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.     

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.     

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.     

Development of a multiallelic SCAR marker for the scab resistance gene Vr1/Vh4/Vx from R12740-7A apple and its utility for molecular breeding

A major scab resistance gene initially called Vr1 was identified in the apple cultivar “Regia” derived from the Malus scab resistance source R12740-7A (Russian seedling, RS). A codominant, multiallelic sequence characterized amplified region (SCAR) marker was developed from a random amplified polymorphic DNA marker identified by bulked-segregant analysis. Additional alleles of the AD13 marker locus proved to be informative for the analysis of genetic relationships within Malus including putative relatives of RS. Separate linkage maps were created for the two families derived from crosses with “Regia”. Using phenotypic data from the greenhouse scab tests, the recombination frequency between Vr1 and AD13-SCAR was between 6 and 17%. The Vr1 locus appeared to be closely linked to the Vx [Hemmat et al. J Am Soc Hortic Sci, 127:365–370, 2002], Vr2 [Patocchi et al. Theor Appl Genet, 109:1087–1092, 2004], and the Vh4 gene [Bus et al. Mol Breed, 15:103–116, 2005a]. Our linkage analysis of the molecular markers identified by Hemmat et al. [J Am Soc Hortic Sci, 127:365–370, 2002] for two scab resistance factors from RS (Vr and Vx) indicate that both genes are separated by a large distance on apple linkage group 2 [Boudichevskaia et al. Acta Hortic, 663:171–175, 2004]. This is in agreement with the results of Bus et al., [Mol Breed, 15:103–116, 2005a] who concluded that (1) the RS-derived gene Vh2 is identical to Vr, (2) the RS-derived gene Vh4 is identical to Vx and Vr1, (3) Vh2/Vr and Vh4/Vr1/Vx map on opposite sides of LG 2. One of our main goals was the verification of the Vr1-SCAR within a practical apple-breeding program. The utility of the AD13-SCAR was evident after 2 years under natural scab infection conditions in both families investigated. This is the first report about the confirmation of a molecular marker for a RS resistance factor in a 2-year field experiment. A multiplex polymerase chain reaction assay based on two codominant SCARs for Vf and Vr1 was tested in an apple progeny segregating for both genes. The result of the two-marker approach is discussed with respect to scab races, which are able to overcome the Vf resistance gene.     

The Vh2 and Vh4 scab resistance genes in two differential hosts derived from Russian apple R12740-7A map to the same linkage group of apple

Russian apple R12740-7A is the designation for an accession grown from seed collected in Russia, which was found to be highly resistant to apple scab. The resistance has historically been attributed to a naturally pyramided complex involving three major genes: one race-nonspecific gene, Vr, conditioning resistance to all known races, plus two race-specific genes. The race-nonspecific gene was identified as an independently segregating gene by Dayton and Williams (1968) and is referred to in this paper as Vr-DW. The first researchers to study the scab resistance gene complex in Russian apple never described the phenotype conditioned by the race-nonspecific gene. Later, Aldwinckle et al. (1976) associated the name Vr with a scab resistance gene conditioning distinctive stellate necrotic reactions, which we refer to as Vr-A in order to distinguish it from Vr-DW. We show that the segregation ratios in progenies from the scab differential hosts 2 and 4 that are derived from Russian apple, crossed with susceptible cultivars were consistent with a single gene conditioning resistance in each host. The genes have been named Vh2 and Vh4, respectively. Resistant segregants from host 2 showed stellate necrotic reactions, while those from host 4 showed hypersensitive reactions. Both the phenotypes and the genetic maps for the genes in the respective hosts were very similar to those of the genes previously named Vr-A and Vx, respectively, in an F1 family of Russian apple. We showed that race 2 of V. inaequalis isolated from host 2 was able to infect resistant descendants of the non-differential accession PRI 442-23 as well as host 2. The descendants of PRI 442-23 were expected to carry the race-nonspecific Vr-DW gene, but in fact carry Vr-A. We conclude that the Vh2 gene in host 2 and Vr-A are the same, and that the Vh4 gene in host 4 and Vx are the same. However, a major finding of this study is that the latter gene mapped to linkage group 2 of apple instead of linkage group 10 as suggested from previous research. With the two race-specific genes from Russian apple defined now, we discuss the nature of the race-nonspecific Vr-DW gene in this accession. We also report the identification of a new scab resistance gene, VT57, from either Golden Delicious or Red Dougherty, which conditions chlorotic resistance reactions and is linked to Vh2.     

