A new quantitative method to validate the expression of homoeologous genes in polyploids

Polyploid plants, which have multiple sets of chromosomes, contain numerous homoeologous genes in their genomes. Although these homoeologous genes often share conserved functions, their expression patterns may diverge during evolution, playing a crucial role in regulating phenotypes. The ability to precisely control the expression of these genes could enhance the breeding of agriculturally improved crops. However, traditional methods such as qRT-PCR and short-read RNA sequencing face challenges in differentiating between highly similar homoeologous genes. A more effective method is needed to analyze their distinct expression patterns. In this study, we developed a hybrid method combining SYBR Green-based qRT-PCR with counting reads from amplicon sequencing which can distinguish among sequence polymorphisms of homoeologs, to validate the expression of each homoeolog in hexaploid S. nigrum. The genomic composition of S. nigrum has changed through evolution, introducing homoeologous genes with varying ploidy levels. We analyzed the sequence similarities of these homoeologous genes and examined their expression profiles based on RNA sequencing results. In genes with high sequence similarity, we observed very high expression correlations between the homoeologous genes, indicating that this correlation may introduce bias when determining expression values. Thus, to reduce this bias, we propose a validation method utilizing qRT-PCR combined with amplicon sequencing to accurately quantify the expression levels of individual homoeologous genes, providing a robust approach for polyploid crop improvement.