Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress

Verona, Italy - 24/27 September, 2003

ISBN 88-900622-4-X

Poster Abstract - 2.48

SINGLE NUCLEOTIDE POLYMORPHISM ISOLATION IN CAPSICUM SPP.

A. ACQUADRO*, D. LEE**, E. CHIAPPARINO**, C. COMINO*, E. PORTIS*, P. DONINI**, S. LANTERI*

*) Di.Va.P.R.A. settore Genetica Agraria, Università di Torino

**) NIAB - Molecular Research Group, Cambridge, UK

SNPs, REF-SSCP, pepper, polymorphism

Single nucleotide polymorphisms (SNPs) are point mutations in which one nucleotide is substituted for another at a particular locus. SNPs are the most common type of sequence differences between alleles and their polymorphism may be used as genetic marker. SNP markers offer great potential for high genome coverage and high throughput analysis in both MAS and basic studies. The identification of SNPs for targeting quality traits, QTLs and disease resistance genes is already advanced on a number of major crops.

We report on a preliminary study aimed at the detection of SNPs in pepper, by REF-SSCP, in genes coding for well-studied enzymes. Fifteen genes from Capsicum annum were selected from the NCBI database for SNP mining. We analysed, for each gene, one fragment of about 600 bp originating from a region overlapping the intron-exon junctions. Fifteen pairs of PCR-primers were designed and employed for the amplification of such sequences in 27 C. annum accessions and 7 pepper species (C. annum L., C. chinense L., C. frutescens L., C. pubescens L., C. chacoense L., C. baccatum L., C. tovari L.). PCR products were analysed by REF-SSCP for the presence of point mutations in different samples, i.e.: the amplification products were digested with MseI or AluI, then denatured to yield single-stranded DNA fragments and rapidly cooled down to 0^oC; the ssDNA fragments were separated on polyacrilamide gels and silver stained.

The polymorphic fragments were purified and directly sequenced in an ABI 3100 sequencer using BigDye™ terminator chemistry. The sequences were aligned with Megalign (DNASTAR) using Clustal algorithm. Putative SNPs were identified and validated by Tetra-primer ARMS-PCR. Primers were designed using the software made available on line http://cedar.genetics.soton.ac.uk/public_html/primer1.html.).

Fifty-seven SNPs were found, of which 34 were in coding region (cSNPs), however only 18 of them caused amino acid changes. Most of the mutations were at the third position of the codon (47%) while 38% and 18% were at the second and the first position respectively.

The frequency of base substitutions between 5 of the 7 Capsicum species in study was 1 SNP per 48 bp while 1 SNP per 1360 bp between C. annuum accessions.

The strategy applied proved to be a reliable and a cost effective method for SNP characterisation. The use of REF-SSCP offers some economic advantages as it is possible to identify and sequence only the polymorphic amplicons. Nevertheless a complete study of nucleotide variability in this species would require a large genome coverage.