Gene duplication and transposition of mobile elements drive evolution of the Rpv3 resistance locus in grapevine.

A wild grape haplotype (Rpv3-1) confers resistance to Plasmopara viticola. We mapped the causal factor for resistance to an interval containing a TIR-NB-LRR (TNL) gene pair that originated 1.6-2.6 million years ago by a tandem segmental duplication. Transient coexpression of the TNL pair in Vitis vinifera leaves activated pathogen-induced necrosis and reduced sporulation compared with control leaves. Even though transcripts of the TNL pair from the wild haplotype appear to be partially subject to nonsense-mediated mRNA decay, mature mRNA levels in a homozygous resistant genotype were individually higher than the mRNA trace levels observed for the orthologous single-copy TNL in sensitive genotypes. Allelic expression imbalance in a resistant heterozygote confirmed that cis-acting regulatory variation promotes expression in the wild haplotype. The movement of transposable elements had a major impact on the generation of haplotype diversity, altering the DNA context around similar TNL coding sequences and the GC-content in their proximal 5'-intergenic regions. The wild and domesticated haplotypes also diverged in conserved single-copy intergenic DNA, but the highest divergence was observed in intraspecific and not in interspecific comparisons. In this case, introgression breeding did not transgress the genetic boundaries of the domesticated species, because haplotypes present in modern varieties sometimes predate speciation events between wild and cultivated species.

Construction of a high-density genetic map and detection of a major QTL of resistance to powdery mildew (Erysiphe necator Sch.) in Caucasian grapes (Vitis vinifera L.).

BACKGROUND: Vitis vinifera L. is the most cultivated grapevine species worldwide. Erysiphe necator Sch., the causal agent of grape powdery mildew, is one of the main pathogens affecting viticulture. V. vinifera has little or no genetic resistances against E. necator and the grape industry is highly dependent on agrochemicals. Some Caucasian V. vinifera accessions have been reported to be resistant to E. necator and to have no genetic relationships to known sources of resistance to powdery mildew. The main purpose of this work was the study and mapping of the resistance to E. necator in the Caucasian grapes 'Shavtsitska' and 'Tskhvedianis tetra'. RESULTS: The Caucasian varieties 'Shavtsitska' and 'Tskhvedianis tetra' showed a strong partial resistance to E. necator which segregated in two cross populations: the resistant genotypes delayed and limited the pathogen mycelium growth, sporulation intensity and number of conidia generated. A total of 184 seedlings of 'Shavtsitska' x 'Glera' population were genotyped through the Genotyping by Sequencing (GBS) technology and two high-density linkage maps were developed for the cross parents. The QTL analysis revealed a major resistance locus, explaining up to 80.15% of the phenotypic variance, on 'Shavtsitska' linkage group 13, which was associated with a reduced pathogen infection as well as an enhanced plant necrotic response. The genotyping of 105 Caucasian accessions with SSR markers flanking the QTL revealed that the resistant haplotype of 'Shavtsitska' was shared by 'Tskhvedianis tetra' and a total of 25 Caucasian grape varieties, suggesting a widespread presence of this resistance in the surveyed germplasm. The uncovered QTL was mapped in the region where the Ren1 locus of resistance to E. necator, identified in the V. vinifera 'Kishmish vatkana' and related grapes of Central Asia, is located. The genetic analysis conducted revealed that the Caucasian grapes in this study exhibit a resistant haplotype different from that of Central Asian grape accessions. CONCLUSIONS: The QTL isolated in 'Shavtsitska' and present in the Caucasian V. vinifera varieties could be a new candidate gene of resistance to E. necator to use in breeding programmes. It co-localizes with the Ren1 locus but shows a different haplotype from that of grapevines of Central Asia. We therefore consider that the Caucasian resistance locus, named Ren1.2, contains a member of a cluster of R-genes, of which the region is rich, and to be linked with, or possibly allelic, to Ren1.

Kernel oil content and oil composition in walnut (Juglans regia L.) accessions from north-eastern Italy.

