Comprehensive Characterization of Simple Sequence Repeats in Eggplant (Solanum melongena L.) Genome and Construction of a Web Resource

Abstract

We have characterized the simple sequence repeat (SSR) markers of the eggplant (Solanum melongena) using a recent high quality sequence of its whole genome. We found nearly 133,000 perfect SSRs, a density of 125.5 SSRs/Mbp, and also about 178,400 imperfect SSRs. Of the perfect SSRs, 15.6% were complex, with two stretches of repeats separated by an intervening block of <100 nt. Di- and trinucleotide SSRs accounted, respectively, for 43 and 37% of the total. The SSRs were classified according to their number of repeats and overall length, and were assigned to their linkage group. We found 2,449 of the perfect SSRs in 2,086 genes, with an overall density of 18.5 SSRs/Mbp across the gene space; 3,524 imperfect SSRs were present in 2,924 genes at a density of 26.7 SSRs/Mbp. Putative functions were assigned via ontology to genes containing at least one SSR. Using this data we developed an "Eggplant Microsatellite DataBase" (EgMiDB) which permits identification of SSR markers in terms of their location on the genome, type of repeat (perfect vs. imperfect), motif type, sequence, repeat number and genomic/gene context. It also suggests forward and reverse primers. We employed an in silico PCR analysis to validate these SSR markers, using as templates two CDS sets and three assembled transcriptomes obtained from diverse eggplant accessions.

Keywords: SSR; Solanum melongena; database; eggplant; genome; microsatellite.

FIGURE 1

The representation of different simple sequence repeat (SSR) motifs across the 14 plant genomes.

FIGURE 2

Perfect SSRs in the eggplant genome. (A) The relative frequency of SSR motif with different length, classified by the number of repeats. (B) The frequency of repeat classes (class I > 30 nt, class II 20–30 nt, class III < 20 nt. (C) The distribution of motif type within each class.

FIGURE 3

The distribution of the major repeat types in the eggplant genome.

FIGURE 4

The intra-chromosomal distribution of SSRs. (A) Relationship between SSR number vs. chromosome length in the eggplant genome. (B) The frequency of mono- to hexanucleotide motifs in eggplant chromosomes.

FIGURE 5

Circos diagram depicting the chromosome-scale SSR distribution (perfect). From outside to inside: repeat density, gene density, all SSRs, SSRs formed by mono-, di-, tri-, tetra-, penta- and hexanucleotides.

FIGURE 6

Distribution of microsatellite sizes in eggplant genome. (A) Non-triplet SSR vs. triplets SSR, and (B) distribution of repeat types within perfect and imperfect SSR motif. (C) Comparison between di- and trinucleotide repeats in both full genomic regions and gene space.

FIGURE 7

Functional analysis (gene ontology) of the set of eggplant genes containing SSRs. (A) GO categorization. The orange bars indicate input genes, while the red bars indicate background genes. (B) REVIGO summary of “biological process” and “molecular function” enriched terms. The bubble size is proportional to the log₁₀ (p-value) of enrichment analyses and its color is also a function of log₁₀ (p-value) of enrichment analyses (blue: low, -red: high p-value). The x and y-axes reflect semantic similarity according to the REVIGO algorithm (similar GO terms appear close together).

FIGURE 8

A worked example of an SSR search and primer design using EgMiDB. (A) EgMiDB Home page. (B) Settings given for chromosome selection and SSR search. (C) The SSR output. (D) Settings given for the design of primers. (E) Suggested primers and the downloading of the result.

FIGURE 9

Simple sequence repeat in silico validation. Relative number of microsatellites showing positive signal following in silico validation of 1000 randomly selected loci in three transcriptomes (Yang et al., 2014; Ramesh et al., 2016; PRJNA247728, unpublished) and two CDS sets (from eggplant genomic projects; Hirakawa et al., 2014; The Eggplant Genome Consortium, 2017).