Haplotype mining panel for genetic dissection and breeding in Eucalyptus

Abstract

To improve our understanding of genetic mechanisms underlying complex traits in plants, a comprehensive analysis of gene variants is required. Eucalyptus is an important forest plantation genus that is highly outbred. Trait dissection and molecular breeding in eucalypts currently relies on biallelic single-nucleotide polymorphism (SNP) markers. These markers fail to capture the large amount of haplotype diversity in these species, and thus multi-allelic markers are required. We aimed to develop a gene-based haplotype mining panel for Eucalyptus species. We generated 17 999 oligonucleotide probe sets for targeted sequencing of selected regions of 6293 genes implicated in growth and wood properties, pest and disease resistance, and abiotic stress responses. We identified and phased 195 834 SNPs using a read-based phasing approach to reveal SNP-based haplotypes. A total of 8915 target regions (at 4637 gene loci) passed tests for Mendelian inheritance. We evaluated the haplotype panel in four Eucalyptus species (E. grandis, E. urophylla, E. dunnii and E. nitens) to determine its ability to capture diversity across eucalypt species. This revealed an average of 3.13-4.52 haplotypes per target region in each species, and 33.36% of the identified haplotypes were shared by at least two species. This haplotype mining panel will enable the analysis of haplotype diversity within and between species, and provide multi-allelic markers that can be used for genome-wide association studies and gene-based breeding approaches.

Keywords: Eucalyptus; gene-centric genotyping; haplotype; multi-allelic markers.

Figure 1

Genome‐wide haplotype and single‐nucleotide polymorphism (SNP) diversity captured by the haplotype marker panel. (a) Distribution of the number of target regions with the given number of SNPs per target region (median = 10). (b) Distribution of the number of target regions with a given number of haplotypes (median = 14). (c) Number of observed haplotypes and corresponding SNPs per target region, and the maximum number of haplotypes possible given the number of SNPs (red line). (d) Distribution of the number of SNPs per target region for the given number of haplotypes.

Figure 2

Haplotype diversity across gene categories in four Eucalyptus species. The number of haplotypes per target region (y‐axis) as recorded for each of the four species (x‐axis). Data are shown as raincloud plots consisting of (from left to right) the raw data points (each point being a target region), a box plot and violin plot, both showing the distribution of the number of observed haplotypes per target region. A total of 20 individuals were analysed per species, making the theoretical maximum number of haplotypes equal to 40 per target region. The mean value shown above each graph is the average number of haplotypes per target region, and n is the number of target regions analysed in each category. A breakdown of haplotype diversity across the four species is provided in Table 1.