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.     

Identification and validation of RAPD and SCAR markers linked to the gene <Emphasis Type="Italic">Er3</Emphasis> conferring resistance to <Emphasis Type="Italic">Erysiphe pisi</Emphasis> DC in pea

Three genes, er1, er2 and Er3, conferring resistance to powdery mildew (Erysiphe pisi) in pea have been described so far. Because single gene-controlled resistance tends to be overcome by evolution of pathogen virulence, accumulation of several resistance genes into a single cultivar should enhance the durability of the resistance. Molecular markers linked to genes controlling resistance to E. pisi may facilitate gene pyramiding in pea breeding programs. Molecular markers linked to er1 and er2 are available. In the present study, molecular markers linked to Er3 have been obtained. A segregating F₂ population derived from the cross between a breeding line carrying the Er3 gene, and the susceptible cultivar ‘Messire’ was developed and genotyped. Bulk Segregant Analysis (BSA) was used to identify Random Amplified Polymorphic DNA (RAPD) markers linked to Er3. Four RAPD markers linked in coupling phase (OPW04_637, OPC04_640, OPF14_1103, and OPAH06_539) and two in repulsion phase (OPAB01_874 and OPAG05_1240), were identified. Two of these, flanking Er3, were converted to Sequence Characterized Amplified Region (SCAR) markers. The SCAR marker SCW4₆₃₇ co-segregated with the resistant gene, allowing the detection of all the resistant individuals. The SCAR marker SCAB1₈₇₄, in repulsion phase with Er3, was located at 2.8 cM from the gene and, in combination with SCW4₆₃₇, was capable to distinguish homozygous resistant individuals from heterozygous with a high efficiency. In addition, the validation for polymorphism in different genetic backgrounds and advanced breeding material confirmed the utility of both markers in marker-assisted selection.     

Identification and fine mapping of <Emphasis Type="Italic">Pi33</Emphasis>, the rice resistance gene corresponding to the Magnaporthe grisea avirulence gene <Emphasis Type="Italic">ACE1</Emphasis>

Rice blast disease is a major constraint for rice breeding. Nevertheless, the genetic basis of resistance remains poorly understood for most rice varieties, and new resistance genes remain to be identified. We identified the resistance gene corresponding to the cloned avirulence gene ACE1 using pairs of isogenic strains of Magnaporthe grisea differing only by their ACE1 allele. This resistance gene was mapped on the short arm of rice chromosome 8 using progenies from the crosses IR64 (resistant) × Azucena (susceptible) and Azucena × Bala (resistant). The isogenic strains also permitted the detection of this resistance gene in several rice varieties, including the differential isogenic line C101LAC. Allelism tests permitted us to distinguish this gene from two other resistance genes [Pi11 and Pi-29(t)] that are present on the short arm of chromosome 8. Segregation analysis in F₂ populations was in agreement with the existence of a single dominant gene, designated as Pi33. Finally, Pi33 was finely mapped between two molecular markers of the rice genetic map that are separated by a distance of 1.6 cM. Detection of Pi33 in different semi-dwarf indica varieties indicated that this gene could originate from either one or a few varieties.Communicated by D.J. Mackill     

The successful application of a marker-assisted wheat breeding strategy

A number of useful marker-trait associations have been reported for wheat. However the number of publications detailing the integrated and pragmatic use of molecular markers in wheat breeding is limited. A previous report by some of these authors showed how marker-assisted selection could increase the genetic gain and economic efficiency of a specific breeding strategy. Here, we present a practical validation of that study. The target of this breeding strategy was to produce wheat lines derived from an elite Australian cultivar ‘Stylet’, with superior dough properties and durable rust resistance donated from ‘Annuello’. Molecular markers were used to screen a BC₁F₁ population produced from a cross between the recurrent parent ‘Stylet’ and the donor parent ‘Annuello’ for the presence of rust resistance genes Lr34/Yr18 and Lr46/Yr29. Following this, marker-assisted selection was applied to haploid plants, prior to chromosome doubling with cochicine, for the rust resistance genes Lr24/Sr24, Lr34/Yr18, height reducing genes, and for the grain protein genes Glu-D1 and Glu-A3. In general, results from this study agreed with those of the simulation study. Genetic improvement for rust resistance was greatest when marker selection was applied on BC₁F₁ individuals. Introgression of both the Lr34/Yr18 and Lr46/Yr29 loci into the susceptible recurrent parent background resulted in substantial improvement in leaf rust and stripe rust resistance levels. Selection for favourable glutenin alleles significantly improved dough resistance and dough extensibility. Marker-assisted selection for improved grain yield, through the selection of recurrent parent genome using anonymous markers, only marginally improved grain yield at one of the five sites used for grain yield assessment. In summary, the integration of marker-assisted selection for specific target genes, particularly at the early stages of a breeding programme, is likely to substantially increase genetic improvement in wheat.     

