Molecular diversity, population structure, and linkage disequilibrium in a worldwide collection of tobacco (Nicotiana tabacum L.) germplasm.

BACKGROUND: The goals of our study were to assess the phylogeny and the population structure of tobacco accessions representing a wide range of genetic diversity; identify a subset of accessions as a core collection capturing most of the existing genetic diversity; and estimate, in the tobacco core collection, the extent of linkage disequilibrium (LD) in seven genomic regions using simple sequence repeat (SSR) markers. To this end, a collection of accessions were genotyped with SSR markers. Molecular diversity was evaluated and LD was analyzed across seven regions of the genome. RESULTS: A genotyping database for 312 tobacco accessions was profiled with 49 SSR markers. Principal Coordinate Analysis (PCoA) and Bayesian cluster analysis revealed structuring of the tobacco population with regard to commercial classes and six main clades were identified, which correspond to "Oriental", Flue-Cured", "Burley", "Dark", "Primitive", and "Other" classes. Pairwise kinship was calculated between accessions, and an overall low level of co-ancestry was observed. A set of 89 genotypes was identified that captured the whole genetic diversity detected at the 49 loci. LD was evaluated on these genotypes, using 422 SSR markers mapping on seven linkage groups. LD was estimated as squared correlation of allele frequencies (r2). The pattern of intrachromosomal LD revealed that in tobacco LD extended up to distances as great as 75 cM with r2 > 0.05 or up to 1 cM with r2 > 0.2. The pattern of LD was clearly dependent on the population structure. CONCLUSIONS: A global population of tobacco is highly structured. Clustering highlights the accessions with the same market class. LD in tobacco extends up to 75 cM and is strongly dependent on the population structure.

A high density genetic map of tobacco (Nicotiana tabacum L.) obtained from large scale microsatellite marker development.

Tobacco (Nicotiana tabacum L.) is a species in the large family of the Solanaceae and is important as an agronomic crop and as a model system in plant biotechnology. Despite its importance, only limited molecular marker resources are available that can be used for genome analysis, genetic mapping and breeding. We report here on the development and characterization of 5,119 new and functional microsatellite markers and on the generation of a high-resolution genetic map for the tetraploid tobacco genome. The genetic map was generated using an F2 mapping population derived from the intervarietal cross of Hicks Broadleaf × Red Russian and merges the polymorphic markers from this new set with those from a smaller set previously used to produce a lower density map. The genetic map described here contains 2,317 microsatellite markers and 2,363 loci, resulting in an average distance between mapped microsatellite markers which is less than 2 million base pairs or 1.5 cM. With this new and expanded marker resource, a sufficient number of markers are now available for multiple applications ranging from tobacco breeding to comparative genome analysis. The genetic map of tobacco is now comparable in marker density and resolution with the best characterized genomes of the Solanaceae: tomato and potato.

Haplotype analysis of vernalization loci in European barley germplasm reveals novel VRN-H1 alleles and a predominant winter VRN-H1/VRN-H2 multi-locus haplotype.

In barley, variation in the requirement for vernalization (an extended period of low temperature before flowering can occur) is determined by the VRN-H1, -H2 and -H3 loci. In European cultivated germplasm, most variation in vernalization requirement is accounted for by alleles at VRN-H1 and VRN-H2 only, but the range of allelic variation is largely unexplored. Here we characterise VRN-H1 and VRN-H2 haplotypes in 429 varieties representing a large portion of the acreage sown to barley in Western Europe over the last 60 years. Analysis of genotype, intron I sequencing data and growth habit tests identified three novel VRN-H1 alleles and determined the most frequent VRN-H1 intron I rearrangements. Combined analysis of VRN-H1 and VRN-H2 alleles resulted in the classification of seventeen VRN-H1/VRN-H2 multi-locus haplotypes, three of which account for 79% of varieties. The molecular markers employed here represent powerful diagnostic tools for prediction of growth habit and assessment of varietal purity. These markers will also allow development of germplasm to test the behaviour of individual alleles with the aim of understanding the relationship between allelic variation and adaptation to specific agri-environments.