BACKGROUND: Walnut oil use is currently limited by its poor oxidative stability due to the high percentage of polyunsaturated fatty acids. Modifying the oil composition may be a goal in walnut breeding to increase interest in this crop. Exploring natural variability and identifying the main environmental factors affecting oil quality are necessary in crop selection. Therefore 190 wild accessions were collected and evaluated during 2013 and 2014 for oil content and its fatty acid profile and compared with five commercial cultivars as references. RESULTS: High variation in kernel oil content and fatty acid composition was found in the native walnut. Kernel oil content ranged from 54.2 to 72.2% (w/w). The major fatty acids were linoleic (range 46.9-68.6%), oleic (10.0-25.1%), linolenic (6.9-17.6%), palmitic (3.9-11.4%) and stearic (1.1-5.2%) acids. Some accessions had oil with a fatty acid ratio very different from the reference commercial cultivars, especially the oleic acid/polyunsaturated fatty acid (PUFA) ratio. A significant linear relationship and positive correlation between the daily minimum temperature and oleic acid content was observed in the wild walnuts. CONCLUSION: The wide variation in fatty acid content and composition allows superior accessions to be selected for diffusion among growers. A suitable strategy would be to make a selection against PUFA content rather than just for high oleic acid. In addition, the selected high oleic accessions, before being utilized per se or as donor parents in breeding programs, have to demonstrate they are not adversely affected by the environment. © 2017 Society of Chemical Industry.

A major QTL controlling apple skin russeting maps on the linkage group 12 of 'Renetta Grigia di Torriana'.

BACKGROUND: Russeting is a disorder developed by apple fruits that consists of cuticle cracking followed by the replacement of the epidermis by a corky layer that protects the fruit surface from water loss and pathogens. Although influenced by many environmental conditions and orchard management practices, russeting is under genetic control. The difficulty in classifying offspring and consequent variable segregation ratios have led several authors to conclude that more than one genetic determinant could be involved, although some evidence favours a major gene (Ru). RESULTS: In this study we report the mapping of a major genetic russeting determinant on linkage group 12 of apple as inferred from the phenotypic observation in a segregating progeny derived from 'Renetta Grigia di Torriana', the construction of a 20 K Illumina SNP chip based genetic map, and QTL analysis. Recombination analysis in two mapping populations restricted the region of interest to approximately 400 Kb. Of the 58 genes predicted from the Golden Delicious sequence, a putative ABCG family transporter has been identified. Within a small set of russeted cultivars tested with markers of the region, only six showed the same haplotype of 'Renetta Grigia di Torriana'. CONCLUSIONS: A major determinant (Ru_RGT) for russeting development putatively involved in cuticle organization is proposed as a candidate for controlling the trait. SNP and SSR markers tightly co-segregating with the Ru_RGT locus may assist the breeder selection. The observed segregations and the analysis of the 'Renetta Grigia di Torriana' haplotypic region in a panel of russeted and non-russeted cultivars may suggest the presence of other determinants for russeting in apple.

Selective sweep at the Rpv3 locus during grapevine breeding for downy mildew resistance.

The Rpv3 locus is a major determinant of downy mildew resistance in grapevine (Vitis spp.). A selective sweep at this locus was revealed by the DNA genotyping of 580 grapevines, which include a highly diverse set of 265 European varieties that predated the spread of North American mildews, 82 accessions of wild species, and 233 registered breeding lines with North American ancestry produced in the past 150 years. Artificial hybridisation and subsequent phenotypic selection favoured a few Rpv3 haplotypes that were introgressed from wild vines and retained in released varieties. Seven conserved haplotypes in five descent groups of resistant varieties were traced back to their founders: (1) 'Munson', a cross between two of Hermann Jaeger's selections of V. rupestris and V. lincecumii made in the early 1880s in Missouri, (2) V. rupestris 'Ganzin', first utilised for breeding in 1879 by Victor Ganzin in France, (3) 'Noah', selected in 1869 from intermingled accessions of V. riparia and V. labrusca by Otto Wasserzieher in Illinois, (4) 'Bayard', a V. rupestris × V. labrusca offspring generated in 1882 by George Couderc in France, and (5) a wild form closely related to V. rupestris accessions in the Midwestern United States and introgressed into 'Seibel 4614' in the 1880s by Albert Seibel in France. Persistence of these Rpv3 haplotypes across many of the varieties generated by human intervention indicates that a handful of vines with prominent resistance have laid the foundation for modern grape breeding. A rampant hot spot of NB-LRR genes at the Rpv3 locus has provided a distinctive advantage for the adaptation of native North American grapevines to withstand downy mildew. The coexistence of multiple resistance alleles or paralogues in the same chromosomal region but in different haplotypes counteracts efforts to pyramidise them in a diploid individual via conventional breeding.

