P-SAMS: a web suite for plant artificial microRNA and synthetic trans-acting small interfering RNA design

[EN] The Plant Small RNA Maker Site (P-SAMS) is a web tool for the simple and automated design of artificial miRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syntasiRNAs) for efficient and specific targeted gene silencing in plants. P-SAMS includes two applications, P-SAMS amiRNA Designer and P-SAMS syn-tasiRNA Designer. The navigation through both applications is wizard-assisted, and the job runtime is relatively short. Both applications output the sequence of designed small RNA(s), and the sequence of the two oligonucleotides required for cloning into `B/c¿ compatible vectors.This work was supported by the National Institutes of Health [grant number AI043288 to J.C.C.]; the National Science Foundation [grants numbers MCB-1231726, MCB-1330562 to J.C.C.]; and the United States Department of Agriculture [fellowship number MOW-2012-01361 to N.F.).Fahlgren, N.; Hill, ST.; Carrington, JC.; Carbonell, A. (2016). P-SAMS: a web suite for plant artificial microRNA and synthetic trans-acting small interfering RNA design. Bioinformatics. 32(1):157-158. https://doi.org/10.1093/bioinformatics/btv534S157158321Ahmed, F., Dai, X., & Zhao, P. X. (2015). Bioinformatics Tools for Achieving Better Gene Silencing in Plants. Plant Gene Silencing, 43-60. doi:10.1007/978-1-4939-2453-0_3Carbonell, A., Takeda, A., Fahlgren, N., Johnson, S. C., Cuperus, J. T., & Carrington, J. C. (2014). New Generation of Artificial MicroRNA and Synthetic Trans-Acting Small Interfering RNA Vectors for Efficient Gene Silencing in Arabidopsis. Plant Physiology, 165(1), 15-29. doi:10.1104/pp.113.234989Carbonell, A., Fahlgren, N., Mitchell, S., Cox, K. L., Reilly, K. C., Mockler, T. C., & Carrington, J. C. (2015). Highly specific gene silencing in a monocot species by artificial micro RNA s derived from chimeric mi RNA precursors. The Plant Journal, 82(6), 1061-1075. doi:10.1111/tpj.12835Fahlgren, N., & Carrington, J. C. (2009). miRNA Target Prediction in Plants. Plant MicroRNAs, 51-57. doi:10.1007/978-1-60327-005-2_4Ossowski, S., Schwab, R., & Weigel, D. (2008). Gene silencing in plants using artificial microRNAs and other small RNAs. The Plant Journal, 53(4), 674-690. doi:10.1111/j.1365-313x.2007.03328.xSchwab, R., Ossowski, S., Riester, M., Warthmann, N., & Weigel, D. (2006). Highly Specific Gene Silencing by Artificial MicroRNAs inArabidopsis. The Plant Cell, 18(5), 1121-1133. doi:10.1105/tpc.105.039834Tiwari, M., Sharma, D., & Trivedi, P. K. (2014). Artificial microRNA mediated gene silencing in plants: progress and perspectives. Plant Molecular Biology, 86(1-2), 1-18. doi:10.1007/s11103-014-0224-7Zhang, Z. J. (2014). Artificial trans-acting small interfering RNA: a tool for plant biology study and crop improvements. Planta, 239(6), 1139-1146. doi:10.1007/s00425-014-2054-

New generation of artificial microRNA and synthetic trans-acting small interfering RNA vectors for efficient gene silencing in Arabidopsis

