Selection and validation of reference genes for real-time qRT-PCR normalization in different tissues of Eucalyptus tereticornis

Abstract

Reference genes are generally used as endogenous normalization factor for relative quantification of target genes in quantitative real-time PCR (qRT-PCR). The present work aimed at identifying suitable reference genes for normalization of qRT-PCR data in tissues of Eucalyptus tereticornis. The expression levels of housekeeping genes like Actin (EtAct2), Isocitrate dehy - drogenase (EtIDH), ribosomal RNA (Et18s rRNA), SAND family protein (EtSAND), Histone protein (EtH2B), α-Tubulin (EtTUB), and eukaryotic initiation factor (EteIF4B) were studied to characterize their normalization stability in different tissues including young leaves, internodes, developing and mature xylem. The expression level of these genes was analyzed using different algorithms like geNorm, NormFinder and Best- Keeper. Among the seven reference genes analyzed, EtAct2 was expressed with less variance and was found to be the most stable reference gene across different tissues using all the three programs, while the least stable gene identified was EtH2B. Further, the normalization efficiency of the reference genes were assessed to predict the expression levels of three primary cell wall specific cellulose synthase transcripts (EtCesAs) in E. tereticornis tissues. The relative expression of EtCesA4, EtCesA5 and EtCesA6 was determined to be 3-19 fold higher in leaf and internode tissues when compared to developing and mature xylem tissues. This study will allow accurate normalization of qRT-PCR experiments across different tissues in E. tereticornis for future genomic research in this tropical eucalypt species.

Reference genes for Eucalyptus spp. under Beauveria bassiana inoculation and subsequently infestation by the galling wasp Leptocybe invasa

“…In other words, the genes that showed the greatest expression stability in the samples evaluated in each study were selected. Thus, the following genes were chosen: PP2A-1 ( Protein phosphatase 2A-2 ), SAND ( SAND family, trafficking protein Mon1 ), UPL7 ( Ubiquitin-protein ligase 7 ), PTB ( Polypyrimidine tract-binding protein 1 ) 38 ; Actin-2 ( Actin 2 ), 18srRNA ( RNA ribosomal 18S ), IDH ( NADP-isocitrate dehydrogenase ) 34 , α-TUB ( α-Tubulin ), UBC ( Ubiquitin C ), EF-1α ( Elongation factor 1-α ) 37 , EUC12 ( Putative RNA binding protein ), and H2B ( Histone H2B ) 36 .…”

“…The results obtained in the present study provides the most suitable reference genes for the normalization of RT-qPCR data in eucalyptus plants under the inoculation by B. bassiana and L. invasa infestation. Although several studies related to the selection of reference genes in eucalyptus have been performed so far 1 , 2 , 34 – 37 , this is the first time that such analysis is conducted in Eucalyptus ( E. tereticornis × E. camaldulensis hybrid) plants under B. bassiana inoculation and L. invasa infestation.…”

“…From this search, the selection of the reference genes followed the recommendation criteria of the article in which they were analyzed, that is, the genes that had the best results (greater expression stability in the sample set used) were prioritized. Following this approach, 12 different genes were selected: PP2A-1 , SAND , UPL7 , PTB 38 ; Actin-2 , 18srRNA , IDH 34 ; α-TUB , UBC , EF-1α 37 ; EUC12 , and H2B 36 .…”

“…Different studies aiming to determine the best eucalyptus reference genes have been performed 1 , 2 , 34 – 39 . However, such studies are still necessary when specific genotypes, tissues or stress conditions are under study 40 , 41 .…”