BACKGROUND
many birch pollen sensitised patients  in central and northern europe suffer from oral allergy symptoms after eating fresh apples  <cit> .
the prevalence of apple allergic individuals mounts up to ~3% in central and northern europe.
this type of apple allergy is caused by cross reactivity of ige antibodies against the major and sensitizing birch pollen allergen bet v  <dig> with mal d  <dig>  the major allergen of apple.
bet v  <dig> and mal d  <dig> are both pathogenesis-related  proteins.
they belong to the pr- <dig> family and share a high degree of homology  <cit> .
from patients' experience it is known for a long time that the severity of allergic reactions to apple was not only related to the specific sensitivity of the individual, but also largely depended on the apple cultivar.
this cultivar dependent allergenicity has also been described in literature.
for instance, mal d  <dig> from the cultivar golden delicious was found highly reactive to specific ige antibodies from allergic patients' sera, whereas mal d  <dig> from the cultivar gloster generally showed much less reactivity  <cit> .
in addition, skin prick testing  with  <dig> different apple cultivars and confirmations for specific cultivars in double-blind placebo-controlled food challenges  and oral challenges of whole apples, revealed a wide range of allergenic reactivity from very high to very low  <cit> .
as a result from these studies, the new cultivar santana was identified as hypo- allergenic for 75% of the patients with a mild apple allergy  <cit> , which is usually assumed to be mal d  <dig> based.
in the netherlands , this cultivar has recently been marketed as 'suited for individuals with mild apple allergy' in order to meet the general desire of apple allergic persons to be able to add this common fruit to their daily diet.
the differences in allergenicity among cultivars raised a crucial question on the origin of this cultivar-specific degree of allergenicity.
allergenicity may depend on the total amount of mal d  <dig> proteins, as suggested by son et al.
<cit>  from their observed ten-fold difference in mal d  <dig> amount between the high-allergenic cultivar golden delicious and the low-allergenic cultivar gloster.
however, there is little evidence supporting this hypothesis because only very few cultivars have been studied and, for these, a linear response between mal d  <dig> protein content and allergenicity estimates is lacking.
on the other hand, qualitative characteristics of the mal d  <dig> proteins could be involved too, as can be argued from the differences in binding capacity of birch pollen-specific ige to two protein variants of mal d  <dig>  <cit> .
to elaborate this latter issue, research on the genetic variation of mal d  <dig> and its expression pattern in the different cultivars is required and should be compared to allergenicity data.
it is known that mal d  <dig> is coded by a large gene family of  <dig> members mapped on three linkage groups of the apple genome  <cit> .
not all of these members are likely to be involved in allergenicity since only a limited number of different mal d  <dig> proteins and mrnas have been traced back in apple fruit so far  <cit> .
research towards the relative importance of the quality and quantity of mal d  <dig> proteins on the allergenicity of apple cultivars is relevant for designing apple breeding programs for low-allergenic apple cultivars of high quality and healthiness.
in this paper, we focused on the genetic diversity of mal d  <dig> genes.
the mal d  <dig> gene family can be subdivided into two major categories: genes with and genes without an intron.
preliminary genetic analyses revealed that the genetic diversity was by far larger in the intron-containing genes.
furthermore, the intron-containing genes cover all three linkage groups that mal d  <dig> loci  <cit> .
therefore, this category of genes has been chosen to start looking for putative qualitative effects of mal d  <dig> proteins in cultivar specific allergenicity.
allelic diversity of the seven intron-containing mal d  <dig> genes was assessed among a set of cultivars chosen for their importance in breeding programs and apple production.
in order to find putative associations with allergenicity, the presence of alleles coding for different protein variants was subsequently compared with the degree of allergenicity for a subset of cultivars for which allergenicity data from spt or dbpcfc tests were available.
RESULTS
diversity of mal d  <dig> genes and deduced proteins
the observed dna polymorphisms in the  <dig> studied cultivars resulted in a total of  <dig> different mal d  <dig> sequences over seven genes .
these sequences were denoted according to the occurrence of 1) dna polymorphisms in the coding region of the gene leading to different protein variants; 2) polymorphism in the coding region that did not affect the protein sequence , and 3) polymorphism in the intron .
although the latter two differences are of minor importance with respect to allergenicity, they provided additional landmarks for the development of sequence specific molecular markers.
avariants refer to different protein sequences.
variant numbers of mal d  <dig> , mal d  <dig>  and mal d  <dig>  are according to the allergen list , those for mal d  <dig>  and mal d  <dig>  are according to gao et al.
<cit> .
bgenome sequences are numbered successively per gene
cgolden delicious , priscilla , ingrid marie , cox , jonathan , red delicious , fuji , discovery , prima and fiesta .
note that different accessions of priscilla seem to exist.
the priscilla used here is a parent of santana as confirmed by  <dig> ssr markers , but can not descend from its supposed mother starking delicious
d indicates the presence of an allele in heterozygous  or homozygous  condition.
avariant numbers of are according to the allergen list , those for mal d  <dig>  and mal d  <dig>  are according to gao et al.
<cit> .
b, c, d as in table 1
e # and * for lg <dig>  alleles that have the same symbol are in coupling phase with each other.
this information was obtained by their co-segregation patterns in mapping progenies ; ingrid marie = cox × open pollinated and fuji = ralls janet × delicious; red delicious is a colour mutant of delicious.
fps <dig> and ps <dig> of mal d  <dig>  refer to pseudo-alleles.
g the allele specific markers for these alleles were derived from table  <dig> of gao et al.
