A repackaged CRISPR platform increases homology-directed repair for yeast engineering.

Inefficient homology-directed repair (HDR) constrains CRISPR-Cas9 genome editing in organisms that preferentially employ nonhomologous end joining (NHEJ) to fix DNA double-strand breaks (DSBs). Current strategies used to alleviate NHEJ proficiency involve NHEJ disruption. To confer precision editing without NHEJ disruption, we identified the shortcomings of the conventional CRISPR platforms and developed a CRISPR platform-lowered indel nuclease system enabling accurate repair (LINEAR)-which enhanced HDR rates (to 67-100%) compared to those in previous reports using conventional platforms in four NHEJ-proficient yeasts. With NHEJ preserved, we demonstrate its ability to survey genomic landscapes, identifying loci whose spatiotemporal genomic architectures yield favorable expression dynamics for heterologous pathways. We present a case study that deploys LINEAR precision editing and NHEJ-mediated random integration to rapidly engineer and optimize a microbial factory to produce (S)-norcoclaurine. Taken together, this work demonstrates how to leverage an antagonizing pair of DNA DSB repair pathways to expand the current collection of microbial factories.

Spirochaete genome identified in red abalone sample represents a novel genus Candidatus Haliotispira gen. nov. within the order Spirochaetales.

A fully assembled spirochaete genome was identified as a contaminating scaffold in our red abalone (Haliotis rufescens) genome assembly. In this paper, we describe the analysis of this bacterial genome. The assembled spirochaete genome is 3.25 Mb in size with 48.5 mol% G+C content. The proteomes of 38 species were compared with the spirochaete genome and it was discovered to form an independent branch within the family Spirochaetaceae on the phylogenetic tree. The comparison of 16S rRNA sequences and average nucleotide identity scores between the spirochaete genome with known species of different families in Spirochaetia indicate that it is an unknown species. Further, the percentage of conserved proteins compared to neighbouring taxa confirm that it does not belong to a known genus within Spirochaetaceae. We propose the name Candidatus Haliotispira prima gen. nov., sp. nov. based on its taxonomic placement and origin. We also tested for the presence of this species in different species of abalone and found that it is also present in white abalone (Haliotis sorenseni). In addition, we highlight the need for better classification of taxa within the class Spirochaetia.

Systematic in vitro specificity profiling reveals nicking defects in natural and engineered CRISPR-Cas9 variants.

Cas9 is an RNA-guided endonuclease in the bacterial CRISPR-Cas immune system and a popular tool for genome editing. The commonly used Streptococcus pyogenes Cas9 (SpCas9) is relatively non-specific and prone to off-target genome editing. Other Cas9 orthologs and engineered variants of SpCas9 have been reported to be more specific. However, previous studies have focused on specificity of double-strand break (DSB) or indel formation, potentially overlooking alternative cleavage activities of these Cas9 variants. In this study, we employed in vitro cleavage assays of target libraries coupled with high-throughput sequencing to systematically compare cleavage activities and specificities of two natural Cas9 variants (SpCas9 and Staphylococcus aureus Cas9) and three engineered SpCas9 variants (SpCas9 HF1, HypaCas9 and HiFi Cas9). We observed that all Cas9s tested could cleave target sequences with up to five mismatches. However, the rate of cleavage of both on-target and off-target sequences varied based on target sequence and Cas9 variant. In addition, SaCas9 and engineered SpCas9 variants nick targets with multiple mismatches but have a defect in generating a DSB, while SpCas9 creates DSBs at these targets. Overall, these differences in cleavage rates and DSB formation may contribute to varied specificities observed in genome editing studies.

CRISPR-Cas12a has widespread off-target and dsDNA-nicking effects.

