The domestication of the Eurasian grape ( Vitis vinifera ssp. sativa ) from its wild ancestor ( Vitis vinifera ssp. sylvestris ) has long been claimed to have occurred in Transcaucasia where its greatest genetic diversity is found and where very early archaeological evidence, including grape pips and artefacts of a 'wine culture', have been excavated. Whether from Transcaucasia or the nearby Taurus or Zagros Mountains, it is hypothesized that this wine culture spread southwards and eventually westwards around the Mediterranean basin, together with the transplantation of cultivated grape cuttings. However, the existence of morphological differentiation between cultivars from eastern and western ends of the modern distribution of the Eurasian grape suggests the existence of different genetic contribution from local sylvestris populations or multilocal selection and domestication of sylvestris genotypes. To tackle this issue, we analysed chlorotype variation and distribution in 1201 samples of sylvestris and sativa genotypes from the whole area of the species' distribution and studied their genetic relationships. The results suggest the existence of at least two important origins for the cultivated germplasm, one in the Near East and another in the western Mediterranean region, the latter of which gave rise to many of the current Western European cultivars. Indeed, over 70% of the Iberian Peninsula cultivars display chlorotypes that are only compatible with their having derived from western sylvestris populations.

The initiation of flowering in plants is controlled by environmental and endogenous signals 1,2 . Molecular analysis of this process in Arabidopsis thaliana indicates that environmental control is exerted through the photoperiod and vernalization pathways, whereas endogenous signals regulate the autonomous and gibberellin pathways. The vernalization and autonomous pathways converge on the negative regulation of FLC 3,4 , a gene encoding a MADS-box protein that inhibits flowering 3,4 . We cloned FVE, a component of the autonomous pathway that encodes AtMSI4, a putative retinoblastomaassociated protein. FVE interacted with retinoblastoma protein in immunoprecipitation assays, and FLC chromatin was enriched in acetylated histones in fve mutants. We conclude that FVE participates in a protein complex repressing FLC transcription through a histone deacetylation mechanism. Our data provide genetic evidence of a new developmental function of these conserved proteins and identify a new genetic mechanism in the regulation of flowering. shown in the upper part, capital letters correspond to the wild-type or mutated nucleotides. In the protein schematic, gray boxes represent WD repeats, the narrow black box represents a putative nuclear localization signal and the asterisk indicates a putative retinoblastoma-binding motif.

AFLP analysis using restriction enzyme isoschizomers that differ in their sensitivity to methylation of their recognition sites has been used to analyse the methylation state of anonymous CCGG sequences in Arabidopsis thaliana. The technique was modified to improve the quality of fingerprints and to visualise larger numbers of scorable fragments. Sequencing of amplified fragments indicated that detection was generally associated with non-methylation of the cytosine to which the isoschizomer is sensitive. Comparison of EcoRI/ HpaII and EcoRI/ MspI patterns in different ecotypes revealed that 35-43% of CCGG sites were differentially digested by the isoschizomers. Interestingly, the pattern of digestion among different plants belonging to the same ecotype is highly conserved, with the rate of intra-ecotype methylation-sensitive polymorphisms being less than 1%. However, pairwise comparisons of methylation patterns between samples belonging to different ecotypes revealed differences in up to 34% of the methylation-sensitive polymorphisms. The lack of correlation between inter-ecotype similarity matrices based on methylation-insensitive or methylation-sensitive polymorphisms suggests that whatever the mechanisms regulating methylation may be, they are not related to nucleotide sequence variation.

SummaryConditions to promote dark morphogenesis and¯ower-ing in Arabidopsis have previously been limited to liquid cultures and to a few laboratory ecotypes. We have obtained development and¯owering of Arabidopsis plants under complete darkness by growing them on vertical Petri dishes containing solid agar medium with sucrose. Under these conditions, all the ecotypes tested were able to develop, giving rise to etiolated plants that¯owered after producing a certain number of leaves. Dark-grown plants showed similarities with phytochrome-de®cient mutants and were different from de-etiolated or constitutive photomorphogenesis mutants such as det and cop. Late-and early-¯owering ecotypes, showing large differences in¯owering time and leaf number under long days,¯owered with a similar number of leaves when grown in the dark. Rapid dark¯owering of late-¯owering ecotypes was not an effect of darkness but the result of the interaction between dark and sucrose availability at the aerial part of the plant, since sucrose also had an effect when plants were grown in the light. Gibberellin-de®cient and insensitive mutants were delayed in the initiation of owers in the dark, indicating a role for these hormones in¯owering promotion in the dark. The late-¯owering phenotype of mutants at different loci of the autonomous and long-day-dependent¯owering induction pathways was rescued in dark growth conditions. However, the late-¯owering phenotype of ft and fwa mutants was not rescued by sucrose either in the dark or in the light, suggesting a different role for these genes in¯owering induction.

