Development of marker sets useful in the early selection of Ren4 powdery mildew resistance and seedlessness for table and raisin grape breeding.

The single, dominant powdery mildew resistance locus Ren4 from Vitis romanetii prevents hyphal growth by Erysiphe necator. Previously, we showed that when introgressed into V. vinifera in the modified BC(2) population 03-3004, Ren4 was linked with the simple sequence repeat marker VMC7f2 on chromosome 18-a marker that is associated with multiple disease resistance and seedlessness. However, in the current study, this marker was monomorphic in related breeding populations 05-3010 and 07-3553. To enhance marker-assisted selection at this locus, we developed multiplexed SNP markers using three approaches: conversion of bulked segregant analysis AFLP markers, sequencing of candidate genes and regions flanking known V. vinifera SNPs, and hybridization to the Vitis9KSNP genotyping array. The Vitis9KSNP array was more cost-efficient than all other approaches tested for marker discovery and genotyping, enabling the genotyping of 1317 informative SNPs within the span of 1 week and at a cost of 11 cents per SNP. From a total of 1,446 high quality, informative markers segregating in 03-3004, we developed a haplotype signature of 15 multiplexed SNP markers linked with Ren4 in 03-3004, 5 of which were linked in 05-3010, and 6 of which were linked in 07-3553. Two of these populations segregated for seedlessness, which was tightly linked with Ren4 in 03-3004 (2 cM) but not in 05-3010 (22 cM). Chromosomal rearrangements were detected among these three populations and the reference genome PN40024. Since this is the first application of the Vitis9KSNP array in a breeding program, some suggestions are provided for application of genotyping arrays. Our results provide novel markers for tracking and pyramiding this unique resistance gene and for further functional characterization of this region on chromosome 18 encoding multiple disease resistance and seedlessness.

Identification of race-specific resistance in North American Vitis spp. limiting Erysiphe necator hyphal growth.

Race-specific resistance against powdery mildews is well documented in small grains but, in other crops such as grapevine, controlled analysis of host-pathogen interactions on resistant plants is uncommon. In the current study, we attempted to confirm powdery mildew resistance phenotypes through vineyard, greenhouse, and in vitro inoculations for test cross-mapping populations for two resistance sources: (i) a complex hybrid breeding line, 'Bloodworth 81-107-11', of at least Vitis rotundifolia, V. vinifera, V. berlandieri, V. rupestris, V. labrusca, and V. aestivalis background; and (ii) Vitis hybrid 'Tamiami' of V. aestivalis and V. vinifera origin. Statistical analysis of vineyard resistance data suggested the segregation of two and three race-specific resistance genes from the two sources, respectively. However, in each population, some resistant progeny were susceptible in greenhouse or in vitro screens, which suggested the presence of Erysiphe necator isolates virulent on progeny segregating for one or more resistance genes. Controlled inoculation of resistant and susceptible progeny with a diverse set of E. necator isolates clearly demonstrated the presence of fungal races differentially interacting with race-specific resistance genes, providing proof of race specificity in the grape powdery mildew pathosystem. Consistent with known race-specific resistance mechanisms, both resistance sources were characterized by programmed cell death of host epidermal cells under appressoria, which arrested or slowed hyphal growth; this response was also accompanied by collapse of conidia, germ tubes, appressoria, and secondary hyphae. The observation of prevalent isolates virulent on progeny with multiple race-specific resistance genes before resistance gene deployment has implications for grape breeding strategies. We suggest that grape breeders should characterize the mechanisms of resistance and pyramid multiple resistance genes with different mechanisms for improved durability.

A single dominant locus, ren4, confers rapid non-race-specific resistance to grapevine powdery mildew.

In the present study we screened the progeny of Vitis vinifera × V. romanetii populations segregating for resistance to powdery mildew and determined the presence of a single, dominant locus, Ren4, conferring rapid and extreme resistance to the grapevine powdery mildew fungus Erysiphe necator. In each of nine Ren4 pseudo-backcross 2 (pBC(2)) and pBC(3) populations (1,030 progeny), resistance fit a 1:1 segregation ratio and overall segregated as 543 resistant progeny to 487 susceptible. In full-sib progeny, microscopic observations revealed the reduction of penetration success rate (as indicated by the emergence of secondary hyphae) from 86% in susceptible progeny to below 10% in resistant progeny. Similarly, extreme differences were seen macroscopically. Ratings for Ren4 pBC(2) population 03-3004 screened using natural infection in a California vineyard and greenhouse and using artificial inoculation of an aggressive New York isolate were fully consistent among all three pathogen sources and environments. From 2006 to 2010, Ren4 pBC(2) and pBC(3) vines were continuously screened in California and New York (in the center of diversity for E. necator), and no sporulating colonies were observed. For population 03-3004, severity ratings on leaves, shoots, berries, and rachises were highly correlated (R(2) = 0.875 to 0.996) in the vineyard. Together, these data document a powdery mildew resistance mechanism not previously described in the Vitaceae or elsewhere, in which a dominantly inherited resistance prevents hyphal emergence and is non-race-specific and tissue-independent. In addition to its role in breeding for durable resistance, Ren4 may provide mechanistic insights into the early events that enable powdery mildew infection.

