Abstract:
Completed genome sequences provide templates for the design of genome analysis tools in orphan species lacking sequence
information. To demonstrate this principle, we designed 384 PCR primer pairs to conserved exonic regions flanking introns,
using Sorghum/Pennisetum expressed sequence tag alignments to the Oryza genome. Conserved-intron scanning primers
(CISPs) amplified single-copy loci at 37% to 80% success rates in taxa that sample much of the approximately 50-million years
of Poaceae divergence. While the conserved nature of exons fostered cross-taxon amplification, the lesser evolutionary
constraints on introns enhanced single-nucleotide polymorphism detection. For example, in eight rice (Oryza sativa) genotypes,
polymorphism averaged 12.1 per kb in introns but only 3.6 per kb in exons. Curiously, among 124 CISPs evaluated across
Oryza, Sorghum, Pennisetum, Cynodon, Eragrostis, Zea, Triticum, and Hordeum, 23 (18.5%) seemed to be subject to rigid
intron size constraints that were independent of per-nucleotide DNA sequence variation. Furthermore, we identified 487
conserved-noncoding sequence motifs in 129 CISP loci. A large CISP set (6,062 primer pairs, amplifying introns from 1,676
genes) designed using an automated pipeline showed generally higher abundance in recombinogenic than in nonrecombinogenic
regions of the rice genome, thus providing relatively even distribution along genetic maps. CISPs are an effective means
to explore poorly characterized genomes for both DNA polymorphism and noncoding sequence conservation on a genomewide
or candidate gene basis, and also provide anchor points for comparative genomics across a diverse range of species.
