222 cultivated (Vitis vinifera) and 22 wild (V. vinifera ssp. sylvestris) grape accessions were analysed for genetic diversity and differentiation at eight microsatellite loci. A total of 94 alleles were detected, with extensive polymorphism among the accessions. Multivariate relationships among accessions revealed 16 genetic groups structured into three clusters, supporting the classical eco-geographic grouping of grape cultivars: occidentalis, pontica and orientalis. French cultivars appeared to be distinct and showed close affinity to the wild progenitor, ssp. sylvestris from south-western France (Pyrenees) and Tunisia, probably reflecting the origin and domestication history of many of the old wine cultivars from France. There was appreciable level of differentiation between table and wine grape cultivars, and the Muscat types were somewhat distinct within the wine grapes. Contingency chi2 analysis indicated significant heterogeneity in allele frequencies among groups at all loci. The observed heterozygosities for different groups ranged from 0.625 to 0.9 with an overall average of 0.771. Genetic relationships among groups suggested hierarchical differentiation within cultivated grape. The gene diversity analysis indicated narrow divergence among groups and that most variation was found within groups (approximately 85%). Partitioning of diversity suggested that the remaining variation is somewhat structured hierarchically at different levels of differentiation. The overall organization of genetic diversity suggests that the germplasm of cultivated grape represents a single complex gene pool and that its structure is determined by strong artificial selection and a vegetative mode of reproduction.

BackgroundGrapes are one of the most economically important fruit crops. There are about 60 species in the genus Vitis. The phylogenetic relationships among these species are of keen interest for the conservation and use of this germplasm. We selected 309 accessions from 48 Vitis species,varieties, and outgroups, examined ~11 kb (~3.4 Mb total) of aligned nuclear DNA sequences from 27 unlinked genes in a phylogenetic context, and estimated divergence times based on fossil calibrations.ResultsVitis formed a strongly supported clade. There was substantial support for species and less for the higher-level groupings (series). As estimated from extant taxa, the crown age of Vitis was 28 Ma and the divergence of subgenera (Vitis and Muscadinia) occurred at ~18 Ma. Higher clades in subgenus Vitis diverged 16 – 5 Ma with overlapping confidence intervals, and ongoing divergence formed extant species at 12 – 1.3 Ma. Several species had species-specific SNPs. NeighborNet analysis showed extensive reticulation at the core of subgenus Vitis representing the deeper nodes, with extensive reticulation radiating outward. Fitch Parsimony identified North America as the origin of the most recent common ancestor of extant Vitis species.ConclusionsPhylogenetic patterns suggested origination of the genus in North America, fragmentation of an ancestral range during the Miocene, formation of extant species in the late Miocene-Pleistocene, and differentiation of species in the context of Pliocene-Quaternary tectonic and climatic change. Nuclear SNPs effectively resolved relationships at and below the species level in grapes and rectified several misclassifications of accessions in the repositories. Our results challenge current higher-level classifications, reveal the abundance of genetic diversity in the genus that is potentially available for crop improvement, and provide a valuable resource for species delineation, germplasm conservation and use.

Next-generation sequencing technologies promise to dramatically accelerate the use of genetic information for crop improvement by facilitating the genetic mapping of agriculturally important phenotypes. The first step in optimizing the design of genetic mapping studies involves large-scale polymorphism discovery and a subsequent genome-wide assessment of the population structure and pattern of linkage disequilibrium (LD) in the species of interest. In the present study, we provide such an assessment for the grapevine (genus Vitis), the world's most economically important fruit crop. Reduced representation libraries (RRLs) from 17 grape DNA samples (10 cultivated V. vinifera and 7 wild Vitis species) were sequenced with sequencing-by-synthesis technology. We developed heuristic approaches for SNP calling, identified hundreds of thousands of SNPs and validated a subset of these SNPs on a 9K genotyping array. We demonstrate that the 9K SNP array provides sufficient resolution to distinguish among V. vinifera cultivars, between V. vinifera and wild Vitis species, and even among diverse wild Vitis species. We show that there is substantial sharing of polymorphism between V. vinifera and wild Vitis species and find that genetic relationships among V. vinifera cultivars agree well with their proposed geographic origins using principal components analysis (PCA). Levels of LD in the domesticated grapevine are low even at short ranges, but LD persists above background levels to 3 kb. While genotyping arrays are useful for assessing population structure and the decay of LD across large numbers of samples, we suggest that whole-genome sequencing will become the genotyping method of choice for genome-wide genetic mapping studies in high-diversity plant species. This study demonstrates that we can move quickly towards genome-wide studies of crop species using next-generation sequencing. Our study sets the stage for future work in other high diversity crop species, and provides a significant enhancement to current genetic resources available to the grapevine genetic community.

The eastern Asian and eastern North American disjunction in Juglans offers an opportunity to estimate the time since divergence of the Eurasian and American lineages and to compare it with paleobotanical evidence. Five chloroplast DNA noncoding spacer (NCS) sequences: trnT−trnF, psbA−trnH, atpB−rbcL, trnV-16S rRNA, and trnS-trnfM and data from earlier studies (matK, ITS, and nuclear RFLP) were used to reconstruct phylogeny and to estimate the divergence time of major lineages. Seventeen taxa from four sections of Juglans and two outgroup taxa, Pterocarya stenoptera and Carya illinoiensis were included. NCS data was congruent only with matK data. Both maximum parsimony (MP) and maximum likelihood (ML) cladograms were concordant at the sectional level and revealed three well-supported monophyletic clades corresponding to sections Juglans, Cardiocaryon, and Rhysocaryon in both NCS and combined analyses. The single extant American butternut, Juglans cinerea was placed within the poorly resolved, but well-supported Rhysocaryon. Placement of taxa within Rhysocaryon and Cardiocaryon were inconsistent between NCS and combined analyses. Overall, the results suggest that: (1) the NCS sequence divergence observed within and between sections of Juglans is low and the addition of matK data only marginally improved resolution within Rhysocaryon;(2) the early divergence of section Juglans in both MP and ML analyses of NCS and combined data implies its ancient origin in contrast to fossil evidence, which suggests the earliest divergence of sections Rhysocaryon and Cardiocaryon; and (3) the extant taxa may not hold the footprints to unravel the evolutionary history of the genus.

