Hypothalamic Transcriptome Response To Simulated Diel Earthen Pond Hypoxia Cycles In Channel Catfish (Ictalurus punctatus).

Commercial culture of channel catfish (Ictalurus punctatus) occurs in earthen ponds that are characterized by diel swings in dissolved oxygen concentration that can fall to severe levels of hypoxia which can suppress appetite and lead to suboptimal growth. Given the significance of the hypothalamus in regulating these processes in other fishes, an investigation into the hypothalamus transcriptome was conducted to identify specific genes and expression patterns responding to hypoxia. Channel catfish in normoxic water were compared to catfish subjected to 12 hours of hypoxia (20% oxygen saturation; 1.8 mg O2/L; 27 °C) followed by 12 hours of recovery in normoxia to mimic 24-hours in a catfish aquaculture pond. Fish were sampled at 0-, 6-, 12-, 18-, and 24-hour time points, with the 6- and 12-hour samplings occurring during hypoxia. A total of 190 genes were differentially expressed during the experiment, with most occurring during hypoxia and returning to baseline values within 6 hours of normoxia. Differentially expressed genes were sorted by function into Gene Ontology biological processes and revealed that most were categorized as "response to hypoxia", "sprouting angiogenesis", and "cellular response to xenobiotic stimulus". The patterns of gene expression reported here suggest that transcriptome responses to hypoxia are broad and quickly reversibly with the onset of normoxia. Although no genes commonly reported to modulate appetite were found to be differentially expressed in this experiment, several candidates were identified for future studies investigating the interplay between hypoxia and appetite in channel catfish, including adm, igfbp1a, igfbp7, and stc2b.

Genome report: chromosome-scale genome assembly of the West Indian fruit fly Anastrepha obliqua (Diptera: Tephritidae).

The West Indian fruit fly, Anastrepha obliqua, is a major pest of mango in Central and South America and attacks more than 60 species of host fruits. To support current genetic and genomic research on A. obliqua, we sequenced the genome using high-fidelity long-read sequencing. This resulted in a highly contiguous contig assembly with 90% of the genome in 10 contigs. The contig assembly was placed in a chromosomal context using synteny with a closely related species, Anastrepha ludens, as both are members of the Anastrepha fraterculus group. The resulting assembly represents the five autosomes and the X chromosome which represents 95.9% of the genome, and 199 unplaced contigs representing the remaining 4.1%. Orthology analysis across the structural annotation sets of high quality tephritid genomes demonstrates the gene annotations are robust, and identified genes unique to Anastrepha species that may help define their pestiferous nature that can be used as a starting point for comparative genomics. This genome assembly represents the first of this species and will serve as a foundation for future genetic and genomic research in support of its management as an agricultural pest.

Two haplotype-resolved genomes reveal important flower traits in bigleaf hydrangea (Hydrangea macrophylla) and insights into Asterid evolution.

The Hydrangea genus belongs to the Hydrangeaceae family, in the Cornales order of flowering plants, which early diverged among the Asterids, and includes several species that are commonly used ornamental plants. Of them, Hydrangea macrophylla is one of the most valuable species in the nursery trade, yet few genomic resources are available for this crop or closely related Asterid species. Two high-quality haplotype-resolved reference genomes of hydrangea cultivars 'Veitchii' and 'Endless Summer' [highest quality at 2.22 gigabase pairs (Gb), 396 contigs, N50 22.8 megabase pairs (Mb)] were assembled and scaffolded into the expected 18 pseudochromosomes. Utilizing the newly developed high-quality reference genomes along with high-quality genomes of other related flowering plants, nuclear data were found to support a single divergence point in the Asterids clade where both the Cornales and Ericales diverged from the euasterids. Genetic mapping with an F1 hybrid population demonstrated the power of linkage mapping combined with the new genomic resources to identify the gene for inflorescence shape, CYP78A5 located on chromosome 4, and a novel gene, BAM3 located on chromosome 17, for causing double flower. Resources developed in this study will not only help to accelerate hydrangea genetic improvement but also contribute to understanding the largest group of flowering plants, the Asterids.

Representing true plant genomes: haplotype-resolved hybrid pepper genome with trio-binning.

