RefFinder: a web-based tool for comprehensively analyzing and identifying reference genes.

Although many genes may serve as reference genes, they may cause different expression patterns by selecting different reference genes because no single gene is expressed consistently in all tested tissues of an organism under all environmental and developmental conditions. Thus, it is becoming increasingly important and necessary to identify suitable reference genes before performing gene expression analysis. Currently, there are several computational tools available for evaluating the stability of candidate reference genes. These tools are based on different statistical algorithms and may produce different rankings in stability within the same reference gene study. To date, the RefFinder is the only web-based tool available for comparing and evaluating housekeeping genes as candidates to be reference genes. In this tool, we integrated the four currently available computational programs (geNorm, NormFinder, BestKeeper, and the comparative ΔCt method) into a web-based tool for evaluating the stability and reliability of reference genes. According to the gene stability rankings derived from the four programs, we assigned an appropriate weight to each gene and calculated the geometric mean of weights for the final rankings. Aside from the overall ranking, a single program or combination of the four programs can be selected for evaluating the ranking of candidate reference genes. This tool has been widely used and validated by many research laboratories around the world. You may use this tool at http://www.heartcure.com.au/reffinder/  or  https://blooge.cn/RefFinder/ . You can also download this algorithm program from https://github.com/fulxie/RefFinder and setup on your own computer. RefFinder is developed by PHP. Users can deploy it to a Php-based server (Apache + PHP) and run it.

Reference gene selection for miRNA and mRNA normalization in potato in response to potato virus Y.

This was the first report on evaluating candidate reference genes for quantifying the expression profiles of both coding (e.g., mRNA) and non-coding (e.g., miRNA) genes in potato response to potato virus Y (PVY) inoculation. The reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) method was employed to quantify the expression profiles of eight selected candidate reference genes; their expression stability was analyzed by four statistical algorithms, i.e., geNorm, BestKeeper, NormFinder and RefFinder. The most stable reference genes were sEF1a, sTUBb and seIF5 with a high stability. The least stable ones were sPP2A, sSUI1 and sGAPDH. The same reference gene allows for normalization of both miRNA and mRNA levels from a single RNA sample using cDNAs synthesized in a single RT reaction, in which a stem-loop primer was used for miRNAs and the oligo (dT) for mRNAs.

Genome of wild olive and the evolution of oil biosynthesis.

Here we present the genome sequence and annotation of the wild olive tree (Olea europaea var. sylvestris), called oleaster, which is considered an ancestor of cultivated olive trees. More than 50,000 protein-coding genes were predicted, a majority of which could be anchored to 23 pseudochromosomes obtained through a newly constructed genetic map. The oleaster genome contains signatures of two Oleaceae lineage-specific paleopolyploidy events, dated at ∼28 and ∼59 Mya. These events contributed to the expansion and neofunctionalization of genes and gene families that play important roles in oil biosynthesis. The functional divergence of oil biosynthesis pathway genes, such as FAD2, SACPD, EAR, and ACPTE, following duplication, has been responsible for the differential accumulation of oleic and linoleic acids produced in olive compared with sesame, a closely related oil crop. Duplicated oleaster FAD2 genes are regulated by an siRNA derived from a transposable element-rich region, leading to suppressed levels of FAD2 gene expression. Additionally, neofunctionalization of members of the SACPD gene family has led to increased expression of SACPD2, 3, 5, and 7, consequently resulting in an increased desaturation of steric acid. Taken together, decreased FAD2 expression and increased SACPD expression likely explain the accumulation of exceptionally high levels of oleic acid in olive. The oleaster genome thus provides important insights into the evolution of oil biosynthesis and will be a valuable resource for oil crop genomics.

Identification, characterization, and gene expression analysis of nucleotide binding site (NB)-type resistance gene homologues in switchgrass.

