Scab Resistance Gene Research Articles

Fine genetic mapping localizes cucumber scab resistance gene Ccu into an R gene cluster

Scab, caused by Cladosporium cucumerinum, is an important disease of cucumber, Cucumis sativus. In this study, we conducted fine genetic mapping of the single dominant scab resistance gene, Ccu, with 148 F(9) recombinant inbred lines (RILs) and 1,944 F(2) plants derived from the resistant cucumber inbred line 9110Gt and the susceptible line 9930, whose draft genome sequence is now available. A framework linkage map was first constructed with simple sequence repeat markers placing Ccu into the terminal 670 kb region of cucumber Chromosome 2. The 9110Gt genome was sequenced at 5× genome coverage with the Solexa next-generation sequencing technology. Sequence analysis of the assembled 9110Gt contigs and the Ccu region of the 9930 genome identified three insertion/deletion (Indel) markers, Indel01, Indel02, and Indel03 that were closely linked with the Ccu locus. On the high-resolution map developed with the F(2) population, the two closest flanking markers, Indel01 and Indel02, were 0.14 and 0.15 cM away from the target gene Ccu, respectively, and the physical distance between the two markers was approximately 140 kb. Detailed annotation of the 180 kb region harboring the Ccu locus identified a cluster of six resistance gene analogs (RGAs) that belong to the nucleotide binding site (NBS) type R genes. Four RGAs were in the region delimited by markers Indel01 and Indel02, and thus were possible candidates of Ccu. Comparative DNA analysis of this cucumber Ccu gene region with a melon (C. melo) bacterial artificial chromosome (BAC) clone revealed a high degree of micro-synteny and conservation of the RGA tandem repeats in this region.

Genetic mapping of 14 avirulence genes in an EU-B04 × 1639 progeny of Venturia inaequalis

Durable resistance to apple scab (Venturia inaequalis (Cke) Wint; anamorph Spilocaea pomi Fries) is one of the major goals of apple (Malus) breeding programs. Since current scab resistance breeding is heavily reliant on genes with gene-for-gene relationships, a good understanding of the genetic basis of host–pathogen interactions needs to be developed for this strategy to be successful. While the genomic organization of apple scab resistance genes has been studied extensively, little is known about the avirulence genes in the pathogen. The progeny of a cross of European V. inaequalis race (1) isolate EU-B04 and race (1,2,8,9) isolate 1639 was used to generate a genetic map based on microsatellite and AFLP markers, and investigated for inheritance of avirulence traits on 20 Malus accessions representing 17 scab resistance genes. The accessions comprised scab differential hosts (0), (1), (2), (8), and (9), and hosts carrying known as well as not previously reported secondary resistance genes, including some identified in crosses that have resistant accessions ‘Geneva’, ‘Dolgo’, Malus baccata jackii, M. micromalus, or ‘Antonovka’ in their pedigree. The latter genes appear to be narrow spectrum genes that showed gene-for-gene relationships as a segregation ratio of Avr:avr=1:1 was observed on 12 accessions, while a ratio of 3:1 was observed on five accessions and a ratio of 7:1 on one host. All progenies were shown to be pathogenic, as all of them were able to infect hosts (0) and (1). A genetic map consisting of 15 major linkage groups (LGs) and spanning 972cM was generated with the aid of 156 markers. The map position of 12 avirulence traits was determined: eight avirulence genes mapped into two separate clusters (1: AvrVdg2, AvrVv1, AvrVu1, AvrVrjrd; and 2: AvrVu2, AvrVh3.2, AvrVs1, AvrVu4), while four avirulence genes (AvrRvi8, AvrVv2, AvrVt57 and AvrVsv) mapped to different LGs. AvrRvi2 and AvrRvi9 also are genetically linked, but showed an interaction with AvrRvi8, the nature of which is unclear. While AvrRvi8 segregated at 1:1 ratio, the other two Avrs segregated at 3:1 ratios. However, all progeny avirulent on hosts (2) and (9) were also avirulent on host (8) and further research is required to determine the avirulence gene relationships. A further two independently segregating loci, AvrRvi1 and AvrRvi6, identified in previous studies, were mapped by inference based on their known linkage to SSR markers. The clustering of avirulence genes in V. inaequalis reflecting the clustering of resistance genes in Malus suggests this pathosystem is a classical example of an “arms race” between host and pathogen. This also seems to apply to the narrow spectrum scab resistance genes, which may imply a larger role in plant defense for these genes than has been assumed to date.

