Isolation and characterization of new polymorphic simple sequence repeat loci in grape (Vitis vinifera L.).

Four new simple sequence repeat (SSR) loci (designated VVMD5, VVMD6, VVMD7, and VVMD8) were characterized in grape and analyzed by silver staining in 77 cultivars of Vitis vinifera. Amplification products ranged in size from 141 to 263 base pairs (bp). The number of alleles observed per locus ranged from 5 to 11 and the number of diploid genotypes per locus ranged from 13 to 27. At each locus at least 75% of the cultivars were heterozygous. Alleles differing in length by only 1 bp could be distinguished by silver staining, and size estimates were within 1 or 2 bp, depending on the locus, of those obtained by fluorescence detection at previously reported loci. Allele frequencies were generally similar in wine grapes and table grapes, with some exceptions. Some alleles were found only in one of the two groups of cultivars. All 77 cultivars were distinguished by the four loci with the exception of four wine grapes considered to be somatic variants of the same cultivar, 'Pinot noir', 'Pinot gris', 'Pinot blanc', and 'Meunier'; two table grapes that are known to be synonymous, 'Keshmesh' and 'Thompson Seedless'; and three table grapes, 'Dattier', 'Rhazaki Arhanon', and 'Markandi', the first two of which have been suggested to be synonymous. Although the high polymorphism at grape SSR loci suggests that very few loci would theoretically be needed to separate all cultivars, the economic and legal significance of grape variety identification requires the increased resolution that can be provided by a larger number of loci. The ease with which SSR markers and data can be shared internationally should encourage their broad use, which will in turn increase the power of these markers for both identification and genetic analysis of grape. Key words : grape, Vitis, microsatellite, simple sequence repeat, DNA typing, identification.     

Genetic diversity and geographical dispersal in grapevine clones revealed by microsatellite markers.

Intravarietal genetic diversification associated with geographical dispersal of a vegetatively propagated species was studied using grapevine Vitis vinifera L. 'Cabernet Sauvignon' as a model. Fifty-nine clonal samples obtained from 7 countries (France, Chile, Spain, Australia, Hungary, USA, and Italy) were analyzed using 84 microsatellite markers. Eighteen polymorphic microsatellite loci (21.4%) were detected, finding 22 different genotypes in the population analyzed with a genetic similarity of over 97%. The presence of chimeric clones was evidenced at locus VMC5g7 by means of a segregation analysis of descendants by self-pollination of a triallelic Chilean clone and by somatic embryogenesis analysis, showing a mutation in L2 cell layer. Only 2 clones (obtained from France and Australia) presented the ancestral genotype, and the most divergent genotype was exhibited by another French clone, which had accumulated 5 somatic mutations. The 2 largest populations considered (from France and Chile) showed a clear divergency in the polymorphisms detected. These antecedents enabled the tracing of geographical dispersal with a phylogenetic hypothesis supporting France as the center of origin of diversification of Cabernet Sauvignon. The results obtained could help to explain diversification processes in other grapevine cultivars. The possibility that this kind of genetic variability occurs in other vegetatively propagated species is discussed, focusing on possible fingerprinting applications.     

Chimerism in grapevines: implications for cultivar identity,ancestry and genetic improvement

In the course of DNA profiling of grapevine cultivars using microsatellite loci we have occasionally observed more than two alleles at a locus in some individuals and have identified periclinal chimerism as the source of such anomalies. This phenomenon in long-lived clonally propagated crops, such as grapevine, which contains historically ancient cultivars, may have a role in clonal differences and affect cultivar identification and pedigree analysis. Here we show that when the two cell layers of a periclinal chimera, Pinot Meunier, are separated by passage through somatic embryogenesis the regenerated plants not only have distinct DNA profiles which are different from those of the parent plant but also have novel phenotypes. Recovery of these phenotypes indicates that additional genetic differences can exist between the two cell layers and that the Pinot Meunier phenotype is due to the interaction of genetically distinct cell layers. It appears that grapevine chimerism can not only modify phenotype but can also impact on grapevine improvement as both genetic transformation and conventional breeding strategies separate mutations in the L1 and L2 cell layers. Received: 14 March 2001 / Accepted: 22 May 2001     