<dig>  <cit>  whereby we took into account that their marker for mal d  <dig> c <dig> actually amplifies mal d  <dig> c <dig> and visa versa, thus harmonizing an inconsistency between their tables  <dig> and  <dig> in the assignment of allele and marker names to the ay <dig> and ay <dig> sequences.
mal d  <dig>  and - <dig>  showed to be highly conserved at the protein level.
the related genes coded for only two variants each that both differed in just a single amino acid  and for which the second variant was found only once.
the other genes were more variable, coding for three  up to six variants .
mal d  <dig>  showed to be special in that two  sequences contained a stop codon in the coding region and were therefore regarded as pseudo alleles .
interestingly, the pseudo alleles occurred frequently as for seven out of the ten cultivars at least one of the alleles was a pseudo allele whereas cultivars priscilla and fuji only contained pseudo alleles of mal d  <dig>  .
allergenicity scores of  <dig> apple cultivars by skin prick test
relative spt responses of  <dig> apple cultivars are given in table  <dig>  fiesta, delblush, pinova and golden delicious were ranked in the high allergenic group .
priscilla and santana showed low spt responses, with wheal areas 30–35% of that of golden delicious.
nine cultivars were intermediate  allergenic.
santana was also identified as low-allergenic in comparison to golden delicious in dbpcfc tests  <cit>  and oral provocation tests  <cit> .
a data for the first three experiments were derived from the original data from three experiments of bolhaar et al.
<cit>  on respectively cultivar screening, intra-cultivar variation and storage, of which we used only the data of patients with mild symptoms.
the forth experiment came from a new cultivar screening experiment.
b the underlying hep values for golden delicious for the four experiments were respectively  <dig> ,  <dig> ,  <dig>  and  <dig> .
general associations from the sequenced cultivars
from the cultivars used to sequence the intron containing mal d  <dig> genes and to perform spt on allergenicity, golden delicious was ranked as the highest allergenic cultivar whereas priscilla was ranked as the lowest .
this difference was not related to protein variant composition of mal d  <dig> ,  <dig> , 106b and  <dig> c because these cultivars have identical protein variants .
in contrast, both mal d  <dig>  and mal d  <dig> a have different protein variants in golden delicious and priscilla.
mal d  <dig>  may contribute to the observed difference in allergenicity between the two cultivars since protein variant  <dig> is present in golden delicious while priscilla is heterozygous for the two pseudo-alleles ps <dig> and ps <dig>  similarly, mal d  <dig> a protein variant - <dig> was found in priscilla whereas variants - <dig> and - <dig> were found in golden delicious.
the intermediate cultivars, jonathan, prima, and fuji, have the putatively high allergenic alleles - <dig> or - <dig> in single dosage, whereas high allergenic cultivars either have both alleles - <dig> and - <dig>  or have - <dig> in homozygous state.
this suggests that allergenicity might be determined by both the protein variant and the gene dosage of highly allergenic alleles.
association analysis by pedigrees: from golden delicious to santana
the identity and origin of genomic alleles and thus protein variants in additional cultivars  could be traced by developed allele specific snap and ssr markers and the use of pedigree information  <cit>  .
for instance, the deduced flow of protein variants over the pedigree of cultivar santana is presented in figure  <dig>  santana and priscilla are low allergenic whereas golden delicious is high allergenic  <cit> .
for mal d  <dig> ,  <dig>  and  <dig>  the same protein variants were found for golden delicious and santana.
in contrast, golden delicious and santana differ in their protein variant composition of mal d. <dig> , - <dig> a and - <dig> b , indicating a possible involvement of these proteins in the observed difference in allergenicity between these cultivars.
santana, like priscilla, has only pseudo alleles for mal d  <dig>  that do not result in protein production, while golden delicious has one functional allele.
the mal d  <dig> a <dig> variant of santana is not present in golden delicious.
associations for mal d  <dig>  and - <dig> a
similarly to santana, pedigree information and sequence specific markers were used to assess the putative protein variant composition for other additional cultivars for which we have spt response data.
this allowed a further validation of the association of the mal d  <dig>  and  <dig> a with allergenicity in a total of  <dig> cultivars .
cultivars delblush and pinova evoked an spt response that was similar to that of golden delicious and also had the same variant compositions as golden delicious for mal d  <dig>  and mal d  <dig> a, namely variant  <dig> for mal d  <dig>   and the two variants  <dig> and  <dig> for mal d  <dig> .
the remaining five cultivars evoking an intermediate allergenic response also had a similar variant composition as the previously identified intermediate cultivars, namely variants  <dig> and  <dig> for mal d  <dig> a, and variant  <dig> of mal d  <dig>  , except for prima, that has two alleles coding for mal d  <dig>  and fuji that contains both pseudo alleles.
the other intron containing genes did not show any association .
a: cultivars in bold were sequenced for their intron containing mal d  <dig> genes
b: in % relative to golden delicious.
discussion
birch pollen induced oral allergy for apple has been the subject in a considerable number of studies.
one of the prominent results has been the presence of cultivar-specific differences in allergenicity.
unfortunately, evidence regarding the causes of cultivar-specific allergenicity is still lacking.
one of the knowledge gaps concerned the number and identity of mal d  <dig> genes and the amount of variation within these genes.
recently, gao et al.
