Use of mRNA-Seq to discriminate contributions to the transcriptome from the constituent genomes of the polyploid crop species Brassica napus

Polyploidy often results in considerable changes in gene expression, both immediately andover evolutionary time. New phenotypes often arise with polyploid formation and maycontribute to the fitness of polyploids in nature or their selection for use in agriculture.Oilseed rape (Brassica napus) is widely used to study the process of polyploidy both inartificially resynthesised and natural forms.

mRNA-Seq, a recently developed approach totranscriptome profiling using deep-sequencing technologies is an alternative to microarraysfor the study of gene expression in a polyploid.

Results: Illumina mRNA-Seq is comparable to microarray analysis for transcript quantification buthas increased sensitivity and, very importantly, the potential to distinguish betweenhomoeologous genes in polyploids. Using a novel curing process, we adapted a referencesequence that was a consensus derived from ESTs from both Brassica A and C genomes toone containing separate A and C genome versions for each of the 94,558 original unigenes.We aligned reads from B.

napus to this cured reference, finding 38% more reads mappingfrom resynthesised lines and 28% more reads mapping from natural lines. Where the A and Cversions differed at single nucleotide positions, termed inter-homoeologue polymorphisms(IHPs), we were able to apportion expression in the polyploid between the A and C genome homoeologues.

43,761 unigenes contained at least one IHP, with a mean frequency of 10.5per kb unigene sequence. 6,350 of the unigenes with IHPs were differentially expressedbetween homoeologous gene pairs in resynthesised B.

napus. 3,212 unigenes showed asimilar pattern of differential expression across a range of natural B.

napus crop varieties and,of these, 995 were in common with resynthesised B. napus.

Functional classification showedover-representation in gene ontology categories not associated with dosage-sensitivity.

Conclusion: mRNA-Seq is the method of choice for measuring transcript abundance in polyploids due toits ability to measure the contributions of homoeologues to gene expression. Theidentification of large numbers of differentially expressed genes in both a newlyresynthesised polyploid and natural B.

napus confirms that there are both immediate andlong-term alterations in the expression of homoeologous gene pairs following polyploidy.

Published on: 2012-06-16

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