Cas12a (Cpf1) is an RNA-guided endonuclease in the bacterial type V-A CRISPR-Cas anti-phage immune system that can be repurposed for genome editing. Cas12a can bind and cut dsDNA targets with high specificity in vivo, making it an ideal candidate for expanding the arsenal of enzymes used in precise genome editing. However, this reported high specificity contradicts Cas12a's natural role as an immune effector against rapidly evolving phages. Here, we employed high-throughput in vitro cleavage assays to determine and compare the native cleavage specificities and activities of three different natural Cas12a orthologs (FnCas12a, LbCas12a, and AsCas12a). Surprisingly, we observed pervasive sequence-specific nicking of randomized target libraries, with strong nicking of DNA sequences containing up to four mismatches in the Cas12a-targeted DNA-RNA hybrid sequences. We also found that these nicking and cleavage activities depend on mismatch type and position and vary with Cas12a ortholog and CRISPR RNA sequence. Our analysis further revealed robust nonspecific nicking of dsDNA when Cas12a is activated by binding to a target DNA. Together, our findings reveal that Cas12a has multiple nicking activities against dsDNA substrates and that these activities vary among different Cas12a orthologs.

Detecting and correcting misclassified sequences in the large-scale public databases.

MOTIVATION: As the cost of sequencing decreases, the amount of data being deposited into public repositories is increasing rapidly. Public databases rely on the user to provide metadata for each submission that is prone to user error. Unfortunately, most public databases, such as non-redundant (NR), rely on user input and do not have methods for identifying errors in the provided metadata, leading to the potential for error propagation. Previous research on a small subset of the NR database analyzed misclassification based on sequence similarity. To the best of our knowledge, the amount of misclassification in the entire database has not been quantified. We propose a heuristic method to detect potentially misclassified taxonomic assignments in the NR database. We applied a curation technique and quality control to find the most probable taxonomic assignment. Our method incorporates provenance and frequency of each annotation from manually and computationally created databases and clustering information at 95% similarity. RESULTS: We found more than two million potentially taxonomically misclassified proteins in the NR database. Using simulated data, we show a high precision of 97% and a recall of 87% for detecting taxonomically misclassified proteins. The proposed approach and findings could also be applied to other databases. AVAILABILITY AND IMPLEMENTATION: Source code, dataset, documentation, Jupyter notebooks and Docker container are available at https://github.com/boalang/nr. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The thick aleurone1 Gene Encodes a NOT1 Subunit of the CCR4-NOT Complex and Regulates Cell Patterning in Endosperm.

Maize (Zea mays) thick aleurone1 (thk1-R) mutants form multiple aleurone layers in the endosperm and have arrested embryogenesis. Prior studies suggest that thk1 functions downstream of defective kernel1 (dek1) in a regulatory pathway that controls aleurone cell fate and other endosperm traits. The original thk1-R mutant contained an ∼2-Mb multigene deletion, which precluded identification of the causal gene. Here, ethyl methanesulfonate mutagenesis produced additional alleles, and RNA sequencing from developing endosperm was used to identify a candidate gene based on differential expression compared with the wild-type progenitor. Gene editing confirmed the gene identity by producing mutant alleles that failed to complement existing thk1 mutants and that produced multiple-aleurone homozygous phenotypes. Thk1 encodes a homolog of NEGATIVE ON TATA-LESS1, a protein that acts as a scaffold for the CARBON CATABOLITE REPRESSION4-NEGATIVE ON TATA-LESS complex. This complex is highly conserved and essential in all eukaryotes for regulating a wide array of gene expression and cellular activities. Maize also harbors a duplicate locus, thick aleurone-like1, which likely accounts for the ability of thk1 mutants to form viable cells. Transcriptomic analysis indicated that THK1 regulates activities involving cell division, signaling, differentiation, and metabolism. Identification of thk1 provides an important new component of the DEK1 regulatory system that patterns cell fate in endosperm.

Response to Persistent ER Stress in Plants: A Multiphasic Process That Transitions Cells from Prosurvival Activities to Cell Death.

