Genomic sequencing of Thinopyrum elongatum chromosome arm 7EL, carrying fusarium head blight resistance, and characterization of its impact on the transcriptome of the introgressed line CS-7EL.

BACKGROUND: The tall wheatgrass species Thinopyrum elongatum carries a strong fusarium head blight (FHB) resistance locus located on the long arm of chromosome 7 (7EL) as well as resistance to leaf and stem rusts, all diseases with a significant impact on wheat production. Towards understanding the contribution of Th. elongatum 7EL to improvement of disease resistance in wheat, the genomic sequence of the 7EL fragment present in the wheat Chinese Spring (CS) telosomic addition line CS-7EL was determined and the contribution and impact of 7EL on the rachis transcriptome during FHB infection was compared between CS and CS-7EL. RESULTS: We assembled the Th. elongatum 7EL chromosome arm using a reference-guided approach. Combining this assembly with the available reference sequence for CS hexaploid wheat provided a reliable reference for interrogating the transcriptomic differences in response to infection conferred by the 7EL fragment. Comparison of the transcriptomes of rachis tissues from CS and CS-7EL showed expression of Th. elongatum transcripts as well as modulation of wheat transcript expression profiles in the CS-7EL line. Expression profiles at 4 days after infection with Fusarium graminearum, the causal agent of FHB, showed an increased in expression of genes associated with an effective defense response, in particular glucan endo-1,3-beta-glucosidases and chitinases, in the FHB-resistant line CS-7EL while there was a larger increase in differential expression for genes associated with the level of fungal infection in the FHB-susceptible line CS. One hundred and seven 7EL transcripts were expressed in the smallest 7EL region defined to carry FHB resistance. CONCLUSION: 7EL contributed to CS-7EL transcriptome by direct expression and through alteration of wheat transcript profiles. FHB resistance in CS-7EL was associated with transcriptome changes suggesting a more effective defense response. A list of candidate genes for the FHB resistance locus on 7EL has been established.

Genome-Wide Identification and Characterization of RNA/DNA Differences Associated with Fusarium graminearum Infection in Wheat.

RNA/DNA difference (RDD) is a post-transcriptional modification playing a crucial role in regulating diverse biological processes in eukaryotes. Although it has been extensively studied in plant chloroplast and mitochondria genomes, RDDs in plant nuclear genomes are not well studied at present. Here, we investigated the RDDs associated with fusarium head blight (FHB) through a novel method by comparing the RNA-seq data between Fusarium-infected and control samples of four wheat genotypes. A total of 187 high-confidence unique RDDs in 36 genes were identified, representing the first landscape of the FHB-responsive RDD in wheat. The majority (26) of these 36 RDD genes were correlated either positively or negatively with FHB levels. Effects of these RDDs on RNA and protein sequences have been identified, their editing frequency and the expression level of the corresponding genes provided, and the prediction of the effect on the minimum folding free energy of mRNA, miRNA binding, and colocation of RDDs with conserved domains presented. RDDs were predicted to induce modifications in the mRNA and protein structures of the corresponding genes. In two genes, TraesCS1B02G294300 and TraesCS3A02G263900, editing was predicted to enhance their affinity with tae-miR9661-5p and tae-miR9664-3p, respectively. To our knowledge, this study is the first report of the association between RDD and FHB in wheat; this will contribute to a better understanding of the molecular basis underlying FHB resistance, and potentially lead to novel strategies to improve wheat FHB resistance through epigenetic methods.

Transfer of fusarium head blight resistance from Thinopyrum elongatum to bread wheat cultivar Chinese Spring.