As sequencing costs decrease and availability of high fidelity long-read sequencing increases, generating experiment specific de novo genome assemblies becomes feasible. In many crop species, obtaining the genome of a hybrid or heterozygous individual is necessary for systems that do not tolerate inbreeding or for investigating important biological questions, such as hybrid vigor. However, most genome assembly methods that have been used in plants result in a merged single sequence representation that is not a true biologically accurate representation of either haplotype within a diploid individual. The resulting genome assembly is often fragmented and exhibits a mosaic of the two haplotypes, referred to as haplotype-switching. Important haplotype level information, such as causal mutations and structural variation is therefore lost causing difficulties in interpreting downstream analyses. To overcome this challenge, we have applied a method developed for animal genome assembly called trio-binning to an intra-specific hybrid of chili pepper (Capsicum annuum L. cv. HDA149 x Capsicum annuum L. cv. HDA330). We tested all currently available softwares for performing trio-binning, combined with multiple scaffolding technologies including Bionano to determine the optimal method of producing the best haplotype-resolved assembly. Ultimately, we produced highly contiguous biologically true haplotype-resolved genome assemblies for each parent, with scaffold N50s of 266.0 Mb and 281.3 Mb, with 99.6% and 99.8% positioned into chromosomes respectively. The assemblies captured 3.10 Gb and 3.12 Gb of the estimated 3.5 Gb chili pepper genome size. These assemblies represent the complete genome structure of the intraspecific hybrid, as well as the two parental genomes, and show measurable improvements over the currently available reference genomes. Our manuscript provides a valuable guide on how to apply trio-binning to other plant genomes.

Long-read, chromosome-scale assembly of Vitis rotundifolia cv. Carlos and its unique resistance to Xylella fastidiosa subsp. fastidiosa.

BACKGROUND: Muscadine grape (Vitis rotundifolia) is resistant to many of the pathogens that negatively impact the production of common grape (V. vinifera), including the bacterial pathogen Xylella fastidiosa subsp. fastidiosa (Xfsf), which causes Pierce's Disease (PD). Previous studies in common grape have indicated Xfsf delays host immune response with a complex O-chain antigen produced by the wzy gene. Muscadine cultivars range from tolerant to completely resistant to Xfsf, but the mechanism is unknown. RESULTS: We assembled and annotated a new, long-read genome assembly for 'Carlos', a cultivar of muscadine that exhibits tolerance, to build upon the existing genetic resources available for muscadine. We used these resources to construct an initial pan-genome for three cultivars of muscadine and one cultivar of common grape. This pan-genome contains a total of 34,970 synteny-constrained entries containing genes of similar structure. Comparison of resistance gene content between the 'Carlos' and common grape genomes indicates an expansion of resistance (R) genes in 'Carlos.' We further identified genes involved in Xfsf response by transcriptome sequencing 'Carlos' plants inoculated with Xfsf. We observed 234 differentially expressed genes with functions related to lipid catabolism, oxidation-reduction signaling, and abscisic acid (ABA) signaling as well as seven R genes. Leveraging public data from previous experiments of common grape inoculated with Xfsf, we determined that most differentially expressed genes in the muscadine response were not found in common grape, and three of the R genes identified as differentially expressed in muscadine do not have an ortholog in the common grape genome. CONCLUSIONS: Our results support the utility of a pan-genome approach to identify candidate genes for traits of interest, particularly disease resistance to Xfsf, within and between muscadine and common grape.

Chromosome-scale genome assembly of the rusty patched bumble bee, Bombus affinis (Cresson) (Hymenoptera: Apidae), an endangered North American pollinator.

The rusty patched bumble bee, Bombus affinis, is an important pollinator in North America and a federally listed endangered species. Due to habitat loss and large declines in population size, B. affinis is facing imminent extinction unless human intervention and recovery efforts are implemented. To better understand B. affinis biology and population genetic and genomic landscapes, we sequenced and assembled the B. affinis genome from a single haploid male. Whole genome HiFi sequencing on PacBio coupled with HiC sequencing resulted in a complete and highly contiguous contig assembly that was scaffolded into a chromosomal context, resolving 18 chromosomes distributed across the 365.1 Mb assembly. All material for both HiFi and HiC sequencing was derived from a single abdominal tissue segment from the single male. These assembly results, coupled with the minimal amount of tissue destructively sampled, demonstrate methods for generating contiguous and complete genomic resources for a rare and endangered species with limited material available and highlight the importance of sample preservation. Precise methods and applications of these methods are presented for potential applications in other species with similar limitations in specimen availability and curation considerations.

