A Brassica Exon Array for Whole-Transcript Gene Expression Profiling

Abstract

Affymetrix GeneChip® arrays are used widely to study transcriptional changes in response to developmental and environmental stimuli. GeneChip® arrays comprise multiple 25-mer oligonucleotide probes per gene and retain certain advantages over direct sequencing. For plants, there are several public GeneChip® arrays whose probes are localised primarily in 3′ exons. Plant whole-transcript (WT) GeneChip® arrays are not yet publicly available, although WT resolution is needed to study complex crop genomes such as Brassica, which are typified by segmental duplications containing paralogous genes and/or allopolyploidy. Available sequence data were sampled from the Brassica A and C genomes, and 142,997 gene models identified. The assembled gene models were then used to establish a comprehensive public WT exon array for transcriptomics studies. The Affymetrix GeneChip® Brassica Exon 1.0 ST Array is a 5 µM feature size array, containing 2.4 million 25-base oligonucleotide probes representing 135,201 gene models, with 15 probes per gene distributed among exons. Discrimination of the gene models was based on an E-value cut-off of 1E−5, with ≤98% sequence identity. The 135 k Brassica Exon Array was validated by quantifying transcriptome differences between leaf and root tissue from a reference Brassica rapa line (R-o-18), and categorisation by Gene Ontologies (GO) based on gene orthology with Arabidopsis thaliana. Technical validation involved comparison of the exon array with a 60-mer array platform using the same starting RNA samples. The 135 k Brassica Exon Array is a robust platform. All data relating to the array design and probe identities are available in the public domain and are curated within the BrassEnsembl genome viewer at http://www.brassica.info/BrassEnsembl/index.html.

The Histone Deacetylase Inhibitor Trichostatin A Promotes Totipotency in the Male Gametophyte

“…The RT-PCR primers are from Malik et al (2007). The quantitative RT-PCR primers for microarray validation were designed based on oligonucleotide probes from the Affymetrix GeneChip Brassica Exon 1.0ST Array (Malik et al, 2007;Love et al, 2010). The Arabidopsis hda T-DNA insertion lines were genotyped using the PCR primers shown in Supplemental Table 2.…”

Regulatory Hotspots Are Associated with Plant Gene Expression under Varying Soil Phosphorus Supply in Brassica rapa

“…Approaches that distinguish between paralogues within the genome, such as the use of high-density oligonucleotide arrays with exonspecific probes or RNAseq, will also increase the accuracy and depth of these analyses in the future (Trick et al, 2009b;Love et al, 2010). …”

“…Furthermore, extensive genetic and genomic resources for B. rapa have now been assembled. These include a rapid-cycling mapping population developed from highly inbred lines of rapid cycling (IMB211) and yellow sarson (R500) B. rapa (IniguezLuy et al, 2009), more than 2M Brassica GenBank sequences, and a B. rapa genome sequence made available in 2011 alongside other reference Brassica genome and resequencing projects (Multinational Brassica Genome Project; www.brassica.info/resource/sequencing.php), as well as oligonucleotide microarrays (Trick et al, 2009a;Love et al, 2010).…”

Genetical and Comparative Genomics of Brassica under Altered Ca Supply Identifies Arabidopsis Ca-Transporter Orthologs

“…This species can accumulate relatively high Ca concentrations in its leaf mesophyll tissues (Rios et al, 2012). Furthermore, B. rapa has a sequenced genome that enables comparative genomic analyses with Arabidopsis and postgenomic resources, including high-density exon-based expression arrays (Love et al, 2010), populations of mutants identified by targeting induced local lesions in genomes (TILLING) (Stephenson et al, 2010;Wang et al, 2012), and mapping lines (Iniguez-Luy et al, 2009). All of these resources were exploited in this study to test the potential of a Brassica-to-Arabidopsis-to-Brassica workflow.…”