A high-throughput apple SNP genotyping platform using the GoldenGate™ assay M. Awais Khan a , Yuepeng Han b , Youfu Frank Zhao c , Schuyler S. Korban a, ⁎ a Department of Natural Resources & Environmental Sciences, University of Illinois, Urbana, IL 61801, USA b Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Moshan, Wuhan, 430074, People's Republic of China c Department of Crop Sciences, University of Illinois, Urbana, IL 61801, USA abstract article info Article history: Accepted 1 December 2011 Available online 24 December 2011 Keywords: Expressed sequence tags (ESTs) Malus Genotyping Single Nucleotide Polymorphisms (SNPs) EST data generated from 14 apple genotypes were downloaded from NCBI and mapped against a reference EST assembly to identify Single Nucleotide Polymorphisms (SNPs). Mapping of these SNPs was undertaken using 90% of sequence similarity and minimum coverage of four reads at each SNP position. In total, 37,807 SNPs were identified with an average of one SNP every 187 bp from a total of 6888 unique EST contigs. Identified SNPs were checked for flanking sequences of ≥60 bp along both sides of SNP alleles for reliable de- sign of a custom high-throughput genotyping assay. A total of 12,299 SNPs, representing 6525 contigs, fit the selected criterion of ≥60 bp sequences flanking a SNP position. Of these, 1411 SNPs were validated using four apple genotypes. Based on genotyping assays, it was estimated that 60% of SNPs were valid SNPs, while 26% of SNPs might be derived from paralogous regions. © 2011 Elsevier B.V. All rights reserved. 1. Introduction In recent years, crop improvement efforts by capitalizing on iden- tification and utilization of genetic diversity have made major strides with the aid of molecular marker technology. Molecular markers are highly reliable in identifying and selecting parents carrying traits of in- terest, advancing breeding schemes, and ultimate crop improvement via either marker-assisted selection/breeding and/or map-based cloning (Han and Korban, 2010). Molecular markers are routinely used to detect and link variations in traits of interest through Quantitative Trait Loci (QTL) mapping. Once DNA markers linked to a trait of interest are identified, and the linkage is close, these markers can be used to select for seedlings possessing the desirable charac- ter(s) in a breeding population using a particular breeding scheme, such as backcrossing (Collard and Mackill, 2008; Moose and Mumm, 2008). Unlike phenotypic evaluation, selection based on DNA markers is not influenced by environmental factors. Moreover, marker-assisted selection can be carried out at the seedling stage, and therefore would alleviate requirements for land space, field evaluations, plant maintenance, along with associated costs, when compared to phenotypic selection (Collard and Mackill, 2008). Among various available molecular marker systems, Microsatellites or Simple Se- quence Repeats (SSRs) have become the marker of choice as these are characterized by 1 to 5 bp repeat motifs, and are highly polymor- phic between and among species (Korban and Tartarini, 2009). More- over, they are multi-allelic, co-dominant, reproducible, and transferable across different species. SSR markers are preferred for QTL mapping studies, marker-assisted selection, and comparative ge- netic analysis (Liebhard et al., 2003). Initial costs for development of SSR markers from genomic regions are usually very high compared to other marker systems. However, for crops wherein expressed se- quence tags (ESTs) are available, SSR development based on EST sequences is easy and cheap. Therefore, EST-SSRs have been recently developed in a wide range of plant species, and are being used for constructing genetic maps and for pursuing phylogenetic studies. Moreover, molecular markers developed from ESTs represent gene- coding regions, and thus are useful tools for bridging functional and structural genomics (Chagné et al., 2008; Korban and Tartarini, 2009; Varshney et al., 2005). To date, DNA sequences for many plant species, particularly those of ESTs, are publically available in large numbers in the NCBI database. Apple genetic maps have been generally constructed using a backbone of genomic SSR markers and populated with RAPDs, RFLPs, and AFLPs for QTL identification (Liebhard et al., 2003; Silfverberg-Dilworth et al., 2006). With avail- ability of apple EST sequences, EST-SSRs have been identified in silico and used for constructing new genetic maps (Han et al., 2011; Naik et al., 2006). Although both genomic SSRs and EST-SSRs offer advan- tages over other marker systems, there are some concerns over their throughput efficacy, identification, genotyping costs, as well as Gene 494 (2012) 196–201 Abbreviations: ESTs, Expressed sequence tags; SNPs, Single Nucleotide Polymor- phisms; QTL, Quantitative Trait Loci; RAPDs, Random Amplified Polymorphic DNAs; RFLPs, Restriction Fragment Length Polymorphisms; AFLPs, Amplified Fragment Length Polymorphisms; SSRs, Microsatellites or Simple Sequence Repeats; HRM, High- resolution Melting; ADT, Assay Design Tool; GC, GenCall; LD, Linkage Disequilibrium. ⁎ Corresponding author. Tel./fax: + 1 217 333 8928. E-mail address: korban@illinois.edu (S.S. Korban). 0378-1119/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2011.12.001 Contents lists available at SciVerse ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene