Genetic variation in high molecular weight glutenin (HMW-GS) genes is tightly linked with the breadmaking quality of wheat. Hundreds of different alleles have been identified in HMW-GS genes worldwide. Such huge variability makes it difficult to distinguish them using conventional genotyping methods (for example, SDS-PAGE, SNP detection, etc.). Here, we exploited the nanopore amplicon sequencing technique (Amplicon-Seq) to uncover genetic variants distributed along the full-length sequence of six HMW-GSs, including the promoter and protein-coding regions. We analyzed 23 wheat accessions for allelic variants of HMW-GSs using the Amplicon-Seq and SDS-PAGE methods. We obtained sufficient (>50×) target gene coverage by ONT reads in just one hour. Using the obtained data, we identified numerous single nucleotide polymorphisms and InDels in the protein coding and promoter regions. Moreover, Amplicon-Seq allowed for the identification of new alleles (Glu-A1x1-T) of the Glu-1Ax gene that could not be recognized by SDS-PAGE. Collectively, our results showed that Amplicon-Seq is a rapid, multiplexed, and efficient method for high-throughput genotyping of full-length genes in large and complex genomes. This opens new avenues for the assessment of target gene variation to select novel alleles and create unique combinations of desirable traits in plant breeding programs.

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.

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 scat-tered 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. Phylogenetic analysis based on SNPs identified five distinct groups in the sunflower collection and multiple alleles for each target wheat gene. The ONT-TAS approach enabled rapid characterization of the genetic diversity of the target genes in the germplasm collections, providing a complete picture of SNPs and InDels along the full-length genes. This method offers a high-throughput, cost-effective solution for genotyping and identifying novel allele variants in plant breeding pro-grams.