Identification of Reference Genes for Quantitative Gene Expression
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Article Identification of Reference Genes for Quantitative Gene Expression Studies in Pinus massoniana and Its Introgression Hybrid Jiaxing Mo 1,2,3 , Jin Xu 1,2,3,* , Wenjing Jin 1,2,3, Liwei Yang 1,2,3, Tongming Yin 1,2,3 and Jisen Shi 1,2,3 1 Key Laboratory of Forest Genetics & Biotechnology of Ministry of Education, Nanjing Forestry University, Nanjing 210037, China 2 Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China 3 College of Forestry, Nanjing Forestry University, Nanjing 210037, China * Correspondence: [email protected]; Tel.: +86-25-8542-7319 Received: 25 July 2019; Accepted: 8 September 2019; Published: 11 September 2019 Abstract: qRT-PCR is a powerful molecular research tool to study the regulation of gene expression. However, to accurately calculate gene expression levels, an experiment should include proper reference genes that show no changes in their expression level. Pinus massoniana, P. hwangshanensis, and their introgression hybrid in Mountain Lushan, China, are an ideal model for studying introgression and speciation. Although some research on reference gene selection for P. massoniana has been reported before, no studies on this subject have been performed where P. massoniana and its introgression hybrid were evaluated simultaneously. Here, we investigated ten genes (upLOC, SDH, ACT, EF, TOC75, DMWD, FBOX, PGK1, UBQ, and CL2417C7) identified from transcriptome data of these two taxa for reference gene potential. These ten genes were then screened across multiple tissues such as cone, young and mature stems, and young needles according to qRT-PCR thermal cycling and dissociation. Correlation coefficient, amplification efficiency, and cycle threshold value (Ct) range were applied to evaluate the reliability of each gene. The stability of candidate reference gene expression was calculated using three algorithms: geNorm, NormFinder, and BestKeeper. Base on the reliability and stability, we then offered a list of genes of recommended and not recommended for seven different tissue type and species. Our results demonstrated that different sample lines require different genes as reference to evaluate. Keywords: Pinus massoniana; reference gene; qRT-PCR; introgression; cone 1. Introduction Masson pine (Pinus massoniana) is an economically important conifer species that is naturally distributed in southern China. Its wood is often used for standing timber and furniture manufacturing. Resin, a secretion from its canals, can be used for the maintenance of musical instrument strings, and is an ingredient in several medicines [1,2]. Because of its fast growth and high biomass, it has been planted extensively in south China during the past fifty years for rapid vegetation recovery and additional ecological purposes. Huangshan pine (P. hwangshanensis) is a species native only to China. Although it grows at a considerably slower rate than P. massoniana, it has ornamental value as a horticultural plant in higher altitude regions. Around the middle and lower reaches of the Yangtze River, P. massoniana mainly grows at an altitude below 700 m (above sea level, a.s.l.), while P. hwangshanensis mainly grows above 900 m (a.s.l.). Forests 2019, 10, 787; doi:10.3390/f10090787 www.mdpi.com/journal/forests Forests 2019, 10, 787 2 of 13 Forests 2019, 10, x FOR PEER REVIEW 2 of 13 On Mountain Lushan, situated in this area, both pines have their own ecological niche according to (a.s.l.). On Mountain Lushan, situated in this area, both pines have their own ecological niche the distribution mentioned above. An introgression hybrid pine (hereafter referred to as the Z pine) according to the distribution mentioned above. An introgression hybrid pine (hereafter referred to as P. massoniana P. hwangshanensis can bethe found Z pine) directly can be adjacentfound directly to the adjacent habitats to the of bothhabitats of both P. massonianaand and P. hwangshanensis(Figure 1). There are(Figure several 1). There morphological are several morphological differences between differencesP. massonianabetween P. massonianaand P. hwangshanensis and P. hwangshanensis[3], while the Z pine[3] displays, while the phenotypic Z pine displays characteristics phenotypic characteristics of both parental of both pines parental [4,5]. pines However, [4,5]. However, it also shows it also novel phenotypes,shows novel such asphenotypes, an ultra-low such pinecone as an ultra-low ripening pinecone and seed ripening germination and seed rate, germination compared rate, to both P. massonianacomparedand toP. both hwangshanensis P. massoniana[ 6and]. This P. hwangshanensis distinct characteristic [6]. This distinct shows thatcharacteristic genetic incompatibilitiesshows