CRISPR-Cas13a-powered electrochemical biosensor for the detection of the L452R mutation in clinical samples of SARS-CoV-2 variants.

Since the end of 2019, a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has deprived numerous lives worldwide, called COVID-19. Up to date, omicron is the latest variant of concern, and BA.5 is replacing the BA.2 variant to become the main subtype rampaging worldwide. These subtypes harbor an L452R mutation, which increases their transmissibility among vaccinated people. Current methods for identifying SARS-CoV-2 variants are mainly based on polymerase chain reaction (PCR) followed by gene sequencing, making time-consuming processes and expensive instrumentation indispensable. In this study, we developed a rapid and ultrasensitive electrochemical biosensor to achieve the goals of high sensitivity, the ability of distinguishing the variants, and the direct detection of RNAs from viruses simultaneously. We used electrodes made of MXene-AuNP (gold nanoparticle) composites for improved sensitivity and the CRISPR/Cas13a system for high specificity in detecting the single-base L452R mutation in RNAs and clinical samples. Our biosensor will be an excellent supplement to the RT-qPCR method enabling the early diagnosis and quick distinguishment of SARS-CoV-2 Omicron BA.5 and BA.2 variants and more potential variants that might arise in the future.

Current status of structural variation studies in plants.

Structural variations (SVs) including gene presence/absence variations and copy number variations are a common feature of genomes in plants and, together with single nucleotide polymorphisms and epigenetic differences, are responsible for the heritable phenotypic diversity observed within and between species. Understanding the contribution of SVs to plant phenotypic variation is important for plant breeders to assist in producing improved varieties. The low resolution of early genetic technologies and inefficient methods have previously limited our understanding of SVs in plants. However, with the rapid expansion in genomic technologies, it is possible to assess SVs with an ever-greater resolution and accuracy. Here, we review the current status of SV studies in plants, examine the roles that SVs play in phenotypic traits, compare current technologies and assess future challenges for SV studies.

Modelling of gene loss propensity in the pangenomes of three Brassica species suggests different mechanisms between polyploids and diploids.

Plant genomes demonstrate significant presence/absence variation (PAV) within a species; however, the factors that lead to this variation have not been studied systematically in Brassica across diploids and polyploids. Here, we developed pangenomes of polyploid Brassica napus and its two diploid progenitor genomes B. rapa and B. oleracea to infer how PAV may differ between diploids and polyploids. Modelling of gene loss suggests that loss propensity is primarily associated with transposable elements in the diploids while in B. napus, gene loss propensity is associated with homoeologous recombination. We use these results to gain insights into the different causes of gene loss, both in diploids and following polyploidization, and pave the way for the application of machine learning methods to understanding the underlying biological and physical causes of gene presence/absence.

First Report of Anthracnose on Camellia japonica Caused by Colletotrichum siamense in Zhejiang Province, China.