<cit>  have shown that mal d  <dig> genes are members of a large gene family by identifying  <dig> different loci that are located in three clusters.
based on sequence identity, these  <dig> genes could be subdivided into intron containing and intronless genes.
in order to create a basis for a better understanding of the genetics of mal d  <dig> genes and their impact on allergenicity, we have studied the allelic diversity of the intron containing genes in  <dig> cultivars that are often used in breeding.
development of sequence specific markers and pedigree information enabled the assessment of putative mal d  <dig> constitutions of other cultivars.
using this information, we assessed the different mal d  <dig> isoforms that cultivars are able to produce and found associations between their putative protein constitutions and spt-responses.
allelic diversity and validity of database sequences
cloning and sequencing of the seven intron-containing mal d  <dig> genes in  <dig> cultivars revealed  <dig> different alleles that coded for  <dig> different mal d  <dig> isoforms.
the variation per gene varied with regard to the number of alleles and deduced proteins.
mal d  <dig>  and mal d  <dig>  were diverse at the gdna level but conserved at the protein level.
for both genes only two protein variants were found, of which the second variant, differing in one amino acid only, was found in just a single cultivar.
for mal d  <dig> , mal d  <dig>  and the three mal d  <dig>  genes, gdnas often coded for different proteins and these genes were therefore more variable at the protein level.
because the examined cultivars are important in the breeding of many modern apple varieties, the set of alleles found in this study likely represents a considerable part of the total variation present in intron containing mal d  <dig> genes of common apple varieties.
although other mal d  <dig> sequences are known from public databases, we suspect that many of these sequences may be artefacts derived through strand switching and pcr mutations.
the problem associated with pcr amplification of a group of closely related sequences, such as the mal d  <dig> gene family, is that besides pcr induced single base pair mutations, in vitro strand switching or re-annealing of incompletely amplified fragments can lead to artefacts as was exemplified by schenk et al.
<cit>  for birch bet v <dig> sequences.
for instance, for mal d  <dig>  one of the most studied mal d  <dig> genes, over  <dig> dna sequences from previous studies are known from public databases  indicating the presence of  <dig> putative protein isoforms.
we know mal d  <dig>  is a single locus gene with maximum two alleles present in a cultivar  <cit> , but four sequences from golden delicious can be found in databases.
from these, only sequence accession af <dig> was identical to one of our two sequences .
the other sequences may be due to artefacts.
firstly, accession af <dig> had one snp at position  <dig>  compared to af <dig>  which is due to the cloning primer used.
similarly, sequences from a number of other cultivars showed this 11a mutation too.
in our study, the cloning primers used were positioned in the 5'-untranslated region thus avoiding this problem.
secondly, accession z <dig> <cit>  that was classified as mal d 1c  <cit>  is quite similar to mal d  <dig> , while the snps found are unique to three other mal d  <dig> genes.
this may be due to an artefact from strand switching during pcr which could explain why we, like other researchers could not retrieve this sequence from golden delicious .
similarly, the first  <dig> nucleotides of af <dig> and af <dig> match to mal d  <dig>  , while the remaining sequence matches to mal d  <dig>  making these accessions also putative erroneous sequences due to strand switching.
therefore, the actual number of mal d  <dig>  variants present among apple cultivars is probably not as high as nine, because at least five of them are likely pcr artefacts.
two protein variants  of mal d  <dig>  could be confirmed in this study.
variants mal d  <dig> ,  <dig>  and  <dig>  are likely artefacts of  <dig>  whereas  <dig>  and  <dig>  are likely artefacts of  <dig> .
a alleles in bold are confirmed by our own sequences .
numbers in brackets indicate genbank accessions of previous sequences.
b cultivar abbreviations additional to those in table 2: ga-gala, jb-jamba, gl-gloster, gd-seedling: seedling from golden delicious.
cultivar names in bold indicate material from this study.
cposition refers to the coding sequence and is presented vertically.
snp nucleotides given in bold are our own observations.
snps in italic are identified errors after cross checking.
dmal d  <dig>  consensus snps compared with mal d  <dig> .
the occurrence of pcr recombination and mutations in sequences from gene families warrants scrutinised assessment of sequences.
the use of two independent pcr-cloning steps for each cultivar may effectively filter out most of these erroneous sequences before database donation since the probability of isolating identical artefacts in independent pcrs is low  <cit> .
sequence specific markers may be used to validate newly found isoforms that have passed this first sifting.
many of the sequences found in this study were either confirmed by identical sequences retrieved from the other cultivars used or by identical sequences previously donated in the databases as well as through the use of sequence specific markers .
cultivar specific allergenicity and its relation to quantitative and qualitative differences in mal d 1
levels of total mal d  <dig> protein can not fully explain allergenicity of cultivars, indicating that other factors are involved as well.
for instance, son et al.
