Comprehensive, integrative genomic analysis of microRNA expression profiles in different tissues of two wheat cultivars with different traits.

Wheat is one of the most important food sources on Earth. MicroRNAs (miRNA) play important roles in wheat productivity. To identify wheat miRNAs, we constructed and sequenced sRNA libraries from leaves and roots of two wheat cultivars (RAC875 and Kukri) with many different traits. Given that available miRNA wheat complement in the plant-specific database PmiREN ( https://pmiren.com ) does not include root tissues and root-associated miRNAs might thus be missing, we performed first the prediction of novel miRNAs using the sRNAbench tool. We found a total of 150 putatively novel miRNA genes with expression of both arms from 289 unique mature sequences and nearly 30% of all miRNA reads in roots corresponded to novel miRNAs. In contrast, this figure in leaves dropped to under 3%, confirming the undersampling of roots in the complement of known miRNAs. By using 120 publicly available wheat datasets, 598 Zea mays small RNA libraries, 64 plant species genomes, wheat degradome library, and functional enrichment analysis, a subset of novel miRNAs were confirmed as bona-fide miRNAs. Of the total 605 miRNAs identified in this study inclusive of 316 known miRNAs, 528 miRNAs were shared by both cultivars, 429 miRNAs were shared by both root tissues and 329 miRNAs were shared by both leaf tissues. In addition, 32 miRNAs were specific to Kukri while 45 miRNAs were specific to RAC875. These miRNAs had diverse functions, such as regulation of gene transcription, protein translation, energy metabolism, and cell cycle progression. Our data provide a genome-wide miRNA expression profile in these two wheat cultivars and help functional studies of wheat genomics.

New roles of DNA-binding and forkhead-associated domains of Fkh1 and Fkh2 in cellular functions.

Two yeast forkhead transcription factors Fkh1 and Fkh2 regulate the transcription of CLB2 cluster genes important for mitosis. Both proteins contain a DNA-binding domain (DBD) and a forkhead-associated domain (FHAD), which are essential for ternary complex formation with transcription factor Mcm1, the transcription of CLB2 cluster genes and the physical interaction with Ndd1 and Clb2. Fkh2 also contains an additional C' domain that contains six consensus Cdk phosphorylation sites, but the function of this domain is dispensable. Here, we found new roles of the DBD, the FHAD, and the C' domain of Fkh1 and Fkh2 in cellular functions. The Fkh2 DBD determines the genetic interaction with NDD1, while both the FHAD and DBD of Fkh1 or Fkh2 determine cell morphology and stability of their own transcripts. Both HFADs, but not DBDs, also mediate physical interaction between Fkh1 and Fkh2. DBD and HFAD of Fkh1 and DBD, but not HFAD, of Fkh2 are also fundamental for nuclear localization. However, the Fkh2-specific C' domain has no role in these aspects except in the stability of some fkh mutant transcripts, which is either increased or decreased in the presence of this domain. These findings reveal that Fkh1 and Fkh2 have multiple cellular functions and function mainly via their DBD and FHAD through a domain-controlled feedback regulation mechanism.

One vector-based method to verify predicted plant miRNAs, target sequences, and function modes.

Many microRNAs (miRNAs) have been predicted from small RNA sequencing data, but little was experimentally verified due to the lack of effective methods. Here, we developed a simple and reliable dual gene expression cassette vector-based method to verify predicted plant miRNAs. We cloned osa-miR528 as a known miRNA, hvu-miRX as a predicted miRNA and TaDREB3 open reading frame as a non-miRNA into the first gene expression cassette and fused their complementary or noncomplementary sequences as predicted target or nontarget sequences with the 3' untranslated region of green fluorescent protein (GFP) in the second one. When these constructs were bombarded into plant cells, only the construct containing both osa-miR528 or hvu-miRX and its complementary sequence did not generate green fluorescence. Stem-loop reverse-transcription polymerase chain reaction detected mature osa-miR528 or mature hvu-miRX in the cells, while northern analysis showed that GFP messenger RNA from the construct containing both osa-miR528 or hvu-miRX and its complementary sequence was degraded. Taken together, the results indicate that hvu-miRX is an authentic miRNA like osa-miR528, miRNA's complementary sequence is its target sequence, and both osa-miR528 and hvu-miRX silenced the GFP expression via a cleavage mode. Our method thus facilitates the verification of predicted plant miRNAs, target sequences, and function modes.