The diploid form of tall wheatgrass, Thinopyrum elongatum (Host) D.R. Dewey (2n = 2x = 14, EE genome), has a high level of resistance to fusarium head blight. The symptoms did not spread beyond the inoculated florets following point inoculation. Using a series of E-genome chromosome additions in a bread wheat cultivar Chinese Spring (CS) background, the resistance was found to be localized to the long arm of chromosome 7E. The CS mutant ph1b was used to induce recombination between chromosome 7E, present in the 7E(7D) substitution and homoeologous wheat chromosomes. Multivalent chromosome associations were detected in the BC1 hybrids, confirming the effectiveness of the ph1b mutant. Genetic markers specific for chromosome 7E were used to estimate the size of the 7E introgression in the wheat genome. Using single sequence repeat (SSR) markers specific for homoeologous wheat chromosome 7, introgressions were detected on wheat chromosomes 7A, 7B, and 7D. Some of the introgression lines were resistant to fusarium head blight.

Introgression of Thinopyrum elongatum DNA fragments carrying resistance to fusarium head blight into Triticum aestivum cultivar Chinese Spring is associated with alteration of gene expression.

The tall wheatgrass species Thinopyrum elongatum carries on the long arm of chromosome 7E, a locus that contributes strongly to resistance to fusarium head blight (FHB), a devastating fungal disease affecting wheat crops in all temperate areas of the world. Introgression of Th. elongatum 7E chromatin into chromosome 7D of wheat was induced by the ph1b mutant of CS. Recombinants between chromosome 7E and wheat chromosome 7D, induced by the ph1b mutation, were monitored by a combination of molecular markers and phenotyping for FHB resistance. Progeny of up to five subsequent generations derived from two lineages, 64-8 and 32-5, were phenotyped for FHB symptoms and genotyped using published and novel 7D- and 7E-specific markers. Fragments from the distal end of 7EL, still carrying FHB resistance and estimated to be less than 114 and 66 Mbp, were identified as introgressed into wheat chromosome arm 7DL of progeny derived from 64-8 and 32-5, respectively. Gene expression analysis revealed variation in the expression levels of genes from the distal ends of 7EL and 7DL in the introgressed progeny. The 7EL introgressed material will facilitate the use of the 7EL FHB resistance locus in wheat breeding programs.

Genome Editing of a Deoxynivalenol-Induced Transcription Factor Confers Resistance to Fusarium graminearum in Wheat.

Deoxynivalenol (DON) is a mycotoxin virulence factor that promotes growth of the Fusarium graminearum fungus in wheat floral tissues. To further our understanding of the effects of DON exposure on plant cell function, we characterized DON-induced transcriptional changes in wheat spikelets. Four hundred wheat genes were differentially expressed during infection with wild-type F. graminearum as compared with a Δtri5 mutant strain that is unable to produce DON. Most of these genes were more induced by the DON-producing strain and included genes involved in secondary metabolism, signaling, transport, and stress responses. DON induction was confirmed for a subset of the genes, including TaNFXL1, by treating tissues with DON directly. Previous work indicates that the NFXL1 ortholog represses trichothecene-induced defense responses and bacterial resistance in Arabidopsis, but the role of the NFXL family has not been studied in wheat. We observed greater DON-induced TaNFXL1 gene expression in a susceptible wheat genotype relative to the F. graminearum-resistant genotype Wuhan 1. Functional testing using both virus-induced gene silencing and CRISPR-mediated genome editing indicated that TaNFXL1 represses F. graminearum resistance. Together, this suggests that targeting the TaNFXL1 gene may help to develop disease resistance in cultivated wheat.

Transcriptional reference map of hormone responses in wheat spikes.