<cit>  found a fourfold higher level of mal d  <dig> protein in granny smith compared to gloster.
although this suggests a correlation between total amount and allergenicity, this relation was contradicted by their results on golden delicious and granny smith, which cultivars were similar allergenic despite of a threefold difference in total mal d  <dig> content.
furthermore, it was demonstrated that different mal d  <dig> isoforms  <cit> , as well as mutants of specific isoforms  <cit> , have different binding affinities to ige indicating that the relative mal d  <dig> protein composition as well as the presence of specific alleles may have a significant effect on allergenicity.
this research is the first to show an association between the genetic mal d  <dig> constitution of apple cultivars and birch pollen related allergenicity.
if the allergenicity is a matter of the variants of a single gene, then mal d  <dig> a will be the most outstanding candidate.
in case of the involvement of multiple genes, also mal d  <dig>  may play a role.
a putative involvement of the intronless genes can not yet be excluded, as their allelic variation has not yet been surveyed.
such a survey will be a challenge considering the high sequence similarity among alleles of different loci, which can be over 98%  <cit> .
with regard to mal d  <dig> , two of the sequenced cultivars showed variant compositions for mal d  <dig>  that did not exactly fit with the relative spt data found.
prima has the functional allele of mal d  <dig>  in duplex but has an intermediate allergenicity, whereas fuji is the only intermediate-allergenic cultivar that had two pseudo-alleles for mal d  <dig>  .
this might indicate that mal d  <dig>  does not show dosage allele effects and cannot explain allergenicity without considering other factors.
alternatively, these two exceptions infer variant  <dig> of mal d  <dig> a to be more allergenic than variant  <dig>  in which case the stronger effect of  <dig> in fuji is counteracted by the absence of a functional allele for mal d  <dig> .
similarly, the milder effect of variant  <dig> of prima is then compensated by the double dosage of a functional mal d  <dig>  allele.
a stronger effect of variant  <dig> fits with the tendency of higher responses of the three heterozygous 01/ <dig> cultivars compared to the two  <dig> homozygous cultivars.
the above described associations could be found due to the presence of allelic variation among the examined apple cultivars and due to performing a complexity reduction of the human variation by only analysing patients with mild spt responses, thus reducing the effect of variation among humans for sensitivity to different allergens.
studies with larger patient sizes may probably benefit from further grouping to also account for genetically determined human variation in sensitivity to different  allergen variants.
such grouping has probably to be based on allergy responses as no knowledge exists on the involved human genes neither on their allelic composition.
the finding that allergenicity depends on the presence and amount of some specific mal d  <dig> isoforms is highly relevant for diagnostics tests and immunotherapy, and justifies additional research on a larger number of apple cultivars as well as atopic individuals.
since the first mal d  <dig> and mal d  <dig> genes have also been recently mapped  <cit>  and the mapping of additional genes of these allergens is in progress, it will become possible to also investigate the effects of allelic composition of these mal d allergens on the allergenicity of cultivars by association studies.
location of amino acid polymorphism in a 3d structure model
for mal d  <dig> a, high-allergenic cultivars have two putative genotypes, homozygous variant  <dig> or heterozygous variant  <dig> together with variant  <dig>  whereas low-allergenic cultivars are homozygous for variant  <dig>  the intermediate-allergenic cultivars contained the low allergenic variant  <dig> in combination with one of the high allergenic variants  <dig> or  <dig>
the three mal d  <dig> a variants differ at two amino acids:  <dig> v/i and 135v/a.
considering the three dimensional structure model of mal d  <dig>  <cit> , the first polymorphism is located in the first loop between the β1-strand and the α1-helix, the second is located in the α3-helix structure motif.
the amino acid changes are all between hydrophobic amino acids but they have different side chains that may have an effect on the 3d structure of the protein and thus on epitope conformation.
expression of mal d  <dig> genes in fruit
for specific mal d  <dig> genes to be involved in allergenicity, expression in apple fruit is a prerequisite.
until now, mrna expression for five genes was observed in mature fruit through both rtpcr  <cit>  and est sequences , representing two genes  on lg  <dig> and three genes  on lg  <dig> , whereas mrna based est sequences of mal d  <dig>  were only found in mature leaves.
because the number of rtpcr studies on and est sequences derived from fruit is still limited and usually only assessed at a single time moment, definitive mrna based conclusions regarding mal d  <dig>  expression in fruit can not yet be made.
recently obtained proof for its expression in the epidermis of fruit  <cit>  is not conclusive as the primers used can probably also amplify mrna of mal d  <dig>  and mal d  <dig> b. from ests identified in cdna libraries of ripe gala fruit, tissue specific expression patterns were found.
mal d  <dig>  and mal d  <dig>  ests were found in both skin and cortex, while mal d  <dig> a,  <dig> b and  <dig> e ests were only found in the skin of apple fruit.
at this moment, there is evidence for the presence of two mal d  <dig> proteins in apple fruit  <cit> , the majority of mal d  <dig> protein is mal d  <dig>   and a minor part is mal d  <dig> a  <cit> .
interestingly, both genes are located on linkage group  <dig> where also mal d  <dig>  is located.
these mrna and protein data thus allow mal d  <dig> a to be involved in differences in allergenicity among cultivars.
the current lack of support for the presence of mal d  <dig>  in fruit might indicate that the observed association is coincidental, but may as well be due to lack of extensive expression studies.
genotyping for mal d  <dig> haplotypes
the mal d  <dig> genes in lg  <dig> are tightly linked to each other  <cit> .
this tight linkage can simplify the genotyping of additional cultivars, at least if their pedigree and the linkage phase of their parental alleles are known.
in these cases, genotyping can be performed by a single representative, multi-allelic marker such as the mal d  <dig> a ssr marker.
as linkage phases of the mal d  <dig> genes of lg  <dig> are known for all  <dig> cultivars of our reference set but discovery , this simple and efficient approach was performed in this study for certain cultivars .