Plant regeneration from leaf protoplasts of apple

Protoplasts were isolated from young leaves or etiolated shoot apices. For initiation of divisions the protoplasts were embedded in sodium alginate and cultivated in MS or MI medium supplemented with 2.2 M BA, 2.6 M NAA and 2.2 M 2,4-dichlorophenoxyacetic acid. The protoplasts of all seven lines tested developed to protocalluses at high frequencies. No genotypic differences were observed. When BA was used in combination with NAA in the regeneration experiments, only a few protocalluses (highest frequency 3%) exhibited shoot organogenesis. When BA was replaced with thidiazuron, the percentage of protocalluses that developed shoots increased in two of three tested lines to 7% and 56%, respectively. Shoot development was achieved under light conditions. The shoots were then rooted and transferred into soil.Abbreviations ABA abscisic acid - BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - FW fresh weight - GA₃ gibberellic acid - IBA indole-3-butyric acid - MES 2-N-morpholinoethane sulphonic acid - NAA -naphthaleneacetic acid     

Identification of co-dominant RAPD markers tightly linked to fruit skin color in apple

A simple genetic basis for the red/yellow skincolor polymorphism in apple was verified using DNA markers. Bulked segregant analysis identified one 10-base oligomer that generated different fragments in each of the bulks. After testing the primer in four populations, two fragments were found to be associated with red skin color and another two fragments associated with yellow skin color. Three of the fragments (1160, 1180, and 1230 bp) were partly sequenced and found to share high sequence homology, suggesting these were generated from the same locus. A pair of universal primers were designed to amplify the fragments. In the Rome Beauty x White Angel population, two fragments were associated with red skin color; one fragment designated as A¹ (1160 bp) was from Rome Beauty and another fragment (A², 1180 bp) was from White Angel. Progeny possessing both fragments, or either one, had red fruit. Both parents displayed an alternate fragment, a¹ (1230 bp), associated with yellowskinned fruit. In three other crosses tested, only fragment A¹ co-segregated with red skin color; two fragments, a¹ and a² (1230 bp and 1320 bp), were associated with yellow skin color. Our results are consistent with the hypothesis that the red/yellow dimorphism is controlled by a monogenic system with the presence of the red anthocyanin pigmentation being dominant. There was no indication that other modifier genes could reverse the effect of the locus (R _f ) linked to the markers. Examination of amplification products in 56 apple cultivars and advanced breeding selections demonstrated that the universal primers could be used to correctly predict fruit skin color in most cases.     

Dissecting apple tree architecture into genetic, ontogenetic and environmental effects: QTL mapping

The present study aimed to dissect tree architectural plasticity into genetic, ontogenetic and environmental effects over the first 4 years of growth of an apple F1 progeny by means of quantitative traits loci (QTL) mapping. Both growth and branching processes were phenotyped on the consecutive annual shoots of different axes within a tree. For each studied trait, predicted values (best linear unbiased predictors, BLUPs) of the genotypic (G) effect or its interaction with tree age (G×A) and climatic year (G×Y) were extracted from mixed linear models of repeated data. These BLUPs, which are independent from autocorrelations between repeated measurements, were used for QTL mapping. QTL detection power was improved by this two-step approach. For each architectural process, numerous QTLs were detected and some particularly interesting co-localised in common genomic regions, for internode lengthening, top diameter, and number and percentage of axillary shoots. When several QTLs were detected for a given trait, global models were estimated, which explained a maximum of 40% of the total variance for both internode length and top diameter and 28% for branching. QTLs detected for BLUPs of G×Y effects were interpreted as resulting from the interaction between genetic maximal potential of growth and climatic factors, while those for G×A effects were interpreted in relation to tree ontogeny. Most of the latter ones were found to be concomitant with key development stages during which the trait average started to decrease, but with different magnitudes depending on genotype. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification and stability of QTLs for fruit quality traits in apple

