Genome sequence of the progenitor of the wheat D genome Aegilops tauschii.

Aegilops tauschii is the diploid progenitor of the D genome of hexaploid wheat (Triticum aestivum, genomes AABBDD) and an important genetic resource for wheat. The large size and highly repetitive nature of the Ae. tauschii genome has until now precluded the development of a reference-quality genome sequence. Here we use an array of advanced technologies, including ordered-clone genome sequencing, whole-genome shotgun sequencing, and BioNano optical genome mapping, to generate a reference-quality genome sequence for Ae. tauschii ssp. strangulata accession AL8/78, which is closely related to the wheat D genome. We show that compared to other sequenced plant genomes, including a much larger conifer genome, the Ae. tauschii genome contains unprecedented amounts of very similar repeated sequences. Our genome comparisons reveal that the Ae. tauschii genome has a greater number of dispersed duplicated genes than other sequenced genomes and its chromosomes have been structurally evolving an order of magnitude faster than those of other grass genomes. The decay of colinearity with other grass genomes correlates with recombination rates along chromosomes. We propose that the vast amounts of very similar repeated sequences cause frequent errors in recombination and lead to gene duplications and structural chromosome changes that drive fast genome evolution.

Insights into the Genetic Architecture and Genomic Prediction of Powdery Mildew Resistance in Flax (Linum usitatissimum L.).

Powdery mildew (PM), caused by the fungus Oidium lini in flax, can cause defoliation and reduce seed yield and quality. To date, one major dominant gene (Pm1) and three quantitative trait loci (QTL) on chromosomes 1, 7 and 9 have been reported for PM resistance. To fully dissect the genetic architecture of PM resistance and identify QTL, a diverse flax core collection of 372 accessions augmented with an additional 75 breeding lines were sequenced, and PM resistance was evaluated in the field for eight years (2010-2017) in Morden, Manitoba, Canada. Genome-wide association studies (GWAS) were performed using two single-locus and seven multi-locus statistical models with 247,160 single nucleotide polymorphisms (SNPs) and the phenotypes of the 447 individuals for each year separately as well as the means over years. A total of 349 quantitative trait nucleotides (QTNs) were identified, of which 44 large-effect QTNs (R2 = 10-30%) were highly stable over years. The total number of favourable alleles per accession was significantly correlated with PM resistance (r = 0.74), and genomic selection (GS) models using all identified QTNs generated significantly higher predictive ability (r = 0.93) than those constructed using the 247,160 genome-wide random SNP (r = 0.69), validating the overall reliability of the QTNs and showing the additivity of PM resistance in flax. The QTNs were clustered on the distal ends of all 15 chromosomes, especially on chromosome 5 (0.4-5.6 Mb and 9.4-16.9 Mb) and 13 (4.7-5.2 Mb). To identify candidate genes, a dataset of 3230 SNPs located in resistance gene analogues (RGAs) was used as input for GWAS, from which an additional 39 RGA-specific QTNs were identified. Overall, 269 QTN loci harboured 445 RGAs within the 200 Kb regions spanning the QTNs, including 45 QTNs located within the RGAs. These RGAs supported by significant QTN/SNP allele effects were mostly nucleotide binding site and leucine-rich repeat receptors (NLRs) belonging to either coiled-coil (CC) NLR (CNL) or toll interleukin-1 (TIR) NLR (TNL), receptor-like kinase (RLK), receptor-like protein kinase (RLP), transmembrane-coiled-coil (TM-CC), WRKY, and mildew locus O (MLO) genes. These results constitute an important genomic tool for resistance breeding and gene cloning for PM in flax.

Chromosome-scale pseudomolecules refined by optical, physical and genetic maps in flax.

