REN1 or other resistance genes might be present in other accessions of V. vinifera grown in Armenia, Moldova, Russia, Georgia, and Uzbekistan (reviewed in [5]). Another cultivar called 'Dzhandzhal kara', also written as 'Djandjal Kara', "Karadzhandal', and 'Janjal kara' in Western literature [13,14] and identified with the synonym 'Semiz kara', has been utilised by breeders as a source of powdery mildew resistance [14,15]. 'Dzhandzhal kara' was first reported in the Uzbek district of Urgut, about 40 km southeast of Samarqand, and a few more stocks also existed in Kitab, within a 10-km range from Shakhrisabz where 'Kishmish vatkana' was also found. 'Dzhandzhal kara' is cultivated for the production of table grapes. 'Dzhandzhal kara' has normal hermaphrodite flowers that develop viable embryos and yield seeded fruit. 'Kishmish vatkana' is regarded as a stenospermocarpic cultivar, because its ovules degenerate soon after fertilisation at the initial stages of embryogenesis. 'Kishmish vatkana' and 'Dzhandzhal kara' survive natural powdery mildew infections better than any V. vinifera cultivar. Under high disease pressure, E. necator occasionally escapes the plant's defence reaction, which leads to microcolony formation and limited conidiation in both cultivars, with 'Dzhandzhal kara' allowing slightly more hyphal growth than 'Kishmish vatkana' (P. Kozma, unpublished results). Adaptive evolution of V. vinifera under the selective pressure of the pathogen was generally disregarded, because this crop is vegetatively propagated, and plants in the wild have probably undergone few mating generations since the arrival of the pathogen to the Eurasian continent. Thus, one might suspect a recent hybridisation with a resistant non-V. vinifera ancestor accidentally present in the area of cultivation, such as a North American rootstock or a wild grape from East Asia brought to the fertile oases of Central Asia along the ancient trade routes to the west [16]. 'Kishmish vatkana' and 'Dzhandzhal kara' are unique within the species V. vinifera in their resistance to powdery mildew, but they are typical among domesticated varieties in all other respects, particularly when compared with varieties grown in the Uzbek oases where 'Kishmish vatkana' and 'Dzhandzhal kara' were found, such as 'Nimrang' and 'Katta kurgan'. 'Nimrang' and 'Katta kurgan' have been imported, utilised, and documented in Western and in Far Eastern literature since the middle of the last century [17-21]. Their pure V. vinifera origin has never been called into question: instead they are credited with some of the most distinguishing features of the V. vinifera subsp. sativa group orientalis subgroup antasiatica [22,23]. Similarly, 'Kishmish vatkana' and 'Dzhandzhal kara' conform to many morphological descriptors that differentiate V. vinifera varieties from wild relatives, and thus do not appear to be the result of a recent genome introgression from feral grapevines.

If the REN1 gene had not moved into the V. vinifera germplasm from outside, the questions remain as to which functional class this gene belongs to and how it has evolved into functionality. Histological analyses have shown that REN1 does not prevent cell entry in 'Kishmish vatkana' but it inhibits the progression of pathogen invasion once haustoria have penetrated the host cells [1]. This mode of action bears a strong resemblance to that of host resistance genes. Many plant resistances to powdery mildews are controlled by NBS-LRR genes, which all confer post penetration resistance [24], including Run1 in the muscadine grape [9]. NBS-LRR genes have proliferated through reiterate tandem duplication in plant genomes, forming gene arrays prone to random shuffling of DNA segments. Genes in this class evolve rapidly, with their clustered organisation facilitating some mutated members to express novel receptors that may match the effectors of a virulent pathogen. In vegetatively propagated crops, R-genes are more likely to develop in wild populations or at the outer margins of the field crops, where plants sexually propagate and generate genetic novelty, some of which might provide a competitive advantage.

