Reverse breeding in Arabidopsis thaliana generates homozygous parental lines from a heterozygous plant

Abstract

Traditionally, hybrid seeds are produced by crossing selected inbred lines. Here we provide a proof of concept for reverse breeding, a new approach that simplifies meiosis such that homozygous parental lines can be generated from a vigorous hybrid individual. We silenced DMC1, which encodes the meiotic recombination protein DISRUPTED MEIOTIC cDNA1, in hybrids of A. thaliana, so that non-recombined parental chromosomes segregate during meiosis. We then converted the resulting gametes into adult haploid plants, and subsequently into homozygous diploids, so that each contained half the genome of the original hybrid. From 36 homozygous lines, we selected 3 (out of 6) complementing parental pairs that allowed us to recreate the original hybrid by intercrossing. In addition, this approach resulted in a complete set of chromosome-substitution lines. Our method allows the selection of a single choice offspring from a segregating population and preservation of its heterozygous genotype by generating homozygous founder lines.

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 .…”

Synthetic Botany

“…Features of Arabidopsis include its phylogenetic relevance, a comparatively rapid life cycle of 2-3 months, small stature, abundant seed production, and relative ease of transformation (Flavell 2009). Although easier to work with than many crop species, Arabidopsis still shows notable levels of genetic redundancy (Briggs et al 2006), and crosses to obtain plants homozygous for integrated transgenes (generally integrated hemizygously) are laborious (Wijnker et al 2012).…”

Review Paper on Approaches in Developing Inbred Lines in Cross-Pollinated Crops

“…Usually this is achieved by introducing GM modifications, which lead to silencing of genes required to initiate meiotic recombination events [30]. In a proof of concept experiment with Arabidopsis thaliana a GM based dominant RNAi approach was used to silence the DMC1 gene to suppress crossover recombination [31]. However other techniques were discussed for achieving this objective among them virus induced gene silencing and grafting on GM rootstock to deliver silencing construct, introduction of dominant-negative alleles and use of chemical inhibitors.…”

“…Other approaches resulting in balanced double-haploid offspring may be used depending on the plant species [31].…”