Genetic diversity of carrot (<Emphasis Type="Italic">Daucus carota</Emphasis> L.) cultivars revealed by analysis of SSR loci

Polymorphism of simple sequence repeat (SSR) loci was assessed in a collection of 88 carrot (Daucus carota L. subsp. sativus Hoffm.) accessions. The collection comprised cultivars and landraces mainly from Asia, Europe, and North America. Plants were grown in the glasshouse and characterized for root color and shape. Thirty SSR loci were fully characterized using parameters derived from allele frequencies, i.e. the number of total, effective and rare alleles, the observed and expected heterozygosity, and fixation index. Using a Bayesian approach, two clusters of 17 and 61 accessions were distinguished, which comprised the Asian and Western type accessions, respectively. Genetic diversity of the Asian gene pool was higher than that of the Western gene pool. The results of SSR analysis were supported by morphological characterization, and are congruent with current knowledge on the history of carrot domestication and breeding.     

AFLP analysis of genetic diversity in leafy kale (<Emphasis Type="Italic">Brassica oleracea</Emphasis> L. convar. <Emphasis Type="Italic">acephala</Emphasis> (DC.) Alef.) landraces,cultivars and wild populations in Europe

AFLP markers were used to characterize diversity and asses the genetic structure among 17 accessions of kale landraces, cultivars and wild populations from Europe. The range of average gene diversity in accessions was 0.11–0.27. Several landraces showed higher levels of diversity than the wild populations and one cultivar had the lowest diversity measures. The landraces that were most genetically diverse were from areas where kales are known to be extensively grown, suggesting in situ conservation in these areas as a supplement to storage of seeds in gene banks. An analysis of molecular variance (AMOVA) showed that 62% of the total variation was found within accessions. For most accessions, genetic distance was not related to geographic distance. Similarities among accessions were probably not caused by recent gene flow since they were widely separated geographically; more likely the relationship among them is due to seed dispersal through human interactions. Our results indicate that a kale population found in a natural habitat in Denmark was probably not truly wild but most likely an escape from a cultivated Danish kale that had subsequently become naturalized.     

Population diversity of the Vicia sativa agg. (Fabaceae) in the flora of the former USSR deduced from RAPD and seed protein analyses

The genetic diversity of 58 wild and weedy populations representing taxa within the V. sativa aggregate from the former USSR, 4 cultivars of V. sativa, 2 accessions of V. cordata and 3 accessions of V. macrocarpa from Mediterranean countries were analysed using randomly amplified DNA fragments (RAPDs) and seed protein electrophoresis (SDS-PAGE). Interspecific variation between taxa in the V. sativa aggregate could readily be detected using both techniques. RAPDs and seed protein patterns were found to be an effective means of identifying accessions that cannot be identified clearly by morphological criteria alone. RAPD and seed protein analysis revealed a clear relationship between observed genetic variation of populations and their geographical distribution. Populations from each region had their own gene pools. Geographical variation was detected in V. segetalis. The degree of genetic divergence between local populations was usually related to proximity. In several locations where wild and weedy populations of different V. sativa agg. taxa grow sympatrically, intermediate forms could be detected at the DNA and protein levels. Both RAPD and seed protein analysis support the view that the V. sativa aggregate consists of 8 taxa warranting recognition at the species level. Several species in this aggregate are evolving intra-specific groups which can readily be detected at the molecular level.     

Dual origin of the cultivated rice based on molecular markers of newly collected annual and perennial strains of wild rice species, Oryza nivara and O. rufipogon

The direct ancestor of rice (Oryza sativa L.) is believed to be AA genome wild relatives of rice in Asia. However, the AA genome wild relatives involve both annual and perennial forms. The distribution of the retrotransposon p-SINE1-r2, a short interspersed nuclear element (SINE) at the waxy locus was analyzed in diverse accessions of the AA genome wild relatives of rice (O. rufipogon sensu lato). Most annual wild rice accessions had this retrotransposon, while most perennial types lacked this element, contradicting results to the previous studies. Results presented here suggest that O. sativa has dual origin that lead to indica-japonica differentiation. Results suggest the indica line of rice varieties evolved from the annual genepool of AA genome and the japonica varieties from the perennial genepool of AA genome wild rice.     

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.     