Identification and characterisation of a previously unknown drought tolerance-associated microRNA in barley.

Drought is the most serious abiotic stress, and causes crop losses on a worldwide scale. The present study identified a previously unknown microRNA (designated as hvu-miRX) of 21 nucleotides (nt) in length in barley. Its precursor (designated pre-miRX) and primary transcript (designated pri-miRX) were also identified, with lengths of 73 and 559 nt, respectively. The identified upstream sequence of pri-miRX contained both the TATA box and the CAAT box, which are both required for initiation of transcription. Transient promoter activation assays showed that the core promoter region of pri-miRX ranged 500 nt from the transcription start site. In transgenic barley overexpression of the wheat DREB3 transcription factor (TaDREB3) caused hvu-miRX to be highly expressed as compared with the same miRNA in non-transgenic barley. However, the high expression was not directly associated with TaDREB3. Genomic analysis revealed that the hvu-miRX gene was a single copy located on the short arm of chromosome 2 and appeared to be only conserved in Triticeae, but not in other plant species. Notably, transgenic barley that overexpressed hvu-miRX showed drought tolerance. Degradome library analysis and other tests showed that hvu-miRX targeted various genes including transcription factors via the cleavage mode. Our data provides an excellent opportunity to develop drought stress tolerant cereals using hvu-miRX.

Decoding common and divergent cellular functions of the domains of forkhead transcription factors Fkh1 and Fkh2.

Forkhead transcription factors play a key role in embryonic patterning during development. In Saccharomyces cerevisiae, two forkhead transcription factors, Fkh1 and Fkh2, regulate the transcription of CLB2 cluster genes important for mitosis. Fkh1 reduces, whereas Fkh2 elevates, the transcription of CLB2 cluster genes. However, the mechanism for this observation remains unclear. Fkh1 and Fkh2 each contain a forkhead domain (DNA-binding domain, DBD) and a forkhead-associated domain (FHAD), whereas Fkh2 possesses an extra C' domain containing six consensus cyclin-dependent kinase phosphorylation sites. In the present study, roles of these domains in protein complexes, the regulation of cell growth and CLB2 cluster genes and protein interactions were investigated using various domain mutants. The result showed that the DBD was vital for ternary complex formation with Mcm1, whereas the FHAD was central for the regulation of cell growth and CLB2 cluster transcription and for interactions with Ndd1 and Clb2. However, the Fkh2 C' domain was dispensable for the above functions. Both DBDs and FHADs had functional divergences in the cell, and Ndd1 functioned via its phosphorylated form. These data provide important insights into the functional mechanism of Fkh1 and Fkh2 in cell cycle control.

Generation of different sizes and classes of small RNAs in barley is locus, chromosome and/or cultivar-dependent.