Status and Perspectives of Clubroot Resistance Breeding in Crucifer Crops

Clubroot disease is a major threat to crops belonging to the Brassicaceae. It is controlled most effectively by the use of resistant cultivars. Plasmodiophora brassicae, the causal agent, shows a wide variation for pathogenicity, which can be displayed by using differential host sets. Except for Brassica juncea and B. carinata, resistant accessions can be found in all major crops. Most resistance sources are race-specific, despite some race-independent resistant accessions which can be found in B. oleracea. European field isolates from P. brassicae display great variation and show a tendency to overcome different resistance sources from either B. rapa or B. oleracea. At present, resistance genes from stubble turnips (B. rapa) are most effective and most widely used in resistance breeding of different Brassica crops. Resistance to P. brassicae from turnips was introduced into Chinese cabbage, oilseed rape, and B. oleracea. Although most turnips carry more than one resistance gene, the resistant cultivars from other crops received primarily a single, dominant resistance gene having a race-specific effect. Populations of P. brassicae that are compatible against most of the used resistance sources have been present in certain European areas for many decades. Such pathogen populations appeared in Japanese Chinese cabbage crops only a few years after the introduction of resistant cultivars. As the spread of virulent P. brassicae pathotypes seems to be slow, resistant cultivars are still a very effective method of control in many cropping areas. Mapping studies have revealed the presence of several clubroot-resistance genes in the Brassica A and C genomes; most of these genes are showing race specificity. Only in B. oleracea was one broad-spectrum locus detected. Two loci from the A genome confer resistance to more than one pathotype, but not to all isolates. Progress made in the determination of resistance loci should be used to formulate and introduce an improved differential set. Future efforts for breeding P. brassicae resistance will focus on durability by broadening the genetic basis of clubroot resistance by using either natural variation or transgenic strategies.     

Screening of wild accessions resistant to gray mold (Botrytis cinerea Pers.) in Lycopersicon

Tomato gray mold (Botrytis cinerea Pers.) is a common disease worldwide, and often causes serious production loss by infecting leaves, stems, flowers and fruits. Presently, no resistant cultivars are available. To find new breeding materials for gray mold resistance, assessment for resistance of the leaflet and stem in six tomato cultivars, 44 wild tomato accessions and a Solanum lycopersicoides accession was performed. Although no correlation was observed (r=−0.127^ns) between resistance of the leaflet and the stem, L. peruvianum LA2745, L. hirsutum LA2314 and L. pimpinellifolium LA1246 showed high resistance both in the leaflet and in the stem. Particularly, in the leaves of LA2745, no lesions were observed even more than two weeks after the inoculation with conidia, and F1s between a cultivated tomato and LA2745 also showed high resistance as observed in LA2745. From these results, LA2745 is thought to be a promising material for breeding gray-mold resistant cultivars.     

Identification of two major resistance genes against race IE-1k of Magnaporthe oryzae in the indica rice cultivar Zhe733