Presence of phylogeographic structure among wild diploid alfalfa accessions (Medicago sativa L. subsp. microcarpa Urb.) with evidence of the center of origin

The genetic structure of wild germplasm on a macrogeographic scale has implications for collection and conservation of wild genetic resources of economically important plants. Cultivated alfalfa is derived from a taxonomic group called the Medicago sativa–falcata complex, which includes a number of morphologically and genetically differentiated diploid and tetraploid subspecies representing the primary gene pool for alfalfa improvement. Although the origin of the taxa included in M. sativa–falcata complex has been described broadly, the center of diversity for each taxon is not clear. In this study, 60 individual genotypes from 16 accessions of the diploid taxon M. sativa subsp. microcarpa Urb. [M. sativa subsp. caerulea (Less. ex Ledeb.) Schmalh.] were obtained from the two proposed centers of diversity, Caucasia and Central Asia, and were genotyped with 89 simple sequence repeat markers. Phylogenetic reconstructions together with population genetics statistics (heterozygosity, allelic diversity, and F_ST) were used to describe the pattern of present genetic diversity, to deduce biogeographic history, and to infer the center of diversity. Our results revealed that the Central Asian accessions were distinct from the Caucasian accessions, and that overall accessions, genetic distance was positively correlated with geographical distance, indicating a spatial genetic structure. The accessions from the Caucasian region had higher mean F_ST values, higher allele diversity and higher heterozygosity, suggesting that the Caucasian region is the likely center of diversity for M. sativa subsp. microcarpa [M. sativa subsp. caerulea] and the accessions recolonized Central Asia from Caucasia after the glacial retreat.     

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.     

Genetic identity and relationships of Iranian apple (Malus?×?domestica Borkh.) cultivars and landraces, wild Malus species and representative old apple cultivars based on simple sequence repeat (SSR) marker analysis

In order to shed light on the role of Iran in apple evolution and domestication, we chose to investigate the relationships of a collection of 159 accessions of wild and domesticated apples including Iranian indigenous apple cultivars and landraces, selected wild species, and old apple scion and rootstock cultivars from different parts of the world. The majority of the wild species belonged to M. sieversii, which is widely believed to be the main maternal wild ancestor of domestic apples, from Kazakhstan and M. orientalis, which is one of the probable minor ancestors of domestic apples, from Turkey and Russia located on the east and west of Iran, respectively. The accessions were assigned into six arbitrary populations for the purpose of generating information on genetic parameters. Nine simple sequence repeat (SSR) loci selected from previous studies in apple were screened over DNA extracted from all the accessions. Results showed that all SSR loci displayed a very high degree of polymorphism with 11–25 alleles per locus. In total, there were 153 alleles across all loci with an average of 17 alleles per locus. The SSR allelic data were then used for estimation of population genetic parameters, including genetic variation statistics, F-statistics, gene flow, genetic identity, genetic distance and then cluster analysis using POPGENE 1.32 software. The F-statistics and gene flow in particular, showed that there was more intra-population than between population variation. The genetic identity and genetic distance estimates, and the dendrogram generated from the un-weighted pair group arithmetic average (UPGMA) method of cluster analysis showed that the Iranian cultivars and landraces were more closely related to M. sieversii from Central Asia (east of Iran) and M. orientalis native to Turkey and Russia than to other accessions of Malus species. Also, the old apple cultivars from different parts of the world have a closer genetic relationship to M. sieversii, M. orientalis and the Iranian apples, than to other wild species. Based on these results, we suggest that the Iranian apples may occupy an intermediate position between the domesticated varieties and wild species. We propose that Iran could be one of the major players in apples’ domestication and transfer from Central Asia to the western countries.     

Variation in primitive landraces of common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) from Argentina

The phenotypic variation found in four common bean (Phaseolus vulgaris L.) complex primitive landraces, among a group of accessions collected in Northwestern Argentina in several missions is described, with particular attention to the wide diversity found in some small areas. It is presented a hypothesis about the maintenance of such diversity in bean mixtures or complex primitive landraces that grow close to their wild relative. Wide diversity regarding to seed type and plant characteristics was displayed by the landraces MCM-SV (composed of 11 lines), MCM-292 (14 lines), MCM-298 (5 lines) and VAV-3716 (14 lines). Food uses of dry seed and fresh pod seemed to be more relevant than the aesthetic use although all of them were presumably considered by humans for centuries resulting in the current phenotypes of these complex primitive landraces. Additionally, some weedy types (intermediate between wild and domesticated types) were detected in the landracesMCM-292 and MCM-298. The four complex landraces described consisted of highly diverse mixtures and they could play a role in breeding to enlarge the genetic basis of domesticated bean varieties belonging to the Andean gene pool.     

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.     

