Abstract
Background Dendrocalamus latiflorus Munro distributes widely in subtropical areas and plays vital roles as valuable natural resources. The transcriptome sequencing for D. latiflorus Munro has been performed and numerous genes especially those predicted to be unique to D. latiflorus Munro were revealed. qRT-PCR has become a feasible approach to uncover gene expression profiling, and the accuracy and reliability of the results obtained depends upon the proper selection of stable reference genes for accurate normalization. Therefore, a set of suitable internal controls should be validated for D. latiflorus Munro.ResultsIn this report, twelve candidate reference genes were selected and the assessment of gene expression stability was performed in ten tissue samples and four leaf samples from seedlings and anther-regenerated plants of different ploidy. The PCR amplification efficiency was estimated, and the candidate genes were ranked according to their expression stability using three software packages: geNorm, NormFinder and Bestkeeper. GAPDH and EF1α were characterized to be the most stable genes among different tissues or in all the sample pools, while CYP showed low expression stability. RPL3 had the optimal performance among four leaf samples. The application of verified reference genes was illustrated by analyzing ferritin and laccase expression profiles among different experimental sets. The analysis revealed the biological variation in ferritin and laccase transcript expression among the tissues studied and the individual plants.ConclusionsgeNorm, NormFinder, and BestKeeper analyses recommended different suitable reference gene(s) for normalization according to the experimental sets. GAPDH and EF1α had the highest expression stability across different tissues and RPL3 for the other sample set. This study emphasizes the importance of validating superior reference genes for qRT-PCR analysis to accurately normalize gene expression of D. latiflorus Munro.
Highlights
Bamboos (Bambusoideae), a unique member of the monophyletic BEP clade (Bambusoideae, Ehrhartoideae, Pooideae) in grass family (Poaceae), comprise various woody and herbaceous varieties [1]
Ten reference genes were selected from D. latiflorus Munro transcriptome dataset and two other genes were attained using homology cloning techniques
The twelve genes mentioned above served as candidates for normalization of gene expression measures and their detailed descriptions are listed on Table 1, including the corresponding accession numbers, gene description and main functions. quantitative real-time polymerase chain reaction (qRT-PCR) was applied to determine the stability of gene expression by quantifying the mRNA level
Summary
Bamboos (Bambusoideae), a unique member of the monophyletic BEP clade (Bambusoideae, Ehrhartoideae, Pooideae) in grass family (Poaceae), comprise various woody and herbaceous varieties [1]. Dendrocalamus latiflorus Munro (D. latiflorus Munro), an evergreen species locally known as ‘tropical giant bamboo’, distributes widely in southern China and southeast Asia and plays vital roles as valuable natural resources [5]. These precious resources, as well as other bamboos, on which animals and human communities closely depend, are threatened, since up to half of the woody bamboo species in the world are in danger of extinction which is aggravated by two reproductive traits: semelparity and mast flowering with long intermast periods [5,6,7,8,9]. A set of suitable internal controls should be validated for D. latiflorus Munro
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