[EN] Artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs) are used for small RNA-based, specific gene silencing or knockdown in plants. Current methods to generate amiRNA or syn-tasiRNA constructs are not well adapted for cost-effective, large-scale production or for multiplexing to specifically suppress multiple targets. Here, we describe simple, fast, and cost-effective methods with high-throughput capability to generate amiRNA and multiplexed syn-tasiRNA constructs for efficient gene silencing in Arabidopsis (Arabidopsis thaliana) and other plant species. amiRNA or syn-tasiRNA inserts resulting from the annealing of two overlapping and partially complementary oligonucleotides are ligated directionally into a zero background BsaI/ccdB-based expression vector. BsaI/ccdB vectors for amiRNA or syn-tasiRNA cloning and expression contain a modified version of Arabidopsis MIR390a or TAS1c precursors, respectively, in which a fragment of the endogenous sequence was substituted by a ccdB cassette flanked by two BsaI sites. Several amiRNA and syn-tasiRNA sequences designed to target one or more endogenous genes were validated in transgenic plants that (1) exhibited the expected phenotypes predicted by loss of target gene function, (2) accumulated high levels of accurately processed amiRNAs or syn-tasiRNAs, and (3) had reduced levels of the corresponding target RNAs.This work was supported by the National Science Foundation (grant nos. MCB-0956526 and MCB-1231726), the National Institutes of Health (grant no. AI043288), the Japan Society for the Promotion of Science (postdoctoral fellowship to A.T.), and the National Institute of Food and Agriculture (postdoctoral fellowship no. MOW-2012-01361 to N.F.)Carbonell, A.; Takeda, A.; Fahlgren, N.; Johnson, SC.; Cuperus, JT.; Carrington, JC. (2014). New generation of artificial microRNA and synthetic trans-acting small interfering RNA vectors for efficient gene silencing in Arabidopsis. PLANT PHYSIOLOGY. 165(1):15-29. https://doi.org/10.1104/pp.113.234989S1529165

Preparation of multiplexed small RNA libraries from plants

[EN] High-throughput sequencing is a powerful tool for exploring small RNA populations in plants. The ever-increasing output from an Illumina Sequencing System allows for multiplexing multiple samples while still obtaining sufficient data for small RNA discovery and characterization. Here we describe a protocol for generating multiplexed small RNA libraries for sequencing up to 12 samples in one lane of an Illumina HiSeq System single-end, 50 base pair run. RNA ligases are used to add the 3¿ and 5¿ adaptors to purified small RNAs; ligation products that lack a small RNA molecule (adaptor-adaptor products) are intentionally depleted. After cDNA synthesis, a linear PCR step amplifies the DNA fragments. The 3¿ PCR primers used here include unique 6- nucleotide sequences to allow for multiplexing up to 12 samples.The original version of this protocol was described in Carbonell et al. (2012). The updated version of the protocol was described in Carbonell et al. (2014). This work was supported by grants from the National Science Foundation (MCB-0956526, MCB-1231726) and National Institutes of Health (AI043288)Gilbert, KB.; Fahlgren, N.; Kasschau, KD.; Chapman, EJ.; Carrington, JC.; Carbonell, A. (2014). Preparation of multiplexed small RNA libraries from plants. Bio-protocol. 4(21):1-17. https://doi.org/10.21769/BioProtoc.1275S11742

Genome-wide profiling of Populus small RNAs

<p>Abstract</p> <p>Background</p> <p>Short RNAs, and in particular microRNAs, are important regulators of gene expression both within defined regulatory pathways and at the epigenetic scale. We investigated the short RNA (sRNA) population (18-24 nt) of the transcriptome of green leaves from the sequenced <it>Populus trichocarpa </it>using a concatenation strategy in combination with 454 sequencing.</p> <p>Results</p> <p>The most abundant size class of sRNAs were 24 nt. Long Terminal Repeats were particularly associated with 24 nt sRNAs. Additionally, some repetitive elements were associated with 22 nt sRNAs. We identified an sRNA hot-spot on chromosome 19, overlapping a region containing both the proposed sex-determining locus and a major cluster of <it>NBS-LRR </it>genes. A number of phased siRNA loci were identified, a subset of which are predicted to target PPR and <it>NBS-LRR </it>disease resistance genes, classes of genes that have been significantly expanded in <it>Populus</it>. Additional loci enriched for sRNA production were identified and characterised. We identified 15 novel predicted microRNAs (miRNAs), including miRNA*sequences, and identified a novel locus that may encode a dual miRNA or a miRNA and short interfering RNAs (siRNAs).</p> <p>Conclusions</p> <p>The short RNA population of <it>P. trichocarpa </it>is at least as complex as that of <it>Arabidopsis thaliana</it>. We provide a first genome-wide view of short RNA production for <it>P. trichocarpa </it>and identify new, non-conserved miRNAs.</p