The unfolded protein response (UPR) is a highly conserved response that protects plants from adverse environmental conditions. The UPR is elicited by endoplasmic reticulum (ER) stress, in which unfolded and misfolded proteins accumulate within the ER. Here, we induced the UPR in maize (Zea mays) seedlings to characterize the molecular events that occur over time during persistent ER stress. We found that a multiphasic program of gene expression was interwoven among other cellular events, including the induction of autophagy. One of the earliest phases involved the degradation by regulated IRE1-dependent RNA degradation (RIDD) of RNA transcripts derived from a family of peroxidase genes. RIDD resulted from the activation of the promiscuous ribonuclease activity of ZmIRE1 that attacks the mRNAs of secreted proteins. This was followed by an upsurge in expression of the canonical UPR genes indirectly driven by ZmIRE1 due to its splicing of Zmbzip60 mRNA to make an active transcription factor that directly upregulates many of the UPR genes. At the peak of UPR gene expression, a global wave of RNA processing led to the production of many aberrant UPR gene transcripts, likely tempering the ER stress response. During later stages of ER stress, ZmIRE1's activity declined, as did the expression of survival modulating genes, Bax inhibitor1 and Bcl-2-associated athanogene7, amid a rising tide of cell death. Thus, in response to persistent ER stress, maize seedlings embark on a course of gene expression and cellular events progressing from adaptive responses to cell death.

Shared data science infrastructure for genomics data.

BACKGROUND: Creating a scalable computational infrastructure to analyze the wealth of information contained in data repositories is difficult due to significant barriers in organizing, extracting and analyzing relevant data. Shared data science infrastructures like Boag is needed to efficiently process and parse data contained in large data repositories. The main features of Boag are inspired from existing languages for data intensive computing and can easily integrate data from biological data repositories. RESULTS: As a proof of concept, Boa for genomics, Boag, has been implemented to analyze RefSeq's 153,848 annotation (GFF) and assembly (FASTA) file metadata. Boag provides a massive improvement from existing solutions like Python and MongoDB, by utilizing a domain-specific language that uses Hadoop infrastructure for a smaller storage footprint that scales well and requires fewer lines of code. We execute scripts through Boag to answer questions about the genomes in RefSeq. We identify the largest and smallest genomes deposited, explore exon frequencies for assemblies after 2016, identify the most commonly used bacterial genome assembly program, and address how animal genome assemblies have improved since 2016. Boag databases provide a significant reduction in required storage of the raw data and a significant speed up in its ability to query large datasets due to automated parallelization and distribution of Hadoop infrastructure during computations. CONCLUSIONS: In order to keep pace with our ability to produce biological data, innovative methods are required. The Shared Data Science Infrastructure, Boag, provides researchers a greater access to researchers to efficiently explore data in new ways. We demonstrate the potential of a the domain specific language Boag using the RefSeq database to explore how deposited genome assemblies and annotations are changing over time. This is a small example of how Boag could be used with large biological datasets.

The genome of the soybean cyst nematode (Heterodera glycines) reveals complex patterns of duplications involved in the evolution of parasitism genes.

BACKGROUND: Heterodera glycines, commonly referred to as the soybean cyst nematode (SCN), is an obligatory and sedentary plant parasite that causes over a billion-dollar yield loss to soybean production annually. Although there are genetic determinants that render soybean plants resistant to certain nematode genotypes, resistant soybean cultivars are increasingly ineffective because their multi-year usage has selected for virulent H. glycines populations. The parasitic success of H. glycines relies on the comprehensive re-engineering of an infection site into a syncytium, as well as the long-term suppression of host defense to ensure syncytial viability. At the forefront of these complex molecular interactions are effectors, the proteins secreted by H. glycines into host root tissues. The mechanisms of effector acquisition, diversification, and selection need to be understood before effective control strategies can be developed, but the lack of an annotated genome has been a major roadblock. RESULTS: Here, we use PacBio long-read technology to assemble a H. glycines genome of 738 contigs into 123 Mb with annotations for 29,769 genes. The genome contains significant numbers of repeats (34%), tandem duplicates (18.7 Mb), and horizontal gene transfer events (151 genes). A large number of putative effectors (431 genes) were identified in the genome, many of which were found in transposons. CONCLUSIONS: This advance provides a glimpse into the host and parasite interplay by revealing a diversity of mechanisms that give rise to virulence genes in the soybean cyst nematode, including: tandem duplications containing over a fifth of the total gene count, virulence genes hitchhiking in transposons, and 107 horizontal gene transfers not reported in other plant parasitic nematodes thus far. Through extensive characterization of the H. glycines genome, we provide new insights into H. glycines biology and shed light onto the mystery underlying complex host-parasite interactions. This genome sequence is an important prerequisite to enable work towards generating new resistance or control measures against H. glycines.

