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.

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.

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.

A high-quality chromosome-level genome assembly of rohu carp, Labeo rohita, and its utilization in SNP-based exploration of gene flow and sex determination.

Labeo rohita (rohu) is a carp important to aquaculture in South Asia, with a production volume close to Atlantic salmon. While genetic improvements to rohu are ongoing, the genomic methods commonly used in other aquaculture improvement programs have historically been precluded in rohu, partially due to the lack of a high-quality reference genome. Here we present a high-quality de novo genome produced using a combination of next-generation sequencing technologies, resulting in a 946 Mb genome consisting of 25 chromosomes and 2,844 unplaced scaffolds. Notably, while approximately half the size of the existing genome sequence, our genome represents 97.9% of the genome size newly estimated here using flow cytometry. Sequencing from 120 individuals was used in conjunction with this genome to predict the population structure, diversity, and divergence in three major rivers (Jamuna, Padma, and Halda), in addition to infer a likely sex determination mechism in rohu. These results demonstrate the utility of the new rohu genome in modernizing some aspects of rohu genetic improvement programs.

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.

Gene disruption by structural mutations drives selection in US rice breeding over the last century.

The genetic basis of general plant vigor is of major interest to food producers, yet the trait is recalcitrant to genetic mapping because of the number of loci involved, their small effects, and linkage. Observations of heterosis in many crops suggests that recessive, malfunctioning versions of genes are a major cause of poor performance, yet we have little information on the mutational spectrum underlying these disruptions. To address this question, we generated a long-read assembly of a tropical japonica rice (Oryza sativa) variety, Carolina Gold, which allowed us to identify structural mutations (>50 bp) and orient them with respect to their ancestral state using the outgroup, Oryza glaberrima. Supporting prior work, we find substantial genome expansion in the sativa branch. While transposable elements (TEs) account for the largest share of size variation, the majority of events are not directly TE-mediated. Tandem duplications are the most common source of insertions and are highly enriched among 50-200bp mutations. To explore the relative impact of various mutational classes on crop fitness, we then track these structural events over the last century of US rice improvement using 101 resequenced varieties. Within this material, a pattern of temporary hybridization between medium and long-grain varieties was followed by recent divergence. During this long-term selection, structural mutations that impact gene exons have been removed at a greater rate than intronic indels and single-nucleotide mutations. These results support the use of ab initio estimates of mutational burden, based on structural data, as an orthogonal predictor in genomic selection.

A long reads-based de-novo assembly of the genome of the Arlee homozygous line reveals chromosomal rearrangements in rainbow trout.

Currently, there is still a need to improve the contiguity of the rainbow trout reference genome and to use multiple genetic backgrounds that will represent the genetic diversity of this species. The Arlee doubled haploid line was originated from a domesticated hatchery strain that was originally collected from the northern California coast. The Canu pipeline was used to generate the Arlee line genome de-novo assembly from high coverage PacBio long-reads sequence data. The assembly was further improved with Bionano optical maps and Hi-C proximity ligation sequence data to generate 32 major scaffolds corresponding to the karyotype of the Arlee line (2 N = 64). It is composed of 938 scaffolds with N50 of 39.16 Mb and a total length of 2.33 Gb, of which ∼95% was in 32 chromosome sequences with only 438 gaps between contigs and scaffolds. In rainbow trout the haploid chromosome number can vary from 29 to 32. In the Arlee karyotype the haploid chromosome number is 32 because chromosomes Omy04, 14 and 25 are divided into six acrocentric chromosomes. Additional structural variations that were identified in the Arlee genome included the major inversions on chromosomes Omy05 and Omy20 and additional 15 smaller inversions that will require further validation. This is also the first rainbow trout genome assembly that includes a scaffold with the sex-determination gene (sdY) in the chromosome Y sequence. The utility of this genome assembly is shown through the improved annotation of the duplicated genome loci that harbor the IGH genes on chromosomes Omy12 and Omy13.

Two New Aspergillus flavus Reference Genomes Reveal a Large Insertion Potentially Contributing to Isolate Stress Tolerance and Aflatoxin Production.

