Wheat-Net: An Automatic Dense Wheat Spike Segmentation Method Based on an Optimized Hybrid Task Cascade Model.

Precise segmentation of wheat spikes from a complex background is necessary for obtaining image-based phenotypic information of wheat traits such as yield estimation and spike morphology. A new instance segmentation method based on a Hybrid Task Cascade model was proposed to solve the wheat spike detection problem with improved detection results. In this study, wheat images were collected from fields where the environment varied both spatially and temporally. Res2Net50 was adopted as a backbone network, combined with multi-scale training, deformable convolutional networks, and Generic ROI Extractor for rich feature learning. The proposed methods were trained and validated, and the average precision (AP) obtained for the bounding box and mask was 0.904 and 0.907, respectively, and the accuracy for wheat spike counting was 99.29%. Comprehensive empirical analyses revealed that our method (Wheat-Net) performed well on challenging field-based datasets with mixed qualities, particularly those with various backgrounds and wheat spike adjacence/occlusion. These results provide evidence for dense wheat spike detection capabilities with masking, which is useful for not only wheat yield estimation but also spike morphology assessments.

Description and functional analysis of the transcriptome from malting barley.

The present study aimed to establish an early model of the malting barley transcriptome, which describes the expression of genes and their ontologies, identify the period during malting with the largest dynamic shift in gene expression for future investigation, and to determine the expression patterns of all starch degrading enzyme genes relevant to the malting and brewing industry. Large dynamic increases in gene expression occurred early in malting with differential expressed genes enriched for cell wall and starch hydrolases amongst many malting related categories. Twenty-five of forty starch degrading enzyme genes were differentially expressed in the malting barley transcriptome including eleven α-amylase genes, six ß-amylase genes, three α-glucosidase genes, and all five starch debranching enzyme genes. Four new or novel α-amylase genes, one ß-amylase gene (Bmy3), three α-glucosidase genes, and two isoamylase genes had appreciable expression that requires further exploration into their potential relevance to the malting and brewing industry.

Identification of Candidate Susceptibility Genes to Puccinia graminis f. sp. tritici in Wheat.

Wheat stem rust disease caused by Puccinia graminis f. sp. tritici (Pgt) is a global threat to wheat production. Fast evolving populations of Pgt limit the efficacy of plant genetic resistance and constrain disease management strategies. Understanding molecular mechanisms that lead to rust infection and disease susceptibility could deliver novel strategies to deploy crop resistance through genetic loss of disease susceptibility. We used comparative transcriptome-based and orthology-guided approaches to characterize gene expression changes associated with Pgt infection in susceptible and resistant Triticum aestivum genotypes as well as the non-host Brachypodium distachyon. We targeted our analysis to genes with differential expression in T. aestivum and genes suppressed or not affected in B. distachyon and report several processes potentially linked to susceptibility to Pgt, such as cell death suppression and impairment of photosynthesis. We complemented our approach with a gene co-expression network analysis to identify wheat targets to deliver resistance to Pgt through removal or modification of putative susceptibility genes.

Hyperspectral imaging and improved feature variable selection for automated determination of deoxynivalenol in various genetic lines of barley kernels for resistance screening.

Fusarium head blight (FHB), a fungus disease of small grain cereal crops, results in reduced yields and diminished value of harvested grain due to the presence of deoxynivalenol (DON), a mycotoxin produced by the causal pathogen Fusarium graminearum. DON and other tricothecene mycotoxins pose serious health risks to both humans and livestock, especially swine. Due to these health concerns, barley used for malting, food or feed is routinely assayed for DON levels. Various methods are available for assaying DON levels in grain samples including enzyme-linked immunosorbent assay (ELISA) and gas chromatography-mass spectrometry (GC-MS). ELISA and GC-MS are very accurate; however, assaying grain samples by these techniques are laborious, expensive and destructive. In this study, we explored the feasibility of using hyperspectral imaging (382-1030 nm) to develop a rapid and non-destructive protocol for assaying DON in barley kernels. Samples of 888 and 116 from various genetic lines were selected for calibration and prediction. Full-wavelength locally weighted partial least squares regression (LWPLSR) achieved high accuracy with the coefficient of determination in prediction (R2P) of 0.728 and root mean square error of prediction (RMSEP) of 3.802. Competitive adaptive reweighted sampling (CARS) was used to choose potential feature wavelengths, and these selected variables were further optimized using the iterative selection of successive projections algorithm (ISSPA). The CARS-ISSPA-LWPLSR model developed using 7 feature variables yielded R2P of 0.680 and RMSEP of 4.213 in DON content prediction. Based on the 7 wavelengths selected by CARS-ISSPA, partial least square discriminant analysis (PLSDA) discriminated barley kernels having lower DON (less than1.25 mg/kg) levels from those with higher levels (including 1.25-3 mg/kg, 3-5 mg/kg, and 5-10 mg/kg), with Matthews correlation coefficient in cross-validation (M-RCV) of as high as 0.931. The results demonstrate that hyperspectral imaging have potential for accelerating non-destructive DON assays of barley samples.

