Dataset Information

Validation of reference genes for gene expression studies in tartary buckwheat (Fagopyrum tataricum Gaertn.) using quantitative real-time PCR.

ABSTRACT: Quantitative real-time reverse transcriptase polymerase chain reaction is a sensitive technique for quantifying gene expression levels. By implementing three distinct algorithms (geNorm, normFinder and BestKeeper), we have validated the stability of the expression of seven candidate reference genes in tartary buckwheat, including FtSAND, FtCACS, FtExpressed1, FtGAPDH, FtActin, FtEF-1a and FtH3. In this study, the results indicated that FtCACS and FtSAND were the best reference genes for 'abiotic cotyledons', FtExpressed1 and FtEF-1? were the best reference genes for aluminium treatment, FtCACS and FtExpressed1 performed the best for the immature seed stage, FtCACS was best for the abiotic treatment, and FtH3 appeared to be the most suitable reference gene for the abiotic treatment in hypocotyls and all samples in this study. In contrast, FtActin and FtGAPDH are unsuitable genes. Our findings offer additional stable reference genes for gene expression research on tartary buckwheat at the immature seed stage and under abiotic treatment.

SUBMITTER: Li C

PROVIDER: S-EPMC6396815 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Validation of reference genes for gene expression studies in tartary buckwheat (Fagopyrum tataricum Gaertn.) using quantitative real-time PCR.

Li Chenglei C Zhao Haixia H Li Maofei M Yao Panfeng P Li Qingqing Q Zhao Xuerong X Wang Anhu A Chen Hui H Tang Zizhong Z Bu Tongliang T Wu Qi Q

PeerJ 20190226

Quantitative real-time reverse transcriptase polymerase chain reaction is a sensitive technique for quantifying gene expression levels. By implementing three distinct algorithms (geNorm, normFinder and BestKeeper), we have validated the stability of the expression of seven candidate reference genes in tartary buckwheat, including FtSAND, FtCACS, FtExpressed1, FtGAPDH, FtActin, FtEF-1a and FtH3. In this study, the results indicated that FtCACS</ ...[more]

PMID: 30834187

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Project description:BackgroundTartary buckwheat (Fagopyrum tataricum Gaertn.) is a widely cultivated medicinal and edible crop with excellent economic and nutritional value. The development of tartary buckwheat seeds is a very complex process involving many expression-dependent physiological changes and regulation of a large number of genes and phytohormones. In recent years, the gene regulatory network governing the physiological changes occurring during seed development have received little attention.ResultsHere, we characterized the seed development of tartary buckwheat using light and electron microscopy and measured phytohormone and nutrient accumulation by using high performance liquid chromatography (HPLC) and by profiling the expression of key genes using RNA sequencing with the support of the tartary buckwheat genome. We first divided the development of tartary buckwheat seed into five stages that include complex changes in development, morphology, physiology and phytohormone levels. At the same time, the contents of phytohormones (gibberellin, indole-3-acetic acid, abscisic acid, and zeatin) and nutrients (rutin, starch, total proteins and soluble sugars) at five stages were determined, and their accumulation patterns in the development of tartary buckwheat seeds were analyzed. Second, gene expression patterns of tartary buckwheat samples were compared during three seed developmental stages (13, 19, and 25 days postanthesis, DPA), and 9 765 differentially expressed genes (DEGs) were identified. We analyzed the overlapping DEGs in different sample combinations and measured 665 DEGs in the three samples. Furthermore, expression patterns of DEGs related to phytohormones, flavonoids, starch, and storage proteins were analyzed. Third, we noted the correlation between the trait (physiological changes, nutrient changes) and metabolites during seed development, and discussed the key genes that might be involved in the synthesis and degradation of each of them.ConclusionWe provided abundant genomic resources for tartary buckwheat and Polygonaceae communities and revealed novel molecular insights into the correlations between the physiological changes and seed development of tartary buckwheat.

Project description:BackgroundTartary buckwheat is an important minor crop species with high nutritional and medicinal value and is widely planted worldwide. Cultivated Tartary buckwheat plants are tall and have hollow stems that lodge easily, which severely affects their yield and hinders the development of the Tartary buckwheat industry.MethodsHeifeng No. 1 seeds were treated with ethylmethanesulfonate (EMS) to generate a mutant library. The dwarf mutant ftdm was selected from the mutagenized population, and the agronomic characteristics giving rise to the dwarf phenotype were evaluated. Ultra-fast liquid chromatography-electrospray ionization tandem mass spectrometry (UFLC-ESI-MS/MS) was performed to determine the factors underlying the different phenotypes between the wild-type (WT) and ftdm plants. In addition, RNA sequencing (RNA-seq) was performed via the HiSeq 2000 platform, and the resulting transcriptomic data were analysed to identify differentially expressed genes (DEGs). Single-nucleotide polymorphism (SNP) variant analysis revealed possible sites associated with dwarfism. The expression levels of the potential DEGs between the WT and ftdm mutant were then measured via qRT-PCR and fragments per kilobase of transcript per million mapped reads (FPKM).ResultThe plant height (PH) of the ftdm mutant decreased to 42% of that of the WT, and compared with the WT, the mutant and had a higher breaking force (BF) and lower lodging index (LI). Lower GA4 and GA7 contents and higher contents of jasmonic acid (JA), salicylic acid (SA) and brassinolactone (BR) were detected in the stems of the ftdm mutant compared with the WT. Exogenous application of GAs could not revert the dwarfism of the ftdm mutant. On the basis of the transcriptomic analysis, 146 homozygous SNP loci were identified. In total, 12 DEGs with nonsynonymous mutations were ultimately identified, which were considered potential candidate genes related to the dwarf trait. When the sequences of eight genes whose expression was downregulated and four genes whose expression was upregulated were compared, SKIP14, an F-box protein whose sequence is 85% homologous to that of SLY1 in Arabidopsis, presented an amino acid change (from Ser to Asn) and was expressed at a lower level in the stems of the ftdm mutant compared with the WT. Hence, we speculated that this amino acid change in SKIP14 resulted in a disruption in GA signal transduction, indirectly decreasing the GA content and downregulating the expression of genes involved in GA biosynthesis or the GA response. Further studies are needed to determine the molecular basis underlying the dwarf phenotype of the ftdm mutant.ConclusionWe report a Tartary buckwheat EMS dwarf mutant, ftdm, suitable for high-density planting and commercial farming. A significant decrease in GA4 and GA7 levels was detected in the ftdm mutant, and 12 DEGs expressed in the stems of the ftdm mutant were selected as candidates of the dwarfing gene. One nonsynonymous mutation was detected in the SKIP14 gene in the ftdm mutant, and this gene had a lower transcript level compared with that in the WT.

Dataset Information

Validation of reference genes for gene expression studies in tartary buckwheat (Fagopyrum tataricum Gaertn.) using quantitative real-time PCR.

Publications

Validation of reference genes for gene expression studies in tartary buckwheat (<i>Fagopyrum tataricum</i> Gaertn.) using quantitative real-time PCR.

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