BACKGROUND: Various small RNA (sRNA) sizes and varieties have been identified, but their relationship as well as relationship with their origins and allocations have not been well understood or investigated. RESULTS: By comparing sRNAs generated from two barley cultivars, Golden Promise (GP) and Pallas, we identified that the generation of different sizes and types of sRNAs in barley was locus-, chromosome- and/or cultivar-dependent. 20-nt sRNAs mainly comprising miRNAs and chloroplast-derived sRNAs were significantly over-expressed in Pallas vs. GP on chromosomes 3H and 6H. MiRNAs-enriched 21-nt sRNAs were significantly over-expressed in Pallas vs. GP only on chromosome 4H. On chromosome 5H this size of sRNAs was significantly under-expressed in Pallas, so were 22-nt sRNAs mainly comprising miRNAs and repeat-derived sRNAs. 24-nt sRNAs mostly derived from repeats were evenly distributed in all chromosomes and expressed similarly between GP and Pallas. Unlike other sizes of sRNAs, 24-nt sRNAs were little conserved in other plant species. Abundant sRNAs were mostly generated from 3' terminal regions of chromosome 1H and 5' terminal regions of chromosome 5H. Over-expressed miRNAs in GP vs. Pallas primarily function in stress responses and iron-binding. CONCLUSIONS: Our study indicates that 23-24-nt sRNAs may be linked to repressive chromatin modifications and function in genome stability while 20-21-nt sRNAs may be important for the cultivar specificity. This study provides a novel insight into the mechanism of sRNA expression and function in barley.

Role of microRNAs in plant drought tolerance.

Drought is a normal and recurring climate feature in most parts of the world and plays a major role in limiting crop productivity. However, plants have their own defence systems to cope with adverse climatic conditions. One of these defence mechanisms is the reprogramming of gene expression by microRNAs (miRNAs). miRNAs are small noncoding RNAs of approximately 22 nucleotides length, which have emerged as important regulators of genes at post-transcriptional levels in a range of organisms. Some miRNAs are functionally conserved across plant species and are regulated by drought stress. These properties suggest that miRNA-based genetic modifications have the potential to enhance drought tolerance in cereal crops. This review summarizes the current understanding of the regulatory mechanisms of plant miRNAs, involvement of plant miRNAs in drought stress responses in barley (Hordeum vulgare L.), wheat (Triticum spp.) and other plant species, and the involvement of miRNAs in plant-adaptive mechanisms under drought stress. Potential strategies and directions for future miRNA research and the utilization of miRNAs in the improvement of cereal crops for drought tolerance are also discussed.

Differential expression of microRNAs and other small RNAs in barley between water and drought conditions.

Drought is a major constraint to crop production, and microRNAs (miRNAs) play an important role in plant drought tolerance. Analysis of miRNAs and other classes of small RNAs (sRNAs) in barley grown under water and drought conditions reveals that drought selectively regulates expression of miRNAs and other classes of sRNAs. Low-expressed miRNAs and all repeat-associated siRNAs (rasiRNAs) tended towards down-regulation, while tRNA-derived sRNAs (tsRNAs) had the tendency to be up-regulated, under drought. Antisense sRNAs (putative siRNAs) did not have such a tendency under drought. In drought-tolerant transgenic barley overexpressing DREB transcription factor, most of the low-expressed miRNAs were also down-regulated. In contrast, tsRNAs, rasiRNAs and other classes of sRNAs were not consistently expressed between the drought-treated and transgenic plants. The differential expression of miRNAs and siRNAs was further confirmed by Northern hybridization and quantitative real-time PCR (qRT-PCR). Targets of the drought-regulated miRNAs and siRNAs were predicted, identified by degradome libraries and confirmed by qRT-PCR. Their functions are diverse, but most are involved in transcriptional regulation. Our data provide insight into the expression profiles of miRNAs and other sRNAs, and their relationship under drought, thereby helping understand how miRNAs and sRNAs respond to drought stress in cereal crops.

Characterization of phosphorus-regulated miR399 and miR827 and their isomirs in barley under phosphorus-sufficient and phosphorus-deficient conditions.

