A systems genomics and genetics approach to identify the genetic regulatory network for lignin content in Brassica napus seeds.

Seed quality traits of oilseed rape, Brassica napus (B. napus), exhibit quantitative inheritance determined by its genetic makeup and the environment via the mediation of a complex genetic architecture of hundreds to thousands of genes. Thus, instead of single gene analysis, network-based systems genomics and genetics approaches that combine genotype, phenotype, and molecular phenotypes offer a promising alternative to uncover this complex genetic architecture. In the current study, systems genetics approaches were used to explore the genetic regulation of lignin traits in B. napus seeds. Four QTL (qLignin_A09_1, qLignin_A09_2, qLignin_A09_3, and qLignin_C08) distributed on two chromosomes were identified for lignin content. The qLignin_A09_2 and qLignin_C08 loci were homologous QTL from the A and C subgenomes, respectively. Genome-wide gene regulatory network analysis identified eighty-three subnetworks (or modules); and three modules with 910 genes in total, were associated with lignin content, which was confirmed by network QTL analysis. eQTL (expression quantitative trait loci) analysis revealed four cis-eQTL genes including lignin and flavonoid pathway genes, cinnamoyl-CoA-reductase (CCR1), and TRANSPARENT TESTA genes TT4, TT6, TT8, as causal genes. The findings validated the power of systems genetics to identify causal regulatory networks and genes underlying complex traits. Moreover, this information may enable the research community to explore new breeding strategies, such as network selection or gene engineering, to rewire networks to develop climate resilience crops with better seed quality.

Enhancing rice growth and yield with weed endophytic bacteria Alcaligenes faecalis and Metabacillus indicus under reduced chemical fertilization.

Endophytic bacteria, recognized as eco-friendly biofertilizers, have demonstrated the potential to enhance crop growth and yield. While the plant growth-promoting effects of endophytic bacteria have been extensively studied, the impact of weed endophytes remains less explored. In this study, we aimed to isolate endophytic bacteria from native weeds and assess their plant growth-promoting abilities in rice under varying chemical fertilization. The evaluation encompassed measurements of mineral phosphate and potash solubilization, as well as indole-3-acetic acid (IAA) production activity by the selected isolates. Two promising strains, tentatively identified as Alcaligenes faecalis (BTCP01) from Eleusine indica (Goose grass) and Metabacillus indicus (BTDR03) from Cynodon dactylon (Bermuda grass) based on 16S rRNA gene phylogeny, exhibited noteworthy phosphate and potassium solubilization activity, respectively. BTCP01 demonstrated superior phosphate solubilizing activity, while BTDR03 exhibited the highest potassium (K) solubilizing activity. Both isolates synthesized IAA in the presence of L-tryptophan, with the detection of nifH and ipdC genes in their genomes. Application of isolates BTCP01 and BTDR03 through root dipping and spraying at the flowering stage significantly enhanced the agronomic performance of rice variety CV. BRRI dhan29. Notably, combining both strains with 50% of recommended N, P, and K fertilizer doses led to a substantial increase in rice grain yields compared to control plants receiving 100% of recommended doses. Taken together, our results indicate that weed endophytic bacterial strains BTCP01 and BTDR03 hold promise as biofertilizers, potentially reducing the dependency on chemical fertilizers by up to 50%, thereby fostering sustainable rice production.

Biological control potential of worrisome wheat blast disease by the seed endophytic bacilli.

Crop production often faces challenges from plant diseases, and biological control emerges as an effective, environmentally friendly, cost-effective, and sustainable alternative to chemical control. Wheat blast disease caused by fungal pathogen Magnaporthe oryzae Triticum (MoT), is a potential catastrophic threat to global food security. This study aimed to identify potential bacterial isolates from rice and wheat seeds with inhibitory effects against MoT. In dual culture and seedling assays, three bacterial isolates (BTS-3, BTS-4, and BTLK6A) demonstrated effective suppression of MoT growth and reduced wheat blast severity when artificially inoculated at the seedling stage. Genome phylogeny identified these isolates as Bacillus subtilis (BTS-3) and B. velezensis (BTS-4 and BTLK6A). Whole-genome analysis revealed the presence of genes responsible for controlling MoT through antimicrobial defense, antioxidant defense, cell wall degradation, and induced systemic resistance (ISR). Taken together, our results suggest that the suppression of wheat blast disease by seed endophytic B. subtilis (BTS-3) and B. velezensis (BTS-4 and BTLK6A) is liked with antibiosis and induced systemic resistance to wheat plants. A further field validation is needed before recommending these endophytic bacteria for biological control of wheat blast.

