Lorestan University of Medical Sciences Herbal Medicines Journal 2538-2144 10 4 2025 10 01 Genome-wide identification, characterization and expression stability assessment of housekeeping genes in saffron (Crocus sativus L) 235275 10.22087/hmj.2025.562106.1028 EN Seyed Sajad Sohrabi Department of Production Engineering and Plant Genetics, Faculty of Agriculture, Lorestan University, Khorramabad, Iran 0000-0002-7528-5886 Seyed Karim Mousavi Department of Plant Protection Research, Lorestan Agricultural and Natural Resources Research Center, AREEO, Khorramabad, Iran 0000-0001-6428-7520 Mohsen Mohammadi Department of Pharmacognosy and Pharmaceutical Biotechnology, Razi Herbal Medicines Research Center, Faculty of Pharmacy, Lorestan University of Medical Sciences, Khorramabad, Iran. 0000-0002-1580-6539 Seyyed Mohsen Sohrabi Department of Production Engineering and Plant Genetics, Faculty of Agriculture, Shahid Chamran University of Ahvaz, Ahvaz, Iran. 0000-0002-7813-0820 Journal Article 2025 07 31 Saffron (Crocus sativus L.) is the only plant known to produce apocarotenoids like crocins, picrocrocin and safranal in significant quantities. Real-time PCR is abundantly used to measure and compare gene expression and also validating the results obtained other transcriptomic data such as like RNA-seq in saffron. Moreover, the accurate estimation of genes transcript abundance levels or gene expression levels depends on the selection of appropriate housekeeping genes. Identify, selection and validation of a set of stably expressed genes or reference genes, for data normalization and differential expression analysis is necessary to obtain reproducible and accurate expression results of target genes. Due to scarce information about the housekeeping genes of saffron to use real-time PCR assays, the present study, for the first time, the whole transcriptome of five saffron tissues, including corm, tepal, leaf, stigma, and stamen, were investigated to find all housekeeping genes using non-parametric methods. Gene stability analysis based on transcriptome data of five saffron tissues led to the identification of 1,211 stable housekeeping genes. According to the stability ranking and Real-time PCR analysis, GAPDH, 28S rRNA, UBQ4, HSP, and TUBA genes were selected as the most stable genes in all saffron tissues, respectively. In conclusion the housekeeping genes identified nay be valuable and used to normalize expression of genes and in a wide range of functional genomics studies in saffron. https://hmj.lums.ac.ir/article_235275_cab43db394ae7b2dee782cfc025b8d1b.pdf