A major resistance gene from Russian apple ‘Antonovka’ conferring field immunity against apple scab is closely linked to the Vf locus

A major scab resistance gene called Va1 was identified in the Russian apple cultivar ‘Antonovka’ (accession APF22) conferring scab resistance under conditions of natural scab infection in the field. After scab scorings over a period of 3 years, a 1:1 segregation was observed in the mapping population 04/214 (‘Golden Delicious’ × ‘Antonovka’). The Va1 resistance gene provides sufficient broad spectrum resistance that is of use in apple resistance breeding and has been assigned Rvi17 according the proposal for a new scab nomenclature (Bus et al., Acta Horticulturae 814:739–746, 2009). Analysis of simple sequence repeats (SSRs) located on the apple linkage group (LG) 1 showed that the Va1 locus is closely linked (1 cM) to SSR CH-Vf1 known to cosegregate with the Vf locus. A tight genetic association was also observed between a specific cleaved amplified polymorphic sequence marker (ARD-CAPS) developed from the HcrVf paralog Vf2ARD present in ‘Antonovka’, but there is no indication yet for a causal relationship with Vf2ARD. Although the whole race spectrum of Va1 is still unknown, it was obvious that it acts against the scab races 6 and 7 which are able to overcome the resistance of Malus floribunda 821. A second resistance factor (named Va2) was studied by race 1-specific scab tests based on grafted 04/214 clones. A 1:1-segregation ratio was observed, too, but 18 “phenotypic recombinants” were found after comparisons with the field scab data of the same genotypes. Va2 was mapped on LG 1 with a genetic distance of about 15 cM above CH-Vf1. The positions of the newly identified ‘Antonovka’ scab resistance factors are compared with previously reported Va mapping approaches and published results from quantitative trait loci analyses performed with different ‘Antonovka’ genotypes.

High-resolution genetic map of the Rvi15 (Vr2) apple scab resistance locus

The Rvi15 (Vr2) apple scab resistance locus found in the GMAL 2473 accession has been previously mapped to the top of the Linkage Group 2 (LG2) by analyzing 89 progeny plants of a cross between ‘Idared’ and GMAL 2473. A new population of 989 progeny plants, derived from a cross between ‘Golden Delicious’ and GMAL 2473, has been analyzed with the two SSR markers CH02c02a and CH02f06, previously found to be associated with Rvi15 (Vr2), and with two published markers derived from NBS sequences (ARGH17 and ARGH37) estimated to map close to the Rvi15 (Vr2) locus. ARGH17 and ARGH37, were found to be the closest markers to the resistance locus, bracketing it within an interval of 1.5 cM. The SSRs mapped one on each side of Rvi15 (Vr2). CH02f06 mapped at 2.9 cM from ARGH37 while CH02a02a mapped at 1.7 from ARGH17. The position of Rvi15 (Vr2) respect to CH02a02a indicates that Rvi15 (Vr2) and Rvi4 (Vh4), a second apple scab gene mapped on the top of LG2, are two different resistance genes. In order to develop even more tightly linked markers to Rvi15 (Vr2), ARGH17 was used as the starting point for chromosome walking through the Rvi15 (Vr2) homolog region of the cv. ‘Florina’. A single ‘Florina’ BAC clone, 36I17, was sufficient to span the homologous locus in the new population’s recombinant progeny. Sequencing of the 36I17 BAC clone allowed identifying seven putative ORFs, including two showing a TIR-NBS-LRR structure. Ten additional markers could be developed mapping within a 1.8 cM interval around the Rvi15 (Vr2) resistance gene. ARGH17 and GmTNL1 markers, the latter also derived from NBS-LRR resistance gene homolog sequence, are the closest markers to Rvi15 (Vr2) bracketing it within a 0.5 cM interval. The availability of 12 markers within the Rvi15 (Vr2) region, all within a small physical distance (kbp) in ‘Florina’, suggests that cloning of the Rvi15 (Vr2) apple scab resistance gene from GMAL 2473 will be possible.

