The Potential of HTS Approaches for Accurate Genotyping in Grapevine (Vitis vinifera L.)

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.

Vitis vinifera genotyping toolbox to highlight diversity and germplasm identification

“…By utilizing HTS to genotype microsatellite markers, it is feasible to compare amplicon length and full amplicon information allele calling procedures to determine changes in the quantity of genetic information produced (De Barba et al, 2017). Kunej et al (2020), compared the two SSR approaches and stated that the HTS, although there is space for improvements in terms of speed, accuracy and price, HTS SSR can replace the CE approach in the years to come. Such an HTS SSR approach may be advantageous, particularly in the case of polyploid species, to resolve allele dosage uncertainty, a problem in which it is impossible to determine how many copies of the allele (one to five) are present, for example, in a hexaploidy species (Cui et al, 2022).…”

“…By utilizing HTS to genotype microsatellite markers, it is feasible to compare amplicon length and full amplicon information allele calling procedures to determine changes in the quantity of genetic information produced ( De Barba et al., 2017 ). Kunej et al. (2020) , compared the two SSR approaches and stated that the HTS, although there is space for improvements in terms of speed, accuracy and price, HTS SSR can replace the CE approach in the years to come.…”

DNA-Based Technologies for Grapevine Biodiversity Exploitation: State of the Art and Future Perspectives

“…As reported above, there are several limitations to genotyping grapevines using conventional gel-based approaches such as a low genome representation, technical variations between assays, the need for relative allele size values that are standardized across laboratories, manual sizing and processing and the necessity of having a good positive control as well as, in a few cases, a lack of suitability for large-scale automation [79]. Recent advances in DNA sequencing have revolutionized the field of genomics, allowing a high-throughput identification of markers at low costs.…”