The race IE-1k of Magnaporthe oryzae recovered from the Southern US overcomes the resistance (R) gene Pita. The objectives of the present study were to identify and tag R genes to IE-1k for rice breeding. TM2, S1, 94071, and B isolates of the race IE-1k were used to identify and map R genes from a resistant indica rice cultivar Zhe733 using a recombinant inbred line population from a cross of the genetic stock KBNTlpa1-1 and Zhe733. The ratio of 3 resistant:1 susceptible in 162 RIL of an F_10-11 KBNTlpa1-1/Zhe733 (K/Z) population indicated that two major R genes in Zhe733 confer resistance to IE-1k. A total of 118 polymorphic simple sequence repeat markers were analyzed in 162 F_10-11 individuals of the K/Z population to determine chromosomal locations of the loci conferring resistance to race IE-1k using composite interval mapping. Two major R genes temporarily designated as Pi42(t) and Pi43(t) each providing complete resistance to IE-1k were identified on chromosomes 8 and 11, respectively. RILs containing Pi42(t) and Pi43(t) were also resistant to other US races IB-1, IB-45, IB-49, IB-54, IC-17, IE-1, IG-1, and IH-1. The Pi42(t) gene was mapped between RM310 and RM72, and the location of Pi43(t) was closely associated with two flanking SSR markers RM1233 and RM224 on chromosome 11 in a chromosomal region carrying the resistance gene Pi1. Two molecular markers RM72 and RM1233 identified in this study should be useful for fine mapping and for facilitating incorporation of Pi42(t) and Pi43(t) into advanced breeding lines by marker-assisted selection. The authors S. Lee and Y. Wamishe contribute equally to this work.     

Identification of SSR markers for a broad-spectrum blast resistance gene Pi20(t) for marker-assisted breeding

The Pi20(t) gene was determined to confer a broad-spectrum resistance against diverse blast pathotypes (races) in China based on inoculation experiments utilizing 160 Chinese Magnaporthe oryzae (formerly Magnaporthe grisea) isolates, among which isolate 98095 can specifically differentiate the Pi20(t) gene present in cv. IR24. Two flanking and three co-segregating simple sequence repeat (SSR) markers for Pi20(t), located near the centromere region of chromosome 12, were identified using 526 extremely susceptible F₂ plants derived from a cross of Asominori, an extremely susceptible cultivar, with resistant cultivar IR24. The SSR OSR32 was mapped at a distance of 0.2 cM from Pi20(t), and the SSR RM28050 was mapped to the other side of Pi20(t) at a distance of 0.4 cM. The other three SSR markers, RM1337, RM5364 and RM7102, co-segregated with Pi20(t). RM1337 and RM5364 were found to be reliable markers of resistance conditioned by Pi20(t) in a wide range of elite rice germplasm in China. As such, they are useful tags in marker-assisted rice breeding programs aimed at incorporating Pi20(t) into advanced rice breeding lines and, ultimately, at obtaining a durable and broad spectrum of resistance to M. oryaze. Wei Li and Cailin Lei contributed equally to this work.     

Consistent detection of QTLs for crown rust resistance in Italian ryegrass (<Emphasis Type="Italic">Lolium multiflorum</Emphasis> Lam.) across environments and phenotyping methods

Crown rust, caused by Puccinia coronata f. sp. lolii, is one of the most important diseases of temperate forage grasses, such as ryegrasses (Lolium spp.), affecting yield and nutritional quality. Therefore, resistance to crown rust is a major goal in ryegrass breeding programmes. In a two-way pseudo-testcross population consisting of 306 Lolium multiflorum individuals, multisite field evaluations as well as alternative methods based on artificial inoculation with natural inoculate in controlled environments were used to identify QTLs controlling resistance to crown rust. Disease scores obtained from glasshouse and leaf segment test (LST) evaluations were highly correlated with scores from a multisite field assessment (r = 0.66 and 0.79, P < 0.01, respectively) and thus confirmed suitability of these methods for crown rust investigations. Moreover, QTL mapping based on a linkage map consisting of 368 amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers revealed similar results across different phenotyping methods. Two major QTLs were consistently detected on linkage group (LG) 1 and LG 2, explaining up to 56% of total phenotypic variance (V _p). Nevertheless, differences between position and magnitude of QTLs were observed among individual field locations and suggested the existence of specific local pathogen populations. The present study not only compared QTL results among crown rust evaluation methods and environments, but also identified molecular markers closely linked to previously undescribed QTLs for crown rust resistance in Italian ryegrass with the potential to be applied in marker-assisted forage crop breeding. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Divergence and allelomorphic relationship of a soybean virus resistance gene based on tightly linked DNA microsatellite and RFLP markers