Camellia japonica is an attractive flowering woody plant with great ornamental and medicinal value in China. However, typical anthracnose lesions on the leaves are usually observed in summer in Zhejiang province. A number of 100 trees have been investigated with over 70% of leaf disease incidence. The symptom initially develops from the tip or edge of the leaf and dark green infected spots appear. The diseased spots expand and become yellow brown. The lesions are covered with abundant, small and black acervuli at the center with yellow edges. The diseased leaves become brittle, cracked, and finally fall off. Sixty leaves with typical anthracnose symptoms were sampled from gardens in Lin'an, Zhejiang province. The diseased tissues were cut into pieces and incubated in moist chambers at 25°C. The spore mass was collected using a sterile needle under dissection microscope and put on 2% malt extract agar (MEA). The cultures were incubated at 25°C in the dark for one week. Thirty single spore cultures were obtained and grown on 2% MEA at 25°C for morphological characterization. White aerial mycelia and black conidiomata with orange masses of conidia developed seven days later. Conidia are cylindrical in shape, 12-19 µm, mean lengths ranging from 15.5 ± 1.0 to 16.0 ± 1.2 µm. The morphological characteristics are consistent with those of Colletotrichum species. DNA was extracted from three selected isolates (HT-71, J-5, J-20) for sequencing. The partial regions of ribosomal internal transcribed spacers (ITS), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin gene (ACT), beta-tubulin (TUB2), Apn2-Mat1-2 intergenic spacer and partial mating type gene (ApMat), and glutamine synthetase (GS) were amplified as described by Liu et al. (2015). Sequences of the above seven loci for the selected isolates were obtained, and deposited in the GenBank database (MZ014901 to MZ014905, MZ514915 to MZ514922, MZ514925 to MZ514930, MZ497332 and MZ497333). BLAST results indicate they represent Colletotricum siamense. Multi-locus phylogenetic analysis including ex-type of C. siamense (ICMP18578=CBS130417) and related species was conducted using Maximum Likelihood method, and C. acutatum (CBS 112996) served as the outgroup. The three obtained isolates clustered with the ex-type isolate of C. siamense. Eight leaves on two Camellia plants were inoculated to confirm the pathogenicity in the field. The leaves were surface sprayed with 75% ethanol and dried with sterilized filter paper. The leaves were inoculated using the wound/drop inoculation method: an aliquot of 10 µL of spore suspension (1.0 × 106 conidia per mL) was dropped on the left side of a leaf after wounding once by pin-pricking with a sterilized needle. The sterile water was dropped on the right side of the same leaf in parallel as control. The initial symptoms were observed seven days later, all inoculated leaves developed lesions similar to those observed in the field, and no symptoms observed in the control. The fungus was successfully re-isolated only from lesions inoculated with spore suspension exhibiting morphological characteristics resembling those in C. siamense, and further confirmed with sequence data. To our knowledge, this represents the first report of anthracnose on C. japonica caused by C. siamense worldwide. Confirmation of this pathogen in the region will be helpful for the disease management on C. japonica, considering previous report of C. camelliae-japonicae on the same host. References Fu, M., et al. 2019. Persoonia. 42: 1. https://doi.org/10.3767/persoonia.2019.42.01 Guarnaccia, V., et al. 2017. Persoonia. 39: 32. https://doi.org/10.3767/persoonia.2017.39.02 Hou, L. W., et al. 2016. Mycosphere. 7: 1111. Doi 10.5943/mycosphere/si/2c/4 Liu, F., et al. 2015. Persoonia. 35: 63. http://dx.doi.org/10.3767/003158515X687597 Vieira, A. D. S., et al. 2019. Mol. Phylogenet. Evol. https://doi.org/10.1016/j.ympev.2019.106694.

Construction and comparison of three reference-quality genome assemblies for soybean.

We report reference-quality genome assemblies and annotations for two accessions of soybean (Glycine max) and for one accession of Glycine soja, the closest wild relative of G. max. The G. max assemblies provided are for widely used US cultivars: the northern line Williams 82 (Wm82) and the southern line Lee. The Wm82 assembly improves the prior published assembly, and the Lee and G. soja assemblies are new for these accessions. Comparisons among the three accessions show generally high structural conservation, but nucleotide difference of 1.7 single-nucleotide polymorphisms (snps) per kb between Wm82 and Lee, and 4.7 snps per kb between these lines and G. soja. snp distributions and comparisons with genotypes of the Lee and Wm82 parents highlight patterns of introgression and haplotype structure. Comparisons against the US germplasm collection show placement of the sequenced accessions relative to global soybean diversity. Analysis of a pan-gene collection shows generally high conservation, with variation occurring primarily in genomically clustered gene families. We found approximately 40-42 inversions per chromosome between either Lee or Wm82v4 and G. soja, and approximately 32 inversions per chromosome between Wm82 and Lee. We also investigated five domestication loci. For each locus, we found two different alleles with functional differences between G. soja and the two domesticated accessions. The genome assemblies for multiple cultivated accessions and for the closest wild ancestor of soybean provides a valuable set of resources for identifying causal variants that underlie traits for the domestication and improvement of soybean, serving as a basis for future research and crop improvement efforts for this important crop species.

runBNG: a software package for BioNano genomic analysis on the command line.

SUMMARY: We developed runBNG, an open-source software package which wraps BioNano genomic analysis tools into a single script that can be run on the command line. runBNG can complete analyses, including quality control of single molecule maps, optical map de novo assembly, comparisons between different optical maps, super-scaffolding and structural variation detection. Compared to existing software BioNano IrysView and the KSU scripts, the major advantages of runBNG are that the whole pipeline runs on one single platform and it has a high customizability. AVAILABILITY AND IMPLEMENTATION: runBNG is written in bash, with the requirement of BioNano IrysSolve packages, GCC, Perl and Python software. It is freely available at https://github.com/appliedbioinformatics/runBNG. CONTACT: dave.edwards@uwa.edu.au. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

BioNanoAnalyst: a visualisation tool to assess genome assembly quality using BioNano data.