Highly specific gene silencing in a monocot species by artificial microRNAs derived from chimeric MIRNA precursors

[EN] Artificial microRNAs (amiRNAs) are used for selective gene silencing in plants. However, current methods to produce amiRNA constructs for silencing transcripts in monocot species are not suitable for simple, cost-effective and large-scale synthesis. Here, a series of expression vectors based on Oryza sativa MIR390 (OsMIR390) precursor was developed for high-throughput cloning and high expression of amiRNAs in monocots. Four different amiRNA sequences designed to target specifically endogenous genes and expressed from OsMIR390-based vectors were validated in transgenic Brachypodium distachyon plants. Surprisingly, amiRNAs accumulated to higher levels and were processed more accurately when expressed from chimeric OsMIR390-based precursors that include distal stem-loop sequences from Arabidopsis thaliana MIR390a (AtMIR390a). In all cases, transgenic plants displayed the predicted phenotypes induced by target gene repression, and accumulated high levels of amiRNAs and low levels of the corresponding target transcripts. Genome-wide transcriptome profiling combined with 5¿-RLM-RACE analysis in transgenic plants confirmed that amiRNAs were highly specific.We thank Goretti Nguyen, Robyn Stevens, Jacob Mreen, Fangfang Ma and Madison Schniers for invaluable technical assistance, and Zacchery R. Smith for his initial contribution to develop the pH7WG2B-OsMIR390-B/c vector. Noah Fahlgren was supported by a USDA AFRI NIFA Postdoctoral Fellowship (MOW-2012-01361). This work was supported by grants from the National Science Foundation (MCB-1231726, MCB-1330562) and National Institutes of Health (AI043288) to James C. Carrington, and from the Department of Energy (DOE DE-SC0006627) to Todd C. Mockler.Carbonell, A.; Fahlgren, N.; Mitchell, S.; Cox, KLJ.; Reilly, KC.; Mockler, TC.; Carrington, JC. (2015). Highly specific gene silencing in a monocot species by artificial microRNAs derived from chimeric MIRNA precursors. The Plant Journal. 82(6):1061-1075. https://doi.org/10.1111/tpj.12835S10611075826Addo-Quaye, C., Eshoo, T. W., Bartel, D. P., & Axtell, M. J. (2008). Endogenous siRNA and miRNA Targets Identified by Sequencing of the Arabidopsis Degradome. Current Biology, 18(10), 758-762. doi:10.1016/j.cub.2008.04.042Alvarez, J. P., Pekker, I., Goldshmidt, A., Blum, E., Amsellem, Z., & Eshed, Y. (2006). Endogenous and Synthetic MicroRNAs Stimulate Simultaneous, Efficient, and Localized Regulation of Multiple Targets in Diverse Species. The Plant Cell, 18(5), 1134-1151. doi:10.1105/tpc.105.040725Arikit, S., Zhai, J., & Meyers, B. C. (2013). Biogenesis and function of rice small RNAs from non-coding RNA precursors. Current Opinion in Plant Biology, 16(2), 170-179. doi:10.1016/j.pbi.2013.01.006Axtell, M. J. (2013). Classification and Comparison of Small RNAs from Plants. Annual Review of Plant Biology, 64(1), 137-159. doi:10.1146/annurev-arplant-050312-120043Axtell, M. J., Jan, C., Rajagopalan, R., & Bartel, D. P. (2006). A Two-Hit Trigger for siRNA Biogenesis in Plants. Cell, 127(3), 565-577. doi:10.1016/j.cell.2006.09.032Bartel, D. P. (2004). MicroRNAs. Cell, 116(2), 281-297. doi:10.1016/s0092-8674(04)00045-5Bernard, P., & Couturier, M. (1992). Cell killing by the F plasmid CcdB protein involves poisoning of DNA-topoisomerase II complexes. Journal of Molecular Biology, 226(3), 735-745. doi:10.1016/0022-2836(92)90629-xBologna, N. G., & Voinnet, O. (2014). The Diversity, Biogenesis, and Activities of Endogenous Silencing Small RNAs in Arabidopsis. Annual Review of Plant Biology, 65(1), 473-503. doi:10.1146/annurev-arplant-050213-035728Bouvier d’Yvoire, M., Bouchabke-Coussa, O., Voorend, W., Antelme, S., Cézard, L., Legée, F., … Sibout, R. (2012). Disrupting thecinnamyl alcohol dehydrogenase 1gene (BdCAD1) leads to altered lignification and improved saccharification inBrachypodium distachyon. The Plant Journal, 73(3), 496-508. doi:10.1111/tpj.12053Butardo, V. M., Fitzgerald, M. A., Bird, A. R., Gidley, M. J., Flanagan, B. M., Larroque, O., … Rahman, S. (2011). Impact of down-regulation of starch branching enzyme IIb in rice by artificial microRNA- and hairpin RNA-mediated RNA silencing. Journal of Experimental Botany, 62(14), 4927-4941. doi:10.1093/jxb/err188Carbonell, A., Fahlgren, N., Garcia-Ruiz, H., Gilbert, K. B., Montgomery, T. A., Nguyen, T., … Carrington, J. C. (2012). Functional Analysis of Three Arabidopsis ARGONAUTES Using Slicer-Defective Mutants  . The Plant Cell, 24(9), 3613-3629. doi:10.1105/tpc.112.099945Carbonell, A., Takeda, A., Fahlgren, N., Johnson, S. C., Cuperus, J. T., & Carrington, J. C. (2014). New Generation of Artificial MicroRNA and Synthetic Trans-Acting Small Interfering RNA Vectors for Efficient Gene Silencing in Arabidopsis. Plant Physiology, 165(1), 15-29. doi:10.1104/pp.113.234989Chen, H., Jiang, S., Zheng, J., & Lin, Y. (2012). Improving panicle exsertion of rice cytoplasmic male sterile line by combination of artificial microRNA and artificial target mimic. Plant Biotechnology Journal, 11(3), 336-343. doi:10.1111/pbi.12019Chen, M., Wei, X., Shao, G., Tang, S., Luo, J., & Hu, P. (2012). Fragrance of the rice grain achieved via artificial microRNA-induced down-regulation ofOsBADH2. Plant Breeding, 131(5), 584-590. doi:10.1111/j.1439-0523.2012.01989.xCuperus, J. T., Carbonell, A., Fahlgren, N., Garcia-Ruiz, H., Burke, R. T., Takeda, A., … Carrington, J. C. (2010). Unique functionality of 22-nt miRNAs in triggering RDR6-dependent siRNA biogenesis from target transcripts in Arabidopsis. Nature Structural & Molecular Biology, 17(8), 997-1003. doi:10.1038/nsmb.1866Cuperus, J. T., Fahlgren, N., & Carrington, J. C. (2011). Evolution and Functional Diversification of MIRNA Genes. The Plant Cell, 23(2), 431-442. doi:10.1105/tpc.110.082784Endo, Y., Iwakawa, H., & Tomari, Y. (2013). Arabidopsis ARGONAUTE7 selects miR390 through multiple checkpoints during RISC assembly. EMBO reports, 14(7), 652-658. doi:10.1038/embor.2013.73Fahlgren, N., & Carrington, J. C. (2009). miRNA Target Prediction in Plants. Plant MicroRNAs, 51-57. doi:10.1007/978-1-60327-005-2_4Felippes, F. F., & Weigel, D. (2009). Triggering the formation of tasiRNAs in Arabidopsis thaliana  : the role of microRNA miR173. EMBO reports, 10(3), 264-270. doi:10.1038/embor.2008.247Gilbert, K., Fahlgren, N., Kasschau, K., Chapman, E., Carrington, J., & Carbonell, A. (2014). Preparation of Multiplexed Small RNA Libraries from Plants. BIO-PROTOCOL, 4(21). doi:10.21769/bioprotoc.1275Guo, Y., Han, Y., Ma, J., Wang, H., Sang, X., & Li, M. (2014). Undesired Small RNAs Originate from an Artificial microRNA Precursor in Transgenic Petunia (Petunia hybrida). PLoS ONE, 9(6), e98783. doi:10.1371/journal.pone.0098783He, G., Zhu, X., Elling, A. A., Chen, L., Wang, X., Guo, L., … Deng, X.-W. (2010). Global Epigenetic and Transcriptional Trends among Two Rice Subspecies and Their Reciprocal Hybrids. The Plant Cell, 22(1), 17-33. doi:10.1105/tpc.109.072041Heisel, S. E., Zhang, Y., Allen, E., Guo, L., Reynolds, T. L., Yang, X., … Roberts, J. K. (2008). Characterization of Unique Small RNA Populations from Rice Grain. PLoS ONE, 3(8), e2871. doi:10.1371/journal.pone.0002871Johnson, C., Kasprzewska, A., Tennessen, K., Fernandes, J., Nan, G.-L., Walbot, V., … Bowman, L. H. (2009). Clusters and superclusters of phased small RNAs in the developing inflorescence of rice. Genome Research, 19(8), 1429-1440. doi:10.1101/gr.089854.108Karimi, M., Inzé, D., & Depicker, A. (2002). GATEWAY™ vectors for Agrobacterium-mediated plant transformation. Trends in Plant Science, 7(5), 193-195. doi:10.1016/s1360-1385(02)02251-3Kozomara, A., & Griffiths-Jones, S. (2013). miRBase: annotating high confidence microRNAs using deep sequencing data. Nucleic Acids Research, 42(D1), D68-D73. doi:10.1093/nar/gkt1181Liang, G., He, H., Li, Y., & Yu, D. (2012). A new strategy for construction of artificial miRNA vectors in Arabidopsis. Planta, 235(6), 1421-1429. doi:10.1007/s00425-012-1610-5Liu, Q., Wang, F., & Axtell, M. J. (2014). Analysis of Complementarity Requirements for Plant MicroRNA Targeting Using a Nicotiana benthamiana Quantitative