# Cost-Effective Detection of SNPs and Structural Variations in Full-Length Genes of Wheat and Sunflower Using Multiplex PCR and Rapid Nanopore Kit

## TL;DR

This paper introduces a fast and affordable method to detect genetic variations in sunflower and wheat genes using PCR and nanopore sequencing, helping plant breeders work more efficiently.

## Contribution

The study introduces a novel, cost-effective method for detecting SNPs and structural variations in full-length plant genes using multiplex PCR and rapid nanopore sequencing.

## Key findings

- The ONT-TAS method successfully identified SNPs and InDels in four sunflower and three wheat genes across multiple genotypes.
- The approach reduced sequencing time to 4.5 hours and cost to USD 3.4 per gene.
- Significant genetic diversity was observed in Ahasl1/Ahasl3 genes in sunflower and Wx-A/Lox-B genes in wheat.

## Abstract

Modern plant breeding relies heavily on harnessing the genetic diversity present in crops, which requires the identification of multiple single-nucleotide polymorphism variants across entire genes of interest. The traditional methods for detecting these variants can be time-consuming, expensive, and often inaccessible for smaller breeding companies and laboratories. In this study, we demonstrate a rapid and cost-effective approach for detecting single-nucleotide polymorphisms and structural variants in full-length target genes by integrating multiplex PCR, a Rapid Barcoding procedure, and nanopore sequencing. We applied this method to analyze genetic variation in four sunflower genes (Ahasl1, Ahasl2, Ahasl3, and FAD2) across 40 genotypes, as well as three wheat genes (Ppo, Wx, and Lox) across 30 genotypes. Our findings provide a comprehensive overview of the genetic variant distribution along these genes, highlighting significant gene diversity within the germplasm collections. This streamlined approach not only enhances efficiency but also reduces costs and labor requirements, making rapid sequencing and genotyping more accessible for plant breeders. By facilitating quicker access to essential genetic information, this method has the potential to accelerate the breeding process and improve crop development outcomes.

The rapid identification of allele variants in target genes is crucial for accelerating marker-assisted selection (MAS) in plant breeding. Although current high-throughput genotyping methods are efficient in detecting known polymorphisms, they are limited when multiple variant sites are scattered along the gene. This study presents a target amplicon sequencing approach using Oxford Nanopore Technologies (ONT-TAS) to rapidly sequence full-length genes and identify allele variants in sunflower and wheat collections. This procedure combines multiplex PCR and a rapid sequencing kit, significantly reducing the time and cost compared to previous methods. The efficiency of the approach was demonstrated by sequencing four genes (Ahasl1, Ahasl2, Ahasl3, and FAD2) in 40 sunflower genotypes and three genes (Ppo, Wx, and Lox) in 30 wheat genotypes. The ONT-TAS revealed a complete picture of SNPs and InDels distributed over the individual alleles, enabling rapid (4.5 h for PCR and sequencing) characterization of the genetic diversity of the target genes in the germplasm collections. The results showed a significant diversity of the Ahasl1/Ahasl3 and Wx-A/Lox-B genes in the sunflower and wheat collections, respectively. This method offers a high-throughput, cost-effective (USD 3.4 per gene) solution for genotyping and identifying novel allele variants in plant breeding programs.