Selection and validation of reference genes for gene expression analysis in apomictic and sexual Cenchrus ciliaris

BMC Research Notes

Peggy Ozias-Akins¹  Joann A Conner¹  Bindu Simon² 

[1] Department of Horticulture, The University of Georgia Tifton Campus, Tifton, GA 31793, USA;Current Address: Burnett School of Biomedical Sciences, University of Central Florida, Florida 32826, USA

关键词: Cenchrus ciliaris;    Buffelgrass;    qRT-PCR;    Reference genes;    Apospory;    Apomixis;   

Others  :  1141490
DOI  :  10.1186/1756-0500-6-397

 received in 2013-04-24, accepted in 2013-09-25,  发布年份 2013

【 摘 要 】

Background

Apomixis is a naturally occurring asexual mode of seed reproduction resulting in offspring genetically identical to the maternal plant. Identifying differential gene expression patterns between apomictic and sexual plants is valuable to help deconstruct the trait. Quantitative RT-PCR (qRT-PCR) is a popular method for analyzing gene expression. Normalizing gene expression data using proper reference genes which show stable expression under investigated conditions is critical in qRT-PCR analysis. We used qRT-PCR to validate expression and stability of six potential reference genes (EF1alpha, EIF4A, UBCE, GAPDH, ACT2 and TUBA) in vegetative and reproductive tissues of B-2S and B-12-9 accessions of C. ciliaris.

Findings

Among tissue types evaluated, EF1alpha showed the highest level of expression while TUBA showed the lowest. When all tissue types were evaluated and compared between genotypes, EIF4A was the most stable reference gene. Gene expression stability for specific ovary stages of B-2S and B-12-9 was also determined. Except for TUBA, all other tested reference genes could be used for any stage-specific ovary tissue normalization, irrespective of the mode of reproduction.

Conclusion

Our gene expression stability assay using six reference genes, in sexual and apomictic accessions of C. ciliaris, suggests that EIF4A is the most stable gene across all tissue types analyzed. All other tested reference genes, with the exception of TUBA, could be used for gene expression comparison studies between sexual and apomictic ovaries over multiple developmental stages. This reference gene validation data in C. ciliaris will serve as an important base for future apomixis-related transcriptome data validation.

