A Comparative Genomics Strategy for Targeted Discovery of Single-Nucleotide Polymorphisms and Conserved-Noncoding Sequences in Orphan Crops

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.

Construction of Genetic Linkage Map and QTL Analysis of Sink-Size Traits in Pearl Millet (Pennisetum glaucum)

“…The development of SSRs based on EST sequences is a fast, efficient, and economic option [35][36][37][38]. In addition, a new generation of marker system termed single-strand conformational polymorphism-single nucleotide polymorphism (SSCP-SNP) has been developed in pearl millet to take advantage of the SSCP technique and the large number of SNPs in the nonexpressed intron regions of genes [39], which are also the target of the conserved intron-spanning primer (CISP) markers [40]. SNP markers provide an inexhaustible source of polymorphic markers for use in high-resolution genetic mapping and are the most abundant type of molecular genetic markers in the genome.…”

Striga

“…In pearl millet, although marker aided-selection (MAS) has not been practiced yet in breeding for Striga resistance, there have been extensive efforts towards development of DNA-based markers including simple sequence repeat (SSR) Budak et al, 2003;Mariac et al, 2006;Qi et al 2001Qi et al , 2004Senthilvel et al, 2004Senthilvel et al, , 2008Jia et al, 2007;Yadav et al, 2007Yadav et al, , 2008Rajaram et al, 2013), diversity array technology (DArT) (Mace et al, 2008(Mace et al, , 2009Supriya et al, 2011) and single nucleotide polymorphism (SNP) (Bertin et al, 2005;Feltus et al, 2006;Kountche, 2013;Moumouni et al, 2015). In most of these studies, a number of genetic maps have been constructed using different crosses, mostly with the aim of mapping specific traits.…”

Gene-Based Marker Systems in Plants: High Throughput Approaches for Marker Discovery and Genotyping

“…In case of well-characterized species, exonintron boundaries can be defined using tools like FEGNESH, etc. and subsequently primer pairs can be designed using the flanking exonic sequence of an intron to amplify intronic region (Feltus et al 2006). By using the genome sequence data of Medicago and Lotus and EST data of soybean and Medicago, >3,000 primer pairs targeting intronic regions have been developed in the laboratory of Doug Cook at UC-Davis, USA (DR Cook, personal communication) to develop gene-based conserved orthologous sequence (COS) markers in legume species.…”

“…In this way, intronic sequence can be identified and exonic sequence of the ESTs are used to design the primer pairs to amplify the intronic region. Based on this concept, after aligning the ESTs of sorghum, pearl millet, Allium and Musa with rice genome, >3,600 primer pairs, called conserved intron spanning primers (CISPs) have been developed for monocot species (Feltus et al 2006;Lohithaswa et al 2007; http://www.plantgenome.uga.edu/CISP/). Following the similar approach, a larger number of gene-based markers have been developed and used for diversity and mapping studies in pearl millet (Bertin et al 2005), lupin (Phan et al 2007), etc.…”