CONCLUSIONS
we have shown that differences in allergenicity among apple cultivars are associated with the allelic composition of two specific genes, mal d  <dig>  and mal d  <dig> a, which are both located on linkage  <dig> of malus domestica.
furthermore, allele dosage effects are found relevant for mal d  <dig> a. our findings indicate the need to reconsider the relevance of merely assessing total amounts of mal d  <dig> protein in allergy research and diagnostic tests and warrant further research on the association of specific mal d  <dig> isoforms and allergenicity among a larger group of cultivars and allergy sufferers.

 many birch pollen sensitised patients in central and northern europe suffer from oral allergy symptoms after eating fresh apples. the prevalence of apple allergic individuals mounts up to ~3% in central and northern europe. this type of apple allergy is caused by cross reactivity of ige antibodies against the major and sensitizing birch pollen allergen bet v with mal d the major allergen of apple. bet v and mal d are both pathogenesis-related proteins. they belong to the pr- family and share a high degree of homology. from patients' experience it is known for a long time that the severity of allergic reactions to apple was not only related to the specific sensitivity of the individual, but also largely depended on the apple cultivar. this cultivar dependent allergenicity has also been described in literature. for instance, mal d from the cultivar golden delicious was found highly reactive to specific ige antibodies from allergic patients' sera, whereas mal d from the cultivar gloster generally showed much less reactivity. in addition, skin prick testing with different apple cultivars and confirmations for specific cultivars in double-blind placebo-controlled food challenges and oral challenges of whole apples, revealed a wide range of allergenic reactivity from very high to very low. as a result from these studies, the new cultivar santana was identified as hypo- allergenic for 75% of the patients with a mild apple allergy, which is usually assumed to be mal d based. in the netherlands, this cultivar has recently been marketed as'suited for individuals with mild apple allergy' in order to meet the general desire of apple allergic persons to be able to add this common fruit to their daily diet. the differences in allergenicity among cultivars raised a crucial question on the origin of this cultivar-specific degree of allergenicity. allergenicity may depend on the total amount of mal d proteins, as suggested by son et al. from their observed ten-fold difference in mal d amount between the high-allergenic cultivar golden delicious and the low-allergenic cultivar gloster. however, there is little evidence supporting this hypothesis because only very few cultivars have been studied and, for these, a linear response between mal d protein content and allergenicity estimates is lacking. on the other hand, qualitative characteristics of the mal d proteins could be involved too, as can be argued from the differences in binding capacity of birch pollen-specific ige to two protein variants of mal d. to elaborate this latter issue, research on the genetic variation of mal d and its expression pattern in the different cultivars is required and should be compared to allergenicity data. it is known that mal d is coded by a large gene family of members mapped on three linkage groups of the apple genome. not all of these members are likely to be involved in allergenicity since only a limited number of different mal d proteins and mrnas have been traced back in apple fruit so far. research towards the relative importance of the quality and quantity of mal d proteins on the allergenicity of apple cultivars is relevant for designing apple breeding programs for low-allergenic apple cultivars of high quality and healthiness. in this paper, we focused on the genetic diversity of mal d genes. the mal d gene family can be subdivided into two major categories: genes with and genes without an intron. preliminary genetic analyses revealed that the genetic diversity was by far larger in the intron-containing genes. furthermore, the intron-containing genes cover all three linkage groups that mal d loci. therefore, this category of genes has been chosen to start looking for putative qualitative effects of mal d proteins in cultivar specific allergenicity. allelic diversity of the seven intron-containing mal d genes was assessed among a set of cultivars chosen for their importance in breeding programs and apple production. in order to find putative associations with allergenicity, the presence of alleles coding for different protein variants was subsequently compared with the degree of allergenicity for a subset of cultivars for which allergenicity data from spt or dbpcfc tests were available.  diversity of mal d genes and deduced proteins the observed dna polymorphisms in the studied cultivars resulted in a total of different mal d sequences over seven genes. these sequences were denoted according to the occurrence of 1) dna polymorphisms in the coding region of the gene leading to different protein variants; 2) polymorphism in the coding region that did not affect the protein sequence, and 3) polymorphism in the intron. although the latter two differences are of minor importance with respect to allergenicity, they provided additional landmarks for the development of sequence specific molecular markers. avariants refer to different protein sequences. variant numbers of mal d, mal d and mal d are according to the allergen list, those for mal d and mal d are according to gao et al.. bgenome sequences are numbered successively per gene cgolden delicious, priscilla, ingrid marie, cox, jonathan, red delicious, fuji, discovery, prima and fiesta. note that different accessions of priscilla seem to exist. the priscilla used here is a parent of santana as confirmed by ssr markers, but can not descend from its supposed mother starking delicious d indicates the presence of an allele in heterozygous or homozygous condition. avariant numbers of are according to the allergen list, those for mal d and mal d are according to gao et al.. b, c, d as in table 1 e # and * for lg alleles that have the same symbol are in coupling phase with each other. this information was obtained by their co-segregation patterns in mapping progenies; ingrid marie = cox × open pollinated and fuji = ralls janet × delicious; red delicious is a colour mutant of delicious. fps and ps of mal d refer to pseudo-alleles. g the allele specific markers for these alleles were derived from table of gao et al. whereby we took into account that their marker for mal d c actually amplifies mal d c and visa versa, thus harmonizing an inconsistency between their tables and in the assignment of allele and marker names to the ay and ay sequences. mal d and - showed to be highly conserved at the protein level. the related genes coded for only two variants each that both differed in just a single amino acid and for which the second variant was found only once. the other