The use of genetically diverse resistance sources is important in breeding for durable disease resistance. Detection and evaluation of resistance genes by conventional inheritance experiments, however, often require laborious screening and genetic testing. In the present study, a marker-assisted screening for resistance sources was initiated in soybean [Glycine max (L.) Merr] using one DNA microsatellite and two RFLP markers tightly linked to a soybean mosaic virus (SMV) resistance gene (Rsv1). The three marker loci were used to screen 67 diverse soybean cultivars, breeding lines, and plant introductions. Five variants were found at the microsatellite locus (HSP176L), and the two RFLP loci (pA186 and pK644a) near Rsv1 show a remarkably higher level of restriction polymorphism than Rsv1-independent RFLP loci. Several specific variants at the three marker loci were found to be correlated with virus resistance, among which HSP176L-2 can be detected by PCR, thus may be useful for germplasm screening. The grouping of the 67 accessions according to their multilocus marker variants agrees with the available pedigree information. When all, or most, of the cultivars within a given group with the same Rsv1-linked marker variant are resistant, their SMV resistance is most likely conferred by Rsv1. These putatively Rsv1-carrying groups contain a total of 38 SMV-resistant lines including six differential cultivars that are known to carry Rsv1. The remaining seven resistant accessions (Columbia, Holladay, Peking, Virginia, FFR-471, PI 507403, and PI 556949) do not carry resistance marker variants, and at least some of them could be sources of resistance genes independent of Rsv1.     

甘蔗与斑茅BC1分子鉴定、抗黑穗病和花叶病初步评价

为了解甘蔗(Saccharum)与斑茅(Erianthus arundinaceus)杂交后代作为抗病亲本的利用价值,通过特异引物PCR鉴定出78份甘蔗与斑茅杂交BC_1真实杂种;通过人工接种花叶病毒和黑穗病菌,初步评价了甘蔗和斑茅杂交BC_1的抗病表现。结果表明,甘蔗和斑茅杂交BC_1的抗花叶病具有普遍性,而黑穗病抗性则出现分离。初步筛选出BC_1无性系YCE01-48、YCE01-71、YCE01-105、YCE01-125、YCE02-184和YCE01-118可同时抗花叶病和黑穗病,有望成为甘蔗杂交利用的高抗病源亲本。     

Development and molecular cytogenetic analysis of wheat-Haynaldia villosa 6VS/6AL translocation lines specifying resistance to powdery mildew

Several Triticum aestivum L.-Haynaldia villosa disomic 6VS/6AL translocation lines with powdery mildew resistance were developed from the hybridization between common wheat cultivar Yangmai 5 and alien substitution line 6V(6A). Mitotic and meiotic C-banding analysis, aneuploid analysis with double ditelosomic stocks, in situ hybridization, as well as the phenotypic assessment of powdery mildew resistance, were used to characterize these lines. The same translocated chromosome, with breakpoints near the centromere, appears to be present in all the lines, despite variation among the lines in their morphology and agronomic characteristics. The resistance gene, conferred by H. villosa and designated as Pm21, is a new and promising source of powdery mildew resistance in wheat breeding.This research was supported by grants from the National High-Tech R and D Program and the National Science and Technology Commission     

Identification of QTLs for resistance to Fusarium head blight, DON accumulation and associated traits in the winter wheat variety Arina

Fusarium head blight (FHB) of wheat has become a serious threat to wheat crops in numerous countries. In addition to loss of yield and quality, this disease is of primary importance because of the contamination of grain with mycotoxins such as deoxynivalenol (DON). The Swiss winter cultivar Arina possesses significant resistance to FHB. The objective of this study was to map quantitative trait loci (QTL) for resistance to FHB, DON accumulation and associated traits in grain in a double haploid (DH) population from a cross between Arina and the FHB susceptible UK variety Riband. FHB resistance was assessed in five trials across different years and locations. Ten QTL for resistance to FHB or associated traits were detected across the trials, with QTL derived from both parents. Very few of the QTL detected in this study were coincident with those reported by authors of two other studies of FHB resistance in Arina. It is concluded that the FHB resistance of Arina, like that of the other European winter wheat varieties studied to date, is conferred by several genes of moderate effect making it difficult to exploit in marker-assisted selection breeding programmes. The most significant and stable QTL for FHB resistance was on chromosome 4D and co-localised with the Rht–D1 locus for height. This association appears to be due to linkage of deleterious genes to the Rht-D1b (Rht2) semi-dwarfing allele rather than differences in height per se. This association may compromise efforts to enhance FHB resistance in breeding programmes using germplasm containing this allele.     

