Measuring global gene expression in polyploidy; a cautionary note from allohexaploid wheat
The number of global gene expression studies has increased significantly in recent years. It is assumed that the different techniques employed report similar levels of gene expression for each sequence type. While this may be true for many species, polyploids containing homoeologous and paralogous gene copies represent a unique situation. In this paper, we describe the comparison of the Affymetrix GeneChip® Wheat Genome Array, an in-house custom-spotted complementary DNA array and quantitative reverse transcription-polymerase chain reaction (PCR) for the study of gene expression in hexaploid wheat. Analysis of the data generated from each platform revealed little concordance and suggested that global comparisons are not possible. Potential causes of these inter-platform discrepancies were investigated and revealed to be due to the inability of the platforms to discriminate between different but related transcripts. Our results also showed that the traditionally used array validation technique, quantitative reverse transcription PCR, differs in its discriminatory ability, resulting in the poor confirmation rates seen in previous polyploid studies. These findings have implications for gene expression studies in polyploid organisms and highlight the need for homoeologous- and paralogous-specific arrays when investigating polyploid gene expression.
KeywordsTriticum aestivum Affymetrix GeneChip array cDNA microarrays Polyploids
We are grateful to the Biotechnology and Biological Sciences Research Council, UK (BBSRC Agri-Food), for providing the main funding for this work (ref. D17385). Ian Wilson was funded by a BBSRC GDB award (ref. BBSB07446) and Gary Barker by a BBSRC GDB award (ref. G20322). Our thanks to Andrew Hughes and Guy Donaldson for their invaluable assistance with the controlled environment growth of wheat plants.
- Akhunov ED, Goodyear AW, Geng S, Qi LL, Echalier B, Gill BS, Miftahudin, Gustafson JP, Lazo G, Chao S, Anderson OD, Linkiewicz AM, Dubcovsky J, La Rota M, Sorrells ME, Zhang D, Nguyen HT, Kalavacharla V, Hossain K, Kianian SF, Peng J, Lapitan NL, Gonzalez-Hernandez JL, Anderson JA, Choi DW, Close TJ, Dilbirliqi M, Gill KS, Walker-Simmons MK, Steber C, Mcguire PE, Qualset CO, Dvorak J (2003) The organization and rate of evolution of wheat genomes are correlated with recombination rates along chromosome arms. Genome Res 13(5):753–763PubMedCrossRefGoogle Scholar
- Brenner S, Johnson M, Bridgham J, Golda G, Johnson D, Luo S, McCurdy S, Foy M, Ewan M, Roth R, George D, Eletr S, Albrecht G, Vermaas E, Williams SR, Moon K, Burcham T, Pallas M, DuBridge RB, Kirchner J, Fearon K, Mao J, Corcoran K (2000) Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays. Nat Biotechnol 18(6):597–598CrossRefGoogle Scholar
- Chantret N, Salse J, Sabot F, Rahman S, Bellec A, Laubin B, Dubois I, Dossat C, Sourdille P, Joudrier P, Gautier MF, Cattolico L, Beckert M, Aubourg S, Weissenbach J, Caboche M, Bernard M, Leroy P, Chalhoub B (2005) Molecular basis of evolutionary events that shaped the hardness locus in diploid and polyploidy wheat species (Triticum and Aegilops). Plant Cell 17:1033–1045PubMedCrossRefGoogle Scholar
- Ishigaki S, Niwa J, Ando Y, Yoshihara T, Sawada K, Doyu M, Yamamoto M, Kato K, Yotsumoto Y, Sobue G (2002) Differentially expressed genes in sporadic amyotrophic lateral sclerosis spinal cords: screening by molecular indexing and subsequent cDNA microarray analysis. FEBS Lett 531(2):354–358PubMedCrossRefGoogle Scholar
- Kawaura K, Mochida K, Yamazaki Y, Ogihara Y (2006) Transcriptome analysis of salinity stress responses in common wheat using a 22 k oligo-DNA microarray. Funct Integr Genomics 276(3):304–312Google Scholar
- Mecham BH, Klus GT, Strovel J, Augustus M, Byrne D, Bozso P, Wetmore DZ, Marriani TJ, Kohane I, Szallasi Z (2004) Sequence-matched probes produce increased cross-platform consistency and more reproducible biological results in microarray-based gene expression measurements. Nucleic Acids Res 32(9):e74PubMedCrossRefGoogle Scholar
- Mitra R, Bhatia CR (1973) Repeated and non-repeated nucleotide sequences in diploid and polyploidy wheat species. Heredity 31:251–262Google Scholar