Defence responses in Rpv3-dependent resistance to grapevine downy mildew.

The Rpv3 locus determines the ability to operate an isolate-specific hypersensitive response (HR) against Plasmopara viticola in grapevines that carry a resistant Rpv3 (+) haplotype. Artificial infection was performed on leaf discs of Rpv3 (+) and Rpv3 (-) grapevines with two distinct isolates of the pathogen (avrRpv3 (+) and avrRpv3 (-)). The plant response, including the establishment of HR and changes in expression of 33 genes, was compared to the development of the pathogen. HR was induced exclusively in the Rpv3 (+) host upon inoculation with the avrRpv3 (+) isolate of the pathogen, which is assumed to use avrRpv3 (+) effectors that are recognised by/through the plant Rpv3 (+) gene product. The limitation imposed on pathogen growth was the result of inducible responses elicited by the Rpv3 (+)-avrRpv3 (+) interaction. This host reaction relied on transcriptional induction of the HR-associated gene HSR1 and salicylic acid-induced pathogenesis-related (PR) genes PR-1 and PR-2 during the initial 24-48 h post-inoculation. These events had no parallel in the Rpv3 (-) host or upon infection with the avrRpv3 (-) isolate. The emerging model for Rpv3-mediated defence, which is dependent upon race-specific recognition, associated with up-regulation of PR-1 and PR-2 genes, and enforced by localised HR-type necrosis, is compatible with the cascade of events initiated by the products of NB-LRR and LRR-kinase receptor-like genes, such as those residing in the Rpv3 locus.

Expansion and subfunctionalisation of flavonoid 3',5'-hydroxylases in the grapevine lineage.

BACKGROUND: Flavonoid 3',5'-hydroxylases (F3'5'Hs) and flavonoid 3'-hydroxylases (F3'Hs) competitively control the synthesis of delphinidin and cyanidin, the precursors of blue and red anthocyanins. In most plants, F3'5'H genes are present in low-copy number, but in grapevine they are highly redundant. RESULTS: The first increase in F3'5'H copy number occurred in the progenitor of the eudicot clade at the time of the γ triplication. Further proliferation of F3'5'Hs has occurred in one of the paleologous loci after the separation of Vitaceae from other eurosids, giving rise to 15 paralogues within 650 kb. Twelve reside in 9 tandem blocks of ~35-55 kb that share 91-99% identity. The second paleologous F3'5'H has been maintained as an orphan gene in grapevines, and lacks orthologues in other plants. Duplicate F3'5'Hs have spatially and temporally partitioned expression profiles in grapevine. The orphan F3'5'H copy is highly expressed in vegetative organs. More recent duplicate F3'5'Hs are predominately expressed in berry skins. They differ only slightly in the coding region, but are distinguished in the structure of the promoter. Differences in cis-regulatory sequences of promoter regions are paralleled by temporal specialisation of gene transcription during fruit ripening. Variation in anthocyanin profiles consistently reflects changes in the F3'5'H mRNA pool across different cultivars. More F3'5'H copies are expressed at high levels in grapevine varieties with 93-94% of 3'5'-OH anthocyanins. In grapevines depleted in 3'5'-OH anthocyanins (15-45%), fewer F3'5'H copies are transcribed, and at lower levels. Conversely, only two copies of the gene encoding the competing F3'H enzyme are present in the grape genome; one copy is expressed in both vegetative and reproductive organs at comparable levels among cultivars, while the other is transcriptionally silent. CONCLUSIONS: These results suggest that expansion and subfunctionalisation of F3'5'Hs have increased the complexity and diversification of the fruit colour phenotype among red grape varieties.