Genome mapping of an apple scab, a powdery mildew and a woolly apple aphid resistance gene from open-pollinated Mildew Immune Selection

Apple is host to a wide range of pests and diseases, with several of these, such as apple scab, powdery mildew and woolly apple aphid, being major causes of damage in most areas around the world. Resistance breeding is an effective way of controlling pests and diseases, provided that the resistance is durable. As the gene pyramiding strategy for increasing durability requires a sufficient supply of resistance genes with different modes of action, the identification and mapping of new resistance genes is an ongoing process in breeding. In this paper, we describe the mapping of an apple scab, a powdery mildew and a woolly apple aphid gene from progeny of open-pollinated mildew immune selection. The scab resistance gene Rvi16 was identified in progeny 93.051 G07-098 and mapped to linkage group 3 of apple. The mildew and woolly aphid genes were identified in accession 93.051 G02-054. The woolly aphid resistance gene Er4 mapped to linkage group 7 to a region close to where previously the genes Sd1 and Sd2, for resistance to the rosy apple leaf-curling aphid, had been mapped. The mildew resistance gene Pl-m mapped to the same region on linkage group 11 where Pl2 had been mapped previously. Flanking markers useful for marker-assisted selection have been identified for each gene.

Genetic Mapping of the Scab Resistance Gene in Cucumber

Scab, caused by Cladosporium cucumerinum Ell. et Arthur, is a prevalent disease of cucumber (Cucumis sativus L.) worldwide. Scab can cause serious losses for cucumber production, especially in protected culture such as high tunnel production. Resistance to cucumber scab is dominant and is controlled by a single gene, Ccu. Breeding for resistant cultivars is the most efficient way to control the disease. Selection for resistance might be made easier if the gene were mapped to linked markers. Thus far, there are no tightly linked (genetic distance less than 1 cM) simple sequence repeat (SSR) markers for the Ccu gene, and no studies on mapping of the Ccu gene in cucumber using SSR markers. The objective of this study was to identify SSR markers for use in molecular breeding of scab resistance. In this study, we used a population of recombinant inbred lines (RILs). The population included 148 individuals derived from the cucumber inbred line 9110 Gt (Ccu Ccu) crossed with line 9930 (ccu ccu). The Ccu gene was mapped to linkage group 2, corresponding to chromosome 2 of cucumber. The flanking markers SSR03084 and SSR17631 were linked to the Ccu gene with distances of 0.7 and 1.6 cM, respectively. The veracity of SSR03084 and SSR17631 was tested using 59 diverse inbred lines and hybrids, and the accuracy rate for the two markers was 98.3%. In conclusion, two SSRs closely linked to scab resistance gene Ccu have been identified and can be used in a cucumber breeding program.

Mapping and functional analysis of four apple receptor-like protein kinases related to LRPKm1 in HcrVf2-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.

RESISTANCE GENE ANALOGUES OF HCRVF2 IN APPLE

The HcrVf2 gene, isolated from the Vf locus, is the first apple scab resistance gene cloned from apple. During the cloning of HcrVf2 it was found that the R-gene belongs to a cluster of four genes and that others HcrVf2-like genes are present in (unknown) loci of the apple genome. The genomic library of the apple cv. 'Florina' (Vf) used to identify HcrVf2 has been screened by hybridization using the whole HcrVf2 sequence as probe to identify all the BAC clones carrying sequences homologous to this gene. Eleven BAC clones not belonging to the contigs assembled to clone HcrVf2 were found. The relative overlapping of the 11 BACs was studied, an SSRs marker was developed from each contig and the SSRs were mapped in the cross 'Fiesta' × 'Discovery'. The analysis of six BAC clones was sufficient to identify all the HcrVf2-like sequences present on these 11 BAC clones. In addition to these BACs, other 7 clones, belonging to the contigs assembled to clone HcrVf2 and known to carrying (unstudied) HcrVf-like sequences, were also investigated. All the 13 BAC clones have been subcloned, subclones carrying HcrVf-like sequences identified, sequenced and for each HcrVf-like sequence a consensus sequence has been generated. Of these 13 BAC clones, 22 sequences showing homologies to HcrVf2 were retrieved. Nine sequences (including HcrVf4) encoded ORFs showing all seven domains found in HcrVf2, and six of them have been shown to be expressed constitutively in young scab uninfected apple leaves of 'Florina' by RT-PCR.