The generation of the first chromosome-level de novo genome assembly and the development and validation of a 50K SNP array for the St. John River aquaculture strain of North American Atlantic salmon.

Atlantic salmon (Salmo salar) in Northeastern US and Eastern Canada has high economic value for the sport fishing and aquaculture industries. Large differences exist between the genomes of Atlantic salmon of European origin and North American (N.A.) origin. Given the genetic and genomic differences between the 2 lineages, it is crucial to develop unique genomic resources for N.A. Atlantic salmon. Here, we describe the resources that we recently developed for genomic and genetic research in N.A. Atlantic salmon aquaculture. Firstly, a new single nucleotide polymorphism (SNP) database for N.A. Atlantic salmon consisting of 3.1 million putative SNPs was generated using data from whole-genome resequencing of 80 N.A. Atlantic salmon individuals. Secondly, a high-density 50K SNP array enriched for the genic regions of the genome and containing 3 sex determination and 61 putative continent of origin markers was developed and validated. Thirdly, a genetic map composed of 27 linkage groups with 36K SNP markers was generated from 2,512 individuals in 141 full-sib families. Finally, a chromosome-level de novo genome assembly from a male N.A. Atlantic salmon from the St. John River aquaculture strain was generated using PacBio long reads. Information from Hi-C proximity ligation sequences and Bionano optical mapping was used to concatenate the contigs into scaffolds. The assembly contains 1,755 scaffolds and only 1,253 gaps, with a total length of 2.83 Gb and N50 of 17.2 Mb. A BUSCO analysis detected 96.2% of the conserved Actinopterygii genes in the assembly, and the genetic linkage information was used to guide the formation of 27 chromosome sequences. Comparative analysis with the reference genome assembly of the European Atlantic salmon confirmed that the karyotype differences between the 2 lineages are caused by a fission in chromosome Ssa01 and 3 chromosome fusions including the p arm of chromosome Ssa01 with Ssa23, Ssa08 with Ssa29, and Ssa26 with Ssa28. The genomic resources we have generated for Atlantic salmon provide a crucial boost for genetic research and for management of farmed and wild populations in this highly valued species.

Genome and Genetic Engineering of the House Cricket (Acheta domesticus): A Resource for Sustainable Agriculture.

Background: The house cricket, Acheta domesticus, is one of the most farmed insects worldwide and the foundation of an emerging industry using insects as a sustainable food source. Edible insects present a promising alternative for protein production amid a plethora of reports on climate change and biodiversity loss largely driven by agriculture. As with other crops, genetic resources are needed to improve crickets for food and other applications. Methods: We present the first high quality annotated genome assembly of A. domesticus from long read data and scaffolded to chromosome level, providing information needed for genetic manipulation. Results: Gene groups related to immunity were annotated and will be useful for improving value to insect farmers. Metagenome scaffolds in the A. domesticus assembly, including Invertebrate Iridescent Virus 6 (IIV6), were submitted as host-associated sequences. We demonstrate both CRISPR/Cas9-mediated knock-in and knock-out of A. domesticus and discuss implications for the food, pharmaceutical, and other industries. RNAi was demonstrated to disrupt the function of the vermilion eye-color gene producing a useful white-eye biomarker phenotype. Conclusions: We are utilizing these data to develop technologies for downstream commercial applications, including more nutritious and disease-resistant crickets, as well as lines producing valuable bioproducts, such as vaccines and antibiotics.

Reference genomes of channel catfish and blue catfish reveal multiple pericentric chromosome inversions.