Single-parent expression drives dynamic gene expression complementation in maize hybrids.

Single-parent expression (SPE) is defined as gene expression in only one of the two parents. SPE can arise from differential expression between parental alleles, termed non-presence/absence (non-PAV) SPE, or from the physical absence of a gene in one parent, termed PAV SPE. We used transcriptome data of diverse Zea mays (maize) inbreds and hybrids, including 401 samples from five different tissues, to test for differences between these types of SPE genes. Although commonly observed, SPE is highly genotype and tissue specific. A positive correlation was observed between the genetic distance of the two inbred parents and the number of SPE genes identified. Regulatory analysis showed that PAV SPE and non-PAV SPE genes are mainly regulated by cis effects, with a small fraction under trans regulation. Polymorphic transposable element insertions in promoter sequences contributed to the high level of cis regulation for PAV SPE and non-PAV SPE genes. PAV SPE genes were more frequently expressed in hybrids than non-PAV SPE genes. The expression of parentally silent alleles in hybrids of non-PAV SPE genes was relatively rare but occurred in most hybrids. Non-PAV SPE genes with expression of the silent allele in hybrids are more likely to exhibit above high parent expression level than hybrids that do not express the silent allele, leading to non-additive expression. This study provides a comprehensive understanding of the nature of non-PAV SPE and PAV SPE genes and their roles in gene expression complementation in maize hybrids.

Localized Genetic and Phenotypic Diversity of Xanthomonas translucens Associated With Bacterial Leaf Streak on Wheat and Barley in Minnesota.

Bacterial leaf streak (BLS) of wheat and barley has been a disease of increasing concern in the Upper Midwest over the past decade. In this study, intra- and interfield genetic and pathogenic diversity of bacteria causing BLS in Minnesota was evaluated. In 2015, 89 strains were isolated from 100 leaf samples collected from two wheat and two barley fields naturally infected with BLS. Virulence assays and multilocus sequence alignments of four housekeeping genes supported pathovar identifications. All wheat strains were pathogenic on wheat and barley and belonged to the same lineage as the Xanthomonas translucens pv. undulosa-type strain. All barley strains were pathogenic on barley but not on wheat. Three lineages of barley strains were detected. The frequency and number of sequence types of each pathovar varied within and between fields. A significant population variance was detected between populations of X. translucens pv. undulosa collected from different wheat fields. Population stratification of X. translucens pv. translucens was not detected. Significant differences in virulence were detected among three dominant sequence types of X. translucens pv. undulosa but not those of X. translucens pv. translucens. Field trials with wheat and barley plants inoculated with strains of known sequence type and virulence did not detect significant race structures within either pathovar. Knowledge of virulence, sequence types, and population structures of X. translucens on wheat and barley can support studies on plant-bacterial interactions and breeding for BLS disease resistance.

Towards Improved Molecular Identification Tools in Fine Fescue (Festuca L., Poaceae) Turfgrasses: Nuclear Genome Size, Ploidy, and Chloroplast Genome Sequencing.

Fine fescues (Festuca L., Poaceae) are turfgrass species that perform well in low-input environments. Based on morphological characteristics, the most commonly-utilized fine fescues are divided into five taxa: three are subspecies within F. rubra L. and the remaining two are treated as species within the F. ovina L. complex. Morphologically, these five taxa are very similar; both identification and classification of fine fescues remain challenging. In an effort to develop identification methods for fescues, we used flow cytometry to estimate genome size and ploidy level and sequenced the chloroplast genome of all five taxa. Fine fescue chloroplast genome sizes ranged from 133,331 to 133,841 bp and contained 113-114 genes. Phylogenetic relationship reconstruction using whole chloroplast genome sequences agreed with previous work based on morphology. Comparative genomics suggested unique repeat signatures for each fine fescue taxon that could potentially be used for marker development for taxon identification.

Emergence of the Ug99 lineage of the wheat stem rust pathogen through somatic hybridisation.