that duringgenetic the process incompatibilities of fertilization during andthe /processor even of embryonic fertilization developmentand/or even embryonic might exist development between both might exist between both parents of the Z pine. Our previous research has shown that a group of parents of the Z pine. Our previous research has shown that a group of differentially expressed genes differentially expressed genes (DEGs) related to reproduction were expressed at a much lower level (DEGs) related to reproduction were expressed at a much lower level in the Z pine than in P. massoniana, in the Z pine than in P. massoniana, possibly causing the abnormal reproductive phenotype in the Z possiblypine causing [7]. the abnormal reproductive phenotype in the Z pine [7]. FigureFigure 1. Schematic 1. Schematic indicating indicating the mainthe main distribution distribution areas areas of ofP. P. massoniana massoniana, ,P.hwangshanensis P.hwangshanensis,, and and their introgressiontheir introgression hybrid, the hybrid, Z pine, the onZ pine, Mt. on Lushan, Mt. Lushan, Jiangxi Jiangxi Province, Province, China. China. a.s.l.: a.s.l. above: above seasea level. UnderstandingUnderstanding how how the the expression expression of certain certain genes genes of interest of interest is regulated is regulated in space, in time, space, and time, and didifferentfferent tissuestissues isis aa crucialcrucial approach to to further further understanding understanding about about their their biological biological function. function. Various methods can be applied to study gene expression: With microarrays, northern blots, RNA- Various methods can be applied to study gene expression: With microarrays, northern blots, RNA-seq, seq, and qRT-PCR (quantitative real-time polymerase chain reaction) being considered some of the and qRT-PCRmore common (quantitative ways, RNA-seq real-time and qRT-PCR polymerase being chain the most reaction) popular. being After considered qRT-PCR was some introduced of the more commonin the ways, late twentieth RNA-seq century and qRT-PCR[8], it has been being widely the applied most popular.to quantify Aftergene expression qRT-PCR in was life science introduced in theresearch late twentieth and medical century diagnosis. [8], it qRT-PCR has been has widely a series applied of advantages, to quantify including gene high expression accuracy, in life sciencesensitivity, research and reproducibility. medical diagnosis. Nevertheless, qRT-PCR due has to aits series high sensitivity, of advantages, the qRT-PCR including assay high might accuracy, sensitivity,be affected and reproducibility.by RNA or cDNA Nevertheless,quality, primer specificity, due to its amplification high sensitivity, efficiency the qRT-PCR[9], etc. Therefore, assay might be affectedoptimal by normalization RNA or cDNA methods quality, are required primer for specificity, sample-to-sample amplification comparison. efficiency A series [9 ],of etc.strategies Therefore, has been introduced for normalizing qRT-PCR data [9–13]. In general, in order to evaluate the optimal normalization methods are required for sample-to-sample comparison. A series of strategies expression quantity of a gene, a reference gene is required for various samples and/or experimental has beenconditions introduced (e.g., developmental for normalizing stages, qRT-PCR different orga datans, [9 treatments,–13]. In general,etc.). A reference in order gene to is evaluate a gene the expressionthat shows quantity unchanging of a gene, expression a reference among gene all samples is required and/or for experimental various samples treatments and /[14]or experimentaland can conditionsthen be (e.g., used developmental to normalize and stages, calibrate di ffresultserent [15] organs,. Therefore, treatments, obtaining etc.). a reliable A reference reference gene gene is is a gene that showsa critical unchanging step before expressionbeing able to amongperform allqRT-PCR samples in further and/or research. experimental treatments [14] and can then be usedHowever, to normalize no single and universal calibrate reference results [15gene]. Therefore,is found acceptable obtaining to a evaluate reliable referenceall species geneor is a criticalsamples. step before Due to being the varying able to PCR perform amplification qRT-PCR efficie in furtherncy of the research. target sequence, a different reference gene could be required. Reference genes with poor availability could lead to significant biases and However, no single universal reference gene is found acceptable to evaluate all species or samples. faulty interpretation of the data. Therefore, determining a reference gene is an essential step before Due tostarting the varying the experimental PCR amplification process. efficiency of the target sequence, a different reference gene