Genomes of varying sizes have been sequenced with next-generation sequencing platforms. However, most reference sequences include draft unordered scaffolds containing chimeras caused by mis-scaffolding. A BioNano genome (BNG) optical map was constructed to improve the previously sequenced flax genome (Linum usitatissimum L., 2n = 30, about 373 Mb), which consisted of 3852 scaffolds larger than 1 kb and totalling 300.6 Mb. The high-resolution BNG map of cv. CDC Bethune totalled 317 Mb and consisted of 251 BNG contigs with an N50 of 2.15 Mb. A total of 622 scaffolds (286.6 Mb, 94.9%) aligned to 211 BNG contigs (298.6 Mb, 94.2%). Of those, 99 scaffolds, diagnosed to contain assembly errors, were refined into 225 new scaffolds. Using the newly refined scaffold sequences and the validated bacterial artificial chromosome-based physical map of CDC Bethune, the 211 BNG contigs were scaffolded into 94 super-BNG contigs (N50 of 6.64 Mb) that were further assigned to the 15 flax chromosomes using the genetic map. The pseudomolecules total about 316 Mb, with individual chromosomes of 15.6 to 29.4 Mb, and cover 97% of the annotated genes. Evidence from the chromosome-scale pseudomolecules suggests that flax has undergone palaeopolyploidization and mesopolyploidization events, followed by rearrangements and deletions or fusion of chromosome arms from an ancient progenitor with a haploid chromosome number of eight.

Evaluation of Genomic Prediction for Pasmo Resistance in Flax.

Pasmo (Septoria linicola) is a fungal disease causing major losses in seed yield and quality and stem fibre quality in flax. Pasmo resistance (PR) is quantitative and has low heritability. To improve PR breeding efficiency, the accuracy of genomic prediction (GP) was evaluated using a diverse worldwide core collection of 370 accessions. Four marker sets, including three defined by 500, 134 and 67 previously identified quantitative trait loci (QTL) and one of 52,347 PR-correlated genome-wide single nucleotide polymorphisms, were used to build ridge regression best linear unbiased prediction (RR-BLUP) models using pasmo severity (PS) data collected from field experiments performed during five consecutive years. With five-fold random cross-validation, GP accuracy as high as 0.92 was obtained from the models using the 500 QTL when the average PS was used as the training dataset. GP accuracy increased with training population size, reaching values >0.9 with training population size greater than 185. Linear regression of the observed PS with the number of positive-effect QTL in accessions provided an alternative GP approach with an accuracy of 0.86. The results demonstrate the GP models based on marker information from all identified QTL and the 5-year PS average is highly effective for PR prediction.

Genome-Wide Association Study and Selection Signatures Detect Genomic Regions Associated with Seed Yield and Oil Quality in Flax.

A genome-wide association study (GWAS) was performed on a set of 260 lines which belong to three different bi-parental flax mapping populations. These lines were sequenced to an averaged genome coverage of 19× using the Illumina Hi-Seq platform. Phenotypic data for 11 seed yield and oil quality traits were collected in eight year/location environments. A total of 17,288 single nucleotide polymorphisms were identified, which explained more than 80% of the phenotypic variation for days to maturity (DTM), iodine value (IOD), palmitic (PAL), stearic, linoleic (LIO) and linolenic (LIN) acid contents. Twenty-three unique genomic regions associated with 33 quantitative trait loci (QTL) for the studied traits were detected, thereby validating four genomic regions previously identified. The 33 QTL explained 48⁻73% of the phenotypic variation for oil content, IOD, PAL, LIO and LIN but only 8⁻14% for plant height, DTM and seed yield. A genome-wide selective sweep scan for selection signatures detected 114 genomic regions that accounted for 7.82% of the flax pseudomolecule and overlapped with the 11 GWAS-detected genomic regions associated with 18 QTL for 11 traits. The results demonstrate the utility of GWAS combined with selection signatures for dissection of the genetic structure of traits and for pinpointing genomic regions for breeding improvement.

Secondary siRNAs from Medicago NB-LRRs modulated via miRNA-target interactions and their abundances.