A Strategy to Investigate the Intravarietal Genetic Variability in Vitis vinifera L. for Clones and Biotypes Identification and to Correlate Molecular Profiles with Morphological Traits or Geographic Origins

Grapevine is the most economically important and widely cultivated fruit crop in the world. Molecular markers have been used on Vitis vinifera to distinguish among both varieties and clones. Microsatellites are used to fingerprint varieties and several other techniques, reported in many papers, are used to analyze the differences among clones, but it is not available in the literature as a well defined strategy to screen a large number of Vitis cultivars. In fact, it is often necessary to use different techniques to investigate the genetic variability in different grapevine varieties and a proposed technique is used to study a cultivar, which is often not suitable for either the study of another cultivar or compare the genetic relationship among various cultivars. We describe here a strategy used for the analysis of several grapevine cultivars to describe a universal method to obtain DNA polymorphisms of Vitis vinifera genotypes from the same cultivar by using amplified fragment length polymorphism (AFLP), selective amplification of microsatellite polymorphic loci (SAMPL), microsatellites AFLP (M-AFLP), and ISSR molecular markers. The strategy here adopted permitted both to identify different biotypes (i.e., Primitivo), accessions (i.e., Garnacha tinta), and clones (i.e., Callet, Manto Negro, Moll) among the variability of same variety and to correlate the genetic differences to their geographical origins (i.e., Garnacha tinta; Malvasia nera di Brindisi/Lecce) or morphological traits (i.e., Malvasia of Candia). Here is also described the application of the protocol that allows to highlight the genetic variability accumulated during centuries of cultivations and selections of the same variety in different environments by vine growers.     

Chloroplast microsatellite polymorphisms in Vitis species.

The use of consensus chloroplast microsatellites primers for dicotyledonous chloroplast genomes revealed the existence of intra and interspecific length variation within the genus Vitis. Three chloroplast microsatellite loci were found to be polymorphic in samples of Vitis vinifera, Vitis berlandieri, Vitis riparia, and Vitis rupestris out of a total of 10 consensus primer pairs tested. These polymorphisms were always due to a variable number of mononucleotide residues within A and (or) T stretches in the amplified regions. Chloroplast microsatellite polymorphisms were used to demonstrate the maternal inheritance of chloroplast in V. vinifera and to characterise the chloroplast haplotypes present in wine grape cultivars of this species grown in Spain and Greece. The different distribution of haplotype frequencies in the two ends of the Mediterranean growth area suggests the existence of independent domestication events for grapevine.     

CHLOROPHYLL-DEFICIENT CELL LINES WHICH ARE GENETICALLY UNCHARACTERIZED CAN BE INAPPROPRIATE FOR USE AS PHENOTYPIC MARKERS IN DEVELOPMENTAL STUDIES

Periclinal chloroplast chimeras are genetic mosaics which possess shoot apices composed of one or more chlorophyll-deficient histogens and can exist as a series of arrangements of normal and mutant layers (A-B-B, A-B-A, etc.). Three periclinal chimeral cultivars of Sansevieria trifasciata L., each of which possesses normal green cell layer(s) but a genetically different chlorophyll-deficient cell layer(s), were utilized to study the effect of genotype on the ability of the cell layers of leaf cuttings and of cultured leaf tissue to regenerate shoots. The epidermis and LI derivatives were apparently incapable of shoot regeneration via leaf cutting, yet in two cultivars produced some shoots in vitro. In two of the cultivars, the chlorophyll-deficient cells never produced shoots. In the third, the capability of chlorophyll-deficient cell layers to produce shoots was less in vitro than in vivo, indicating that when determining morphogenic potential, direct comparisons between in vitro and in vivo systems may not be valid. Results also demonstrate that because genetically different albino cell layers can differ in their morphogenic response, utilizing a series of periclinal chimeras is useful only if the series is composed of the same two genotypes.     