BACKGROUND: Switchgrass (Panicum virgatum L.) is a warm-season perennial grass that can be used as a second generation bioenergy crop. However, foliar fungal pathogens, like switchgrass rust, have the potential to significantly reduce switchgrass biomass yield. Despite its importance as a prominent bioenergy crop, a genome-wide comprehensive analysis of NB-LRR disease resistance genes has yet to be performed in switchgrass. RESULTS: In this study, we used a homology-based computational approach to identify 1011 potential NB-LRR resistance gene homologs (RGHs) in the switchgrass genome (v 1.1). In addition, we identified 40 RGHs that potentially contain unique domains including major sperm protein domain, jacalin-like binding domain, calmodulin-like binding, and thioredoxin. RNA-sequencing analysis of leaf tissue from 'Alamo', a rust-resistant switchgrass cultivar, and 'Dacotah', a rust-susceptible switchgrass cultivar, identified 2634 high quality variants in the RGHs between the two cultivars. RNA-sequencing data from field-grown cultivar 'Summer' plants indicated that the expression of some of these RGHs was developmentally regulated. CONCLUSIONS: Our results provide useful insight into the molecular structure, distribution, and expression patterns of members of the NB-LRR gene family in switchgrass. These results also provide a foundation for future work aimed at elucidating the molecular mechanisms underlying disease resistance in this important bioenergy crop.

Identification and characterization of microRNAs in the plant parasitic root-knot nematode Meloidogyne incognita using deep sequencing.

The root-knot nematode Meloidogyne incognita is among the most damaging plant-parasitic pests of several crops including cotton (Gossypium hirsutum) and tomato (Lycopersicon escultentum). Recently, a genome has become available for M. incognita, which greatly facilitates investigation of the interactions between M. incognita and its plant hosts at the molecular level and enables formation of hypotheses concerning development at the cellular level. MicroRNAs (miRNAs) are a class of small RNA molecules that serve as endogenous gene regulators. They regulate many biological processes including reproduction, the sequencing of morphological development, and potentially of parasitism as well. Certain miRNAs regulate fundamental metabolism pathways and stress responses in M. incognita. Since a list of miRNAs has not been generated for M. incognita, we employed a bioinformatics tool called mirDeepFinder to identify miRNAs from the small RNA database of M. incognita (GSM611102) that was generated from deep sequencing. A total of 254 conserved miRNAs belonging to 161 miRNA families were identified, as were 35 novel miRNAs belonging to 31 families. The 16 most commonly found miRNAs in order of abundance were min-miR-100a, min-miR-124, min-miR-71a, min-miR-1, min-miR-228, min-miR-92, min-miR-72, min-miR-49b, min-miR-58, min-miR-252, min-miR-lin-4, min-miR-87, min-miR-2a, min-miR-34a, min-miR-50a, and min-miR-279a. The length of the pre-miRNAs varied greatly from 50 to 197 nt, with an average of 88 ± 39 nt. The average minimal folding free energy (MFE) and MFE index (MFEI) of the identified miRNAs were -30.3 Kcal/mol and 0.92, respectively, indicating that these miRNAs can readily fold into a typical hairpin secondary structure.

microRNA evolution and expression analysis in polyploidized cotton genome.

Cotton (Gossypium hirsutum L.), the most important fibre plant in the world, is a tetraploid species, originating from the reunion of two ancestral cotton species ~1-2 million years ago. It has been reported that a great number of genes were quickly erased or preferentially remained after whole-genome duplication, ultimately leading to morphogenesis evolution. However, microRNAs (miRNAs), a new class of gene regulators, have not been well studied in polyploidization. Here, we systematically investigated miRNA evolution amongst cultivated upland cotton G. hirsutum (AADD) and its two ancestors, G. arboreum (AA) and G. raimondii (DD). Our results show that certain highly conserved miRNAs were likely to be lost, whereas certain were remained after genome polyploidization. Cotton-specific miRNAs might undergo remarkably expansion, resulting in overall miRNA increase in upland cotton. Based on the sequenced genomes of G. arboreum and G. raimondii, we are capable for the first time to categorize the origin of miRNAs and coding genes in upland cotton. Different genome-derived miRNAs and miRNA*s displayed asymmetric expression pattern, implicating their diverse functions in upland cotton. No miRNA targeting preference was observed between different genome-derived miRNAs. The origin of miRNAs and coding genes has no impact on becoming miRNAs and their targets, despite some miRNAs and their targets are extremely conserved in the three cotton species. GO- and KEGG-based analysis of conserved miRNAs show that conserved miRNAs and their targets participate in a series of important biological processes and metabolism pathways. Additionally, A-derived miRNAs might be more responsible for ovule and fibre development.