Insights into teleost sex determination from the Seriola dorsalis genome assembly.

BACKGROUND: The assembly and annotation of a genome is a valuable resource for a species, with applications ranging from conservation genomics to gene discovery. Genomic resource development is especially important for species in culture, such as the California Yellowtail (Seriola dorsalis), the likely candidate for the establishment of commercial offshore aquaculture production in southern California. Genomic resource development for this species will improve the understanding of sex and other phenotypic traits, and allow for rapid increases in genetic improvement for and economic gain in culture production. RESULTS: We describe the assembly and annotation of the S. dorsalis genome, and present resequencing data from 45 male and 45 female wild-caught S. dorsalis used to identify a sex-determining region and marker in this species. The genome assembly captured approximately 93% of the total 685 MB genome with an average coverage depth of 180×. Using the assembled genome, resequencing data from the 90 fish were aligned to place boundaries on the sex-determining region. Sex-specific markers were developed based on a female-specific, 61 nucleotide deletion identified in that region. We hypothesize that Estradiol 17-beta-dehydrogenase is the putative sex-determining gene and propose a plausible genetic mechanism for ZW sex determination in S. dorsalis involving a female-specific deletion of a transcription factor binding motif that may be targeted by Sox3. CONCLUSIONS: Understanding the mechanism of sex determination and development of assays to determine sex is critical both for management of wild fisheries and for development of efficient and sustainable aquaculture practices. In addition, this genome assembly for S. dorsalis will be a substantial resource for a variety of future research applications.

Aquaculture genomics, genetics and breeding in the United States: current status, challenges, and priorities for future research.

Advancing the production efficiency and profitability of aquaculture is dependent upon the ability to utilize a diverse array of genetic resources. The ultimate goals of aquaculture genomics, genetics and breeding research are to enhance aquaculture production efficiency, sustainability, product quality, and profitability in support of the commercial sector and for the benefit of consumers. In order to achieve these goals, it is important to understand the genomic structure and organization of aquaculture species, and their genomic and phenomic variations, as well as the genetic basis of traits and their interrelationships. In addition, it is also important to understand the mechanisms of regulation and evolutionary conservation at the levels of genome, transcriptome, proteome, epigenome, and systems biology. With genomic information and information between the genomes and phenomes, technologies for marker/causal mutation-assisted selection, genome selection, and genome editing can be developed for applications in aquaculture. A set of genomic tools and resources must be made available including reference genome sequences and their annotations (including coding and non-coding regulatory elements), genome-wide polymorphic markers, efficient genotyping platforms, high-density and high-resolution linkage maps, and transcriptome resources including non-coding transcripts. Genomic and genetic control of important performance and production traits, such as disease resistance, feed conversion efficiency, growth rate, processing yield, behaviour, reproductive characteristics, and tolerance to environmental stressors like low dissolved oxygen, high or low water temperature and salinity, must be understood. QTL need to be identified, validated across strains, lines and populations, and their mechanisms of control understood. Causal gene(s) need to be identified. Genetic and epigenetic regulation of important aquaculture traits need to be determined, and technologies for marker-assisted selection, causal gene/mutation-assisted selection, genome selection, and genome editing using CRISPR and other technologies must be developed, demonstrated with applicability, and application to aquaculture industries.Major progress has been made in aquaculture genomics for dozens of fish and shellfish species including the development of genetic linkage maps, physical maps, microarrays, single nucleotide polymorphism (SNP) arrays, transcriptome databases and various stages of genome reference sequences. This paper provides a general review of the current status, challenges and future research needs of aquaculture genomics, genetics, and breeding, with a focus on major aquaculture species in the United States: catfish, rainbow trout, Atlantic salmon, tilapia, striped bass, oysters, and shrimp. While the overall research priorities and the practical goals are similar across various aquaculture species, the current status in each species should dictate the next priority areas within the species. This paper is an output of the USDA Workshop for Aquaculture Genomics, Genetics, and Breeding held in late March 2016 in Auburn, Alabama, with participants from all parts of the United States.