Genome diversity and gene haplotypes in the grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L.), as revealed by single nucleotide polymorphisms

EST (expressed sequence tags) sequencing, SNP (single nucleotide polymorphisms) development and haplotype assessment are powerful tools for the support of marker-assisted selection. The grapevine genome is currently being scavenged in our laboratory using an EST-SNP approach. Nine parental genotypes, used to create five inter- or intra-specific hybrids, have been tested to evaluate the degree of polymorphism between Vitis vinifera, Vitis riparia and a further intraspecific hybrid, measuring their nucleotide diversity. The SNPs were analysed on cDNA sequences of 4 functional classes of genes based on homology with genes present in a public database: sugar metabolism, cell signalling, anthocyanin metabolism and defence related. Primer pairs were deduced and used to amplify corresponding genomic sequences. Almost 12,000 bp of DNA have been scanned revealing differences among genotypes of up to 247 SNPs, with the highest rate of one SNP occurring every 78 bp when clones of different Vitis species are compared. Re-sequencing allowed the definition of haplotypes in the nine genotypes studied and these were confirmed by analysing segregating populations. The efficiency of SSCP, in comparison with re-sequencing, was considered for 25 gene fragments of the same 9 genotypes.these two authors contributed equally to this work     

Chromosome Replacement and Deletion Lead to Clonal Polymorphism of Berry Color in Grapevine

Clonal polymorphism mainly results from somatic mutations that occur naturally during plant growth. In grapevine, arrays of clones have been selected within varieties as a valuable source of diversity, among them clones showing berry color polymorphism. To identify mutations responsible for this color polymorphism, we studied a collection of 33 clones of Pinot noir, Pinot gris, and Pinot blanc. Haplotypes of the L2 cell layer of nine clones were resolved by genotyping self-progenies with molecular markers along a 10.07 Mb region of chromosome 2, including the color locus. We demonstrated that at least six haplotypes could account for the loss of anthocyanin biosynthesis. Four of them resulted from the replacement of sections of the ‘colored’ haplotype, sized from 31 kb to 4.4 Mb, by the homologous sections of the ‘white’ haplotype mutated at the color locus. This transfer of information between the two homologous sequences resulted in the partial homozygosity of chromosome 2, associated in one case with a large deletion of 108 kb-long. Moreover, we showed that, in most cases, somatic mutations do not affect the whole plant; instead, they affect only one cell layer, leading to periclinal chimeras associating two genotypes. Analysis of bud sports of Pinot gris support the hypothesis that cell layer rearrangements in the chimera lead to the homogenization of the genotype in the whole plant. Our findings shed new light on the way molecular and cellular mechanisms shape the grapevine genotypes during vegetative propagation, and enable us to propose a scheme of evolutionary mechanism of the Pinot clones.     

Genetic chimerism of <Emphasis Type="Italic">Vitis vinifera</Emphasis>cv. Chardonnay 96 is maintained through organogenesis but not somatic embryogenesis

Background  

Grapevine can be a periclinal chimera plant which is composed at least of two distinct cell layers (L1, L2). When the cell layers of this plant are separated by passage through somatic embryogenesis, regenerated plants could show distinct DNA profiles and a novel phenotype which proved different from that of the parent plant.     

Molecular genetic diversity of the French-American grapevine hybrids cultivated in North America.