Transient Assay  . The Plant Cell, 26(2), 741-753. doi:10.1105/tpc.113.120972Mi, S., Cai, T., Hu, Y., Chen, Y., Hodges, E., Ni, F., … Qi, Y. (2008). Sorting of Small RNAs into Arabidopsis Argonaute Complexes Is Directed by the 5′ Terminal Nucleotide. Cell, 133(1), 116-127. doi:10.1016/j.cell.2008.02.034Montgomery, T. A., Howell, M. D., Cuperus, J. T., Li, D., Hansen, J. E., Alexander, A. L., … Carrington, J. C. (2008). Specificity of ARGONAUTE7-miR390 Interaction and Dual Functionality in TAS3 Trans-Acting siRNA Formation. Cell, 133(1), 128-141. doi:10.1016/j.cell.2008.02.033Ossowski, S., Schwab, R., & Weigel, D. (2008). Gene silencing in plants using artificial microRNAs and other small RNAs. The Plant Journal, 53(4), 674-690. doi:10.1111/j.1365-313x.2007.03328.xOster, U., Tanaka, R., Tanaka, A., & Rüdiger, W. (2000). Cloning and functional expression of the gene encoding the key enzyme for chlorophyll b biosynthesis (CAO) from Arabidopsis thaliana. The Plant Journal, 21(3), 305-310. doi:10.1046/j.1365-313x.2000.00672.xPhilippar, K., Geis, T., Ilkavets, I., Oster, U., Schwenkert, S., Meurer, J., & Soll, J. (2007). Chloroplast biogenesis: The use of mutants to study the etioplast-chloroplast transition. Proceedings of the National Academy of Sciences, 104(2), 678-683. doi:10.1073/pnas.0610062104Rapaport, F., Khanin, R., Liang, Y., Pirun, M., Krek, A., Zumbo, P., … Betel, D. (2013). Comprehensive evaluation of differential gene expression analysis methods for RNA-seq data. Genome Biology, 14(9), R95. doi:10.1186/gb-2013-14-9-r95Schwab, R., Ossowski, S., Riester, M., Warthmann, N., & Weigel, D. (2006). Highly Specific Gene Silencing by Artificial MicroRNAs inArabidopsis. The Plant Cell, 18(5), 1121-1133. doi:10.1105/tpc.105.039834Takeda, A., Iwasaki, S., Watanabe, T., Utsumi, M., & Watanabe, Y. (2008). The Mechanism Selecting the Guide Strand from Small RNA Duplexes is Different Among Argonaute Proteins. Plant and Cell Physiology, 49(4), 493-500. doi:10.1093/pcp/pcn043Tanaka, A., Ito, H., Tanaka, R., Tanaka, N. K., Yoshida, K., & Okada, K. (1998). Chlorophyll a oxygenase (CAO) is involved in chlorophyll b formation from chlorophyll a. Proceedings of the National Academy of Sciences, 95(21), 12719-12723. doi:10.1073/pnas.95.21.12719Thole, V., Peraldi, A., Worland, B., Nicholson, P., Doonan, J. H., & Vain, P. (2011). T-DNA mutagenesis in Brachypodium distachyon. Journal of Experimental Botany, 63(2), 567-576. doi:10.1093/jxb/err333Tiwari, M., Sharma, D., & Trivedi, P. K. (2014). Artificial microRNA mediated gene silencing in plants: progress and perspectives. Plant Molecular Biology, 86(1-2), 1-18. doi:10.1007/s11103-014-0224-7Trabucco, G. M., Matos, D. A., Lee, S. J., Saathoff, A. J., Priest, H. D., Mockler, T. C., … Hazen, S. P. (2013). Functional characterization of cinnamyl alcohol dehydrogenase and caffeic acid O-methyltransferase in Brachypodium distachyon. BMC Biotechnology, 13(1). doi:10.1186/1472-6750-13-61Vogel, J., & Hill, T. (2007). High-efficiency Agrobacterium-mediated transformation of Brachypodium distachyon inbred line Bd21-3. Plant Cell Reports, 27(3), 471-478. doi:10.1007/s00299-007-0472-yWang, L., Si, Y., Dedow, L. K., Shao, Y., Liu, P., & Brutnell, T. P. (2011). A Low-Cost Library Construction Protocol and Data Analysis Pipeline for Illumina-Based Strand-Specific Multiplex RNA-Seq. PLoS ONE, 6(10), e26426. doi:10.1371/journal.pone.0026426Warthmann, N., Chen, H., Ossowski, S., Weigel, D., & Hervé, P. (2008). Highly Specific Gene Silencing by Artificial miRNAs in Rice. PLoS ONE, 3(3), e1829. doi:10.1371/journal.pone.0001829Zeng, L.-R., Qu, S., Bordeos, A., Yang, C., Baraoidan, M., Yan, H., … Wang, G.-L. (2004). Spotted leaf11, a Negative Regulator of Plant Cell Death and Defense, Encodes a U-Box/Armadillo Repeat Protein Endowed with E3 Ubiquitin Ligase Activityw⃞. The Plant Cell, 16(10), 2795-2808. doi:10.1105/tpc.104.025171Zhang, X., Niu, D., Carbonell, A., Wang, A., Lee, A., Tun, V., … Jin, H. (2014). 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Genome-Wide Profiling and Analysis of Arabidopsis siRNAs