BACKGROUND: Reference genome assemblies are valuable, as they provide insights into gene content, genetic evolution and domestication. The higher the quality of a reference genome assembly the more accurate the downstream analysis will be. During the last few years, major efforts have been made towards improving the quality of genome assemblies. However, erroneous and incomplete assemblies are still common. Complementary to DNA sequencing technologies, optical mapping has advanced genomic studies by facilitating the production of genome scaffolds and assessing structural variation. However, there are few tools available to comprehensively examine misassemblies in reference genome sequences using optical map data. RESULTS: We present BioNanoAnalyst, a software package to examine genome assemblies based on restriction endonuclease cut sites and optical map data. A graphical user interface (GUI) allows users to assess reference genome sequences on different computer platforms without the requirement of programming knowledge. The zoom function makes visualisation convenient, while a GFF3 format output file gives an option to directly visualise questionable assembly regions by location and nucleotides following import into a local genome browser. CONCLUSIONS: BioNanoAnalyst is a tool to identify misassemblies in a reference genome sequence using optical map data. With the reported information, users can rapidly identify assembly errors and correct them using other software tools, which could facilitate an accurate downstream analysis.

An advanced reference genome of Trifolium subterraneum L. reveals genes related to agronomic performance.

Subterranean clover is an important annual forage legume, whose diploidy and inbreeding nature make it an ideal model for genomic analysis in Trifolium. We reported a draft genome assembly of the subterranean clover TSUd_r1.1. Here we evaluate genome mapping on nanochannel arrays and generation of a transcriptome atlas across tissues to advance the assembly and gene annotation. Using a BioNano-based assembly spanning 512 Mb (93% genome coverage), we validated the draft assembly, anchored unplaced contigs and resolved misassemblies. Multiple contigs (264) from the draft assembly coalesced into 97 super-scaffolds (43% of genome). Sequences longer than >1 Mb increased from 40 to 189 Mb giving 1.4-fold increase in N50 with total genome in pseudomolecules improved from 73 to 80%. The advanced assembly was re-annotated using transcriptome atlas data to contain 31 272 protein-coding genes capturing >96% of the gene content. Functional characterization and GO enrichment confirmed gene expression for response to water deprivation, flavonoid biosynthesis and embryo development ending in seed dormancy, reflecting adaptation to the harsh Mediterranean environment. Comparative analyses across Papilionoideae identified 24 893 Trifolium-specific and 6325 subterranean-clover-specific genes that could be mined further for traits such as geocarpy and grazing tolerance. Eight key traits, including persistence, improved livestock health by isoflavonoid production in addition to important agro-morphological traits, were fine-mapped on the high-density SNP linkage map anchored to the assembly. This new genomic information is crucial to identify loci governing traits allowing marker-assisted breeding, comparative mapping and identification of tissue-specific gene promoters for biotechnological improvement of forage legumes.

Assembly and comparison of two closely related Brassica napus genomes.

As an increasing number of plant genome sequences become available, it is clear that gene content varies between individuals, and the challenge arises to predict the gene content of a species. However, genome comparison is often confounded by variation in assembly and annotation. Differentiating between true gene absence and variation in assembly or annotation is essential for the accurate identification of conserved and variable genes in a species. Here, we present the de novo assembly of the B. napus cultivar Tapidor and comparison with an improved assembly of the Brassica napus cultivar Darmor-bzh. Both cultivars were annotated using the same method to allow comparison of gene content. We identified genes unique to each cultivar and differentiate these from artefacts due to variation in the assembly and annotation. We demonstrate that using a common annotation pipeline can result in different gene predictions, even for closely related cultivars, and repeat regions which collapse during assembly impact whole genome comparison. After accounting for differences in assembly and annotation, we demonstrate that the genome of Darmor-bzh contains a greater number of genes than the genome of Tapidor. Our results are the first step towards comparison of the true differences between B. napus genomes and highlight the potential sources of error in future production of a B. napus pangenome.

A pangenome analysis pipeline provides insights into functional gene identification in rice.

BACKGROUND: A pangenome aims to capture the complete genetic diversity within a species and reduce bias in genetic analysis inherent in using a single reference genome. However, the current linear format of most plant pangenomes limits the presentation of position information for novel sequences. Graph pangenomes have been developed to overcome this limitation. However, bioinformatics analysis tools for graph format genomes are lacking. RESULTS: To overcome this problem, we develop a novel strategy for pangenome construction and a downstream pangenome analysis pipeline (PSVCP) that captures genetic variants' position information while maintaining a linearized layout. Using PSVCP, we construct a high-quality rice pangenome using 12 representative rice genomes and analyze an international rice panel with 413 diverse accessions using the pangenome as the reference. We show that PSVCP successfully identifies causal structural variations for rice grain weight and plant height. Our results provide insights into rice population structure and genomic diversity. We characterize a new locus (qPH8-1) associated with plant height on chromosome 8 undetected by the SNP-based genome-wide association study (GWAS). CONCLUSIONS: Our results demonstrate that the pangenome constructed by our pipeline combined with a presence and absence variation-based GWAS can provide additional power for genomic and genetic analysis. The pangenome constructed in this study and the associated genome sequence and genetic variants data provide valuable genomic resources for rice genomics research and improvement in future.

