Linkage map positions and allelic diversity of two Mal d 3 (non-specific lipid transfer protein) genes in the cultivated apple (Malus domestica)

Abstract

Non-specific lipid transfer proteins (nsLTPs) of Rosaceae fruits, such as peach, apricot, cherry, plum and apple, represent major allergens for Mediterranean atopic populations. As a first step in elucidating the genetics of nsLTPs, we directed the research reported here towards identifying the number and location of nsLTP (Mal d 3) genes in the apple genome and determining their allelic diversity. PCR cloning was initially performed on two cultivars, Prima and Fiesta, parents of a core apple mapping progeny in Europe, based on two Mal d 3 sequences (AF221502 and AJ277164) in the GenBank. This resulted in the identification of two distinct sequences (representing two genes) encoding the mature nsLTP proteins. One is identical to accession AF221502 and has been named Mal d 3.01, and the other is new and has been named Mal d 3.02. Subsequent genome walking in the upstream direction and DNA polymorphism analysis revealed that these two genes are intronless and that they could be mapped on two homoeologous segments of linkage groups 12 and 4, respectively. Further cloning and sequencing of the coding and upstream region of both Mal d 3 genes in eight cultivars was performed to identify allelic variation. Assessment of the deduced nsLTP amino acid sequences gave a total of two variants at the protein level for Mal d 3.01 and three for Mal d 3.02. The consequences of our results for allergen nomenclature and the breeding of low allergenic apple cultivars are discussed.

Genomic cloning and linkage mapping of the Mal d 1 (PR-10) gene family in apple (Malus domestica)

“…) DNA isolation, PCR cloning, and sequencing Genomic DNA was extracted using the CTAB-based, large-scale nuclei-isolation method (Roche et al 1997). The PCR cloning and sequencing procedures have been described previously (Gao et al 2005). Here we mention only the key points and some changes.…”

“…Designing and testing of sequence-specific markers Two types of molecular markers were used to distinguish a specific sequence or allele in the context of the PM · FS or the JO · PM population: single nucleotide amplification polymorphism (SNAP) markers (Drenkard et al 2000;Gao et al 2005) and simple sequence repeat (SSR) markers. The SNAP markers were tested first on gDNA of PM, FS, and eight individuals of their population to confirm PCR conditions, expected product size and segregation pattern.…”

“…Genes with more than 95% DNA sequence identity that are also clustered on the same linkage group (LG) were denoted by adding a capital letter to their isoallergen name, such as Gao et al (2005).…”

Genomic characterization and linkage mapping of the apple allergen genes Mal d 2 (thaumatin-like protein) and Mal d 4 (profilin)

“…They are the same cultivars that had been chosen for mapping Mal d 1 (Gao et al 2005a) and Mal d 3 (Gao et al 2005b). Two primer pairs located in the untranslated region (UTR) were used to amplify the targeted Mal d 2 genomic sequences (Table 1).…”

Production of hypoallergenic plant foods by selection, breeding and genetic modification

“…Assessment of the deduced nsLTP aminoacid sequences in 10 genetically unrelated apple cultivars gave a total of two variants for the one, and three variants for the other gene. This indicates that the variations in the expressed proteins are very minor and that differences in Mal d 3 allergenicity among apple cultivars will mainly depend on the content of Mal d 3 (Gao et al 2005).…”