Small RNAs are a class of non-coding RNAs that are of great importance in gene expression regulatory networks. Different families of small RNAs are generated via distinct biogenesis pathways. One such family specific to plants is that of phased, secondary siRNAs (phasiRNAs); these require RDR6, DCL4, and (typically) a microRNA (miRNA) trigger for their biogenesis. Protein-encoding genes are an important source of phasi-RNAs. The model legume Medicago truncatula generates phasiRNAs from many PHAS loci, and we aimed to investigate their biogenesis and mechanism by which miRNAs trigger these molecules. We modulated miRNA abundances in transgenic tissues showing that the abundance of phasiRNAs correlates with the levels of both miRNA triggers and the target, precursor transcripts. We identified sets of phasiRNAs or PHAS loci that predominantly and substantially increase in response to miRNA overexpression. In the process of validating targets from miRNA overexpression tissues, we found that in the miRNA-mRNA target pairing, the 3' terminal nucleotide (the 22nd position), but not the 10th position, is important for phasiRNA production. Mutating the single 3' terminal nucleotide dramatically diminishes phasiRNA production. Ectopic expression of Medicago NB-LRR-targeting miRNAs in Arabidopsis showed that only a few NB-LRRs are capable of phasiRNA production; our data indicate that this might be due to target inaccessibility determined by sequences flanking target sites. Our results suggest that target accessibility is an important component in miRNA-target interactions that could be utilized in target prediction, and the evolution of mRNA sequences flanking miRNA-target sites may be impacted.

RGAugury: a pipeline for genome-wide prediction of resistance gene analogs (RGAs) in plants.

BACKGROUND: Resistance gene analogs (RGAs), such as NBS-encoding proteins, receptor-like protein kinases (RLKs) and receptor-like proteins (RLPs), are potential R-genes that contain specific conserved domains and motifs. Thus, RGAs can be predicted based on their conserved structural features using bioinformatics tools. Computer programs have been developed for the identification of individual domains and motifs from the protein sequences of RGAs but none offer a systematic assessment of the different types of RGAs. A user-friendly and efficient pipeline is needed for large-scale genome-wide RGA predictions of the growing number of sequenced plant genomes. RESULTS: An integrative pipeline, named RGAugury, was developed to automate RGA prediction. The pipeline first identifies RGA-related protein domains and motifs, namely nucleotide binding site (NB-ARC), leucine rich repeat (LRR), transmembrane (TM), serine/threonine and tyrosine kinase (STTK), lysin motif (LysM), coiled-coil (CC) and Toll/Interleukin-1 receptor (TIR). RGA candidates are identified and classified into four major families based on the presence of combinations of these RGA domains and motifs: NBS-encoding, TM-CC, and membrane associated RLP and RLK. All time-consuming analyses of the pipeline are paralleled to improve performance. The pipeline was evaluated using the well-annotated Arabidopsis genome. A total of 98.5, 85.2, and 100 % of the reported NBS-encoding genes, membrane associated RLPs and RLKs were validated, respectively. The pipeline was also successfully applied to predict RGAs for 50 sequenced plant genomes. A user-friendly web interface was implemented to ease command line operations, facilitate visualization and simplify result management for multiple datasets. CONCLUSIONS: RGAugury is an efficiently integrative bioinformatics tool for large scale genome-wide identification of RGAs. It is freely available at Bitbucket: https://bitbucket.org/yaanlpc/rgaugury .

Pseudogenes and Their Genome-Wide Prediction in Plants.

Pseudogenes are paralogs generated from ancestral functional genes (parents) during genome evolution, which contain critical defects in their sequences, such as lacking a promoter, having a premature stop codon or frameshift mutations. Generally, pseudogenes are functionless, but recent evidence demonstrates that some of them have potential roles in regulation. The majority of pseudogenes are generated from functional progenitor genes either by gene duplication (duplicated pseudogenes) or retro-transposition (processed pseudogenes). Pseudogenes are primarily identified by comparison to their parent genes. Bioinformatics tools for pseudogene prediction have been developed, among which PseudoPipe, PSF and Shiu's pipeline are publicly available. We compared these three tools using the well-annotated Arabidopsis thaliana genome and its known 924 pseudogenes as a test data set. PseudoPipe and Shiu's pipeline identified ~80% of A. thaliana pseudogenes, of which 94% were shared, while PSF failed to generate adequate results. A need for improvement of the bioinformatics tools for pseudogene prediction accuracy in plant genomes was thus identified, with the ultimate goal of improving the quality of genome annotation in plants.