Results of molecular analysis of an archaeological hemp (<Emphasis Type="Italic">Cannabis sativa</Emphasis> L.) DNA sample from North West China

Hemp (Cannabis sativa L.) cultivation and utilization is an ancient practice to human civilization. There are some controversies on the origin and subsequent spread of this species. Ancient plant DNA has proven to be a powerful tool to solve phylogenetic problems. In this study, ancient DNA was extracted from an archaeological specimen of Cannabis sativa associated with archaeological human remains from China. Ribosomal and Cannabis specific chloroplast DNA regions were PCR amplified. Sequencing of a species-specific region and subsequent comparison with published sequences were performed. Successful amplification, sequencing and sequence comparison with published data suggested the presence of hemp specific DNA in the archeological specimen. The role of Humulus japonicus Sieb. et Zucc. in the evolution of Cannabis is also indicated. The identification of ancient DNA of 2500 years old C. sativa sample showed that C. sativa races might have been introduced into China from the European–Siberian center of diversity.     

Patterns of diversity in Cannabis

Summary A Cannabis germplasm collection was evaluated for traits relevant to the use of hemp as an arable source of paper pulp. The traits were related to stem yield and quality, psychoactive potency, resistance to root-knot nematodes and plant morphology. The present study is directed at diversity for these traits considered together. For the interpretation of diversity patterns, accessions were grouped on the basis of the presumed purpose and status of domestication resulting in the recognition of four plant-use groups: fibre cultivars, fibre landraces, drug strains, and truly wild or naturalized populations. Principal component analysis showed that stem quality, phenological development, psychoactive potency and host reactions to root-knot nematodes explained most of the total variation among accessions. The plant-use groups were reasonably well separated in the principal component space. Discriminant analysis showed that the a priori defined plant-use groups could be discriminated quite well on the basis of linear combinations of all agronomic and morphological traits. The contents of bark fibre and cannabinoids had the highest grouping value. Reallocations concerned mainly old fibre cultivars which were placed in the group of fibre landraces due to a low content of bark fibre.A genetic characterization of accessions was based on electrophoretic patterns of seed proteins. Isoelectric ocusing patterns of bulk seed extracts showed variation for the presence of six out of 45 protein bands in the H range from 4.75 to 6.40. Banding patterns were however independent from any grouping based on origin or gronomic and morphological traits and did not reflect expected common ancestry of accessions.     

The fragrance (fgr) gene in natural populations of wild rice (Oryza rufipogon Griff.)

The Asian cultivated rice, Oryza sativa L. (spp. indica or japonica), is assumed to have originated from one or both of the two wild Asian species, O. rufipogon Griff. and O. nivara Sharma and Shastry. They occur throughout the monsoon Asia and west Oceania. Fragrance is the most important trait among the domesticated characters of basmati and jasmine rice of Asia. The gene for fragrance in a scented rice shows the presence of a mutated portion (i.e., an eight base pair deletion in exon 7) that result in its loss of fragrance. In the present study, 229 wild rice O. rufipogon accessions were genotyped for this locus using a PCR assay. The wild rice species contained the mutated allele of the fgr gene at a low frequencies of 0.23. The surveyed populations were in Hardy–Weinberg equilibrium. This observation supports the hypothesis that the allele for fragrance was already present in the wild rice, and that this trait appeared in scented rice cultivars because of selection by the farmers of genotypes possessing this character during the process of domestication.     

Phylogenetic relationships among cultivated types of <Emphasis Type="Italic">Brassica rapa</Emphasis> L. em. Metzg. as revealed by AFLP analysis

The cultivated types of Brassica rapa L. em. Metzg. consist of morphologically distinct subspecies such as turnip, turnip rape, Chinese cabbage, pak choi and pot herb mustard which are classified as ssp. rapa, ssp. oleifera, ssp. pekinensis, ssp. chinensis and ssp. nipposinica (syn. ssp. japonica), respectively. We attempted to elucidate the phylogenetic relationships among the cultivated types of B. rapa. Thirty-two accessions from the Eurasian Continent were analyzed using AFLP markers with a cultivar of B. oleracea as an outgroup. In total, 455 bands were detected in the ingroup and 392 (86.6%) were polymorphic. The Neighbor-Joining tree based on the AFLP markers indicated that the accessions of B. rapa were congregated into two groups according to geographic origin. One group consisted of ssp. rapa and ssp. oleifera of Europe and Central Asia and the other included all the subspecies of East Asia. Our results suggest that cultivars from East Asia were probably derived from a primitive cultivated type, which originated in Europe or in Central Asia and migrated to East Asia. This primitive cultivated type was probably a common ancestor of ssp. rapa and ssp. oleifera. The Neighbor-Joining tree also shows that leafy vegetables in East Asia such as ssp. pekinensis, ssp. chinensis and ssp. nipposinica were differentiated several times from the distinct cultivars of ssp. oleifera in East Asia.     

Geographical variation of nuclear genome RFLPs and genetic differentiation in foxtail millet, Setaria italica (L.) P. Beauv.

