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

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 (<i>Ahasl1</i>, <i>Ahasl2</i>, <i>Ahasl3</i>, and <i>FAD2</i>) in 40 sunflower genotypes and three genes (<i>Ppo</i>, <i>Wx</i>, and <i>Lox</i>) 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 <i>Ahasl1</i>/<i>Ahasl3</i> and <i>Wx-A</i>/<i>Lox-B</i> 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.