Fruit size and seedlessness are highly relevant traits in many fruit crop species, and both are primary targets of breeding programs for table grapes. In this work we performed a quantitative genetic analysis of size and seedlessness in an F1 segregating population derived from the cross between a classical seeded (Vitis vinifera L. 'Dominga') and a newly bred seedless ('Autumn Seedless') cultivar. Fruit size was scored as berry weight (BW), and for seedlessness we considered both seed fresh weight (SFW) and the number of seeds and seed traces (SN) per berry. Quantitative trait loci (QTL) analysis of BW detected 3 QTLs affecting this trait and accounting for up to 67% of the total phenotypic variance. QTL analysis for seedlessness detected 3 QTLs affecting SN (explaining up to 35% of total variance) and 6 affecting SFW (explaining up to 90% of total variance). Among them, a major effect QTL explained almost half of the phenotypic variation for SFW. Comparative analysis of QTLs for these traits reduced the number of grapevine genomic regions involved, one of them being a major effect QTL for seedlessness. Association analyses showed that microsatellite locus VMC7F2, closely linked to this QTL, is a useful marker for selection of seedlessnes.

We have analyzed double mutants that combine late-flowering mutations at four flowering-time loci (FVE, FPA, FWA, and FT) with mutations at the LEAFY (LFY), APETALA1 (AP1), and TERMINAL FLOWER1 (TFL1) loci involved in the floral initiation process (FLIP). Double mutants between ft-1 or fwa-1 and lfy-6 completely lack flowerlike structures, indicating that both FWA and FT act redundantly with LFY to control AP1. Moreover, the phenotypes of ft-1 ap1-1 and fwa-1 ap1-1 double mutants are reminiscent of the phenotype of ap1-1 cal-1 double mutants, suggesting that FWA and FT could also be involved in the control of other FLIP genes. Such extreme phenotypes were not observed in double mutants between fve-2 or fpa-1 and lfy-6 ap1-1. Each of these showed a phenotype similar to that of ap1-1 or lfy-6 mutants grown under noninductive photoperiods, suggesting a redundant interaction with FLIP genes. Finally, the phenotype of double mutants combining the late-flowering mutations with tfl1-2 were also consistent with the different roles of flowering-time genes.

The genetics and biochemistry of anthocyanins and flavonol biosynthesis and their role in plant organ pigmentation is well established in model species. However, the genetic basis of colour variation is species specific and understanding this variation is very relevant in many fruit and flower crop species. Among grape cultivars, there is a wide genetic variation for berry colour ranging from yellow-green ("white" cultivars) to dark blue berries. Berry colour results from the synthesis and accumulation of anthocyanins in the berry skin, which in plants is commonly regulated by transcription factors belonging to the MYB and bHLH families. In this work, we aimed to identify the major genetic determinants of berry colour variation in a large collection of table grape cultivars and somatic variants. The genetic analyses of berry colour in a few grape segregating progenies had previously identified a single locus on linkage group 2 responsible for colour variation. Furthermore, somatic variation for berry skin colour in cultivar Italia had been associated with the presence of a Gret1 retrotransposon in the promoter region of VvmybA1, a Myb gene whose expression is associated to skin colouration. The results show that VvmybA1 is the gene underlying the mapped locus controlling berry colour in grape. Additionally, the molecular analyses indicate that genetic and somatic berry colour variation can be associated to molecular variation at VvmybA1 in more than 95% of the analyzed cultivars. Thus, VvmybA1 is a major determinant of berry colour variation in table grape and its instability is the major cause of somatic variation for this trait.

BackgroundRapid and consistent genotyping is an important requirement for cultivar identification in many crop species. Among them grapevine cultivars have been the subject of multiple studies given the large number of synonyms and homonyms generated during many centuries of vegetative multiplication and exchange. Simple sequence repeat (SSR) markers have been preferred until now because of their high level of polymorphism, their codominant nature and their high profile repeatability. However, the rapid application of partial or complete genome sequencing approaches is identifying thousands of single nucleotide polymorphisms (SNP) that can be very useful for such purposes. Although SNP markers are bi-allelic, and therefore not as polymorphic as microsatellites, the high number of loci that can be multiplexed and the possibilities of automation as well as their highly repeatable results under any analytical procedure make them the future markers of choice for any type of genetic identification.ResultsWe analyzed over 300 SNP in the genome of grapevine using a re-sequencing strategy in a selection of 11 genotypes. Among the identified polymorphisms, we selected 48 SNP spread across all grapevine chromosomes with allele frequencies balanced enough as to provide sufficient information content for genetic identification in grapevine allowing for good genotyping success rate. Marker stability was tested in repeated analyses of a selected group of cultivars obtained worldwide to demonstrate their usefulness in genetic identification.ConclusionsWe have selected a set of 48 stable SNP markers with a high discrimination power and a uniform genome distribution (2-3 markers/chromosome), which is proposed as a standard set for grapevine (Vitis vinifera L.) genotyping. Any previous problems derived from microsatellite allele confusion between labs or the need to run reference cultivars to identify allele sizes disappear using this type of marker. Furthermore, because SNP markers are bi-allelic, allele identification and genotype naming are extremely simple and genotypes obtained with different equipments and by different laboratories are always fully comparable.