French-American hybrid grapevines are most popular in eastern and mid-western North America: they are hardy cultivars derived from crosses between the European Vitis vinifera and American wild vines. The aim of this study was to characterize their genetic background using 6 microsatellite (SSR) markers and a set of 33 diagnostic RAPD markers. The latter were reproducible with different PCR thermal cyclers. Two SSR loci were found to be synonymous, VrZAG47 and VVMD27. The DNA profile frequencies estimated for each cultivar were much lower with multi-locus SSR data than that obtained from multi-fragment RAPD data. There was no significant correlation between the multi-locus DNA profile frequencies derived from SSRs and those from RAPDs. Estimates of genetic diversity derived from SSRs were generally higher and the average similarity between cultivars was generally lower than values reported for subgroups of V. vinifera, in accordance with expectations for hybrid cultivars. The phenetic relationships depicted by UPGMA (unweighted pair-group method with arithmetic averaging) and neighbor-joining analyses of microsatellite data were congruent and, to a large extent, in agreement with the known pedigree or history of each cultivar. A major dichotomy was observed between one group where the known genetic background was dominated by the North American Vitis riparia and Vitis labrusca, and another one where the genetic background was dominated by the European V. vinifera. Two Kulhmann varieties thought to be synonymous were found to be different, though closely related.     

Biochemical and Genetic Polymorphisms for Carboxylesterase and Acetylesterase in Grape Clones of Vitis vinifera L. (Vitaceae) Cultivars

Native polyacrylamide gel electrophoresis (PAGE) was employed to show the highest number of esterase loci and to detect alpha- and beta-esterase polymorphisms in leaf buds of Vitis vinifera cultivars. A total of 16 esterase isozymes were detected in leaf buds from 235 plants including Italia, Rubi, Benitaka, and Brasil cultivars. Biochemical characterization of the grape esterases using ester substrates revealed alpha-, beta-, and alpha/beta-esterases with inhibitor tests distinguishing both carboxylesterases (EST-2, EST-3, EST-5, EST-6, EST-7, EST-8, EST-9, EST-10, and EST-16 isozymes) and acetylesterases (EST-4, EST-11, EST-12, EST-13, EST-14, EST-15 isozymes). No allele variation for alpha-, beta-, and alpha/beta-esterases was detected; however, EST-3 alpha-carboxylesterase was absent in 61.7% of vines, and EST-4 alpha/beta-acetylesterase was absent in one vine of Rubi cv. Null EST-3 carboxylesterase phenotype (61.7%) cannot be explained in this article, but the high genetic polymorphism in four V. vinifera clones is a positive aspect for genetic selection and development of new clones with different characteristics.     

Genetic relationship among cultivated and wild grapevine accessions from Tunisia.

We have used nuclear and chloroplast molecular markers to genotype cultivated and wild accessions of Vitis vinifera L. from Tunisia and assess their genetic relationships. Fifty-five distinct genotypes were identified among 80 cultivated accessions, including 18 genotypic groups containing between 2 and 5 accessions per group. They could represent a total of 60 distinct cultivars owing to berry colour variation found within identical genotype groups. Most of the 55 genotypes represent unique table grape genotypes except for one of them that was found identical to the genotype of table grape cultivar Rosseti. Hybridization among cultivars as well as self pollinations seems to have played an important role in their origin since several groups of closely related cultivars were observed. Furthermore, a parentage analysis showed a high probability for a parent hybrid relationship within two groups of three cultivars. No strong genetic similarities were found between cultivated and wild samples indicating that the cultivated accessions do not derive from local Vitis vinifera L. populations but could have been introduced from other regions in historic times.     

SOMATIC GENETIC ANALYSIS OF THE APICAL LAYERS OF CHIMERAL SPORTS IN CHRYSANTHEMUM BY EXPERIMENTAL PRODUCTION OF ADVENTITIOUS SHOOTS

Many commercial chrysanthemum cultivars display unusual somatic variability. The ‘Indianapolis’ family of chrysanthemum sports was analyzed for the genetic potential for color of each of the three layers in the apical meristem of their shoots. Populations of each cultivar were grown and sectors and off-color plants recorded. The location of the pigment within cells and between tissues was determined by microscopic examination of free-hand sections of fresh petals. Adventitious buds were forced from the stems of each cultivar by excising all normal lateral buds. These observations, showed 12 of the 16 ‘Indianapolis’ cultivars to be periclinal chimeras. Adventitious shoots often originated from two or more cells, derived from at least two different apical layers, and thus were themselves periclinal chimeras. While somatic mutation is the ultimate source of the variability in ‘Indianapolis’ chrysanthemums, the most frequent type of sporting resulted from the loss in mitosis of a chromosome carrying a supressor for the formation of yellow chromoplasts, giving a yellow sector or shoot. Sectors resulting from rearrangement of layers in the periclinal chimeras were less frequent than the sectors from chromosome loss.     

