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Table 7 Description of nine sequence-specific SNAP markers and one SSR marker used for the genotyping of cultivars (in bold: SNP, underlined are deliberate introduced SNP to increase specificity).

From: Assessment of allelic diversity in intron-containing Mal d 1 genes and their association to apple allergenicity

Marker namea

Primers 5'-3'

SNPs detectedb

Product (nt)

Tmc

Cyclesd

Mal d 1.010502

F: TGAAGCACAGGATTGACGCA

R: CCACACAACCTTCGACTCA

390A

346

56

35

Mal d 1.010501

F: AGCTGAAATCCTTGAACGAA

R: CAATGTTTCCCTTGGTGAGA

- 528T

426

55/53

10/30

Mal d 1.010503

F: TGAAGCACAGGATTGACACG

R: CAATGTTTCCCTTGGTGTC G

390G 528C

177

57/55

10/30

Mal d 1.0201-1

F: TCACTTTTGGTGAAGGTCTG

R: AGGCGTATGAGTAGTTTACC

- 402G

252

57

35

Mal d 1.0201-4

F: ATTATTTAGATGGTTTCGCTA T

R: TATGCGTCGGGGTGTCCCTA G

227T 624C

439

55

35

Mal d 1.0201-3

F: GCCCTGGAACCATCAAG TAG

R: GGCTGCCTGTGAGACAACTG

165G, 168G 342C

213

61

35

Mal d 1.04-6/7

F: CATCCCGAAGATTGCTCCG T

R: CCTTAGCATGGTAGTGGCGAG

109T 464C

395

57

35

Mal d 1.04-4

F: CAAGGAAGAGCATGTTAAAGT

R: AGGGTAACACACAAATTACATG

516A, 518T

136

60

35

Mal d 1.06C0201-2

F: CCACCATTTTCTCCATTAACTTCA

R: GCCTTAACATGCTCTTCCTTGATT

221A

360

62/60

5/35

Mal d 1.06A-SSRe

F: GGTGAAGGTTAGTTTAATTTCCACA

R: GTTTCACATAGCTGTATTCACTCCCTf

(CA) SSR

 

60

30

  1. a Names are according to Table 1;
  2. bPosition refers to the genomic sequence counted from the ATG starting codon;
  3. c,d PCR annealing temperature (Tm) and number of cycles, respectively. In case of two values, a touch-down PCR was performed in two steps.
  4. eNote that although the SSR marker for Mal d 1.06A is gene specific, its is not sequence specific. In cases where a marker allele could be derived from both parents, linked markers have been used to assess the inheritance of protein variants to cultivars.
  5. f Nucleotides that are italicized were added to the 5' end of the initial primer to obtain the sequence GTTT, which serves a pig-tail [31].