1. The Potential of HTS Approaches for Accurate Genotyping in Grapevine (Vitis vinifera L.)
- Author
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Valérie Laucou, Urban Kunej, Nataša Štajner, Aida Dervishi, Jernej Jakše, University of Ljubljana, University of Tirana, Diversité, Adaptation et Amélioration de la Vigne [AGAP] (DAAV), Amélioration génétique et adaptation des plantes méditerranéennes et tropicales (UMR AGAP), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), and Slovenian Research Agency - Slovenia P4-0077
- Subjects
0106 biological sciences ,0301 basic medicine ,Vitis vinifera L ,lcsh:QH426-470 ,genotipiziranje ,Computational biology ,Biology ,01 natural sciences ,microsatellites ,DNA sequencing ,03 medical and health sciences ,Capillary electrophoresis ,Genetics ,Typing ,Vitis vinifera ,vinska trta ,Genotyping ,Genetics (clinical) ,mikrosateliti ,[SDV.GEN]Life Sciences [q-bio]/Genetics ,SSR markers ,high-throughput sequencing ,sorte ,sekvenciranje ,Amplicon ,SSR markerji ,udc:577.2 ,lcsh:Genetics ,030104 developmental biology ,genotyping ,Microsatellite ,Primer (molecular biology) ,molekularni markerji ,010606 plant biology & botany - Abstract
The main challenge associated with genotyping based on conventional length polymorphisms is the cross-laboratory standardization of allele sizes. This step requires the inclusion of standards and manual sizing to avoid false results. Capillary electrophoresis (CE) approaches limit the information to the length polymorphism and do not allow the determination of a complete marker sequence. As an alternative, high-throughput sequencing (HTS) offers complete information regarding marker sequences and their flanking regions. In this work, we investigated the suitability of a semi-quantitative sequencing approach for microsatellite genotyping using Illumina paired-end technology. Twelve microsatellite loci that are well established for grapevine CE typing were analysed on 96 grapevine samples from six different countries. We redesigned primers to the length of the amplicon for short sequencing (~100 bp). The primer pair was flanked with a 10 bp overhang for the introduction of barcodes on both sides of the amplicon to enable high multiplexing. The highest data peaks were determined as simple sequence repeat (SSR) alleles and compared with the CE dataset based on 12 reference samples. The comparison showed that HTS SSR genotyping can successfully replace the CE system in further experiments. We believe that, with next-generation sequencing, genotyping can be improved in terms of its speed, accuracy, and price.
- Published
- 2020
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