BACKGROUND: Phytohormones are key regulators of plant growth, development, and signalling networks involved in responses to diverse biotic and abiotic stresses. Transcriptional reference maps of hormone responses have been reported for several model plant species such as Arabidopsis thaliana, Oryza sativa, and Brachypodium distachyon. However, because of species differences and the complexity of the wheat genome, these transcriptome data are not appropriate reference material for wheat studies. RESULTS: We comprehensively analysed the transcriptomic responses in wheat spikes to seven phytohormones, including indole acetic acid (IAA), gibberellic acid (GA), abscisic acid (ABA), ethylene (ET), cytokinin (CK), salicylic acid (SA), and methyl jasmonic acid (MeJA). A total of 3386 genes were differentially expressed at 24 h after the hormone treatments. Furthermore, 22.7% of these genes exhibited overlapping transcriptional responses for at least two hormones, implying there is crosstalk among phytohormones. We subsequently identified genes with expression levels that were significantly and differentially induced by a specific phytohormone (i.e., hormone-specific responses). The data for these hormone-responsive genes were then compared with the transcriptome data for wheat spikes exposed to biotic (Fusarium head blight) and abiotic (water deficit) stresses. CONCLUSION: Our data were used to develop a transcriptional reference map of hormone responses in wheat spikes.

Characterization of QTL and eQTL controlling early Fusarium graminearum infection and deoxynivalenol levels in a Wuhan 1 x Nyubai doubled haploid wheat population.

BACKGROUND: Fusarium head blight (FHB) is a major disease of cereal crops, caused by the fungal pathogen Fusarium graminearum and related species. Breeding wheat for FHB resistance contributes to increase yields and grain quality and to reduce the use of fungicides. The identification of genes and markers for FHB resistance in different wheat genotypes has nevertheless proven challenging. RESULTS: In this study, early infection by F. graminearum was analyzed in a doubled haploid population derived from the cross of the moderately resistant wheat genotypes Wuhan 1 and Nyubai. Three quantitative trait loci (QTL) were identified: 1AL was associated with lower deoxynivalenol content, and 4BS and 5A were associated with reduced F. graminearum infection at 2 days post inoculation. Early resistance alleles were inherited from Wuhan 1 for QTL 1AL and 4BS and inherited from Nyubai for the 5A QTL. Cis and trans expression QTL (eQTL) were identified using RNA-seq data from infected head samples. Hotspots for trans eQTL were identified in the vicinity of the 1AL and 4BS QTL peaks. Among differentially expressed genes with cis eQTL within the QTL support intervals, nine genes had higher expression associated with FHB early resistance, and four genes had higher expression associated with FHB early susceptibility. CONCLUSIONS: Our analysis of genotype and gene expression data of wheat infected by F. graminearum identified three QTL associated with FHB early resistance, and linked genes with eQTL and differential expression patterns to those QTL. These findings may have applications in breeding wheat for early resistance to FHB.

Correction to: Identification and mapping of expressed genes associated with the 2DL QTL for fusarium head blight resistance in the wheat line Wuhan 1.

Following publication of the original article [1], we have been notified that some important information was omitted by the authors in the Copyright note. The Copyright note should read as below.

Identification and mapping of expressed genes associated with the 2DL QTL for fusarium head blight resistance in the wheat line Wuhan 1.

BACKGROUND: Fusarium head blight (FHB) is a problem of great concern in small grain cereals, especially wheat. A quantitative trait locus (QTL) for FHB resistance (FHB_SFI) located on the long arm of chromosome 2D in the spring wheat genotype Wuhan 1 is a resistance locus which has potential to improve the FHB resistance of bread wheat since it confers effective resistance to wheat breeding lines. Recently, differentially expressed genes (DEG) have been identified by comparing near isogenic lines (NIL) carrying the susceptible and resistant alleles for the 2DL QTL, using RNA-Seq. In the present study, we aimed to identify candidate genes located within the genetic interval for the 2DL QTL for FHB resistance, as assessed by single floret inoculation (FHB_SFI), and possibly contributing to it. RESULTS: Combining previous and additional bioinformatics analyses, 26 DEG that were located on chromosome arm 2DL were selected for further characterization of their expression profile by RT-qPCR. Seven of those DEG showed a consistent differential expression profile between either three pairs of near isogenic lines or other genotypes carrying the R and S alleles for the 2DL QTL for FHB resistance. UN25696, which was identified in previous expression work using microarray was also confirmed to have a differential expression pattern. Those eight candidate genes were further characterized in 85 lines of a double haploid mapping population derived from the cross Wuhan 1/Nyubai, the population where the 2DL QTL was originally identified. The expression QTL for gene Traes_2DL_179570792 overlapped completely with the mapping interval for the 2DL QTL for FHB_SFI while the expression QTL for UN25696 mapped near the QTL, but did not overlap with it. None of the other genes had a significant eQTL on chromosome 2DL. Higher expression of Traes_2DL_179570792 and UN25696 was associated with the resistant allele at that locus. CONCLUSIONS: Of the 26 DEG from the 2DL chromosome further characterized in this study, only two had an expression QTL located in or near the interval for the 2DL QTL. Traes_2DL_179570792 is the first expression marker identified as associated with the 2DL QTL.

