary test. Eighteen primer pairs selected randomly were further used to assess genetic relationship among 20 Malus species or cultivars. As a result, these primers displayed high level of polymorphism with a mean of 6.94 alleles per locus and UPGMA cluster analysis grouped twenty Malus accessions into five groups at the similarity level of 0.6800 that were largely congruent to the traditional taxonomy. Subsequently, all of the 94 primer pairs were tested on four accessions of Pyrus to evaluate the transferability of the markers, and 40 of 72 functional SSRs produced polymorphic amplicons from which 8 SSR loci selected randomly were employed to analyze genetic diversity and relationship among a collection of Pyrus. The 8 primer pairs produced expected bands with the similar size in apples with an average of 7.375 alleles per locus. The observed heterozygosity of different loci ranged from 0.29 (MES₉₆) to 0.83 (MES₁₃₈), with a mean of 0.55 which is lower than 0.63 reported in genome-derived SSR marker analysis in Pyrus. The UPGMA dendrogram was similar to the previous results obtained by using RAPD and AFLP markers. Our results showed that these EST-SSR markers displayed reliable amplification and considerable polymorphism in both Malus and Pyrus, and will contribute to the knowledge of genetic study of Malus and genetically closed genera.     

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.     

Analysis of genetic variability in <Emphasis Type="Italic">Couepia</Emphasis> accessions using AFLP markers

Couepia is a genus distributed in tropical regions of America. The nut of some Couepia species is used as fresh fruit and oil source for local communities. Despite its critical situation of conservation and its economic potential, there is a lack of information on the genetic variability of Couepia species. This study examines AFLP variation among 40 accessions of Couepia collected in the Colombian Amazonian region, representing two species: Couepia dolichopoda and Couepia subcordata. The individuals were examined for 96 markers generated from four EcoRI/MseI primer pairs, with 80% polymorphism across all accessions studied. According to cluster analysis, 40 accessions were grouped into two major clusters, corresponding to the two species analyzed, except one case whose situation is discussed. In C. dolichopoda accessions, significant correlation between the clustering pattern and the geographical origin was detected; the extent of variation within and among its collect sites was examined by AMOVA. The knowledge about the genetic variability of the accessions examined contributes to development of Couepia conservational efforts.     

Genetic diversity of the D-genome in <Emphasis Type="Italic">T. aestivum</Emphasis> and <Emphasis Type="Italic">Aegilops</Emphasis> species using SSR markers

Simple sequence repeats (SSRs), highly dispersed nucleotide sequences in genomes, were used for germplasm analysis and estimation of the genetic relationship of the D-genome among 52 accessions of T. aestivum (AABBDD), Ae. tauschii (D^tD^t), Ae. cylindrica (CCD^cD^c) and Ae. crassa (MMD^cr1D^cr1), collected from 13 different sites in Iran. A set of 21 microsatellite primers, from various locations on the seven D-genome chromosomes, revealed a high level of polymorphism. A total of 273 alleles were detected across all four species and the number of alleles per each microsatellite marker varied from 3 to 27. The highest genetic diversity occurred in Ae. tauschii followed by Ae. crassa, and the genetic distance was the smallest between Ae. tauschii and Ae. cylindrica. Data obtained in this study supports the view that genetic variability in the D-genome of hexaploid wheat is less than in Ae. tauschii. The highest number of unique alleles was observed within Ae. crassa accessions, indicating this species as a great potential source of novel genes for bread wheat improvement. Knowledge of genetic diversity in Aegilops species provides different levels of information which is important in the management of germplasm resources.     

On evolutionary pathways of <Emphasis Type="Italic">Avena</Emphasis> species

This article presents literary review and results analysis of evaluation of representative set of oat accessions of all Avena L. species. Results of complex study of major morphological characters and utilization of the karyotype structure data confirmed by the results of RAPD and avenin spectrum analysis are presented. Relationships of genomes of different Avena species at each ploidy level are discussed. Two genomes form the base of all Avena species, namely the A and C genomes. Results of the evaluation of several characteristics of the oat species and their geographical distribution are analysed. Probable evolutionary pathway of Avena species are suggested. Most likely the centres of origin of genus Avena L. are determined.     

