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Reference Genes for RT-qPCR Analysis of Environmentally and Developmentally Regulated Gene Expression in Alfalfa

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ABSTRACT
Reverse transcription quantitative PCR (RT-qPCR) is a highly sensitive technique that has become the standard for the analysis of differences in gene expression in response to experimental treatments or among genetic sources. The accuracy of the RT-qPCR results can be significantly affected by uncontrolled sources of variation that can be accounted for normalization with so-called reference genes stably expressed under various conditions. In this study we assessed the stability of 21 reference gene candidates in crowns of two alfalfa cultivars (Apica and Evolution) exposed to various environmental conditions (cold, water stress and photoperiod) and from above ground biomass of the cultivar Orca sampled at three developmental stages (vegetative, full bloom and mature pods). Candidates were selected based on their previous identification in other plant species or their stable expression in a differential hybridization of alfalfa ESTs with cDNA from non-acclimated and cold-acclimated alfalfa. Genes encoding ubiquitin protein ligase 2a (UBL-2a), actin depolymerizing factor (ADF) and retention in endoplasmic reticulum 1 protein (Rer1) were the most stable across experimental conditions. Conversely β-actin (Act), α-tubulin (Tub) and glyce-raldehyde 3-phosphate dehydrogenase (GAPDH) frequently used as “housekeeping genes” in gene expression studies showed poor stability. No more than two reference genes were required to normalize the gene expression data under each condition. Normalization of the expression of genes of interest with unstable reference genes led to observations that were conflicting with those made with validated reference genes and that were in some cases inconsistent with the current knowledge of the trait. The reference genes identified in this study are strong candidates for normalization of gene expression in cultivated alfalfa.
 
Cite this paper
Castonguay, Y. , Michaud, J. and Dubé, M. (2015) Reference Genes for RT-qPCR Analysis of Environmentally and Developmentally Regulated Gene Expression in Alfalfa. American Journal of Plant Sciences, 6, 132-143. doi: 10.4236/ajps.2015.61015.
 
References
[1]Russelle, M.P. (2001) Alfalfa. American Scientist, 89, 252-261. http://dx.doi.org/10.1511/2001.3.252
 
[2]Han, Y., Kang, Y., Torres-Jerez, I., Cheung, F., Town, C., et al. (2011) Genome-Wide SNP Discovery in Tetraploid Alfalfa Using 454 Sequencing and High Resolution Melting Analysis. BMC Genomics, 12, 350. http://dx.doi.org/10.1186/1471-2164-12-350
 
[3]Verenosi, F., Brummer, E.C. and Hyuyghe, C. (2010) Alfalfa. In: Boller, B., Posselt, U.K., Veronesi, F., Eds., Fodder Crops and Amenity Grasses, Handbook of Plant Breeding, Vol. 5, Springer, New York, 395-437.
 
[4]Castonguay, Y., Laberge, S., Brummer, E.C. and Volenec, J.J. (2006) Alfalfa Winter Hardiness: A Research Retrospective and Integrated Perspective. Advances in Agronomy, 90, 203-265.
http://dx.doi.org/10.1016/S0065-2113(06)90006-6
 
[5]Jung, H.J.G., Samac, D.A. and Sarath, G. (2012) Modifying Crops to Increase Cell Wall Digestibility. Plant Science, 185-186, 65-77. http://dx.doi.org/10.1016/j.plantsci.2011.10.014
 
[6]Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J., et al. (2009) The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments. Clinical Chemistry, 55, 611-622. http://dx.doi.org/10.1373/clinchem.2008.112797
 
[7]Guénin, S., Mauriat, M., Pelloux, J., Van Wuytswinkel, O., Bellini, C., et al. (2009) Normalization of qRT-PCR Data: The Necessity of Adopting a Systematic, Experimental Conditions-Specific, Validation of References. Journal of Experimental Botany, 60, 487-493. http://dx.doi.org/10.1093/jxb/ern305
 
[8]Chang, E., Shi, S., Liu, J., Cheng, T., Xue, L., et al. (2012) Selection of Reference Genes for Quantitative Gene Expression Studies in Platycladus orientalis (Cupressaceae) Using Real-Time PCR. PLoS ONE, 7, e33278.
 
[9]Gutierrez, L., Mauriat, M., Guénin, S., Pelloux, J., Lefebvre, J.-F., et al. (2008) The Lack of a Systematic Validation of Reference Genes: A Serious Pitfall Undervalued in Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis in Plants. Plant Biotechnology Journal, 6, 609-618.
http://dx.doi.org/10.1111/j.1467-7652.2008.00346.x
 
[10]Bin, W.S., Wei, L.K., Ping, D.W., Li, Z., Wei, G., et al. (2012) Evaluation of Appropriate Reference Genes for Gene Expression Studies in Pepper by Quantitative Real-Time PCR. Molecular Breeding, 30, 1393-1400.
 
