A 48 SNP set for grapevine cultivar identification.

BACKGROUND: Rapid 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. RESULTS: We 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. CONCLUSIONS: We 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.

Overexpression of a gibberellin inactivation gene alters seed development, KNOX gene expression, and plant development in Arabidopsis.

We have examined the role of gibberellins (GAs) in plant development by expression of the pea GA 2-oxidase2 (PsGA2ox2) cDNA, which encodes a GA inactivating enzyme, under the control of the MEDEA (MEA) promoter. Expression of MEA:PsGA2ox2 in Arabidopsis caused seed abortion, demonstrating that active GAs in the endosperm are essential for normal seed development. MEA:PsGA2ox2 plants had reduced ovule number per ovary and exhibited defects in phyllotaxy and leaf morphology which were partly suppressed by GA treatment. The leaf architecture and phyllotaxy defects of MEA:PsGA2ox2 plants were also restored by sly1-d which reduces DELLA protein stability to increase GA response. MEA:PsGA2ox2 seedlings had increased expression of the KNOTTED1-like homeobox (KNOX) genes, BP, KNAT2 and KNAT6, which are known to control plant architecture. The expression of KNOX genes is also altered in wild-type plants treated with GA. These results support the conclusion that GAs can suppress the effects of elevated KNOX gene expression, and raise the possibility that localized changes in GA levels caused by PsGA2ox2 alter the expression of KNOX genes to modify plant architecture.

Identification of grapevine MLO gene candidates involved in susceptibility to powdery mildew.

The European cultivated grapevine, Vitis vinifera L., is a host for the powdery mildew pathogen Erisyphe necator, which is the most economically important fungal disease of viticulture. MLO proteins mediate powdery mildew susceptibility in the model plant species Arabidopsis and the crop plants barley and tomato. Seven VvMLO cDNA sequences were isolated from grapevine and were subsequently identified as part of a 17 member VvMLO gene family within the V. vinifera genome. Phylogenetic analysis of the 17 VvMLO genes in the grape genome indicated that the proteins they encode fall into six distinct clades. The expression of representative VvMLOs from each clade were analysed in a range of grape tissues, as well as in response to a range of biotic and abiotic factors. The VvMLOs investigated have unique, but overlapping tissue expression patterns. Expression analysis of VvMLO genes following E. necator infection identified four upregulated VvMLOs which are orthologous to the Arabidopsis AtMLO2, AtMLO6 and AtMLO12 and tomato SlMLO1 genes required for powdery mildew susceptibility. This suggests a degree of functional redundancy between the proteins encoded by these genes in terms of susceptibility to powdery mildew, and, as such, represent potential targets for modification to generate powdery mildew resistant grapevines.

Preliminary development of a genetic strategy to prevent transgene escape by blocking effective pollen flow from transgenic plants.

Genetic modification (GM) of plants has great potential in the production of food and industrial compounds, and in molecular pharming. One of the greatest public concerns regarding this technology is effective pollen flow, in which wind- or insect-borne transgenic pollen is able to fertilise either non-GM crops of the same species, or closely related weed species, and lead to viable seed formation. In this paper we describe a novel concept, based on epigenetic inheritance (imprinting) and post-transcriptional gene silencing (PTGS)/RNA interference (RNAi), designed to prevent transgene escape via pollen flow from transgenic plants. A key advantage of this strategy is that it would allow all seeds from self-pollinated transgenic plants to be harvested and re-sown, without the need for specific treatments, while retaining all of the transgenes present in the parent. Thus, this strategy is not a Genetic Use Restriction Technology (GURT) and if implemented would not prevent seed saving by end-users.

Gibberellins are required for seed development and pollen tube growth in Arabidopsis.

Gibberellins (GAs) are tetracyclic diterpenoids that are essential endogenous regulators of plant growth and development. GA levels within the plant are regulated by a homeostatic mechanism that includes changes in the expression of a family of GA-inactivating enzymes known as GA 2-oxidases. Ectopic expression of a pea GA 2-oxidase2 cDNA caused seed abortion in Arabidopsis, extending and confirming previous observations obtained with GA-deficient mutants of pea, suggesting that GAs have an essential role in seed development. A new physiological role for GAs in pollen tube growth in vivo also has been identified. The growth of pollen tubes carrying the 35S:2ox2 transgene was reduced relative to that of nontransgenic pollen, and this phenotype could be reversed partially by GA application in vitro or by combining with spy-5, a mutation that increases GA response. Treatment of wild-type pollen tubes with an inhibitor of GA biosynthesis in vitro also suggested that GAs are required for normal pollen tube growth. These results extend the known physiological roles of GAs in Arabidopsis development and suggest that GAs are required for normal pollen tube growth, a physiological role for GAs that has not been established previously.