Genetic diversity among <Emphasis Type="Italic">Jatropha</Emphasis> species as revealed by RAPD markers

The genus Jatropha is native of tropical America with more than 200 species that are widely distributed in tropics with a promise for use as an oil crop for biodiesel. This investigation was carried out to assess the genetic diversity of 12 Jatropha species based on random amplified polymorphic DNA markers. From 26 random primers used, 18 primers gave reproducible amplification banding patterns of 112 polymorphic bands out of 134 bands scored accounting for 80.2% polymorphism across the genotypes. Three primers viz., OPA 4, OPF 11, and OPD 14 generated 100% polymorphic patterns. The polymorphic information content was highest for the primer OPD 14 (0.50) followed by the primers OPF 11 and OPAD 11 (0.48). Jaccard’s coefficient of similarity varied from 0.00 to 0.85, indicative of high level of genetic variation among the genotypes studied. UPGMA cluster analysis indicated three distinct clusters, one comprising all accessions of J. curcas L., while second included six species viz., J. ramanadensis Ramam., J. gossypiifolia L., J. podagrica Hook., J. tanjorensis J. L. Ellis et Saroja J. villosa Wight and J. integerrima Jacq. J. glandulifera Roxb. remained distinct and formed third cluster indicating its higher genetic distinctness from other species. The overall grouping pattern of clustering corresponds well with principal component analysis confirming patterns of genetic diversity observed among the species. The result provides valid guidelines for collection, conservation and characterization of Jatropha genetic resources.     

The Genetic Diversity and Similarities Among <Emphasis Type="Italic">Kengyilia</Emphasis> Species Based on Random Amplified Microsatellite Polymorphism (RAMP)

The genetic diversity and similarities among 32 Kengyilia accessions, distributed to 14 species and one variety were analyzed by using random amplified microsatellite polymorphism (RAMP) markers. Of the 160 RAMP primer combinations tested, 40 (25%) produced polymorphic and clear bands. A total of 264 bands were produced by 40 primer combinations, among which 231 out of 264 bands (87.5%) were polymorphic. Two to 11 polymorphic bands could be amplified from each primer combination, with an average of 5.8 bands. The data of 264 bands were used for RAMP assay. By NTSYS-pc program, genetic similarity coefficients were generated and dendrogram was constructed using UPGMA. The genetic similarity coefficients ranged from 0.477 to 0.965 with the mean of 0.714. The results showed as follows: (1) distinct genetic differences were present among the different species; (2) the different accessions in a species were clustered together, respectively, which had larger genetic similarities and closer relations; (3) the species with similar morphological characters and the species from the same areas or neighboring geographical regions were clustered together; (4) the lowest genetic similarity was found between K. hirsuta (PI531618) and K. laxiflora (PI531631), while the highest genetic similarity was observed between K. hirsuta (Y2364) and K. hirsuta (Y2368); (5) RAMP results are basically comparable with those obtained from studies on morphology and cytology. It is a useful method for analysis of the genetic diversity and similarities in Kengyilia.     

Determining genetic diversity between lines of <Emphasis Type="Italic">Vigna unguiculata</Emphasis> subspecies by AFLP and SSR markers

AFLP (Amplified Fragment Length Polymorphisms) and SSR (Simple Sequence Repeat) markers were utilized to assess genetic diversity and relatedness between Vigna unguiculata subspecies. Three AFLP primer combinations and 10 SSR primer sets successfully identified closely related accessions, and the presence of heterogeneity in some accessions. AFLP methodology was successful in separating different species of Vigna. However, the level of intra-subspecies variation was as great as was the interspecies variation with both marker methods. The number of markers employed was insufficient to successfully group the subspecies into distinct clades.     

DNA markers and pollen morphology reveal that <Emphasis Type="Italic">Praecitrullus fistulosus</Emphasis> is more closely related to <Emphasis Type="Italic">Benincasa hispida</Emphasis> than to <Emphasis Type="Italic">Citrullus</Emphasis> spp.