Synteny analysis in Rosids with a walnut physical map reveals slow genome evolution in long-lived woody perennials.

BACKGROUND: Mutations often accompany DNA replication. Since there may be fewer cell cycles per year in the germlines of long-lived than short-lived angiosperms, the genomes of long-lived angiosperms may be diverging more slowly than those of short-lived angiosperms. Here we test this hypothesis. RESULTS: We first constructed a genetic map for walnut, a woody perennial. All linkage groups were short, and recombination rates were greatly reduced in the centromeric regions. We then used the genetic map to construct a walnut bacterial artificial chromosome (BAC) clone-based physical map, which contained 15,203 exonic BAC-end sequences, and quantified with it synteny between the walnut genome and genomes of three long-lived woody perennials, Vitis vinifera, Populus trichocarpa, and Malus domestica, and three short-lived herbs, Cucumis sativus, Medicago truncatula, and Fragaria vesca. Each measure of synteny we used showed that the genomes of woody perennials were less diverged from the walnut genome than those of herbs. We also estimated the nucleotide substitution rate at silent codon positions in the walnut lineage. It was one-fifth and one-sixth of published nucleotide substitution rates in the Medicago and Arabidopsis lineages, respectively. We uncovered a whole-genome duplication in the walnut lineage, dated it to the neighborhood of the Cretaceous-Tertiary boundary, and allocated the 16 walnut chromosomes into eight homoeologous pairs. We pointed out that during polyploidy-dysploidy cycles, the dominant tendency is to reduce the chromosome number. CONCLUSION: Slow rates of nucleotide substitution are accompanied by slow rates of synteny erosion during genome divergence in woody perennials.

Disease Resistance Gene Analogs (RGAs) in Plants.

Plants have developed effective mechanisms to recognize and respond to infections caused by pathogens. Plant resistance gene analogs (RGAs), as resistance (R) gene candidates, have conserved domains and motifs that play specific roles in pathogens' resistance. Well-known RGAs are nucleotide binding site leucine rich repeats, receptor like kinases, and receptor like proteins. Others include pentatricopeptide repeats and apoplastic peroxidases. RGAs can be detected using bioinformatics tools based on their conserved structural features. Thousands of RGAs have been identified from sequenced plant genomes. High-density genome-wide RGA genetic maps are useful for designing diagnostic markers and identifying quantitative trait loci (QTL) or markers associated with plant disease resistance. This review focuses on recent advances in structures and mechanisms of RGAs, and their identification from sequenced genomes using bioinformatics tools. Applications in enhancing fine mapping and cloning of plant disease resistance genes are also discussed.

Data-Based Optimal Switching and Control With Admissibility Guaranteed Q -Learning.

This article addresses the data-based optimal switching and control codesign for discrete-time nonlinear switched systems via a two-stage approximate dynamic programming (ADP) algorithm. Through offline policy improvement and policy evaluation, the proposed algorithm iteratively determines the optimal hybrid control policy using system input/output data. Moreover, a strict proof of the convergence is given for the two-stage ADP algorithm. Admissibility, an essential property of the hybrid control policy must be ensured for practical application. To this end, the properties of the hybrid control policies are analyzed and an admissibility criterion is obtained. To realize the proposed Q -learning algorithm, an actor-critic neural network (NN) structure that employs multiple NNs to approximate the Q -functions and control policies for different subsystems is adopted. By applying the proposed admissibility criterion, the obtained hybrid control policy is guaranteed to be admissible. Finally, two numerical simulations verify the effectiveness of the proposed algorithm.

Rice RNA-dependent RNA polymerase 6 acts in small RNA biogenesis and spikelet development.

