The genomic diversification of grapevine clones

Abstract

Background: Vegetatively propagated clones accumulate somatic mutations. The purpose of this study was to better appreciate clone diversity and involved defining the nature of somatic mutations throughout the genome. Fifteen Zinfandel winegrape clone genomes were sequenced and compared to one another using a highly contiguous genome reference produced from one of the clones, Zinfandel 03.

Results: Though most heterozygous variants were shared, somatic mutations accumulated in individual and subsets of clones. Overall, heterozygous mutations were most frequent in intergenic space and more frequent in introns than exons. A significantly larger percentage of CpG, CHG, and CHH sites in repetitive intergenic space experienced transition mutations than in genic and non-repetitive intergenic spaces, likely because of higher levels of methylation in the region and because methylated cytosines often spontaneously deaminate. Of the minority of mutations that occurred in exons, larger proportions of these were putatively deleterious when they occurred in relatively few clones.

Conclusions: These data support three major conclusions. First, repetitive intergenic space is a major driver of clone genome diversification. Second, clones accumulate putatively deleterious mutations. Third, the data suggest selection against deleterious variants in coding regions or some mechanism by which mutations are less frequent in coding than noncoding regions of the genome.

Keywords: Clonal propagation; DNA methylation; Genome diversification; Somatic mutations; Structural variation; Transposable elements.

Fig. 1

Structural variation between Zin03 haplotypes. a. Distribution of structural variation sizes. Boxplots show the 25th quartile, median, and 75th quartile for each type of SV. Whiskers are 1.5^{Inter-Quartile Range}. Diamonds indicate the mean log₁₀(length) of each type of SV; b,c,d. Examples of heterozygous structural variants between haplotypes that intersect genes. For each reported structural variation, (from top to bottom) the coverage, haplotype-resolved alignment of reads, and the genes annotated in the region are shown; b. 4 kbp heterozygous deletion of two genes; c. 11 kbp heterozygous deletion of two genes; d. 22 kbp inversion that intersects a single gene. Triangles indicate boundaries of the inversion. A gap is shown rather than the center of the inverted region

Fig. 2

Gene content and structural variability between Zin03 and Cabernet Sauvignon. a. Distribution of structural variation sizes. Boxplots show the 25th quartile, median, and 75th quartile for each type of SV. Whiskers are 1.5^{Inter-Quartile Range}. Diamonds indicate the mean log₁₀(length) of each type of SV; b,c. Selected deletions in Cabernet Sauvignon relative to Zin03 that intersect genes. For each reported deletion, (from top to bottom) the coverage of reads over the region by long Zinfandel and Cabernet Sauvignon reads, haplotype-resolved alignment of the reads, and the genes annotated in the region are shown; b. Two genes are completely deleted in Cabernet Sauvignon relative to Zinfandel and are deleted in one Zinfandel haplotype; c. One gene contains a homozygous partial deletion in Cabernet Sauvignon

Fig. 3

The relationships between Zinfandel selections based on SNVs and sites at which all clones were called by GATK a. Principal component analysis of Zinfandel selections based on SNVs. Zin03 was not included in the analysis; b. Kinship analysis of Zinfandel selections and other cultivars with known relationships. The Kinship coefficient, PHI, is shown, as well as a dendrogram constructed by hierarchically clustering genotypes using their kinship coefficients

Fig. 4

Characterization of variants and their frequency among Zinfandel selections and other vinifera cultivars (Pinot Noir, Chardonnay, Merlot, Cabernet Franc, and Sauvignon Blanc). Only variant sites at which all samples were called by GATK (All non-Zinfandel clones and Zin03, left-hand column; All clones, right-hand column) were used. The normalized rate of variants (number of variants divided by the total feature length in the genome * 1 k) by type (SNV, INDEL), feature (Intergenic, Intron, Exon), and genotype (Non-Zinfandel cultivars, Zinfandel clones). Boxplots show the 25th quartile, median, and 75th quartile

Fig. 5

The abundance and impact of shared and unique heterozygous mutations among Zinfandel clones. Only loci at which all clones were called by GATK were used. a. The number of heterozygous SNVs, INDELs, and SVs shared by only N Zinfandel clone(s); b. The number of SNVs and INDELs shared by only N clone(s) in exons, introns, intergenic repeats (“Repeats”), and non-repetitive intergenic space; c. The ratio of transitions (Tr) to transversions (Tv) for heterozygous SNVs that uniquely occur in single Zinfandel clones and in different genome features. Different letters indicate significant differences in Tr/Tv rates between features (Tukey HSD, p < 0.01). The mean is shown as a blue circle; d. The mean percentages of CpG, CHG, and CHH in exons, introns, intergenic repeats (“Repeats”), and non-repetitive intergenic space that experience transition mutations. Standard error is shown. Heterozygous SNVs that uniquely occur in a single Zinfandel clone were used. Different letters indicate significant differences (Tukey HSD, p < 0.01); e. Proportion of exonic SNVs and INDELs that are putatively deleterious and shared by only N Zinfandel clone(s)

Fig. 6

Transposable element insertions among Zinfandel selections. a. Transposable element insertions shared by only N Zinfandel selection(s) relative to PN40024; b. Types of transposable element insertions shared by only N Zinfandel selection(s) relative to PN40024; c. The proximity of intergenic transposable element insertions to PN40024 genes