Transcriptome dynamics associated with resistance and susceptibility against fusarium head blight in four wheat genotypes.

BACKGROUND: Fusarium head blight (FHB) of wheat in North America is caused mostly by the fungal pathogen Fusarium graminearum (Fg). Upon exposure to Fg, wheat initiates a series of cellular responses involving massive transcriptional reprogramming. In this study, we analyzed transcriptomics data of four wheat genotypes (Nyubai, Wuhan 1, HC374, and Shaw), at 2 and 4 days post inoculation (dpi) with Fg, using RNA-seq technology. RESULTS: A total of 37,772 differentially expressed genes (DEGs) were identified, 28,961 from wheat and 8811 from the pathogen. The susceptible genotype Shaw exhibited the highest number of host and pathogen DEGs, including 2270 DEGs associating with FHB susceptibility. Protein serine/threonine kinases and LRR-RK were associated with susceptibility at 2 dpi, while several ethylene-responsive, WRKY, Myb, bZIP and NAC-domain containing transcription factors were associated with susceptibility at 4 dpi. In the three resistant genotypes, 220 DEGs were associated with resistance. Glutathione S-transferase (GST), membrane proteins and distinct LRR-RKs were associated with FHB resistance across the three genotypes. Genes with unique, high up-regulation by Fg in Wuhan 1 were mostly transiently expressed at 2 dpi, while many defense-associated genes were up-regulated at both 2 and 4 dpi in Nyubai; the majority of unique genes up-regulated in HC374 were detected at 4 dpi only. In the pathogen, most genes showed increased expression between 2 and 4 dpi in all genotypes, with stronger levels in the susceptible host; however two pectate lyases and a hydrolase were expressed higher at 2 dpi, and acetyltransferase activity was highly enriched at 4 dpi. CONCLUSIONS: There was an early up-regulation of LRR-RKs, different between susceptible and resistant genotypes; subsequently, distinct sets of genes associated with defense response were up-regulated. Differences in expression profiles among the resistant genotypes indicate genotype-specific defense mechanisms. This study also shows a greater resemblance in transcriptomics of HC374 to Nyubai, consistent with their sharing of two FHB resistance QTLs on 3BS and 5AS, compared to Wuhan 1 which carries one QTL on 2DL in common with HC374.

Multiple metabolic pathways for metabolism of l-tryptophan in Fusarium graminearum.

Fusarium graminearum is a plant pathogen that can cause the devastating cereal grain disease fusarium head blight in temperate regions of the world. Previous studies have shown that F. graminearum can synthetize indole-3-acetic acid (auxin) using l-tryptophan (L-TRP)-dependent pathways. In the present study, we have taken a broader approach to examine the metabolism of L-TRP in F. graminearum liquid culture. Our results showed that F. graminearum was able to transiently produce the indole tryptophol when supplied with L-TRP. Comparative gene expression profiling between L-TRP-treated and control cultures showed that L-TRP treatment induced the upregulation of a series of genes with predicted function in the metabolism of L-TRP via anthranilic acid and catechol towards the tricarboxylic acid cycle. It is proposed that this metabolic activity provides extra energy for 15-acetyldeoxynivalenol production, as observed in our experiments. This is the first report of the use of L-TRP to increase energy resources in a Fusarium species.