BACKGROUND: miR399 and miR827 are both involved in conserved phosphorus (P) deficiency signalling pathways. miR399 targets the PHO2 gene encoding E2 enzyme that negatively regulates phosphate uptake and root-to-shoot allocation, while miR827 targets SPX-domain-containing genes that negatively regulate other P-responsive genes. However, the response of miR399 and miR827 to P conditions in barley has not been investigated. RESULTS: In this study, we investigated the expression profiles of miR399 and miR827 in barley (Hordeum vulagre L.) under P-deficient and P-sufficient conditions. We identified 10 members of the miR399 family and one miR827 gene in barley, all of which were significantly up-regulated under deficient P. In addition, we found many isomirs of the miR399 family and miR827, most of which were also significantly up-regulated under deficient P. Several isomirs of miR399 members were found to be able to cleave their predicted targets in vivo. Surprisingly, a few small RNAs (sRNAs) derived from the single-stranded loops of the hairpin structures of MIR399b and MIR399e-1 were also found to be able to cleave their predicted targets in vivo. Many antisense sRNAs of miR399 and a few for miR827 were also detected, but they did not seem to be regulated by P. Intriguingly, the lowest expressed member, hvu-miR399k, had four-fold more antisense sRNAs than sense sRNAs, and furthermore under P sufficiency, the antisense sRNAs are more frequent than the sense sRNAs. We identified a potential regulatory network among miR399, its target HvPHO2 and target mimics HvIPS1 and HvIPS2 in barley under P-deficient and P-sufficient conditions. CONCLUSIONS: Our data provide an important insight into the mechanistic regulation and function of miR399, miR827 and their isomirs in barley under different P conditions.

Genome-Wide Analysis of microRNA Expression Profile in Roots and Leaves of Three Wheat Cultivars under Water and Drought Conditions.

Wheat is one of the most important food sources on Earth. MicroRNAs (miRNAs) play important roles in wheat productivity. To identify wheat miRNAs as well as their expression profiles under drought condition, we constructed and sequenced small RNA (sRNA) libraries from the leaves and roots of three wheat cultivars (Kukri, RAC875 and Excalibur) under water and drought conditions. A total of 636 known miRNAs and 294 novel miRNAs were identified, of which 34 miRNAs were tissue- or cultivar-specific. Among these, 314 were significantly regulated under drought conditions. miRNAs that were drought-regulated in all cultivars displayed notably higher expression than those that responded in a cultivar-specific manner. Cultivar-specific drought response miRNAs were mainly detected in roots and showed significantly different drought regulations between cultivars. By using wheat degradome library, 6619 target genes were identified. Many target genes were strongly enriched for protein domains, such as MEKHLA, that play roles in drought response. Targeting analysis showed that drought-downregulated miRNAs targeted more genes than drought-upregulated miRNAs. Furthermore, such genes had more important functions. Additionally, the genes targeted by drought-downregulated miRNAs had multiple interactions with each other, while the genes targeted by drought-upregulated miRNAs had no interactions. Our data provide valuable information on wheat miRNA expression profiles and potential functions in different tissues, cultivars and drought conditions.

Phosphate utilization efficiency correlates with expression of low-affinity phosphate transporters and noncoding RNA, IPS1, in barley.

Genetic variation in phosphorus (P) efficiency exists among wheat (Triticum aestivum) and barley (Hordeum vulgare) genotypes, but the underlying mechanisms for the variation remain elusive. High- and low-affinity phosphate (Pi) PHT1 transporters play an indispensable role in P acquisition and remobilization. However, little is known about genetic variation in PHT1 gene expression and association with P acquisition efficiency (PAE) and P utilization efficiency (PUE). Here, we present quantitative analyses of transcript levels of high- and low-affinity PHT1 Pi transporters in four barley genotypes differing in PAE. The results showed that there was no clear pattern in the expression of four paralogs of the high-affinity Pi transporter HvPHT1;1 among the four barley genotypes, but the expression of a low-affinity Pi transporter, HvPHT1;6, and its close homolog HvHPT1;3 was correlated with the genotypes differing in PUE. Interestingly, the expression of HvPHT1;6 and HvPHT1;3 was correlated with the expression of HvIPS1 (for P starvation inducible; noncoding RNA) but not with HvIPS2, suggesting that HvIPS1 plays a distinct role in the regulation of the low-affinity Pi transporters. In addition, high PUE was found to be associated with high root-shoot ratios in low-P conditions, indicating that high carbohydrate partitioning into roots occurs simultaneously with high PUE. However, high PUE accompanying high carbon partitioning into roots could result in low PAE. Therefore, the optimization of PUE through the modification of low-affinity Pi transporter expression may assist further improvement of PAE for low-input agriculture systems.

