High Daytime Temperature Responsive MicroRNA Profiles in Developing Grains of Rice Varieties with Contrasting Chalkiness.

High temperature impairs starch biosynthesis in developing rice grains and thereby increases chalkiness, affecting the grain quality. Genome encoded microRNAs (miRNAs) fine-tune target transcript abundances in a spatio-temporal specific manner, and this mode of gene regulation is critical for a myriad of developmental processes as well as stress responses. However, the role of miRNAs in maintaining rice grain quality/chalkiness during high daytime temperature (HDT) stress is relatively unknown. To uncover the role of miRNAs in this process, we used five contrasting rice genotypes (low chalky lines Cyp, Ben, and KB and high chalky lines LaGrue and NB) and compared the miRNA profiles in the R6 stage caryopsis samples from plants subjected to prolonged HDT (from the onset of fertilization through R6 stage of caryopsis development). Our small RNA analysis has identified approximately 744 miRNAs that can be grouped into 291 families. Of these, 186 miRNAs belonging to 103 families are differentially regulated under HDT. Only two miRNAs, Osa-miR444f and Osa-miR1866-5p, were upregulated in all genotypes, implying that the regulations greatly varied between the genotypes. Furthermore, not even a single miRNA was commonly up/down regulated specifically in the three tolerant genotypes. However, three miRNAs (Osa-miR1866-3p, Osa-miR5150-3p and canH-miR9774a,b-3p) were commonly upregulated and onemiRNA (Osa-miR393b-5p) was commonly downregulated specifically in the sensitive genotypes (LaGrue and NB). These observations suggest that few similarities exist within the low chalky or high chalky genotypes, possibly due to high genetic variation. Among the five genotypes used, Cypress and LaGrue are genetically closely related, but exhibit contrasting chalkiness under HDT, and thus, a comparison between them is most relevant. This comparison revealed a general tendency for Cypress to display miRNA regulations that could decrease chalkiness under HDT compared with LaGrue. This study suggests that miRNAs could play an important role in maintaining grain quality in HDT-stressed rice.

Systematic prediction of EMS-induced mutations in a sorghum mutant population.

The precise detection of causal DNA mutations (deoxyribonucleic acid) is very crucial for forward genetic studies. Several sources of errors contribute to false-positive detections by current variant-calling algorithms, which impact associating phenotypes with genotypes. To improve the accuracy of mutation detection, we implemented a binning method for the accurate detection of likely ethyl methanesulfonate (EMS)-induced mutations in a sequenced mutant population. We also implemented a clustering algorithm for detecting likely false negatives with high accuracy. Sorghum bicolor is a very valuable crop species with tremendous potential for uncovering novel gene functions associated with highly desirable agronomical traits. We demonstrate the precision of the described approach in the detection of likely EMS-induced mutations from the publicly available low-cost sequencing of the M3 generation from 600 sorghum BTx623 mutants. The approach detected 3,274,606 single nucleotide polymorphisms (SNPs), of which 96% (3,141,908) were G/C to A/T DNA substitutions, as expected by EMS-mutagenesis mode of action. We demonstrated the general applicability of the described method and showed a high concordance, 94% (3,074,759) SNPs overlap between SAMtools-based and GATK-based variant-calling algorithms. Our clustering algorithm uncovered evidence for an additional 223,048 likely false-negative shared EMS-induced mutations. The final 3,497,654 SNPs represent an 87% increase in SNPs detected from the previous analysis of the mutant population, with an average of one SNP per 125 kb in the sorghum genome. Annotation of the final SNPs revealed 10,263 high-impact and 136,639 moderate-impact SNPs, including 7217 stop-gained mutations, which averages 12 stop-gained mutations per mutant, and four high- or medium-impact SNPs per sorghum gene. We have implemented a public search database for this new genetic resource of 30,285 distinct sorghum genes containing medium- or high-impact EMS-induced mutations. Seedstock for a select 486 of the 600 described mutants are publicly available in the Germplasm Resources Information Network (GRIN) database.

Mutation of the nuclear pore complex component, aladin1, disrupts asymmetric cell division in Zea mays (maize).

The nuclear pore complex (NPC) regulates the movement of macromolecules between the nucleus and cytoplasm. Dysfunction of many components of the NPC results in human genetic diseases, including triple A syndrome (AAAS) as a result of mutations in ALADIN. Here, we report a nonsense mutation in the maize ortholog, aladin1 (ali1-1), at the orthologous amino acid residue of an AAAS allele from humans, alters plant stature, tassel architecture, and asymmetric divisions of subsidiary mother cells (SMCs). Crosses with the stronger nonsense allele ali1-2 identified complex allele interactions for plant height and aberrant SMC division. RNA-seq analysis of the ali1-1 mutant identified compensatory transcript accumulation for other NPC components as well as gene expression consequences consistent with conservation of ALADIN1 functions between humans and maize. These findings demonstrate that ALADIN1 is necessary for normal plant development, shoot architecture, and asymmetric cell division in maize.