The round melon Praecitrullus fistulosus (Stocks) Pangalo has been cultivated in Asia since ancient times and has been considered an underexploited crop in the western world. In the USA, there is an increased interest in using P. fistulosus as a commercial vegetable, and possibly as a rootstock for grafting watermelon, melon, or cucumber. However, the taxonomic classification of P. fistulosus is incomplete and for many years it has been considered a close relative of watermelon [Citrullus lanatus subsp. vulgaris (Schrad. ex Eckl. et Zeyh.) Fursa] and was previously classified as Citrullus lanatus subsp. fistulosus (Stocks) Duthie et J.B. Fuller. Here, we used two sets of DNA markers to assess the genetic similarity of P. fistulosus in relation to Citrullus spp. {including Citrullus lanatus subsp. vulgaris, C. lanatus subsp. lanatus, Citroides group [also known as C. lanatus (Thunb.) Matsum. et Nakai subsp. lanatus var. citroides (Bailey) Mansf. ex Greb.], and C. colocynthis (L.) Schrad.}, Cucumis spp. (including C. melo, C. sativus, C. anguria, C. meeusei, C. zeyheri), Benincasa hispida (Thunb.) Cogn., Lagenaria siceraria (Mol.) Standl. and Cucurbita spp. (including C. moschata Duchesne and the winter squash C. maxima Duchesne). The first marker set comprised 501 markers that were produced by 38 primer pairs derived from watermelon expressed sequenced tags (ESTs) containing simple sequence repeat (SSR) motifs (designated as EST-SSR primers; produced 311 markers), and by 18 primer pairs derived from ESTs that do not contain SSR motives (designated here as EST-PCR primers; produced 190 markers). The second marker set comprised 628 markers that were produced by 18 sequence related amplified polymorphism (SRAP) primer pairs. The phylogenetic data indicated that among these cucurbit species, the wax gourd B. hispida is the closest to the P. fistulosus. Pollen observations, using light microscopy, indicated that each of the cucurbit genera examined here has unique pollen morphology. The Cucurbita spp. have globular pollen grains with a stigmatic surface. The L. siceraria has polygonal pollen grains with symmetrical boundaries, while the Citrullus spp. and Cucumis spp. have ovular (conical) and triangular shaped pollen grains (respectively). The B. hispida and P. fistulosus have spherical or semispherical pollen grains. These pollen features appear to be in agreement with the phylogenetic relationships of these two species based on DNA markers. Analysis with 12 SRAP primer pairs revealed low genetic diversity among 18 United States Plant Introductions (PIs) of P. fistulosus, indicating the need to expand the germplasm collection of this cucurbit crop.     

Genetic diversity in oil and vegetable mustard (<Emphasis Type="Italic">Brassica</Emphasis><Emphasis Type="Italic">juncea</Emphasis>) landraces revealed by SRAP markers

Mustard (Brassica juncea) is an important crop in both ancient and modern world. It has a broad resource of genetic diversity that is used primarily as oilseed but as vegetables, condiment and medicines also. Its superior tolerance to adverse environments, e.g., drought, high temperature and low fertility suggests its better adaptability in future possible harsh environments. Chinese vegetable mustard displays a wide spectrum of morphotypes. A collection of 95 accessions of B. juncea representing oil and vegetable mustards from China, France, India, Pakistan, and Japan were assessed to determine diversity at the molecular level using sequence-related amplified polymorphism (SRAP). Eight SRAP primer combinations identified a total of 326 scorable fragments of which 161 were polymorphic (49.39%). The percentage of polymorphism for each primer combination varied from 21.88 to 66.67%. Both Shannon-Weaver and Simpson genetic diversity index indicated that the level of genetic diversity within vegetable mustard is much higher than within oil mustard, and also winter oil mustards are genetically more diverse than spring oil mustards. Based on the Cluster and Principal Coordinates analysis, which were conducted on the similarity matrix of SRAP marker data, vegetable, spring oil and winter oil mustard were clearly divided into three distinct groups and among these three groups, spring and winter oil mustard are geneticlly closer than vegetable mustard. This suggests that bilateral gene exchange between oil and vegetable gene pools in the breeding program will effectively elevate the genetic potential in developing higher yields, more disease resistance, better quality and better adapted lines.     

<Emphasis Type="Italic">In situ</Emphasis> conservation and genetic diversity of three populations of <Emphasis Type="Italic">Gossypium mustelinum</Emphasis> Miers ex Watt

Gossypium mustelinum Miers ex Watt is a native from Northeastern Brazil and belongs to the primary gene pool of the cultivated cotton. The unique places where the species was known to occur were visited to plan ex situ and in situ preservation. Two populations at Caicó became extinct, and only eleven individuals were found. At Jaguarari, although one population became extinct, a new one was localized with approximately 500 adult plants, where a fence avoided cattle feeding. The population from Macururé consisted of 28 plants protected from animals by thorns of Bromelia laciniosa Mart. ex Schult. f. Thirteen SSR primer pairs amplified 40 alleles, among which 30% were exclusive of one of the populations. The genetic differences between populations represented 58.3% of the total genetic variation observed. The high genetic distances are likely to be caused by geographical isolation as well as by the small number of individuals which contribute to new generations, and consequential genetic drift.     

Development and utilization of diagnostic DAMD-PCR markers for <Emphasis Type="Italic">Capsicum</Emphasis> accessions

A polymerase chain reaction (PCR) based approach involving the directed amplification of minisatellite DNA region (DAMD-PCR) was used to identify accession specific DNA markers and study genetic relationships between and within 15 accessions corresponding to 11 species in genus Capsicum. A touch down PCR profile and unique chemical concentration of ingredients resulted in reproducible and reliable DNA amplifications. The number of amplified products varied from 1 to 12 fragments depending on the template DNA and the primers. The DAMD-PCR technique provided a total of 38 accession specific DNA markers (diagnostic DAMD-PCR) which can be utilized in accession identification, preservation and genetic studies of Capsicum germplasm. Based on 1,292 polymorphic and monomorphic DNA markers directed with 22 minisatellite specific primers, accessions were divided into four major groups, three of which corresponded to the three distinct Capsicum complexes. Capsicum chacoense was found to be the most distinct species.     