CRISPR interference and priming varies with individual spacer sequences.

CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) systems allow bacteria to adapt to infection by acquiring 'spacer' sequences from invader DNA into genomic CRISPR loci. Cas proteins use RNAs derived from these loci to target cognate sequences for destruction through CRISPR interference. Mutations in the protospacer adjacent motif (PAM) and seed regions block interference but promote rapid 'primed' adaptation. Here, we use multiple spacer sequences to reexamine the PAM and seed sequence requirements for interference and priming in the Escherichia coli Type I-E CRISPR-Cas system. Surprisingly, CRISPR interference is far more tolerant of mutations in the seed and the PAM than previously reported, and this mutational tolerance, as well as priming activity, is highly dependent on spacer sequence. We identify a large number of functional PAMs that can promote interference, priming or both activities, depending on the associated spacer sequence. Functional PAMs are preferentially acquired during unprimed 'naïve' adaptation, leading to a rapid priming response following infection. Our results provide numerous insights into the importance of both spacer and target sequences for interference and priming, and reveal that priming is a major pathway for adaptation during initial infection.

Genome, transcriptome and methylome sequencing of a primitively eusocial wasp reveal a greatly reduced DNA methylation system in a social insect.

Comparative genomics of social insects has been intensely pursued in recent years with the goal of providing insights into the evolution of social behaviour and its underlying genomic and epigenomic basis. However, the comparative approach has been hampered by a paucity of data on some of the most informative social forms (e.g. incipiently and primitively social) and taxa (especially members of the wasp family Vespidae) for studying social evolution. Here, we provide a draft genome of the primitively eusocial model insect Polistes dominula, accompanied by analysis of caste-related transcriptome and methylome sequence data for adult queens and workers. Polistes dominula possesses a fairly typical hymenopteran genome, but shows very low genomewide GC content and some evidence of reduced genome size. We found numerous caste-related differences in gene expression, with evidence that both conserved and novel genes are related to caste differences. Most strikingly, these -omics data reveal a major reduction in one of the major epigenetic mechanisms that has been previously suggested to be important for caste differences in social insects: DNA methylation. Along with a conspicuous loss of a key gene associated with environmentally responsive DNA methylation (the de novo DNA methyltransferase Dnmt3), these wasps have greatly reduced genomewide methylation to almost zero. In addition to providing a valuable resource for comparative analysis of social insect evolution, our integrative -omics data for this important behavioural and evolutionary model system call into question the general importance of DNA methylation in caste differences and evolution in social insects.

Transcriptome profiling of soybean (Glycine max) roots challenged with pathogenic and non-pathogenic isolates of Fusarium oxysporum.

BACKGROUND: Fusarium oxysporum is one of the most common fungal pathogens causing soybean root rot and seedling blight in U.S.A. In a recent study, significant variation in aggressiveness was observed among isolates of F. oxysporum collected from roots in Iowa, ranging from highly pathogenic to weakly or non-pathogenic isolates. RESULTS: We used RNA-seq analysis to investigate the molecular aspects of the interactions of a partially resistant soybean genotype with non-pathogenic/pathogenic isolates of F. oxysporum at 72 and 96 h post inoculation (hpi). Markedly different gene expression profiles were observed in response to the two isolates. A peak of highly differentially expressed genes (HDEGs) was triggered at 72 hpi in soybean roots and the number of HDEGs was about eight times higher in response to the pathogenic isolate compared to the non-pathogenic one (1,659 vs. 203 HDEGs, respectively). Furthermore, the magnitude of induction was much greater in response to the pathogenic isolate. This response included a stronger activation of defense-related genes, transcription factors, and genes involved in ethylene biosynthesis, secondary and sugar metabolism. CONCLUSIONS: The obtained data provide an important insight into the transcriptional responses of soybean-F. oxysporum interactions and illustrate the more drastic changes in the host transcriptome in response to the pathogenic isolate. These results may be useful in the developing new methods of broadening resistance of soybean to F. oxysporum, including the over-expression of key soybean genes.