Global microRNA modification in cotton (Gossypium hirsutum L.).

MicroRNAs (miRNAs) are small noncoding RNAs participating in versatile biological processes via post-transcriptionally gene regulation. However, how miRNAs are modified or degraded remains unknown, despite years of studies have unravelled much details of miRNA biogenesis and function. Here, we systematically investigated miRNA modification using six small RNA sequencing libraries generated from cotton seedling as well as cotton fibre at five developmental stages. Our results show that 1-2-nt truncation and addition on both 5' and 3' ends of miRNAs are the major modification forms. The 5' and 3' end miRNA modification was almost equal in the six development stages. Truncation was more common than addition on both 5' and 3' end. Structure analysis of the 5' and 3' ends of miRNAs and isomiRs shows that uridine is the preferential nucleotide at the first position of both 5' and 3' ends. According to analysis of nucleotides truncated and tailed from miRNAs, both miRNAs and isomiRs share a similar positional structure distribution at their 5' and 3' ends, respectively. Furthermore, opposite to previous reports, cytodine is more frequently truncated and tailed from the two ends of isomiRs, implying existence of a complex cytodine balance in isomiRs. Comparison of isomiR expression shows differential miRNA modification amongst the six developmental stages in terms of selective modification form, development-dependent modification and differential expression abundance. Our results globally uncovered miRNA modification features in cotton, which could contribute us to understanding miRNA's postmature modification and its regulatory function.

Small RNA sequencing identifies miRNA roles in ovule and fibre development.

MicroRNAs (miRNAs) have been found to be differentially expressed during cotton fibre development. However, which specific miRNAs and how they are involved in fibre development is unclear. Here, using deep sequencing, 65 conserved miRNA families were identified and 32 families were differentially expressed between leaf and ovule. At least 40 miRNAs were either leaf or ovule specific, whereas 62 miRNAs were shared in both leaf and ovule. qRT-PCR confirmed these miRNAs were differentially expressed during fibre early development. A total of 820 genes were potentially targeted by the identified miRNAs, whose functions are involved in a series of biological processes including fibre development, metabolism and signal transduction. Many predicted miRNA-target pairs were subsequently validated by degradome sequencing analysis. GO and KEGG analyses showed that the identified miRNAs and their targets were classified to 1027 GO terms including 568 biological processes, 324 molecular functions and 135 cellular components and were enriched to 78 KEGG pathways. At least seven unique miRNAs participate in trichome regulatory interaction network. Eleven trans-acting siRNA (tasiRNA) candidate genes were also identified in cotton. One has never been found in other plant species and two of them were derived from MYB and ARF, both of which play important roles in cotton fibre development. Sixteen genes were predicted to be tasiRNA targets, including sucrose synthase and MYB2. Together, this study discovered new miRNAs in cotton and offered evidences that miRNAs play important roles in cotton ovule/fibre development. The identification of tasiRNA genes and their targets broadens our understanding of the complicated regulatory mechanism of miRNAs in cotton.

Deep sequencing reveals important roles of microRNAs in response to drought and salinity stress in cotton.

Drought and salinity are two major environmental factors adversely affecting plant growth and productivity. However, the regulatory mechanism is unknown. In this study, the potential roles of small regulatory microRNAs (miRNAs) in cotton response to those stresses were investigated. Using next-generation deep sequencing, a total of 337 miRNAs with precursors were identified, comprising 289 known miRNAs and 48 novel miRNAs. Of these miRNAs, 155 miRNAs were expressed differentially. Target prediction, Gene Ontology (GO)-based functional classification, and Kyoto Encyclopedia of Genes and Genomes (KEGG)-based functional enrichment show that these miRNAs might play roles in response to salinity and drought stresses through targeting a series of stress-related genes. Degradome sequencing analysis showed that at least 55 predicted target genes were further validated to be regulated by 60 miRNAs. CitationRank-based literature mining was employed to determinhe the importance of genes related to drought and salinity stress. The NAC, MYB, and MAPK families were ranked top under the context of drought and salinity, indicating their important roles for the plant to combat drought and salinity stress. According to target prediction, a series of cotton miRNAs are associated with these top-ranked genes, including miR164, miR172, miR396, miR1520, miR6158, ghr-n24, ghr-n56, and ghr-n59. Interestingly, 163 cotton miRNAs were also identified to target 210 genes that are important in fibre development. These results will contribute to cotton stress-resistant breeding as well as understanding fibre development.