BACKGROUND: Channel catfish and blue catfish are the most important aquacultured species in the USA. The species do not readily intermate naturally but F1 hybrids can be produced through artificial spawning. F1 hybrids produced by mating channel catfish female with blue catfish male exhibit heterosis and provide an ideal system to study reproductive isolation and hybrid vigor. The purpose of the study was to generate high-quality chromosome level reference genome sequences and to determine their genomic similarities and differences. RESULTS: We present high-quality reference genome sequences for both channel catfish and blue catfish, containing only 67 and 139 total gaps, respectively. We also report three pericentric chromosome inversions between the two genomes, as evidenced by long reads across the inversion junctions from distinct individuals, genetic linkage mapping, and PCR amplicons across the inversion junctions. Recombination rates within the inversional segments, detected as double crossovers, are extremely low among backcross progenies (progenies of channel catfish female × F1 hybrid male), suggesting that the pericentric inversions interrupt postzygotic recombination or survival of recombinants. Identification of channel catfish- and blue catfish-specific genes, along with expansions of immunoglobulin genes and centromeric Xba elements, provides insights into genomic hallmarks of these species. CONCLUSIONS: We generated high-quality reference genome sequences for both blue catfish and channel catfish and identified major chromosomal inversions on chromosomes 6, 11, and 24. These perimetric inversions were validated by additional sequencing analysis, genetic linkage mapping, and PCR analysis across the inversion junctions. The reference genome sequences, as well as the contrasted chromosomal architecture should provide guidance for the interspecific breeding programs.

Detecting Cotton Leaf Curl Virus Resistance Quantitative Trait Loci in Gossypium hirsutum and iCottonQTL a New R/Shiny App to Streamline Genetic Mapping.

Cotton leaf curl virus (CLCuV) causes devastating losses to fiber production in Central Asia. Viral spread across Asia in the last decade is causing concern that the virus will spread further before resistant varieties can be bred. Current development depends on screening each generation under disease pressure in a country where the disease is endemic. We utilized quantitative trait loci (QTL) mapping in four crosses with different sources of resistance to identify single nucleotide polymorphism (SNP) markers associated with the resistance trait to allow development of varieties without the need for field screening every generation. To assist in the analysis of multiple populations, a new publicly available R/Shiny App was developed to streamline genetic mapping using SNP arrays and to also provide an easy method to convert and deposit genetic data into the CottonGen database. Results identified several QTL from each cross, indicating possible multiple modes of resistance. Multiple sources of resistance would provide several genetic routes to combat the virus as it evolves over time. Kompetitive allele specific PCR (KASP) markers were developed and validated for a subset of QTL, which can be used in further development of CLCuV-resistant cotton lines.

Chromosome-scale genome assembly of the pink bollworm, Pectinophora gossypiella, a global pest of cotton.

The pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), is a major global pest of cotton. Current management practices include chemical insecticides, cultural strategies, sterile insect releases, and transgenic cotton producing crystalline (Cry) protein toxins of the bacterium Bacillus thuringiensis (Bt). These strategies have contributed to the eradication of P. gossypiella from the cotton-growing areas of the United States and northern Mexico. However, this pest has evolved resistance to Bt cotton in Asia, where it remains a critical pest, and the benefits of using transgenic Bt crops have been lost. A complete annotated reference genome is needed to improve global Bt resistance management of the pink bollworm. We generated the first chromosome-level genome assembly for pink bollworm from a Bt-susceptible laboratory strain (APHIS-S) using PacBio continuous long reads for contig generation, Illumina Hi-C for scaffolding, and Illumina whole-genome re-sequencing for error correction. The pseudo-haploid assembly consists of 29 autosomes and the Z sex chromosome. The assembly exceeds the minimum Earth BioGenome Project quality standards, has a low error rate, is highly contiguous at both the contig and scaffold levels (L/N50 of 18/8.26 MB and 14/16.44 MB, respectively), and is complete, with 98.6% of lepidopteran single-copy orthologs represented without duplication. The genome was annotated with 50% repeat content and 14,107 protein-coding genes, further assigned to 41,666 functional annotations. This assembly represents the first publicly available complete annotated genome of pink bollworm and will serve as the foundation for advancing molecular genetics of this important pest species.

Whole-genome resequencing data support a single introduction of the invasive white pine sawfly, Diprion similis.

