Developing SSCP markers in two Pinus species

Plomion, C.; Hurme, P.; Frigerio, J.M.; Ridolfi, M.; Pot, D.; Pionneau, C.; Avila, C.; Gallardo, F.; Daṿid, Ḥ; Neutelings, G.

Developing SSCP markers in two Pinus species

1999

This study demonstrates the feasibility of generating sequence-based markers in Pinus species, from data available in electronic databases. Nucleotide sequences from 23 partially or fully characterized cDNAs or genomic sequences of pines were used to design PCR primers for amplifying targeted fragments of genomic DNA from Maritime and Scots pine. Various template DNA and MgCl2 concentrations, annealing temperatures, and buffer compositions were used to optimize the PCR amplifications. The polymorphism of 16 sequences was then investigated in a tree-generation inbred pedigree of Maritime pine and in a two-generation pedigree of Scots pine, using single-stranded DNA conformation polymorphism (SSCP) on polymerase chain reaction (PCR) products. The level of polymorphism was shown to be independent of (1) fragment size, (2) the presence or absence of introns in the amplified product and (3) temperature during electrophoresis. Mendelian segregation was tested for 5 SSCP markers in each species. Chromosomal locations of five genes were identified by linkage analysis with previously mapped markers in a genetic map of Maritime pine. The use of SSCP is recommended for constructing a transcriptional map for comparative mapping studies among pines and to provide useful 'candidate genes' for characterizing quantitative trait loci.

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