Mapping QTL for vernalization requirement identified adaptive divergence of the candidate gene Flowering Locus C in polyploid Camelina sativa.

Vernalization requirement is an integral component of flowering in winter-type plants. The availability of winter ecotypes among Camelina species facilitated the mapping of quantitative trait loci (QTL) for vernalization requirement in Camelina sativa. An inter and intraspecific crossing scheme between related Camelina species, where one spring and two different sources of winter-type habit were used, resulted in the development of two segregating populations. Linkage maps generated with sequence-based markers identified three QTLs associated with vernalization requirement in C. sativa; two from the interspecific (chromosomes 13 and 20) and one from the intraspecific cross (chromosome 8). Notably, the three loci were mapped to different homologous regions of the hexaploid C. sativa genome. All three QTLs were found in proximity to Flowering Locus C (FLC), variants of which have been reported to affect the vernalization requirement in plants. Temporal transcriptome analysis for winter-type Camelina alyssum demonstrated reduction in expression of FLC on chromosomes 13 and 20 during cold treatment, which would trigger flowering, since FLC would be expected to suppress floral initiation. FLC on chromosome 8 also showed reduced expression in the C. sativa ssp. pilosa winter parent upon cold treatment, but was expressed at very high levels across all time points in the spring-type C. sativa. The chromosome 8 copy carried a deletion in the spring-type line, which could impact its functionality. Contrary to previous reports, all three FLC loci can contribute to controlling the vernalization response in C. sativa and provide opportunities for manipulating this requirement in the crop.

Improvement of growth, yield and associated bacteriome of rice by the application of probiotic Paraburkholderia and Delftia.

Plant probiotic bacteria enhance growth and yield of crop plants when applied at the appropriate time and dose. Two rice probiotic bacteria, Paraburkholderia fungorum strain BRRh-4 and Delftia sp. strain BTL-M2 promote growth and yield of plants. However, no information is available on application of these two bacteria on growth, yield, and diversity and population of bacteriome in roots and rhizosphere soils of the treated rice plants. This study aimed to assess the effect of BRRh-4 and BTL-M2 application on growth, yield and bacteriome in roots and rhizosphere soil of rice under varying doses of N, P and K fertilizers. Application of BRRh-4 and BTL-M2 strains significantly (p < 0.05) increased seed germination, growth and yield of rice compared to an untreated control. Interestingly, the grain yield of rice by these bacteria with 50% less of the recommended doses of N, P, and K fertilizers were statistically similar to or better than the rice plants treated with 100% doses of these fertilizers. Targeted amplicon (16S rRNA) sequence-based analysis revealed significant differences (PERMANOVA, p = 0.00035) in alpha-diversity between the root (R) and rhizosphere soil (S) samples, showing higher diversity in the microbial ecosystem of root samples. Additionally, the bacteriome diversity in the root of rice plants that received both probiotic bacteria and chemical fertilizers were significantly higher (PERMANOVA, p = 0.0312) compared to the rice plants treated with fertilizers only. Out of 185 bacterial genera detected, Prevotella, an anaerobic and Gram-negative bacterium, was found to be the predominant genus in both rhizosphere soil and root metagenomes. However, the relative abundance of Prevotella remained two-fold higher in the rhizosphere soil metagenome (52.02%) than in the root metagenome (25.04%). The other predominant bacterial genera detected in the rice root metagenome were Bacillus (11.07%), Planctomyces (4.06%), Faecalibacterium (3.91%), Deinococcus (2.97%), Bacteroides (2.61%), and Chryseobacterium (2.30%). On the other hand, rhizosphere soil metagenome had Bacteroides (12.38%), Faecalibacterium (9.50%), Vibrio (5.94%), Roseomonas (3.40%), and Delftia (3.02%). Interestingly, we found the presence and/or abundance of specific genera of bacteria in rice associated with the application of a specific probiotic bacterium. Taken together, our results indicate that improvement of growth and yield of rice by P. fungorum strain BRRh-4 and Delftia sp. strain BTL-M2 is likely linked with modulation of diversity, structures, and signature of bacteriome in roots and rhizosphere soils. This study for the first time demonstrated that application of plant growth promoting bacteria significantly improve growth, yield and increase the diversity of bacterial community in rice.

