RAPD markers linked to the Vf gene for scab resistance in apple

Scab (Venturia inaequalis) is one of the most harmful diseases of apple, significantly affecting world apple production. The identification and early selection of resistant genotypes by molecular markers would greatly improve breeding strategies. Bulked segregant analysis was chosen for the identification of RAPD markers linked to the Vf scab resistant gene. Five different RAPD markers, derived from the wild species Malus floribunda. 821, were identified, and their genetic distance from Vf gene was estimated. The markers OPAM19₂₂₀₀ and OPAL07₅₈₀ were found to be very closely linked to the Vf gene. This result was indirectly confirmed by the analysis of resistant genotypes collected from various breeding programmes. Except for cv Murray, which carries the Vm gene, all these resistant genotypes showed the markers OPAM19₂₂₀₀ and OPAL07₅₈₀.     

Identification and mapping of the novel apple scab resistance gene Vd3

Apple scab, caused by the fungal pathogen Venturia inaequalis, is one of the most devastating diseases for the apple growing in temperate zones with humid springs and summers. Breeding programs around the world have been able to identify several sources of resistance, the Vf from Malus floribunda 821 being the most frequently used. The appearance of two new races of V. inaequalis (races 6 and 7) in several European countries that are able to overcome the resistance of the Vf gene put in evidence the necessity of the combination of different resistance genes in the same genotype (pyramiding). Here, we report the identification and mapping of a new apple scab resistance gene (Vd3) from the resistant selection “1980-015-25” of the apple breeding program at Plant Research International, The Netherlands. This selection contains also the Vf gene and the novel V25 gene for apple scab resistance. We mapped Vd3 on linkage group 1, 1 cM to the south of Vf in repulsion phase to it. Based on pedigree analysis and resistance tests, it could be deduced that 1980-015-25 had inherited Vd3 from the founder “D3.” This gene provides resistance to the highly virulent EU-NL-24 strain of race 7 of V. inaequalis capable of overcoming the resistance from Vf and Vg. JMS and SGJ contributed equally to this work     

Phenotypic Reaction and Genetic Analysis Using AFLP-derived SCARs for Resistance to Apple Scab

Six sequence‐characterized amplified region (SCAR) markers linked to the apple scab resistance gene Vf were evaluated for their utility in marker‐assisted selection (MAS) in apple breeding. Of the six SCARs used in this study, ACS‐6 was located left of the Vf gene, ACS‐7 and ACS‐9 co‐segregated with Vf, and ACS‐8, ACS‐4, ACS‐5 were located right of the Vf gene. Three families derived from crosses between scab‐resistant and scab‐susceptible cultivars, including ‘Liberty’ × ‘Deljub’, ‘Liberty’ × ‘Delcorf’, and ‘Florina’ בDelcorf’, previously screened for scab resistance following greenhouse inoculation with the fungal pathogen Venturia inaequalis, were genotyped and compared with phenotypic reactions to scab infection in the field. For each family, a subset progeny of 30 seedlings (propagated onto Malling 9 rootstock and of 7 years old) was selected based on fungal sporulation according to the following scheme. Ten seedlings with no visible scab sporulation on leaves were given phenotypic scores of 0 (deemed resistant); 10 seedlings with moderate scab sporulation were given phenotypic scores of 1.0 (deemed moderately resistant); and 10 seedlings with heavy sporulation were given phenotypic scores of 2.0 (deemed susceptible). DNA was isolated from leaf tissue collected from all 90 seedlings, parents and Malus floribunda 821, the original source of the Vf gene, and screened with all six SCARs. All six SCARs were present in the two scab‐resistant parents, ‘Liberty’ and ‘Florina’, and M. floribunda 821; while, the two scab‐susceptible parents, ‘Deljub’ and ‘Delcorf’, lacked all SCARs. All SCARs were either present or absent in varying numbers of seedlings in each progeny with phenotypic ratings of either 0 (resistant) or 1.0 (moderately resistant); while all seedlings with phenotypic ratings of 2.0 (susceptible) lacked all SCARs. The inconsistencies between phenotypic scab ratings and SCAR marker data are discussed.     