Discovery of barley miRNAs through deep sequencing of short reads.

BACKGROUND: MicroRNAs are important components of the regulatory network of biological systems and thousands have been discovered in both animals and plants. Systematic investigations performed in species with sequenced genomes such as Arabidopsis, rice, poplar and Brachypodium have provided insights into the evolutionary relationships of this class of small RNAs among plants. However, miRNAs from barley, one of the most important cereal crops, remain unknown. RESULTS: We performed a large scale study of barley miRNAs through deep sequencing of small RNAs extracted from leaves of two barley cultivars. By using the presence of miRNA precursor sequences in related genomes as one of a number of supporting criteria, we identified up to 100 miRNAs in barley. Of these only 56 have orthologs in wheat, rice or Brachypodium that are known to be expressed, while up to 44 appear to be specifically expressed in barley. CONCLUSIONS: Our study, the first large scale investigation of small RNAs in barley, has identified up to 100 miRNAs. We demonstrate that reliable identification of miRNAs via deep sequencing in a species whose genome has not been sequenced requires a more careful analysis of sequencing errors than is commonly performed. We devised a read filtering procedure for dealing with errors. In addition, we found that the use of a large dataset of almost 35 million reads permits the use of read abundance distributions along putative precursor sequences as a practical tool for isolating miRNAs in a large background of reads originating from other non-coding and coding RNAs. This study therefore provides a generic approach for discovering novel miRNAs where no genome sequence is available.

Development of genomic resources for the narrow-leafed lupin (Lupinus angustifolius): construction of a bacterial artificial chromosome (BAC) library and BAC-end sequencing.

BACKGROUND: Lupinus angustifolius L, also known as narrow-leafed lupin (NLL), is becoming an important grain legume crop that is valuable for sustainable farming and is becoming recognised as a potential human health food. Recent interest is being directed at NLL to improve grain production, disease and pest management and health benefits of the grain. However, studies have been hindered by a lack of extensive genomic resources for the species. RESULTS: A NLL BAC library was constructed consisting of 111,360 clones with an average insert size of 99.7 Kbp from cv Tanjil. The library has approximately 12 × genome coverage. Both ends of 9600 randomly selected BAC clones were sequenced to generate 13985 BAC end-sequences (BESs), covering approximately 1% of the NLL genome. These BESs permitted a preliminary characterisation of the NLL genome such as organisation and composition, with the BESs having approximately 39% G:C content, 16.6% repetitive DNA and 5.4% putative gene-encoding regions. From the BESs 9966 simple sequence repeat (SSR) motifs were identified and some of these are shown to be potential markers. CONCLUSIONS: The NLL BAC library and BAC-end sequences are powerful resources for genetic and genomic research on lupin. These resources will provide a robust platform for future high-resolution mapping, map-based cloning, comparative genomics and assembly of whole-genome sequencing data for the species.

BAC library resources for map-based cloning and physical map construction in barley (Hordeum vulgare L.).