The coordinated regulation of early meiotic stages is dominated by non-coding RNAs and stage-specific transcription in wheat.

Reproductive success hinges on precisely coordinated meiosis, yet our understanding of how structural rearrangements of chromatin and phase transitions during meiosis are transcriptionally regulated is limited. In crop plants, detailed analysis of the meiotic transcriptome could identify regulatory genes and epigenetic regulators that can be targeted to increase recombination rates and broaden genetic variation, as well as provide a resource for comparison among eukaryotes of different taxa to answer outstanding questions about meiosis. We conducted a meiotic stage-specific analysis of messenger RNA (mRNA), small non-coding RNA (sncRNA), and long intervening/intergenic non-coding RNA (lincRNA) in wheat (Triticum aestivum L.) and revealed novel mechanisms of meiotic transcriptional regulation and meiosis-specific transcripts. Amidst general repression of mRNA expression, significant enrichment of ncRNAs was identified during prophase I relative to vegetative cells. The core meiotic transcriptome was comprised of 9309 meiosis-specific transcripts, 48 134 previously unannotated meiotic transcripts, and many known and novel ncRNAs differentially expressed at specific stages. The abundant meiotic sncRNAs controlled the reprogramming of central metabolic pathways by targeting genes involved in photosynthesis, glycolysis, hormone biosynthesis, and cellular homeostasis, and lincRNAs enhanced the expression of nearby genes. Alternative splicing was not evident in this polyploid species, but isoforms were switched at phase transitions. The novel, stage-specific regulatory controls uncovered here challenge the conventional understanding of this crucial biological process and provide a new resource of requisite knowledge for those aiming to directly modulate meiosis to improve crop plants. The wheat meiosis transcriptome dataset can be queried for genes of interest using an eFP browser located at https://bar.utoronto.ca/efp_wheat/cgi-bin/efpWeb.cgi?dataSource=Wheat_Meiosis.

Sequencing of Camelina neglecta, a diploid progenitor of the hexaploid oilseed Camelina sativa.

Camelina neglecta is a diploid species from the genus Camelina, which includes the versatile oilseed Camelina sativa. These species are closely related to Arabidopsis thaliana and the economically important Brassica crop species, making this genus a useful platform to dissect traits of agronomic importance while providing a tool to study the evolution of polyploids. A highly contiguous chromosome-level genome sequence of C. neglecta with an N50 size of 29.1 Mb was generated utilizing Pacific Biosciences (PacBio, Menlo Park, CA) long-read sequencing followed by chromosome conformation phasing. Comparison of the genome with that of C. sativa shows remarkable coincidence with subgenome 1 of the hexaploid, with only one major chromosomal rearrangement separating the two. Synonymous substitution rate analysis of the predicted 34 061 genes suggested subgenome 1 of C. sativa directly descended from C. neglecta around 1.2 mya. Higher functional divergence of genes in the hexaploid as evidenced by the greater number of unique orthogroups, and differential composition of resistant gene analogs, might suggest an immediate adaptation strategy after genome merger. The absence of genome bias in gene fractionation among the subgenomes of C. sativa in comparison with C. neglecta, and the complete lack of fractionation of meiosis-specific genes attests to the neopolyploid status of C. sativa. The assembled genome will provide a tool to further study genome evolution processes in the Camelina genus and potentially allow for the identification and exploitation of novel variation for Camelina crop improvement.

Quantitative evaluation of nonlinear methods for population structure visualization and inference.

Population structure (also called genetic structure and population stratification) is the presence of a systematic difference in allele frequencies between subpopulations in a population as a result of nonrandom mating between individuals. It can be informative of genetic ancestry, and in the context of medical genetics, it is an important confounding variable in genome-wide association studies. Recently, many nonlinear dimensionality reduction techniques have been proposed for the population structure visualization task. However, an objective comparison of these techniques has so far been missing from the literature. In this article, we discuss the previously proposed nonlinear techniques and some of their potential weaknesses. We then propose a novel quantitative evaluation methodology for comparing these nonlinear techniques, based on populations for which pedigree is known a priori either through artificial selection or simulation. Based on this evaluation metric, we find graph-based algorithms such as t-SNE and UMAP to be superior to principal component analysis, while neural network-based methods fall behind.