Parasexuality contributes to diversity and adaptive evolution of haploid (monokaryotic) fungi. However, non-sexual genetic exchange mechanisms are not defined in dikaryotic fungi (containing two distinct haploid nuclei). Newly emerged strains of the wheat stem rust pathogen, Puccinia graminis f. sp. tritici (Pgt), such as Ug99, are a major threat to global food security. Here, we provide genomics-based evidence supporting that Ug99 arose by somatic hybridisation and nuclear exchange between dikaryons. Fully haplotype-resolved genome assembly and DNA proximity analysis reveal that Ug99 shares one haploid nucleus genotype with a much older African lineage of Pgt, with no recombination or chromosome reassortment. These findings indicate that nuclear exchange between dikaryotes can generate genetic diversity and facilitate the emergence of new lineages in asexual fungal populations.

Dynamic Patterns of Transcript Abundance of Transposable Element Families in Maize.

Transposable Elements (TEs) are mobile elements that contribute the majority of DNA sequences in the maize genome. Due to their repetitive nature, genomic studies of TEs are complicated by the difficulty of properly attributing multi-mapped short reads to specific genomic loci. Here, we utilize a method to attribute RNA-seq reads to TE families rather than particular loci in order to characterize transcript abundance for TE families in the maize genome. We applied this method to assess per-family expression of transposable elements in >800 published RNA-seq libraries representing a range of maize development, genotypes, and hybrids. While a relatively small proportion of TE families are transcribed, expression is highly dynamic with most families exhibiting tissue-specific expression. A large number of TE families were specifically detected in pollen and endosperm, consistent with reproductive dynamics that maintain silencing of TEs in the germ line. We find that B73 transcript abundance is a poor predictor of TE expression in other genotypes and that transcript levels can differ even for shared TEs. Finally, by assessing recombinant inbred line and hybrid transcriptomes, complex patterns of TE transcript abundance across genotypes emerged. Taken together, this study reveals a dynamic contribution of TEs to maize transcriptomes.

Transposable elements contribute to dynamic genome content in maize.

Transposable elements (TEs) are ubiquitous components of eukaryotic genomes and can create variation in genome organization and content. Most maize genomes are composed of TEs. We developed an approach to define shared and variable TE insertions across genome assemblies and applied this method to four maize genomes (B73, W22, Mo17 and PH207) with uniform structural annotations of TEs. Among these genomes we identified approximately 400 000 TEs that are polymorphic, encompassing 1.6 Gb of variable TE sequence. These polymorphic TEs include a combination of recent transposition events as well as deletions of older TEs. There are examples of polymorphic TEs within each of the superfamilies of TEs and they are found distributed across the genome, including in regions of recent shared ancestry among individuals. There are many examples of polymorphic TEs within or near maize genes. In addition, there are 2380 gene annotations in the B73 genome that are located within variable TEs, providing evidence for the role of TEs in contributing to the substantial differences in annotated gene content among these genotypes. TEs are highly variable in our survey of four temperate maize genomes, highlighting the major contribution of TEs in driving variation in genome organization and gene content. OPEN RESEARCH BADGES: This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data is available at https://github.com/SNAnderson/maizeTE_variation; https://mcstitzer.github.io/maize_TEs.

Classifying cold-stress responses of inbred maize seedlings using RGB imaging.

Increasing the tolerance of maize seedlings to low-temperature episodes could mitigate the effects of increasing climate variability on yield. To aid progress toward this goal, we established a growth chamber-based system for subjecting seedlings of 40 maize inbred genotypes to a defined, temporary cold stress while collecting digital profile images over a 9-daytime course. Image analysis performed with PlantCV software quantified shoot height, shoot area, 14 other morphological traits, and necrosis identified by color analysis. Hierarchical clustering of changes in growth rates of morphological traits and quantification of leaf necrosis over two time intervals resulted in three clusters of genotypes, which are characterized by unique responses to cold stress. For any given genotype, the set of traits with similar growth rates is unique. However, the patterns among traits are different between genotypes. Cold sensitivity was not correlated with the latitude where the inbred varieties were released suggesting potential further improvement for this trait. This work will serve as the basis for future experiments investigating the genetic basis of recovery to cold stress in maize seedlings.

De Novo Assembly and Phasing of Dikaryotic Genomes from Two Isolates of Puccinia coronata f. sp. avenae, the Causal Agent of Oat Crown Rust.