Breakdown of resistance to grapevine downy mildew upon limited deployment of a resistant variety.

BACKGROUND: Natural disease resistance is a cost-effective and environmentally friendly way of controlling plant disease. Breeding programmes need to make sure that the resistance deployed is effective and durable. Grapevine downy mildew, caused by the Oomycete Plasmopara viticola, affects viticulture and it is controlled with pesticides. Downy mildew resistant grapevine varieties are a promising strategy to control the disease, but their use is currently restricted to very limited acreages. The arising of resistance-breaking isolates under such restricted deployment of resistant varieties would provide valuable information to design breeding strategies for the deployment of resistance genes over large acreages whilst reducing the risks of the resistance being defeated. The observation of heavy downy mildew symptoms on a plant of the resistant variety Bianca, whose resistance is conferred by a major gene, provided us with a putative example of emergence of a resistance-breaking isolate in the interaction between grapevine and P. viticola. RESULTS: In this paper we describe the emergence of a P. viticola isolate (isolate SL) that specifically overcomes Rpv3, the major resistance gene carried by Bianca at chromosome 18. We show that isolate SL has the same behaviour as two P. viticola isolates avirulent on Bianca (isolates SC and SU) when inoculated on susceptible plants or on resistant plants carrying resistances derived from other sources, suggesting there is no fitness cost associated to the virulence. Molecular analysis shows that all three isolates are genetically closely related. CONCLUSIONS: Our results are the first description of a resistance-breaking isolate in the grapevine/P. viticola interaction, and show that, despite the reduced genetic variability of P. viticola in Europe compared to its basin of origin and the restricted use of natural resistance in European viticulture, resistance-breaking isolates overcoming monogenic resistances may arise even in cases where deployment of the resistant varieties is limited to small acreages. Our findings represent a warning call for the use of resistant varieties and an incentive to design breeding programmes aiming to optimize durability of the resistances.

The SSR-based molecular profile of 1005 grapevine (Vitis vinifera L.) accessions uncovers new synonymy and parentages, and reveals a large admixture amongst varieties of different geographic origin.

A collection of 1005 grapevine accessions was genotyped at 34 microsatellite loci (SSR) with the aim of analysing genetic diversity and exploring parentages. The comparison of molecular profiles revealed 200 groups of synonymy. The removal of perfect synonyms reduced the database to 745 unique genotypes, on which population genetic parameters were calculated. The analysis of kinship uncovered 74 complete pedigrees, with both parents identified. Many of these parentages were not previously known and are of considerable historical interest, e.g. Chenin blanc (Sauvignon × Traminer rot), Covè (Harslevelu selfed), Incrocio Manzoni 2-14 and 2-15 (Cabernet franc × Prosecco), Lagrein (Schiava gentile × Teroldego), Malvasia nera of Bolzano (Perera × Schiava gentile), Manzoni moscato (Raboso veronese × Moscato d'Amburgo), Moscato violetto (Moscato bianco × Duraguzza), Muscat of Alexandria (Muscat blanc à petit grain × Axina de tres bias) and others. Statistical robustness of unexpected pedigrees was reinforced with the analysis of an additional 7-30 SSRs. Grouping the accessions by profile resulted in a weak correlation with their geographical origin and/or current area of cultivation, revealing a large admixture of local varieties with those most widely cultivated, as a result of ancient commerce and population flow. The SSRs with tri- to penta-nucleotide repeats adopted for the present study showed a great capacity for discriminating amongst accessions, with probabilities of identity by chance as low as 1.45 × 10(-27) and 9.35 × 10(-12) for unrelated and full sib individuals, respectively. A database of allele frequencies and SSR profiles of 32 reference cultivars are provided.

Targeted Mutagenesis of the Female-Suppressor SyGI Gene in Tetraploid Kiwifruit by CRISPR/CAS9.