Genome sequencing and analysis of the paclitaxel-producing endophytic fungus Penicillium aurantiogriseum NRRL 62431.

BACKGROUND: Paclitaxel (Taxol™) is an important anticancer drug with a unique mode of action. The biosynthesis of paclitaxel had been considered restricted to the Taxus species until it was discovered in Taxomyces andreanae, an endophytic fungus of T. brevifolia. Subsequently, paclitaxel was found in hazel (Corylus avellana L.) and in several other endophytic fungi. The distribution of paclitaxel in plants and endophytic fungi and the reported sequence homology of key genes in paclitaxel biosynthesis between plant and fungi species raises the question about whether the origin of this pathway in these two physically associated groups could have been facilitated by horizontal gene transfer. RESULTS: The ability of the endophytic fungus of hazel Penicillium aurantiogriseum NRRL 62431 to independently synthesize paclitaxel was established by liquid chromatography-mass spectrometry and proton nuclear magnetic resonance. The genome of Penicillium aurantiogriseum NRRL 62431 was sequenced and gene candidates that may be involved in paclitaxel biosynthesis were identified by comparison with the 13 known paclitaxel biosynthetic genes in Taxus. We found that paclitaxel biosynthetic gene candidates in P. aurantiogriseum NRRL 62431 have evolved independently and that horizontal gene transfer between this endophytic fungus and its plant host is unlikely. CONCLUSIONS: Our findings shed new light on how paclitaxel-producing endophytic fungi synthesize paclitaxel, and will facilitate metabolic engineering for the industrial production of paclitaxel from fungi.

Expression of microRNAs and their targets regulates floral development in tobacco (Nicotiana tabacum).

MicroRNAs (miRNAs) are an extensive class of endogenous posttranscriptional gene regulators that function to mediate gene expression by cleaving target mRNAs or by preventing protein translation. Because of their importance in mediating gene regulation, identifying and elucidating the function of miRNAs have been the primary focus of many researchers. Now that many miRNAs have been identified and assessed for their functionality, the next step is to create expression profiles for miRNAs, so that gene expression studies can be further enhanced with knowledge of the basal expression levels of miRNAs and their targets. In a previous study, 259 putative miRNAs were identified in tobacco, in which 11 of them were confirmed. The primary goal of this study was to further expand on that study and create an expression profile for nine miRNAs and their targets in a tissue-specific manner in tobacco. We chose to study miRNAs that largely play a role in floral development and nutrient stress response. The results of our study show that all tested miRNAs and their targets were expressed in a differential manner. The results of our study also show that out of the tested miRNAs and their targets, miR159, miR157, miR167, miR172, and superoxide dismutase were expressed the highest, suggesting that these genes may play a vital role in the growth and development of tobacco. Corrected expression of miRNAs and their targets regulates floral development.

High-throughput deep sequencing shows that microRNAs play important roles in switchgrass responses to drought and salinity stress.

MicroRNAs (miRNAs) are an important class of small regulatory RNAs. The goal of this study was to analyse stress-responsive miRNAs in switchgrass (Panicum virgatum), the emerging biofuel crop, to facilitate choosing gene targets for improving biomass and biofuel yield. After sequencing three small RNA libraries constructed from control, salt- and drought-treated switchgrass using Illumina sequencing technology, we identified 670 known miRNA families from a total of more than 50 million short reads. A total of 273 miRNAs were identified with precursors: 126 conserved miRNAs and 147 novel miRNAs. Of them, 265 miRNAs were found to have their opposite sequences (miRNA*) with 2-nt overhang on the 3' end. Of them, 194 were detected in switchgrass transcriptome sequences generated from 31 high-throughput RNA sequencing (RNA-Seq) data sets in NCBI. Many miRNAs were differentially or uniquely expressed during salinity or drought stress treatment. We also discovered 11 miRNA clusters containing 29 miRNAs. These identified miRNAs potentially targeted 28549 genes with a various function, including transcription factors, stress-response proteins and cellulose biosynthesis-related proteins. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the identified miRNAs and their targets were classified to 3779 GO terms including 1534 molecular functions, 1851 biological processes and 394 cellular components and were enriched to 147 KEGG pathways. Interestingly, 195 miRNA families and 450 targets were involved in the biosynthesis pathways of carbon, glucose, starch, fatty acid and lignin and in xylem formation, which could aid in designing next-generation switchgrass for biomass and biofuel.

