Comprehensive Identification of Reliable Reference Genes for qRT-PCR Normalization of Fusarium oxysporum-Resistant Genes’ Expressions in Lilium sargentiae Wilson

Abstract

Fusarium wilt (caused by Fusarium oxysporum f. sp. Lilii) is one of the most damaging diseases in lily (Lilium sargentiae Wilson). Although some F. oxysporum-resistant lily varieties have been identified and are being utilized in resistant breeding, the regulation network of the resistance-associated mechanisms is yet to be studied due to the lack of reliable reference genes for qRT-PCR (quantitative reverse transcription PCR) normalization. The reliability of results by qRT-PCR relies mainly on the stability of the reference genes. This study investigated the reliability of nine candidate reference genes (CYP, EF1-α, GAPDH, TUB, UBQ, AQP, HIS, PGK, and RPL13) for qRT-PCR analysis of F. oxysporum-resistant genes. Expression stability analysis via common programs GeNorm, BestKeeper, and NormFinder, at different time points post-inoculation of F. oxysporum, revealed that all nine genes met the basic requirements of reference genes. Amongst them, HIS and GAPDH displayed the highest and the lowest expression stability, respectively. The reliability of HIS was further validated by analyzing the expression levels of four resistance-related candidate genes. The expression patterns of the four target genes were consistent with their responses to pathogenetic fungi in other plants. Our results show that HIS is the most suitable reference gene for accurately normalizing F. oxysporum-resistant genes’ expressions in L. sargentiae.

Identification and Validation of Reference Genes for qRT-PCR Analysis of Petal-Color-Related Genes in Rosa praelucens

“…The expression pattern of six petal-color-regulated genes, determined via qRT-PCR analysis, using EEF1A as reference gene for normalization, were consistent with the transcriptome sequencing result, confirming the reliability and suitability of applying it as the reference gene in R. praelucens . The selected new reference gene in this study, EEF1A , was different from those that had previously been screened and validated for the petal-color-related gene expression of either Asiatic hybrid lilies, i.e., TUB , [ 49 ], or Anemone obtusiloba , i.e., UBQ [ 50 ], which reconfirmed that suitable reference genes had to be detected for each species and for each experiment [ 28 , 30 , 31 ].…”