Leucine metabolism regulates TRI6 expression and affects deoxynivalenol production and virulence in Fusarium graminearum.

TRI6 is a positive regulator of the trichothecene gene cluster and the production of trichothecene mycotoxins [deoxynivalenol (DON)] and acetylated forms such as 15-Acetyl-DON) in the cereal pathogen Fusarium graminearum. As a global transcriptional regulator, TRI6 expression is modulated by nitrogen-limiting conditions, sources of nitrogen and carbon, pH and light. However, the mechanism by which these diverse environmental factors affect TRI6 expression remains underexplored. In our effort to understand how nutrients affect TRI6 regulation, comparative digital expression profiling was performed with a wild-type F. graminearum and a Δtri6 mutant strain, grown in nutrient-rich conditions. Analysis showed that TRI6 negatively regulates genes of the branched-chain amino acid (BCAA) metabolic pathway. Feeding studies with deletion mutants of MCC, encoding methylcrotonyl-CoA-carboxylase, one of the key enzymes of leucine metabolism, showed that addition of leucine specifically down-regulated TRI6 expression and reduced 15-ADON accumulation. Constitutive expression of TRI6 in the Δmcc mutant strain restored 15-ADON production. A combination of cellophane breach assays and pathogenicity experiments on wheat demonstrated that disrupting the leucine metabolic pathway significantly reduced disease. These findings suggest a complex interaction between one of the primary metabolic pathways with a global regulator of mycotoxin biosynthesis and virulence in F. graminearum.

Candidate Genes Expressed in Tolerant Common Wheat With Resistant to English Grain Aphid.

The English grain aphid, Sitobion avenae (F.) (Hemiptera: Aphididae), is a common worldwide pest of wheat (Triticum aestivum L.). The use of improved resistant cultivars by the farmers is the most effective and environmentally friendly method to control this aphid in the field. The winter wheat genotypes 98-10-35 and Amigo are resistant to S. avenae. To identify genes responsible for resistance to S. avenae in these genotypes, differential-display reverse transcription-polymerase chain reaction was used to identify the corresponding differentially expressed sequences in current study. Two backcross progenies were obtained by crossing the two resistant genotypes with the susceptible genotype 1376. Six potential expected-differential bands were sequenced. Lengths of the expressed sequence tags ranged from 128 to 532 bp. Although these expressed sequences were likely associated with S. avenae resistance, there was one expressed sequence tag located on 7DL chromosome, and its potential function may associate with the ability to maintain photosynthesis in wheat. That serves as an active way for tolerant common wheat with resistant to S. avenae. Cloning the full length of these sequences would help us thoroughly understand the mechanism of wheat resistance to S. avenae and be valuable for breeding cultivars with S. avenae resistance.

The molecular diversity of α-gliadin genes in the tribe Triticeae.

Many of the unique properties of wheat flour are derived from seed storage proteins such as the α-gliadins. In this study these α-gliadin genes from diploid Triticeae species were systemically characterized, and divided into 3 classes according to the distinct organization of their protein domains. Our analyses indicated that these α-gliadins varied in the number of cysteine residues they contained. Most of the α-gliadin genes were grouped according to their genomic origins within the phylogenetic tree. As expected, sequence alignments suggested that the repetitive domain and the two polyglutamine regions were responsible for length variations of α-gliadins as were the insertion/deletion of structural domains within the three different classes (I, II, and III) of α-gliadins. A screening of celiac disease toxic epitopes indicated that the α-gliadins of the class II, derived from the Ns genome, contain no epitope, and that some other genomes contain much fewer epitopes than the A, S(B) and D genomes of wheat. Our results suggest that the observed genetic differences in α-gliadins of Triticeae might indicate their use as a fertile ground for the breeding of less CD-toxic wheat varieties.