An integrated genetic linkage map of chickpea (Clcei) has been developed that consists of 9 morphological, 27 isozyme, 10 RFLP, and 45 RAPD markers covering 550 cM. The map was made from segregation data from populations of three interspecific crosses of cultivated chickpea (C. arietlnum, 2n = 16) and a closely related wild species (C. retlculatum, 2n = 16). The linkage map has 10 linkage groups representing the eight chromosomes of chickpea. Interspecific crosses were chosen for mapping because of the extremely low level of polymorphism found within the cultivated chickpea species. Several regions of the genome were found to be slightly skewed from the expected Mendellan ratios of alleles. The map was compared with published maps for pea (Plsum) and lentil (Lens). Five regions of the chickpea map have gene orders that are similar to those found in the pea genome. The degree of similarity is somewhat less than that found between pea and lentil, which is consistent with the evolutionary distances between these three genera. We have also observed that lentil genomlc DNA RFLP probes hybridize poorly to chickpea DNA, Indicating considerable divergence of these genomes at the sequence level.

Fusarium wilt caused by Fusarium oxysporum Schlechtend.:Fr. f. sp. ciceris (Padwick) Matuo & K. Sato is the most widely spread soilborne disease of chickpea (Cicer arietinum L.). To advance our understanding of the genetics of wilt resistance and aid chickpea breeding programs, we developed a set of F6 recombinant inbred lines (RILs) between Fusarium wilt susceptible (C‐104) and resistant 315) parents. Prior screening of selected F3 plants identified two primers (UBC‐170 and CS‐27) that produced random amplified polymorphic DNA {RAPD) markers associated with Fusarium wilt race 1 resistance. Analysis of the RILs with these primers yielded an estimate of 7% recombination between the two markers and the locus for wilt resistance, and 6% recombination between the loci corresponding to the two RAPD markers. The DNA fragments were cloned and sequenced in order to construct primers that would amplify only the markers of interest. Primer pair CS‐27F/CS‐27R amplified a fragment linked to the allele for susceptibility to race 1 of Fusarium wilt and thus constitute allele specific associated primers (ASAPs), whereas UBC‐170FFLIBC‐170R produced a single band for both resistant and susceptible genotypes, thus demonstrating locus specificity rather than allele specificity. The use of markers generated by the RAPD or ASAP approaches can facilitate the introgression of resistance genes into susceptible lines and expedite the screening of chickpea germplasm resources and will be useful in extending the genetic map of chickpea.

Understanding relationships among species is a fundamental goal of evolutionary biology. Single nucleotide polymorphisms (SNPs) identified through next generation sequencing and related technologies enable phylogeny reconstruction by providing unprecedented numbers of characters for analysis. One approach to SNP-based phylogeny reconstruction is to identify SNPs in a subset of individuals, and then to compile SNPs on an array that can be used to genotype additional samples at hundreds or thousands of sites simultaneously. Although powerful and efficient, this method is subject to ascertainment bias because applying variation discovered in a representative subset to a larger sample favors identification of SNPs with high minor allele frequencies and introduces bias against rare alleles. Here, we demonstrate that the use of hybridization intensity data, rather than genotype calls, reduces the effects of ascertainment bias. Whereas traditional SNP calls assess known variants based on diversity housed in the discovery panel, hybridization intensity data survey variation in the broader sample pool, regardless of whether those variants are present in the initial SNP discovery process. We apply SNP genotype and hybridization intensity data derived from the Vitis9kSNP array developed for grape to show the effects of ascertainment bias and to reconstruct evolutionary relationships among Vitis species. We demonstrate that phylogenies constructed using hybridization intensities suffer less from the distorting effects of ascertainment bias, and are thus more accurate than phylogenies based on genotype calls. Moreover, we reconstruct the phylogeny of the genus Vitis using hybridization data, show that North American subgenus Vitis species are monophyletic, and resolve several previously poorly known relationships among North American species. This study builds on earlier work that applied the Vitis9kSNP array to evolutionary questions within Vitis vinifera and has general implications for addressing ascertainment bias in array-enabled phylogeny reconstruction.

Adult females of the coccinellid predator Hippodamia convergens (Say) spent more time walking and less time grooming on a line of peas, Pisum sativum L., that has reduced waxbloom on all parts of the plant (due to the mutation well compared with a near-isogenic sister line with normal waxbloom. H. convergens walking was distributed over all parts of the low-wax plants, whereas on normal-wax plants walking occurred mostly on stems and the edges ofleaves and stipules. The beetles were able to generate 30 times the adhesive traction force on leaf surfaces ofJow-wax plants compared with normal-wax plants. In cage studies, H. convergens (4 adults per plant) were more effective at reducing population growth of pea aphid, Acyrthosiphon pisum (Harris), on low-wax plants than on normal-wax plants, but only at initial aphid densities of 10 aphids per plant. At higher initial densities (20 and 40 aphids per plant), differential impact of H. convergens was not observed or disappeared after 4-5 d. The results indicate that reduced waxbloom in peas could improve the effectiveness of H. convergens on peas at low prey densities.KEY WORDS Acyrthosiphon pisum, pea aphid, ladybird beetles, plant waxes, predator behavior, tritrophic interactions 0046-225X/98/0902-0909$02.00/0