Vf scab resistance of Malus

The apple production in temperate regions with spring rains, the Scab caused by the fungus Venturia inaequalis is the most important constraint. To produce spotless apples and avoid damage that develops during storage, growers apply fungicide on a regular or weather-determined basis. All major apple cultivars are highly susceptible to this disease. To limit the need for fungicide applications, apple breeders are currently introgressing disease resistance from wild Malus accessions into commercial lines. The first attempts to do this were made 100 years ago. As apples are self-incompatible, pseudo-backcrossing is used to eliminate unwanted traits from wild Malus and select new cultivars that are attractive to both producers and consumers. This process, from the first cross of a commercial cultivar with a wild, disease-resistant Malus, is extremely long due to apple’s long juvenile phase, the need for more than seven backcross steps and the high heterozygosity of this genus. Therefore, most of today’s scab-resistant cultivars rely on a single introduction of scab resistance from Malus floribunda 821, referred to as Vf. In this paper, we trace the history of Vf from its initial identification through its use in breeding and commercial production. We sum up the literature describing how and where Vf resistance has been overcome by new pathotypes of V. inaequalis. Finally, we describe the current knowledge of the genes behind Vf resistance, its mode of action and the use of Vf genes in gene technology.     

Introgression of the Vf source of scab resistance and distribution of linked marker alleles within the Malus gene pool

A chromosomal region originating from Malus floribunda 821 confers Vf scab resistance to many isolates of Venturia inaequalis. Twelve DNA markers located in this region were used to scan the equivalent of 31 cM in 98 Malus accessions. This allowed a molecular diagnosis of a source of resistance in apple germplasm with the aid of pedigree information, and in the context of a limited marker survey representing other chromosomes. At least five marker alleles were present in all scab-resistant breeding selections or varieties arising from M. floribunda. The validity of findings based on RAPD markers was confirmed with SCAR assays and Southern-hybridisation experiments. The order of markers determined in previous mapping studies was confirmed and sets of recombinants identified that establish reliable fine-mapping orders within 0.7 cM of the resistance locus. None of the marker alleles were present in the accessions that are either susceptible or possess weak polygenic resistance to scab. The presence of some alleles corresponding to those present at least 5.3 cM from Vf in M. floribunda was detected in some accessions. Other major sources of scab resistance do not appear to possess alleles in common with the Vf region, which will simplify future allelism tests. The results are discussed in the context of the introgression of resistance loci together with marker-assisted selection. The use of breeding pedigrees to assist in fine-scale mapping and map-based cloning is discussed. Received: 16 February 1999 / Accepted: 11 March 1999     

Narrowing down the region of the<Emphasis Type="Italic"> Vf</Emphasis> locus for scab resistance in apple using AFLP-derived SCARs

A narrow-down strategy to restrict the Vf region, which controls resistance to the fungal disease apple scab in apple, to a genetic distance of 0.4 cM is presented. Using 11 AFLP-derived SCARs and three RAPD-derived SCARs, all linked to the Vf gene, we subjected 1,412 scab-resistant individuals from 16 mapping populations to genotype analysis. Eleven recombinant individuals were identified within a genetic distance of 0.9 cM around the Vf gene. Using these 11 recombinants, we achieved fine-resolution of several AFLP-derived SCAR markers surrounding the Vf gene, resulting in the following genetic linkage map: ACS-6 and ACS are located left of the Vf gene at genetic distances of 0.2 cM and 0.1 cM, respectively; ACS-7 and ACS-9 are inseparable from the Vf gene; ACS-8, ACS-10, and ACS-4 are located to the right of the Vf gene at genetic distances of 0.1 cM, 0.4 cM, and 0.5 cM, respectively; the remaining five SCARs—ACS-11, ACS-5, ACS-2, ACS-1, and AL07—are inseparable and are located right of the Vf gene at a genetic distance of 0.7 cM. By integrating this linkage data with our previous physical map, we generated a revised map of the narrowed-down region of Vf.Communicated by P. Langridge     

Identification of functional apple scab resistance gene promoters

Apple scab (Venturia inaequalis) is one of the most damaging diseases affecting commercial apple production. Some wild Malus species possess resistance against apple scab. One gene, HcrVf2, from a cluster of three genes derived from the wild apple Malus floribunda clone 821, has recently been shown to confer resistance to apple scab when transferred into a scab-susceptible apple variety. For this proof-of-function experiment, the use of the 35S promoter from Cauliflower mosaic virus was reliable and appropriate. However, in order to reduce the amount of non-plant DNA in genetically modified apple to a minimum, with the aim of increasing genetically modified organism acceptability, these genes would ideally be regulated by their own promoters. In this study, sequences from the promoter region of the three members of the HcrVf gene family were compared. Promoter constructs containing progressive 5 deletions were prepared and used for functional analyses. Qualitative assessment confirmed promoter activity in apple. Quantitative promoter comparison was carried out in tobacco (Nicotiana glutinosa) and led to the identification of several promoter regions with different strengths from a basal level to half the strength of the 35S promoter from Cauliflower mosaic virus.     

