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

Natalia Aguirre,Martín García,Máximo Rivarola,María Martínez,Pamela Villalba,Sergio González,Norma Paniego,Cintia Acuña,Andrea Puebla,Susana Marcucci Poltri,Carla Filippi,Giusi Zaina,Michele Morgante,Horacio Hopp,Juan Rivas

doi:10.3390/agronomy9090484

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.

Highlights

Efficient plant genome sampling, with sufficient and informative genetic markers, plays a key role in breeding, conservation, and evolution studies
Restriction-site associated DNA sequencing methodologies are becoming the most popular strategies in genomic data generation for a variety of applications related to crop and tree species breeding and genetics [15]
Restriction site-associated DNA sequencing (RADseq) and genotyping by sequencing (GBS)-based methodologies have the potential to avoid this type of bias [30], but the experience in Eucalyptus reported to date is not encouraging

Summary

Introduction

With sufficient and informative genetic markers, plays a key role in breeding, conservation, and evolution studies. Researchers have developed different types of useful molecular markers, nowadays SNPs have become the markers of choice. This selection is based on their high abundance in genomes, stability, co-dominance, and automation of the genotyping process [1]. SNP arrays are high-throughput and cost-effective tools, with the extra advantage of generating relatively reduced amount of missing data. These features make them one of the most popular. The development of a novel SNP array is costly, making them unaffordable for non-commercial plant species.

Methods

Results

Conclusion