genes were more variable, coding for three up to six variants. mal d showed to be special in that two sequences contained a stop codon in the coding region and were therefore regarded as pseudo alleles. interestingly, the pseudo alleles occurred frequently as for seven out of the ten cultivars at least one of the alleles was a pseudo allele whereas cultivars priscilla and fuji only contained pseudo alleles of mal d. allergenicity scores of apple cultivars by skin prick test relative spt responses of apple cultivars are given in table fiesta, delblush, pinova and golden delicious were ranked in the high allergenic group. priscilla and santana showed low spt responses, with wheal areas 30–35% of that of golden delicious. nine cultivars were intermediate allergenic. santana was also identified as low-allergenic in comparison to golden delicious in dbpcfc tests and oral provocation tests. a data for the first three experiments were derived from the original data from three experiments of bolhaar et al. on respectively cultivar screening, intra-cultivar variation and storage, of which we used only the data of patients with mild symptoms. the forth experiment came from a new cultivar screening experiment. b the underlying hep values for golden delicious for the four experiments were respectively,, and. general associations from the sequenced cultivars from the cultivars used to sequence the intron containing mal d genes and to perform spt on allergenicity, golden delicious was ranked as the highest allergenic cultivar whereas priscilla was ranked as the lowest. this difference was not related to protein variant composition of mal d,, 106b and c because these cultivars have identical protein variants. in contrast, both mal d and mal d a have different protein variants in golden delicious and priscilla. mal d may contribute to the observed difference in allergenicity between the two cultivars since protein variant is present in golden delicious while priscilla is heterozygous for the two pseudo-alleles ps and ps similarly, mal d a protein variant - was found in priscilla whereas variants - and - were found in golden delicious. the intermediate cultivars, jonathan, prima, and fuji, have the putatively high allergenic alleles - or - in single dosage, whereas high allergenic cultivars either have both alleles - and - or have - in homozygous state. this suggests that allergenicity might be determined by both the protein variant and the gene dosage of highly allergenic alleles. association analysis by pedigrees: from golden delicious to santana the identity and origin of genomic alleles and thus protein variants in additional cultivars could be traced by developed allele specific snap and ssr markers and the use of pedigree information. for instance, the deduced flow of protein variants over the pedigree of cultivar santana is presented in figure santana and priscilla are low allergenic whereas golden delicious is high allergenic. for mal d, and the same protein variants were found for golden delicious and santana. in contrast, golden delicious and santana differ in their protein variant composition of mal d., - a and - b, indicating a possible involvement of these proteins in the observed difference in allergenicity between these cultivars. santana, like priscilla, has only pseudo alleles for mal d that do not result in protein production, while golden delicious has one functional allele. the mal d a variant of santana is not present in golden delicious. associations for mal d and - a similarly to santana, pedigree information and sequence specific markers were used to assess the putative protein variant composition for other additional cultivars for which we have spt response data. this allowed a further validation of the association of the mal d and a with allergenicity in a total of cultivars. cultivars delblush and pinova evoked an spt response that was similar to that of golden delicious and also had the same variant compositions as golden delicious for mal d and mal d a, namely variant for mal d and the two variants and for mal d. the remaining five cultivars evoking an intermediate allergenic response also had a similar variant composition as the previously identified intermediate cultivars, namely variants and for mal d a, and variant of mal d, except for prima, that has two alleles coding for mal d and fuji that contains both pseudo alleles. the other intron containing genes did not show any association. a: cultivars in bold were sequenced for their intron containing mal d genes b: in % relative to golden delicious. discussion birch pollen induced oral allergy for apple has been the subject in a considerable number of studies. one of the prominent results has been the presence of cultivar-specific differences in allergenicity. unfortunately, evidence regarding the causes of cultivar-specific allergenicity is still lacking. one of the knowledge gaps concerned the number and identity of mal d genes and the amount of variation within these genes. recently, gao et al. have shown that mal d genes are members of a large gene family by identifying different loci that are located in three clusters. based on sequence identity, these genes could be subdivided into intron containing and intronless genes. in order to create a basis for a better understanding of the genetics of mal d genes and their impact on allergenicity, we have studied the allelic diversity of the intron containing genes in cultivars that are often used in breeding. development of sequence specific markers and pedigree information enabled the assessment of putative mal d constitutions of other cultivars. using this information, we assessed the different mal d isoforms that cultivars are able to produce and found associations between their putative protein constitutions and spt-responses. allelic diversity and validity of database sequences cloning and sequencing of the seven intron-containing mal d genes in cultivars revealed different alleles that coded for different mal d isoforms. the variation per gene varied with regard to the number of alleles and deduced proteins. mal d and mal d were diverse at the gdna level but conserved at the protein level. for both genes only two protein variants were found, of which the second variant, differing in one amino acid only, was found in just a single cultivar. for mal d, mal d and the three mal d genes, gdnas often coded for different proteins and these genes were therefore more variable at the protein level. because the examined cultivars are important in the breeding of many modern apple varieties, the set of alleles found in this study likely represents a considerable part of the total variation present in intron containing mal d genes of common apple varieties. although other mal d sequences are known from public databases, we suspect that many of these sequences may be artefacts derived through strand switching and pcr mutations. the problem associated with pcr amplification of a group of closely related sequences, such as the mal d gene family, is that besides pcr induced single base pair mutations, in