Optimizing ddRADseq in non-model species: a case study in Eucalyptus dunnii maiden
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.
Main Authors: | , , , , , , , , , , , , , , |
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MDPI
2019-08
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Subjects: | Single Nucleotide Polymorphism, Genotypes, Genetic Markers, Marcadores Genéticos, DNA Sequence, Polimorfismo de un Solo Nucleótido, Genotipos, Eucalyptus, Secuencia de ADN, Eucalyptus dunnii, |
Online Access: | https://www.mdpi.com/2073-4395/9/9/484/htm http://hdl.handle.net/20.500.12123/6254 https://doi.org/10.3390/agronomy9090484 |
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Single Nucleotide Polymorphism Genotypes Genetic Markers Marcadores Genéticos DNA Sequence Polimorfismo de un Solo Nucleótido Genotipos Eucalyptus Secuencia de ADN Eucalyptus dunnii Single Nucleotide Polymorphism Genotypes Genetic Markers Marcadores Genéticos DNA Sequence Polimorfismo de un Solo Nucleótido Genotipos Eucalyptus Secuencia de ADN Eucalyptus dunnii |
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Single Nucleotide Polymorphism Genotypes Genetic Markers Marcadores Genéticos DNA Sequence Polimorfismo de un Solo Nucleótido Genotipos Eucalyptus Secuencia de ADN Eucalyptus dunnii Single Nucleotide Polymorphism Genotypes Genetic Markers Marcadores Genéticos DNA Sequence Polimorfismo de un Solo Nucleótido Genotipos Eucalyptus Secuencia de ADN Eucalyptus dunnii Aguirre, Natalia Cristina Filippi, Carla Valeria Zaina, Giusi Rivas, Juan Gabriel Acuña, Cintia Vanesa Villalba, Pamela Victoria Garcia, Martin Nahuel Gonzalez, Sergio Rivarola, Maximo Lisandro Martinez, Maria Carolina Puebla, Andrea Fabiana Morgante, Michele Hopp, Horacio Esteban Paniego, Norma Beatriz Marcucci Poltri, Susana Noemi Optimizing ddRADseq in non-model species: a case study in Eucalyptus dunnii maiden |
description |
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. |
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info:ar-repo/semantics/artículo |
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Single Nucleotide Polymorphism Genotypes Genetic Markers Marcadores Genéticos DNA Sequence Polimorfismo de un Solo Nucleótido Genotipos Eucalyptus Secuencia de ADN Eucalyptus dunnii |
author |
Aguirre, Natalia Cristina Filippi, Carla Valeria Zaina, Giusi Rivas, Juan Gabriel Acuña, Cintia Vanesa Villalba, Pamela Victoria Garcia, Martin Nahuel Gonzalez, Sergio Rivarola, Maximo Lisandro Martinez, Maria Carolina Puebla, Andrea Fabiana Morgante, Michele Hopp, Horacio Esteban Paniego, Norma Beatriz Marcucci Poltri, Susana Noemi |
author_facet |
Aguirre, Natalia Cristina Filippi, Carla Valeria Zaina, Giusi Rivas, Juan Gabriel Acuña, Cintia Vanesa Villalba, Pamela Victoria Garcia, Martin Nahuel Gonzalez, Sergio Rivarola, Maximo Lisandro Martinez, Maria Carolina Puebla, Andrea Fabiana Morgante, Michele Hopp, Horacio Esteban Paniego, Norma Beatriz Marcucci Poltri, Susana Noemi |
author_sort |
Aguirre, Natalia Cristina |
title |
Optimizing ddRADseq in non-model species: a case study in Eucalyptus dunnii maiden |
title_short |
Optimizing ddRADseq in non-model species: a case study in Eucalyptus dunnii maiden |
title_full |
Optimizing ddRADseq in non-model species: a case study in Eucalyptus dunnii maiden |
title_fullStr |
Optimizing ddRADseq in non-model species: a case study in Eucalyptus dunnii maiden |
title_full_unstemmed |
Optimizing ddRADseq in non-model species: a case study in Eucalyptus dunnii maiden |
title_sort |
optimizing ddradseq in non-model species: a case study in eucalyptus dunnii maiden |
publisher |
MDPI |
publishDate |
2019-08 |
url |
https://www.mdpi.com/2073-4395/9/9/484/htm http://hdl.handle.net/20.500.12123/6254 https://doi.org/10.3390/agronomy9090484 |
work_keys_str_mv |
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oai:localhost:20.500.12123-62542019-10-31T15:43:12Z Optimizing ddRADseq in non-model species: a case study in Eucalyptus dunnii maiden Aguirre, Natalia Cristina Filippi, Carla Valeria Zaina, Giusi Rivas, Juan Gabriel Acuña, Cintia Vanesa Villalba, Pamela Victoria Garcia, Martin Nahuel Gonzalez, Sergio Rivarola, Maximo Lisandro Martinez, Maria Carolina Puebla, Andrea Fabiana Morgante, Michele Hopp, Horacio Esteban Paniego, Norma Beatriz Marcucci Poltri, Susana Noemi Single Nucleotide Polymorphism Genotypes Genetic Markers Marcadores Genéticos DNA Sequence Polimorfismo de un Solo Nucleótido Genotipos Eucalyptus Secuencia de ADN Eucalyptus dunnii 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. Instituto de Biotecnología Fil: Aguirre, Natalia Cristina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Filippi, Carla Valeria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Zaina, Giusi. University of Udine. Department of Agricultural, Food, Environmental and Animal Sciences; Italia Fil: Rivas, Juan Gabriel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Acuña, Cintia Vanesa. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Villalba, Pamela Victoria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Garcia, Martin Nahuel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Gonzalez, Sergio Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Rivarola, Maximo Lisandro. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Martinez, Maria Carolina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Puebla, Andrea Fabiana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Morgante, Michele. University of Udine. Department of Agricultural, Food, Environmental and Animal Sciences; Italia Fil: Hopp, Horacio Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Paniego, Norma Beatriz. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Marcucci Poltri, Susana Noemi. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina 2019-10-31T15:36:37Z 2019-10-31T15:36:37Z 2019-08 info:ar-repo/semantics/artículo info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion https://www.mdpi.com/2073-4395/9/9/484/htm http://hdl.handle.net/20.500.12123/6254 2073-4395 https://doi.org/10.3390/agronomy9090484 eng info:eu-repograntAgreement/INTA/PNBIO/1131042/AR./Genómica aplicada al mejoramiento molecular. info:eu-repo/semantics/openAccess application/pdf MDPI Agronomy 9 (9) : 484 (Agosto 2019) |