Deoxynivalenol accumulation and detoxification in cereals and its potential role in wheat-Fusarium graminearum interactions.

Deoxynivalenol (DON) is a prominent mycotoxin showing significant accumulation in cereal plants during infection by the phytopathogen Fusarium graminearum. It is a virulence factor that is important in the spread of F. graminearum within cereal heads, and it causes serious yield losses and significant contamination of cereal grains. In recent decades, genetic and genomic studies have facilitated the characterization of the molecular pathways of DON biosynthesis in F. graminearum and the environmental factors that influence DON accumulation. In addition, diverse scab resistance traits related to the repression of DON accumulation in plants have been identified, and experimental studies of wheat-pathogen interactions have contributed to understanding detoxification mechanisms in host plants. The present review illustrates and summarizes the molecular networks of DON mycotoxin production in F. graminearum and the methods of DON detoxification in plants based on the current literature, which provides molecular targets for crop improvement programs. This review also comprehensively discusses recent advances and challenges related to genetic engineering-mediated cultivar improvements to strengthen scab resistance. Furthermore, ongoing advancements in genetic engineering will enable the application of these molecular targets to develop more scab-resistant wheat cultivars with DON detoxification traits.

RNA-Seq Data Processing in Plant-Pathogen Interaction System: A Case Study.

In RNA-seq data processing, short reads are usually aligned from one species against its own genome sequence; however, in plant-pathogen interaction systems, reads from both host and pathogen samples are blended together. In contrast with single-genome analyses, both pathogen and host reference genomes are involved in the alignment process. In such circumstances, the order in which the alignment is carried out, whether the host or pathogen is aligned first, or if both genomes are aligned simultaneously, influences the read counts of certain genes. This is a problem, especially at advanced infection stages. It is crucial to have an appropriate strategy for aligning the reads to their respective genomes, yet the existing strategies of either sequential or parallel alignment become problematic when mapping mixed reads to their corresponding reference genomes. The challenge lies in the determination of which reads belong to which species, especially when homology exists between the host and pathogen genomes. This chapter proposes a combo-genome alignment strategy, which was compared with existing alignment scenarios. Simulation results demonstrated that the degree of discrepancy in the results is correlated with phylogenetic distance of the two species in the mixture which was attributable to the extent of homology between the two genomes involved. This correlation was also found in the analysis using two real RNA-seq datasets of Fusarium-challenged wheat plants. Comparisons of the three RNA-seq processing strategies on three simulation datasets and two real Fusarium-infected wheat datasets showed that an alignment to a combo-genome, consisting of both host and pathogen genomes, improves mapping quality as compared to sequential alignment procedures.

Differential Expression Feature Extraction (DEFE): A Case Study in Wheat FHB RNA-Seq Data Analysis.

In differential gene expression data analysis, one objective is to identify groups of co-expressed genes from a large dataset in order to detect the association between such a group of genes and an experimental condition. This is often done through a clustering approach, such as k-means or bipartition hierarchical clustering, based on particular similarity measures in the grouping process. In such a dataset, the gene differential expression itself is an innate attribute that can be used in the feature extraction process. For example, in a dataset consisting of multiple treatments versus their controls, the expression of a gene in each treatment would have three possible behaviors, upregulated, downregulated, or unchanged. We present in this chapter, a differential expression feature extraction (DEFE) method by using a string consisting of three numerical values at each character to denote such behavior, i.e., 1 = up, 2 = down, and 0 = unchanged, which results in up to 3B differential expression patterns across all B comparisons. This approach has been successfully applied in many research projects, and among these, we demonstrate the strength of DEFE in a case study on RNA-sequencing (RNA-seq) data analysis of wheat challenged with the phytopathogenic fungus, Fusarium graminearum. Combinations of multiple schemes of DEFE patterns revealed groups of genes putatively associated with resistance or susceptibility to FHB.