Eukaryotes contain a diversified set of small RNA-guided pathways that control genes, repeated sequences, and viruses at the transcriptional and posttranscriptional levels. Genome-wide profiles and analyses of small RNAs, particularly the large class of 24-nucleotide (nt) short interfering RNAs (siRNAs), were done for wild-type Arabidopsis thaliana and silencing pathway mutants with defects in three RNA-dependent RNA polymerase (RDR) and four Dicer-like (DCL) genes. The profiling involved direct analysis using a multiplexed, parallel-sequencing strategy. Small RNA-generating loci, especially those producing predominantly 24-nt siRNAs, were found to be highly correlated with repetitive elements across the genome. These were found to be largely RDR2- and DCL3-dependent, although alternative DCL activities were detected on a widespread level in the absence of DCL3. In contrast, no evidence for RDR2-alternative activities was detected. Analysis of RDR2- and DCL3-dependent small RNA accumulation patterns in and around protein-coding genes revealed that upstream gene regulatory sequences systematically lack siRNA-generating activities. Further, expression profiling suggested that relatively few genes, proximal to abundant 24-nt siRNAs, are regulated directly by RDR2- and DCL3-dependent silencing. We conclude that the widespread accumulation patterns for RDR2- and DCL3-dependent siRNAs throughout the Arabidopsis genome largely reflect mechanisms to silence highly repeated sequences