FIRST EVIDENCE OF D-HPLC EFFICIENCY FOR AN AUTOMATED CDNA-AFLP IN THE APPLE SCAB RESISTANCE MODEL

Apple scab resistance is one of the most well-characterized plant-pathogen interactions in woody plant species. After the cloning of the apple scab resistance gene HcrVf2, we aimed at identifying the network of genes that are differentially expressed after pathogen challenge by cDNA-AFLP. As electrophoretic analyses are labour-intensive with only limited potential for automation and the recovery of DNA fragments from gels is cumbersome, we investigated the possibility of performing the cDNA-AFLP analysis by d-HPLC (denaturing high performance liquid chromatography) and automating the DNA fragments collection using the Transgenomic WAVE System. Direct sequencing of the collected DNA fragments was also carried out and our preliminary results are reported.

DEVELOPMENT OF CISGENIC APPLES WITH DURABLE RESISTANCE TO APPLE SCAB

Most of the apple (Malus × domestica) growers are facing serious disease problems with apple scab which is caused by the fungus Venturia inaequalis. Developing a resistant variety in apple through classical breeding is very slow and inefficient. So, we aim at improving existing apple varieties through a new concept called cisgenesis which saves time and effort compared to classical breeding. Malus floribunda proved to be a good source of natural scab resistance genes. The genes HcrVf1 and HcrVf2, consisting of promoter, coding and terminator sequences in their natural configuration, were isolated from Malus floribunda and cloned into the binary vector pMF1. Apple cv. 'Gala' was transformed with pMF1 containing HcrVf1 and HcrVf2, individually or in combination. pMF1 can be used to obtain marker-free plants by recombinase-based excision of a fragment carrying undesired gene sequences, such as antibiotic-selection marker genes, leaving behind only the gene(s)-of-interest and one recombination site. Using this vector it is therefore possible to stack several genes by retransformation using the same selection procedure. In order to obtain durable resistance, we have the intention to combine different resistance genes from Malus either by stacking them one by one or by introducing them all together in one T-DNA. Performance of all different types of cisgenic plants will be evaluated by monitoring scab resistance levels phenotypically and by determining gene expression profiles through quantitative RT-PCR.

Towards improvement of marker assisted selection of apple scab resistant cultivars: Venturia inaequalis virulence surveys and standardization of molecular marker alleles associated with resistance genes

Molecular breeding for pathogen resistance faces two major problems that delay its widespread adoption, resistance breakdown and difficulties in unambiguously identifying the alleles of the markers associated with specific resistance genes. Since the breakdown of the Rvi6 (Vf) gene in the Northern part of Europe breeders have intensified the search for new resistance sources to be introduced into their breeding programs. Alternative major genes to Rvi6 are available (e.g. Rvi2, Rvi4, Rvi5, Rvi10; Rvi11, Rvi12, Rvi13, and Rvi15, respectively Vh2, Vh4, Vm, Va, Vbj, Vb, Vd, Vr2 according to the old apple scab resistance gene nomenclature) but, with few exceptions (i.e., Rvi4, Rvi5 and, Rvi13), they have so far not been incorporated in commercial varieties. Pyramiding, i.e., combining several of these major resistance genes (R-genes) in individual plants, is one of the most promising strategies currently available to develop apple cultivars with durable apple scab resistance. But, which genes are the best suited to produce such new cultivars? Although the most interesting genes are surely those whose resistance so far has not been broken by the pathogen, genes with resistance that has been overcome coupled with only limited spread of the virulence may also be used in the pyramiding process. However, obtaining information on whether an R-gene is overcome and if so, the extent of the spread of the virulence is difficult and time consuming. Furthermore, often such reports are not up-to-date and the correctness of the data is difficult to verify. To solve these problems, the initiative “Monitoring of Venturia inaequalis virulences” has been proposed. The monitoring is based on a network of orchards of selected differential hosts. Incidence and severity of scab on these genotypes will be collected yearly; and after validation, the data will be published through the homepage of the project (www.vinquest.ch). Here, we present an outline of this initiative. A second major obstacle for broad adoption of marker assisted selection is the lack of tools to align marker analyzes performed in different laboratories to unambiguously identify the alleles linked to specific resistances. The identification of the alleles of the markers in coupling with the resistance genes is often very difficult, if the same genotype used to develop the markers is not simultaneously analyzed. In this paper we present an approach to standardize the size of the alleles in coupling with the resistance genes, using easily accessible cultivars. The proposed procedure has been applied to selected markers for the apple scab resistance genes Rvi2, Rvi4, Rvi5, Rvi6, Rvi11, Rvi12, Rvi13, Rvi14 and Rvi15 (respectively Vh2, Vh4, Vm, Vf, Vbj, Vb, Vd, Rvi14 and Vr2 according to the old nomenclature).

IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF VF-LIKE CANDIDATE GENES IN CULTIVATED APPLES AND SELECTIONS FROM MALUS SIEVERSII

The genetic locus for resistance to apple scab most frequently used in apple breeding is Vf, derived from Malus floribunda 821. For the Vf locus a cluster of four resistance gene paralogs encoding receptor-like proteins (RLP) with similarity to the tomato Cf resistance genes is known. Based on nucleotide alignments of published sequences of HcrVf1, HcrVf2, and HcrVf3, PCR primers were designed from the variable leucine-rich repeat (LRR) C1 subdomain. PCR products with high amino acid identity (85 to 100%) to HcrVf1 and HcrVf2 were obtained not only from M. floribunda 821 and Vf cultivars, but also from other apple scab resistance sources, such as ‘Russian Seedling’ R12740-7A or some ‘Antonovka’ accessions tested. A series of thirteen HcrVf candidate genes have been partly cloned from the PCR fragments spanning N-terminal LRRs 20 - 30. Expression analyses and mapping were focused on two specific Vf-homologous candidate genes, Vf1RSA and Vf2ARD, identified in resistant Malus genotypes known for carrying other scab resistance genes than Vf. RT-PCR experiments showed that Vf1RSA as well as the Vf2ARD are expressed under pathogen-free conditions. The results of a quantitative PCR-based transcription profiling performed for Vf2ARD suggest that this gene is scab-inducible in some resistant cultivars. Vf1RSA has been mapped, so far, without any recombination to the known Vf gene cluster on linkage group LG 1. The second candidate gene Vf2ARD is separated from the Vf gene cluster with a genetic distance of about 2 cM and might be a member of a second Vf-like locus on apple linkage group LG 1.

CISGENESIS IS A PROMISING APPROACH FOR FAST, ACCEPTABLE AND SAFE BREEDING OF PIP FRUIT

Introgression of traits from wild germplasm into pip fruit cultivars by means of classical breeding is painstakingly slow. Introgression of e.g., the apple scab resistance gene Vf from Malus floribunda 821 into marketable high quality apple cultivars took approximately 50 years. In the mean time the Vf resistance is being broken down in Europe. For durable resistance, different resistance genes should be accumulated. However, this may take another series of decades. This slow tempo is caused mainly by the long juvenile period of apple and the phenomenon that not only the allele of interest is inherited by the progeny, but also hundreds of unwanted alleles. The process would be much faster if only the allele of interest was inserted, without unwanted alleles. This can be achieved by cisgenesis. We defined cisgenesis as genetic modification of plants, inserting alleles of the plant itself or from crossable relatives. The allele should contain its native introns and should be flanked by its native promoter and terminator in sense-orientation. If the plant is equipped with foreign genes from outside the gene pool of the conventional breeder, the plant is named transgenic. Inquiries indicate that cisgenic plants are more acceptable to consumers than transgenic plants. As the phenotypic result of cisgenesis can, in principle, also be obtained by means of conventional breeding or translocation breeding, cisgenic plants are as safe as plants from conventional breeding or mutation breeding. Therefore we have proposed to treat cisgenic plants like conventionally bred plants, by exempting cisgenic plants from the GMO regulation. The number of isolated, functionally analysed genes and their alleles from fruit tree crops is increasing. Also technologies are available for introduction of these alleles without leaving selection genes behind. Cisgenesis is combining the knowledge of native alleles with marker free technologies. Cisgenesis is a promising path for utilizing the wealth of knowledge on plant genes to the benefit of the society in a fast, safe, and acceptable way.

