Reference genes selection for Calotropis procera under different salt stress conditions

Abstract

Calotropis procera is a perennial Asian shrub with significant adaptation to adverse climate conditions and poor soils. Given its increased salt and drought stress tolerance, C . procera stands out as a powerful candidate to provide alternative genetic resources for biotechnological approaches. The qPCR (real-time quantitative polymerase chain reaction), widely recognized among the most accurate methods for quantifying gene expression, demands suitable reference genes (RGs) to avoid over- or underestimations of the relative expression and incorrect interpretation. This study aimed at evaluating the stability of ten RGs for normalization of gene expression of root and leaf of C . procera under different salt stress conditions and different collection times. The selected RGs were used on expression analysis of three target genes. Three independent experiments were carried out in greenhouse with young plants: i) Leaf 100 = leaf samples collected 30 min, 2 h, 8 h and 45 days after NaCl-stress (100 mM NaCl); ii) Root 50 and iii) Root 200 = root samples collected 30 min, 2 h, 8 h and 1day after NaCl-stress (50 and 200 mM NaCl, respectively). Stability rank among the three algorithms used showed high agreement for the four most stable RGs. The four most stable RGs showed high congruence among all combination of collection time, for each software studied, with minor disagreements. CYP23 was the best RG (rank of top four) for all experimental conditions (Leaf 100 , Root 50, and Root 200 ). Using appropriated RGs, we validated the relative expression level of three differentially expressed target genes ( NAC78 , CNBL4 , and ND1 ) in Leaf 100 and Root 200 samples. This study provides the first selection of stable reference genes for C . procera under salinity. Our results emphasize the need for caution when evaluating the stability RGs under different amplitude of variable factors.

Reference genes for quantitative real-time PCR normalization of Cenostigma pyramidale roots under salt stress and mycorrhizal association

“…RT-qPCR is currently the most reliable method to validate gene expression results obtained by other high throughput methodologies, such as RNA-Seq. Thus, selecting suitable reference genes is a fundamental requirement to mitigate potential errors in RT-qPCR results ( Coêlho et al ., 2019 )⁠. Furthermore, the use of inappropriate RGs may overestimate or underestimate the relative expression of target transcripts ( Sinha et al ., 2015 )⁠.…”

Comparative Transcriptomics Analysis of Roots and Leaves under Cd Stress in Calotropis gigantea L.

“…Specific primers were designed on the Integrated DNA Technologies website ( , 30 August 2021), and specific primer information used in this study is listed in Supplementary Table S1 . According to previous research, CYP23 (Asia05T000290) was used as the internal reference gene [ 63 ]. qRT-PCR was carried out using the Bio–Rad CFX96 fluorescence quantitative PCR System.…”

BIOCHEMICAL PROFILE OF Calotropis procera FLOWERS

“…1). Being a drought and salt tolerant plant, it profusely grows and can survive under harsh environmental conditions (Coêlho et al, 2019). It is commonly found along watercourses, roadsides, coastal dunes, overgrazed pastures and river flats.…”