Screening and verification of internal reference genes by real time quantitative PCR analysis in Carya cathayensis

Abstract: 【Objective】Hickory (Carya cathayensis Sarg.) is a perennial deciduous nut tree, belonging to Juglans of walnut family. It is a unique nut and woody oil tree species in China, with high nutritional and economic value. In order to explore the internal molecular regulatory mechanism for growth, development, fruit quality and stress response, our research team has completed the whole genome sequencing and assembly of hickory and analyzed the expression of some genes using actin as an internal reference gene. However, it remains greatly unknown about the best internal reference gene of Carya cathayensis. The aim of the present study is to select the best internal reference gene in Carya Cathayensis. 【Methods】The root, stem, leaf, stigma, pericarp and embryos at different developmental stages were used as experimental materials for selecting the candidate internal reference genes at tissue levels. To further screen the internal reference genes under stress conditions, one-year-old hickory seedlings were treated with 100 mmol·L^-1 NaCl for 0 h, 12 h and 24 h, and 25% PEG 8000 for 0 h, 12 h and 24 h, and afterward, the leaves were collected for real time quantitative PCR (RT-qPCR) analysis. Furthermore,10 kinds of different plant hormones including IAA (100 μmol·L^-1), IBA (100 μmol·L^-1), NAA (100 μmol·L^-1), NPA (10 μmol·L^-1), KT (100 μmol·L^-1), ABA (100 μmol·L^-1), SA (200 μmol·L^-1), GA3 (200μmol·L^-1), ETH (1 mmol·L^-1) and MeJA (100 mmol·L^-1) were sprayed on the leaf surface of hickory, and the leaves were sampled after 3 h for RT-qPCR analysis. For grafting treatment, two-year-old seed  lings and the young shoots were used as rootstocks and scions, respectively, and both were collected at 0, 3, 7 and 14 d for RT-qPCR analysis. The RNA extraction method of each sample was in accordance with the instruction manual of Takara minibest plant RNA extraction kit, and was reversely transcribed into cDNA according to the instructions of PrimerScriptTMRT Master Mix (Perfect Real Time) Master Mix (perfect real time). Primer Premier 5 software was used to design primers of the 19 candidate internal reference genes in 10 housekeeping gene families, including ADP-ribosylation factor (ARF), Actin1 (ACT1), Actin7(ACT7), Metalloprotease (MPS), Ubiquitin conjugating enzyme 9 (UBC9), Cyclophilin (CYP), 60S ribosomal protein (60SRP), Elongation factor1α (EF-1α), Glyceraldehyde-3-phosphate dehydrogenase (GAPHD) and Histone (HIS), based on the related literatures of internal reference genes and the transcriptome sequencing data of Carya cathayensis. To determine the specificity of the primers, the obtained PCR products were detected by agarose gel (1.5%) electrophoresis, and the melting curves of the samples were measured by RT-qPCR. The RT-qPCR procedure was adjusted according to the instructions of reagent Kit and was performed on Bio-Rad CFX96. Relative expressions of candidate genes were calculated using the comparative threshold cycle (2^-ΔCT) method. Three biological replicates were conducted. Using geNorm, NormFinder, BestKeeper and RefFinder, the stability of the expressions of 19 candidate internal reference genes was comprehensively analyzed to select the best internal reference gene in Carya Cathayensis.【Results】The CT values of 19 candidate genes in 6 kinds of tissues of Carya cathayensis had certain changes, and the average CT values were between 22.49 and 26.06, of which, the highest was UBC9-5, and the lowest was EF-1α-2. By comparing the stability of internal reference gene expressions in different tissues, the top five genes (ARF-1, 60SRP, ACT7-1, ACT1-1 and UBC9-5) were selected and the expression of ARF-1 and 60SRP genes were the most stable. Under salt and drought stresses, the average CT values of the 5 candidate top genes ranged from 23.73 to 26.42, of which, the highest was UBC9- 5, and the lowest was 60SRP. With different hormone treatments, the average CT values of Carya cathayensis leaves were within 21.39 to 23.71, and the highest was UBC9-5, while the lowest was ACT7-1. Under the grafting condition, the average CT values ranged from 22.24 to 27.23, and ACT1-1 was the highest expression gene and 60SRP was the lowest. Together, the average CT values ranged from 22.32 to 25.25, of which, UBC9-5 was the highest and 60SRP was the lowest. GeNorm analysis further showed that the 60SRP gene was the most stable in response to salt and drought stress as well as grafted treatment; UBC9-5 was the most stable in response to different hormones treatments, while ACT7-1 and UBC9-5 were the most stable in all the sample groups. GeNorm analysis also displayed that two internal reference genes of hickory were enough to ensure the accuracy of the results under stress and with different hormone treatments but at least three under grafting conditions. The results of NormFinder analysis were basically consistent with those of geNorm. BestKeeper analysis indicated that the expression of ACT7-1 gene was the most stable under salt and drought stresses as well as with different hormone treatments, which was the least stable while UBC9-5 was just the opposite with grafting treatment, and ARF-1 was the most stable gene in all tissues and treatments. Comprehensive Reffinder analysis showed that 60SRP was the most stable under salt stress and drought conditions, UBC9-5 was the most stable gene with different hormone treatments, both of which were the most stable after grafting, and UBC9-5 was also the most stable gene in all tissues and treatments.【Conclusions】Taken together, the optimal internal reference genes in hickory were different in various tissues and different experimental treatments, which was affected by analytical approaches. This study provided important theoretical guidance for selecting internal reference genes in RT-qPCR analysis of gene expression in Carya cathayensis.