By identifying two grapevines that share REN1 by descent, more information has been gained about the genealogy of these resistant grapevines. A likelihood-based kinship analysis, which predicts parent-offspring and second-degree relationships without prior knowledge of the parents [41], provided evidence that one of the parents of 'Kishmish vatkana' is 'Sultanina', also known as 'Thompson Seedless'. This parent-offspring relationship is not surprising, since both cultivars produce stenospermocarpic fruit, and 'Sultanina' has been widely cultivated for centuries in Uzbekistan and Northern Afghanistan, with the local synonym of 'Ak-Kishmish'. Since 'Kishmish vatkana' but not 'Sultanina' has a second degree relationship with 'Dzhandzhal kara', and the resistant and seedless 'Kishmish vatkana' displays a combination of biological features that are uncoupled in the parents (the pollen parent 'Sultanina' does not produce viable embryos and it is susceptible to powdery mildew, the unknown resistant parent must have been able to produce seeds), the direction of the relationship could also be established. It is not known whether the cross was made deliberately or occurred by chance in a promiscuous vineyard. However, the fact that 'Kishmish vatkana' is poorly documented in chronicles and historical records, in spite of its attractiveness as a seedless and powdery mildew resistant table grape, suggests that this hybridisation occurred rather recently.

'Dzhandzhal kara' has a longer recorded history and was already disseminated worldwide by the early 1970's, which might suggest that the seedling 'Dzhandzhal kara' is more ancient than 'Kishmish vatkana'. A grape accession catalogued as Vitis vinifera subsp. vinifera 'Karadzhandal' in a US collection in 1965 is currently maintained at the USDA-ARS National Germplasm Repository in Davis, California. A variety named 'Kara dzandzal' was mentioned in a trial conducted in Japan in 1969 on 74 varieties imported from the former USSR, and recommended for breeding in a strict list of five top cultivars, including the typical Central Asian table grapes 'Katta kurgan' and 'Nimrang' [18]. A variety spelled 'Djandjal Kara' was included in a privileged set of accessions collected in Australian germplasm repositories and used in a seminal work on cultivar DNA fingerprinting in the 1990's [13].

Second-degree relationships could not be identified between 'Dzhandzhal kara' and any of the few other Central Asian grapes analysed in this paper, except for 'Kishmish vatkana'. However, the alleles carried by 'Dzhandzhal kara' at 19 SSR loci sampled over all chromosomes and the 'Kishmish vatkana' alleles at the same loci not shared with 'Sultanina' seem not to be foreign to the gene pool of V. vinifera ssp. sativa proles pontica and orientalis, which include domesticated cultivars from Central Asia and around the Caspian Sea. Thus, the kinship data, in addition to the phenotypic features of these plants, argue against close relatedness to exotic germplasm and the introduction of REN1 from outside of V. vinifera, such as East Asian genotypes or rootstocks of North American origin. Populations of Vitis vinifera ssp. sylvestris, the grapevines that climb trees in natural environments, extend far into Tajikistan and the Western Himalayas. The possibility of an infusion of genes, including resistance alleles if they exist, from indigenous and undomesticated grapevines into Central Asian varieties can not be excluded. The existence of continuous gene flow between natural and cultivated compartments of V. vinifera and the frequency with which these grapevines spontaneously hybridise has been recently estimated in a few Western European sites [42].

An inquiry further back into the genealogy of the powdery mildew-resistant genotypes could provide additional information regarding the origin of REN1. This might be accomplished by DNA analysis of more exhaustive collections of the Asian V. vinifera germplasm. Grape germplasm is very rich with indigenous and introduced accessions in the geographical region where the REN1 resistant genotypes have been found. Viticulture in fertile oases of northern Afghanistan and Uzbekistan was founded by Greek colonists in the fourth century B.C. and had already flourished by the second century B.C., at the time when General Zhang Qian brought viticulture back to China from his expeditions in the far provinces of the Western Han realm and in the modern-day Uzbekistan [43]. The geographical location of Uzbek oases on the harsh northern routes of the Silk Road accelerated the introduction of varieties traded from commercial hubs in the eastern Mediterranean basin (such as 'Sultanina', which is thought to have originated in Iran or Turkey), varieties from the Black and Caspian Seas, and grapes collected in the Xinjiang province of modern China and the Turfan depression where V. vinifera cultivars have been cultivated for over 2,000 years [see Additional file 10]. All of this transiting material might have mixed and cross-pollinated with locally domesticated grapes or indigenous forms of V. vinifera subsp. sylvestris. In particular, Samarqand and its surroundings, where 'Kishmish vatkana' and 'Dzhandzhal kara' were discovered, was an important hub for caravans that headed south crossing the Kashkadarya valley to Termez, the gate to northern Afghanistan, and to the junctions with the southern trade routes [see Additional file 10].

