Genome skimming-based simple sequence repeat (SSR) marker discovery and characterization in Grevillea robusta

Abstract

Proteaceae, a largely southern hemisphere family consisting of 80 genera distributed in Australia and southern Africa as its centres of greatest diversity, also extends well in northern and southern America. Under this family, Grevillea robusta is a fast-growing species got popularity in farm and avenue plantations. Despite the ecological and economic importance, the species has not yet been investigated for its genetic improvement and genome-based studies. Only a few molecular markers are available for the species or its close relatives, which hinders genomic and population genetics studies. Genetic markers have been intensively applied for the main strategies in breeding programs, especially for the economically important traits. Hence, it is of utmost priority to develop genomic database resources and species-specific markers for studying quantitative genetics in G. robusta. Given this, the present study aimed to develop de novo genome sequencing, robust microsatellites markers, sequence annotation and their validation in different stands of G. robusta in northern India. Library preparation and sequencing were carried out using Illumina paired-end sequencing technology. Approximately, ten gigabases (Gb) sequence data with 70.87 million raw reads assembled into 425,923 contigs (read mapped to 76.48%) comprising 455 Mb genome size (23 × coverage) generated through genome skimming approach. In total, 9421 simple sequence repeat (SSR) primer pairs were successfully designed from 13,335 microsatellite repeats. Afterward, a subset of 161 primer pairs was randomly selected, synthesized and validated. All the tested primers showed successful amplification but only 13 showed polymorphisms. The polymorphic SSRs were further used to estimate the measures of genetic diversity in 12 genotypes each from the states of Punjab, Haryana, Himachal Pradesh and Uttarakhand. Importantly, the average number of alleles (N_a), observed heterozygosity (H_o), expected heterozygosity (H_e), and the polymorphism information content (PIC) were recorded as 2.69, 0.356, 0.557 and 0.388, respectively. The availability of sequence information and newly developed SSR markers could potentially be used in various genetic analyses and improvements through molecular breeding strategies for G. robusta.

Supplementary information: The online version contains supplementary material available at 10.1007/s12298-021-01035-w.

Keywords: Genetic diversity; Genome sequencing; Grevillea robusta; Microsatellite markers.

Fig. 1

The simple sequence repeat (SSR) types generated through Illumina sequencing: a Radar indicates frequency of all types of SSR motifs; and b–d most predominant repeat motifs, i.e. di-, tri- and tetra-nucleotides; and e the distribution of identified microsatellite motifs in different genomic regions of G. robusta

Fig. 2

Representative sample* through SSRs showing polymorphic banding pattern in G. robusta: a GRGMS 9118, and b GRGMS 826. *Where, M: 100 bp DNA ladder; 1–48 representing 12 genotypes each from 4 stands

Fig. 3

Functional hierarchies obtained through KEGG BRITE

Fig. 4

The hierarchical clustering of the genes assigned to a particular process in GO

Fig. 5

The UPGMA dendrogram unbiased measures of genetic distance among 48 genotypes representing 4 stands of G. robusta