Ab initio Discovery of Indel and SNP Markers Without a priori Knowledge of Map Position

One disadvantage to developing SSR or SNP markers in transcribed sequences is their low polymorphism between closely related tomato species and within cultivated germplasm. The sequence of coding regions is more conserved than non-coding regions. Therefore, the design of primers corresponding to the 5' or 3' UTR provides one approach to increase the chance for polymorphic SSR, indel or SNP detection (Cato et al. 2001). Another approach for detection of polymorphisms is to mine intron sequences. This approach is feasible because intron position is conserved between diverged species, especially for highly conserved genic sequences. Using conserved orthologous sequences (COS) derived for tomato (Fulton et al. 2002b; Wu et al. 2006) the position of introns can sometimes be predicted by comparison to the Arabidopsis genome (Timms et al. 2006; Van Deynze et al. 2006; Wu et al. 2006). Tools to find the position of introns are available from several genome projects including the Com-positae Project Python blast viewer and the SGN In-tron Finder (Table 5). For tomato, the intron mining approach resulted in the identification of 593 SNPs in 161 loci and 294indels in 122 loci within breeding lines, and 1,113 SNPs in 274loci and 206indels in 96 loci between cultivated varieties and S. pimpinellifolium (Van Deynze, van der Knaap and Francis, unpublished).

A promising method to identify SNPs is through oligo-based microarray hybridization of fluorescently labeled target sequences. Both cDNA and genomic DNA can be used as labeled targets during hybridization (Cui et al. 2005). For tomato, cDNA made from

RNA extracted from various tissues of S. lycopersicum cv. Ohio7814 and S. pimpinellifolium LA1589 was hybridized to a NimbleGen array consisting of 15,270 tomato unigenes (Sim et al. 2007). In the Nimble-Gen design, each gene was represented by 12 perfect match and 12 mismatch oligonucleotide probes of 24 bp in length. Probes identified as outliers are potential single feature polymorphisms (SFPs). For the Ohio7814 and LA1589 hybridizations, 1,296 putative SFPs were identified. The verification of these SFPs was conducted by sequencing alleles for 216 putative SFPs, and 52% of the features identified as outliers were confirmed as polymorphic. SNPs accounted for 86% of the confirmed polymorphisms while indels accounted for 14% (Francis et al. unpublished).

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