Rapid creation of Arabidopsis doubled haploid lines for quantitative trait locus mapping

Abstract

Quantitative trait loci (QTL) mapping is a powerful tool for investigating the genetic basis of natural variation. QTL can be mapped using a number of different population designs, but recombinant inbred lines (RILs) are among the most effective. Unfortunately, homozygous RIL populations are time consuming to construct, typically requiring at least six generations of selfing starting from a heterozygous F 1 . Haploid plants produced from an F 1 combine the two parental genomes and have only one allele at every locus. Converting these sterile haploids into fertile diploids (termed "doubled haploids," DHs) produces immortal homozygous lines in only two steps. Here we describe a unique technique for rapidly creating recombinant doubled haploid populations in Arabidopsis thaliana: centromere-mediated genome elimination. We generated a population of 238 doubled haploid lines that combine two parental genomes and genotyped them by reduced representation Illumina sequencing. The recombination rate and parental allele frequencies in our population are similar to those found in existing RIL sets. We phenotyped this population for traits related to flowering time and for petiole length and successfully mapped QTL controlling each trait. Our work demonstrates that doubled haploid populations offer a rapid, easy alternative to RILs for Arabidopsis genetic analysis.

A haploid genetics toolbox for Arabidopsis thaliana

“…Characterization of sex-lethal transmission after haploidization should add useful information on the function and regulation of these genes. These findings add to our previous work in which we demonstrated that haploids can be used to rapidly generate mapping populations 46 , chromosome substitution lines 47 , parental lines for reverse breeding 47 and for engineering clonal reproduction through seeds 48 . Finally, like in haploid yeast, it is possible to measure meiotic crossover and gene conversion using Arabidopsis haploids 49 and to study meiotic recombination events in the absence of a homologous partner during haploid meiosis 50 .…”

Genetic Architecture of Natural Variation of Telomere Length inArabidopsis thaliana

“…To begin elucidating factors that lead to telomere lengthening in a natural background, a new mapping population was constructed using the cenh3 haploid inducer system (Ravi and Chan 2010). The major advantage of this system includes the fast generation time of RIL populations from doubled haploids (Seymour et al 2012). Results from F 1 crosses showed bimodal telomere length, suggesting that telomeres inherited from both parents are not immediately adjusted to the new genetically set homeostasis.…”

“…Producing RIL populations is notoriously time-consuming, and selfing of recombinant lines to achieve homozygosity at all loci can take 2 years to complete. Centromere-mediated genome elimination has recently been shown to enable rapid generation of RIL populations through the production of doubled haploid lines (Ravi and Chan 2010;Seymour et al 2012). Using this system, a RIL population derived from a cross between Col-0 and Pro-0 was produced ( Figure 3A).…”

“…To identify loci that contribute to telomere length variation, we decided to use quantitative trait loci (QTL) mapping approaches with established RILs to map causative loci. In addition to using published RIL populations, one population targeting elongated telomeres was created by centromeremediated genome elimination, a new haploidization tool for Arabidopsis shown to facilitate the rapid generation of RILs (Ravi and Chan 2010;Seymour et al 2012). Results from three separate RIL populations indicate that a number of shared and unique QTL underlie natural telomere length variation.…”

Determining Mutation Density Using Restriction Enzyme Sequence Comparative Analysis (RESCAN)

“…The RESCAN method has been successfully employed to assess mutation density in rice mutants derived from sodium azide mutagenesis with and without the use of the Nipponbare reference genome sequence . RESCAN has also been used in various genotyping applications including QTL mapping (Seymour et al 2012), evaluation of genetic diversity (Kim and Tai 2013a) and identifying introgressed regions in advanced backcross lines (Kim and Tai 2013b).…”