Oat crown rust, caused by the fungus Pucinnia coronata f. sp. avenae, is a devastating disease that impacts worldwide oat production. For much of its life cycle, P. coronata f. sp. avenae is dikaryotic, with two separate haploid nuclei that may vary in virulence genotype, highlighting the importance of understanding haplotype diversity in this species. We generated highly contiguous de novo genome assemblies of two P. coronata f. sp. avenae isolates, 12SD80 and 12NC29, from long-read sequences. In total, we assembled 603 primary contigs for 12SD80, for a total assembly length of 99.16 Mbp, and 777 primary contigs for 12NC29, for a total length of 105.25 Mbp; approximately 52% of each genome was assembled into alternate haplotypes. This revealed structural variation between haplotypes in each isolate equivalent to more than 2% of the genome size, in addition to about 260,000 and 380,000 heterozygous single-nucleotide polymorphisms in 12SD80 and 12NC29, respectively. Transcript-based annotation identified 26,796 and 28,801 coding sequences for isolates 12SD80 and 12NC29, respectively, including about 7,000 allele pairs in haplotype-phased regions. Furthermore, expression profiling revealed clusters of coexpressed secreted effector candidates, and the majority of orthologous effectors between isolates showed conservation of expression patterns. However, a small subset of orthologs showed divergence in expression, which may contribute to differences in virulence between 12SD80 and 12NC29. This study provides the first haplotype-phased reference genome for a dikaryotic rust fungus as a foundation for future studies into virulence mechanisms in P. coronata f. sp. avenaeIMPORTANCE Disease management strategies for oat crown rust are challenged by the rapid evolution of Puccinia coronata f. sp. avenae, which renders resistance genes in oat varieties ineffective. Despite the economic importance of understanding P. coronata f. sp. avenae, resources to study the molecular mechanisms underpinning pathogenicity and the emergence of new virulence traits are lacking. Such limitations are partly due to the obligate biotrophic lifestyle of P. coronata f. sp. avenae as well as the dikaryotic nature of the genome, features that are also shared with other important rust pathogens. This study reports the first release of a haplotype-phased genome assembly for a dikaryotic fungal species and demonstrates the amenability of using emerging technologies to investigate genetic diversity in populations of P. coronata f. sp. avenae.

Natural variation for gene expression responses to abiotic stress in maize.

Plants respond to abiotic stress through a variety of physiological, biochemical, and transcriptional mechanisms. Many genes exhibit altered levels of expression in response to abiotic stress, which requires concerted action of both cis- and trans-regulatory features. In order to study the variability in transcriptome response to abiotic stress, RNA sequencing was performed using 14-day-old maize seedlings of inbreds B73, Mo17, Oh43, PH207 and B37 under control, cold and heat conditions. Large numbers of genes that responded differentially to stress between parental inbred lines were identified. RNA sequencing was also performed on similar tissues of the F1 hybrids produced by crossing B73 and each of the three other inbred lines. By evaluating allele-specific transcript abundance in the F1 hybrids, we were able to measure the abundance of cis- and trans-regulatory variation between genotypes for both steady-state and stress-responsive expression differences. Although examples of trans-regulatory variation were observed, cis-regulatory variation was more common for both steady-state and stress-responsive expression differences. The genes with cis-allelic variation for response to cold or heat stress provided an opportunity to study the basis for regulatory diversity.

Transposable element influences on gene expression in plants.

Transposable elements (TEs) comprise a major portion of many plant genomes and bursts of TE movements cause novel genomic variation within species. In order to maintain proper gene function, plant genomes have evolved a variety of mechanisms to tolerate the presence of TEs within or near genes. Here, we review our understanding of the interactions between TEs and gene expression in plants by assessing three ways that transposons can influence gene expression. First, there is growing evidence that TE insertions within introns or untranslated regions of genes are often tolerated and have minimal impact on expression level or splicing. However, there are examples in which TE insertions within genes can result in aberrant or novel transcripts. Second, TEs can provide novel alternative promoters, which can lead to new expression patterns or original coding potential of an alternate transcript. Third, TE insertions near genes can influence regulation of gene expression through a variety of mechanisms. For example, TEs may provide novel cis-acting regulatory sites behaving as enhancers or insert within existing enhancers to influence transcript production. Alternatively, TEs may change chromatin modifications in regions near genes, which in turn can influence gene expression levels. Together, the interactions of genes and TEs provide abundant evidence for the role of TEs in changing basic functions within plant genomes beyond acting as latent genomic elements or as simple insertional mutagens. This article is part of a Special Issue entitled: Plant Gene Regulatory Mechanisms and Networks, edited by Dr. Erich Grotewold and Dr. Nathan Springer.

Draft Assembly of Elite Inbred Line PH207 Provides Insights into Genomic and Transcriptome Diversity in Maize.