[11]Castro, P., Román, B., Rubio, J. and Die, J.V. (2012) Selection of Reference Genes for Expression Studies in Cicer arietinum L.: Analysis of cyp81E3 Gene Expression against Ascochyta rabiei. Molecular Breeding, 29, 261-274. http://dx.doi.org/10.1007/s11032-010-9544-8
 
[12]Chen, L., Zhong, H.-Y., Kuang, J.-F., Li, J.-G., Lu, W.-J. and Chen, J.-Y. (2011) Validation of Reference Genes for RT-qPCR Studies of Gene Expression in Banana Fruit under Different Experimental Conditions. Planta, 234, 377-390. http://dx.doi.org/10.1007/s00425-011-1410-3
 
[13]Fernández, M., Villarroel, C., Balbontín, C. and Valenzuela, S. (2010) Validation of Reference Genes for Real-Time qRT-PCR Normalization during Cold Acclimation in Eucalyptus globulus. Trees, 24, 1109-1116. http://dx.doi.org/10.1007/s00468-010-0483-0
 
[14]Li, H., Qin, Y., Xiao, X. and Tang, C. (2011) Screening of Valid Reference Genes for Real-Time RT-PCR Data Normalization in Hevea brasiliensis and Expression Validation of a Sucrose Transporter Gene HbSUT3. Plant Science, 181, 132-139. http://dx.doi.org/10.1016/j.plantsci.2011.04.014
 
[15]Maroufi, A., Van Bockstaele, E. and De Loose, M. (2010) Validation of Reference Genes for Gene Expression Analysis in Chicory (Cichorium intybus) Using Quantitative Real-Time PCR. BMC Molecular Biology, 11, 15. http://dx.doi.org/10.1186/1471-2199-11-15
 
[16]Park, S.-C., Kim, Y.-H., Ji, C.Y., Park, S., Jeong, J.C., Lee, H.-S. and Kwak, S.-S. (2012) Stable Internal Reference Genes for the Normalization of Real-Time PCR in Different Sweetpotato Cultivars Subjected to Abiotic Stress Conditions. PLoS ONE, 7, e51502. http://dx.doi.org/10.1371/journal.pone.0051502
 
[17]Goulao, L., Fortunato, A.C. and Ramalho, J. (2012) Selection of Reference Genes for Normalizing Quantitative Real-Time PCR Gene Expression Data with Multiple Variables in Coffea spp. Plant Molecular Biology Reporter, 30, 741-759. http://dx.doi.org/10.1007/s11105-011-0382-6
 
[18]Rapacz, M., Stepień, A. and Skorupa, K. (2012) Internal Standards for Quantitative RT-PCR Studies of Gene Expression under Drought Treatment in Barley Hordeum vulgare: The Effects of Developmental Stage and Leaf Age. Acta Physiologiae Plantarum, 34, 1723-1733.
http://dx.doi.org/10.1007/s11738-012-0967-1
 
[19]Saha, G.C. and Vandemark, G.J. (2013) Stability of Expression of Reference Genes among Different Lentil (Lens culinaris) Genotypes Subjected to Cold Stress, White Mold Disease, and Aphanomyces Root Rot. Plant Molecular Biology Reporter, 31, 1109-1115. http://dx.doi.org/10.1007/s11105-013-0579-y
 
[20]Shi, J., Liu, M., Shi, J.N., Zheng, G., Wang, Y., Wang, J.Y., et al. (2012) Reference Gene Selection for qPCR in Ammopiptanthus mongolicus under Abiotic Stresses and Expression Analysis of Seven ROS-Scavenging Enzyme Genes. Plant Cell Reports, 31 1245-1254. http://dx.doi.org/10.1007/s00299-012-1245-9
 
[21]Zhu, J.F., Zhang, L., Li, W.F., Han, S.Y., Yang, W.H. and Qi, L.W. (2013) Reference Gene Selection for Quantitative Real-Time PCR Normalization in Caragana intermedia under Different Abiotic Stress Conditions. PLoS ONE, 8, e53196. http://dx.doi.org/10.1371/journal.pone.0053196
 
[22]Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N. and De Paepe, A. (2002) Accurate Normalization of Real-Time Quantitative RT-PCR Data by Geometric Averaging of Multiple Internal Control Genes. Genome Biology, 3, RESEARCH0034.
 