Mutations in sorghum SBEIIb and SSIIa affect alkali spreading value, starch composition, thermal properties and flour viscosity.

KEY MESSAGE: Seven novel alleles of SBEIIb and one allele of SSIIa co-segregated with the ASV phenotype and contributed to distinct starch quality traits important for food-processing applications. Sorghum is an important food crop for millions of people in Africa and Asia. Whole-genome re-sequencing of sorghum EMS mutants exhibiting an alkali spreading value (ASV) phenotype revealed candidate SNPs in Sobic.004G163700 and Sobic.010G093400. Comparative genomics identified Sobic.010G093400 as a starch synthase IIa and Sobic.004G163700 as a starch branching enzyme IIb. Segregation analyses showed that mutations in Sobic.010G093400 or Sobic.004G163700 co-segregated with the ASV phenotype. Mutants in SSIIa exhibited no change in amylose content but expressed lower final viscosity and lower starch gelatinization temperature (GT) than starches from non-mutant plants. The sbeIIb mutants exhibited significantly higher amylose levels and starch GT and lower viscosity compared to non-mutant starches and ssIIa mutants. Mutations in SBEIIb had a dosage-dependent effect on amylose content. Double mutants of sbeIIb and ssIIa resembled their sbeIIb parent in amylose content, starch thermal properties and viscosity profiles. These variants will provide opportunities to produce sorghum varieties with modified starch end-use qualities important for the beer brewing and baking industries and specialty foods for humans with diabetes.

Whole-Genome Sequence Accuracy Is Improved by Replication in a Population of Mutagenized Sorghum.

The accurate detection of induced mutations is critical for both forward and reverse genetics studies. Experimental chemical mutagenesis induces relatively few single base changes per individual. In a complex eukaryotic genome, false positive detection of mutations can occur at or above this mutagenesis rate. We demonstrate here, using a population of ethyl methanesulfonate (EMS)-treated Sorghum bicolor BTx623 individuals, that using replication to detect false positive-induced variants in next-generation sequencing (NGS) data permits higher throughput variant detection with greater accuracy. We used a lower sequence coverage depth (average of 7×) from 586 independently mutagenized individuals and detected 5,399,493 homozygous single nucleotide polymorphisms (SNPs). Of these, 76% originated from only 57,872 genomic positions prone to false positive variant calling. These positions are characterized by high copy number paralogs where the error-prone SNP positions are at copies containing a variant at the SNP position. The ability of short stretches of homology to generate these error-prone positions suggests that incompletely assembled or poorly mapped repeated sequences are one driver of these error-prone positions. Removal of these false positives left 1,275,872 homozygous and 477,531 heterozygous EMS-induced SNPs, which, congruent with the mutagenic mechanism of EMS, were >98% G:C to A:T transitions. Through this analysis, we generated a collection of sequence indexed mutants of sorghum. This collection contains 4035 high-impact homozygous mutations in 3637 genes and 56,514 homozygous missense mutations in 23,227 genes. Each line contains, on average, 2177 annotated homozygous SNPs per genome, including seven likely gene knockouts and 96 missense mutations. The number of mutations in a transcript was linearly correlated with the transcript length and also the G+C count, but not with the GC/AT ratio. Analysis of the detected mutagenized positions identified CG-rich patches, and flanking sequences strongly influenced EMS-induced mutation rates. This method for detecting false positive-induced mutations is generally applicable to any organism, is independent of the choice of in silico variant-calling algorithm, and is most valuable when the true mutation rate is likely to be low, such as in laboratory-induced mutations or somatic mutation detection in medicine.

Genome-wide association study identifies a major gene for beech bark disease resistance in American beech (Fagus grandifolia Ehrh.).