Kiwifruit belong to the genus Actinidia with 54 species apparently all functionally dioecious. The sex-determinants of the type XX/XY, with male heterogametic, operate independently of the ploidy level. Recently, the SyGI protein has been described as the suppressor of female development. In the present study, we exploited the CRISPR/Cas9 technology by targeting two different sites in the SyGI gene in order to induce a stable gene knock-out in two tetraploid male accessions of Actinidia chinensis var. chinensis. The two genotypes showed a regenerative efficiency of 58% and 73%, respectively. Despite not yet being able to verify the phenotypic effects on the flower structure, due to the long time required by tissue-cultured kiwifruit plants to flower, we obtained two regenerated lines showing near fixation of a unique modification in their genome, resulting in both cases in the onset of a premature stop codon, which induces the putative gene knock-out. Evaluation of gRNA1 locus for both regenerated plantlets resulted in co-amplification of a minor variant differing from the target region for a single nucleotide. A genomic duplication of the region in proximity of the Y genomic region could be postulated.

Resistance to Plasmopara viticola in grapevine 'Bianca' is controlled by a major dominant gene causing localised necrosis at the infection site.

Downy mildew resistance is a quantitative trait in grapevines of the genus Vitis. The grapevine 'Bianca' has retained resistance, originally present in its North American ancestors, through several cycles of backcrossing with susceptible cultivars of Vitis vinifera followed by phenotypic selection. The genetic control of the trait was studied using 116 full-siblings from the cross 'Chardonnay' x 'Bianca' and parental genetic maps consisting of 298 and 312 markers, respectively. Ratings of resistance and histological identification of the stage of interaction, when pathogen development is impaired in resistant individuals, were performed using leaf disc inoculation assays with two isolates of Plasmopara viticola collected in Italian and French vineyards. 'Bianca' and 59% of its offspring were heterozygous for a dominant gene, located in a 2.9 cM interval at the Rpv3 locus on chromosome 18, responsible for the onset of a hypersensitive response (HR) at the infection sites within 2 days post inoculation (dpi). Localised necrosis was the earliest phenotypic difference compared to susceptible individuals, it did not halt pathogen growth, but it was associated with a significant reduction of pathogen performance and disease symptoms from 3 to 6 dpi. QTL peaks for quantitative ratings revealed the strongest effects being caused by the Rpv3 locus: extent of mesophyll colonisation (LOD 3.1, percentage of explained phenotypic variance 16.2%), sporulation density (29.7, 74.3%), and symptom severity expressed by the OIV452 descriptor recommended by the Office International de la Vigne et du Vin (28.3, 74.6%). Strong correlation was observed between the ability of a seedling to mount an HR under controlled experimental conditions and quantitative resistance of the adult plant exposed to natural infections in the field, which was expressed by the number of leaves with fungal sporulation, in two consecutive years of observations.

The powdery mildew resistance gene REN1 co-segregates with an NBS-LRR gene cluster in two Central Asian grapevines.

BACKGROUND: Grape powdery mildew is caused by the North American native pathogen Erysiphe necator. Eurasian Vitis vinifera varieties were all believed to be susceptible. REN1 is the first resistance gene naturally found in cultivated plants of Vitis vinifera. RESULTS: REN1 is present in 'Kishmish vatkana' and 'Dzhandzhal kara', two grapevines documented in Central Asia since the 1920's. These cultivars have a second-degree relationship (half sibs, grandparent-grandchild, or avuncular), and share by descent the chromosome on which the resistance allele REN1 is located. The REN1 interval was restricted to 1.4 cM using 38 SSR markers distributed across the locus and the segregation of the resistance phenotype in two progenies of collectively 461 offspring, derived from either resistant parent. The boundary markers delimit a 1.4-Mbp sequence in the PN40024 reference genome, which contains 27 genes with known functions, 2 full-length coiled-coil NBS-LRR genes, and 9 NBS-LRR pseudogenes. In the REN1 locus of PN40024, NBS genes have proliferated through a mixture of segmental duplications, tandem gene duplications, and intragenic recombination between paralogues, indicating that the REN1 locus has been inherently prone to producing genetic variation. Three SSR markers co-segregate with REN1, the outer ones confining the 908-kb array of NBS-LRR genes. Kinship and clustering analyses based on genetic distances with susceptible cultivars representative of Central Asian Vitis vinifera indicated that 'Kishmish vatkana' and 'Dzhandzhal kara' fit well into local germplasm. 'Kishmish vatkana' also has a parent-offspring relationship with the seedless table grape 'Sultanina'. In addition, the distant genetic relatedness to rootstocks, some of which are derived from North American species resistant to powdery mildew and have been used worldwide to guard against phylloxera since the late 1800's, argues against REN1 being infused into Vitis vinifera from a recent interspecific hybridisation. CONCLUSION: The REN1 gene resides in an NBS-LRR gene cluster tightly delimited by two flanking SSR markers, which can assist in the selection of this DNA block in breeding between Vitis vinifera cultivars. The REN1 locus has multiple layers of structural complexity compared with its two closely related paralogous NBS clusters, which are located some 5 Mbp upstream and 4 Mbp downstream of the REN1 interval on the same chromosome.

