Whole genome re-sequencing of date palms yields insights into diversification of a fruit tree crop

Abstract

Date palms (Phoenix dactylifera) are the most significant perennial crop in arid regions of the Middle East and North Africa. Here, we present a comprehensive catalogue of approximately seven million single nucleotide polymorphisms in date palms based on whole genome re-sequencing of a collection of 62 cultivars. Population structure analysis indicates a major genetic divide between North Africa and the Middle East/South Asian date palms, with evidence of admixture in cultivars from Egypt and Sudan. Genome-wide scans for selection suggest at least 56 genomic regions associated with selective sweeps that may underlie geographic adaptation. We report candidate mutations for trait variation, including nonsense polymorphisms and presence/absence variation in gene content in pathways for key agronomic traits. We also identify a copia-like retrotransposon insertion polymorphism in the R2R3 myb-like orthologue of the oil palm virescens gene associated with fruit colour variation. This analysis documents patterns of post-domestication diversification and provides a genomic resource for this economically important perennial tree crop.

Figure 1. Summary of single nucleotide polymorphisms in 62 date palm cultivars.

(a) Circos plot of the 50 longest scaffolds (18.4% of the cv. Khalas assembly). Tracks from outer to inner are θ_W (grid lines are drawn at 0.01 intervals) and SNP density (grid lines are drawn at 250-SNP intervals) in non-overlapping 25-kb bins. Gene density and transposable element densities in sliding windows of 100 kb with step size 20 kb. A zoom in scaffold S000001 showing π and SNP density in non-overlapping 10-kb bins (b) Distribution of SNPs among functional effect classes compared with the proportion of sites in the reference (cv. Khalas) genome. (c) Minor allele site-frequency spectrum among selected coding region site classes. (d) Decay of linkage disequilibrium measured as the squared correlation coefficient (r²) by physical distance in 62 cultivars.

Figure 2. Population structure in cultivated date palm.

(a) Principal component (PC) analysis of SNP genotypes based on ∼25,000 SNPs for 62 cultivars. PC1, 2 and 3 axes account for 11.96, 4.03 and 3.69% of the variation, respectively. (b) Neighbour-joining tree based on the distance metric of Gronau et al. using 7,176,238 SNPs. (c) Population stratification based on STRUCTURE for K=2. Pie charts represent admixture proportions (that is, percentage of genome composition associated with each ancestral subpopulation cluster), and are placed on the map in the traditionally recognized country of origin of each cultivar.

Figure 3. Evidence of inbreeding in date palm genomes.

(a) Density distribution of the proportion of heterozygous genotypes in 50 kb windows for four date palm cultivars. Peaks in the distributions at low heterozygosity are apparent in the more inbred samples (b) An example run of homozygosity (ROH) in cv. Ajwa compared with cv. Mazafati on genome assembly scaffold S000023. Lines are loess fits to the proportion of heterozygous genotypes in non-overlapping 20 kb windows.

Figure 4. Identification of candidate selective sweep regions.

The highlighted region on scaffold S000007 shows a reduction in diversity in samples from the Middle East in a region containing a pectin lyase gene, which is a candidate fruit ripening locus. Shown are the Z-score transformation of log₂(θ_{Middle East}/θ_{North Africa}) in 5-kb windows across the longest 30 scaffolds in the genome assembly. Points from the negative half of the Z-score distribution are shown. θ_w and F_ST tracks show estimates from 5 kb and 500-bp windows. The θ_W track shows estimates in sliding windows of 5 kb with step size of 500 bp. The F_ST track shows estimates in 500-bp non-overlapping windows.

Figure 5. Variation in selected genes and pathways of interest in date palm.

(a) Major effect mutations in selected pathways and large gene families in the date palm genome. (b) Evidence of elevated neutral polymorphism in regions containing NBS–LRR, CC–NBS–LRR and RLP genes consistent with long-term balancing selection acting on these gene families. Bars represent mean θ_W in 10-kb windows that contain at least one putative disease resistance gene in the indicated class compared with windows without these genes. P values are based on a two-tailed Wilcoxon rank-sum test (***P<10⁻¹⁰; **P<10⁻⁵). (c) Date palm cultivars with predicted deletion alleles at resistance/immunity-related gene loci in African (dark orange) and Middle Eastern (dark blue) samples. Samples without deletions are colored light yellow and light blue.

Figure 6. Fruit colour polymorphism in date palm is likely controlled by a dominant negative mutation in an R2R3 myb-like transcription factor.

(a) Khalal stage fruits in cv. Barhee and cv. Khisab illustrating the red/yellow fruit colour polymorphism in date palm, respectively. (b) Phylogeny of selected members of the R2R3 myb-like transcription factor family. The analysis supports the orthology of date palm and oil palm VIR genes. (c) Alignment of the C-terminal end of the VIR alleles in date palm and the orthologous gene in oil palm. (d) Representation of VIR⁺ and VIR^copia alleles found at the date palm orthologue of the oil palm R2R3 myb-like VIR gene, defined based on a pre-mature stop codon in exon 3 that truncates the protein in the cv. Khalas reference genome relative to the wild-type (VIR⁺) allele. The stop codon is introduced by a copia-like retrotransposon element insertion. (e) Genotype counts based on the alleles in (d) showing perfect concordance between VIR genotype and the fruit colour phenotype. Yellow represents a range of intermediate colours between yellow and red, including golden yellow and orange yellow (Supplementary Table 10). Samples with missing genotypes or whose fruit colour cannot be verified were excluded. This suggests that yellow fruit colour in both oil palm and date palm is caused by truncated alleles that act as dominant negative mutations.