Biological introductions are unintended "natural experiments" that provide unique insights into evolutionary processes. Invasive phytophagous insects are of particular interest to evolutionary biologists studying adaptation, as introductions often require rapid adaptation to novel host plants. However, adaptive potential of invasive populations may be limited by reduced genetic diversity-a problem known as the "genetic paradox of invasions." One potential solution to this paradox is if there are multiple invasive waves that bolster genetic variation in invasive populations. Evaluating this hypothesis requires characterizing genetic variation and population structure in the invaded range. To this end, we assemble a reference genome and describe patterns of genetic variation in the introduced white pine sawfly, Diprion similis. This species was introduced to North America in 1914, where it has rapidly colonized the thin-needled eastern white pine (Pinus strobus), making it an ideal invasion system for studying adaptation to novel environments. To evaluate evidence of multiple introductions, we generated whole-genome resequencing data for 64 D. similis females sampled across the North American range. Both model-based and model-free clustering analyses supported a single population for North American D. similis. Within this population, we found evidence of isolation-by-distance and a pattern of declining heterozygosity with distance from the hypothesized introduction site. Together, these results support a single-introduction event. We consider implications of these findings for the genetic paradox of invasion and discuss priorities for future research in D. similis, a promising model system for invasion biology.

A Chromosome-Scale Genome Assembly of a Helicoverpa zea Strain Resistant to Bacillus thuringiensis Cry1Ac Insecticidal Protein.

Helicoverpa zea (Lepidoptera: Noctuidae) is an insect pest of major cultivated crops in North and South America. The species has adapted to different host plants and developed resistance to several insecticidal agents, including Bacillus thuringiensis (Bt) insecticidal proteins in transgenic cotton and maize. Helicoverpa zea populations persist year-round in tropical and subtropical regions, but seasonal migrations into temperate zones increase the geographic range of associated crop damage. To better understand the genetic basis of these physiological and ecological characteristics, we generated a high-quality chromosome-level assembly for a single H. zea male from Bt-resistant strain, HzStark_Cry1AcR. Hi-C data were used to scaffold an initial 375.2 Mb contig assembly into 30 autosomes and the Z sex chromosome (scaffold N50 = 12.8 Mb and L50 = 14). The scaffolded assembly was error-corrected with a novel pipeline, polishCLR. The mitochondrial genome was assembled through an improved pipeline and annotated. Assessment of this genome assembly indicated 98.8% of the Lepidopteran Benchmark Universal Single-Copy Ortholog set were complete (98.5% as complete single copy). Repetitive elements comprised approximately 29.5% of the assembly with the plurality (11.2%) classified as retroelements. This chromosome-scale reference assembly for H. zea, ilHelZeax1.1, will facilitate future research to evaluate and enhance sustainable crop production practices.

Analysis of a tetraploid cotton line Mac7 transcriptome reveals mechanisms underlying resistance against the whitefly Bemisia tabaci.

Whitefly inflicts both direct and indirect losses to cotton crop. Whitefly resistant cotton germplasm is a high priority and considered among the best possible solutions to mitigate this issue. In this study, we evaluated cotton leaf curl disease (CLCuD) resistant cotton line Mac7 under whitefly stress. Furthermore, we utilized the already available transcriptome data of Mac7 concerning whitefly stress to elucidate associated mechanisms and identify functionally important genes in cotton. In transcriptomic data analysis, differentially expressed genes (DEGs) were found involved in complex relay pathways, activated on whitefly exposure. The response implicates signalling through resistance genes (R-genes), MAPK, ROS, VQs or RLKs, transcription factors, which leads to the activation of defence responses including, Ca2+messengers, phytohormonal cross-talk, gossypol, flavonoids, PhasiRNA and susceptibility genes (S-genes). The qRT-PCR assay of 10 functionally important genes also showed their involvement in differential responses at 24 and 48 h post whitefly infestation. Briefly, our study helps in understanding the resistant nature of Mac7 under whitefly stress.

Transcriptomic Responses of Fusarium verticillioides to Lactam and Lactone Xenobiotics.