Genetic mapping reveals a single major QTL for bacterial wilt resistance in Italian ryegrass (Lolium multiflorum Lam.)

Bacterial wilt caused by Xanthomonas translucens pv. graminis (Xtg) is a major disease of economically important forage crops such as ryegrasses and fescues. Targeted breeding based on seedling inoculation has resulted in cultivars with considerable levels of resistance. However, the mechanisms of inheritance of resistance are poorly understood and further breeding progress is difficult to obtain. This study aimed to assess the relevance of the seedling screening in the glasshouse for adult plant resistance in the field and to investigate genetic control of resistance to bacterial wilt in Italian ryegrass (Lolium multiflorum Lam.). A mapping population consisting of 306 F₁ individuals was established and resistance to bacterial wilt was assessed in glasshouse and field experiments. Highly correlated data (r = 0.67–0.77, P < 0.01) between trial locations demonstrated the suitability of glasshouse screens for phenotypic selection. Analysis of quantitative trait loci (QTL) based on a high density genetic linkage map consisting of 368 amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers revealed a single major QTL on linkage group (LG) 4 explaining 67% of the total phenotypic variance (Vp). In addition, a minor QTL was observed on LG 5. Field experiments confirmed the major QTL on LG 4 to explain 43% (in 2004) to 84% (in 2005) of Vp and also revealed additional minor QTLs on LG 1, LG 4 and LG 6. The identified QTLs and the closely linked markers represent important targets for marker-assisted selection of Italian ryegrass.     

Marker-assisted pyramiding of two cereal cyst nematode resistance genes from <Emphasis Type="Italic">Aegilops variabilis</Emphasis> in wheat

The cereal cyst nematode (CCN) Heterodera avenae, is a significant pathogen of wheat. The wild grass Aegilops variabilis Accession No.1 has been found to be resistant to pathotypes of CCN; at least two genes transferred to wheat, designated as CreX and CreY, are involved in the resistance response. The CreY gene may be the same as Rkn-mn1, which confers resistance to root knot nematode (RKN) Meloidogyne naasi. The objective of this work was to pyramid the two CCN resistance genes in a wheat background through marker-assisted selection. As a first step, molecular markers flanking CreX were identified. The completely linked RAPD marker of Rkn-mn1 (CreY), OpY16-_1065, previously obtained, was converted into a SCAR. All these dominant markers were used to incorporate in the same genotype the two Ae. variabilis chromosome segments carrying the two genes for resistance. CCN bioassays with the Ha12 pathotype showed that the level of resistance of the pyramided line was significantly higher than that of CreX and CreY single introgression lines, but lower than that of Ae. variabilis. This study thus illustrates the utilization of molecular markers in breeding for host resistance.     

Genetic and QTL analysis of resistance to <Emphasis Type="Italic">Xiphinema index</Emphasis> in a grapevine cross

Resistance to the dagger nematode Xiphinema index has been an important objective in grape rootstock breeding programs. This nematode not only causes severe feeding damage to the root system, but it also vectors grapevine fanleaf virus (GFLV), the causal agent of fanleaf degeneration and one of the most severe viral diseases of grape. The established screening procedures for dagger nematode resistance are time consuming and can produce inconsistent results. A fast and reliable greenhouse-based system for screening resistance to X. index that is suitable for genetic studies and capable of evaluating breeding populations is needed. In this report, the dynamics of nematode numbers, gall formation, and root weight loss were investigated using a variety of soil mixes and pot sizes over a 52-week period. Results indicated that the number of galls formed was correlated with the size of the nematode population and with the degree of root weight loss. After inoculation with 100 nematodes, gall formation could be reliably evaluated in 4–8 weeks in most plant growth conditions and results were obtained 6 months more rapidly than past evaluation methods. This modified X. index resistance screening method was successfully applied to 185 of the 188 F₁ progeny from a cross of D8909-15 × F8909-17 (the 9621 population), which segregates for a form of X. index resistance originally derived from Vitis arizonica. Quantitative trait loci (QTL) analysis was carried out on both parental genetic maps of 255 markers using MapQTL 4.0. Results revealed that X. index resistance is controlled by a major QTL, designated Xiphinema index Resistance 1 (XiR1), near marker VMC5a10 on chromosome 19. The XiR1 QTL was supported by a LOD score of 36.9 and explained 59.9% of the resistance variance in the mapping population. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Herbicide response polymorphisms in wild emmer wheat: ecological and isozyme correlations