BACKGROUND: The American Beech tree (Fagus grandifolia Ehrh.), native to eastern North America, is ecologically important and provides high quality wood products. This species is susceptible to beech bark disease (BBD) and is facing high rates of mortality in North America. The disease occurs from an interaction between the woolly beech scale insect (Cryptococcus fagisuga), one of two species of the fungus Neonectria (N. faginata or N. ditissima), and American Beech trees. METHODS: In this case-control genome-wide association study (GWAS), we tested 16 K high quality SNPs using the Affymetrix Axiom 1.5 K - 50 K assay to genotype an association population of 514 individuals. We also conducted linkage analysis in a full-sib family of 115 individuals. Fisher's exact test and logistic regression tests were performed to test associations between SNPs and phenotypes. RESULTS: Association tests revealed four highly significant SNPs on chromosome (Chr) 5 for a single gene (Mt), which encodes a mRNA for metallothionein-like protein (metal ion binding) in Fagus sylvatica. Metallothioneins represent Cys-rich metal chelators able to coordinate metal atoms and may play an important role in the resistance mechanisms against beech scale insect. CONCLUSION: The GWAS study has identified a single locus of major effect contributing to beech bark disease resistance. Knowledge of this genetic locus contributing to resistance might be used in applied breeding, conservation and restoration programs.

Forward Genetics by Sequencing EMS Variation-Induced Inbred Lines.

In order to leverage novel sequencing techniques for cloning genes in eukaryotic organisms with complex genomes, the false positive rate of variant discovery must be controlled for by experimental design and informatics. We sequenced five lines from three pedigrees of ethyl methanesulfonate (EMS)-mutagenized Sorghum bicolor, including a pedigree segregating a recessive dwarf mutant. Comparing the sequences of the lines, we were able to identify and eliminate error-prone positions. One genomic region contained EMS mutant alleles in dwarfs that were homozygous reference sequences in wild-type siblings and heterozygous in segregating families. This region contained a single nonsynonymous change that cosegregated with dwarfism in a validation population and caused a premature stop codon in the Sorghum ortholog encoding the gibberellic acid (GA) biosynthetic enzyme ent-kaurene oxidase. Application of exogenous GA rescued the mutant phenotype. Our method for mapping did not require outcrossing and introduced no segregation variance. This enables work when line crossing is complicated by life history, permitting gene discovery outside of genetic models. This inverts the historical approach of first using recombination to define a locus and then sequencing genes. Our formally identical approach first sequences all the genes and then seeks cosegregation with the trait. Mutagenized lines lacking obvious phenotypic alterations are available for an extension of this approach: mapping with a known marker set in a line that is phenotypically identical to starting material for EMS mutant generation.

Re-Evaluation of Reportedly Metal Tolerant Arabidopsis thaliana Accessions.

Santa Clara, Limeport, and Berkeley are Arabidopsis thaliana accessions previously identified as diversely metal resistant. Yet these same accessions were determined to be genetically indistinguishable from the metal sensitive Col-0. We robustly tested tolerance for Zn, Ni and Cu, and genetic relatedness by growing these accessions under a range of Ni, Zn and Cu concentrations for three durations in multiple replicates. Neither metal resistance nor variance in growth were detected between them and Col-0. We re-sequenced the genomes of these accessions and all stocks available for each accession. In all cases they were nearly indistinguishable from the standard laboratory accession Col-0. As Santa Clara was allegedly collected from the Jasper Ridge serpentine outcrop in California, USA we investigated the possibility of extant A. thaliana populations adapted to serpentine soils. Botanically vouchered Arabidopsis accessions in the Jepson database were overlaid with soil maps of California. This provided no evidence of A. thaliana collections from serpentine sites in California. Thus, our work demonstrates that the Santa Clara, Berkeley and Limeport accessions are not metal tolerant, not genetically distinct from Col-0, and that there are no known serpentine adapted populations or accessions of A. thaliana.

miRNA-mediated auxin signalling repression during Vat-mediated aphid resistance in Cucumis melo.

Resistance to Aphis gossypii in melon is attributed to the presence of the single dominant R gene virus aphid transmission (Vat), which is biologically expressed as antibiosis, antixenosis and tolerance. However, the mechanism of resistance is poorly understood at the molecular level. Aphid-induced transcriptional changes, including differentially expressed miRNA profiles that correspond to resistance interaction have been reported in melon. The potential regulatory roles of miRNAs in Vat-mediated aphid resistance were further revealed by identifying the specific miRNA degradation targets. A total of 70 miRNA:target pairs, including 28 novel miRNA:target pairs, for the differentially expressed miRNAs were identified: 11 were associated with phytohormone regulation, including six miRNAs that potentially regulate auxin interactions. A model for a redundant regulatory system of miRNA-mediated auxin insensitivity is proposed that incorporates auxin perception, auxin modification and auxin-regulated transcription. Chemically inhibiting the transport inhibitor response-1 (TIR-1) auxin receptor in susceptible melon tissues provides in vivo support for the model of auxin-mediated impacts on A. gossypii resistance.

Expression of small RNA in Aphis gossypii and its potential role in the resistance interaction with melon.