Remarks on genetic diversity and relationship of <Emphasis Type="Italic">Punica protopunica</Emphasis> and <Emphasis Type="Italic">P. granatum</Emphasis> assessed by molecular analyses

Here, two Punica species, viz., P. protopunica Balf. fil., reported as native to Socotra, and P. granatum L., were compared for the first time. Analysis of one P. protopunica and eleven P. granatum accessions was performed using three molecular markers, i.e., sequence related amplified polymorphism (SRAP), target region amplification polymorphism (TRAP), and intron targeted amplified polymorphism (ITAP), along with analysis of pgWD40 sequences, a gene involved in anthocyanin biosynthesis. All markers revealed the relationship between the two species and placed them at 33% similarity. SRAP, TRAP, and ITAP generated a total of 299, 260, and 160 bands, respectively. Of these, 78, 74, and 41 bands were specific for P. protopunica, and 92, 85, and 57 bands, respectively, were shared between both species. Sequence analysis of pgWD40~870 bp amplicons exhibited 100% identity among P. granatum accessions and 98% identity to that of P. protopunica. Phylogenetic analysis of WD40 sequences from monocot and dicot species, including both Punica species confirmed the relation between P. protopunica and P. granatum, supporting earlier reports that P. protopunica could be an ancestral species of P. granatum. Furthermore, the genetic diversity among and within P. granatum accessions from Egypt (3), Mexico (5), and Yemen (3) was assessed. Molecular marker-based relationships among region-bulked accessions was approximately the same (~90% similarity), whereas the degree of genetic variation was altered within each region. Specific bands (alleles) for accessions of each region along with those shared among them were identified. Thus, these bands could be used for pomegranate genotyping and breeding programs.     

Genetic and bioclimatic variation in <Emphasis Type="Italic">Solanum pimpinellifolium</Emphasis>

Solanum pimpinellifolium, due to its close relationship to S. lycopersicum, has been a genetic source for many commercially important tomato traits. It is a wild species found in the coastal areas of Peru and Ecuador. In this study, the genetic variation of S. pimpinellifolium was studied using the diversity found in 10 microsatellites in 248 plants spread throughout its entire distribution area, including Ecuador, which has been underrepresented in previous studies. Peruvian and Ecuadorian accessions are genetically quite differentiated. A possible cause of these differences could be the non-uniform nature of the coastal Ecuadorian and Peruvian climates, seeing as an important correlation between genetic differentiation and climate has been found. In addition, Ecuadorian and south Peruvian accessions have a lower genetic diversity and a higher homozygosity due to their higher autogamy, lower population size, and possible colonization bottlenecks. The Galápagos Islands population is an extreme case, with no diversity, likely caused by a recent colonization from the northern continental Ecuadorian region where genetically identical plants have been found. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Elena Zuriaga and José M. Blanca contributed equally to this work.     

AFLP-based genetic diversity assessment among Chinese vegetable mustards (<Emphasis Type="Italic">Brassica juncea</Emphasis> (L.) Czern.)

Amplified fragment length polymorphism (AFLP) analysis was used to evaluate the genetic relationships and diversities of Chinese vegetable mustards. Fourteen pairs of primers generated a total of 366 scorable fragments among 16 accessions of Brassica juncea studied, of which 296 bands were polymorphic with an average of 21.1% polymorphic bands per primer combination. Genetic similarities were obtained using Nei and Li similarity coefficients, and a dendrogram of the 16 accessions was made by UPGMA clustering method. The Nei and Li Similarity coefficient value ranged from 0.63 to 0.88. This result indicated that the 16 accessions of B. juncea possessed high level genetic variations. The cluster analysis showed that the vegetable mustards could be grouped into two main groups and some minor rami, which was partially in accordance with the traditional classification that based on different edible organs of vegetable mustards. The incongruity between morphological and molecular classification might be attributed to the high selection pressure during domestication of Chinese vegetable mustards, producing some accessions with similar genetic backgrounds evolving into abundant morphological variations. The great diversification among Chinese vegetable mustards not only provides an excellent object for molecular evolution research of B. juncea but also is of great value for widening the genetic basis of breeding programs and breeding materials selection. Besides, our study also indicates that AFLP are informative and can provide significant insights for genetic diversity research in B. juncea.