【 授权许可】

   
2013 Simon et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

【 参考文献 】

• [1]Grimanelli D, Leblanc O, Perotti E, Grossniklaus U: Developmental genetics of gametophytic apomixis. Trends Genet 2001, 17:597-604.
• [2]Ozias-Akins P, Van-Dijk PJ: Mendelian genetics of apomixis in plants. Annu Rev Genet 2007, 11:509-537.
• [3]Spillane C, Curtis MD, Grossniklaus U: Apomixis technology development–virgin births in farmers’ fields? Nat Biotechnol 2004, 22:687-691.
• [4]Asker SE, Jerling L: Apomixis in Plants. Boca Raton, FL: CRC Press; 1992.
• [5]Richards AJ: Agamospery. In Plant Breeding Systems. Edited by Richards AJ. Boston: George Allen & Unwin; 1986:403-509.
• [6]Koltunow AM, Grossniklaus U: Apomixis: A developmental perspective. Annu Rev Plant Biol 2003, 54:547-574.
• [7]Carman JG: Asynchronous expression of duplicate genes in angiosperms may cause apomixis, bispory, tetraspory, and polyembryony. Biol J Linnean Soc 1997, 61:51-94.
• [8]Hignight KW, Bashaw EC, Hussey MA: Cytological and morphological diversity of native apomictic buffelgrass, Pennisetum ciliare (L.) Link. Bot Gaz 1991, 152:214-218.
• [9]Akiyama Y, Conner JA, Goel S, Morishige DT, Mullet JE, DeBarry J, Yang L, Bennetzen JL, Klein PE, Ozias-Akins P: High-resolution physical mapping in Pennisetum squamulatum reveals extensive chromosomal heteromorphism of the genomic region associated with apomixis. Plant Physiol 2004, 134:1733-1741.
• [10]Akiyama Y, Hanna WW, Ozias-Akins P: High-resolution physical mapping reveals that the apospory-specific genomic region (ASGR) in Cenchrus ciliaris is located on a heterochromatic and hemizygous region of a single chromosome. Theor Appl Genet 2005, 111:1042-1051.
• [11]Goel S, Chen Z, Conner JA, Akiyama Y, Hanna WW, Ozias-Akins P: Physical evidence that a single hemizygous chromosomal region is sufficient to confer aposporous embryo sac formation in Pennisetum squamulatum and Cenchrus ciliaris. Genetics 2003, 163:1069-1082.
• [12]Goel S, Chen Z, Akiyama Y, Conner JA, Basu M, Gualtieri G, Hanna WW, Ozias-Akins P: Comparative physical mapping of the apospory-specific genomic region in two apomictic grasses: Pennisetum squamulatum and Cenchrus ciliaris. Genetics 2006, 173:389-400.
• [13]Ozias-Akins P, Roche D, Hanna WW: Tight clustering and hemizygosity of apomixis-linked molecular markers in Pennisetum squamulatum implies genetic control of apospory by a divergent locus which may have no allelic form in sexual genotypes. Proc Natl Acad Sci U S A 1998, 95:5127-5132.
• [14]Roche D, Cong P, Chen ZB, Hanna WW, Gustine DL, Sherwood RT, Ozias-Akins P: An apospory-specific genomic region is conserved between buffelgrass (Cenchrus ciliaris L.) and Pennisetum squamulatum Fresen. Plant J 1999, 19:203-208.
• [15]Nogler GA: Gametophytic apomixis. In Embryology of Angiosperms. Edited by Johri BM. Berlin, Heidelberg, New York, Tokyo: Springer; 1984:475-518.
• [16]Vielle J-P, Burson BL, Bashaw EC, Hussey MA: Early fertilization events in the sexual and aposporous egg apparatus of Pennisetum ciliare (L.) Link. Plant J 1995, 8:309-316.
• [17]Bashaw EC: Apomixis and sexuality in buffelgrass. Crop Sci 1962, 2:412-415.
• [18]Gustine DL, Sherwood RT, Huff DR: Apospory-linked molecular markers in buffelgrass. Crop Sci 1997, 37:947-951.
• [19]Conner JA, Goel S, Gunawan G, Cordonnier-Pratt MM, Johnson VE, Liang C, Wang H, Pratt LH, Mullet JE, Debarry J, Yang L, Bennetzen JL, Klein PE, Ozias-Akins P: Sequence analysis of bacterial artificial chromosome clones from the apospory-specific genomic region of Pennisetum and Cenchrus. Plant Physiol 2008, 147:1396-1411.
• [20]Pellino M, Sharbel TF, Mau M, Amiteye S, Corral JM: Selection of reference genes for quantitative real-time PCR expression studies of microdissected reproductive tissues in apomictic and sexual Boechera. BMC Res Notes 2011, 4:303. BioMed Central Full Text
• [21]Sharbel TF, Voigt ML, Corral JM, Thiel T, Varshney A, Kumlehn J, Vogel H, Rotter B: Molecular signatures of apomictic and sexual ovules in the Boechera holboellii complex. Plant J 2009, 58:870-882.
• [22]Sharbel TF, Voigt ML, Corral JM, Galla G, Kumlehn J, Klukas C, Schreiber F, Vogel H, Rotter B: Apomictic and sexual ovules of Boechera display heterochronic global gene expression patterns. Plant Cell 2010, 22:655.
• [23]Hong SY, Seo PJ, Yang MS, Xiang F, Park CM: Exploring valid reference genes for gene expression studies in Brachypodium distachyon by real-time PCR. BMC Plant Biol 2008, 8:112. BioMed Central Full Text
