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Open Access Methodology article

Quantitative RT-PCR based platform for rapid quantification of the transcripts of highly homologous multigene families and their members during grain development

Agnieszka Kaczmarczyk1, Steve Bowra2, Zoltan Elek3 and Eva Vincze1*

Author affiliations

1 Department of Genetics and Biotechnology, Aarhus University, Research Centre Flakkebjerg, Forsøgsvej 1, Slagelse, DK-4200, Denmark

2 Verzyme (UK) Ltd., Plas Gogerddan, Aberystwyth, Wales, SY23 3EB, United Kingdom

3 MTA-ELTE-MTM Ecology Research Group, Biological Institute, Eötvös Loránd University, Pázmány Péter sétány 1C, Budapest, H-1117, Hungary

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Citation and License

BMC Plant Biology 2012, 12:184  doi:10.1186/1471-2229-12-184

Published: 9 October 2012

Abstract

Background

Cereal storage proteins represent one of the most important sources of protein for food and feed and they are coded by multigene families. The expression of the storage protein genes exhibits a temporal fluctuation but also a response to environmental stimuli. Analysis of temporal gene expression combined with genetic variation in large multigene families with high homology among the alleles is very challenging.

Results

We designed a rapid qRT-PCR system with the aim of characterising the variation in the expression of hordein genes families. All the known D-, C-, B-, and γ-hordein sequences coding full length open reading frames were collected from commonly available databases. Phylogenetic analysis was performed and the members of the different hordein families were classified into subfamilies. Primer sets were designed to discriminate the gene expression level of whole families, subfamilies or individual members. The specificity of the primer sets was validated before successfully applying them to a cDNA population derived from developing grains of field grown Hordeum vulgare cv. Barke. The results quantify the number of moles of transcript contributed to a particular gene family and its subgroups. More over the results indicate the genotypic specific gene expression.

Conclusions

Quantitative RT-PCR with SYBR Green labelling can be a useful technique to follow gene expression levels of large gene families with highly homologues members. We showed variation in the temporal expression of genes coding for barley storage proteins. The results imply that our rapid qRT-PCR system was sensitive enough to identify the presence of alleles and their expression profiles. It can be used to check the temporal fluctuations in hordein expressions or to find differences in their response to environmental stimuli. The method could be extended for cultivar recognition as some of the sequences from the database originated from cv. Golden Promise were not expressed in the studied barley cultivar Barke although showed primer specificity with their cloned DNA sequences.

Keywords:
SYBR Green; High homology multigene families; Transcript abundance; Hordeins