Optimizing ddRADseq in Non-Model Species: A Case Study in Eucalyptus dunnii Maiden

Abstract

Restriction site-associated DNA sequencing (RADseq) and its derived protocols, such as double digest RADseq (ddRADseq), offer a flexible and highly cost-effective strategy for efficient plant genome sampling. This has become one of the most popular genotyping approaches for breeding, conservation, and evolution studies in model and non-model plant species. However, universal protocols do not always adapt well to non-model species. Herein, this study reports the development of an optimized and detailed ddRADseq protocol in Eucalyptus dunnii, a non-model species, which combines different aspects of published methodologies. The initial protocol was established using only two samples by selecting the best combination of enzymes and through optimal size selection and simplifying lab procedures. Both single nucleotide polymorphisms (SNPs) and simple sequence repeats (SSRs) were determined with high accuracy after applying stringent bioinformatics settings and quality filters, with and without a reference genome. To scale it up to 24 samples, we added barcoded adapters. We also applied automatic size selection, and therefore obtained an optimal number of loci, the expected SNP locus density, and genome-wide distribution. Reliability and cross-sequencing platform compatibility were verified through dissimilarity coefficients of 0.05 between replicates. To our knowledge, this optimized ddRADseq protocol will allow users to go from the DNA sample to genotyping data in a highly accessible and reproducible way.

Genetic Diversity, Population Structure and Linkage Disequilibrium Assessment among International Sunflower Breeding Collections

“…The He values obtained here (~0.452) are higher than those reported by Mandel et al [9] using a 10K Illumina SNP chip on the same USDA-UBC accessions (~0.404), suggesting that our ddRADseq method provides enough informative markers to conduct population studies in sunflower. In addition to generating a SNP panel with similar power to that of the chip, our ddRADseq strategy also allows new marker discovery avoiding ascertainment bias in new germplasm [20].…”

“…Bowtie2 aligner with default parameters [18] was used to align the reads to the reference genome (XRQ inbred line, GCA_002127325.1, [12], retrieved from plants.ensembl.org, [19]). Single nucleotide polymorphisms (SNPs) were called using the ref_map routine implemented in Stacks software [17], as described in Aguirre et al [20]. Additional cleaning of ambiguous alleles and/or putative sequencing errors was carried out with the rxstacks module by removing SNP calls with the likelihood below -10 and accepting loci with a maximum of 50% sample carrying confounding alleles (i.e., excess of alleles or matching more than one catalog locus).…”

ddRAD sequencing-based genotyping for population structure analysis in cultivated tomato provides new insights into the genomic diversity of Mediterranean ‘da serbo’ type long shelf-life germplasm

“…1 Geographical provenance of the 288 tomato accessions presented in this study. Circle colors define the type while their size is proportional to the number of samples plant species 28 . Hence, this combination was used to generate fragments within the 288 tomato samples.…”

Genomic Predictions Using Low-Density SNP Markers, Pedigree and GWAS Information: A Case Study with the Non-Model Species Eucalyptus cladocalyx

“…Silva-Junior et al [7], for instance, developed a genome-wide SNP chip for multiple species of Eucalyptus, which has been effective for genomic studies in a wide variety of economically important eucalypt species and their hybrids, including Eucalyptus grandis, Eucalyptus urophylla, Eucalyptus nitens and Eucalyptus globulus [8][9][10][11][12]. However, despite the versatility of this SNP array, it does not perform as well in terms of genome coverage or number of available SNPs for species which are more-distantly related to those for which the chip was developed [13].…”