The Genetic Puzzle of Multicopy Genes: Challenges and Troubleshooting

Background: Studies with multicopy genes impose challenges related to gene redundancy and sequence similarity among copies. Recent advances in molecular biology and genomics tools associated with dedicated databases facilitate their study. Thus, the present work emphasizes the need for rigorous methodologies and standardized approaches to interpret RT-qPCR results accurately. Results: The present work in Physcomitrium patens provides a comprehensive five-step protocol, using thiamine thiazole synthase (THI1) and sucrose 6-phosphate phosphohydrolase (S6PP) genes as proof of concept, to showcase a systematic workflow for studying multicopy genes. Beyond examining genes of interest, we highlight the critical role of choosing appropriate internal controls in the analytical process for accurately interpreting gene expression patterns. We emphasized the importance of identifying the most relevant orthologous gene, recognizing the inherent challenges in determining the most functional copy for subsequent studies. Our objective is to enhance comprehension of gene redundancy by dissecting multicopy genes' genomic landscape and its characteristics. Furthermore, we address the decision-making process surrounding the quantification of expression levels of multicopy genes. Conclusions: The study of multicopy genes discloses early events of functional adaptation. We emphasize the significance of multicopy genes in plant biology and provide a practical protocol for their study. Plant systems are strongly influenced by light/dark cycles, and the challenges inherent to this process are acknowledged. In conclusion, our comprehensive approach aims to advance the understanding of multicopy gene dynamics, offering practical methodologies and contributing valuable insights to the scientific community.