• Cloning
• DNAstar
• Mal d
• Malus domestica
• Molecular markers
• OAS
• Sequencing
• SNP
• Thaumatin like protein

Apple is the most important fruit in people’s daily life. Its scientific name is Malus domestica. The majority of apple cultivars is diploid (2n=34). Although apple is healthy fruit to most people, some individuals can not eat apple because it provokes allergic reactions. Apple allergy is common in Europe and affects about 2% of the population. The most common symptom of apple allergy is oral allergy syndrome (OAS). Symptoms occur in other organs like skin, nose and lungs as well. In apple, four classes of allergens (Mal d 1, -2, -3 and -4) have been identified through immunoblotting, protein and DNA sequencing. Mal d 2 is researched and discussed in this thesis. The Mal d 2 genes coding to thaumatin-like protein, a homologue of the thaumatin-like protein (TLP), is one of the major protein constituents of mature apple fruit.
TLPs belong to the PR-5 families of pathogenesis-related proteins. Several researchers provided evidence that TLPs play a role in plant defence against pathogens.
TLP is one of the major protein constituents of mature apple fruit. It is homologous to an intensely sweet tasting protein, isolated from the fruit of Thaumatococcus daniellii. TLPs can be categorized into three subclasses based on their isoelectric point (pI). Most TLPs have 16 conserved cysteines that form eight disulfide bonds contributing to the protein’s resistance to proteolysis and heat.
Its cDNA (AF090143) has been isolated and characterized from apple fruit. More recently, a TLP 23.2-kDa, deduced from a full-length cDNA clone (AJ243427) isolated from ripe apple fruits, it was classified as Mal d 2 allergen and characterized as an antifungal protein. The purified recombinant Mal d 2 appeared to have a double band at 31–32 kDa on SDS-PAGE, while MALDI-TOF mass spectrometry revealed a dominant peak close to the calculated 23 kDa, showing a characteristic pattern of 16 cysteine residues, forming 8 disulfide bonds that are responsible for protein stability and compactness. This stabilized structure contributes to the protein’s resistance to low pH conditions, heat-induced denaturation, and proteolysis. Numerous studies have been performed on the frequency of sensitization to Mal d 2, but only limited information on the biochemical and immunological properties of the purified natural protein is available.
The haploid genome of apple contains 17 chromosomes, but chromosomes are highly duplicated according to the information indicated from SSR and RFLP markers, for instance, linkage group (LG) 2 and LG7, LG8 and part of LG15, LG9 and LG17 are duplicated chromosomes. To date, two Mal d 2.01 genes have been mapped on a linkage map (Mal d 2.01A-B in linkage group PM9 from PMXFS, Mal d 2.01B in linkage group FS9 from PMXFS, Mal d 2.01B in linkage group DS9 from FSXDS).
In order to find in new cultivars the Mal d 2.01 genes a database survey will be performed, and based on these Mal d 2.01 sequence data.
The sequencing reaction will be performed to find in new cultivars the Mal d 2.01 genes. The sequences found in NCBI and other papers will be aligned through DNAstar® software.
Through sequence alignment and identification of polymorphism, afterwards the sequence specific primers can be designed as single nucleotide polymorphism (SNP) markers and then tested on a segregating apple population. With the data of this segregating population these marker can be subsequently mapped.