Polkhovskaya Ekaterina, Moskalev Evgeniy, Merkulov Pavel, Dudnikova Ksenia, Dudnikov Maxim, Gruzdev Ivan, Demurin Yakov, Soloviev Alexander, Kirov Ilya
All-Russia Research Institute of Agricultural Biotechnology, Timiryazevskaya Str. 42, 127550 Moscow, Russia.
Pustovoit All-Russia Research Institute of Oilseed Crops, Filatova St. 17, 350038 Krasnodar, Russia.
Biology (Basel). 2025 Jan 29;14(2):138. doi: 10.3390/biology14020138.
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 (, , , and ) in 40 sunflower genotypes and three genes (, , and ) 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 / and / 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.
快速鉴定目标基因中的等位基因变异对于加速植物育种中的标记辅助选择（MAS）至关重要。尽管当前的高通量基因分型方法在检测已知多态性方面效率很高，但当多个变异位点分散在基因中时，它们就会受到限制。本研究提出了一种使用牛津纳米孔技术的目标扩增子测序方法（ONT-TAS），用于对向日葵和小麦群体中的全长基因进行快速测序并鉴定等位基因变异。该方法结合了多重PCR和快速测序试剂盒，与以前的方法相比，显著减少了时间和成本。通过对40个向日葵基因型中的四个基因（，，，和）以及30个小麦基因型中的三个基因（，，和）进行测序，证明了该方法的有效性。ONT-TAS揭示了分布在各个等位基因上的单核苷酸多态性（SNP）和插入缺失（InDel）的全貌，能够快速（PCR和测序共4.5小时）表征种质群体中目标基因的遗传多样性。结果表明，向日葵和小麦群体中的/和/基因分别具有显著的多样性。该方法为植物育种计划中的基因分型和鉴定新的等位基因变异提供了一种高通量、经济高效（每个基因3.4美元）的解决方案。