Breeding for fruit quality traits is complex due to the polygenic (quantitative) nature of the genetic control of these traits. Therefore, to improve the speed and efficiency of genotype selection, attention in recent years has focused on the identification of quantitative trait loci (QTLs) and molecular markers associated with these QTLs. However, despite the huge potential of molecular markers in breeding programmes, their implementation in practice has been limited by the lack of information on the stability of QTLs across different environments and within different genetic backgrounds. Here, we present the results from a comprehensive analysis of the inheritance of fruit quality traits within a population derived from a cross between the apple cultivars ‘Telamon’ and ‘Braeburn’ over two successive seasons. A total of 74 different QTLs were identified for all the major fruit physiological traits including fruit height, diameter, weight and stiffness, flesh firmness, rate of flesh browning, acidity, the oBrix content and harvest date. Seventeen of these QTLs were ‘major’ QTLs, accounting for over 20% of the observed population variance of the trait. However, only one third (26) of the identified QTLs were stable over both harvest years, and of these year-stable QTLs only one was a major QTL. A direct comparison with published QTL results obtained using other populations (King et al., Theor Appl Genet 102:1227–1235, 2001; Liebhard et al., Plant Mol Biol 52:511–526, 2003) is difficult because the linkage maps do not share a sufficient number of common markers and due to differences in the trait evaluation protocols. Nonetheless, our results suggest that for the six fruit quality traits which were measured in all populations, nine out of a total of 45 QTLs were common or stable across all population × environments combinations. These results are discussed in the framework of the development and application of molecular markers for fruit quality trait improvement.     

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     

Repeat flowering in apple caused by water stress or defoliation

Summary A series of experiments involving defoliation or water stress at different dates indicated that either of these treatments can make potted apple trees flower a second time in any one year, as long as the treatment is given near the end of July. The results suggest that the reflowering after a period of water stress was primarily a result of the loss of leaves that occurred when the plants were subsequently rewatered. Reflowering normally occurred only if flower primordia had already differentiated at the time of the treatment. There was an indication that in early July water stress was more effective than defoliation at stimulating reflowering.     

Factors that effect leaf regeneration efficiency in apple,and effect of antibiotics in morphogenesis

Several factors that affect the frequency of organogenesis in apple leaf explants were examined for the scion cultivars Empire, Freedom, Golden Delicious, Liberty, McIntosh, and Mutsu and for the rootstocks Malling 7A and Malling 26. The main factors affecting morphogenesis were BA concentration, basal medium, leaf explant origin and maturity, explant orientation, and photosynthetic photon flux. Depending on the genotype, optimal regeneration was obtained using either 22.2 or 31.1 M BA and the N6 basal medium, with the exception of Golden Delicious which regenerated better on MS medium. After 6 weeks, the average number of shoots per segment varied from 5 to 16, and the percentage of regeneration between 70 and 100%, depending on the genotype tested and the maturity of the explant. Regeneration capacity increased dramatically from the tip towards the base of the leaf, and was higher from the middle to the proximal end.Cefotaxime and carbenicillin, two antibiotics commonly used during transformation studies to eliminate Agrobacterium tumefaciens from plant tissue, were tested to determine their effect on morphogenesis. Cefotaxime at a dose of 250 mg 1^-1 enhanced regeneration and shoot development, whereas carbenicillin at a dose of 500 mg l^-1 induced abundant callus formation and inhibited regeneration. Kanamycin, a widely used selection agent for plant transformation, strongly inhibited regeneration even at very low doses. Schemes for selection and recovery of transgenic apple plants when kanamycin is used as the selection agent are discussed.Abbreviations BA benzyladenine - Cef cefotaxime - Crb carbenicillin - IBA indolebutyric acid - Kan kanamycin - LS Linsmaier and Skoog (1965) medium - M Malling - MS Murashige and Skoog (1962) medium - NAA naphthaleneacetic acid - N6 medium (Chu et al. 1975) as modified by Welander (1988) - PPF photosynthetic photon flux     

