Acid phosphatase-1, a tightly linked molecular marker for root-knot nematode resistance in tomato: from protein to gene, using PCR and degenerate primers containing deoxyinosine

With a view to cloning the root-knot nematode resistance gene Mi in tomato by chromosome walking, we have developed a molecular probe for the tightly linked acid phosphatase-1 (Aps-1) locus. The acid phosphatase-1 allozyme (APS-1(1)), encoded by the Aps-1(1) allele originating from Lycopersicon peruvianum, was purified to apparent homogeneity from tomato roots andsuspension cells. Microsequencing of CNBr and tryptic peptides generated from APS-1(1) provided a partialamino acid sequence, which accounted for approximately 23% of the protein and revealed two stretchesof homology with soybean proteins KSH3 and VSP27, comprising 22 matches within 26 amino acidresidues. The partial amino acid sequence information enabled us to isolate a 2.4 kb genomicAps-1(1) sequence by means of the polymerase chain reaction (PCR), primed by degenerate pools of oligodeoxyribonucleotides, synthesized on the basis of the amino acid sequences. Synthesis of the 2.4 kb PCR productwas specific for genomic templates carrying the L. peruvianum Aps-1(1) allele. Crucial to thepriming specificity and the synthesis of the 2.4 kb genomic sequence was the use of degenerate primer pools in whichthe number of different primer species was limited by incorporating deoxyinosine phosphate residues atthree and four base ambiguities. In using cDNA as a template, a 490 bp sequence was obtained, indicating ahigh proportion of intron sequences in the 2.4 kb genomic Aps-1(1) sequence. The Aps-l(1) origin ofthe PCR product was confirmed by RFLP (restriction fragment length polymorphism) analysis, using both a chromosome 6 substitution line and a pair of nearly isogenic lines, differing for a smallchromosomal region around the Aps-l/Mi loci.