The restriction of the genetic locus in the resistant genotypes and the investigation of the genes contained in the homologous interval of the PN40024 reference sequence provided clues to the possible molecular nature of the REN1 gene and to the structural organisation of the locus. Functional candidates in the interval belong to two gene categories: NBS-LRR genes and cinnamyl alcohol dehydrogenases. NBS-LRRs are by far the most abundant class of plant resistance genes, in particular in the context of host-resistance and in response to biotrophs. The presence of this gene category in the REN1 locus is in agreement with the type of histological responses observed in inoculated leaves of 'Kishmish vatkana' [1]. The necrosis of penetrated host cells resembles the hypersensitive response (HR) triggered by a gene-for-gene type of plant-pathogen recognition. By contrast, it is not known if the presence of CAD genes in the REN1 locus is accidental or if it might have some biological significance. Cinnamyl alcohol dehydrogenase catalyzes the last step in the synthesis of the monomeric precursors of lignin. Different CAD classes are involved specifically in wood development or in responses to biotic stresses.

CAD inhibition partially suppresses papilla-associated cell wall appositions and jeopardises penetration resistance in powdery mildew-cereal interactions [44,45]. CADs are also involved in post-penetration resistance. Intact CAD activity is necessary for expressing an epidermal cell HR in several plant-pathogen interactions [46,47]. This observation has been explained by the role of CADs in phenylpropanoid biosynthesis products derived from cinnamyl alcohol, such as phenolic compounds and free radicals, which are integral to oxidative processes and lignin cell wall encrustations associated with HR. In grapevines, CAD transcript levels were constitutively higher in the powdery mildew resistant V. aestivalis 'Norton' than in the susceptible 'Cabernet Sauvignon' [48]. It is noteworthy that evolution has frequently favoured physical clustering of genes belonging to widely different pathways but with related or complementary functions in defence responses (reviewed in [49]). The accumulation of favourable alleles of physically linked NBS-LRR and CAD genes that trigger and contribute to exerting HR, respectively, would provide an evolutionary advantage to the individual that inherited that block of DNA.

NBS-LRR and CAD genes and pseudogenes in the REN1 locus were generated through several rounds of duplication events, which appear more chaotic than duplications in the most closely related paralogous loci of the grape genome. This DNA block appears inherently prone to structural rearrangements, which makes it somewhat similar to the powdery mildew Mla resistance locus in barley [49], the Rps1-k locus in soybean [50], and Rp1 complex in maize [51]. In addition to segmental duplications and extensive invasion of transposable elements, NBS genes in the REN1 interval also evolved through intragenic recombination. The gene NBSj displayed a sharp variation of nucleotide identity immediately downstream of the MHDV site, the 3' border of the NBS domain, when compared to its paralogues NBSc and NBSk. NBSj is highly similar to the 5' end of NBSc and to the 3' end of NBSk, but substantially dissimilar to these in the complementary parts. The level of identity with either paralogue extends homogeneously beyond each end of the coding region, indicating that NBSj is more likely the result of intragenic recombination between tandemly arrayed paralogues rather than the result of domain-specific accumulation of advantageous mutations. Unequal crossing-over and gene conversion are events of nonreciprocal exchange between similar sequences, which can generate hybrid genes with novel functions by domain swapping (reviewed in [52]). Intragenic recombination frequently occurs between NBS-LRR genes and promotes diversification by reassorting domains between functional paralogues and by recruiting fragments from pseudogenes that may have accumulated mutations in the absence of selective pressure [53,54]. Either intrachromosomal gene conversions or unequal crossing-over rearranged NBS-LRR paralogues in Arabidopsis thaliana [53] and at the maize Rp1 locus [51], respectively. Both events are favoured by close physical vicinity between the partners, by high sequence similarity, and by direct orientation of the gene pair [55], all features that occur in the REN1 locus. In some instances, it was proven that reassortment of domains by unequal crossing over generate chimeric NBS-LRR variants associated with novel race specificities (reviewed in [51]).