Genetic Diversity and Population Structure of a Camelina sativa Spring Panel

There is a need to explore renewable alternatives (e.g., biofuels) that can produce energy sources to help reduce the reliance on fossil oils. In addition, the consumption of fossil oils adversely affects the environment and human health via the generation of waste water, greenhouse gases, and waste solids. Camelina sativa, originated from southeastern Europe and southwestern Asia, is being re-embraced as an industrial oilseed crop due to its high seed oil content (36–47%) and high unsaturated fatty acid composition (\u3e90%), which are suitable for jet fuel, biodiesel, high-value lubricants and animal feed. C. sativa’s agronomic advantages include short time to maturation, low water and nutrient requirements, adaptability to adverse environmental conditions and resistance to common pests and pathogens. These characteristics make it an ideal crop for sustainable agricultural systems and regions of marginal land. However, the lack of genetic and genomic resources has slowed the enhancement of this emerging oilseed crop and exploration of its full agronomic and breeding potential. Here, a core of 213 spring C. sativa accessions was collected and genotyped. The genotypic data was used to characterize genetic diversity and population structure to infer how natural selection and plant breeding may have affected the formation and differentiation within the C. sativa natural populations, and how the genetic diversity of this species can be used in future breeding efforts. A total of 6,192 high-quality single nucleotide polymorphisms (SNPs) were identified using genotypingby- sequencing (GBS) technology. The average polymorphism information content (PIC) value of 0.29 indicate moderate genetic diversity for the C. sativa spring panel evaluated in this report. Population structure and principal coordinates analyses (PCoA) based on SNPs revealed two distinct subpopulations. Sub-population 1 (POP1) contains accessions that mainly originated from Germany while the majority of POP2 accessions (\u3e75%) were collected from Eastern Europe. Analysis of molecular variance (AMOVA) identified 4% variance among and 96% variance within subpopulations, indicating a hig

Functional analysis of three Arabidopsis ARGONAUTES using slicer-defective mutants