BACKGROUND: Although second generation sequencing (2GS) technologies allow re-sequencing of previously gold-standard-sequenced genomes, whole genome shotgun sequencing and de novo assembly of large and complex eukaryotic genomes is still difficult. Availability of a genome-wide physical map is therefore still a prerequisite for whole genome sequencing for genomes like barley. To start such an endeavor, large insert genomic libraries, i.e. Bacterial Artificial Chromosome (BAC) libraries, which are unbiased and representing deep haploid genome coverage, need to be ready in place. RESULT: Five new BAC libraries were constructed for barley (Hordeum vulgare L.) cultivar Morex. These libraries were constructed in different cloning sites (HindIII, EcoRI, MboI and BstXI) of the respective vectors. In order to enhance unbiased genome representation and to minimize the number of gaps between BAC contigs, which are often due to uneven distribution of restriction sites, a mechanically sheared library was also generated. The new BAC libraries were fully characterized in depth by scrutinizing the major quality parameters such as average insert size, degree of contamination (plate wide, neighboring, and chloroplast), empty wells and off-scale clones (clones with <30 or >250 fragments). Additionally a set of gene-based probes were hybridized to high density BAC filters and showed that genome coverage of each library is between 2.4 and 6.6 X. CONCLUSION: BAC libraries representing >20 haploid genomes are available as a new resource to the barley research community. Systematic utilization of these libraries in high-throughput BAC fingerprinting should allow developing a genome-wide physical map for the barley genome, which will be instrumental for map-based gene isolation and genome sequencing.

The N-terminal 12 amino acids of tomato aspermy virus 2b protein function in infection and recombination.

The roles for various regions of the 2b protein in infection, hypervirulence and recombination were examined by introducing stop codons in a chimeric virus containing RNA 1 from the cucumber mosaic virus (CMV strain Q), RNA 3 from the tomato aspermy virus (TAV) and RNA 2 of CMV with a 2b gene from TAV. Chimeric virus expressing the intact 2b protein induced severe symptoms in inoculated Nicotiana clevelandii and Nicotiana glutinosa and facilitated CMV-TAV recombination, while chimeric viruses not expressing 2b protein did not infect plants systemically. Chimeric viruses expressing either the N-terminal 43 or 12 aa of the 2b protein infected both plant species systemically and facilitated CMV-TAV recombination, but induced mild symptoms and no symptoms in the infected plants, respectively. These data suggest that oligopeptides can have important functions in the biology of viruses and prompt a re-examination of existing small ORFs in sequenced virus genomes.

The cucumovirus 2b gene drives selection of inter-viral recombinants affecting the crossover site, the acceptor RNA and the rate of selection.

RNA-RNA recombination is an important pathway in virus evolution and has been described for many viruses. However, the factors driving recombination or promoting the selection of recombinants are still unclear. Here, we show that the small movement protein (2b) was able to promote selection of RNA 1/2-RNA 3 recombinants within a chimeric virus having RNAs 1 and 2 from cucumber mosaic virus, and RNA 3 from the related tomato aspermy virus, along with heterologous 2b genes. The source of the 2b also determined the selection of the acceptor RNA and the crossover site, as well as affecting the rate of selection of the recombinant RNAs. The nature of the RNA 3 also influenced the selection of the recombinant RNAs. A 163-nt tandem repeat in RNA 3 significantly affected the rate of selection of the recombinant RNA, while a single nucleotide within the repeat affected the crossover site. The recombination occurred in a non-random manner, involved no intermediates and probably was generated via a copy-choice mechanism during (+) strand RNA synthesis.

Stability and competitiveness of interviral recombinant RNAs derived from a chimeric cucumovirus.

We previously described interviral recombinant RNAs derived from a chimeric virus having RNAs 1 and 2 of cucumber mosaic virus (CMV) with RNA 3 from the related tomato aspermy virus (TAV) and the 2b gene from either TAV or another strain of CMV. Here, we show that these interviral recombinant RNAs 3 were stable in the infected plants and could co-exist with their wild-type parental viral RNAs in the same plants, but their de novo generations were inhibited in the presence of the wild-type parental viral RNAs. The recombinant viral genomes did not prevent the replication of other viral RNAs or vice versa, but one of the interviral recombinant viruses induced different symptoms in Physalis floridana from those induced by the parental chimeric virus without the interviral RNA 3 recombinant. Factors such as the nature of the 2b gene and/or the presence or absence of competing wild-type parental RNAs influenced the generation of the recombinant RNAs described. Our data provide additional mechanistic insight into generation, stabilization and competition of recombinant viral RNA in infected host plants.