Expression profiling in HcrVf2-transformed apple plants in response to Venturia inaequalis

Apple scab resistance is one of the most well-characterized plant–pathogen interactions in a woody plant species. While the HcrVf2 gene from the wild apple Malus floribunda 821 has proved capable of conferring scab resistance to the susceptible cv. Gala after genetic transformation, its identification represents only the first step in understanding the molecular mechanisms and, hence, the network of genes underlying the defence response. We used a PCR-based suppression subtractive hybridization to identify apple genes that are differentially expressed after Venturia inaequalis inoculation. Subtractive hybridization was performed between cDNA from challenged leaves of HcrVf2-resistant transgenic Gala and susceptible cv. Gala plants. A library of 523 unigenes was constructed and characterized by assigning a putative function via comparison with public databases. This set of pathogen-modulated apple genes includes many defence-related genes and is therefore an important source of information for understanding the molecular basis of the Malus–V. inaequalis interaction. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Construction of a 550 kb BAC contig spanning the genomic region containing the apple scab resistance gene Vf

A positional cloning project was started in apple with the aim of isolating the Vf resistance gene of Malus floribunda 821. Vf confers resistance against apple scab, the most important disease in apple orchards. A chromosome walk starting from two molecular markers (M18-CAPS and AM19-SCAR) flanking Vf was performed, using a bacterial artificial chromosome (BAC) library containing inserts of the cultivar Florina, which is heterozygous for Vf. Thirteen BAC clones spanning the region between the two markers were identified in nine chromosome walking steps. The size of the resulting contig is approximately 550 kb. In order to map the Vf region in more detail, we analyzed over 2000 plants from different populations segregating for Vf with markers produced from BAC end sequences. In this way, we were able to restrict the possible location of the Vf gene to a minimum of five clones spanning an interval of approximately 350 kb. Received: 4 July 1999 / Accepted: 16 September 1999     

Genetics of resistance in apple against <Emphasis Type="Italic">Venturia inaequalis</Emphasis> (Wint.) Cke

Apple (Malus × domestica) is the third important fruit in terms of production and consumption worldwide. Apple scab caused by Venturia inaequalis is the most devastating disease of apple. In the apple-growing regions, many fungicides are sprayed to control the disease leading to increase in the production cost. Development of scab-resistant cultivars is the long-lasting solution to control the disease. In apples, more than 20 major scab resistance genes have been identified in various cultivars and few wild relatives. Of all these genes, Rvi6 derived from Malus floribunda has been most extensively used in different breeding programs. Gene for gene interactions of these resistance genes with the avirulence genes from V. inaequalis have been understood in many cases. QTL-based polygenic resistance has also been characterized in apple. Nucleotide Binding Site Leucine-Rich Repeats (NBS-LRR) have been identified from the apple genome and many of them have been characterized from the scab resistance region. Molecular markers associated with most of the major scab resistance genes have been identified and their position has been mapped on different linkage groups. Marker-assisted selection (MAS) can be helpful in speeding up and accurately identifying the scab-resistant parents and progeny. Pyramiding of several major resistance genes can be undertaken for more durable resistance against apple scab. The present paper reviews the Malus-Venturia pathosystem, current status of knowledge about scab resistance genes, and their application in breeding against apple scab.     

Multiple field and glasshouse assessments increase the reliability of linkage mapping of the Vf source of scab resistance in apple

 Apple scab, caused by the fungus Venturia inaequalis (Cke.) Wint., is an important disease in commercial apple production. A mapping population of 155 individuals, derived from a cross between the apple varieties ‘Prima’ (resistant)בFiesta’ (susceptible), was scored for response to the disease in replicated field and glasshouse trials throughout Europe. Twenty data sets were selected and cluster analysis was used to form a consensus score for the population fitting a 1 : 1 segregation ratio of resistance:susceptibility. The progeny were scored with molecular markers. A detailed map covering 54 cM of the ‘Prima’ linkage group containing the Vf gene for scab resistance was constructed using 24 molecular markers linked to the resistance gene. One isoenzyme marker (Pgm-1), six RFLP markers and 17 RAPD markers formed a linkage group with the consensus measure of resistance to scab. Four marker bridges were established with the corresponding ‘Fiesta’ linkage group with additional markers (one isozyme, one RFLP, three RAPD and one AFLP). A low chi-square value indicated a good fit of the marker ordering, which was in close agreement with previously reported linkage positions for some of the markers and Vf. Differences were observed in the ability of different scoring methods to resolve susceptible and resistant classes. The results obtained for the consensus classification of resistance to scab for the population may suggest the presence of virulent inocula at some sites, which could overcome the Vf gene for resistance. The consequences of relying on individual scoring occasions for studying Vf scab resistance are discussed in the context of linkage analysis, conventional breeding selection, and marker-assisted selection. Received: 23 July 1997 / Accepted: 31 October 1997     