Evaluation of reference genes for miRNA and mRNA normalization in tobacco infected with PVYNTN, PVYN-Wi and PVYZ-NTN strains.

This is the first report on identification of the most suitable reference genes for RT-qPCR quantification of miRNA and mRNA in tobacco response to the prevalent recombinant potato virus Y (PVY) strains PVYNTN, PVYN-Wi and the newly identified PVYZ-NTN. Of 10 tested genes, the expression levels of neIF5C, nU2af and nPP2A were the most stable in samples taken from non-inoculated, mock-inoculated, and infected plants at three days post-inoculation (dpi) and 14 dpi. While the homologues of eIF5 were most stably expressed in tobacco in this study and in potato in our previous study (Yin et al., 2021) following inoculation with the same three PVY strains, the homologues of other two genes PP2A and U2af were stably expressed only in tobacco but unstable in potato. The tobacco homologue of PP2A, which was the most stably expressed one in tobacco interaction with PVYNTN, PVYN-Wi and PVYZ-NTN strains in this study, was the least stable one in tobacco interaction with the non-recombinant PVYO strain in a previous study (Baek et al., 2017). This study provides evidence on the influence of host species on expression of housekeeping genes and points out virus strain as a new factor influencing expression stability of reference gene. Caution should be taken when choosing reference genes in gene expression study in Solanaceae hosts response to different PVY strains.

miRDeepFinder: a miRNA analysis tool for deep sequencing of plant small RNAs.

miRDeepFinder is a software package developed to identify and functionally analyze plant microRNAs (miRNAs) and their targets from small RNA datasets obtained from deep sequencing. The functions available in miRDeepFinder include pre-processing of raw data, identifying conserved miRNAs, mining and classifying novel miRNAs, miRNA expression profiling, predicting miRNA targets, and gene pathway and gene network analysis involving miRNAs. The fundamental design of miRDeepFinder is based on miRNA biogenesis, miRNA-mediated gene regulation and target recognition, such as perfect or near perfect hairpin structures, different read abundances of miRNA and miRNA*, and targeting patterns of plant miRNAs. To test the accuracy and robustness of miRDeepFinder, we analyzed a small RNA deep sequencing dataset of Arabidopsis thaliana published in the GEO database of NCBI. Our test retrieved 128 of 131 (97.7%) known miRNAs that have a more than 3 read count in Arabidopsis. Because many known miRNAs are not associated with miRNA*s in small RNA datasets, miRDeepFinder was also designed to recover miRNA candidates without the presence of miRNA*. To mine as many miRNAs as possible, miRDeepFinder allows users to compare mature miRNAs and their miRNA*s with other small RNA datasets from the same species. Cleaveland software package was also incorporated into miRDeepFinder for miRNA target identification using degradome sequencing analysis. Using this new computational tool, we identified 13 novel miRNA candidates with miRNA*s from Arabidopsis and validated 12 of them experimentally. Interestingly, of the 12 verified novel miRNAs, a miRNA named AC1 spans the exons of two genes (UTG71C4 and UGT71C3). Both the mature AC1 miRNA and its miRNA* were also found in four other small RNA datasets. We also developed a tool, "miRNA primer designer" to design primers for any type of miRNAs. miRDeepFinder provides a powerful tool for analyzing small RNA datasets from all species, with or without the availability of genome information. miRDeepFinder and miRNA primer designer are freely available at http://www.leonxie.com/DeepFinder.php and at http://www.leonxie.com/miRNAprimerDesigner.php , respectively. A program (called RefFinder: http://www.leonxie.com/referencegene.php ) was also developed for assessing the reliable reference genes for gene expression analysis, including miRNAs.