Transcriptional analysis of phloem-associated cells of potato.

BACKGROUND: Numerous signal molecules, including proteins and mRNAs, are transported through the architecture of plants via the vascular system. As the connection between leaves and other organs, the petiole and stem are especially important in their transport function, which is carried out by the phloem and xylem, especially by the sieve elements in the phloem system. The phloem is an important conduit for transporting photosynthate and signal molecules like metabolites, proteins, small RNAs, and full-length mRNAs. Phloem sap has been used as an unadulterated source to profile phloem proteins and RNAs, but unfortunately, pure phloem sap cannot be obtained in most plant species. RESULTS: Here we make use of laser capture microdissection (LCM) and RNA-seq for an in-depth transcriptional profile of phloem-associated cells of both petioles and stems of potato. To expedite our analysis, we have taken advantage of the potato genome that has recently been fully sequenced and annotated. Out of the 27 k transcripts assembled that we identified, approximately 15 k were present in phloem-associated cells of petiole and stem with greater than ten reads. Among these genes, roughly 10 k are affected by photoperiod. Several RNAs from this day length-regulated group are also abundant in phloem cells of petioles and encode for proteins involved in signaling or transcriptional control. Approximately 22 % of the transcripts in phloem cells contained at least one binding motif for Pumilio, Nova, or polypyrimidine tract-binding proteins in their downstream sequences. Highlighting the predominance of binding processes identified in the gene ontology analysis of active genes from phloem cells, 78 % of the 464 RNA-binding proteins present in the potato genome were detected in our phloem transcriptome. CONCLUSIONS: As a reasonable alternative when phloem sap collection is not possible, LCM can be used to isolate RNA from specific cell types, and along with RNA-seq, provides practical access to expression profiles of phloem tissue. The combination of these techniques provides a useful approach to the study of phloem and a comprehensive picture of the mechanisms associated with long-distance signaling. The data presented here provide valuable insights into potentially novel phloem-mobile mRNAs and phloem-associated RNA-binding proteins.

Changes in twelve homoeologous genomic regions in soybean following three rounds of polyploidy.

With the advent of high-throughput sequencing, the availability of genomic sequence for comparative genomics is increasing exponentially. Numerous completed plant genome sequences enable characterization of patterns of the retention and evolution of genes within gene families due to multiple polyploidy events, gene loss and fractionation, and differential evolutionary pressures over time and across different gene families. In this report, we trace the changes that have occurred in 12 surviving homoeologous genomic regions from three rounds of polyploidy that contributed to the current Glycine max genome: a genome triplication before the origin of the rosids (~130 to 240 million years ago), a genome duplication early in the legumes (~58 million years ago), and a duplication in the Glycine lineage (~13 million years ago). Patterns of gene retention following the genome triplication event generally support predictions of the Gene Balance Hypothesis. Finally, we find that genes in networks with a high level of connectivity are more strongly conserved than those with low connectivity and that the enrichment of these highly connected genes in the 12 highly conserved homoeologous segments may in part explain their retention over more than 100 million years and repeated polyploidy events.

A chromosome-level genome assembly of the disco clam, Ctenoides ales.