The important cereal crops of maize, rye, and wheat constitutively produce precursors to 2-benzoxazolinone, a phytochemical having antifungal effects towards many Fusarium species. However, Fusarium verticillioides can tolerate 2-benzoxazolinone by converting it into non-toxic metabolites through the synergism of two previously identified gene clusters, FDB1 and FDB2. Inspired by the induction of these two clusters upon exposure to 2-benzoxazolinone, RNA sequencing experiments were carried out by challenging F. verticillioides individually with 2-benzoxazolinone and three related chemical compounds, 2-oxindole, 2-coumaranone, and chlorzoxazone. These compounds all contain lactam and/or lactone moieties, and transcriptional analysis provided inferences regarding the degradation of such lactams and lactones. Besides induction of FDB1 and FDB2 gene clusters, four additional clusters were identified as induced by 2-benzoxazolinone exposure, including a cluster thought to be responsible for biosynthesis of pyridoxine (vitamin B6), a known antioxidant providing tolerance to reactive oxygen species. Three putative gene clusters were identified as induced by challenging F. verticillioides with 2-oxindole, two with 2-coumaranone, and two with chlorzoxazone. Interestingly, 2-benzoxazolinone and 2-oxindole each induced two specific gene clusters with similar composition of enzymatic functions. Exposure to 2-coumranone elicited the expression of the fusaric acid biosynthetic gene cluster. Another gene cluster that may encode enzymes responsible for degrading intermediate catabolic metabolites with carboxylic ester bonds was induced by 2-benzoxazolinone, 2-oxindole, and chlorzoxazone. Also, the induction of a dehalogenase encoding gene during chlorzoxazone exposure suggested its role in the removal of the chlorine atom. Together, this work identifies genes and putative gene clusters responsive to the 2-benzoxazolinone-like compounds with metabolic inferences. Potential targets for future functional analyses are discussed.

Transcriptomic Response of Fusarium verticillioides to Variably Inhibitory Environmental Isolates of Streptomyces.

Fusarium verticillioides is a mycotoxigenic fungus that is a threat to food and feed safety due to its common infection of maize, a global staple crop. A proposed strategy to combat this threat is the use of biological control bacteria that can inhibit the fungus and reduce mycotoxin contamination. In this study, the effect of multiple environmental isolates of Streptomyces on F. verticillioides was examined via transcriptome analysis. The Streptomyces strains ranged from inducing no visible response to dramatic growth inhibition. Transcriptionally, F. verticillioides responded proportionally to strain inhibition with either little to no transcript changes to thousands of genes being differentially expressed. Expression changes in multiple F. verticillioides putative secondary metabolite gene clusters was observed. Interestingly, genes involved in the fusaric acid gene cluster were suppressed by inhibitory strains of Streptomyces. A F. verticillioides beta-lactamase encoding gene (FVEG_13172) was found to be highly induced by specific inhibitory Streptomyces strains and its deletion increased visible response to those strains. This study demonstrates that F. verticillioides does not have an all or nothing response to bacteria it encounters but rather a measured response that is strain specific and proportional to the strength of inhibition.

Microscopic and Transcriptomic Analyses of Dalbergoid Legume Peanut Reveal a Divergent Evolution Leading to Nod-Factor-Dependent Epidermal Crack-Entry and Terminal Bacteroid Differentiation.

Root nodule symbiosis (RNS) is the pillar behind sustainable agriculture and plays a pivotal role in the environmental nitrogen cycle. Most of the genetic, molecular, and cell-biological knowledge on RNS comes from model legumes that exhibit a root-hair mode of bacterial infection, in contrast to the Dalbergoid legumes exhibiting crack-entry of rhizobia. As a step toward understanding this important group of legumes, we have combined microscopic analysis and temporal transcriptome to obtain a dynamic view of plant gene expression during Arachis hypogaea (peanut) nodule development. We generated comprehensive transcriptome data by mapping the reads to A. hypogaea, and two diploid progenitor genomes. Additionally, we performed BLAST searches to identify nodule-induced yet-to-be annotated peanut genes. Comparison between peanut, Medicago truncatula, Lotus japonicus, and Glycine max showed upregulation of 61 peanut orthologs among 111 tested known RNS-related genes, indicating conservation in mechanisms of nodule development among members of the Papilionoid family. Unlike model legumes, recruitment of class 1 phytoglobin-derived symbiotic hemoglobin (SymH) in peanut indicates diversification of oxygen-scavenging mechanisms in the Papilionoid family. Finally, the absence of cysteine-rich motif-1-containing nodule-specific cysteine-rich peptide (NCR) genes but the recruitment of defensin-like NCRs suggest a diverse molecular mechanism of terminal bacteroid differentiation. In summary, our work describes genetic conservation and diversification in legume-rhizobia symbiosis in the Papilionoid family, as well as among members of the Dalbergoid legumes.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Initiation of genomics-assisted breeding in Virginia-type peanuts through the generation of a de novo reference genome and informative markers.

