A systematic PCR-based framework for amplification of long and multi-exonic genes in non-model insects: : A case study of Bemisia tabaci Asia II 1.

backgroundAmplification of long gene fragments is essential for full-length gene characterization, functional annotation, and comparative genomics, particularly in non-model organisms with limited genomic resources. Ion channel genes such as the ryanodine receptor (RYR), Transient Receptor Potential (TRP) channels, and ionotropic receptors (IRs) are structurally complex and often difficult to amplify due to their large size and variable expression levels. In cryptic species like Bemisia tabaci Asia II 1, standardized and reproducible strategies for long-fragment amplification remain limited. METHODS AND

resultsWe developed a comprehensive PCR-based workflow for amplifying the ~ 15.3 kb RYR gene, 14 TRP channel genes (1.8–4.9 kb), and four IR genes (1.6–2.9 kb) from B. tabaci Asia II 1. A two-step homology-based screening approach was employed to identify candidate genes from model and non-model insects. The RYR gene was amplified using a segmentation strategy comprising four overlapping cDNA fragments. Gradient PCR was used to optimize annealing temperatures, followed by nested and touchdown PCR for low-expression or difficult targets. Amplification efficiency improved significantly using undiluted cDNA as template. Amplicons were Sanger sequenced and assembled into full-length contigs using CAP3, with open reading frames validated through NCBI ORF Finder and AUGUSTUS gene prediction. The workflow enabled high-fidelity amplification and accurate structural annotation of ion channel gene families.

conclusionsThis optimized methodology provides a robust and reproducible framework for long-fragment gene amplification in non-model insects. The approach facilitates accurate structural characterization of complex ion channel genes and can be readily adapted for broader functional genomics and molecular entomology studies.