Higher plants have evolved multiple RNA-dependent RNA polymerases (RDRs), which work with Dicer-like (DCL) proteins to produce different classes of small RNAs with specialized molecular functions. Here we report that OsRDR6, the rice (Oryza sativa L.) homolog of Arabidopsis RDR6, acts in the biogenesis of various types and sizes of small RNAs. We isolated a rice osrdr6-1 mutant, which was temperature sensitive and showed spikelet defects. This mutant displays reduced accumulation of tasiR-ARFs, the conserved trans-acting siRNAs (tasiRNAs) derived from the TAS3 locus, and ectopic expression of tasiR-ARF target genes, the Auxin Response Factors (including ARF2 and ARF3/ETTIN). The loss of tasiR-mediated repression of ARFs in osrdr6-1 can explain its morphological defects, as expression of two non-targeted ARF3 gene constructs (ARF3muts) in a wild-type background mimics the osrdr6 and osdcl4-1 mutant phenotypes. Small RNA high-throughput sequencing also reveals that besides tasiRNAs, 21-nucleotide (nt) phased small RNAs are also largely dependent on OsRDR6. Unexpectedly, we found that osrdr6-1 has a strong impact on the accumulation of 24-nt phased small RNAs, but not on unphased ones. Our work uncovers the key roles of OsRDR6 in small RNA biogenesis and directly illustrates the crucial functions of tasiR-ARFs in rice development.

Roles of DCL4 and DCL3b in rice phased small RNA biogenesis.

Higher plants have evolved multiple proteins in the RNase III family to produce and regulate different classes of small RNAs with specialized molecular functions. In rice (Oryza sativa), numerous genomic clusters are targeted by one of two microRNAs (miRNAs), miR2118 and miR2275, to produce secondary small interfering RNAs (siRNAs) of either 21 or 24 nucleotides in a phased manner. The biogenesis requirements or the functions of the phased small RNAs are completely unknown. Here we examine the rice Dicer-Like (DCL) family, including OsDCL1, -3a, -3b and -4. By deep sequencing of small RNAs from different tissues of the wild type and osdcl4-1, we revealed that the processing of 21-nucleotide siRNAs, including trans-acting siRNAs (tasiRNA) and over 1000 phased small RNA loci, was largely dependent on OsDCL4. Surprisingly, the processing of 24-nucleotide phased small RNA requires the DCL3 homolog OsDCL3b rather than OsDCL3a, suggesting functional divergence within DCL3 family. RNA ligase-mediated 5' rapid amplification of cDNA ends and parallel analysis of RNA ends (PARE)/degradome analysis confirmed that most of the 21- and 24-nucleotide phased small RNA clusters were initiated from the target sites of miR2118 and miR2275, respectively. Furthermore, the accumulation of the two triggering miRNAs requires OsDCL1 activity. Finally, we show that phased small RNAs are preferentially produced in the male reproductive organs and are likely to be conserved in monocots. Our results revealed significant roles of OsDCL4, OsDCL3b and OsDCL1 in the 21- and 24-nucleotide phased small RNA biogenesis pathway in rice.

Novel Evaluation Method for Flushing Efficiency Based on the Principle of Wall Shear Rate Equality under High Temperature and High Pressure.

An excellent flushing efficiency evaluation device is indispensable for studying the cementing flushing fluid. However, the existing flushing efficiency evaluation device cannot simulate the formation and flushing process of the downhole mud cake. Therefore, this paper proposes a novel flushing efficiency evaluation device that can simulate the formation and flushing of downhole mud cakes. The rotational speed of the device during flushing and the rotor's diameter is deduced based on the principle of equal wall shear rate. The evaluation device and method can be used to quantitatively evaluate the flushing efficiency of the flushing liquid on the mud cake under high temperatures and high pressures. This device analyzed the effects of the annular gap, temperature, construction displacement, and flushing fluids on flushing efficiency. The results show that the smaller the annular gap, the higher the temperature, the larger the displacement, the higher the scouring efficiency, and the higher the shear bond strength. Fiber flushing has the dual function of mechanical and chemical flushing, so its flushing efficiency is 14.75% higher than that of heavy flushing fluid at 10 min. The surfactant in the emulsified rinse leads to a sudden increase in rinse efficiency in the middle and late stages. The reduced flushing efficiency of the flushing fluid is due to the reduced ζ potential.

