Dataset Information

Development and Validation of a Gene-Targeted dCAPS Marker for Marker-Assisted Selection of Low-Alkaloid Content in Seeds of Narrow-Leafed Lupin (Lupinus angustifolius L.).

ABSTRACT: Low-alkaloid content is an important breeding target to improve the quality of lupin seeds. An APETALA2/ethylene response transcription factor, RAP2-7, is likely a candidate gene for the major alkaloid locus iucundus, and plays a crucial role in regulation of seed alkaloid content in narrow-leafed lupin (NLL; Lupinus angustifolius L.). Here, we exploited a single-nucleotide polymorphism within RAP2-7 credibly associated with seed alkaloid content, to develop the co-dominant derived cleaved amplified polymorphic sequence (dCAPS) marker iuc_RAP2-7. Marker validation in 202 NLL accessions demonstrated that seed alkaloid content ?0.9% of the seed dry weight was associated with the high-alkaloid marker band (Iucundus genotypes), whereas alkaloid content up to 0.5% of the seed dry weight was associated with the low-alkaloid marker band (iucundus genotypes). Within a given detection limit, iuc_RAP2-7 unambiguously identified all but three low-alkaloid accessions. The latter accessions apparently have a different regulatory mechanism for seed alkaloid content because the RAP2-7 gene/putative promoter sequence and expression of alkaloid-associated genes in the leaves of the three ambiguous accessions were similar to those of bitter Iucundus lines. We consider the iuc_RAP2-7 marker is a powerful tool that will facilitate NLL marker-assisted selection by rapid rejection of bitter Iucundus genotypes and thus accelerate development of new low-alkaloid cultivars.

SUBMITTER: Kroc M

PROVIDER: S-EPMC6628303 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Publications

Development and Validation of a Gene-Targeted dCAPS Marker for Marker-Assisted Selection of Low-Alkaloid Content in Seeds of Narrow-Leafed Lupin (Lupinus angustifolius L.).

Kroc Magdalena M Czepiel Katarzyna K Wilczura Paulina P Mokrzycka Monika M Święcicki Wojciech W

Genes 20190604 6

Low-alkaloid content is an important breeding target to improve the quality of lupin seeds. An APETALA2/ethylene response transcription factor, RAP2-7, is likely a candidate gene for the major alkaloid locus iucundus, and plays a crucial role in regulation of seed alkaloid content in narrow-leafed lupin (NLL; Lupinus angustifolius L.). Here, we exploited a single-nucleotide polymorphism within RAP2-7 credibly associated with seed alkaloid content, to develop th ...[more]

PMID: 31167507

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Project description: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.

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Dataset Information

Development and Validation of a Gene-Targeted dCAPS Marker for Marker-Assisted Selection of Low-Alkaloid Content in Seeds of Narrow-Leafed Lupin (Lupinus angustifolius L.).

Publications

Development and Validation of a Gene-Targeted dCAPS Marker for Marker-Assisted Selection of Low-Alkaloid Content in Seeds of Narrow-Leafed Lupin (<i>Lupinus angustifolius</i> L.).

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