The application of SSRs characterized for grape (Vitis vinifera) to conservation studies in Vitaceae

The use of microsatellite loci developed from a single plant species across a number of related taxa is becoming increasingly widespread. This approach can provide highly informative markers even for species for which microsatellites have not been characterized. As a number of expressed sequence tag (EST)-derived and enrichment-derived microsatellites are available for grape (Vitis vinifera), this study set out to assess transferability of nine such loci across 25 species from five different Vitaceae genera. Intergeneric transfer success in Vitaceae was high (51.1%) and EST-derived loci performed better than enrichment-derived loci. Six loci were further tested across two Australian native species, Cissus hypoglauca and C. sterculiifolia, in order to assess the conservation of microsatellite repeats and their flanking sequences. Polymorphism of these selected loci was successfully tested for each species across a small, isolated rain forest population from northern New South Wales (Australia). Results from this preliminary investigation suggest that it is possible to use grape-derived simple sequence repeats (SSR) loci for population studies on Vitaceae. As Vitaceae are an important component of rain forest flora, the availability of such highly informative loci will be beneficial to future studies aimed at determining the genetic consequences of rain forest fragmentation.     

Endophytic colonization of Vitis vinifera L. by plant growth-promoting bacterium Burkholderia sp. strain PsJN

Patterns of colonization of Vitis vinifera L. cv. Chardonnay plantlets by a plant growth-promoting bacterium, Burkholderia sp. strain PsJN, were studied under gnotobiotic conditions. Wild-type strain PsJN and genetically engineered derivatives of this strain tagged with gfp (PsJN::gfp2x) or gusA (PsJN::gusA11) genes were used to enumerate and visualize tissue colonization. The rhizospheres of 4- to 5-week-old plantlets with five developed leaves were inoculated with bacterial suspensions. Epiphytic and endophytic colonization patterns were then monitored by dilution plating assays and microscopic observation of organ sections. Bacteria were chronologically detected first on root surfaces, then in root internal tissues, and finally in the fifth internode and the tissues of the fifth leaf. Analysis of the PsJN colonization patterns showed that this strain colonizes grapevine root surfaces, as well as cell walls and the whole surface of some rhizodermal cells. Cells were also abundant at lateral root emergence sites and root tips. Furthermore, cell wall-degrading endoglucanase and endopolygalacturonase secreted by PsJN explained how the bacterium gains entry into root internal tissues. Host defense reactions were observed in the exodermis and in several cortical cell layers. Bacteria were not observed on stem and leaf surfaces but were found in xylem vessels of the fifth internode and the fifth leaf of plantlets. Moreover, bacteria were more abundant in the fifth leaf than in the fifth internode and were found in substomatal chambers. Thus, it seems that Burkholderia sp. strain PsJN induces a local host defense reaction and systemically spreads to aerial parts through the transpiration stream.     

An improved procedure for the propagation in vitro of grapevine (t Vitis vinifera cv. Pinot noir) using axillary-bud microcuttings

A method has been developed for rapid propagation in vitro of Vitis vinifera cv. 'Pinot noir' from axillary-bud microcuttings harvested from plantlets initially cultured in vitro. The requirement of plant growth regulators for the different stages of the micropropagation was examined. BA at 8.9 μM added to MS basal medium was found to be optimal for culture establishment, while for subcultures, the concentration of BA was reduced to 4.4 μM to prevent hyperhydricity. Among the two auxins (NAA and IBA) tested for rooting, IBA at 2.5 μM was found to induce a good rooting- system in 100% of the shoots. The advantages of this method, using microcuttings from established plants in vitro, is discussed. This revised version was published online in June 2006 with corrections to the Cover Date.