Citrus (Rutaceae) SNP markers based on Competitive Allele‐Specific PCR; transferability across the Aurantioideae subfamily

Single nucleotide polymorphisms (SNPs) are the most frequent type of DNA sequence polymorphism. Their abundance and uniform distribution in genomes make them very powerful genetic markers. Several SNP genotyping methods have been developed. For low‐to‐medium throughput genotyping, the KBioscience Competitive Allele‐Specific PCR genotyping system (KASPar; KBioscience Ltd., Hoddesdon, United Kingdom) appears to be an interesting approach (Cuppen, 2007) that has been successfully applied in animals and plants (Nijman et al., 2008; Bauer et al., 2009; Cortes et al., 2011). For genetic diversity studies with SNP markers, it is very important to determine the representativeness of the discovery panel (Albrechtsen et al., 2010). Ascertainment bias of the SNP markers affects the evaluation of genetic parameters, as was observed for the Citrus L. genus using SNP markers mined in a single Clementine cultivar (Ollitrault et al., 2012). Recently, Garcia‐Lor et al. (2013) sequenced 27 amplified nuclear gene fragments for 45 genotypes of Citrus, which resulted in the identification of 1097 SNPs. Taking advantage of these previously obtained SNP data, the objective of this work was to implement a set of polymorphic SNP markers for systematic germplasm bank characterization within the Citrus genus and to investigate their transferability across the Aurantioideae [Engler] subfamily. More generally, the objective was to estimate the usefulness of SNP markers developed using KASPar technology, which were selected from a limited intrageneric discovery panel, for broader diversity analysis at the intra‐ and intergeneric levels.

METHODS AND RESULTS

The 42 SNP markers used in this study were selected from SNPs identified by Garcia‐Lor et al. (2013) in 27 nuclear genes. Most cultivated citrus (except for C. aurantifolia (Christm.) Swingle) arose from interspecific hybridization of three ancestral taxa: C. medica L., C. reticulata Blanco, and C. maxima (Burm.) Merr. (Nicolosi et al., 2000; Barkley et al., 2006; Garcia‐Lor et al., 2012). Therefore, we selected SNPs between and within these three taxa (based on seven C. reticulata, five C. maxima, and five C. medica accessions). Primers were defined by KBioscience (http://www.kbioscience.co.uk/) from each SNP‐locus flanking sequence (Appendix S1). Two allele‐specific oligonucleotides and one common oligonucleotide were defined for each locus (Table 1). The KASPar system uses...