The potential of HTS approaches for accurate genotyping in grapevine (Vitis vinifera L.)

Kunej, Urban (author), Dervishi, Aida (author), Laucou, Valérie (author), Jakše, Jernej (author), Štajner, Nataša (author)

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.

• Language: English
• Keywords: Vitis vinifera L., microsatellites, high-throughput sequencing, SSR markers, genotyping
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: BF - Biotechnical Faculty
• Publication status: Published
• Publication version: Version of Record
• Year: 2020
• Number of pages: 16 str.
• Numbering: Vol. 11, iss. 8, art. 917
• PID: 20.500.12556/RUL-134355
• UDC: 577.2
• ISSN on article: 2073-4425
• DOI: 10.3390/genes11080917
• COBISS.SI-ID: 24967939
• Publication date in RUL: 10.01.2022

Record is a part of a journal

• Title: Genes
• Shortened title: Genes
• Publisher: Multidisciplinary Digital Publishing Institute (MDPI)
• ISSN: 2073-4425
• COBISS.SI-ID: 523100185

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.
• Licensing start date: 10.08.2020

Secondary language

• Language: Slovenian
• Keywords: Vitis vinifera, sorte, vinska trta, mikrosateliti, SSR markerji, genotipiziranje, sekvenciranje, molekularni markerji

Projects

• Funder: ARRS - Slovenian Research Agency
• Project number: P4-0077
• Name: Kmetijske rastline - genetika in sodobne tehnologije