vitro strand switching or re-annealing of incompletely amplified fragments can lead to artefacts as was exemplified by schenk et al. for birch bet v sequences. for instance, for mal d one of the most studied mal d genes, over dna sequences from previous studies are known from public databases indicating the presence of putative protein isoforms. we know mal d is a single locus gene with maximum two alleles present in a cultivar, but four sequences from golden delicious can be found in databases. from these, only sequence accession af was identical to one of our two sequences. the other sequences may be due to artefacts. firstly, accession af had one snp at position compared to af which is due to the cloning primer used. similarly, sequences from a number of other cultivars showed this 11a mutation too. in our study, the cloning primers used were positioned in the 5'-untranslated region thus avoiding this problem. secondly, accession z that was classified as mal d 1c is quite similar to mal d, while the snps found are unique to three other mal d genes. this may be due to an artefact from strand switching during pcr which could explain why we, like other researchers could not retrieve this sequence from golden delicious. similarly, the first nucleotides of af and af match to mal d, while the remaining sequence matches to mal d making these accessions also putative erroneous sequences due to strand switching. therefore, the actual number of mal d variants present among apple cultivars is probably not as high as nine, because at least five of them are likely pcr artefacts. two protein variants of mal d could be confirmed in this study. variants mal d, and are likely artefacts of whereas and are likely artefacts of. a alleles in bold are confirmed by our own sequences. numbers in brackets indicate genbank accessions of previous sequences. b cultivar abbreviations additional to those in table 2: ga-gala, jb-jamba, gl-gloster, gd-seedling: seedling from golden delicious. cultivar names in bold indicate material from this study. cposition refers to the coding sequence and is presented vertically. snp nucleotides given in bold are our own observations. snps in italic are identified errors after cross checking. dmal d consensus snps compared with mal d. the occurrence of pcr recombination and mutations in sequences from gene families warrants scrutinised assessment of sequences. the use of two independent pcr-cloning steps for each cultivar may effectively filter out most of these erroneous sequences before database donation since the probability of isolating identical artefacts in independent pcrs is low. sequence specific markers may be used to validate newly found isoforms that have passed this first sifting. many of the sequences found in this study were either confirmed by identical sequences retrieved from the other cultivars used or by identical sequences previously donated in the databases as well as through the use of sequence specific markers. cultivar specific allergenicity and its relation to quantitative and qualitative differences in mal d 1 levels of total mal d protein can not fully explain allergenicity of cultivars, indicating that other factors are involved as well. for instance, son et al. found a fourfold higher level of mal d protein in granny smith compared to gloster. although this suggests a correlation between total amount and allergenicity, this relation was contradicted by their results on golden delicious and granny smith, which cultivars were similar allergenic despite of a threefold difference in total mal d content. furthermore, it was demonstrated that different mal d isoforms, as well as mutants of specific isoforms, have different binding affinities to ige indicating that the relative mal d protein composition as well as the presence of specific alleles may have a significant effect on allergenicity. this research is the first to show an association between the genetic mal d constitution of apple cultivars and birch pollen related allergenicity. if the allergenicity is a matter of the variants of a single gene, then mal d a will be the most outstanding candidate. in case of the involvement of multiple genes, also mal d may play a role. a putative involvement of the intronless genes can not yet be excluded, as their allelic variation has not yet been surveyed. such a survey will be a challenge considering the high sequence similarity among alleles of different loci, which can be over 98%. with regard to mal d, two of the sequenced cultivars showed variant compositions for mal d that did not exactly fit with the relative spt data found. prima has the functional allele of mal d in duplex but has an intermediate allergenicity, whereas fuji is the only intermediate-allergenic cultivar that had two pseudo-alleles for mal d. this might indicate that mal d does not show dosage allele effects and cannot explain allergenicity without considering other factors. alternatively, these two exceptions infer variant of mal d a to be more allergenic than variant in which case the stronger effect of in fuji is counteracted by the absence of a functional allele for mal d. similarly, the milder effect of variant of prima is then compensated by the double dosage of a functional mal d allele. a stronger effect of variant fits with the tendency of higher responses of the three heterozygous 01/ cultivars compared to the two homozygous cultivars. the above described associations could be found due to the presence of allelic variation among the examined apple cultivars and due to performing a complexity reduction of the human variation by only analysing patients with mild spt responses, thus reducing the effect of variation among humans for sensitivity to different allergens. studies with larger patient sizes may probably benefit from further grouping to also account for genetically determined human variation in sensitivity to different allergen variants. such grouping has probably to be based on allergy responses as no knowledge exists on the involved human genes neither on their allelic composition. the finding that allergenicity depends on the presence and amount of some specific mal d isoforms is highly relevant for diagnostics tests and immunotherapy, and justifies additional research on a larger number of apple cultivars as well as atopic individuals. since the first mal d and mal d genes have also been recently mapped and the mapping of additional genes of these allergens is in progress, it will become possible to also investigate the effects of allelic composition of these mal d allergens on the allergenicity of cultivars by association studies. location of amino acid polymorphism in a 3d structure model for mal d a, high-allergenic cultivars have two putative genotypes, homozygous variant or heterozygous variant together with variant whereas low-allergenic cultivars are homozygous for variant the intermediate-allergenic cultivars contained the low allergenic variant in combination with one of the high allergenic variants or the three mal d a variants differ at two amino acids: v/i and 135v/a. considering