Genomic analysis and characterization of new loci associated with seed protein and oil content in soybeans.

Breeding for increased protein without a reduction in oil content in soybeans [Glycine max (L.) Merr.] is a challenge for soybean breeders but an expected goal. Many efforts have been made to develop new soybean varieties with high yield in combination with desirable protein and/or oil traits. An elite line, R05-1415, was reported to be high yielding, high protein, and low oil. Several significant quantitative trait loci (QTL) for protein and oil were reported in this line, but many of them were unstable across environments or genetic backgrounds. Thus, a new study under multiple field environments using the Infinium BARCSoySNP6K BeadChips was conducted to detect and confirm stable genomic loci for these traits. Genetic analyses consistently detected a single major genomic locus conveying these two traits with remarkably high phenotypic variation explained (R2 ), varying between 24.2% and 43.5%. This new genomic locus is located between 25.0 and 26.7 Mb, distant from the previously reported QTL and did not overlap with other commonly reported QTL and the recently cloned gene Glyma.20G085100. Homolog analysis indicated that this QTL did not result from the paracentric chromosome inversion with an adjacent genomic fragment that harbors the reported QTL. The pleiotropic effect of this QTL could be a challenge for improving protein and oil simultaneously; however, a further study of four candidate genes with significant expressions in the seed developmental stages coupled with haplotype analysis may be able to pinpoint causative genes. The functionality and roles of these genes can be determined and characterized, which lay a solid foundation for the improvement of protein and oil content in soybeans.

Near-gapless genome assemblies of Williams 82 and Lee cultivars for accelerating global soybean research.

Complete, gapless telomere-to-telomere chromosome assemblies are a prerequisite for comprehensively investigating the architecture of complex regions, like centromeres or telomeres and removing uncertainties in the order, spacing, and orientation of genes. Using complementary genomics technologies and assembly algorithms, we developed highly contiguous, nearly gapless, genome assemblies for two economically important soybean [Glycine max (L.) Merr] cultivars (Williams 82 and Lee). The centromeres were distinctly annotated on all the chromosomes of both assemblies. We further found that the canonical telomeric repeats were present at the telomeres of all chromosomes of both Williams 82 and Lee genomes. A total of 10 chromosomes in Williams 82 and eight in Lee were entirely reconstructed in single contigs without any gap. Using the combination of ab initio prediction, protein homology, and transcriptome evidence, we identified 58,287 and 56,725 protein-coding genes in Williams 82 and Lee, respectively. The genome assemblies and annotations will serve as a valuable resource for studying soybean genomics and genetics and accelerating soybean improvement.

Applications of Optical Mapping for Plant Genome Assembly and Structural Variation Detection.

Optical mapping plays an important role in plant genomics, particularly in plant genome assembly and large-scale structural variation detection. While DNA sequencing provides base-by-base nucleotide information, optical mapping shows the physical locations of selected enzyme restriction sites in a genome. The long single-molecule maps produced by optical mapping make it a useful auxiliary technique to DNA sequencing, which generally cannot span large and complex genomic regions. Although optical mapping, therefore, offers unique advantages to researchers, there are few dedicated tools to assist in optical mapping analyses. In this chapter, we present runBNG2, a successor of runBNG to help optical-mapping data analysis for diverse datasets.

Copy Number Variation among Resistance Genes Analogues in Brassica napus.

Copy number variations (CNVs) are defined as deletions, duplications and insertions among individuals of a species. There is growing evidence that CNV is a major factor underlining various autoimmune disorders and diseases in humans; however, in plants, especially oilseed crops, the role of CNVs in disease resistance is not well studied. Here, we investigate the genome-wide diversity and genetic properties of CNVs in resistance gene analogues (RGAs) across eight Brassica napus lines. A total of 1137 CNV events (704 deletions and 433 duplications) were detected across 563 RGAs. The results show CNVs are more likely to occur across clustered RGAs compared to singletons. In addition, 112 RGAs were linked to a blackleg resistance QTL, of which 25 were affected by CNV. Overall, we show that the presence and abundance of CNVs differ between lines, suggesting that in B. napus, the distribution of CNVs depends on genetic background. Our findings advance the understanding of CNV as an important type of genomic structural variation in B. napus and provide a resource to support breeding of advanced canola lines.