Sources of genomic diversity in the self-fertile plant pathogen, Sclerotinia sclerotiorum, and consequences for resistance breeding.

The ascomycete, Sclerotinia sclerotiorum, has a broad host range and causes yield loss in dicotyledonous crops world wide. Genomic diversity was determined in a population of 127 isolates obtained from individual canola (Brassica napus) fields in western Canada. Genotyping with 39 simple sequence repeat (SSR) markers revealed each isolate was a unique haplotype. Analysis of molecular variance showed 97% was due to isolate and 3% due to geographical location. Testing of mycelium compatibility among 133 isolates identified clones of mutually compatible isolates with 86-95% similar SSR haplotype, whereas incompatible isolates were highly diverse. In the Province of Manitoba, 61% of isolates were compatible forming clones and stings of pairwise compatible isolates not described before. In contrast, only 35% of isolates were compatible in Alberta without forming clones and strings, while 39% were compatible in Saskatchewan with a single clone, but no strings. These difference can be explained by wetter growing seasons and more susceptible crop species in Manitoba favouring frequent mycelium interaction and more life cycles over time, which might also explain similar differences observed in other geographical areas and host crops. Analysis of linkage disequilibrium rejected random recombination, consistent with a self-fertile fungus, restricted outcrossing due to mycelium incompatibility, and only a single annual opportunity for genomic recombination during meiosis in the ascospore stage between non-sister chromatids in the rare event nuclei from different isolates come together. More probable sources of genomic diversity is slippage during DNA replication and point mutation affecting single nucleotides that accumulate and likely increase mycelium incompatibility in a population over time. A phylogenetic tree based on SSR haplotype grouped isolates into 17 sub-populations. Aggressiveness was tested by inoculating one isolate from each sub-population onto B. napus lines with quantitative resistance. Analysis of variance was significant for isolate, line, and isolate by line interaction. These isolates represent the genomic and pathogenic diversity in western Canada, and are suitable for resistance screening in canola breeding programs.

Whole-genome sequencing of a year-round fruiting jackfruit (Artocarpus heterophyllus Lam.) reveals high levels of single nucleotide variation.

Jackfruit (Artocarpus heterophyllus Lam.) is the national fruit of Bangladesh and produces fruit in the summer season only. However, jackfruit is not commercially grown in Bangladesh because of an extremely high variation in fruit quality, short seasonal fruiting (June-August) and susceptibility to abiotic stresses. Conversely, a year-round high yielding (ca. 4-fold higher than the seasonal variety) jackfruit variety, BARI Kanthal-3 developed by the Bangladesh Agricultural Research Institute (BARI) derived from a wild accession found in Ramgarh of Chattogram Hiltracts of Bangladesh, provides fruits from September to June. This study aimed to generate a draft whole-genome sequence (WGS) of BARI Kanthal-3 to obtain molecular insights including genes associated with year-round fruiting trait of this important unique variety. The estimated genome size of BARI Kanthal-3 was 1.04-gigabase-pair (Gbp) with a heterozygosity rate of 1.62%. De novo assembly yielded a scaffolded 817.7 Mb genome while a reference-guided approach, yielded 843 Mb of genome sequence. The estimated GC content was 34.10%. Variant analysis revealed that BARI Kanthal-3 included 5.7 M (35%) and 10.4 M (65%) simple and heterozygous single nucleotide polymorphisms (SNPs), and about 90% of all these polymorphisms are in inter-genic regions. Through BUSCO assessment, 97.2% of the core genes were represented in the assembly with 1.3% and 1.5% either fragmented or missing, respectively. By comparing identified orthologous gene groups in BARI Kanthal-3 with five closely and one distantly related species of 10,092 common orthogroups were found across the genomes of the six species. The phylogenetic analysis of the shared orthogroups showed that A. heterophyllus was the closest species to BARI Kanthal-3 and orthogroups related to flowering time were found to be more highly prevalent in BARI Kanthal-3 compared to the other Arctocarpus spp. The findings of this study will help better understanding the evolution, domestication, phylogenetic relationships, year-round fruiting of this highly nutritious fruit crop as well as providing a resource for molecular breeding.

Deep neural networks for genomic prediction do not estimate marker effects.