Summary We demonstrate that the scores and frequencies of chlortoluron (CT) and metoxuron (MX) resistance and susceptible phenotypes of wild emmer wheat, Triticum dicoccoides, are correlated with ecological factors and allozyme markers. Some isozyme markers located on chromosome 6B (e.g. Adh,Est-4 and Got), which also harbours the CT and MX resistance gene, provide good genetic markers for herbicide resistance breeding. Significant correlations between herbicide and photosynthetic characters suggest that the evolution of herbicide resistance polymorphisms may be related to the process of photosynthesis in nature and predated domestication of cultivated wheat.     

A genetic linkage map for an apricot (<Emphasis Type="Italic">Prunus armeniaca</Emphasis> L.) BC<Subscript>1</Subscript> population mapping <Emphasis Type="Italic">plum pox virus</Emphasis> resistance

Plum pox virus (sharka; PPV) can cause severe crop loss in economically important Prunus species such as peach, plum, apricot, and cherry. Of these species, certain apricot cultivars (‘Stark Early Orange’, ‘Goldrich’, ‘Harlayne’) display significant levels of resistance to the disease and are the genetic substrate for studies of several xlaboratories working cooperatively to genetically characterize and mark the resistance locus or loci for marker-assisted breeding. The goals of the work presented in this communication are the characterization of the genetics of PPV resistance in ‘Stark Early Orange’ and the development of co-dominant molecular markers for marker-assisted selection (MAS) in PPV resistance breeding. We present the first genetic linkage map for an apricot backcross population of ‘Stark Early Orange’ and the susceptible cultivar ‘Vestar’ that segregates for resistance to PPV. This map is comprised of 357 loci (330 amplified fragment length polymorphisms (AFLPs), 26 simple sequence repeats (SSRs), and 1 morphological marker for PPV resistance) assigned to eight linkage groups. Twenty-two of the mapped SSRs are shared in common with genetic reference map for Prunus (T × E; Joobeur et al. 1998) and anchor our apricot map to the general Prunus map. A PPV resistance locus was mapped in linkage group 1 and four AFLP markers segregating with the PPV resistance trait, identified through bulk segregant analysis, facilitated the development of SSRs in this region. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Lalli, D.A. and Salava, J. contributed equally to this work.     

Genome mapping of three major resistance genes to woolly apple aphid (Eriosoma lanigerum Hausm.)

Woolly apple aphid (WAA; Eriosoma lanigerum Hausm.) can be a major economic problem to apple growers in most parts of the world, and resistance breeding provides a sustainable means to control this pest. We report molecular markers for three genes conferring WAA resistance and placing them on two linkage groups (LG) on the genetic map of apple. The Er1 and Er2 genes derived from ‘Northern Spy’ and ‘Robusta 5,’ respectively, are the two major genes that breeders have used to date to improve the resistance of apple rootstocks to this pest. The gene Er3, from ‘Aotea 1’ (an accession classified as Malus sieboldii), is a new major gene for WAA resistance. Genetic markers linked to the Er1 and Er3 genes were identified by screening random amplification of polymorphic deoxyribonucleic acid (DNA; RAPD) markers across DNA bulks from resistant and susceptible plants from populations segregating for these genes. The closest RAPD markers were converted into sequence-characterized amplified region markers and the genome location of these two genes was assigned to LG 08 by aligning the maps around the genes with a reference map of ‘Discovery’ using microsatellite markers. The Er2 gene was located on LG 17 of ‘Robusta 5’ using a genetic map developed in a M.9 × ‘Robusta 5’ progeny. Markers for each of the genes were validated for their usefulness for marker-assisted selection in separate populations. The potential use of the genetic markers for these genes in the breeding of apple cultivars with durable resistance to WAA is discussed.