Sequencing the USDA core soybean collection reveals gene loss during domestication and breeding.

The gene content of plants varies between individuals of the same species due to gene presence/absence variation, and selection can alter the frequency of specific genes in a population. Selection during domestication and breeding will modify the genomic landscape, though the nature of these modifications is only understood for specific genes or on a more general level (e.g., by a loss of genetic diversity). Here we have assembled and analyzed a soybean (Glycine spp.) pangenome representing more than 1,000 soybean accessions derived from the USDA Soybean Germplasm Collection, including both wild and cultivated lineages, to assess genomewide changes in gene and allele frequency during domestication and breeding. We identified 3,765 genes that are absent from the Lee reference genome assembly and assessed the presence/absence of all genes across this population. In addition to a loss of genetic diversity, we found a significant reduction in the average number of protein-coding genes per individual during domestication and subsequent breeding, though with some genes and allelic variants increasing in frequency associated with selection for agronomic traits. This analysis provides a genomic perspective of domestication and breeding in this important oilseed crop.

CRISPR-Cas12a-Empowered Electrochemical Biosensor for Rapid and Ultrasensitive Detection of SARS-CoV-2 Delta Variant.

Coronavirus disease 2019 (COVID-19) is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The gold standard method for the diagnosis of SARS-CoV-2 depends on quantitative reverse transcription-polymerase chain reaction till now, which is time-consuming and requires expensive instrumentation, and the confirmation of variants relies on further sequencing techniques. Herein, we first proposed a robust technique-methodology of electrochemical CRISPR sensing with the advantages of rapid, highly sensitivity and specificity for the detection of SARS-CoV-2 variant. To enhance the sensing capability, gold electrodes are uniformly decorated with electro-deposited gold nanoparticles. Using DNA template identical to SARS-CoV-2 Delta spike gene sequence as model, our biosensor exhibits excellent analytical detection limit (50 fM) and high linearity (R2 = 0.987) over six orders of magnitude dynamic range from 100 fM to 10 nM without any nucleic-acid-amplification assays. The detection can be completed within 1 h with high stability and specificity which benefits from the CRISPR-Cas system. Furthermore, based on the wireless micro-electrochemical platform, the proposed biosensor reveals promising application ability in point-of-care testing.

Advances in optical mapping for genomic research.

Recent advances in optical mapping have allowed the construction of improved genome assemblies with greater contiguity. Optical mapping also enables genome comparison and identification of large-scale structural variations. Association of these large-scale genomic features with biological functions is an important goal in plant and animal breeding and in medical research. Optical mapping has also been used in microbiology and still plays an important role in strain typing and epidemiological studies. Here, we review the development of optical mapping in recent decades to illustrate its importance in genomic research. We detail its applications and algorithms to show its specific advantages. Finally, we discuss the challenges required to facilitate the optimization of optical mapping and improve its future development and application.

Legume Pangenome Construction Using an Iterative Mapping and Assembly Approach.

A pangenome is a collection of genomic sequences found in the entire species rather than a single individual. It allows for comprehensive, species-wide characterization of genetic variations and mining of variable genes which may play important roles in phenotypes of interest. Recent advances in sequencing technologies have facilitated draft genome sequence construction and have made pangenome constructions feasible. Here, we present a reference genome-based iterative mapping and assembly method to construct a pangenome for a legume species.

Method for Genome-Wide Association Study: A Soybean Example.

Genome-wide association studies (GWAS) are a valuable approach to identify single nucleotide polymorphisms (SNPs) associated with a phenotype of interest. There are now a variety of R-packages and command line tools available to perform GWAS. Here, we provide an example downloading and filtering SNP data, followed by GWAS analysis using the R-package rMVP.

Single-Cell Genomic Analysis in Plants.

Individual cells in an organism are variable, which strongly impacts cellular processes. Advances in sequencing technologies have enabled single-cell genomic analysis to become widespread, addressing shortcomings of analyses conducted on populations of bulk cells. While the field of single-cell plant genomics is in its infancy, there is great potential to gain insights into cell lineage and functional cell types to help understand complex cellular interactions in plants. In this review, we discuss current approaches for single-cell plant genomic analysis, with a focus on single-cell isolation, DNA amplification, next-generation sequencing, and bioinformatics analysis. We outline the technical challenges of analysing material from a single plant cell, and then examine applications of single-cell genomics and the integration of this approach with genome editing. Finally, we indicate future directions we expect in the rapidly developing field of plant single-cell genomic analysis.