Genomic prediction is a promising technology for advancing both plant and animal breeding, with many different prediction models evaluated in the literature. It has been suggested that the ability of powerful nonlinear models, such as deep neural networks, to capture complex epistatic effects between markers offers advantages for genomic prediction. However, these methods tend not to outperform classical linear methods, leaving it an open question why this capacity to model nonlinear effects does not seem to result in better predictive capability. In this work, we propose the theory that, because of a previously described principle called shortcut learning, deep neural networks tend to base their predictions on overall genetic relatedness rather than on the effects of particular markers such as epistatic effects. Using several datasets of crop plants [lentil (Lens culinaris Medik.), wheat (Triticum aestivum L.), and Brassica carinata A. Braun], we demonstrate the network's indifference to the values of the markers by showing that the same network, provided with only the locations of matches between markers for two individuals, is able to perform prediction to the same level of accuracy.

Alternative splicing dynamics and evolutionary divergence during embryogenesis in wheat species.

Among polyploid species with complex genomic architecture, variations in the regulation of alternative splicing (AS) provide opportunities for transcriptional and proteomic plasticity and the potential for generating trait diversities. However, the evolution of AS and its influence on grain development in diploid grass and valuable polyploid wheat crops are poorly understood. To address this knowledge gap, we developed a pipeline for the analysis of alternatively spliced transcript isoforms, which takes the high sequence similarity among polyploid wheat subgenomes into account. Through analysis of synteny and detection of collinearity of homoeologous subgenomes, conserved and specific AS events across five wheat and grass species were identified. A global analysis of the regulation of AS in diploid grass and polyploid wheat grains revealed diversity in AS events not only between the endosperm, pericarp and embryo overdevelopment, but also between subgenomes. Analysis of AS in homoeologous triads of polyploid wheats revealed evolutionary divergence between gene-level and transcript-level regulation of embryogenesis. Evolutionary age analysis indicated that the generation of novel transcript isoforms has occurred in young genes at a more rapid rate than in ancient genes. These findings, together with the development of comprehensive AS resources for wheat and grass species, advance understanding of the evolution of regulatory features of AS during embryogenesis and grain development in wheat.

Long-read sequence assembly: a technical evaluation in barley.

Sequence assembly of large and repeat-rich plant genomes has been challenging, requiring substantial computational resources and often several complementary sequence assembly and genome mapping approaches. The recent development of fast and accurate long-read sequencing by circular consensus sequencing (CCS) on the PacBio platform may greatly increase the scope of plant pan-genome projects. Here, we compare current long-read sequencing platforms regarding their ability to rapidly generate contiguous sequence assemblies in pan-genome studies of barley (Hordeum vulgare). Most long-read assemblies are clearly superior to the current barley reference sequence based on short-reads. Assemblies derived from accurate long reads excel in most metrics, but the CCS approach was the most cost-effective strategy for assembling tens of barley genomes. A downsampling analysis indicated that 20-fold CCS coverage can yield very good sequence assemblies, while even five-fold CCS data may capture the complete sequence of most genes. We present an updated reference genome assembly for barley with near-complete representation of the repeat-rich intergenic space. Long-read assembly can underpin the construction of accurate and complete sequences of multiple genomes of a species to build pan-genome infrastructures in Triticeae crops and their wild relatives.

A high-contiguity Brassica nigra genome localizes active centromeres and defines the ancestral Brassica genome.

It is only recently, with the advent of long-read sequencing technologies, that we are beginning to uncover previously uncharted regions of complex and inherently recursive plant genomes. To comprehensively study and exploit the genome of the neglected oilseed Brassica nigra, we generated two high-quality nanopore de novo genome assemblies. The N50 contig lengths for the two assemblies were 17.1 Mb (12 contigs), one of the best among 324 sequenced plant genomes, and 0.29 Mb (424 contigs), respectively, reflecting recent improvements in the technology. Comparison with a de novo short-read assembly corroborated genome integrity and quantified sequence-related error rates (0.2%). The contiguity and coverage allowed unprecedented access to low-complexity regions of the genome. Pericentromeric regions and coincidence of hypomethylation enabled localization of active centromeres and identified centromere-associated ALE family retro-elements that appear to have proliferated through relatively recent nested transposition events (<1 Ma). Genomic distances calculated based on synteny relationships were used to define a post-triplication Brassica-specific ancestral genome, and to calculate the extensive rearrangements that define the evolutionary distance separating B. nigra from its diploid relatives.

