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Geographical distribution and diversity of current plant species have been strongly shaped by climatic oscillations during the Quaternary. Analysing the resulting divergence among species and differentiation within species is crucial to understand the evolution of taxa like the Vitis genus, which provides very useful genetic resources for grapevine improvement and might reveal original recolonization patterns due to growth habit and dispersal mode. Here, we studied the genetic structure in natural populations of three species from eastern North America: Vitis aestivalis, V. cinerea and V. riparia using different marker types. Vitis aestivalis and V. cinerea showed higher diversity than V. riparia. The two former species are less differentiated, confirming an earlier divergence of V. riparia. V. aestivalis and V. riparia exhibited different genetic groups on both sides of the Appalachian Mountains that could mirror different recolonization routes from southern refugia. Genetic structure was stronger in V. cinerea, for which two varieties (var. berlandieri and var. cinerea) are morphologically recognized. Our results confirm this distinction and suggest the existence of three other lineages within var. cinerea. These discontinuities appear linked to adaptation of var. berlandieri to dry and limy areas of Texas and partially to the Mississippi River Valley. Rapid range expansions from refugia upon climate warming are also suggested by the low linkage disequilibrium values observed. Furthermore, large variation for downy mildew resistance was observed in the three species. Our findings appeared consistent with the vegetation history of eastern North America.

Aim : Vitis subg. Vitis provides an example of a plant disjunction occurring in the Northern Hemisphere. It shows broad morphological variation but is assumed to be a species complex with limited genetic differentiation. Based on a comprehensive sampling of taxa and polymorphism in both chloroplast and nuclear DNA, we assessed genetic variation within this subgenus. Our aims were to clarify the relationships among species and to examine their historical biogeography. Location: Asia, Europe, North America. Methods: We analysed a total of 30 species and putative hybrids from subgenus Vitis and examined the infra-specific variation in some species. Polymorphism in chloroplast DNA was assessed in trnL and trnH-psbA-trnK sequences (c. 2170 bp) and in 15 microsatellite loci. We also obtained nuclear data for size variation at 24 microsatellite loci. Phylogenetic inference was performed with Bayesian analyses. A maximum parsimony network was constructed to depict the evolutionary relationships among haplotypes, and microsatellite data were also subjected to hierarchical clustering analysis using the Ward distance. In addition, we assessed size homoplasy by sequencing both chloroplast and nuclear microsatellite loci. Results Chloroplast polymorphisms resolved subgenus Vitis as a monophyletic group with limited genetic variation. The ancestral haplotypes were found in Eurasia. American taxa all harboured derived haplotypes. Most of them formed a monophyletic group that did not include Vitis californica. The four main haplotypes in Vitis vinifera corresponded to two different origins. Nuclear microsatellites indicated that genetic variation was especially large in North America. Asian species exhibited a lower level of nuclear divergence and the European V. vinifera corresponded to a differentiated nuclear lineage. Main conclusions: We obtained some evidence that subgenus Vitis has an Asian origin and then dispersed to Europe and North America. Geographic separation was followed by diversification, presumably during the Pleistocene, resulting in phylogeographic patterns similar to other biota. In contrast to chloroplast DNA, nuclear DNA shows a larger than expected genetic variation. Our molecular data also highlight the need to re-examine certain aspects of the current subgeneric classification

All protocols for Next Generation Sequencers (NGS) start with the preparation of a DNA library from extracted DNA. Quality, number, and length of the sequences produced by NGS all depend on the quality of the library that was prepared, itself dependent on the DNA quality. Generally, when using NGS, one would wish to obtain the genomic information contained in the nucleus without an excessive proportion of cytoplasmic DNA. Multiple copies of cytoplasmic genomes are present in every cell (11 to 70 copies, depending on the developmental stage and/or physiological status) ( Tymms et al., 1983 ). The chloroplast DNA of plants, therefore, represents 17 - 23% of their total DNA ( Boffey and Leech, 1982 ). In this study, we developed a protocol that strongly reduces the amount of cytoplasmic DNA. The improvement in quality makes the method perfectly adapted to library construction for NGS. We applied this extraction method to the grapevine ( Vitis vinifera L.) and coffee tree ( Coffea canephora Pierre ex A. Froehner). These species are known to contain many secondary metabolites, which can generate diffi culties for DNA extraction ( Peterson et al., 1997 ; Mattivi et al., 2006 ). To establish the protocol, we were partially inspired by a classic protocol for nuclear DNA plant extraction destined for the construction of BAC (Bacterial Artifi cial Chromosome) libraries ( Peterson et al., 1997 ). The two stages of the protocol are: (i) isolation of nuclei and (ii) nuclear DNA extraction. Nuclear DNA obtained with this protocol was then sequenced using 454/GS-FLX technology (Roche, Basel, Switzerland) with the Titanium kit. All protocols for Next Generation Sequencers (NGS) start with the preparation of a DNA library from extracted DNA. Quality, number, and length of the sequences produced by NGS all depend on the quality of the library that was prepared, itself dependent on the DNA quality. Generally, when using NGS, one would wish to obtain the genomic information contained in the nucleus without an excessive proportion of cytoplasmic DNA. Multiple copies of cytoplasmic genomes are present in every cell (11 to 70 copies, depending on the developmental stage and/or physiological status) ( Tymms et al., 1983 ). The chloroplast DNA of plants, therefore, represents 17 - 23% of their total DNA ( Boffey and Leech, 1982 ). In this study, we developed a protocol that strongly reduces the amount of cytoplasmic DNA. The improvement in quality makes the method perfectly adapted to library construction for NGS. We applied this extraction method to the grapevine ( Vitis vinifera L.) and coffee tree ( Coffea canephora Pierre ex A. Froehner). These species are known to contain many secondary metabolites, which can generate diffi culties for DNA extraction ( Peterson et al., 1997 ; Mattivi et al., 2006 ). To establish the protocol, we were partially inspired by a classic protocol for nuclear DNA plant extraction destined for the construction of BAC (Bacterial Artifi cial Chromosome) libraries ( Peterson et al., 1997 ). The two stages of the protocol are: (i) isolation of nuclei and (ii) nuclear DNA extraction. Nuclear DNA obtained with this protocol was then sequenced using 454/GS-FLX technology (Roche, Basel, Switzerland) with the Titanium kit.