Fine mapping of biomass QTLs by increasing meiotic crossovers in Arabidopsis

Abstract

During meiosis homologous chromosomes undergo reciprocal exchanges of genetic materials, called crossovers. Meiotic crossover is essential for segregation of homologous chromosomes and contributes to genetic diversity in progeny. While at least one obligate crossover forms per chromosome pair, multiple crossovers are rare and non-randomly spaced apart by crossover interference, which limits QTL (quantitative trait locus) mapping and breeding in plants. However, the number of crossovers in Arabidopsis can be elevated by genetic disruption of anti-crossover factors such as RECQ4 helicase. Here, we demonstrate that fine mapping of QTLs for biomass in Arabidopsis can be accelerated by increasing crossovers in a QTL mapping population. The resolution of QTL mapping depends on marker density, population size, and positions of crossovers that break genetic linkages. Increasing crossovers in QTL mapping population allows for more breakages of genetic linkage blocks. We used CRISPR-Cas9 to mutate RECQ4A/4B genes in Arabidopsis C24 and Ler accessions, resulting in approximately 3-fold in crossovers during meiosis of C24/Ler F1 hybrid plants. In the F1 generation, an increase in leaf area was observed due to hybrid vigor. In wild-type and recq4 C24/Ler F2 individuals we measured a wide range of leaf sizes using image analysis and determined the locations of crossovers through genotyping-by-sequencing. Subsequently, we performed QTL analysis for leaf area by integrating the leaf area phenotype with the genotyping-by-sequencing results. Following the identification of QTL peaks, we selected candidate genetic variations for the size of the leaf area. We will investigate these genetic variations to determine their specific effect on leaf area size.