BACKGROUND: The regulatory role of small RNAs (sRNAs) in various biological processes is an active area of investigation; however, there has been limited information available on the role of sRNAs in plant-insect interactions. This study was designed to identify sRNAs in cotton-melon aphid (Aphis gossypii) during the Vat-mediated resistance interaction with melon (Cucumis melo). METHODOLOGY/PRINCIPAL FINDINGS: The role of miRNAs was investigated in response to aphid herbivory, during both resistant and susceptible interactions. sRNA libraries made from A. gossypii tissues feeding on Vat⁺ and Vat⁻ plants revealed an unexpected abundance of 27 nt long sRNA sequences in the aphids feeding on Vat⁺ plants. Eighty-one conserved microRNAs (miRNAs), twelve aphid-specific miRNAs, and nine novel candidate miRNAs were also identified. Plant miRNAs found in the aphid libraries were most likely ingested during phloem feeding. The presence of novel miRNAs was verified by qPCR experiments in both resistant Vat⁺ and susceptible Vat⁻ interactions. The comparative analyses revealed that novel miRNAs were differentially regulated during the resistant and susceptible interactions. Gene targets predicted for the miRNAs identified in this study by in silico analyses revealed their involvement in morphogenesis and anatomical structure determination, signal transduction pathways, cell differentiation and catabolic processes. CONCLUSION/SIGNIFICANCE: In this study, conserved and novel miRNAs were reported in A. gossypii. Deep sequencing data showed differences in the abundance of miRNAs and piRNA-like sequences in A. gossypii. Quantitative RT-PCR revealed that A. gossypii miRNAs were differentially regulated during resistant and susceptible interactions. Aphids can also ingest plant miRNAs during phloem feeding that are stable in the insect.

Transcriptome-wide identification of microRNA targets in rice.

MicroRNA (miRNA)-guided target RNA expression is vital for a wide variety of biological processes in eukaryotes. Currently, miRBase (version 13) lists 142 and 353 miRNAs from Arabidopsis and rice (Oryza sativa), respectively. The integration of miRNAs in diverse biological networks relies upon the confirmation of their RNA targets. In contrast with the well-characterized miRNA targets that are cleaved in Arabidopsis, only a few such targets have been confirmed in rice. To identify small RNA targets in rice, we applied the 'degradome sequencing' approach, which globally identifies the remnants of small RNA-directed target cleavage by sequencing the 5' ends of uncapped RNAs. One hundred and sixty targets of 53 miRNA families (24 conserved and 29 rice-specific) and five targets of TAS3-small interfering RNAs (siRNAs) were identified. Surprisingly, an additional conserved target for miR398, which has not been reported so far, has been validated. Besides conserved homologous transcripts, 23 non-conserved genes for nine conserved miRNAs and 56 genes for 29 rice-specific miRNAs were also identified as targets. Besides miRNA targets, the rice degradome contained fragments derived from MIRNA precursors. A closer inspection of these fragments revealed a unique pattern distinct from siRNA-producing loci. This attribute can serve as one of the ancillary criteria for separating miRNAs from siRNAs in plants.

Sliced microRNA targets and precise loop-first processing of MIR319 hairpins revealed by analysis of the Physcomitrella patens degradome.

Expression profiling of the 5' ends of uncapped mRNAs ("degradome" sequencing) can be used to empirically catalog microRNA (miRNA) targets, to probe patterns of miRNA hairpin processing, to examine mRNA decay, and to analyze accumulation of endogenous short interfering RNA (siRNA) precursors. We sequenced and analyzed the degradome of the moss Physcomitrella patens, an important model system for functional genomic analyses in plant evolution. A total of 52 target mRNAs of 27 different Physcomitrella miRNA families were identified. Many targets of both more conserved and less conserved miRNA families encoded putative regulatory proteins. Remnants of MIRNA hairpin processing also populated the degradome data and indicated an unusual "loop-first" mode of precise processing for the MIR319 gene family. Precise loop-first processing was confirmed for native Physcomitrella, rice, and Arabidopsis MIR319 hairpins, as well as an Arabidopsis artificial MIRNA (aMIRNA) based upon a MIR319 backbone. MIR319 is thus a conserved exception to the general rule of loop-last processing of MIRNA hairpins. Loop-first MIR319 processing may contribute to the high efficacy of a widely used MIR319-based strategy for aMIRNA production in plants.

CleaveLand: a pipeline for using degradome data to find cleaved small RNA targets.