Sex change in kiwifruit (Actinidia chinensis Planch.): a developmental framework for the bisexual to unisexual floral transition.

KEY MESSAGE: The developmental morphology of male and female kiwifruit flowers is tracked to delimit a framework of events to aid the study of divergence in floral gene expression. The transition from hermaphrodite to unisexual development of kiwifruit (Actinidia chinensis Planch) flowers has been reported previously, but differences in gene expression controlling sexual development for this species have not been associated with the major developmental changes occurring within pistils. We investigated the key stages in male and female flower development to define the point at which meristematic activities diverge in the two sexes. A combination of scanning electron microscopy and light microscopy was used to investigate pistil development from the earliest stages. We identified seven distinct stages characterized by differences in ovary size and shape, macrosporogenesis, ovule primordium development, anther locule lengthening, microspore wall thickening, and pollen degeneration. Sex differences were evident from the initial stage of development, with a laterally compacted gynoecium in male flowers. However, the key developmental stage, at which tissue differentiation clearly deviated between the two sexes, was stage 3, when flowers were 3.5 to 4.5 mm in length at approximately 10 d from initiation of stamen development. At this stage, male flowers lacked evident carpel meristem development as denoted by a lack of ovule primordium formation. Pollen degeneration in female flowers, probably driven by programmed cell death, occurred at the late stage 6, while the final stage 7 was represented by pollen release. As the seven developmental stages are associated with specific morphological differences, including flower size, the scheme suggested here can provide the required framework for the future study of gene expression during the regulation of flower development in this crop species.

Resistance to Sharka in Apricot: Comparison of Phase-Reconstructed Resistant and Susceptible Haplotypes of 'Lito' Chromosome 1 and Analysis of Candidate Genes.

Sharka, a common disease among most stone fruit crops, is caused by the Plum Pox Virus (PPV). Resistant genotypes have been found in apricot (Prunus armeniaca L.), one of which-the cultivar 'Lito' heterozygous for the resistance-has been used to map a major quantitative trait locus (QTL) on linkage group 1, following a pseudo-test-cross mating design with 231 individuals. In addition, 19 SNP markers were selected from among the hundreds previously developed, which allowed the region to be limited to 236 kb on chromosome 1. A 'Lito' bacterial artificial chromosome (BAC) library was produced, screened with markers of the region, and positive BAC clones were sequenced. Resistant (R) and susceptible (S) haplotypes were assembled independently. To refine the assembly, the whole genome of 'Lito' was sequenced to high coverage (98×) using PacBio technology, enabling the development of a detailed assembly of the region that was able to predict and annotate the genes in the QTL region. The selected cultivar 'Lito' allowed not only to discriminate structural variants between the two haplotypic regions but also to distinguish specific allele expression, contributing towards mining the PPVres locus. In light of these findings, genes previously indicated (i.e., MATHd genes) to have a possible role in PPV resistance were further analyzed, and new candidates were discussed. Although the results are not conclusive, the accurate and independent assembly of R and S haplotypes of 'Lito' is a valuable resource to predict and test alternative transcription and regulation mechanisms underpinning PPV resistance.

