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Double Digest Restriction-Site Associated DNA Sequencing (ddRADseq) Technology.

Natalia Cristina Aguirre1, Carla Valeria Filippi2,3, Pablo Alfredo Vera2

  • 1Instituto de Agrobiotecnología y Biología Molecular (IABiMo), Unidad Ejecutora de Doble Dependencia Instituto Nacional de Tecnología Agropecuaria (INTA) - Consejo Nacional de Ciencia y Técnica (CONICET), Hurlingham, Argentina. aguirre.natalia@inta.gob.ar.

Methods in Molecular Biology (Clifton, N.J.)
|February 13, 2023
PubMed
Summary

Double digest restriction-site associated DNA sequencing (ddRADseq) offers a powerful method for generating thousands of genetic markers. This protocol enables simultaneous genotyping of many plant samples for diverse applications.

Keywords:
Genotyping by sequencingInDelsMolecular markersNext generation sequencingRADseqReduced representation librariesSNPSSR

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Area of Science:

  • Genomics
  • Molecular Biology
  • Plant Science

Background:

  • Double digest restriction-site associated DNA sequencing (ddRADseq) is a next-generation sequencing technique for genome-wide genetic marker discovery.
  • It enables the simultaneous genotyping of numerous samples, providing a reduced representation of the genome.

Purpose of the Study:

  • To describe a 96-plex derived ddRADseq protocol.
  • To demonstrate its flexibility in obtaining variable coverage depths per locus.
  • To highlight its applicability for both model and non-model plant species.

Main Methods:

  • Utilizes double digestion with two restriction enzymes for genome reduction.
  • Employs next-generation sequencing (NGS) to generate sequence data.
  • A 96-plex protocol is detailed for high-throughput sample processing.

Main Results:

  • Generates thousands of genetic markers, including Single Nucleotide Polymorphisms (SNPs), Simple Sequence Repeats (SSRs), and Insertions/Deletions (InDels).
  • Allows for flexible control over locus coverage depth.
  • Successfully applicable to a wide range of plant species.

Conclusions:

  • The described ddRADseq protocol is an efficient and versatile tool for genetic marker analysis in plants.
  • It facilitates large-scale genotyping for population genetics, breeding, and evolutionary studies.
  • The method is adaptable for species with and without existing genomic resources.