Genome-wide association mapping of date palm fruit traits

Abstract

Date palms (Phoenix dactylifera) are an important fruit crop of arid regions of the Middle East and North Africa. Despite its importance, few genomic resources exist for date palms, hampering evolutionary genomic studies of this perennial species. Here we report an improved long-read genome assembly for P. dactylifera that is 772.3 Mb in length, with contig N50 of 897.2 Kb, and use this to perform genome-wide association studies (GWAS) of the sex determining region and 21 fruit traits. We find a fruit color GWAS at the R2R3-MYB transcription factor VIRESCENS gene and identify functional alleles that include a retrotransposon insertion and start codon mutation. We also find a GWAS peak for sugar composition spanning deletion polymorphisms in multiple linked invertase genes. MYB transcription factors and invertase are implicated in fruit color and sugar composition in other crops, demonstrating the importance of parallel evolution in the evolutionary diversification of domesticated species.

Fig. 1

BC4 male date palm genome assembly. a Genome assembly work flow. b Total (anchored + unplaced) numbers of contigs. c Total (anchored + unplaced) contig lengths. d Length distribution of haplotigs and primary contigs generated by FALCON-Unzip. e Gene counts in the original (All) gene set and high confidence (Final) set. Pie diagrams indicate the percentage of genes with Pfam domains in each set. f Summary of BUSCO analysis of assembly completeness. g Circos diagram of 18 linkage groups showing gene density (inner) and linkage group ideograms (outer). Linkage groups are numbered in clockwise order beginning with LG1. Gene densities were calculated in sliding windows of 200 kb with step size of 50 kb. The source data underlying Fig. 1b–g are provided as a Source Data file

Fig. 2

GWAS analysis of the sex determination region of date palm. a PCA of genetic structure in the GWAS panel. Points are colored based on whether the ancestry of the variety traces to Middle Eastern (blue) or North African (orange) date populations. The shapes of the points indicate the location of the tree in Al-Shuwaib (triangle) or Ras Al-Khaimah (dot), b decay of linkage disequilibrium with physical distance in the GWAS mapping population. The half decay distance is 22.9 kb, c GWAS of sex determination in date palm (n = 157 date palms). Top: Manhattan plot using the randomly downsampled SNP set (392,948 SNPs) for all linkage groups and unplaced scaffolds, below: Manhattan plot using the full SNP set on LG 12. Corresponding quantile-quantile plot can be found in Supplementary Fig. 4. Source data are provided as a Source Data file

Fig. 3

The genetics of fruit color variation in date palms. a Color variation in khalal stage fruits among date palm varieties. b Fruit color measured by a color index (a, b) as a function of anthocyanin level with points colored according to the RGB score of the fruit color. Each point represents the average of eight fruits per cultivar. c GWAS of fruit color in date palms (n = 157). Top: Manhattan plot using the randomly down-sampled SNP set (392,948 SNPs) for all linkage groups and unplaced scaffolds. Below: Manhattan plot using the full SNP set on LG 4. The dotted lines in each panel represent the Bonferroni significance threshold. d Gene models of VIR alleles. The VIR^IM allele is shown with the Ibn Majid retrotransposon insertion which introduces a premature stop in exon 3 and vir^saf allele is shown with the start codon polymorphism caused by a G to A change. e Boxplot distributions of fruit color phenotypes by Ibn Majid genotype (n = 145 date palms). Center line is the median, bounds of box represent the frst and third quartiles, the upper and lower whiskers extend from the hinge to the largest or smallest value, respectively, no further than 1.5 * IQR from the hinge (where IQR is the inter-quartile range, or distance between the first and third quartiles). f RNA-Seq analysis of VIR expression across fruit development in two varieties with red and yellow fruits. Each point is the mean of normalized expression counts across three or more replicates. Error bars represent s.e.m. g Variation in fruit colors by two-locus genotype. Loci are defined by the Ibn Majid and start codon mutations in VIR. Points in e–g are colored as in (b). Points in g are jittered randomly on the x-axis. The source data underlying Fig. 3b, c, e–g are provided as a Source Data file

Fig. 4

Date palm fruit sugar composition phenotypes in the GWAS panel. a Sugar composition (g/100 g dry matter) of reducing-type and sucrose-type varieties Khalas and Soukar Iraqi at the tamar stage (Suc = sucrose, Fru = fructose, Glu = glucose). b Distribution of the percentage sucrose to total sugar in the mapping population. c Correlations of sugar concentrations across varieties. Source data are provided as a Source Data file

Fig. 5

GWAS mapping of sugar composition in date palm fruit and functional characterization of candidate genes. a Top: Manhattan plot GWAS results for tamar stage fruit sugar composition measured as [sucrose]/[sucrose + glucose + fructose]. The plot is based on analysis conducted with the randomly downsampled SNP call set. Middle: Manhattan plot of the region on LG 14 containing significant associations based on analysis of all SNPs on this linkage group. Dotted lines represent the Bonferroni significance threshold. Gray vertical lines represent boundaries of the regions highlighted below. Bottom: Close-up of two highlighted regions with invertase genes in the vicinity of the GWAS peak. From top to bottom, gene models, normalized coverage depth in 500 bp windows for a sucrose-type variety (Soukar Iraqi), a reducing sugar-type variety (Khalas), and a heatmap of normalized coverage depth (low coverage is dark blue to high coverage light blue; zero coverage = gray) of the ten most extreme sugar composition phenotypes at each end of the sucrose-type and reducing-type distributions (see Supplementary Figs. 12 and 13 for all samples). Numbers in parenthesis next to variety names are the sucrose to total sugar rank among the 125 varieties with sugar measurements, where (1) is highest sucrose. b Expression of three candidate invertase genes in developing fruit at different stages post pollination (mean ± s.e.m.) in the Khenezi variety. c–e Differential gene expression of khalal stage fruit between four sucrose-type and four reducing-type varieties at three candidate invertase genes (mean ± s.e.m.). f Invertase enzyme activity in khalal stage fruit in two reducing-type and two sucrose-type varieties (mean ± s.d.). Source data are provided as a Source Data file