RNA‐seq‐based selection of reference genes for RT‐qPCR analysis of pitaya

Nong, Quandong; Yang, Yongchao; Zhang, Mingyong; Zhang, Mei; Chen, Jiantong; Jian, Shuguang; Lü, Hongfang; Xia, Kuaifei

doi:10.1002/2211-5463.12678

Cited by 17 publications

(11 citation statements)

References 37 publications

(109 reference statements)

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“…The relative expression of each gene was analyzed using the comparative Ct (ΔΔCt) method with β-actin as a reference gene for normalization. The reference gene was selected according to previous studies [56]. Briefly, gene-specific primers were designed by Primer Premier 5 and checked by melting curve after amplification.…”

Section: Identification Of Differentially Expressed Mrnasmentioning

confidence: 99%

Small Auxin Up RNAs influence the distribution of indole-3-acetic acid and play a potential role in increasing seed size in Euryale ferox Salisb

et al. 2020

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Background: Aquatic Euryale ferox Salisb. is an economically important crop in China and India. Unfortunately, low yield limitations seriously hinder market growth. Unveiling the control of seed size is of remarkable importance in improvement of crops. Here, we generated a new hybrid line (HL) with larger seeds by crossing South Gordon Euryale and North Gordon Euryale (WT) which hasn't been reported before. However, the functional genes and molecular mechanisms controlling the seed size in Euryale ferox Salisb. remain unclear. In this study, we focused on the differentially expressed genes in the auxin signal transduction pathway during fruit development between HL and WT to explore candidate regulatory genes participated in regulating seed size. Results: Both concentration and localization of indole-3-acetic acid (IAA) at two growth stages of fruits of WT and HL were detected by LC-MS and immunofluorescence. Although IAA content between the two lines did not differ, IAA distribution was significantly different. To elucidate the mechanism and to seek the key genes underlying this difference, RNA-seq was performed on young fruits at the two selected growth stages, and differentially expressed genes related to the auxin transduction pathway were selected for further analysis. Conclusion: Hybrid Euryale ferox Salisb. expressed significant heterosis, resulting in non-prickly, thin-coated, large seeds, which accounted for the significantly larger yield of HL than that of WT. Our study indicated that Small Auxin Up RNAs (SAURs)-mediated localization of IAA regulates seed size in Euryale ferox Salisb. We found that some SAURs may act as a positive mediator of the auxin transduction pathway, thereby contributing to the observed heterosis.

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Section: Identification Of Differentially Expressed Mrnasmentioning

confidence: 99%

Small Auxin Up RNAs influence the distribution of indole-3-acetic acid and play a potential role in increasing seed size in Euryale ferox Salisb

et al. 2020

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“…However, an advantage of RNA-seq is that it does not require preliminary knowledge or assumptions about the genes of interest. Thus, it has been successfully used in transcriptomic studies (e.g., [85][86][87]). In some cases, RNA-seq has been applied to validate data [88,89].…”

Section: Discussionmentioning

confidence: 99%

Identification of Rf Genes in Hexaploid Wheat (Triticumaestivum L.) by RNA-Seq and Paralog Analyses

Tyrka

Bakera

Szeliga

et al. 2021

IJMS

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Among the natural mechanisms used for wheat hybrid breeding, the most desirable is the system combining the cytoplasmic male sterility (cms) of the female parent with the fertility-restoring genes (Rf) of the male parent. The objective of this study was to identify Rf candidate genes in the wheat genome on the basis of transcriptome sequencing (RNA-seq) and paralog analysis data. Total RNA was isolated from the anthers of two fertility-restorer (Primépi and Patras) and two non-restorer (Astoria and Grana) varieties at the tetrad and late uninucleate microspore stages. Of 36,912 differentially expressed genes (DEGs), 21 encoding domains in known fertility-restoring proteins were selected. To enrich the pool of Rf candidates, 52 paralogs (PAGs) of the 21 selected DEGs were included in the analyses. The expression profiles of most of the DEGs and PAGs determined bioinformatically were as expected (i.e., they were overexpressed in at least one fertility-restorer variety). However, these results were only partially consistent with the quantitative real-time PCR data. The DEG and PAG promoters included cis-regulatory elements common among PPR-encoding genes. On the basis of the obtained results, we designated seven genes as Rf candidate genes, six of which were identified for the first time in this study.

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“…Unstable reference genes can substantially affect the results of such analyses and can even lead to erroneous conclusions 38 . Primer amplification efficiency represents the amplicon doubling rate during the PCR process, which also affects the accuracy of qRT-PCR 16 . A good primer has an E value ranging from 90 to 105% 23 .…”

Section: Discussionmentioning

confidence: 99%

Comprehensive evaluation of candidate reference genes for quantitative real-time PCR-based analysis in Caucasian clover

Yin

et al. 2021

Sci Rep

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The forage species Caucasian clover (Trifolium ambiguum M. Bieb.), a groundcover plant, is resistant to both cold and drought. However, reference genes for qRT-PCR-based analysis of Caucasian clover are lacking. In this study, 12 reference genes were selected on the basis of transcriptomic data. These genes were used to determine the most stably expressed genes in various organs of Caucasian clover under cold, salt and drought stress for qRT-PCR-based analysis. Reference gene stability was analyzed by geNorm, NormFinder, BestKeeper, the ∆Ct method and RefFinder. Under salt stress, RCD1 and PPIL3 were the most stable reference genes in the leaves, and NLI1 and RCD1 were the most stable references genes in the roots. Under low-temperature stress, APA and EFTu-GTP were the most stable reference genes in the leaves, and the RCD1 and NLI2 genes were highly stable in the roots. Under 10% PEG-6000 stress, NLI1 and NLI2 were highly stable in the leaves, and RCD1 and PPIL3 were the most stable in the roots. Overall, RCD1 and NLI2 were the most stable reference genes in organs under normal conditions and across all samples. The most and least stable reference genes were validated by assessing their appropriateness for normalization via WRKY genes.

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RNA‐seq‐based selection of reference genes for RT‐qPCR analysis of pitaya

Cited by 17 publications

References 37 publications

Small Auxin Up RNAs influence the distribution of indole-3-acetic acid and play a potential role in increasing seed size in Euryale ferox Salisb

Small Auxin Up RNAs influence the distribution of indole-3-acetic acid and play a potential role in increasing seed size in Euryale ferox Salisb

Identification of Rf Genes in Hexaploid Wheat (Triticumaestivum L.) by RNA-Seq and Paralog Analyses

Comprehensive evaluation of candidate reference genes for quantitative real-time PCR-based analysis in Caucasian clover

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