Abstract
With the next generation sequencing technology, RNA-Seq (RNA sequencing) becomes one of the most powerful tools in quantification of global transcriptomes, discovery of new transcripts and alternative isoforms, as well as detection of single nucleotide polymorphisms (SNPs). RNA-Seq is advantageous over hybridization-based gene quantification methods: (1) it does not require prior information about genomic sequences, (2) it avoids high background problem caused by cross-hybridization, and (3) it is highly sensitive and avoids background and saturation of signals; and finally it is capable of detecting allelic expression differences in hybrids and allopolyploids. We used the RNA-Seq method to determine the genome-wide transcriptome changes in Arabidopsis allotetraploids and their parents, A. thaliana and A. arenosa. The use of this approach allows us to quantify transcriptome from these species and more importantly, to identify allelic or homoeologous-specific gene expression that plays a role in morphological evolution of allopolyploids. The computational pipelines developed are also applicable to the analysis of chromatin immunoprecipitation sequencing (ChIP-seq) data in Arabidopsis-related species, hybrids, and allopolyploids. Comparative analysis of RNA-Seq and ChIP-Seq data will allow us to determine the effects of chromatin modifications on nonadditive gene expression in hybrids and allopolyploids.
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References
Wang J, Tian L, Lee HS, Wei NE, Jiang H, Watson B, Madlung A, Osborn TC, Doerge RW, Comai L, Chen ZJ (2006) Genomewide nonadditive gene regulation in Arabidopsis allotetraploids. Genetics 172:507–517
Wang J, Tian L, Madlung A, Lee HS, Chen M, Lee JJ, Watson B, Kagochi T, Comai L, Chen ZJ (2004) Stochastic and epigenetic changes of gene expression in Arabidopsis polyploids. Genetics 167:1961–1973
Flagel L, Udall J, Nettleton D, Wendel J (2008) Duplicate gene expression in allopolyploid Gossypium reveals two temporally distinct phases of expression evolution. BMC Biol 6:16
Hegarty MJ, Barker GL, Wilson ID, Abbott RJ, Edwards KJ, Hiscock SJ (2006) Transcriptome shock after interspecific hybridization in senecio is ameliorated by genome duplication. Curr Biol 16:1652–1659
Pumphrey M, Bai J, Laudencia-Chingcuanco D, Anderson O, Gill BS (2009) Nonadditive expression of homoeologous genes is established upon polyploidization in hexaploid wheat. Genetics 181:1147–1157
Chague V, Just J, Mestiri I, Balzergue S, Tanguy AM, Huneau C, Huteau V, Belcram H, Coriton O, Jahier J, Chalhoub B (2010) Genome-wide gene expression changes in genetically stable synthetic and natural wheat allohexaploids. New Phytol 187:1181–1194
Osborn TC, Pires JC, Birchler JA, Auger DL, Chen ZJ, Lee HS, Comai L, Madlung A, Doerge RW, Colot V, Martienssen RA (2003) Understanding mechanisms of novel gene expression in polyploids. Trends Genet 19:141–147
Chen ZJ (2007) Genetic and epigenetic mechanisms for gene expression and phenotypic variation in plant polyploids. Annu Rev Plant Biol 58:377–406
Chen ZJ, Ni Z (2006) Mechanisms of genomic rearrangements and gene expression changes in plant polyploids. Bioessays 28:240–252
Comai L, Tyagi AP, Winter K, Holmes-Davis R, Reynolds SH, Stevens Y, Byers B (2000) Phenotypic instability and rapid gene silencing in newly formed Arabidopsis allotetraploids. Plant Cell 12:1551–1568
Jackson S, Chen ZJ (2010) Genomic and expression plasticity of polyploidy. Curr Opin Plant Biol 13:153–159
Ni Z, Kim ED, Ha M, Lackey E, Liu J, Zhang Y, Sun Q, Chen ZJ (2009) Altered circadian rhythms regulate growth vigour in hybrids and allopolyploids. Nature 457:327–331
Hoheisel JD (2006) Microarray technology: beyond transcript profiling and genotype analysis. Nat Rev Genet 7:200–210
Sobek J, Bartscherer K, Jacob A, Hoheisel JD, Angenendt P (2006) Microarray technology as a universal tool for high-throughput analysis of biological systems. Comb Chem High Throughput Screen 9:365–380
Marioni JC, Mason CE, Mane SM, Stephens M, Gilad Y (2008) RNA-seq: an assessment of technical reproducibility and comparison with gene expression arrays. Genome Res 18:1509–1517
Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B (2008) Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 5:621–628
Pepke S, Wold B, Mortazavi A (2009) Computation for ChIP-seq and RNA-seq studies. Nat Methods 6:S22–S32
Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10:57–63
Gautier L, Cope L, Bolstad BM, Irizarry RA (2004) affy—analysis of Affymetrix GeneChip data at the probe level. Bioinformatics 20:307–315
Quackenbush J (2001) Computational analysis of microarray data. Nat Rev Genet 2:418–427
Bloom JS, Khan Z, Kruglyak L, Singh M, Caudy AA (2009) Measuring differential gene expression by short read sequencing: quantitative comparison to 2-channel gene expression microarrays. BMC Genomics 10:221
Wang L, Feng Z, Wang X, Zhang X (2010) DEGseq: an R package for identifying differentially expressed genes from RNA-seq data. Bioinformatics 26:136–138
Eriksson N, Pachter L, Mitsuya Y, Rhee SY, Wang C, Gharizadeh B, Ronaghi M, Shafer RW, Beerenwinkel N (2008) Viral population estimation using pyrosequencing. PLoS Comput Biol 4:e1000074
Do CB, Batzoglou S (2008) What is the expectation maximization algorithm? Nat Biotechol 26:897–899
Saleh A, Alvarez-Venegas R, Avramova Z (2008) An efficient chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in Arabidopsis plants. Nat Protoc 3:1018–1025
Acknowledgements
We thank Luca Comai and his lab members for sharing A. arenosa SNP databases for mapping the reads. The work was supported by grants from the Plant Genome Research Program of the National Science Foundation (DBI0733857 to Z.J.C.).
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Ng, D.WK., Shi, X., Nah, G., Chen, Z.J. (2014). High-Throughput RNA-Seq for Allelic or Locus-Specific Expression Analysis in Arabidopsis-Related Species, Hybrids, and Allotetraploids. In: Spillane, C., McKeown, P. (eds) Plant Epigenetics and Epigenomics. Methods in Molecular Biology, vol 1112. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-773-0_3
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DOI: https://doi.org/10.1007/978-1-62703-773-0_3
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