Mapping quantitative trait loci associated with leaf rust resistance in five spring wheat populations using single nucleotide polymorphism markers.

Growing resistant wheat (Triticum aestivum L) varieties is an important strategy for the control of leaf rust, caused by Puccinia triticina Eriks. This study sought to identify the chromosomal location and effects of leaf rust resistance loci in five Canadian spring wheat cultivars. The parents and doubled haploid lines of crosses Carberry/AC Cadillac, Carberry/Vesper, Vesper/Lillian, Vesper/Stettler and Stettler/Red Fife were assessed for leaf rust severity and infection response in field nurseries in Canada near Swift Current, SK from 2013 to 2015, Morden, MB from 2015 to 2017 and Brandon, MB in 2016, and in New Zealand near Lincoln in 2014. The populations were genotyped with the 90K Infinium iSelect assay and quantitative trait loci (QTL) analysis was performed. A high density consensus map generated based on 14 doubled haploid populations and integrating SNP and SSR markers was used to compare QTL identified in different populations. AC Cadillac contributed QTL on chromosomes 2A, 3B and 7B (2 loci), Carberry on 1A, 2B (2 loci), 2D, 4B (2 loci), 5A, 6A, 7A and 7D, Lillian on 4A and 7D, Stettler on 2D and 6B, Vesper on 1B, 1D, 2A, 6B and 7B (2 loci), and Red Fife on 7A and 7B. Lillian contributed to a novel locus QLr.spa-4A, and similarly Carberry at QLr.spa-5A. The discovery of novel leaf rust resistance QTL QLr.spa-4A and QLr.spa-5A, and several others in contemporary Canada Western Red Spring wheat varieties is a tremendous addition to our present knowledge of resistance gene deployment in breeding. Carberry demonstrated substantial stacking of genes which could be supplemented with the genes identified in other cultivars with the expectation of increasing efficacy of resistance to leaf rust and longevity with little risk of linkage drag.

Assessing Diversity in the Camelina Genus Provides Insights into the Genome Structure of Camelina sativa.

Camelina sativa (L.) Crantz an oilseed crop of the Brassicaceae family is gaining attention due to its potential as a source of high value oil for food, feed or fuel. The hexaploid domesticated C. sativa has limited genetic diversity, encouraging the exploration of related species for novel allelic variation for traits of interest. The current study utilized genotyping by sequencing to characterize 193 Camelina accessions belonging to seven different species collected primarily from the Ukrainian-Russian region and Eastern Europe. Population analyses among Camelina accessions with a 2n = 40 karyotype identified three subpopulations, two composed of domesticated C. sativa and one of C. microcarpa species. Winter type Camelina lines were identified as admixtures of C. sativa and C. microcarpa Eighteen genotypes of related C. microcarpa unexpectedly shared only two subgenomes with C. sativa, suggesting a novel or cryptic sub-species of C. microcarpa with 19 haploid chromosomes. One C. microcarpa accession (2n = 26) was found to comprise the first two subgenomes of C. sativa suggesting a tetraploid structure. The defined chromosome series among C. microcarpa germplasm, including the newly designated C. neglecta diploid née C. microcarpa, suggested an evolutionary trajectory for the formation of the C. sativa hexaploid genome and re-defined the underlying subgenome structure of the reference genome.

WheatCRISPR: a web-based guide RNA design tool for CRISPR/Cas9-mediated genome editing in wheat.

BACKGROUND: CRISPR/Cas9 gene editing has become a revolutionary technique for crop improvement as it can facilitate fast and efficient genetic changes without the retention of transgene components in the final plant line. Lack of robust bioinformatics tools to facilitate the design of highly specific functional guide RNAs (gRNAs) and prediction of off-target sites in wheat is currently an obstacle to effective application of CRISPR technology to wheat improvement. DESCRIPTION: We have developed a web-based bioinformatics tool to design specific gRNAs for genome editing and transcriptional regulation of gene expression in wheat. A collaborative study between the Broad Institute and Microsoft Research used large-scale empirical evidence to devise algorithms (Doech et al., 2016, Nature Biotechnology 34, 184-191) for predicting the on-target activity and off-target potential of CRISPR/SpCas9 (Streptococcus pyogenes Cas9). We applied these prediction models to determine on-target specificity and potential off-target activity for individual gRNAs targeting specific loci in the wheat genome. The genome-wide gRNA mappings and the corresponding Doench scores predictive of the on-target and off-target activities were used to create a gRNA database which was used as a data source for the web application termed WheatCRISPR. CONCLUSION: The WheatCRISPR tool allows researchers to browse all possible gRNAs targeting a gene or sequence of interest and select effective gRNAs based on their predicted high on-target and low off-target activity scores, as well as other characteristics such as position within the targeted gene. It is publicly available at https://crispr.bioinfo.nrc.ca/WheatCrispr/ .