Intense artificial selection over the last 100 years has produced elite maize (Zea mays) inbred lines that combine to produce high-yielding hybrids. To further our understanding of how genome and transcriptome variation contribute to the production of high-yielding hybrids, we generated a draft genome assembly of the inbred line PH207 to complement and compare with the existing B73 reference sequence. B73 is a founder of the Stiff Stalk germplasm pool, while PH207 is a founder of Iodent germplasm, both of which have contributed substantially to the production of temperate commercial maize and are combined to make heterotic hybrids. Comparison of these two assemblies revealed over 2500 genes present in only one of the two genotypes and 136 gene families that have undergone extensive expansion or contraction. Transcriptome profiling revealed extensive expression variation, with as many as 10,564 differentially expressed transcripts and 7128 transcripts expressed in only one of the two genotypes in a single tissue. Genotype-specific genes were more likely to have tissue/condition-specific expression and lower transcript abundance. The availability of a high-quality genome assembly for the elite maize inbred PH207 expands our knowledge of the breadth of natural genome and transcriptome variation in elite maize inbred lines across heterotic pools.

RNA-directed DNA methylation enforces boundaries between heterochromatin and euchromatin in the maize genome.

The maize genome is relatively large (∼ 2.3 Gb) and has a complex organization of interspersed genes and transposable elements, which necessitates frequent boundaries between different types of chromatin. The examination of maize genes and conserved noncoding sequences revealed that many of these are flanked by regions of elevated asymmetric CHH (where H is A, C, or T) methylation (termed mCHH islands). These mCHH islands are quite short (∼ 100 bp), are enriched near active genes, and often occur at the edge of the transposon that is located nearest to genes. The analysis of DNA methylation in other sequence contexts and several chromatin modifications revealed that mCHH islands mark the transition from heterochromatin-associated modifications to euchromatin-associated modifications. The presence of an mCHH island is fairly consistent in several distinct tissues that were surveyed but shows some variation among different haplotypes. The presence of insertion/deletions in promoters often influences the presence and position of an mCHH island. The mCHH islands are dependent upon RNA-directed DNA methylation activities and are lost in mop1 and mop3 mutants, but the nearby genes rarely exhibit altered expression levels. Instead, loss of an mCHH island is often accompanied by additional loss of DNA methylation in CG and CHG contexts associated with heterochromatin in nearby transposons. This suggests that mCHH islands and RNA-directed DNA methylation near maize genes may act to preserve the silencing of transposons from activity of nearby genes.

Genomic limitations to RNA sequencing expression profiling.

The field of genomics has grown rapidly with the advent of massively parallel sequencing technologies, allowing for novel biological insights with regards to genomic, transcriptomic, and epigenomic variation. One widely utilized application of high-throughput sequencing is transcriptional profiling using RNA sequencing (RNAseq). Understanding the limitations of a technology is critical for accurate biological interpretations, and clear interpretation of RNAseq data can be difficult in species with complex genomes. To understand the limitations of accurate profiling of expression levels we simulated RNAseq reads from annotated gene models in several plant species including Arabidopsis, brachypodium, maize, potato, rice, soybean, and tomato. The simulated reads were aligned using various parameters such as unique versus multiple read alignments. This allowed the identification of genes recalcitrant to RNAseq analyses by having over- and/or under-estimated expression levels. In maize, over 25% of genes deviated by more than 20% from the expected count values, suggesting the need for cautious interpretation of RNAseq data for certain genes. The reasons identified for deviation from expected expression varied between species due to differences in genome structure including, but not limited to, genes encoding short transcripts, overlapping gene models, and gene family size. Utilizing existing empirical datasets we demonstrate the potential for biological misinterpretation resulting from inclusion of 'flagged genes' in analyses. While RNAseq is a powerful tool for understanding biology, there are limitations to this technology that need to be understood in order to improve our biological interpretations.

Reduced representation approaches to interrogate genome diversity in large repetitive plant genomes.

Technology and software improvements in the last decade now provide methodologies to access the genome sequence of not only a single accession, but also multiple accessions of plant species. This provides a means to interrogate species diversity at the genome level. Ample diversity among accessions in a collection of species can be found, including single-nucleotide polymorphisms, insertions and deletions, copy number variation and presence/absence variation. For species with small, non-repetitive rich genomes, re-sequencing of query accessions is robust, highly informative, and economically feasible. However, for species with moderate to large sized repetitive-rich genomes, technical and economic barriers prevent en masse genome re-sequencing of accessions. Multiple approaches to access a focused subset of loci in species with larger genomes have been developed, including reduced representation sequencing, exome capture and transcriptome sequencing. Collectively, these approaches have enabled interrogation of diversity on a genome scale for large plant genomes, including crop species important to worldwide food security.