MAPPING 1000 ABIOTIC STRESS RESPONSIVE GENES IN BARLEY USING SINGLE NUCLEOTIDE- AND SINGLE FEATURE-POLYMORHISM

Jan T. Svensson¹, Jin Xu², Nils Rostoks³, Pascal Condamine¹,Nils Stein⁴, Steve Wanamaker¹, Rajeev Varshney⁴, Andreas Graner⁴, Robbie Waugh³, Mikeal L. Roose¹, Xinping Cui², and Timothy J. Close¹

¹Dept. of Botany & Plant Sciences, University of California, Riverside, CA, 92521, USA

²Dept. of Computer Sciences, University of California, Riverside, CA, 92521, USA

³Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, Scotland, UK

⁴Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 3, D-06466, Gatersleben, Germany

*Corresponding Author: TJC: (951) 827-3318; E-mail: timothy.close@ucr.edu

To couple the physical and genetic map of barley, we aim to map 1000 abiotic stress related genes. To create an extended list of abiotic stress responsive genes, microarray analyses were done on ABA-, drought-, salt- and low temperature-treated barley. A total of 7735 abiotic stress responsive probe sets were found representing 7422 unigenes. To map a subset of these unigenes we are making use of single nucleotide polymorphisms (SNPs) evident in EST sequence data and single feature polymorphisms (SFPs) defined by data from the Affymetrix GeneChip.

SNP discovery among EST sequences at UC Riverside made use of HarvEST:Barley assembly #32, which is based on more permissive CAP3 parameters than the assembly that was used as the basis of the Affymetrix GeneChip. A total of 38 different genotype by genotype comparisons resulted in a list of 12,615 eSNPs in 3509 unigenes. To check the eSNP method we sequenced a small subset predicted from the Barke by Morex EST comparison, and 29 of 32 (91%) were validated. In addition, SNP discovery solely by genomic amplicon sequencing (dSNPs) at the Scottish Crop Research Institute (SCRI) and the Institute of Plant Genetics and Crop Plant Research (IPK) identified 565 and 217 unigenes containing SNPs, respectively. The complete eSNP and dSNP compilation was used for design of an Illumina Oligo Pool Assay (OPA), designed to detect 1526 SNPs. Our plan is to genotype 96 maplines each from Steptoe x Morex, Barke x Morex, and Oregon Wolfe Barley (OWB) parents. Also 96 different cultivars, landraces and other elite lines will be examined.

To map additional loci for which nucleotide polymorphism information is not available, or polymorphisms that are not compatible with the Illumina platform, our second approach involves SFPs defined using the Affymetrix Barley1 GeneChip. We hybridized the chip with labeled cRNA from five parental genotypes to derive lists of SFPs related to the same three mapping populations that are to be used for the Illumina platform. Our SFP detection method, developed at UC Riverside, uses a robustified projection pursuit (RPP) method to identify single probes that distinguish between two genotypes. A random sample of 72 predicted SFPs yielded 59 (82%) that were validated by genomic amplicon sequencing. In total, 2107 SFPs were detected, of which 1203 belonged to 995 abiotic stress responsive probe sets. These were divided into two groups (1) EST supported and (2) non-EST supported, and a Nimblegen array was used to further investigate about 650 of these SFPs. From the data, we have defined an optimized path for SFP discovery and representation on 25-mer-based chips.