the three dimensional structure model of mal d, the first polymorphism is located in the first loop between the β1-strand and the α1-helix, the second is located in the α3-helix structure motif. the amino acid changes are all between hydrophobic amino acids but they have different side chains that may have an effect on the 3d structure of the protein and thus on epitope conformation. expression of mal d genes in fruit for specific mal d genes to be involved in allergenicity, expression in apple fruit is a prerequisite. until now, mrna expression for five genes was observed in mature fruit through both rtpcr and est sequences, representing two genes on lg and three genes on lg, whereas mrna based est sequences of mal d were only found in mature leaves. because the number of rtpcr studies on and est sequences derived from fruit is still limited and usually only assessed at a single time moment, definitive mrna based conclusions regarding mal d expression in fruit can not yet be made. recently obtained proof for its expression in the epidermis of fruit is not conclusive as the primers used can probably also amplify mrna of mal d and mal d b. from ests identified in cdna libraries of ripe gala fruit, tissue specific expression patterns were found. mal d and mal d ests were found in both skin and cortex, while mal d a, b and e ests were only found in the skin of apple fruit. at this moment, there is evidence for the presence of two mal d proteins in apple fruit, the majority of mal d protein is mal d and a minor part is mal d a. interestingly, both genes are located on linkage group where also mal d is located. these mrna and protein data thus allow mal d a to be involved in differences in allergenicity among cultivars. the current lack of support for the presence of mal d in fruit might indicate that the observed association is coincidental, but may as well be due to lack of extensive expression studies. genotyping for mal d haplotypes the mal d genes in lg are tightly linked to each other. this tight linkage can simplify the genotyping of additional cultivars, at least if their pedigree and the linkage phase of their parental alleles are known. in these cases, genotyping can be performed by a single representative, multi-allelic marker such as the mal d a ssr marker. as linkage phases of the mal d genes of lg are known for all cultivars of our reference set but discovery, this simple and efficient approach was performed in this study for certain cultivars.  we have shown that differences in allergenicity among apple cultivars are associated with the allelic composition of two specific genes, mal d and mal d a, which are both located on linkage of malus domestica. furthermore, allele dosage effects are found relevant for mal d a. our findings indicate the need to reconsider the relevance of merely assessing total amounts of mal d protein in allergy research and diagnostic tests and warrant further research on the association of specific mal d isoforms and allergenicity among a larger group of cultivars and allergy sufferers. 
in this study, the allelic diversity of the seven intron-containing mal d genes was assessed among a set of apple cultivars by sequencing or indirectly through pedigree genotyping. protein variant compositions of mal d and - a and, in case of mal d a, allele doses are associated with the differences in allergenicity among fourteen apple cultivars. protein variant constitutions were subsequently compared with skin prick test responses to study the association of deduced protein variants with allergenicity in a set of cultivars. mal d, mal d and mal d a, b and c were more variable, coding for three to six different protein variants. comparison of mal d allelic composition between the high-allergenic cultivar golden delicious and the low-allergenic cultivars santana and priscilla, which are linked in pedigree, showed an association between the protein variants coded by the mal d and - a genes with allergenicity. conversely, no associations were observed for the protein variants coded by the mal d, -, - b, - c genes, nor by the mal d gene. results from this study have implications for medical diagnostics, immunotherapy, clinical research and breeding schemes for new hypo-allergenic cultivars. from the seven intron-containing mal d genes investigated, mal d and mal d were highly conserved, as nine out of ten cultivars coded for the same protein variant, while only one cultivar coded for a second variant. this information indicates the involvement of qualitative as well as quantitative factors in allergenicity and warrants further research in the relative importance of quantitative and qualitative aspects of mal d gene expression on allergenicity. in addition, mal d a allele dosage effects associated with the degree of allergenicity based on prick to prick testing. the mal d gene family is known to have at least intron-containing and intronless members that have been mapped in clusters on three linkage groups.
 mal d is a major apple allergen causing food allergic symptoms of the oral allergy syndrome in birch-pollen sensitised patients. the mal d gene family is known to have at least intron-containing and intronless members that have been mapped in clusters on three linkage groups. in this study, the allelic diversity of the seven intron-containing mal d genes was assessed among a set of apple cultivars by sequencing or indirectly through pedigree genotyping. protein variant constitutions were subsequently compared with skin prick test responses to study the association of deduced protein variants with allergenicity in a set of cultivars.  from the seven intron-containing mal d genes investigated, mal d and mal d were highly conserved, as nine out of ten cultivars coded for the same protein variant, while only one cultivar coded for a second variant. mal d, mal d and mal d a, b and c were more variable, coding for three to six different protein variants. comparison of mal d allelic composition between the high-allergenic cultivar golden delicious and the low-allergenic cultivars santana and priscilla, which are linked in pedigree, showed an association between the protein variants coded by the mal d and - a genes with allergenicity. this association was confirmed in other cultivars. in addition, mal d a allele dosage effects associated with the degree of allergenicity based on prick to prick testing. conversely, no associations were observed for the protein variants coded by the mal d, -, - b, - c genes, nor by the mal d gene.  protein variant compositions of mal d and - a and, in case of mal d a, allele doses are associated with the differences in allergenicity among fourteen apple cultivars. this information indicates the involvement of qualitative as well as quantitative factors in allergenicity and warrants further research in the relative importance of quantitative and qualitative aspects of mal d gene expression on allergenicity. results from this study have implications for medical diagnostics, immunotherapy, clinical research and breeding schemes for new hypo-allergenic cultivars. 