[EN] In RNA-directed silencing pathways, ternary complexes result from small RNA-guided ARGONAUTE (AGO) associating with target transcripts. Target transcripts are often silenced through direct cleavage (slicing), destabilization through slicer-independent turnover mechanisms, and translational repression. Here, wild-type and active-site defective forms of several Arabidopsis thaliana AGO proteins involved in posttranscriptional silencing were used to examine several AGO functions, including small RNA binding, interaction with target RNA, slicing or destabilization of target RNA, secondary small interfering RNA formation, and antiviral activity. Complementation analyses in ago mutant plants revealed that the catalytic residues of AGO1, AGO2, and AGO7 are required to restore the defects of Arabidopsis ago1-25, ago2-1, and zip-1 (AGO7-defective) mutants, respectively. AGO2 had slicer activity in transient assays but could not trigger secondary small interfering RNA biogenesis, and catalytically active AGO2 was necessary for local and systemic antiviral activity against Turnip mosaic virus. Slicer-defective AGOs associated with miRNAs and stabilized AGO-miRNA-target RNA ternary complexes in individual target coimmunoprecipitation assays. In genome-wide AGO-miRNA-target RNA coimmunoprecipitation experiments, slicer-defective AGO1-miRNA associated with target RNA more effectively than did wild-type AGO1-miRNA. These data not only reveal functional roles for AGO1, AGO2, and AGO7 slicer activity, but also indicate an approach to capture ternary complexes more efficiently for genome-wide analyses.We thank Goretti Nguyen for excellent technical assistance. A. C. was supported by a postdoctoral fellowship from the Ministerio de Ciencia e Innovacion (BMC-2008-0188). H.G.-R. was the recipient of a Helen Hay Whitney Postdoctoral fellowship (F-972). This work was supported by grants from the National Science Foundation (MCB-1231726), the National Institutes of Health (AI043288), and Monsanto Corporation.Carbonell, A.; Fahlgren, N.; García-Ruíz, H.; Gilbert, KB.; Montgomery, TA.; Nguyen, T.; Cuperus, JT.... (2012). Functional analysis of three Arabidopsis ARGONAUTES using slicer-defective mutants. The Plant Cell. 24(9):3613-3629. https://doi.org/10.1105/tpc.112.099945S36133629249Allen, E., Xie, Z., Gustafson, A. M., & Carrington, J. C. (2005). microRNA-Directed Phasing during Trans-Acting siRNA Biogenesis in Plants. Cell, 121(2), 207-221. doi:10.1016/j.cell.2005.04.004Aukerman, M. J., & Sakai, H. (2003). Regulation of Flowering Time and Floral Organ Identity by a MicroRNA and Its APETALA2-Like Target Genes. The Plant Cell, 15(11), 2730-2741. doi:10.1105/tpc.016238Axtell, M. J., Jan, C., Rajagopalan, R., & Bartel, D. P. (2006). A Two-Hit Trigger for siRNA Biogenesis in Plants. 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An automated, high-throughput method for standardizing image color profiles to improve image-based plant phenotyping

High-throughput phenotyping has emerged as a powerful method for studying plant biology. Large image-based datasets are generated and analyzed with automated image analysis pipelines. A major challenge associated with these analyses is variation in image quality that can inadvertently bias results. Images are made up of tuples of data called pixels, which consist of R, G, and B values, arranged in a grid. Many factors, for example image brightness, can influence the quality of the image that is captured. These factors alter the values of the pixels within images and consequently can bias the data and downstream analyses. Here, we provide an automated method to adjust an image-based dataset so that brightness, contrast, and color profile is standardized. The correction method is a collection of linear models that adjusts pixel tuples based on a reference panel of colors. We apply this technique to a set of images taken in a high-throughput imaging facility and successfully detect variance within the image dataset. In this case, variation resulted from temperature-dependent light intensity throughout the experiment. Using this correction method, we were able to standardize images throughout the dataset, and we show that this correction enhanced our ability to accurately quantify morphological measurements within each image. We implement this technique in a high-throughput pipeline available with this paper, and it is also implemented in PlantCV

Specificity of ARGONAUTE7-miR390 Interaction and Dual Functionality in TAS3 Trans-Acting siRNA Formation

SummaryTrans-acting siRNA form through a refined RNAi mechanism in plants. miRNA-guided cleavage triggers entry of precursor transcripts into an RNA-DEPENDENT RNA POLYMERASE6 pathway, and sets the register for phased tasiRNA formation by DICER-LIKE4. Here, we show that miR390-ARGONAUTE7 complexes function in distinct cleavage or noncleavage modes at two target sites in TAS3a transcripts. The AGO7 cleavage, but not the noncleavage, function could be provided by AGO1, the dominant miRNA-associated AGO, but only when AGO1 was guided to a modified target site through an alternate miRNA. AGO7 was highly selective for interaction with miR390, and miR390 in turn was excluded from association with AGO1 due entirely to an incompatible 5′ adenosine. Analysis of AGO1, AGO2, and AGO7 revealed a potent 5′ nucleotide discrimination function for some, although not all, ARGONAUTEs. miR390 and AGO7, therefore, evolved as a highly specific miRNA guide/effector protein pair to function at two distinct tasiRNA biogenesis steps