Introduction: Virginia-type peanut, Arachis hypogaea subsp. hypogaea, is the second largest market class of peanut cultivated in the United States. It is mainly used for large-seeded, in-shell products. Historically, Virginia-type peanut cultivars were developed through long-term recurrent phenotypic selection and wild species introgression projects. Contemporary genomic technologies represent a unique opportunity to revolutionize the traditional breeding pipeline. While there are genomic tools available for wild and cultivated peanuts, none are tailored specifically to applied Virginia-type cultivar development programs. Methods and respective results: Here, the first Virginia-type peanut reference genome, "Bailey II", was assembled. It has improved contiguity and reduced instances of manual curation in chromosome arms. Whole-genome sequencing and marker discovery was conducted on 66 peanut lines which resulted in 1.15 million markers. The high marker resolution achieved allowed 34 unique wild species introgression blocks to be cataloged in the A. hypogaea genome, some of which are known to confer resistance to one or more pathogens. To enable marker-assisted selection of the blocks, 111 PCR Allele Competitive Extension assays were designed. Forty thousand high quality markers were selected from the full set that are suitable for mid-density genotyping for genomic selection. Genomic data from representative advanced Virginia-type peanut lines suggests this is an appropriate base population for genomic selection. Discussion: The findings and tools produced in this research will allow for rapid genetic gain in the Virginia-type peanut population. Genomics-assisted breeding will allow swift response to changing biotic and abiotic threats, and ultimately the development of superior cultivars for public use and consumption.

Legacy genetics of Arachis cardenasii in the peanut crop shows the profound benefits of international seed exchange.

The narrow genetics of most crops is a fundamental vulnerability to food security. This makes wild crop relatives a strategic resource of genetic diversity that can be used for crop improvement and adaptation to new agricultural challenges. Here, we uncover the contribution of one wild species accession, Arachis cardenasii GKP 10017, to the peanut crop (Arachis hypogaea) that was initiated by complex hybridizations in the 1960s and propagated by international seed exchange. However, until this study, the global scale of the dispersal of genetic contributions from this wild accession had been obscured by the multiple germplasm transfers, breeding cycles, and unrecorded genetic mixing between lineages that had occurred over the years. By genetic analysis and pedigree research, we identified A. cardenasii-enhanced, disease-resistant cultivars in Africa, Asia, Oceania, and the Americas. These cultivars provide widespread improved food security and environmental and economic benefits. This study emphasizes the importance of wild species and collaborative networks of international expertise for crop improvement. However, it also highlights the consequences of the implementation of a patchwork of restrictive national laws and sea changes in attitudes regarding germplasm that followed in the wake of the Convention on Biological Diversity. Today, the botanical collections and multiple seed exchanges which enable benefits such as those revealed by this study are drastically reduced. The research reported here underscores the vital importance of ready access to germplasm in ensuring long-term world food security.

"Jumping Jack": Genomic Microsatellites Underscore the Distinctiveness of Closely Related Pseudoperonospora cubensis and Pseudoperonospora humuli and Provide New Insights Into Their Evolutionary Past.

Downy mildews caused by obligate biotrophic oomycetes result in severe crop losses worldwide. Among these pathogens, Pseudoperonospora cubensis and P. humuli, two closely related oomycetes, adversely affect cucurbits and hop, respectively. Discordant hypotheses concerning their taxonomic relationships have been proposed based on host-pathogen interactions and specificity evidence and gene sequences of a few individuals, but population genetics evidence supporting these scenarios is missing. Furthermore, nuclear and mitochondrial regions of both pathogens have been analyzed using microsatellites and phylogenetically informative molecular markers, but extensive comparative population genetics research has not been done. Here, we genotyped 138 current and historical herbarium specimens of those two taxa using microsatellites (SSRs). Our goals were to assess genetic diversity and spatial distribution, to infer the evolutionary history of P. cubensis and P. humuli, and to visualize genome-scale organizational relationship between both pathogens. High genetic diversity, modest gene flow, and presence of population structure, particularly in P. cubensis, were observed. When tested for cross-amplification, 20 out of 27 P. cubensis-derived gSSRs cross-amplified DNA of P. humuli individuals, but few amplified DNA of downy mildew pathogens from related genera. Collectively, our analyses provided a definite argument for the hypothesis that both pathogens are distinct species, and suggested further speciation in the P. cubensis complex.