Sixteen RFLP loci in 62 landraces were assayed to study geneticdifferentiation in foxtail millet, Setariaitalica (L.) P. Beauv. Among 52 bands, 47 werepolymorphic among foxtail millet landraces. A dendrogram constructedbased on RFLPs was divided into five major clusters (clusterI–V). Clusters I and II contained strains mainly from EastAsia. Cluster III consisted of strains from subtropical and tropicalregions in Asia such as Nansei Islands of Japan, Taiwan and thePhilippines and India and cluster IV consisted of some strains fromEast Asia, a strain from Nepal and a strain from Myanmar. Cluster Vcontained strains from central and western regions of Eurasia such asAfghanstan, Central Asia and Europe. Chinese landraces wereclassified into four clusters. These results indicate that foxtailmillet landraces have differentiated genetically between differentregions and that Chinese landraces were highly variable.     

Genetic Relationship and Diversity of Four MangiferaSpecies Revealed through AFLP Analysis

We used AFLP analysis to explore the genetic relationship and diversity between and within 4 Mangifera species. We analyzed 35 accessions comprising 8 cultivars and 3 landraces of M. indica L., 11 landraces of M. odorata Griff., 7 landraces of M. foetida Lour., and 6 landraces of M. caesia Jack. Using 8 primer combinations produced a total of 518 bands, 499 (96.3%) of which were polymorphic among the 35 accessions. Clustering analysis showed that all 35 accessions were basically classified into 4 groups corresponding to the 4 Mangifera species. Our results indicate that the genetic relationship of these 4 Mangifera species based on AFLP analysis is in good agreement with their classification by classic methods. In addition, it was clearly revealed the genetic diversity between and within 4 Mangifera species. The findings obtained in this study are useful for the breeding in Mangifera species.     

Relationships Among <Emphasis Type="Italic">Brassica napus</Emphasis> (L.) Germplasm from Spain and Great Britain as Determined by RAPD Markers

The genetic diversity and the relationships among a collection of Brassica napus L. European populations were evaluated using random amplified polymorphic DNA markers. The study included 33 accessions of B. napus collected from Galicia (northwestern Spain) and 18 British cultivars, 16 accessions of B. napus and two accessions of Brassica oleracea L. used as controls. DNA from 25 individuals per population was analyzed using 18 decamer primers. One hundred thirty-eight amplification products were scored of which 105 were polymorphic. These bands ranged in size from 350 to 2500 base pairs. Similarity coefficients and cluster analysis were computed and six groups were obtained. Cluster I was the largest and included all the landraces from northwestern Spain, except two accessions that grouped separately into Clusters III and IV, respectively. A low level of genetic variability was detected among the B. napus Spanish genotypes, while considerable diversity was present among the British ones, which grouped into three groups, two main clusters and one group formed by one accession. Cluster II included all commercial varieties grown in Great Britain whereas Cluster V grouped local varieties maintained by the growers for many years. Cluster VI was a singularity formed by one entry. British accessions of B. oleracea had the greatest dissimilarity with all the other populations and grouped separately in Clusters VII and VIII. As conclusion, B. napus landraces used in northwestern Spain as leafy-green vegetable probably have an independent origin from B. napus crops grown in other European regions. Besides, separate domestication in northwestern Spain and Great Britain for a different end use might have led to two distinct gene pools.     

Characterization of Some Italian Common Bean (<Emphasis Type="Italic">Phaseolus Vulgaris</Emphasis> L.) Landraces by RAPD,Semi-random and ISSR Molecular Markers

Randomly amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and a semi-random PCR system were used to analyze the genetic diversity of 16 Italian common bean landraces and their relationship to four commercial cultivars. Of the primers tested, 8 ISSR, 6 RAPD and 7 semi-random primers produced polymorphic and reproducible DNA fragments. A higher proportion of polymorphic bands were observed using ISSR (85%) and semi-random (90%) primers than RAPD (69%) method. The combination of any two semi-random markers allowed the identification of all 20 bean genotypes. In contrast ISSR (except for primer (CAC)₃GC) and RAPD markers appeared to be less informative as more than two markers were necessary to achieve the same diagnostic level. Moreover, 7 ISSR, 2 RAPD and 8 semi-random exclusive bands were identified as putative population-specific markers. Semi-random and ISSR derived dendrograms showed similar tendencies in terms of genetic relatedness, whereas clustering of genotypes within groups was not similar when compared with the RAPD technique. Despite the different ability to resolve genetic variation among the investigated landraces, two major clusters with less than 60% (ISSR) and 40% (RAPD and semi-random) genetic similarity were formed with all three marker systems. The two groups were correlated with the phaseolin patterns and seed size of the landraces. The analysis showed that the cultivar ȁ8Lingua di Fuocoȁ9 and most of the landraces (13 out of 16) collected in Italy belong to the Andean gene pool, whereas only the three populations from Pratomagno belong to the Middle American gene pool.