UNLABELLED: MicroRNAs (miRNAs) are approximately 20- to 22-nt long endogenous RNA sequences that play a critical role in the regulation of gene expression in eukaryotic genomes. Confident identification of miRNA targets is vital to understand their functions. Currently available computational algorithms for miRNA target prediction have diverse degrees of sensitivity and specificity and as a consequence each predicted target generally requires experimental confirmation. miRNAs and other small RNAs that direct endonucleolytic cleavage of target mRNAs produce diagnostic uncapped, polyadenylated mRNA fragments. Degradome sequencing [also known as PARE (parallel analysis of RNA ends) and GMUCT (genome-wide mapping of uncapped transcripts)] samples the 5'-ends of uncapped mRNAs and can be used to discover in vivo miRNA targets independent of computational predictions. Here, we describe a generalizable computational pipeline, CleaveLand, for the detection of cleaved miRNA targets from degradome data. CleaveLand takes as input degradome sequences, small RNAs and an mRNA database and outputs small RNA targets. CleaveLand can thus be applied to degradome data from any species provided a set of mRNA transcripts and a set of query miRNAs or other small RNAs are available. AVAILABILITY: The code and documentation for CleaveLand is freely available under a GNU license at http://www.bio.psu.edu/people/faculty/Axtell/AxtellLab/Software.html

Physcomitrella patens DCL3 is required for 22-24 nt siRNA accumulation, suppression of retrotransposon-derived transcripts, and normal development.

Endogenous 24 nt short interfering RNAs (siRNAs), derived mostly from intergenic and repetitive genomic regions, constitute a major class of endogenous small RNAs in flowering plants. Accumulation of Arabidopsis thaliana 24 nt siRNAs requires the Dicer family member DCL3, and clear homologs of DCL3 exist in both flowering and non-flowering plants. However, the absence of a conspicuous 24 nt peak in the total RNA populations of several non-flowering plants has raised the question of whether this class of siRNAs might, in contrast to the ancient 21 nt microRNAs (miRNAs) and 21-22 nt trans-acting siRNAs (tasiRNAs), be an angiosperm-specific innovation. Analysis of non-miRNA, non-tasiRNA hotspots of small RNA production within the genome of the moss Physcomitrella patens revealed multiple loci that consistently produced a mixture of 21-24 nt siRNAs with a peak at 23 nt. These Pp23SR loci were significantly enriched in transposon content, depleted in overlap with annotated genes, and typified by dense concentrations of the 5-methyl cytosine (5 mC) DNA modification. Deep sequencing of small RNAs from two independent Ppdcl3 mutants showed that the P. patens DCL3 homolog is required for the accumulation of 22-24 nt siRNAs, but not 21 nt siRNAs, at Pp23SR loci. The 21 nt component of Pp23SR-derived siRNAs was also unaffected by a mutation in the RNA-dependent RNA polymerase mutant Pprdr6. Transcriptome-wide, Ppdcl3 mutants failed to accumulate 22-24 nt small RNAs from repetitive regions while transcripts from two abundant families of long terminal repeat (LTR) retrotransposon-associated reverse transcriptases were up-regulated. Ppdcl3 mutants also displayed an acceleration of leafy gametophore production, suggesting that repetitive siRNAs may play a role in the development of P. patens. We conclude that intergenic/repeat-derived siRNAs are indeed a broadly conserved, distinct class of small regulatory RNAs within land plants.

Endogenous siRNA and miRNA targets identified by sequencing of the Arabidopsis degradome.

MicroRNAs (miRNAs) regulate the expression of target mRNAs in plants and animals [1]. Plant miRNA targets have been predicted on the basis of their extensive and often conserved complementarity to the miRNAs [2-4], as well as on miRNA overexpression experiments [5]; many of these target predictions have been confirmed by isolation of the products of miRNA-directed cleavage. Here, we present a transcriptome-wide experimental method, called "degradome sequencing," to directly detect cleaved miRNA targets without relying on predictions or overexpression. The 5' ends of polyadenylated, uncapped mRNAs from Arabidopsis were directly sampled, resulting in an empirical snapshot of the degradome. miRNA-mediated-cleavage products were easily discerned from an extensive background of degraded mRNAs, which collectively covered the majority of the annotated transcriptome. Many previously known Arabidopsis miRNA targets were confirmed, and several novel targets were also discovered. Quantification of cleavage fragments revealed that those derived from TAS transcripts, which are unusual in their production of abundant secondary small interfering RNAs (siRNAs), accumulated to very high levels. A subset of secondary siRNAs are also known to direct cleavage of targets in trans[6]; degradome sequencing revealed many cleaved targets of these trans-acting siRNAs (ta-siRNAs). This empirical method is broadly applicable to the discovery and quantification of cleaved targets of small RNAs without a priori predictions.