De novo sequencing and a comprehensive analysis of purple sweet potato (Impomoea batatas L.) transcriptome.

High-throughput RNA sequencing was performed for comprehensively analyzing the transcriptome of the purple sweet potato. A total of 58,800 unigenes were obtained and ranged from 200 nt to 10,380 nt with an average length of 476 nt. The average expression of one unigene was 34 reads per kb per million reads (RPKM) with a maximum expression of 1,935 RPKM. At least 40,280 (68.5%) unigenes were identified to be protein-coding genes, in which 11,978 and 5,184 genes were homologous to Arabidopsis and rice proteins, respectively. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analysis showed that 19,707 (33.5%) unigenes were classified to 1,807 terms of GO including molecular functions, biological processes, and cellular components and 9,970 (17.0%) unigenes were enriched to 11,119 KEGG pathways. We found that at least 3,553 genes may be involved in the biosynthesis pathways of starch, alkaloids, anthocyanin pigments, and vitamins. Additionally, 851 potential simple sequence repeats (SSRs) were identified in all unigenes. Transcriptome sequencing on tuberous roots of the sweet potato yielded substantial transcriptional sequences and potentially useful SSR markers which provide an important data source for sweet potato research. Comparison of two RNA-sequence datasets from the purple and the yellow sweet potato showed that UDP-glucose-flavonoid 3-O-glucosyltransferase was one of the key enzymes in the pathway of anthocyanin biosynthesis and that anthocyanin-3-glucoside might be one of the major components for anthocyanin pigments in the purple sweet potato. This study contributes to the molecular mechanisms of sweet potato development and metabolism and therefore that increases the potential utilization of the sweet potato in food nutrition and pharmacy.

Transcriptome-wide identification and stress properties of the 14-3-3 gene family in cotton (Gossypium hirsutum L.).

14-3-3s are a class of conserved proteins in eukaryotes involved in almost all cellular processes. Published studies have shown that this family plays a role in response to stress conditions. In this study, comparative genomics identified thirty-one 14-3-3 cDNAs encoding 25 unique proteins with transcripts that were sufficiently divergent in the coding regions to define different 14-3-3 members. Phylogenetic analysis indicated that the cotton 14-3-3 family was similar to the family in the plant model species Arabidopsis and rice. The multiple copies of 14-3-3 genes in cotton are suggested to be the result of a recent duplication event after the divergence of cotton from its progenitor species. 14-3-3s are differentially expressed in a range of cotton organs as well as under different environmental conditions. Salinity and drought stress significantly induced the altered expression of 14-3-3 genes, suggesting that 14-3-3s play a role in cotton response to environmental abiotic stress. The 14-3-3 gene CGF14-4 was more sensitive to both drought and salinity stress with a 10.2-fold change under 1% of PEG treatment, while other 14-3-3s responded only to either drought or salinity stress. These results implied that specific isoforms of 14-3-3s play different regulatory roles in stress response besides their role in development. 14-3-3s were more sensitive to abiotic stress in roots than in leaves, suggesting that the root is more sensitive to stress treatment than leaves. The differential expression pattern of 14-3-3s also suggests the occurrence of functional divergence among specific isoforms.

Target-align: a tool for plant microRNA target identification.

MOTIVATION: MicroRNAs (miRNAs) are important regulatory molecules. A critical step in elucidating miRNA function is identifying potential miRNA targets. However, few reliable tools have been developed for identifying miRNA targets in plants. RESULTS: Here, we developed a Smith-Waterman-like alignment tool in order to accurately predict miRNA targets. Dynamic programming was used to build a score matrix based on the complementarity of nucleotides in order to trace the optimal local alignments. Important parameters, such as maximum mismatches and maximum consecutive mismatches between miRNAs and their targets, were also used for filtering the optimal local alignments. Almost all of the parameters in this alignment tool can be adjusted by users. Compared to other target prediction tools, Target-align exhibits strong sensitivity and accuracy for identifying miRNA targets. More importantly, Target-align can identify multi-target sites as well potential for non-cleaved targets sites by change the default settings. Windows, web and command-line versions were developed to better serve different users. AVAILABILITY: http://www.leonxie.com/targetAlign.php. CONTACT: zhangb@ecu.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Identification and characterization of microRNAs and their targets in the bioenergy plant switchgrass (Panicum virgatum).