Efforts in genome sequencing in the Aspergillus genus have led to the development of quality reference genomes for several important species including A. nidulans, A. fumigatus, and A. oryzae However, less progress has been made for A. flavus As part of the effort of the USDA-ARS Annual Aflatoxin Workshop Fungal Genome Project, the isolate NRRL3357 was sequenced and resulted in a scaffold-level genome released in 2005. Our goal has been biologically driven, focusing on two areas: isolate variation in aflatoxin production and drought stress exacerbating aflatoxin production by A. flavus Therefore, we developed two reference pseudomolecule genome assemblies derived from chromosome arms for two isolates: AF13, a MAT1-2, highly stress tolerant, and highly aflatoxigenic isolate; and NRRL3357, a MAT1-1, less stress tolerant, and moderate aflatoxin producer in comparison to AF13. Here, we report these two reference-grade assemblies for these isolates through a combination of PacBio long-read sequencing and optical mapping, and coupled them with comparative, functional, and phylogenetic analyses. This analysis resulted in the identification of 153 and 45 unique genes in AF13 and NRRL3357, respectively. We also confirmed the presence of a unique 310 Kb insertion in AF13 containing 60 genes. Analysis of this insertion revealed the presence of a bZIP transcription factor, named atfC, which may contribute to isolate pathogenicity and stress tolerance. Phylogenomic analyses comparing these and other available assemblies also suggest that the species complex of A. flavus is polyphyletic.

Self-illuminating quantum dots for non-invasive bioluminescence imaging of mammalian gametes.

BACKGROUND: The fertility performance of animals is still a mystery and the full comprehension of mammalian gametes maturation and early embryonic development remains to be elucidated. The recent development in nanotechnology offers a new opportunity for real-time study of reproductive cells in their physiological environments. As a first step toward that goal, we evaluated the effectiveness of a fluorescent and luminescent nanoparticle for in vitro and ex vivo imaging of porcine gametes. METHODS: Freshly harvested boar sperm were labeled with red-shifted (655 nm) quantum dot nanoparticles conjugated (QD+) or not (QD-) with plasminogen antibody and evaluated. Subsets of labeled spermatozoa were loaded into straws and placed within the lumen of gilt reproductive tracts for ex vivo intra-uterine imaging. Porcine cumulus-oocyte complexes (COCs) were matured in the presence of QD- or QD+. Ovarian follicles were microinjected with QD- or QD+ and placed in culture for up to 4 days. After labeling, all samples were supplemented with coelenterazine, the luciferase substrate, and immediately submitted to bioluminescence analysis, followed by fluorescence and hyperspectral imaging. Data were analyzed with ANOVA and P < 0.05 indicated significant differences. RESULTS: All labeled-samples revealed bioluminescence emission that was confirmed by fluorescence and hyperspectral imaging of the QD localization within the cells and tissues. Over 76% of spermatozoa and both immature and mature COCs were successfully labeled with QD- or QD+. The QD- fluorescence appeared homogenously distributed in the oocytes, while found in the entire sperm length with a higher accumulation within the mid-piece. Labeled-follicles exhibited a progressive migration of QD nanoparticles within the follicle wall during culture. In contrast, QD+ fluorescence signals appeared condensed and stronger in the follicle cells, sperm head, and sub-plasma membrane area of mature oocytes. Weaker QD+ signals were detected in the cumulus cells. Fluorescence and hyperspectral microscope imaging showed comparable intracellular QD localization. Ex-vivo intra-uterine bioluminescence imaging of labeled spermatozoa revealed stronger signals captured over the oviducts, with uterine body allowing the lowest signal detection. CONCLUSION: Findings indicate that conjugated and non-conjugated fluorescent nanoparticles can be used for effective labeling of mammalian gametes for in vitro monitoring and potential in vivo targeted-imaging.

The use of a whole animal biophotonic model as a screen for the angiogenic potential of estrogenic compounds.

