Bridging the Gap Between Platforms: Comparing Grape Phylloxera Daktulosphaira vitifoliae (Fitch) Microsatellite Allele Size and DNA Sequence Variation

TL;DR

This paper compares different methods for analyzing grape phylloxera microsatellites to ensure consistent results across research platforms.

Contribution

The study introduces standardized methods for phylloxera microsatellite genotyping and identifies reference samples to ensure compatibility between platforms.

Findings

Capillary genotyping results most closely match high-throughput sequencing allele sizes.

Polyacrylamide-based allele sizes differ by up to three base pairs due to uncharacterized DNA sequence indels.

Seven clonal lineages are proposed as reference samples for cross-platform calibration.

Abstract

Grape phylloxera is a serious insect pest of grapevines worldwide. The past two decades have seen genotypic identification of phylloxera using microsatellite markers become an integral part of control, informing the selection of resistant rootstocks and implementation of quarantine zones to prevent the spread of highly virulent genotypes. Here, we assessed three different molecular methods for screening phylloxera microsatellites, providing comparisons with previous data using newer laboratory approaches, including phylloxera whole-genome DNA sequence data. These comparisons and the standard laboratory protocols presented will allow molecular diagnostic results to be consistently obtained between different research groups and maintain compatibility of future work with valuable historic datasets.

Grape phylloxera, Daktulosphaira vitifoliae (Fitch), is an economically significant pest of grapevines. Identification of phylloxera genotypes is an important aspect of management as genotypes differ in virulence and susceptibility to control using resistant rootstocks. Microsatellite markers developed on polyacrylamide gel systems have been the most widely used molecular method for phylloxera genotype identification, but this approach has been superseded by fluorescent capillary-based genotyping. The current study presents new laboratory methods for amplifying a standard set of eight phylloxera microsatellite markers using PCR-incorporated fluorescently labelled primers, genotyped on an ABI capillary platform. Comparison of allele size data scored on (i) polyacrylamide, (ii) capillary, and (iii) high-throughput sequencing (HTS) platforms revealed that the capillary genotyping most closely matched the HTS allele sizes, while alleles of loci originally scored on a polyacrylamide platform differ in size by up to three base pairs, mostly due to the presence of previously uncharacterised DNA sequence indels. Seven common clonal lineages of phylloxera known from Australia are proposed as reference samples for use in calibrating genotyping systems between platforms and laboratories to ensure universal scoring of allele sizes, providing a critical link for accurately matching previous phylloxera genotype studies with current research.

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