Mapping quantitative trait loci associated with common bunt resistance in a spring wheat (Triticum aestivum L.) variety Lillian.

KEY MESSAGE: Based on their consistency over environments, two QTL identified in Lillian on chromosomes 5A and 7A could be useful targets for marker assisted breeding of common bunt resistance. Common bunt of wheat (Triticum aestivum L.) caused by Tilletia tritici and T. laevis is an economically important disease because of losses in grain yield and reduced grain quality. Resistance can be quantitative, under the control of multiple small effect genes. The Canada Western Red Spring wheat variety Lillian is moderately resistant to common bunt races found on the Canadian prairies. This study was conducted to identify and map quantitative trait loci (QTL) conferring resistance against common bunt in Lillian. A doubled haploid population comprising 280 lines was developed from F1 plants of the cross of Lillian by Vesper. The lines were inoculated at seeding with the two races L16 (T. laevis) and T19 (T. tritici), grown in field near Swift Current, SK, in 2014, 2015 and 2016 and assessed for disease incidence. The lines were genotyped with the 90 K iSelect SNP genotyping assay, and a high-density genetic map was constructed. Quantitative trait locus analysis was performed with MapQTL.6® software. Two relatively stable common bunt resistance QTL, detected in two of the 3 years, were identified on chromosomes 5A and 7A from Lillian. In addition, three less stable QTL, appearing in one out of 3 years, were identified: one was contributed by Lillian on chromosome 3D and two were contributed by Vesper on chromosomes 1D and 2A. Epistatic interaction was identified for the bunt incidence between 3D and 7A resulting in greater bunt resistance. Future bunt resistance breeding will benefit from combining these QTL through gene pyramiding.

Correction to: MeioCapture: an efficient method for staging and isolation of meiocytes in the prophase I sub-stages of meiosis in wheat.

Following publication of the original article [1], a reader spotted an incorrect citation of the reference 14 [2] in the 'Background'. The male meiocyte isolation work described in this article [2] was carried out in rice and not in Brassica as originally stated in the 'Background' [1]. Thus, the following amendment to the Background section should be noted.

MeioCapture: an efficient method for staging and isolation of meiocytes in the prophase I sub-stages of meiosis in wheat.

BACKGROUND: Molecular analysis of meiosis has been hindered by difficulties in isolating high purity subpopulations of sporogenous cells representing the succeeding stages of meiosis. Isolation of purified male meiocytes from defined meiotic stages is crucial in discovering meiosis specific genes and associated regulatory networks. RESULTS: We describe an optimized method termed MeioCapture for simultaneous isolation of uncontaminated male meiocytes from wheat (Triticum spp.), specifically from the pre-meiotic G2 and the five sub-stages of meiotic prophase I. The MeioCapture protocol builds on the traditional anther squash technique and the capillary collection method, and involves extrusion of intact sporogenous archesporial columns (SACs) containing meiocytes. This improved method exploits the natural meiotic synchrony between anthers of the same floret, the correlation between the length of anthers and meiotic stage, and the occurrence of meiocytes in intact SACs largely free of somatic cells. The main advantage of MeioCapture, compared to previous methods, is that it allows simultaneous collection of meiocytes from different sub-stages of prophase I at a very high level of purity, through correlation of stages with anther sizes. A detailed description is provided for all steps, including the collection of tissue, isolation and size sorting of anthers, extrusion of intact SACs, and staging of meiocytes. Precautions for individual steps throughout the procedure are also provided to facilitate efficient isolation of pure meiocytes. The proof-of-concept was successfully established in wheat, and a light microscopic atlas of meiosis, encompassing all stages from pre-meiosis to telophase II, was developed. CONCLUSION: The MeioCapture method provides an essential technique to study the molecular basis of chromosome pairing and exchange of genetic information in wheat, leading to strategies for manipulating meiotic recombination frequencies. The method also provides a foundation for similar studies in other crop species.