BACKGROUND: Vascular endothelial growth factor (VEGF) is essential for normal vascular growth and development during wound repair. VEGF is estrogen responsive and capable of regulating its own receptor, vascular endothelial growth factor receptor-2 (VEGFR-2). Several agricultural pesticides (e.g., methoxychlor) have estrogenic potential that can initiate inappropriate physiological responses in estrogenic-sensitive tissues following exposure in vivo. Thus, the current study was designed to determine whether the VEGFR-2-Luciferase (Luc) reporter transgenic mouse is a useful model for evaluating estrogenic tendencies of methoxychlor by monitoring wound healing via VEGFR-2-mediated gene expression using bioluminescence and real-time imaging technology. RESULTS: VEGFR-2-Luc gene activity peaked by d 7 (P<0.001) in all groups but was not different (P>0.05) between control and estrogen/methoxychlor exposed mice. CONCLUSIONS: Changes in VEGFR-2-Luc gene activity associated with the dermal wound healing process were able to be measured via photonic emission. The increase in vasculature recruitment and formation is paralleled by the increase of VEGFR-2-Luc activity with a peak on day 7. However, estrogen/methoxychlor did not significantly alter wound healing mediated VEGFR-2-Luc gene expression patterns compared to controls. This suggests that the VEGFR-2-Luc transgenic mouse wound model tested in this study may not be optimal for use as a screen for the angiogenic potential of estrogenic compounds.

Application of quantum dot nanoparticles for potential non-invasive bio-imaging of mammalian spermatozoa.

BACKGROUND: Various obstacles are encountered by mammalian spermatozoa during their journey through the female genital tract, and only few or none will reach the site of fertilization. Currently, there are limited technical approaches for non-invasive investigation of spermatozoa migration after insemination. As the knowledge surrounding sperm behavior throughout the female genital tract still remains elusive, the recent development of self-illuminating quantum dot nanoparticles may present a potential means for real-time in vitro and in vivo monitoring of spermatozoa. RESULTS: Here, we show the ability of boar spermatozoa to harmlessly interact and incorporate bioluminescent resonance energy transfer-conjugated quantum dot (BRET-QD) nanoparticles. The confocal microscope revealed in situ fluorescence of BRET-QD in the entire spermatozoon, while the ultra-structural analysis using the transmission electron microscope indicated BRET-QD localization on the sperm plasma membrane and intracellular compartment. In controlled-in vitro assays, bioluminescent imaging demonstrated that spermatozoa incubated with BRET-QD and luciferase substrate (coelenterazine) emit light (photons/sec) above the background, which confirmed the in situ fluorescence imaging. Most importantly, sperm motility, viability, and fertilizing potential were not affected by the BRET-QD incorporation when used at an appropriated ratio. CONCLUSIONS: Our results demonstrate that pig spermatozoa can incorporate BRET-QD nanoparticles without affecting their motility and capacity to interact with the oocyte when used at an appropriated balance. We anticipate that our study will enable in-depth exploration of the male components of in vivo migration, fertilization, and embryonic development at the molecular level using this novel approach.

Photonic monitoring in real time of vascular endothelial growth factor receptor 2 gene expression under relaxin-induced conditions in a novel murine wound model.

Relaxin is known to promote vascular endothelial growth factor (VEGF) expression in reproductive tissue, and successful wound healing depends on good vascularization of wound sites, a process that relaxin may facilitate. Thus, the objective of this study was to evaluate the efficacy of relaxin on the development of vascular tissue at wound sites in a novel VEGF receptor 2-luc (VEGFR2-luc) transgenic mouse wound model by monitoring the rate of VEGFR2-luc-mediated gene expression using bioluminescence and real-time imaging. To this end, 12 FVB/N VEGFR2-luc transgenic male mice were assigned to treatments (six per group): saline alone or relaxin (1 g/6 h/14 days) administered intraperitoneally (i.p.). On day 0, a set of full-thickness wounds (6-mm punch) were generated under anesthesia on the dorsal aspect of each mouse. Photonic emissions were recorded (5-min collection of photons) from wound sites 10 min after the administration of luciferin (150 mg/kg i.p.) on day 0 and on days 1, 2, 4, 7, 9, 11, and 14 postwounding to quantify luciferase activity using an IVIS 100 biophotonic imaging system. Animals were sacrificed (three per group) on day 7 or 14, and wound tissue specimens were recovered for molecular and histologic analyses. Although photonic emission from wound sites increased (P < .001) over time with peak values obtained by day 7, no significant (P > .05) effect of relaxin treatment on VEGFR2-luc gene expression was noted at wound sites. Whereas measuring relaxin's effect on angiogenesis indirectly via the VEGFR2 model was not successful, photonic imaging provides an exciting new tool using alternative models (i.e., VEGF-luc mouse) to study relaxin-induced gene expression in normal (i.e., wound healing) or tumorigenic tissues in real time.