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.

Pyramidisieren von Schorfresistenzen in der Apfelzüchtung

High quality disease resistant apples are the main breeding objective in the programme at Agroscope Changins-Wadenswil (ACW). Apple scab caused by the fungus Venturia inaequalis is still the most important disease in apple production. To ensure the durability of scab resistance we are combining different resistance genes (pyramiding). Moreover, we are incorporating also resistances against powdery mildew (Podosphaera leucotricha) and fire blight (Erwinia amylovora). Principles and methods of resistance breeding are outlined and pyramiding of resistances is illustrated by analysing crosses between two cultivars carrying different scab resistance genes (Vf and Vh2). Crosses are being analysed applying marker-assisted selection (MAS) to detect plants carrying multiple resistances. Phenotypic screening of seedlings for scab symptoms and MAS results are compared and the observed segregations into resistant and susceptible progenies discussed.

HcrVfparalogs are present on linkage groups 1 and 6 ofMalus

Molecular markers derived from resistance gene analogs of HcrVf2, the first apple resistance gene cloned, may pave the way to the cloning of additional apple scab resistance genes. The Malus xdomestica 'Florina' (Vf) bacterial artificial chromosome (BAC) genomic library was screened by hybridization using HcrVf2 as a probe. Positive BAC clones were assembled into contigs and microsatellite markers developed from each contig mapped. Only linkage groups 1 and 6 contained HcrVf2 paralogs. On linkage group 1, five loci in addition to the Vf locus were identified. A single locus was detected on linkage group 6. Representative BAC clones of these loci including the Vf locus were sequenced and the gene structure compiled. A total of 22 sequences, showing high sequence similarity to HcrVf2, were identified. Nine sequences were predicted to encode all seven protein domains described in HcrVf2, while three were truncated. Transcriptional analysis indicated that six genes with a complete HcrVf-like structure were constitutively expressed in young uninfected leaves of 'Florina'. The map position of each HcrVf analog was compared with the location of the major apple scab resistance genes. None of the major genes conferring scab resistance co-localized with HcrVf paralogs, indicating that they are unlikely to belong to the leucine-rich repeat - transmembrane class, which includes the Vf gene.

Identification and molecular analysis of candidate genes homologous toHcrVfgenes for scab resistance in apple

AbstractThe genetic locus for resistance to apple scab most frequently used in apple breeding isVf, derived fromMalus floribunda821. For theVflocus a cluster of four resistance gene paralogs (called asHcrVfgenes) encoding receptor‐like proteins (RLP) with similarity to the tomatoCfresistance genes is known. Based on published sequences forHcrVf1andHcrVf2PCR primers were designed from the domain B and the variable leucine‐rich repeat (LRR) C1 subdomain. PCR products with high amino acid identity (85–100%) toHcrVf1andHcrVf2were obtained not only fromM. floribunda821 andVfcultivars but also from other apple scab resistance sources, such as ‘Russian Seedling’ R12740‐7A (Vrresistance) or ‘Antonovka polutorafuntovaya’ (VAresistance). A series of 13HcrVfcandidate genes have been partly cloned from the PCR fragments spanning N‐terminal LRRs 20–30. A considerable number of amino acid exchanges within the solvent‐exposed xxLxLxx structural motives were detected among the homologous sequences. Expression analyses and mapping focused on a selectedVf‐homologous candidate gene (calledVf2ARD) identified in resistantMalusgenotypes known for carrying other scab resistance genes thanVf. RT‐PCR experiments showed thatVf2ARDis expressed under pathogen‐free conditions. The results of a quantitative PCR‐based transcription profiling suggest that this gene is scab‐inducible in some resistant cultivars.Vf2ARDhas been mapped on linkage group LG 1. It is separated from theVfgene cluster with a genetic distance of about 2 cM and might be a member of a secondVf ‐like locus on apple linkage group LG 1.

