Open Access Methodology article

Development of a next-generation NIL library in Arabidopsis thaliana for dissecting complex traits

Richard S Fletcher12, Jack L Mullen1, Seth Yoder1, William L Bauerle3, Gretchen Reuning3, Saunak Sen4, Eli Meyer5, Thomas E Juenger6 and John K McKay1*

Author Affiliations

1 Department of Bioagricultural Sciences & Pest Management, Colorado State University, 80523 Fort Collins, CO, USA

2 Cargill Specialty Seeds & Oils, 80525 Fort Collins, CO, USA

3 Department of Horticulture and Landscape Architecture, Colorado State University, F80523 Fort Collins, CO, USA

4 Department of Epidemiology and Biostatistics, University of California San Francisco, 94143 San Francisco, CA, USA

5 Department of Zoology, Oregon State University Corvallis, 97331 Corvallis, OR, USA

6 Section of Integrative Biology & Institute of Cellular and Molecular Biology, University of Texas, 78712 Austin, TX, USA

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BMC Genomics 2013, 14:655  doi:10.1186/1471-2164-14-655

Published: 25 September 2013



The identification of the loci and specific alleles underlying variation in quantitative traits is an important goal for evolutionary biologists and breeders. Despite major advancements in genomics technology, moving from QTL to causal alleles remains a major challenge in genetics research. Near-isogenic lines are the ideal raw material for QTL validation, refinement of QTL location and, ultimately, gene discovery.


In this study, a population of 75 Arabidopsis thaliana near-isogenic lines was developed from an existing recombinant inbred line (RIL) population derived from a cross between physiologically divergent accessions Kas-1 and Tsu-1. First, a novel algorithm was developed to utilize genome-wide marker data in selecting RILs fully isogenic to Kas-1 for a single chromosome. Seven such RILs were used in 2 generations of crossing to Tsu-1 to create BC1 seed. BC1 plants were genotyped with SSR markers so that lines could be selected that carried Kas-1 introgressions, resulting in a population carrying chromosomal introgressions spanning the genome. BC1 lines were genotyped with 48 genome-wide SSRs to identify lines with a targeted Kas-1 introgression and the fewest genomic introgressions elsewhere. 75 such lines were selected and genotyped at an additional 41 SNP loci and another 930 tags using 2b-RAD genotyping by sequencing. The final population carried an average of 1.35 homozygous and 2.49 heterozygous introgressions per line with average introgression sizes of 5.32 and 5.16 Mb, respectively. In a simple case study, we demonstrate the advantage of maintaining heterozygotes in our library whereby fine-mapping efforts are conducted simply by self-pollination. Crossovers in the heterozygous interval during this single selfing generation break the introgression into smaller, homozygous fragments (sub-NILs). Additionally, we utilize a homozygous NIL for validation of a QTL underlying stomatal conductance, a low heritability trait.


The present results introduce a new and valuable resource to the Brassicaceae research community that enables rapid fine-mapping of candidate loci in parallel with QTL validation. These attributes along with dense marker coverage and genome-wide chromosomal introgressions make this population an ideal starting point for discovery of genes underlying important complex traits of agricultural and ecological significance.

2b-RAD; Fine-mapping; Quantitative trait loci; Stomatal conductance