DNA markers linked to Malus floribunda 821 scab resistance

Breeding resistant apple plants is an alternative way to control fungal pathogens reducing the environmental impact due to the use of pesticides. The breeding of apple cultivars resistant to Venturia inaequalis could be much improved by marker-assisted selection. A molecular marker closely linked to the resistance locus called Vf could replace selection based on infection studies. To find such molecular markers, DNA of progenies from crossings of a resistant and a susceptible apple tree was subject to bulked segregant analysis. Two markers were found with a genetic distance of 10.6% and 19.7% recombination frequency to the Vf locus.     

A detailed linkage map around an apple scab resistance gene demonstrates that two disease resistance classes both carry the V f gene

A detailed genetic map has been constructed in apple (Malus x domestica Borkh.) in the region of the v _f gene. This gene confers resistance to the apple scab fungus Venturia inaequalis (Cooke) Wint. Linkage data on four RAPD (random amplified polymorphic DNA) markers and the isoenzyme marker PGM-1, previously reported to be linked to the v _f gene, are integrated using two populations segregating for resistance to apple scab. Two new RAPD markers linked to v _f (identified by bulked segregant analysis) and a third marker previously reported as being present in several cultivars containing v _f are also placed on the map. The map around v _f now contains eight genetic markers spread over approximately 28 cM, with markers on both sides of the resistance gene. The study indicates that RAPD markers in the region of crab apple DNA introgressed with resistance are often transportable between apple clones carrying resistance from the same source. Analysis of co-segregation of the resistance classes 3A (weakly resistant) and 3B (weakly susceptible) with the linked set of genetic markers demonstrates that progeny of both classes carry the resistance gene.This work was supported in part by grants from the New Zealand Foundation for Research Science and Technology (FoRST) Programme 94-HRT-07-366 and ENZA New Zealand (International)     

Molecular selection in apple for resistance to scab caused by Venturia inaequalis

Large-scale marker-assisted selection requires highly reproducible, consistent and simple markers. The use of genetic markers is important in woody plant breeding in general, and in apple in particular, because of the high level of heterozygosity present in Malus species. We present here the transformation of two RAPD markers, which we found previously to be linked to the major scab resistance gene Vf, into more reliable and reproducible markers that can be applied directly to apple breeding. We give an example of how the use of such markers can speed up selection for the introduction of scab resistance genes into the same plant, reducing labour and avoiding time-consuming test crosses. We discuss the nature and relationship of the scab resistance gene Vf to the one present in Nova Easygro, thought to be Vr.     

Construction and characterization of a bacterial artificial chromosome library of apple

 A bacterial artificial chromosome (BAC) library has been constructed from apple (Malus×domestica Borkh.) using the variety “Florina”, which is resistant to scab (Venturia inaequalis) by virtue of the Vf gene. Since apple leaves are rich in polyphenols, high-molecular-weight DNA was extracted from leaf nuclei with a protocol adapted to apple. The nuclei were then embedded in agarose microbeads and partially digested by varying ratios of EcoRI to EcoRI methylase. The resulting DNA fragments were size-selected by pulsed-field gel electrophoresis, ligated to the BAC cloning vector pECBAC1 and transformed into Escherichia coli cells by electroporation. A total of 36 864 recombinant clones (BACs) were obtained. The library has an average insert size of 120 kb and represents approximately 5×apple haploid-genome equivalents. It was screened with six cDNA probes using the chemiluminescent DIG system. An average of 4.4 clones was detected for each locus. The apple BAC library will be used to isolate the Vf scab resistance gene through map-based cloning. In this connection the library was screened with a marker closely linked to the Vf gene and six positive clones have been isolated. This library should thus be well suited for map-based gene cloning, in particular for the isolation of the Vf gene and for the construction of a physical map of the apple genome. Received: 19 February 1998 / Accepted: 30 April 1998     

Stability of RAPD fingerprints in potato: Effect of source tissue and primers

Variations in random amplified polymorphic DNA (RAPD) profiles from leaf, stem, root, and tuber tissues were observed in case of two glasshouse grown potato cultivars using 40 decamer primers suggesting possible danger of cultivar misidentification. Genomic DNA extracted from the above four tissues of four in vitro grown potato cultivars, however, produced more uniform RAPD fingerprints. A significant effect of random primers on fingerprint uniformity was observed in case of both glasshouse and in vitro grown samples. A new concept of stability index for random primers based on homogeneity of RAPD profiles obtained from different tissues of a single plant have been introduced. It is concluded that RAPD analysis of genomic DNA extracted from any tissue of in vitro grown potato plants using 14 selected decamer primers could be used to develop RAPD fingerprints for identification of Indian potato cultivars.     

Mapping and functional analysis of four apple receptor-like protein kinases related to <Emphasis Type="Italic">LRPKm1</Emphasis> in <Emphasis Type="Italic">HcrVf2</Emphasis>-transgenic and wild-type apple plants

The Malus–Venturia inaequalis interaction is the most studied plant–pathogen interaction involving a woody species. Besides the cloning of an apple scab resistance gene HcrVf2, several sequences have been recently identified that are modulated after pathogen recognition in Vf-resistant genotypes. Among these, there is a putative leucine-rich repeat receptor-like protein kinase from the apple scab-resistant cv. Florina, named LRPKm1 that is induced after V. inaequalis inoculation and salicylic acid treatment. In this work, the isolation, characterization, and mapping of four new genes belonging to the LRPKm multigene family are reported. According to their cumulative expression profiles in HcrVf2-transgenic and wild-type apple plants treated with V. inaequalis, LRPKm genes have been divided in two groups. LRPKm1 and LRPKm3, giving a response related to the presence of HcrVf2, are probably involved in the recognition of pathogen-derived signals. LRPKm2 and LRPKm4, with an expression profile unrelated to the HcrVf2 gene, are putatively involved in the plant basal defense. Furthermore, we have localized LRPKm proteins at the cytological level in the plasma membrane of epidermal cells in resistant genotypes following pathogen challenge, thus confirming software predictions and molecular results. The possible involvement of LRPKm proteins in apple scab resistance and in the plant basal defense makes them attractive for a better comprehension of the molecular mechanisms of the signal transduction pathways after pathogen recognition.     

Towards the map-based cloning of Vf: fine and physical mapping of the Vf Region

A map-based cloning scheme is being used to isolate the Vf resistance gene of apple against the fungus Venturia inaequalis. Vf is a major dominant gene that is inherited in a Mendelian manner and influenced by minor genes that modify its activity. The two recently published markers M18 and AL07, bracketing Vf, were tested on 1179 progeny plants of three crosses to fine-map Vf. M18 and AL07 were positioned at 0.2 cM and 1.1 cM from Vf respectively, for a total distance between the two markers of 1.3 cM. Physical mapping by pulsed-field gel electrophoresis, using M18 and AL07 as probes, demonstrated that both markers hybridize to a common 870 kb NotI restriction fragment. We therefore found a relationship between physical and genetic distance of 670 kb/cM in the Vf region. This led us to the conclusion that a chromosome walk using the recently published apple BAC library starting from M18 and AL07 is feasible. Received: 8 February 1999 / Accepted: 16 March 1999     

Molecular characterization of cisgenic lines of apple ‘Gala’ carrying the Rvi6 scab resistance gene

Using resistance genes from a crossable donor to obtain cultivars resistant to diseases and the use of such cultivars in production appears an economically and environmentally advantageous approach. In apple, introgression of resistance genes by classical breeding results in new cultivars, while introducing cisgenes by biotechnological methods maintains the original cultivar characteristics. Recently, plants of the popular apple ‘Gala’ were genetically modified by inserting the apple scab resistance gene Rvi6 (formerly HcrVf2) under control of its own regulatory sequences. This gene is derived from the scab‐resistant apple ‘Florina’ (originally from the wild apple accession Malus floribunda 821). The vector used for genetic modification allowed a postselection marker gene elimination to achieve cisgenesis. In this work, three cisgenic lines were analysed to assess copy number, integration site, expression level and resistance to apple scab. For two of these lines, a single insertion was observed and, despite a very low expression of 0.07‐ and 0.002‐fold compared with the natural expression of ‘Florina’, this was sufficient to induce plant reaction and reduce fungal growth by 80% compared with the scab‐susceptible ‘Gala’. Similar results for resistance and expression analysis were obtained also for the third line, although it was impossible to determine the copy number and TDNA integration site–such molecular characterization is requested by the (EC) Regulation No. 1829/2003, but may become unnecessary if cisgenic crops become exempt from GMO regulation.