Reference genes for quantitative real-time PCR normalization of Cenostigma pyramidale roots under salt stress and mycorrhizal association.

Gabriella Frosi,João Pacífico Bezerra-Neto,Ana Maria Benko-Iseppon,Mauro Guida Santos,Ederson Akio Kido,Valesca Pandolfi,José Ribamar Costa Ferreira-Neto,David Anderson De Lima Morais,Laís Luana De Lima,Leonor Costa Maia

doi:10.1590/1678-4685-gmb-2020-0424

Gabriella Frosi, João Pacífico Bezerra-Neto + Show 8 more

Open Access

https://doi.org/10.1590/1678-4685-gmb-2020-0424

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Abstract

Cenostigma pyramidale is a native legume of the Brazilian semiarid region which performs symbiotic association with arbuscular mycorrhizal fungi (AMF), being an excellent model for studying genes associated with tolerance against abiotic and biotic stresses. In RT-qPCR approach, the use of reference genes is mandatory to avoid incorrect interpretation of the relative expression. This study evaluated the stability of ten candidate reference genes (CRGs) from C. pyramidale root tissues under salt stress (three collection times) and associated with AMF (three different times of salinity). The de novo transcriptome was obtained via RNA-Seq sequencing. Three algorithms were used to calculate the stability of CRGs under different conditions: (i) global (Salt, Salt+AMF, AMF and Control, and collection times), (ii) only non-inoculated plants, and (iii) AMF (only inoculated plants). HAG2, SAC1, aRP3 were the most stable CRGs for global and AMF assays, whereas HAG2, SAC1, RHS1 were the best for salt stress assay. This CRGs were used to validate the relative expression of two up-regulated transcripts in Salt2h (RAP2-3 and PIN8). Our study provides the first set of reference genes for C. pyramidale under salinity and AMF, supporting future researches on gene expression with this species.

Highlights

In the Brazilian semiarid region, there is a seasonally dry tropical forest called Caatinga
Ten transcripts including: Actin-Related Protein (ARP3); Suppressor of Actin (SAC1); F-Box Proteine (VFB1); LysyltRNA Synthetase (ATKRS-1); Tubulin (TUBB2); Serine/ threonine-proteinkinase (GRIK2); Transcription repressor (MYB4); Zinc finger protein (CpOXS2); Relat/SPOT homolog1 (RSH1) and Histone acetyltransferase (HAG2) were chosen as reference genes (RGs) candidate in RT- qPCR assays of C. pyramidale roots submitted to salinity and associated with arbuscular mycorrhizal fungi (AMF) (Table 1)
All primer pairs amplified a single PCR product with the expected size, as indicated by melting curves generated by RT-qPCR (Figure S1)

Summary

Introduction

In the Brazilian semiarid region, there is a seasonally dry tropical forest called Caatinga. Cenostigma pyramidale (Tul.) L.P. Queiroz (previously called Poincianella pyramidalis), popularly known as ‘Catingueira’, is a woody legume tree and figure as one of the most representative species in the Caatinga drylands. Other aspects of C. pyramidale have been studied, such as genetic diversity (Santos et al, 2012) and phenotypic plasticity on ecological succession gradients in the semiarid area (Falcão et al, 2015)⁠ Recent studies on this species revealed an association of arbuscular mycorrhizal fungi (AMFs) with most efficient physiological and biochemical responses under drought (Frosi et al, 2016) ⁠and salinity (Frosi et al, 2018)

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