Use of adenoviral vectors to target chemotherapy to tumor vascular endothelial cells suppresses growth of breast cancer and melanoma.

To target chemotherapy to tumor vascular endothelial cells (TVECs), we created the AdTie2RprCDFib(knob-RGD+) vector by inserting into an AdEasy adenoviral vector (Ad) backbone: (i) the cytosine deaminase (CD) gene driven by the Tie2 receptor promoter (Tie2Rpr) into the E1 region of Ad; (ii) mutations that reduce binding of the fiber knob to the Coxsackie adenovirus receptor (CAR); and (iii) the RGD peptide into the H1 loop of fiber for binding to the alpha(V)beta(3) integrin receptors on TVECs. To reduce uptake of the AdTie2RprCDFib(knob-RGD+) by reticuloendothelial (RE) and liver cells, we intravenously (i.v.) injected Hetastarch and low-dose Ad (one million vector particles (VPs)) prior to i.v. injection of a therapeutic dose (one billion VPs) of the AdTie2RprCDFib(knob-RGD+) vector. This treatment induced regressions of N202 breast cancer and B16 melanoma without toxicity to normal tissues. We showed that the tumor regression was induced by infection of the TVECs and not by the infection of tumor cells by the AdTie2RprCDFib(knob-RGD+) vector.

Small RNA-directed epigenetic natural variation in Arabidopsis thaliana.

Progress in epigenetics has revealed mechanisms that can heritably regulate gene function independent of genetic alterations. Nevertheless, little is known about the role of epigenetics in evolution. This is due in part to scant data on epigenetic variation among natural populations. In plants, small interfering RNA (siRNA) is involved in both the initiation and maintenance of gene silencing by directing DNA methylation and/or histone methylation. Here, we report that, in the model plant Arabidopsis thaliana, a cluster of approximately 24 nt siRNAs found at high levels in the ecotype Landsberg erecta (Ler) could direct DNA methylation and heterochromatinization at a hAT element adjacent to the promoter of FLOWERING LOCUS C (FLC), a major repressor of flowering, whereas the same hAT element in ecotype Columbia (Col) with almost identical DNA sequence, generates a set of low abundance siRNAs that do not direct these activities. We have called this hAT element MPF for Methylated region near Promoter of FLC, although de novo methylation triggered by an inverted repeat transgene at this region in Col does not alter its FLC expression. DNA methylation of the Ler allele MPF is dependent on genes in known silencing pathways, and such methylation is transmissible to Col by genetic crosses, although with varying degrees of penetrance. A genome-wide comparison of Ler and Col small RNAs identified at least 68 loci matched by a significant level of approximately 24 nt siRNAs present specifically in Ler but not Col, where nearly half of the loci are related to repeat or TE sequences. Methylation analysis revealed that 88% of the examined loci (37 out of 42) were specifically methylated in Ler but not Col, suggesting that small RNA can direct epigenetic differences between two closely related Arabidopsis ecotypes.

Aegilops tauschii genome assembly Aet v5.0 features greater sequence contiguity and improved annotation.