MicroRNAs (miRNAs) are a class of non-coding small endogenous RNAs with lengths of approximately 22 nucleotides (nt) that have been shown to regulate gene expression at the post-transcriptional levels by targeting mRNAs for degradation or by inhibiting protein translation. Although thousands of miRNAs have been identified in many species, miRNAs have not yet been identified in switchgrass (Panicum virgatum), one of the most important bioenergy crops in the United States and around the world. In this study, we identified 121 potential switchgrass miRNAs, belonging to 44 families, using a well-defined comparative genome-based computational approach. We also identified miRNA clusters and antisense miRNAs in switchgrass expressed sequences tags. These identified miRNAs potentially target 839 protein-coding genes, which can act as transcription factors, and take part in multiple biological and metabolic processes including sucrose and fat metabolism, signal transduction, stress response, and plant development. Gene ontology (GO) analysis, based on these targets, showed that 527 biological processes were involved. Twenty-five of these processes were demonstrated to participate in the metabolism of carbon, glucose, starch, fatty acid, and lignin and in xylem formation. According to pathway enrichment analysis based on Kyoto Encyclopedia of Genes and Genomes (KEGG), 118 metabolism networks were found. These networks are involved in sucrose metabolism, fat metabolism, carbon fixation, hormone regulation, oxidative stress response, and the processing of other secondary metabolites.

Identification and characterization of microRNAs and their target genes in tobacco (Nicotiana tabacum).

microRNAs (miRNAs) are a recently discovered class of small (~21 nt) endogenous gene regulators that have been shown to play an important role in plant growth and development by aiding in organ maturation, hormone signaling, tissue differentiation, and plant tolerance to environmental stress. Since a list of miRNAs has never been generated for tobacco, we employed genome survey sequence analysis to computationally identify 259 potentially conserved tobacco miRNAs, belonging to 65 families, and validated 11 of these miRNAs using qRT-PCR. The 65 miRNA families were dramatically different in size. miRNA precursor (pre-miRNA) sequence analysis showed that tobacco pre-miRNAs greatly varied from 45 to 635 nt in length with an average of 141 ± 108 nt. We were also able to determine the presence of antisense miRNAs as well as miRNA clusters in tobacco. Using previously established protocols, a total of 1,225 potential target genes were predicted for the newly identified tobacco miRNAs. These target genes include transcription factors, DNA replication proteins, metabolic enzymes, as well as other gene targets necessary for proper plant maturation. The results of this study show that conserved miRNAs exist in tobacco and suggest that these miRNAs may play an important role in tobacco growth and development.

microRNA response in potato virus Y infected tobacco shows strain-specificity depending on host and symptom severity.

The present study demonstrates how different potato virus Y (PVY) strains affect the miRNA balance in tobacco cv. Samsun. The two prevalent strains PVYNTN and PVYN-Wi caused severe and mild veinal necrosis (VN) respectively, and the unique PVYZ-NTN strain induced milder vein clearing (VCl) in the upper non-inoculated leaves. A single amino acid polymorphisms (SAPs) I252V and a Q412 to R412 substitution in the HC-Pro cistron of the PVYZ-NTN strain might relate to the loss of VN in tobacco. The abundance of 18 out of the 26 tested miRNAs was increased upon infection by the severe strains PVYNTN and PVYN-Wi. Expression of a group of defense related transcripts were increased accordingly. Two miRNAs, nta-miR6020a-5p and nta-miR6164a/b, which target the TIR-NBS-LRR type resistant TMV N genes involving in signal transduction, might correlate with the PVYNTN and PVYN-Wi induced VN. The down-regulated mRNAs, e.g., RAP2-7 and TOE3, PXC3, LRR-RLK, ATHB-14 and TCP4 targeted by nta-miR172, nta-miR390, nta-miR482, nta-miR166 and nta-miR319/159 respectively, were related to regulation of transcription, protein phosphorylation and cell differentiation. The observed strain-specific alteration of miRNAs and their targets are host dependent and corresponds to the symptom severity and the viral HC-Pro RNA levels.