Genome-Wide Mapping of Structural Variations Reveals a Copy Number Variant That Determines Reproductive Morphology in Cucumber

Abstract

Structural variations (SVs) represent a major source of genetic diversity. However, the functional impact and formation mechanisms of SVs in plant genomes remain largely unexplored. Here, we report a nucleotide-resolution SV map of cucumber (Cucumis sativas) that comprises 26,788 SVs based on deep resequencing of 115 diverse accessions. The largest proportion of cucumber SVs was formed through nonhomologous end-joining rearrangements, and the occurrence of SVs is closely associated with regions of high nucleotide diversity. These SVs affect the coding regions of 1676 genes, some of which are associated with cucumber domestication. Based on the map, we discovered a copy number variation (CNV) involving four genes that defines the Female (F) locus and gives rise to gynoecious cucumber plants, which bear only female flowers and set fruit at almost every node. The CNV arose from a recent 30.2-kb duplication at a meiotically unstable region, likely via microhomology-mediated break-induced replication. The SV set provides a snapshot of structural variations in plants and will serve as an important resource for exploring genes underlying key traits and for facilitating practical breeding in cucumber.

Figure 1.

SV Formation Mechanisms in the Cucumber Genome. (A) Relative contribution of different SV formation mechanisms inferred from deletions with nucleotide resolution. Outer circle: number of deletions derived from different mechanisms. Inner circle: cumulative size of the deletions derived from different mechanisms. NAHR, nonallelic homologous recombination; MMBIR (5-19), MMBIR with homologous fragments from 5 to 19 bp; MMBIR (2-4), 2- to 4-bp identical flanking nucleotides at the two sides of the breakpoint; IR, inverted repeat; NHR, nonhomologous rearrangement. (B) Distribution of TE-related deletions and TEs across the seven cucumber chromosomes. (C) Example of a deleted full-length LTR retrotransposon in the genomes of Indian and Xishuangbanna cucumbers, as well as melon, compared with the 9930 reference genome.

Figure 2.

Relationship between SVs and Nucleotide Diversity (π). Distribution of nucleotide diversity of the 2-kb upstream and the 2-kb downstream regions (bin size: 100 bp) of deletions, insertions, inversions, and duplications (A), SVs of different sizes (<100 bp, 100 to 500 bp, 500 to 1000 bp, and >1000 bp) (B), and SVs with different MAF (<0.1, 0.1 to 0.2, 0.2 to 0.3, and 0.3 to 0.4) (C).

Figure 3.

A 30.2-kb Duplicated Sequence Is Responsible for the Gynoecious Cucumber Phenotype. (A) Phenotypes of gynoecious (WI1983G) and monoecious (WI1983GM) cucumbers. WI1983GM is a mutant of WI1983G with a predominance of male flowers. (B) Manhattan plot of GWAS using the SV set for the gynoecy phenotype. The arrow points to the SV that is significantly associated with gynoecy. (C) Schematic illustration of the mapped reads from gynoecious accession (CG5278) on the 9930 reference genome. Read depth is represented by reads marked by black arrows, abnormally mapped RPs are indicated in orange, and SRs are indicated in light blue. (D) Structural organization of the 30.2-kb duplicated region in gynoecious and monoecious lines. ACS1, Csa6G496450; A1, Csa6G496950; B1, Csa6G496960; BCAT, Csa6G496970. (E) Copy numbers of the 30.2-kb region and geographical distribution of the core collection and six additional gynoecious lines (GCA07, GCA09, HAU106, IVF204, HAU107, and SJ08). The 115 accessions were ordered as listed in Supplemental Data Set 1. The copy numbers of the duplicated 30.2-kb region for the cucumbers from Indian, Xishuangbanna, Eurasian, and East Asian groups are denoted as green, red, orange, and blue bars, respectively. The six additional lines (GCA07, GCA09, HAU106, IVF204, HAU107, and SJ08) from Europe are represented by the orange bars on the far right of the histogram. (F) DNA gel blot analysis of the duplicated region of 9930 (monoecious), WI1983G (gynoecious), WI1983GM (mutant of WI1983G, monoecious), G06 (gynoecious), G06M (mutant of G06, monoecious), and IVF204 (gynoecious). (G) PCR validation of the duplication.