Development of AFLP and CAPS markers linked to the scab resistance gene, Rvn2, in an inter-specific hybrid pear (Pyrus spp.)

SummaryScab, caused by Venturia nashicola, is one of the most damaging diseases in pear. The development of varieties resistant to scab has long been an aim of Asian pear breeding programmes. The application of molecular markers to select scab-resistant plants will increase the efficiency of breeding programmes. To identify DNA markers linked to the scab-resistance gene named Rvn2 in PS2-93-3-98, the progeny of a cross between PS2-93-3-98 and ‘Yali’ pear was studied. As scab-resistance clearly segregated 1:1 in the F1 progeny, Rvn2 is a single dominant gene. Bulked segregant analysis and amplified fragment length polymorphism (AFLP) analysis were then applied to identify markers associated with Rvn2. Three AFLP markers linked to Rvn2 were identified. Mapping of the markers indicated that the three selected markers, E-AGT/M-CCA234, E-ATT/M-CCG328, and E-GGT/M-TCT217, were located 4.9, 3.2, and 0.8 cM from the Rvn2 locus, respectively. Rvn2 was found to be located in a different linkage group from previously identified scab-resistance gene loci. For marker-assisted selection of scab resistance, two of the AFLP markers (E-AGT/M-CCA234 and E-GGT/M-TCT217) were converted into cleaved amplified polymorphic sequence (CAPS) markers. These CAPS markers, designated PSC217-Xho I and PSC234-Hae III could distinguish between resistant and susceptible individuals.These CAPS markers therefore have the potential to increase the efficiency of selection for scab-resistance in pear breeding programmes.

The Validity of Marker-assisted Selection Using DNA Markers Linked to a Pear Scab Resistance Gene (Vnk) in Two Populations

Marker-assisted selection (MAS) using 5 DNA markers linked to the pear scab resistance derived from ‘Kinchaku’ (Vnk) was examined in a pear breeding program. The relationship between DNA markers and scab resistance was investigated. All individuals without both DNA markers of STS-OPW2 and STS-OPO9 derived from ‘Kinchaku’ showed scab symptoms with heavy sporulation on leaves. Individuals possessing these markers exhibited no symptoms, or necrosis with or without weak sporulation on petioles which was localized and did not affect growth, and were classified as resistant. Hence, elimination of individuals that have neither STS-OPW2 nor STS-OPO9 would be a suitable strategy to select for scab resistance in Japanese pear breeding when introgressing Vnk from ‘Kinchaku’.

Use of Phenotypic and MAS Selection Based on Bulk Segregant Analysis to Reveal the Genetic Variability Induced by Artificial Hybridization in Apple

Phenotypic selection in apple seedling populations (F1), derived from semidiallel hybridizations between genitors with different peculiarities (Florina and Liberty, resistant to apple scab attack; Starkrimson and Golden spur, with spur ideotype) was completed with Marker Assisted Selection (MAS), based on Bulk Segregant Analysis (BSA) technique. Molecular analyses performing by Bulk Segregant Analysis were intended to indentifying markers for scab resistance, powdery mildew resistance and architectural tree structure (ideotype) with bulks of five to six plants per groups, and testing the bulks with RAPD polymorphic primers. The polymorphic bands between the bulks were mostly associated to apple scab resistance genes. Concerning powdery mildew resistance and architectural tree structure analysis the results are inconclusive. Although it seems that the primer 54 showed a polymorphic band in a resistant to powdery mildew hybrid, which is missing in the sensitive hybrids, the result is only apparently favourable since this band is present in both parental forms (Starkrimson, tolerant to powdery mildew and Liberty, relative susceptible). Molecular profile of the standard ideotype and spur ideotype, resulted in hybrids originated in standard parental form (Florina) and spur parental form (Golden spur), were usually monomorphic. Genetic association among the parental molecular polymorphism for scab resistance and the other two important traits (powdery mildew resistance and ideotype), was not conclusive.