Leonor Ruiz-García

Regulation of flowering time by FVE, a retinoblastoma-associated protein

The initiation of flowering in plants is controlled by environmental and endogenous signals. 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, a gene encoding a MADS-box protein that inhibits flowering. We cloned FVE, a component of the autonomous pathway that encodes AtMSI4, a putative retinoblastoma-associated 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.

Analysis of DNA methylation in Arabidopsis thaliana based on methylation-sensitive AFLP markers

Abstract. 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.

Different roles of flowering-time genes in the activation of floral initiation genes in Arabidopsis.

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.

Molecular genetics of berry colour variation in table grape

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.

DNA methylation increases throughout Arabidopsis development

We used amplified fragment length polymorphisms (AFLP) to analyze the stability of DNA methylation throughout Arabidopsis development. AFLP can detect genome-wide changes in cytosine methylation produced by DNA demethylation agents, such as 5-azacytidine, or specific mutations at the DDM1 locus. In both cases, cytosine demethylation is associated with a general increase in the presence of amplified fragments. Using this approach, we followed DNA methylation at methylation sensitive restriction sites throughout Arabidopsis development. The results show a progressive DNA methylation trend from cotyledons to vegetative organs to reproductive organs.

A 48 SNP set for grapevine cultivar identification

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.