Dataset Information

Identifying Stable Reference Genes for qRT-PCR Normalisation in Gene Expression Studies of Narrow-Leafed Lupin (Lupinus angustifolius L.).

ABSTRACT: Quantitative Reverse Transcription PCR (qRT-PCR) is currently one of the most popular, high-throughput and sensitive technologies available for quantifying gene expression. Its accurate application depends heavily upon normalisation of gene-of-interest data with reference genes that are uniformly expressed under experimental conditions. The aim of this study was to provide the first validation of reference genes for Lupinus angustifolius (narrow-leafed lupin, a significant grain legume crop) using a selection of seven genes previously trialed as reference genes for the model legume, Medicago truncatula. In a preliminary evaluation, the seven candidate reference genes were assessed on the basis of primer specificity for their respective targeted region, PCR amplification efficiency, and ability to discriminate between cDNA and gDNA. Following this assessment, expression of the three most promising candidates [Ubiquitin C (UBC), Helicase (HEL), and Polypyrimidine tract-binding protein (PTB)] was evaluated using the NormFinder and RefFinder statistical algorithms in two narrow-leafed lupin lines, both with and without vernalisation treatment, and across seven organ types (cotyledons, stem, leaves, shoot apical meristem, flowers, pods and roots) encompassing three developmental stages. UBC was consistently identified as the most stable candidate and has sufficiently uniform expression that it may be used as a sole reference gene under the experimental conditions tested here. However, as organ type and developmental stage were associated with greater variability in relative expression, it is recommended using UBC and HEL as a pair to achieve optimal normalisation. These results highlight the importance of rigorously assessing candidate reference genes for each species across a diverse range of organs and developmental stages. With emerging technologies, such as RNAseq, and the completion of valuable transcriptome data sets, it is possible that other potentially more suitable reference genes will be identified for this species in future.

SUBMITTER: Taylor CM

PROVIDER: S-EPMC4752343 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Identifying Stable Reference Genes for qRT-PCR Normalisation in Gene Expression Studies of Narrow-Leafed Lupin (Lupinus angustifolius L.).

Taylor Candy M CM Jost Ricarda R Erskine William W Nelson Matthew N MN

PloS one 20160212 2

Quantitative Reverse Transcription PCR (qRT-PCR) is currently one of the most popular, high-throughput and sensitive technologies available for quantifying gene expression. Its accurate application depends heavily upon normalisation of gene-of-interest data with reference genes that are uniformly expressed under experimental conditions. The aim of this study was to provide the first validation of reference genes for Lupinus angustifolius (narrow-leafed lupin, a significant grain legume crop) usi ...[more]

PMID: 26872362

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Project description:Lupins, like other legumes, have a unique biosynthesis scheme of 5-deoxy-type flavonoids and isoflavonoids. A key enzyme in this pathway is chalcone isomerase (CHI), a member of CHI-fold protein family, encompassing subfamilies of CHI1, CHI2, CHI-like (CHIL), and fatty acid-binding (FAP) proteins. Here, two Lupinus angustifolius (narrow-leafed lupin) CHILs, LangCHIL1 and LangCHIL2, were identified and characterized using DNA fingerprinting, cytogenetic and linkage mapping, sequencing and expression profiling. Clones carrying CHIL sequences were assembled into two contigs. Full gene sequences were obtained from these contigs, and mapped in two L. angustifolius linkage groups by gene-specific markers. Bacterial artificial chromosome fluorescence in situ hybridization approach confirmed the localization of two LangCHIL genes in distinct chromosomes. The expression profiles of both LangCHIL isoforms were very similar. The highest level of transcription was in the roots of the third week of plant growth; thereafter, expression declined. The expression of both LangCHIL genes in leaves and stems was similar and low. Comparative mapping to reference legume genome sequences revealed strong syntenic links; however, LangCHIL2 contig had a much more conserved structure than LangCHIL1. LangCHIL2 is assumed to be an ancestor gene, whereas LangCHIL1 probably appeared as a result of duplication. As both copies are transcriptionally active, questions arise concerning their hypothetical functional divergence. Screening of the narrow-leafed lupin genome and transcriptome with CHI-fold protein sequences, followed by Bayesian inference of phylogeny and cross-genera synteny survey, identified representatives of all but one (CHI1) main subfamilies. They are as follows: two copies of CHI2, FAPa2 and CHIL, and single copies of FAPb and FAPa1. Duplicated genes are remnants of whole genome duplication which is assumed to have occurred after the divergence of Lupinus, Arachis, and Glycine.

Project description:Narrow-leafed lupin (Lupinus angustifolius L.) is a moderate-yielding legume crop known for its high grain protein content and contribution to soil improvement. It is cultivated under photoperiods ranging from 9 to 17 h, as a spring-sown (in colder locations) or as an autumn-sown crop (in warmer regions). Wild populations require a prolonged cold period, called vernalization, to induce flowering. The key achievement of L. angustifolius domestication was the discovery of two natural mutations (named Ku and Jul) conferring vernalization independence. These mutations are overlapping deletion variants in the promoter of LanFTc1, a homolog of the Arabidopsis thaliana FLOWERING LOCUS T (FT) gene. The third deletion, named here as Pal, was recently found in primitive germplasm. In this study, we genotyped L. angustifolius germplasm that differs in domestication status and geographical origin for LanFTc1 alleles, which we then phenotyped to establish flowering time and vernalization responsiveness. The Ku and Jul lines were vernalization-independent and early flowering, wild (ku) lines were vernalization-dependent and late flowering, whereas the Pal line conferred intermediate phenotype. Three lines representing ku, Pal, and Ku alleles were subjected to gene expression surveys under 8- and 16-h photoperiods. FT homologs (LanFTa1, LanFTa2, LanFTc1, and LanFTc2) and some genes selected by recent expression quantitative trait loci mapping were analyzed. Expression profiles of LanFTc1 and LanAGL8 (AGAMOUS-like 8) matched observed differences in flowering time between genotypes, highlighted by high induction after vernalization in the ku line. Moreover, these genes revealed altered circadian clock control in Pal line under short days. LanFD (FD) and LanCRLK1 (CALCIUM/CALMODULIN-REGULATED RECEPTOR-LIKE KINASE 1) were negatively responsive to vernalization in Ku and Pal lines but positively responsive or variable in ku, whereas LanUGT85A2 (UDP-GLUCOSYL TRANSFERASE 85A2) was significantly suppressed by vernalization in all lines. Such a pattern suggests the opposite regulation of these gene pairs in the vernalization pathway. LanCRLK1 and LanUGT85A2 are homologs of A. thaliana genes involved in the FLOWERING LOCUS C (FLC) vernalization pathway. Lupins, like many other legumes, do not have any FLC homologs. Therefore, candidate genes surveyed in this study, namely LanFTc1, LanAGL8, LanCRLK1, and LanUGT85A2, may constitute anchors for further elucidation of molecular components contributing to vernalization response in legumes.

Dataset Information

Identifying Stable Reference Genes for qRT-PCR Normalisation in Gene Expression Studies of Narrow-Leafed Lupin (Lupinus angustifolius L.).

Publications

Identifying Stable Reference Genes for qRT-PCR Normalisation in Gene Expression Studies of Narrow-Leafed Lupin (Lupinus angustifolius L.).

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