A microsatellite marker based linkage map of tobacco.

We report the first linkage map of tobacco (Nicotiana tabacum L.) generated through microsatellite markers. The microsatellite markers were predominantly derived from genomic sequences of the Tobacco Genome Initiative (TGI) through bioinformatics screening for microsatellite motives. A total of 684 primer pairs were screened for functionality in a panel of 16 tobacco lines. Of those, 637 primer pairs were functional. Potential parents for mapping populations were evaluated for their polymorphism level through genetic similarity analysis. The similarity analysis revealed that the known groups of tobacco varieties (Burley, Flue-cured, Oriental and Dark) form distinct clusters. A mapping population, based on a cross between varieties Hicks Broad Leaf and Red Russian, and consisting of 186 F2 individuals, was selected for mapping. A total of 282 functional microsatellite markers were polymorphic in this population and 293 loci could be mapped together with the morphological trait flower color. Twenty-four tentative linkage groups spanning 1,920 cM could be identified. This map will provide the basis for the genetic mapping of traits in tobacco and for further analyses of the tobacco genome.

DNA extraction and analysis from processed coffee beans.

The authenticity of coffee is an important issue for both producers and consumers. Premium Arabica material is especially prone to being adulterated, and a number of different techniques have been employed to determine the quality of both roasted and instant coffee. Currently, assessment of coffee authenticity relies on chemical methods which can discriminate between coffee species, but not varieties. Several genetic markers are available for assessing coffee origin, but their suitability to testing commercial coffee is limited by the ability to extract DNA from highly processed beans. In this paper, we demonstrate that PCR-grade DNA may be obtained from roasted beans and even instant coffee. This would allow analysis of commercial samples, provided that suitable markers for species/variety identification are found.

Development and characterization of microsatellite markers in Cynara cardunculus L.

Cynara cardunculus L. is a species native to the Mediterranean basin that comprises 2 crops, globe artichoke (var. scolymus L.) and cultivated cardoon (var. altilis DC), as well as wild cardoon (var. sylvestris (Lamk) Fiori). Globe artichoke represents an important component of the South European agricultural economy but is also cultivated in North Africa, the Near East, South America, the United States, and China. Breeding activities and molecular marker studies have been, to date, extremely limited. Better knowledge of the genome of the species might be gained by developing a range of molecular markers. Here, we report on the development of 14 microsatellites (simple sequence repeats (SSRs)) through a novel approach that we have defined as the microsatellite amplified library (MAL). The approach represents a combination of amplified fragment length polymorphism and a primer extension based enriched library, is rapid, and requires no hybridization enrichment steps. The technique provided a approximately 40-fold increase in the efficiency of SSR identification compared with conventional library procedures. The developed SSRs were applied for genotyping 36 accessions of C. cardunculus, including a core of 27 varietal types of globe artichoke, 3 accessions of cultivated cardoon, and 6 Sicilian accessions of wild cardoon. Principal coordinates analysis made it possible to differentiate both cultivated and wild forms from each other.

Tubulin-based polymorphism (TBP): a new tool, based on functionally relevant sequences, to assess genetic diversity in plant species.

TBP (tubulin-based polymorphism) is a new molecular marker based tool that relies on the presence of intron-specific DNA polymorphisms of the plant beta-tubulin gene family. The multifunctional and essential role of the tubulin proteins is reflected in the conservation of regions within their primary amino acid sequence. The ubiquitous nature of this gene family can be exploited using primers that amplify the first intron of different beta-tubulin isotypes, revealing specific fingerprints. The method is rapid, simple, and reliable and does not require preliminary sequence information of the plant genome of interest. The ability of TBP to discriminate between accessions and species in oilseed rape, coffee, and lotus is shown. In all cases, TBP was able to detect specific genetic polymorphisms in the context of a simplified and readily appreciable pattern of DNA amplification. The application of TBP for assessing genetic diversity and genome origins in disseminated plant landraces rather than in highly inbred cultivated species is also discussed.