Centromere Locations in Brassica A and C Genomes Revealed Through Half-Tetrad Analysis

Abstract

Locating centromeres on genome sequences can be challenging. The high density of repetitive elements in these regions makes sequence assembly problematic, especially when using short-read sequencing technologies. It can also be difficult to distinguish between active and recently extinct centromeres through sequence analysis. An effective solution is to identify genetically active centromeres (functional in meiosis) by half-tetrad analysis. This genetic approach involves detecting heterozygosity along chromosomes in segregating populations derived from gametes (half-tetrads). Unreduced gametes produced by first division restitution mechanisms comprise complete sets of nonsister chromatids. Along these chromatids, heterozygosity is maximal at the centromeres, and homologous recombination events result in homozygosity toward the telomeres. We genotyped populations of half-tetrad-derived individuals (from Brassica interspecific hybrids) using a high-density array of physically anchored SNP markers (Illumina Brassica 60K Infinium array). Mapping the distribution of heterozygosity in these half-tetrad individuals allowed the genetic mapping of all 19 centromeres of the Brassica A and C genomes to the reference Brassica napus genome. Gene and transposable element density across the B. napus genome were also assessed and corresponded well to previously reported genetic map positions. Known centromere-specific sequences were located in the reference genome, but mostly matched unanchored sequences, suggesting that the core centromeric regions may not yet be assembled into the pseudochromosomes of the reference genome. The increasing availability of genetic markers physically anchored to reference genomes greatly simplifies the genetic and physical mapping of centromeres using half-tetrad analysis. We discuss possible applications of this approach, including in species where half-tetrads are currently difficult to isolate.

Keywords: Brassica; centromeres; molecular karyotyping; recombination; unreduced gametes.

Figure 1

Overview of the methods used to obtain unreduced gamete-derived individuals to map the Brassica A- and C-genome centromere locations.

Figure 2

Percentage of heterozygosity as assessed by presence of both parental alleles at a single locus in the diploid genome in individuals derived from (A) microspore culture of B. juncea × B. napus (2n = AABC) interspecific hybrids; (B) microspore culture of B. napus × B. carinata (2n = CCAB) interspecific hybrids; and (C) test crosses between B. napus × B. carinata (2n = CCAB) interspecific hybrids and B. juncea.

Figure 3

Gene density, TE density, and percentage of heterozygosity (the latter in a population derived from first-division restitution-type unreduced gametes) along each B. napus chromosome represented in a Circos plot (Krzywinski et al. 2009). The B. napus chromosomes belonging to the A and C subgenomes are in blue and red, respectively. The size of each chromosome in megabase pairs is indicated above each chromosome and a ruler is drawn underneath each chromosome, with larger tick marks every 10 Mbp and smaller tick marks every 2 Mbp. Locations of active centromeres are visible as peaks of increased heterozygosity, increased TE density, and decreased gene density. Dots indicate BLAST-located centromeric and pericentromere sequences: CentBr1 sequences (red), CentBr2 sequences (yellow), TR238 sequences (green), and TR805 sequences (blue).