Genetic diversity of wild grapevine populations in Spain and their genetic relationships with cultivated grapevines.

The wild grapevine, Vitis vinifera L. ssp. sylvestris (Gmelin) Hegi, considered as the ancestor of the cultivated grapevine, is native from Eurasia. In Spain, natural populations of V. vinifera ssp. sylvestris can still be found along river banks. In this work, we have performed a wide search of wild grapevine populations in Spain and characterized the amount and distribution of their genetic diversity using 25 nuclear SSR loci. We have also analysed the possible coexistence in the natural habitat of wild grapevines with naturalized grapevine cultivars and rootstocks. In this way, phenotypic and genetic analyses identified 19% of the collected samples as derived from cultivated genotypes, being either naturalized cultivars or hybrid genotypes derived from spontaneous crosses between wild and cultivated grapevines. The genetic diversity of wild grapevine populations was similar than that observed in the cultivated group. The molecular analysis showed that cultivated germplasm and wild germplasm are genetically divergent with low level of introgression. Using a model-based approach implemented in the software structure, we identified four genetic groups, with two of them fundamentally represented among cultivated genotypes and two among wild accessions. The analyses of genetic relationships between wild and cultivated grapevines could suggest a genetic contribution of wild accessions from Spain to current Western cultivars.

Multiple origins of cultivated grapevine (Vitis vinifera L. ssp. sativa) based on chloroplast DNA polymorphisms.

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.

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.

Analysis of differential messenger RNA expression between bovine blastocysts produced in different culture systems: implications for blastocyst quality.

Using reverse transcriptase-amplified fragment length polymorphism (RT-AFLP) analysis of differential mRNA expression and semiquantitative reverse transcriptase-polymerase chain reaction, we compared mRNA expression in bovine blastocysts from 4 sources, known to differ in quality in terms of their ability to withstand cryopreservation: 1) in vitro culture in synthetic oviduct fluid of in vitro-matured (IVM)/in vitro fertilized (IVF) zygotes; 2) in vitro culture in TCM-199 supplemented with granulosa cells (coculture) of IVM/IVF zygotes; 3) in vivo culture in the ewe oviduct of IVM/IVF zygotes; or 4) superovulation, artificial insemination, and nonsurgical embryo recovery. Total mRNA was isolated from pools of blastocysts and reverse transcription was performed. Triplicate reactions from each sample were displayed, and only consistent banding variations were recorded. Using AFLP-differential display assay, we found that cDNA banding patterns are highly conserved between the 4 groups of blastocysts studied; however, there was a difference of 7% in bands either missing or expressed across the groups. Fifty bands were reamplified, and a sequence comparison search revealed similarity of 14 isolated fragments to ribosomal and mitochondrial genes, 16 matched to described cDNA, and 20 corresponded to unknown sequences that may represent novel genes. The study of 7 differentially expressed mRNAs known to be involved in developmental process in the embryo suggests roles for apoptosis, oxidative stress, gap junctions, and differentiation in the determination of embryo quality. The aberrant transcription patterns detected in in vitro-produced bovine embryos compared with those produced in vivo may explain their reduced quality in terms of viability after cryopreservation.

Genetic structure of natural populations of the grass endophyte Epichloë festucae in semiarid grasslands.

Plants of red fescue (Festuca rubra), a commercially important turf grass, are infected by the fungal endophyte Epichloë festucae in semiarid natural grasslands, known as dehesas, in western Spain. We used amplified fragment length polymorphism (AFLP) markers to analyse the genetic polymorphism existing in two natural populations of Epichloë festucae. Linkage disequilibrium and the presence of clonal lineages indicated that nonrecombinant asexual reproduction predominates in both populations. However, most genetic variation detected was found to occur within populations, with only a moderate amount of genetic differentiation between populations (F(ST): 0.197). Overall, the study suggests that dehesa grasslands are useful reservoirs of Epichloë festucae endophytes, and provides information on population structure which is relevant to design sampling strategies.

Recombination and spontaneous mutation at the major cluster of resistance genes in lettuce (Lactuca sativa).

Two sets of overlapping experiments were conducted to examine recombination and spontaneous mutation events within clusters of resistance genes in lettuce. Multiple generations were screened for recombinants using PCR-based markers flanking Dm3. The Dm3 region is not highly recombinagenic, exhibiting a recombination frequency 18-fold lower than the genome average. Recombinants were identified only rarely within the cluster of Dm3 homologs and no crossovers within genes were detected. Three populations were screened for spontaneous mutations in downy mildew resistance. Sixteen Dm mutants were identified corresponding to spontaneous mutation rates of 10(-3) to 10(-4) per generation for Dm1, Dm3, and Dm7. All mutants carried single locus, recessive mutations at the corresponding Dm locus. Eleven of the 12 Dm3 mutations were associated with large chromosome deletions. When recombination could be analyzed, deletion events were associated with exchange of flanking markers, consistent with unequal crossing over; however, although the number of Dm3 paralogs was changed, no novel chimeric genes were detected. One mutant was the result of a gene conversion event between Dm3 and a closely related homolog, generating a novel chimeric gene. In two families, spontaneous deletions were correlated with elevated levels of recombination. Therefore, the short-term evolution of the major cluster of resistance genes in lettuce involves several genetic mechanisms including unequal crossing over and gene conversion.

