Identification of Suitable Meloidogyne spp. Housekeeping Genes

Abstract

Gene expression studies often require reliable housekeeping (HK) genes to accurately capture gene expression levels under given conditions. This is especially true for root-knot nematodes (RKN, Meloidogyne spp.), whose drastic developmental changes are strongly dependent upon their environment. Here we utilized a publicly available M. hapla RNAseq database to identify putative HK genes throughout the nematode lifecycle. We then validated these candidate HK genes on M. incognita in order to develop a small library of suitable HK genes for RKN. Seven putative HK genes were selected for validation based on high expression level and ease of primer design. The expression of these genes was quantified by qPCR at different developmental stages to capture the entire life cycle of M. incognita which included eggs and naive infective juveniles through 3-wk post inoculation. Two algorithms, geNorm and Normfinder, identified three genes (Disu, Poly, and Skinase) constitutively and uniformly expressed throughout the entire life cycle of RKN. We believe these genes are superior HK genes suitable to be used as internal reference genes at all stages of RKN. Importantly, while we identified Actin, a commonly used HK gene, as a candidate gene within our RNAseq analyses, our qPCR results did not demonstrate stable expression throughout the nematode life cycle of this gene. This study successfully validated suitable HK genes utilizing both RNAseq data and standard qPCR methods across two species of RKN; suitable HK genes are likely applicable to other species of RKN, or even plant-parasitic nematodes. Additional lists of potential HK genes are also provided if the nematode of interest does not have homologues of the three superior reference genes described here. Gene expression studies on RKN should use validated HK genes to ensure accurate representation of transcript abundance.

The Centromere Histone Is Conserved and Associated with Tandem Repeats Sharing a Conserved 19-bp Box in the Holocentromere of Meloidogyne Nematodes

“…The exception was female stage where transcription of αCenH3 is decreased to the level of other CenH3 . To test the quality of analyzed M. incognita transcriptomes expression of the reference gene disulfide-isomerase ( Disu ) ( Hu and DiGennaro 2019 ) was performed. The results showed a comparable amount of Disu transcripts in all M. incognita developmental stages ( fig.…”

“…2017 ), and M. hapla (PRJEB14142) were used. The relative expressions of CenH3 genes and reference gene Disu ( Hu and DiGennaro 2019 ) were analyzed using Bowtie2 v.2.3.0 mapper ( Langmead and Salzberg 2012 ). Single-end reads were mapped with parameters -a and –very-sensitive for each transcriptome separately to CenH3 genes.…”

Satellitome analyses in nematodes illuminate complex species history and show conserved features in satellite DNAs

“…Satellites that showed either high expression (relative to GAPDH) or differential expression were singled out. Separate analysis of the expression of housekeeping genes was performed in M. incognita based on previously validated candidates [65]. Reproducibility of our analysis was tested by comparing satDNA expression on two RNA-seq data from M. incognita juvenile stage 2 using the tools Bowtie2 and BBMap v35.82 [66].…”

“…Table S4. Expression analyses of 4 house-keeping genes in M. incognita based on previously validated candidates [65]. For mapping of two juvenile (J2) transcriptome databases (ERR790027 and ERR790028), coding sequences (CDS) of reference genes downloaded from WormBase ParaSite (https:// paras ite.…”

Reference Genes Screening and Gene Expression Patterns Analysis Involved in Gelsenicine Biosynthesis under Different Hormone Treatments in Gelsemium elegans

“…Among these, GeNorm is used to rank potential reference genes by the mean pairwise change in expression of a potential reference gene compared to every other gene in the set, and to assess the variability between genes [15]. NormFinder compares candidate reference genes in pairs within and between groups, and BestKeeper uses an analytical procedure based on the mean cycle threshold (Ct) values of candidate reference genes [16][17][18]. The ∆CT method compares the relative expression levels of pairs of genes in each sample [19].…”