Aegilops tauschii is the donor of the D subgenome of hexaploid wheat and an important genetic resource. The reference-quality genome sequence Aet v4.0 for Ae. tauschii acc. AL8/78 was therefore an important milestone for wheat biology and breeding. Further advances in sequencing acc. AL8/78 and release of the Aet v5.0 sequence assembly are reported here. Two new optical maps were constructed and used in the revision of pseudomolecules. Gaps were closed with Pacific Biosciences long-read contigs, decreasing the gap number by 38,899. Transposable elements and protein-coding genes were reannotated. The number of annotated high-confidence genes was reduced from 39,635 in Aet v4.0 to 32,885 in Aet v5.0. A total of 2245 biologically important genes, including those affecting plant phenology, grain quality, and tolerance of abiotic stresses in wheat, was manually annotated and disease-resistance genes were annotated by a dedicated pipeline. Disease-resistance genes encoding nucleotide-binding site domains, receptor-like protein kinases, and receptor-like proteins were preferentially located in distal chromosome regions, whereas those encoding transmembrane coiled-coil proteins were dispersed more evenly along the chromosomes. Discovery, annotation, and expression analyses of microRNA (miRNA) precursors, mature miRNAs, and phasiRNAs are reported, including miRNA target genes. Other small RNAs, such as hc-siRNAs and tRFs, were characterized. These advances enhance the utility of the Ae. tauschii genome sequence for wheat genetics, biotechnology, and breeding.

Identification and expression analysis of miRNAs from nitrogen-fixing soybean nodules.

miRNAs are approximately 21nt non-coding RNAs and play important roles in plant development and response to stress. Symbiotic nitrogen fixation (SNF) is agronomically important for reducing the need of nitrogen fertilizers. The soybean root nodule is the place where SNF takes place. To identify miRNAs that are possibly involved in nitrogen fixation in soybean functional nitrogen-fixing nodules, a small library of RNAs was constructed from the functional nodules harvested 28 days after inoculation with rhizobium. Thirty-two small RNA sequences were identified as belonging to 11 miRNA families. Eight miRNAs are conserved across plant species, twenty are specific to soybean, and the four remaining miRNAs are novel. Expression analysis revealed that miRNAs were differentially expressed in the different tissues. Combinatorial miRNA target prediction identified genes that are involved in multiple biological processes. The results suggest that miRNAs play critical and diverse roles in SNF, nutrient acquisition, and plant development.

Developing Chloroplast Genomic Resources from 25 Avena Species for the Characterization of Oat Wild Relative Germplasm.

Chloroplast (cp) genomics will play an important role in the characterization of crop wild relative germplasm conserved in worldwide gene banks, thanks to the advances in genome sequencing. We applied a multiplexed shotgun sequencing procedure to sequence the cp genomes of 25 Avena species with variable ploidy levels. Bioinformatics analysis of the acquired sequences generated 25 de novo genome assemblies ranging from 135,557 to 136,006 bp. The gene annotations revealed 130 genes and their duplications, along with four to six pseudogenes, for each genome. Little differences in genome structure and gene arrangement were observed across the 25 species. Polymorphism analyses identified 1313 polymorphic sites and revealed an average of 277 microsatellites per genome. Greater nucleotide diversity was observed in the short single-copy region. Genome-wide scanning of selection signals suggested that six cp genes were under positive selection on some amino acids. These research outputs allow for a better understanding of oat cp genomes and evolution, and they form an essential set of cp genomic resources for the studies of oat evolutionary biology and for oat wild relative germplasm characterization.

Fine-Tuning of MiR528 Accumulation Modulates Flowering Time in Rice.

In plants, microRNA (miRNA) functions in the post-transcriptional repression of target mRNAs have been well explored. However, the mechanisms regulating the accumulation of miRNAs remain poorly understood. Here, we report that distinct mechanisms regulate accumulation of a monocot-specific miRNA, rice (Oryza sativa) miR528. At the transcriptional level, miR528 accumulated to higher levels in older plants than in young seedlings and exhibited aging-modulated gradual accumulation and diurnal rhythms in leaves; at the post-transcriptional level, aging also modulated miR528 levels by enhancing pri-miR528 alternative splicing. We found that miR528 promotes rice flowering under long-day conditions by targeting RED AND FAR-RED INSENSITIVE 2 (OsRFI2). Moreover, natural variations in the MIR528 promoter region caused differences in miR528 expression among rice varieties, which are correlated with their different binding affinities with the transcription factor OsSPL9 that activates the expression of miR528. Taken together, our findings reveal rice plants have evolved sophisticated modes fine-tuning miR528 levels and provide insight into the mechanisms that regulate MIRNA expression in plants.