QTL analysis for aphid resistance and growth traits in apple

The rosy apple aphid (Dysaphis plantaginea), the leaf-curling aphid (Dysaphis cf. devecta) and the green apple aphid (Aphis pomi) are widespread pest insects that reduce growth of leaves, fruits and shoots in apple (Malus × domestica). Aphid control in apple orchards is generally achieved by insecticides, but alternative management options like growing resistant cultivars are needed for a more sustainable integrated pest management (IPM). A linkage map available for a segregating F₁-cross of the apple cultivars ‘Fiesta’ and ‘Discovery’ was used to investigate the genetic basis of resistance to aphids. Aphid infestation and plant growth characteristics were repeatedly assessed for the same 160 apple genotypes in three different environments and 2 consecutive years. We identified amplified fragment length polymorphism (AFLP) markers linked to quantitative trait loci (QTLs) for resistance to D. plantaginea (‘Fiesta’ linkage group 17, locus 57.7, marker E33M35–0269; heritability: 28.3%), and to D. cf. devecta (‘Fiesta’ linkage group 7, locus 4.5, marker E32M39–0195; heritability: 50.2%). Interactions between aphid species, differences in climatic conditions and the spatial distribution of aphid infestation were identified as possible factors impeding the detection of QTLs. A pedigree analysis of simple sequence repeat (SSR) marker alleles closely associated with the QTL markers revealed the presence of the alleles in other apple cultivars with reported aphid resistance (‘Wagener’, ‘Cox’s Orange Pippin’), highlighting the genetic basis and also the potential for gene pyramiding of aphid resistance in apple. Finally, significant QTLs for shoot length and stem diameter were identified, while there was no relationship between aphid resistance and plant trait QTLs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

CO2-Enrichment and photosynthetic photon flux affect the growth of in vitro-cultured apple plantlets

Micro-cuttings (shoots with two small leaves) of cultivar M9 apple were cultured in-vitro for 40 d under CO₂-enriched and non-enriched (i.e., ambient air) conditions, and at a PPF of 40 or 100 μmol m^-2 s^-1 Afterward, shoot length, number of leaves, leaf area, chlorophyll content, shoot and root fresh weights, and % survival were recorded. Those plant-lets grown under CO₂- and PPF-enriched treatments were healthy and vigorous, and showed higher values for their growth parameters. In contrast, those grown without supplemental CO₂ or PPF often showed hyperhydricity. We also demonstrated that CO₂ enrichment and a relatively high PPF during in-vitro culture promoted normal photosynthesis and growth after ex-vitro transplantation.     

The influence of cation and gelling agent concentrations on vitrification of apple cultivars in vitro

Shoot tips of York and Vermont Spur Delicious apples (Malus domestica Borkh.) were cultured in vitro to test the influence of K⁺, Mg⁺⁺ and gelling agent concentrations on vitrification. These concentrations were 20.05, 14.05 and 8.05 mM K⁺, 1.5 and 3.0 mM Mg⁺⁺, 7.0 g/l Difco Bacto agar and 1.0, 1.5 and 2.0 g/l Gelrite. The lowest K⁺ level produced a higher percentage of vitrified shoots, affected tissue appearance, reduced shoot number and shoot elongation and apparently altered shoot metabolic activity. Gelrite consistently produced vitrified leaves and stems, even though media gelled with 1.5 g/l Gelrite presented the same apparent gel firmness as using 7 g/l Difco Bacto agar, which did not induce vitrification. Less shoot elongation, fewer total shoots, and more usable shoots of York were obtained on Bacto-agar, while similar but less noticeable effects were obtained with Vermont Spur Delicious. The results presented here show that vitrification can be studied in a standardized system in which the only change is substitution of one gelling agent for another.     

Molecular tagging and mapping of the erect panicle gene in rice

Erect panicle (EP) is one of the more important traits of the proposed ideotype of high-yielding rice. Several rice cultivars with the EP phenotype, which has been reported to be controlled by a dominant gene, have been successfully developed and released for commercial production in North China. To analyze the inheritance of the EP trait, we generated segregating F₂ and BC₁F₁ populations by crossing an EP-type variety, Liaojing 5, and a curved-panicle-type variety, Fengjin. Our results confirmed that a dominant gene controls the EP trait. Simple-sequence repeat (SSR) and bulked segregant analyses of the F₂ population revealed that the EP gene is located on chromosome 9, between two newly developed SSR markers, RM5833-11 and RM5686-23, at a genetic distance of 1.5 and 0.9 cM, respectively. Markers closer to the EP gene were developed by amplified fragment length polymorphism (AFLP) analysis with 128 AFLP primer combinations. Three AFLP markers were found to be linked to the EP gene, and the nearest marker, E-TA/M-CTC₂₀₀, was mapped to the same location as SSR marker RM5686-23, 1.5 cM from the EP gene. A local map around the EP gene comprising nine SSR and one AFLP marker was constructed. These markers will be useful for marker-assisted selection (MAS) for the EP trait in rice breeding programs.