The bivalve subclass Pteriomorphia, which includes the economically important scallops, oysters, mussels, and ark clams, exhibits extreme ecological, morphological, and behavioral diversity. Among this diversity are five morphologically distinct eye types, making Pteriomorphia an excellent setting to explore the molecular basis for the evolution of novel traits. Of pteriomorphian bivalves, Limida is the only order lacking genomic resources, greatly limiting the potential phylogenomic analyses related to eyes and phototransduction. Here, we present a limid genome assembly, the disco clam, Ctenoides ales, which is characterized by invaginated eyes, exceptionally long tentacles, and a flashing light display. This genome assembly was constructed with PacBio long reads and Dovetail Omni-CTM proximity-ligation sequencing. The final assembly is ∼2.3Gb and over 99% of the total length is contained in 18 pseudomolecule scaffolds. We annotated 41,064 protein coding genes and report a BUSCO completeness of 91.9% for metazoa_obd10. Additionally, we report a complete and annotated mitochondrial genome, which also had been lacking from Limida. The ∼20Kb mitogenome has 12 protein coding genes, 22 tRNAs, 2 rRNA genes, and a 1,589 bp duplicated sequence containing the origin of replication. The C. ales nuclear genome size is substantially larger than other pteriomorphian genomes, mainly accounted for by transposable element sequences. We inventoried the genome for opsins, the signaling proteins that initiate phototransduction, and found that, unlike its closest eyed-relatives, the scallops, C. ales lacks duplication of the rhabdomeric Gq-protein coupled opsin that is typically used for invertebrate vision. In fact, C. ales has uncharacteristically few opsins relative to the other pteriomorphian families, all of which have unique expansions of xenopsins, a recently discovered opsin subfamily. This chromosome-level assembly, along with the mitogenome, will be valuable resources for comparative genomics and phylogenetics in bivalves and particularly for the understudied but charismatic limids.

Chromosome level assembly and annotation of the genome of the endangered giant Patagonian bumble bee Bombus dahlbomii.

This article describes a genome assembly and annotation for Bombus dahlbomii, the giant Patagonian bumble bee. DNA from a single, haploid male collected in Argentina was used for PacBio (HiFi) sequencing and HiC technology was then used to map chromatin contacts. Using Juicer and manual curation, the genome was scaffolded into 18 main pseudomolecules, representing a high quality, near chromosome-level assembly. The sequenced genome size is estimated at 265Mb. The genome was annotated based on RNA-sequencing data of another male from Argentina, and BRAKER3 produced 15,767 annotated genes. The genome and annotation show high completeness, with >95% BUSCO scores for both the genome and annotated genes (based on conserved genes from Hymenoptera). This genome provides a valuable resource for studying the biology of this iconic and endangered species, as well as for understanding the impacts of its decline and designing strategies for its preservation.

Identification of candidate genes underlying an iron efficiency quantitative trait locus in soybean.

Prevalent on calcareous soils in the United States and abroad, iron deficiency is among the most common and severe nutritional stresses in plants. In soybean (Glycine max) commercial plantings, the identification and use of iron-efficient genotypes has proven to be the best form of managing this soil-related plant stress. Previous studies conducted in soybean identified a significant iron efficiency quantitative trait locus (QTL) explaining more than 70% of the phenotypic variation for the trait. In this research, we identified candidate genes underlying this QTL through molecular breeding, mapping, and transcriptome sequencing. Introgression mapping was performed using two related near-isogenic lines in which a region located on soybean chromosome 3 required for iron efficiency was identified. The region corresponds to the previously reported iron efficiency QTL. The location was further confirmed through QTL mapping conducted in this study. Transcriptome sequencing and quantitative real-time-polymerase chain reaction identified two genes encoding transcription factors within the region that were significantly induced in soybean roots under iron stress. The two induced transcription factors were identified as homologs of the subgroup lb basic helix-loop-helix (bHLH) genes that are known to regulate the strategy I response in Arabidopsis (Arabidopsis thaliana). Resequencing of these differentially expressed genes unveiled a significant deletion within a predicted dimerization domain. We hypothesize that this deletion disrupts the Fe-DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT)/bHLH heterodimer that has been shown to induce known iron acquisition genes.