Identification of novel α-gliadin genes.

Ten novel α-gliadin genes (Gli-ta, Gli-turg1, Gli-turg2, Gli-turg3, Gli-turg4, Gli-turg5, Gli-turg6, Gli-cs1, Gli-cs2, and Gli-cs3) with unique characteristics were isolated from wheat (Triticum aestivumL.), among which Gli-cs1, Gli-cs2, Gli-cs3, and Gli-turg6 were pseudogenes. Gli-cs3 and nine other sequences were much larger and smaller, respectively, than the typical α-gliadins. This variation was caused by insertion or deletion of the unique domain I and a polyglutamine region, possibly the result of illegitimate recombination. Consequently, Gli-cs3 contained 10 cysteine residues, whereas there were 2 cysteine residues only in the other nine sequences. Gli-ta/Gli-ta-like α-gliadin genes are normally expressed during the development of seeds. SDS-PAGE analysis showed that in-vitro-expressed Gli-ta could form intermolecular disulphide bonds and could be chain extenders. A protein band similar in size to Gli-ta has been observed in seed extracts, and mass spectrometry results confirm that the band contains small molecular mass α-gliadins, which is a characteristic of the novel α-gliadins. Mass spectrometry results also indicated that the two cysteine residues of Gli-ta/Gli-ta-like proteins participated in the formation of intermolecular disulphide bonds in vivo.

Wheat-Fusarium graminearum Interactions Under Sitobion avenae Influence: From Nutrients and Hormone Signals.

The English grain aphid Sitobion avenae and phytopathogen Fusarium graminearum are wheat spike colonizers. "Synergistic" effects of the coexistence of S. avenae and F. graminearum on the wheat spikes have been shown in agroecosystems. To develop genetic resistance in diverse wheat cultivars, an important question is how to discover wheat-F. graminearum interactions under S. avenae influence. In recent decades, extensive studies have typically focused on the unraveling of more details on the relationship between wheat-aphids and wheat-pathogens that has greatly contributed to the understanding of these tripartite interactions at the ecological level. Based on the scientific production available, the working hypotheses were synthesized from the aspects of environmental nutrients, auxin production, hormone signals, and their potential roles related to the tripartite interaction S. avenae-wheat-F. graminearum. In addition, this review highlights the relevance of preexposure to the herbivore S. avenae to trigger the accumulation of mycotoxins, which stimulates the infection process of F. graminearum and epidemic of Fusarium head blight (FHB) in the agroecosystems.

Genome-wide identification and evaluation of novel internal control genes for Q-PCR based transcript normalization in wheat.

To accurately quantify gene expression using quantitative PCR amplification, it is vital that one or more ideal internal control genes are used to normalize the samples to be compared. Ideally, the expression level of those internal control genes should vary as little as possible between tissues, developmental stages and environmental conditions. In this study, 32 candidate genes for internal control were obtained from the analysis of nine independent experiments which included 333 Affymetrix GeneChip Wheat Genome arrays. Expression levels of the selected genes were then evaluated by quantitative real-time PCR with cDNA samples from different tissues, stages of development and environmental conditions. Finally, fifteen novel internal control genes were selected and their respective expression profiles were compared using NormFinder, geNorm, Pearson correlation coefficients and the twofold-change method. The novel internal control genes from this study were compared with thirteen traditional ones for their expression stability. It was observed that seven of the novel internal control genes were better than the traditional ones in expression stability under all the tested cDNA samples. Among the traditional internal control genes, the elongation factor 1-alpha exhibited strong expression stability, whereas the 18S rRNA, Alpha-tubulin, Actin and GAPDH genes had very poor expression stability in the range of wheat samples tested. Therefore, the use of the novel internal control genes for normalization should improve the accuracy and validity of gene expression analysis.