A set of microsatellite markers with long core repeat optimized for grape (Vitis spp.) genotyping.

BACKGROUND: Individual fingerprinting based on molecular markers has become a popular tool for studies of population genetics and analysis of genetic diversity in germplasm collections, including the solution of synonymy/homonymy and analysis of paternity and kinship. Genetic profiling of individuals is nowadays based on SSR (Simple Sequence Repeat) markers, which have a number of positive features that make them superior to any other molecular marker developed so far. In humans, SSRs with core repeats three to five nucleotides long are preferred because neighbour alleles are more easily separated and distinguished from each other; while in plants, SSRs with shorter repeats, namely two-nucleotides long, are still in use although they suffer lower separation of neighbour alleles and uncomfortable stuttering. RESULTS: New microsatellite markers, containing tri-, tetra-, and penta-nucleotide repeats, were selected from a total of 26,962 perfect microsatellites in the genome sequence of nearly homozogous grapevine PN40024, assembled from reads covering 8.4 X genome equivalents. Long nucleotide repeats were selected for fingerprinting, as previously done in many species including humans. The new grape SSR markers were tested for their reproducibility and information content in a panel of 48 grape cultivars. Allelic segregation was tested in progenies derived from two controlled crosses. CONCLUSION: A list of 38 markers with excellent quality of peaks, high power of discrimination, and uniform genome distribution (1-3 markers/chromosome), is proposed for grape genotyping. The reasons for exclusion are given for those that were discarded. The construction of marker-specific allelic ladders is also described, and their use is recommended to harmonise allelic calls and make the data obtained with different equipment and by different laboratories fully comparable.

A physical map of the heterozygous grapevine 'Cabernet Sauvignon' allows mapping candidate genes for disease resistance.

BACKGROUND: Whole-genome physical maps facilitate genome sequencing, sequence assembly, mapping of candidate genes, and the design of targeted genetic markers. An automated protocol was used to construct a Vitis vinifera 'Cabernet Sauvignon' physical map. The quality of the result was addressed with regard to the effect of high heterozygosity on the accuracy of contig assembly. Its usefulness for the genome-wide mapping of genes for disease resistance, which is an important trait for grapevine, was then assessed. RESULTS: The physical map included 29,727 BAC clones assembled into 1,770 contigs, spanning 715,684 kbp, and corresponding to 1.5-fold the genome size. Map inflation was due to high heterozygosity, which caused either the separation of allelic BACs in two different contigs, or local mis-assembly in contigs containing BACs from the two haplotypes. Genetic markers anchored 395 contigs or 255,476 kbp to chromosomes. The fully automated assembly and anchorage procedures were validated by BAC-by-BAC blast of the end sequences against the grape genome sequence, unveiling 7.3% of chimerical contigs. The distribution across the physical map of candidate genes for non-host and host resistance, and for defence signalling pathways was then studied. NBS-LRR and RLK genes for host resistance were found in 424 contigs, 133 of them (32%) were assigned to chromosomes, on which they are mostly organised in clusters. Non-host and defence signalling genes were found in 99 contigs dispersed without a discernable pattern across the genome. CONCLUSION: Despite some limitations that interfere with the correct assembly of heterozygous clones into contigs, the 'Cabernet Sauvignon' physical map is a useful and reliable intermediary step between a genetic map and the genome sequence. This tool was successfully exploited for a quick mapping of complex families of genes, and it strengthened previous clues of co-localisation of major NBS-LRR clusters and disease resistance loci in grapevine.

Colour variation in red grapevines (Vitis vinifera L.): genomic organisation, expression of flavonoid 3'-hydroxylase, flavonoid 3',5'-hydroxylase genes and related metabolite profiling of red cyanidin-/blue delphinidin-based anthocyanins in berry skin.