Molecular diversity at the major cluster of disease resistance genes in cultivated and wild Lactuca spp.

Diversity was analyzed in wild and cultivated Lactuca germplasm using molecular markers derived from resistance genes of the NBS-LRR type. Three molecular markers, one microsatellite marker and two SCAR markers that amplified LRR-encoding regions, were developed from sequences of resistance gene homologs at the main resistance gene cluster in lettuce. Variation for these markers were assessed in germplasm including accessions of cultivated lettuce, Lactuca sativa L. and three wild Lactuca spp., L. serriola L., L. saligna and L. virosa L. Diversity was also studied within and between natural populations of L. serriola from Israel and California; the former is close to the center of diversity for Lactuca spp. while the latter is an area of more recent colonization. Large numbers of haplotypes were detected indicating the presence of numerous resistance genes in wild species. The diversity in haplotypes provided evidence for gene duplication and unequal crossing-over during the evolution of this cluster of resistance genes. However, there was no evidence for duplications and deletions within the LRR-encoding regions studied. The three markers were highly correlated with resistance phenotypes in L. sativa. They were able to discriminate between accessions that had previously been shown to be resistant to all known isolates of Bremia lactucae. Therefore, these markers will be highly informative for the establishment of core collections and marker-aided selection. A hierarchical analysis of the population structure of L. serriola showed that countries, as well as locations, were significantly differentiated. These differences may reflect local founder effects and/or divergent selection.

A transgenic mutant of Lactuca sativa (lettuce) with a T-DNA tightly linked to loss of downy mildew resistance.

One hundred and ninety-two independent primary transformants of lettuce cv. Diana were obtained by co-cultivation with Agrobacterium tumefaciens carrying constructs containing maize Ac transposase and Ds. R2 families were screened for mutations at four genes (Dm) for resistance to downy mildew. One family, designated dm3t524, had lost resistance to an isolate of Bremia lactucae expressing the avirulence gene Avr3. Loss of resistance segregated as a single recessive allele of Dm3. The mutation was not due to a large deletion as all molecular markers flanking Dm3 were present. Loss of Dm3 activity co-segregated with a T-DNA from which Ds had excised. Genomic DNA flanking the right border of this T-DNA was isolated by inverse polymerase chain reaction. This genomic sequence was present in four to five copies in wild-type cv. Diana. One copy was missing in all eight deletion mutants of Dm3 and altered in dm3t524, indicating tight physical linkage to Dm3. Three open reading frames (ORFs) occurred in a 6.6-kb region flanking the insertion site; however, expression of these ORFs was not detected. No similarities were detected between these ORFs and resistance genes cloned from other species. Transgenic complementation with 11-to 27-kb genomic fragments of Diana spanning the insertion site failed to restore Dm3 function to two ethyl methanesulfonate (EMS)-induced mutants of Dm3 or to cv. Cobham Green, which naturally lacks Dm3 activity. Therefore, either the T-DNA inserted extremely close to, but not within, Dm3 and the mutation may have been caused by secondary movement of Ds, or Dm3 activity is encoded by a gene extending beyond the fragments used for complementation.

Molecular analysis of irradiation-induced and spontaneous deletion mutants at a disease resistance locus in Lactuca sativa.

The major cluster of disease resistance genes in lettuce (Lactuca sativa) contains at least nine downy mildew resistance genes (Dm) spanning a genetic distance of 20cM and a physical distance of at least 6 Mb. Nine molecular markers that were genetically tightly linked to Dm3 were used to analyze nine independent deletion mutants and construct a map of the region surrounding Dm3. This analysis identified a linear order of deletion breakpoints and markers along the chromosome. There was no evidence for chromosomal rearrangements associated with the deletions. The region is not highly recombinagenic and the deletion breakpoints provided greater genetic resolution than meiotic recombinants. The region contains a mixture of high-and low-copy number sequences; no single-copy sequences were detected. Three markers hybridized to low-copy-number families of sequences that are duplicated predominantly close to Dm3. This was not true for sequences related to the triose-phosphate isomerase gene; these had been shown previously to be linked to Dm3, as well as to two independent clusters of Dm genes, and elsewhere in the genome. Two spontaneous mutants of Dm3 were identified; several markers flanking Dm3 are absent in one of these two mutants. The stability of the Dm3 region was also studied by analyzing the genotypes of diverse related cultivars. The 1.5 Mb region surrounding Dm3 has remained stable through many generations of breeding with and without selection for Dm3 activity.