Identification of Suitable Reference Genes for Expression Studies in Pomegranate Under Different Biotic and Abiotic Stress Conditions

Pomegranate (Punica granatum) is an important economic fruit crop, facing many biotic and abiotic challenges during cultivation. Several research programs are in progress to understand both biotic and abiotic stress factors and mitigate these challenges using gene expression studies based on the qPCR approach. However, research publications are not available yet to select the standard reference gene for normalizing target gene expression values in pomegranate. The most suitable candidate reference gene is required to ensure precise and reliable results for qPCR analysis. In the current research, eight candidate reference genes' stability was evaluated under different stress conditions using different algorithms such as ∆Ct, geNorm, BestKeeper, NormFinder and RefFinder. The various algorithms revealed that EFA1 and 18S rRNA were common and most stable reference genes (RGs) under abiotic and wilt stress. Whereas comprehensive ranking by RefFinder showed GAPDH and CYPF were the most stable RGs under combined biotic (pooled samples of all biotic stress) and bacterial blight samples. The two most stable reference genes are adequate for normalizing target gene expression under wilt, nematode, bacterial blight and abiotic stress conditions using qPCR. The above data provide comprehensive details for the selection of a candidate reference gene in various stresses in pomegranate

Selection of reference genes for quantitative real-time PCR analysis in halophytic plantRhizophora apiculata

Rhizophora apiculatais a halophytic, small mangrove tree distributed along the coastal regions of the tropical and subtropical areas of the world. They are natural genetic reservoirs of salt adaptation genes and offer a unique system to explore adaptive mechanisms under salinity stress. However, there are no reliable studies available on selection and validation of reference genes for quantitative real-time polymerase chain reaction (qRT-PCR) inR. apiculataphysiological tissues and in salt stress conditions. The selection of appropriate candidate reference gene for normalization of qRT-PCR data is a crucial step towards relative analysis of gene expression. In the current study, seven genes such as elongation factor 1α (EF1α), Ubiquitin (UBQ), β-tubulin (β-TUB), Actin (ACT), Ribulose1,5-bisphosphate carboxylase/oxygenase (rbcL), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and 18S rRNA (18S) were selected and analyzed for their expression stability. Physiological tissues such as leaf, root, stem, and flower along with salt stress leaf samples were used for selection of candidate reference genes. The high-quality expression data was obtained from biological replicates and further analyzed using five different programs such as geNorm, NormFinder, BestKeeper, Delta Ct and RefFinder. All algorithms comprehensively rankedEF1α followed byACTas the most stable candidate reference genes inR. apiculataphysiological tissues. Moreover, β-TUBand 18S were ranked as moderately stable candidate reference genes, while GAPDH andrbcLwere least stable reference genes. Under salt stress,EF1α was comprehensively recommended top-ranked candidate reference gene followed byACTand 18S. In order to validate the identified most stable candidate reference genes,EF1α,ACT, 18S andUBQwere used for relative gene expression level of sodium/proton antiporter (NHX) gene under salt stress. The expression level ofNHXvaried according to the internal control which showed the importance of selection of appropriate reference gene. Taken together, this is the first ever systematic attempt of selection and validation of reference gene for qRT-PCR inR. apiculataphysiological tissues and in salt stress. This study would promote gene expression profiling of salt stress tolerance related genes inR. apiculata.

Reference Gene Selection for Quantitative Real-Time PCR of Mycelia fromLentinula edodesunder High-Temperature Stress

Housekeeping genes are important for measuring the transcription expression of functional genes; 10 traditional reference genes,TUB, TUA, GADPH, EF1, 18S, GTP, ACT, UBI, UBC,andH2A, were tested for their adequacy inLentinula edodes(L. edodes). Using specific primers, mRNA levels of these candidate housekeeping genes were evaluated in mycelia ofL. edodes, which were treated with high-temperature stress at 37°C for 0, 4, 8, 12, 18, and 24 hours. After treatment, expression stability of candidate genes was evaluated using three statistical software programs: geNorm, NormFinder, and BestKeeper. According to geNorm,TUBhad the lowest M values inL. edodesstrains 18 and 18N44. Using NormFinder, the best candidate reference gene in strain 18 wasTUB(0.030), and the best candidate reference gene in strain 18N44 wasUBI(0.047). In BestKeeper analysis, the standard deviation (SD) values ofUBC,TUA,H2A,EF1,ACT,18S, andGTPin strain 18 and those ofGADPHandGTPin strain 18N44 were greater than 1; thus, these genes were disqualified as reference genes. Taken together, onlyUBIandTUBwere found to be desirable reference genes by BestKeeper software. Based on the results of three software analyses,TUBwas the most stable gene under all conditions and was verified as an appropriate reference gene for quantitative real-time polymerase chain reaction inL. edodesmycelia under high-temperature stress.