Identifying and validating housekeeping hybrid Prunus spp. genes for root gene-expression studies

Area of Science:

• Plant Science
• Molecular Biology
• Genetics

Background:

• Prunus rootstocks are vital for fruit production, with increasing use of inter-species hybrids for enhanced stress resistance.
• Accurate gene expression analysis using RT-qPCR is essential for understanding traits in these hybrids.
• A lack of validated housekeeping genes hinders reliable expression studies in hybrid Prunus rootstocks.

Purpose of the Study:

• To identify and validate stable housekeeping genes for accurate RT-qPCR analysis in hybrid Prunus rootstocks.
• To expand the repertoire of reliable reference genes for gene expression studies in diverse Prunus genotypes and under various stress conditions.

Main Methods:

• Candidate housekeeping genes were pre-selected from RNAseq data comparing root transcriptomes under hypoxia.
• Gene expression stability was assayed using RT-qPCR under hypoxia, cold, heat, and nitrogen deficiency treatments.
• NormFinder, geNorm, and BestKeeper software were employed to analyze gene stability across different conditions and genotypes.

Main Results:

• Seven housekeeping genes showed high stability under hypoxia treatment: Unknown 3, Unknown 7 (low expression), and GTB 1, TUA 3, ATPase P, PRT 6, RP II (medium expression).
• Gene stability rankings varied based on treatment, genotype, and analysis software, with GTB 1 frequently showing the highest stability.
• No single optimal set of reference genes was identified across all tested conditions and genotypes, indicating condition-specific validation is necessary.

Conclusions:

• The study successfully identified several stable housekeeping genes for RT-qPCR in Prunus rootstocks, enhancing the reliability of gene expression analysis.
• The findings underscore the importance of selecting reference genes tailored to specific stress conditions and genotypes within Prunus spp. rootstocks.
• This research provides a valuable resource for plant breeders and researchers studying stress responses and trait development in Prunus rootstocks.