Single Primer Enrichment Technology (SPET) for High-Throughput Genotyping in Tomato and Eggplant Germplasm

Abstract

Single primer enrichment technology (SPET) is a new, robust, and customizable solution for targeted genotyping. Unlike genotyping by sequencing (GBS), and like DNA chips, SPET is a targeted genotyping technology, relying on the sequencing of a region flanking a primer. Its reliance on single primers, rather than on primer pairs, greatly simplifies panel design, and allows higher levels of multiplexing than PCR-based genotyping. Thanks to the sequencing of the regions surrounding the target SNP, SPET allows the discovery of thousands of closely linked, novel SNPs. In order to assess the potential of SPET for high-throughput genotyping in plants, a panel comprising 5k target SNPs, designed both on coding regions and introns/UTRs, was developed for tomato and eggplant. Genotyping of two panels composed of 400 tomato and 422 eggplant accessions, comprising both domesticated material and wild relatives, generated a total of 12,002 and 30,731 high confidence SNPs, respectively, which comprised both target and novel SNPs in an approximate ratio of 1:1.6, and 1:5.5 in tomato and eggplant, respectively. The vast majority of the markers was transferrable to related species that diverged up to 3.4 million years ago ( Solanum pennellii for tomato and S. macrocarpon for eggplant). Maximum Likelihood phylogenetic trees and PCA outputs obtained from the whole dataset highlighted genetic relationships among accessions and species which were congruent with what was previously reported in literature. Better discrimination among domesticated accessions was achieved by using the target SNPs, while better discrimination among wild species was achieved using the whole SNP dataset. Our results reveal that SPET genotyping is a robust, high-throughput technology for genetic fingerprinting, with a high degree of cross-transferability between crops and their cultivated and wild relatives, and allows identification of duplicates and mislabeled accessions in genebanks.

Accelerating Tomato Breeding by Exploiting Genomic Selection Approaches

“…Alternately, the cDNA-based GBS technique (RAR-seq restriction site-associated RNA sequencing) may detect conserved SNPs associated with a candidate mutation directly at the expression level [ 83 ]. Recently, a customizable method for tomato targeted genotyping, named single primer enrichment technology (SPET) was developed for improving the panel design and increasing the multiplexing levels of tomato genotyping [ 84 ]. Previous GS data can help to design an optimized suite of markers for the next steps.…”

SILEX: A fast and inexpensive high-quality DNA extraction method suitable for multiple sequencing platforms and recalcitrant plant species

“…In order to evaluate the suitability of the gDNA obtained with SILEX for high-throughput genotyping, 1,380 tomato samples were genotyped using SPET [6]. The reads obtained showed excellent Phred-quality scores along the 150 bp, with a mean value of 33.1 ( Figure 4).…”

“…In the last decade, sequencing and genotyping technologies have become routine, allowing to generate a wealth of genomic information even in non-model plant species and neglected crops [1,2]. Nowadays, genome sequencing, as well as the most common high-throughput genotyping strategies, like Genotyping-by-Sequencing (GBS [3]), Restriction Associated DNA Sequencing (RADseq [4]) and Single Primer Enrichment Technology (SPET [5,6]), are conducted using next-generation sequencing (NGS) platforms. However, despite the advances, DNA quality is still a main bottleneck, mostly for the thirdgeneration sequencing platforms where high-molecular-weight DNA free of contaminants is required [7].…”

Applications and Trends of Machine Learning in Genomics and Phenomics for Next-Generation Breeding

“…RAD-seq proved to be more appropriate to profile and define the genetic differentiation of S. pimpinellifolium accessions as well as to estimate linkage disequilibrium. Another targeted genotyping methodology used to investigate the diversity in tomato and eggplant is the Single Primer Enrichment Technology [21].…”

“…The method requires a priori information on target sequences for probe design. This approach is a valid alternative to random complexity reduction methods and arrays, being able to give better discrimination among domesticated and wild species with respect to previous methodologies and with a high degree of transferability between closely related species [21]. Moreover, it allows users to customize the panel of target markers.…”