A simple TAE-based method to generate large insert BAC libraries from plant species.

Large insert libraries are valuable tools for the positional cloning of genes of interest, physical mapping of chromosomes, comparative genomics, and molecular breeding. There are five types of large DNA insert libraries: cosmid, yeast artificial chromosomes (YACs), bacteriophage P1, bacterial artificial chromosomes (BACs) and P1-derived artificial chromosomes (PACs) libraries. Of these libraries, BAC libraries are the most widely used due to their ease of manipulation, large insert size, and stability. This chapter reports on a simplified method for plant BAC library construction. This method involves isolation and partial digestion of intact nuclei, selection of appropriate size of DNA via pulsed-field gel (PFG) electrophoresis, elution of DNA from agarose gels, ligation of DNA into the BAC vector, electroporation of the ligation mix into Escherichia coli cells, and estimation of insert sizes. The whole process takes 1-3 months depending on the genome size and coverage required. We have used this method to produce BAC libraries from different plant species including sunolgrass (Phalaris coerulescens L.), barley (Hordeum vulgare L.), lupin (Lupinus angustifolias L.) and rye (Secale cereale L.).

Comparative study of genes expressed from rice fungus-resistant and susceptible lines during interactions with Magnaporthe oryzae.

Rice blast disease caused by Magnaporthe oryzae is the most important fungal disease of rice. To understand the molecular basis of interaction between the fungus and rice, we constructed a cDNA library from a rice-resistant line inoculated with M. oryzae. One hundred and fifty-three cDNA clones were sequence analyzed, of which 129 exhibited significant nucleotide sequence homology to known genes, 21 were homologous to unknown genes, while three clones did not match to any database. However, these three unmatched clones showed sequence homology at protein level in the protein databases and one of them encoded a disease resistance-related protein kinase and was abundant in the EST collection. Northern analysis showed that this disease resistance-related protein kinase gene was induced by inoculation and only expressed in the rice-resistant, but not susceptible, lines. Southern analysis showed that this gene was present in a single copy in the rice genome and co-segregated with the M. oryzae resistance in the cross of the resistant and susceptible lines. This study illustrates that sequencing of ESTs from inoculated resistant plants can reveal genes responsive to pathogen infection, which could help understand plant defense mechanisms.

Identification of reference genes for quantitative expression analysis of microRNAs and mRNAs in barley under various stress conditions.

For accurate and reliable gene expression analysis using quantitative real-time reverse transcription PCR (qPCR), the selection of appropriate reference genes as an internal control for normalization is crucial. We hypothesized that non-coding, small nucleolar RNAs (snoRNAs)would be stably expressed in different barley varieties and under different experimental treatments,in different tissues and at different developmental stages of plant growth and therefore might prove to be suitable reference genes for expression analysis of both microRNAs (miRNAs)and mRNAs. In this study, we examined the expression stability of ten candidate reference genes in six barley genotypes under five experimental stresses, drought, fungal infection,boron toxicity, nutrient deficiency and salinity. We compared four commonly used housekeeping genes; Actin (ACT), alpha-Tubulin (α-TUB), Glycolytic glyceraldehyde-3-phosphate dehydrogenase(GAPDH), ADP-ribosylation factor 1-like protein (ADP), four snoRNAs; (U18,U61, snoR14 and snoR23) and two microRNAs (miR168, miR159) as candidate reference genes. We found that ADP, snoR14 and snoR23 were ranked as the best of these candidates across diverse samples. Additionally, we found that miR168 was a suitable reference gene for expression analysis in barley. Finally, we validated the performance of our stable and unstable candidate reference genes for both mRNA and miRNA qPCR data normalization under different stress conditions and demonstrated the superiority of the stable candidates. Our data demonstrate the suitability of barley snoRNAs and miRNAs as potential reference genes form iRNA and mRNA qPCR data normalization under different stress treatments [corrected].