BACKGROUND: Structural genes of the phenyl-propanoid pathway which encode flavonoid 3'- and 3',5'-hydroxylases (F3'H and F3'5'H) have long been invoked to explain the biosynthesis of cyanidin- and delphinidin-based anthocyanin pigments in the so-called red cultivars of grapevine. The relative proportion of the two types of anthocyanins is largely under genetic control and determines the colour variation among red/purple/blue berry grape varieties and their corresponding wines. RESULTS: Gene fragments of VvF3'H and VvF3'5'H, that were isolated from Vitis vinifera 'Cabernet Sauvignon' using degenerate primers designed on plant homologous genes, translated into 313 and 239 amino acid protein fragments, respectively, with up to 76% and 82% identity to plant CYP75 cytochrome P450 monooxygenases. Putative function was assigned on the basis of sequence homology, expression profiling and its correlation with metabolite accumulation at ten different ripening stages. At the onset of colour transition, transcriptional induction of VvF3'H and VvF3'5'H was temporally coordinated with the beginning of anthocyanin biosynthesis, the expression being 2-fold and 50-fold higher, respectively, in red berries versus green berries. The peak of VvF3'5'H expression was observed two weeks later concomitantly with the increase of the ratio of delphinidin-/cyanidin-derivatives. The analysis of structural genomics revealed that two copies of VvF3'H are physically linked on linkage group no. 17 and several copies of VvF3'5'H are tightly clustered and embedded into a segmental duplication on linkage group no. 6, unveiling a high complexity when compared to other plant flavonoid hydroxylase genes known so far, mostly in ornamentals. CONCLUSION: We have shown that genes encoding flavonoid 3'- and 3',5'-hydroxylases are expressed in any tissues of the grape plant that accumulate flavonoids and, particularly, in skin of ripening red berries that synthesise mostly anthocyanins. The correlation between transcript profiles and the kinetics of accumulation of red/cyanidin- and blue/delphinidin-based anthocyanins indicated that VvF3'H and VvF3'5'H expression is consistent with the chromatic evolution of ripening bunches. Local physical maps constructed around the VvF3'H and VvF3'5'H loci should help facilitate the identification of the regulatory elements of each isoform and the future manipulation of grapevine and wine colour through agronomical, environmental and biotechnological tools.

Mapping with a few plants: using selective mapping for microsatellite saturation of the Prunus reference map.

The concept of selective (or bin) mapping is used here for the first time, using as an example the Prunus reference map constructed with an almond x peach F2 population. On the basis of this map, a set of six plants that jointly defined 65 possible different genotypes for the codominant markers mapped on it was selected. Sixty-three of these joint genotypes corresponded to a single chromosomal region (a bin) of the Prunus genome, and the two remaining corresponded to two bins each. The 67 bins defined by these six plants had a 7.8-cM average length and a maximum individual length of 24.7 cM. Using a unit of analysis composed of these six plants, their F1 hybrid parent, and one of the parents of the hybrid, we mapped 264 microsatellite (or simple-sequence repeat, SSR) markers from 401 different microsatellite primer pairs. Bin mapping proved to be a fast and economic strategy that could be used for further map saturation, the addition of valuable markers (such as those based on microsatellites or ESTs), and giving a wider scope to, and a more efficient use of, reference mapping populations.

Observation of eight ancient olive trees (Olea europaea L.) growing in the Garden of Gethsemane.

For thousands of years, olive trees (Olea europaea L.) have been a significant presence and a symbol in the Garden of Gethsemane, a place located at the foot of the Mount of Olives, Jerusalem, remembered for the agony of Jesus Christ before his arrest. This investigation comprises the first morphological and genetic characterization of eight olive trees in the Garden of Gethsemane. Pomological traits, morphometric, and ultrastructural observations as well as SSR (Simple Sequence Repeat) analysis were performed to identify the olive trees. Statistical analyses were conducted to evaluate their morphological variability. The study revealed a low morphological variability and minimal dissimilarity among the olive trees. According to molecular analysis, these trees showed the same allelic profile at all microsatellite loci analyzed. Combining the results of the different analyses carried out in the frame of the present work, we could conclude that the eight olive trees of the Gethsemane Garden have been propagated from a single genotype.