[23]Castonguay, Y., Michaud, R., Nadeau, P. and Bertrand, A. (2009) An Indoor Screening Method for Improvement of Freezing Tolerance in Alfalfa. Crop Science, 49, 809-818.
http://dx.doi.org/10.2135/cropsci2008.09.0539
 
[24]Castonguay, Y., Bertrand, A., Michaud, R. and Laberge, S. (2011) Cold-Induced Biochemical and Molecular Changes in Afalfa Populations Selectively Improved for Freezing Tolerance. Crop Science, 51, 2132-2144. http://dx.doi.org/10.2135/cropsci2011.02.0060
 
[25]Duceppe, M.-O., Bertrand, A., Pattathil, S., Miller, J., Castonguay, Y., Hahn, M.G., et al. (2012) Assessment of Genetic Variability of Cell Wall Degradability for the Selection of Alfalfa with Improved Saccharification Efficiency. BioEnergy Research, 5, 904-914. http://dx.doi.org/10.1007/s12155-012-9204-4
 
[26]Dubé, M.P., Castonguay, Y., Cloutier, J., Michaud, J. and Bertrand, A. (2013) Characterization of Two Novel Cold-Inducible K3 Dehydrin Genes from Alfalfa (Medicago sativa spp. sativa L.). Theoretical and Applied Genetics, 126, 823-835. http://dx.doi.org/10.1007/s00122-012-2020-6
 
[27]Hellemans, J., Mortier, G., De Paepe, A., Speleman, F. and Vandesompele, J. (2007) qBase Relative Quantification Framework and Software for Management and Automated Analysis of Real-Time Quantitative PCR Data. Genome Biology, 8, R19. http://dx.doi.org/10.1186/gb-2007-8-2-r19
 
[28]Bustin, S.A., Benes, V., Nolan, T. and Pfaffl, M.W. (2005) Quantitative Real-Time RT-PCR—A Perspective. Journal of Molecular Endocrinology, 34, 597-601. http://dx.doi.org/10.1677/jme.1.01755
 
[29]Derveaux, S., Vandesompele, J. and Hellemans, J. (2010) How to Do Successful Gene Expression Analysis Using Real-Time PCR. Methods, 50, 227-230. http://dx.doi.org/10.1016/j.ymeth.2009.11.001
 
[30]Guerriero, G., Legay, S. and Hausman, J.-F. (2014) Alfalfa Cellulose Synthase Gene Expression under Abiotic Stress: A Hitchhiker’s Guide to RT-qPCR Normalization. PLoS ONE, 9, e103808.
http://dx.doi.org/10.1371/journal.pone.0103808
 
[31]Pembleton, K.G. and Sathish, P. (2014) Giving Drought the Cold Shoulder: A Relationship between Drought Tolerance and Fall Dormancy in an Agriculturally Important Crop. AoB PLANTS, 6, plu012.
 
[32]Xia, W., Liu, Z., Yang, Y., Xiao, Y., Mason, A.S., Zhao, S.L. and Ma, Z.L. (2014) Selection of Reference Genes for Quantitative Real-Time PCR in Cocos nucifera during Abiotic Stress. Botany, 92, 179-186. http://dx.doi.org/10.1139/cjb-2013-0212
 
[33]Czechowski, T., Stitt, M., Altmann, T., Udvardi, M.K. and Scheible, W.R. (2005) Genome-Wide Identification and Testing of Superior Reference Genes for Transcript Normalization in Arabidopsis. Plant Physiology, 139, 5-17. http://dx.doi.org/10.1104/pp.105.063743
 
[34]Libault, M., Thibivilliers, S., Bilgin, D.D., Radwan, O., Benitez, M., Clough, S.J. and Stacey, G. (2008) Identification of Four Soybean Reference Genes for Gene Expression Normalization. Plant Genome, 1, 44-54. http://dx.doi.org/10.3835/plantgenome2008.02.0091
 
[35]Lee, H., Kim, J.H., Park, M., Kim, I.C., Yim, J.H., et al. (2010) Reference Genes Validation for qPCR Normalization in Deschampsia antarctica during Abiotic Stresses. Antarctic Science, 22, 477-484. http://dx.doi.org/10.1017/S0954102009990782
 
[36]Janská, A., Hodek, J., Svoboda, P., Zámecník, J., PráŠil, I.T., Vlasáková, E., et al. (2013) The Choice of Reference Gene Set for Assessing Gene Expression in Barley (Hordeum vulgare L.) under Low Temperature and Drought Stress. Molecular Genetics and Genomics, 288, 639-649.
http://dx.doi.org/10.1007/s00438-013-0774-4
 
[37]Castonguay, Y. and Nadeau, P. (1998) Enzymatic Control of Soluble Carbohydrate Accumulation in Cold-Acclimated Crowns of Alfalfa. Crop Science, 38, 1183-1189.
http://dx.doi.org/10.2135/cropsci1998.0011183X003800050012x
 
[38]Szabados, L. and Savouré, A. (2010) Proline: A Multifunctional Amino Acid. Trends in Plant Science, 15, 89-97. http://dx.doi.org/10.1016/j.tplants.2009.11.009                                                                   eww150116lx
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