Identification of suitable reference genes by quantitative real-time PCR for gene expression normalization in sunflower

Abstract

Quantitative real-time PCR (qPCR) is currently the most accurate method for detecting differential gene expression, but depends greatly on normalization with stably expressed housekeeping genes. Transcriptomics analyses and experimental validation in different plant species have shown that the reliability of these endogenous controls can be influenced by, growth conditions and organs/tissues examined. Thus, reliable validation of reference genes is required to ensure proper normalization. This paper presents a systematic comparison of ten potential reference genes in sunflower: five commonly used genes (Actin, Elongation Factor1, Plastid-encode RNA polymerase, Tubulin, and Ubiquitin, as ACT, EF1, PEP, TUB, and UBQ respectively), as well as five new candidates (Translation initiation factor, MicroRNA precursors 171 and 156, Ask-interacting protein, and Protein of unknown function, as ETIF5, MIR171, MIR156, SKIP, and UNK2 respectively). Reference gene expression stability was examined by qPCR across 20 biological samples, representing different tissues at various developmental stages. Expression of all 10 genes was variable to some extent, but that of ACT, UNK2, and EF1 was overall the most stable. A combination of ETIF5/UNK2/EF1 would be appropriate to use as a reference panel for normalizing gene expression data among vegetative tissues, whereas the combination of ACT/MIR156/UNK2 is most suitable for reproductive tissues. Reference genes selected in this study were further validated by examining relative expression of ahas1, one of three acetohydroxyacid synthase genes of sunflower. Our identification and validation of suitable normalizer genes will be of use to ensure accurate results in future transcriptomics studies in this crop.

Identification and Functional Characterization of Genes Involved in the Biosynthesis of Caffeoylquinic Acids in Sunflower (Helianthus annuus L.)

“…Then, qRT-PCR was performed using gene-specific primers (Supplementary Table S1). Eukaryotic translation initiation factor 5A ( ETIF5A ; XM_022156448.1), elongation factor 2 ( EF2 ; XM_022137686.1), and actin 7 ( ACT7 ; XM_022154554.1) of sunflower were used as reference genes (Ochogavía et al, 2017). Reactions were conducted in volumes of 10 μl in a 96-well PCR plate using Luna® universal qPCR master mix (New England Biolabs®, USA).…”

Partial Male Sterility in Imisun Sunflower: Imazapyr Treatment in Advanced Vegetative Stages Decreases Pollen Yield and Alters ahas Gene Expression

“…The oligonucleotides sequences of the ahas genes have been previously designed and assayed by our group and published in Breccia et al (2013). The reference genes has also been selected and validated by our research group (ACT, MIR156, and UNK2) and were published as the optimal reference panel for reproductive tissue expression normalization of H. annuus (Ochogavía et al, 2017). The primers and amplification conditions are available in Ochogavía et al (2017).…”

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

“…species, such as rice [14], rubber tree [15], sunflower [16], black locust [17], grapevine [18], and annual ryegrass [19]. species, such as rice [14], rubber tree [15], sunflower [16], black locust [17], grapevine [18], and annual ryegrass [19].…”