Genome expansion and gene loss in powdery mildew fungi reveal tradeoffs in extreme parasitism.

Powdery mildews are phytopathogens whose growth and reproduction are entirely dependent on living plant cells. The molecular basis of this life-style, obligate biotrophy, remains unknown. We present the genome analysis of barley powdery mildew, Blumeria graminis f.sp. hordei (Blumeria), as well as a comparison with the analysis of two powdery mildews pathogenic on dicotyledonous plants. These genomes display massive retrotransposon proliferation, genome-size expansion, and gene losses. The missing genes encode enzymes of primary and secondary metabolism, carbohydrate-active enzymes, and transporters, probably reflecting their redundancy in an exclusively biotrophic life-style. Among the 248 candidate effectors of pathogenesis identified in the Blumeria genome, very few (less than 10) define a core set conserved in all three mildews, suggesting that most effectors represent species-specific adaptations.

Genomic amplification of the Gret1 retroelement in white-fruited accessions of wild vitis and interspecific hybrids.

Retrotransposons are retrovirus-related mobile sequences that have the potential to replicate via RNA intermediates and increase the genome size by insertion into new sites. The retroelement, Gret1, has been identified as playing a key role in generating fruit color variation in cultivated grape (Vitis vinifera L.) due to its insertion into the promoter of VvMybA1. Fruit color variation is an important distinguishing feature of cultivated grapes and virtually no fruit color variation is observed in wild grape species. The presence and relative copy number of Gret1 was assessed using quantitative PCR on 22 different Vitis species, only four of which (plus interspecific hybrids) are known to contain white accessions. Gret1 copy number was observed to vary by species as well as by color within species and was significantly higher in white-fruited accessions across all taxa tested. Additionally, genomic regions surrounding Gret1 insertion were sequenced in white V. vinifera, hybrid, V. labrusca, V. aestivalis, and V. riparia accessions.

Wine grape (Vitis vinifera L.) color associates with allelic variation in the domestication gene VvmybA1.

During the process of crop domestication and early selection, numerous changes occur in the genetic and physiological make-up of crop plants. In grapevine (Vitis vinifera) numerous changes have occurred as a result of human selection, including the emergence of hermaphroditism and greatly increased variation in berry color. This report examines the effect of human selection on variable skin color by examining the variation present in the gene VvmybA1, a transcriptional regulator of anthocyanin biosynthesis. In over 200 accessions of V. vinifera, the insertion of the retroelement Gret1 in the promoter region of VvmybA1 was in strong association with the white-fruited phenotype. This retroelement was inserted at the same location for each individual in which it was present. Additional polymorphisms in the VvmybA1 gene were also strongly associated with red or pink fruited accessions, including variation that was generated by the excision of Gret1 from the promoter of VvmybA1. Differences in nucleotide diversity were observed between the white and pigmented alleles of VvmybA1, suggesting that the white allele arose only once or a limited number of times. Rarely, association of Gret1 with the white fruited phenotype was not observed, suggesting that the white phenotype can also be obtained through mutation in additional genes. These results provide evidence that variation in one transcriptional regulator has generated an allelic series strongly associated with fruit color variation in cultivated grapevine. These findings provide information about the evolution of grapes since domestication and have direct implications for the regulation of fruit and wine quality of this important crop plant.

Differential gene expression in Phaseolus vulgaris I locus NILs challenged with Bean common mosaic virus.

The Phaseolus vulgaris I locus-Bean common mosaic virus (BCMV; Potyviridae) pathosystem is of critical importance to bean geneticists, breeders and pathologists because of the worldwide distribution of both the virus and germplasm containing this resistance gene. In order to learn more about the molecular responses characteristic of this resistance gene, a cDNA-AFLP screen was conducted on homozygous NILs of P. vulgaris variety 'Black Turtle Soup' (BT), containing either the I locus allele for resistance (BT(II)) or susceptibility (BT(ii)) to BCMV. Eight conditions were compared in a factorial analysis: BT(II) versus BT(ii); mock inoculated versus BCMV inoculated; 26 versus 34 degrees C. Transcripts induced in response to viral infection and that were further responsive to temperature, genotype or both were isolated and cloned. Sequence analysis of the resultant clones revealed several classes of putative genes, including transcription-related and signal transduction-related genes. Review of disease resistance literature suggests further avenues of research involving the candidates isolated in this screen.