• [24]Pfaffl MW: Quantification strategies in real time PCR. In A-Z of quantitative PCR. Edited by Bustin SA. La Jolla, CA: International University Line; 2004:1-20.
• [25]Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP: Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper–Excel-based tool using pair-wise correlations. Biotechnol Lett 2004, 26:509-515.
• [26]Nolan T, Hands RE, Bustin SA: Quantification of mRNA using real-time RT-PCR. Nat Protoc 2006, 1:1559-1582.
• [27]VanGuilder HD, Vrana KE, Freeman WM: Twenty-five years of quantitative PCR for gene expression analysis. Biotechniques 2008, 44:619-626.
• [28]Expósito-Rodríguez M, Borges AA, Borges-Pérez A, Pérez JA: Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process. BMC Plant Biol 2008, 8:131. BioMed Central Full Text
• [29]Huggett J, Dheda K, Bustin SA: Normalization. In Real-Time PCR. Edited by Dorak MT. New York: BIOS Advanced Methods; 2006:83-91.
• [30]Thellin O, Zorzi W, Lakaye B, De-Borman B, Coumans B, Hennen G, Grisar T, Igout A, Heinen E: Housekeeping genes as internal standards: use and limits. J Biotechnol 1999, 75:291-295.
• [31]Vandesompele J, De-Preter K, Pattyn F, Poppe B, Van-Roy N, De-Paepe A, Speleman F: Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002, 3:0034.1-0034.11.
• [32]Andersen CL, Jensen JL, Orntoft TF: Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 2004, 64:5245-5250.
• [33]Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible WR: Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol 2005, 139:5-17.
• [34]Dean JD, Goodwin PH, Hsiang T: Comparison of relative RT-PCR and northern blot analyses to measure expression of β-1,3-glucanase in Nicotiana benthamiana infected with Colletotrichum destructivum. Plant Mol Biol Rep 2002, 20:347-356.
• [35]Orsel M, Krapp A, Daniel-Vedele F: Analysis of the NRT2 nitrate transporter family in Arabidopsis: Structure and gene expression. Plant Physiol 2002, 129:886-888.
• [36]Ozturk ZN, Talamé V, Deyholos M, Michalowski CB, Galbraith DW, Gozukirmizi N, Tuberosa R, Bohnert HJ: Monitoring large-scale changes in transcript abundance in drought- and salt-stressed barley. Plant Mol Biol 2002, 48:551-573.
• [37]Stürzenbaum SR, Kille P: Control genes in quantitative molecular biological techniques: the variability of invariance. Comp Biochem Physiol B 2007, 130:281-289.
• [38]Thomas C, Meyer D, Wolff M, Himber C, Alioua M, Steinmetz A: Molecular characterization and spatial expression of the sunflower ABP1 gene. Plant Mol Biol 2003, 52:1025-1036.
• [39]Silveira ED, Alves-Ferreira M, Guimarães LA, Da-Silva FR, Carneiro VTC: Selection of reference genes for quantitative real-time PCR expression studies in the apomictic and sexual grass Brachiaria brizantha. BMC Plant Biol 2009, 9:84. BioMed Central Full Text
• [40]Sahu PP, Gupta S, Malaviya DR, Roy AK, Kaushal P, Prasad M: Transcriptome analysis of differentially expressed genes during embryo sac development in apomeiotic non-parthenogenetic interspecific hybrid of Pennisetum glaucum. Mol Biotechnol 2012, 51:262-271.
• [41]Zeng Y, Conner J, Ozias-Akins P: Identification of ovule transcripts from the Apospory-Specific Genomic Region (ASGR)-carrier chromosome. BMC Genomics 2011, 12:206. BioMed Central Full Text
• [42]Libus J, Štorchová H: Quantification of cDNA generated by reverse transcription of total RNA provides a simple alternative tool for quantitative RT-PCR normalization. Biotechniques 2006, 41:156-164.
• [43]Rozen S, Skaletsky HJ: Primer3 on the WWW for general users and for biologist programmers. In Bioinformatics Methods and Protocols: Methods in Molecular Biology. Edited by Krawetz S, Misener S. Totowa, NJ: Humana Press; 2000:365-386.
• [44]Zhao SFR: Comprehensive algorithm for quantitative real-time polymerase chain reaction. J Comp Biol 2005, 12:1047-1064.
• [45]Bustin SA, Nolan T: Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. J Biomol Tech 2004, 15:155-156.
• [46]Yan J, Yuan F, Long G, Qin L, Deng Z: Selection of reference genes for quantitative real-time RT-PCR analysis in citrus. Mol Biol Rep 2012, 39:1831-1838.
• [47]Demidenko NV, Logacheva MD, Penin AA: Selection and validation of reference genes for quantitative real-time PCR in buckwheat (Fagopyrum esculentum) based on transcriptome sequence data. PLoS One 2011, 6:e19434.
• [48]Mehta R, Birerdinc A, Hossain N, Afendy A, Chandhoke V, Younossi Z, Baranova A: Validation of endogenous reference genes for qRT-PCR analysis of human visceral adipose samples. BMC Mol Biol 2010, 11:39. BioMed Central Full Text