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.

Gossypium hirsutum gene of unknown function Gohir.A02G161000 encodes a potential transmembrane Root UVB Sensitive 4 Protein with a putative protein-protein interaction interface.

A gene of unknown function, Gohir.A02G161000.1, identified in Gossypium hirsutum was studied using computational sequence and structure bioinformatics tools. The associated protein GhRUS4-A0A1U8JPV7 (UniProt A0A1U8JPV7) is predicted to be a plastid-localized, transmembrane root UVB-sensitive 4 (RUS4) protein with a newly identified potential dimerization surface. Evidence from homology and sequence conservation suggest involvement in auxin transport and pollen maturation.

Gossypium hirsutum gene of unknown function Gohir.A02G131900 encodes a potential plant-specific, dual-domain exo-1,3-ß-glucosidase.

A gene of unknown function, Gohir.A02G131900.1, identified in Gossypium hirsutum was studied using computational sequence and structure bioinformatic tools. The encoded protein GhGH5BG-A0A1U8NW40 (UniProt A0A1U8NW40) is predicted to be secreted and localized to the cell wall. Homology and conserved residues indicate it belongs to a plant-specific subgroup of the glycoside hydrolase family 5 and likely has exo-1,3-ß-glucosidase activity. This subgroup is unique in containing a fascin-like domain which may have evolved a unique glucan binding site of interest for further research.

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.

Gossypium hirsutum gene of unknown function, Gohir.A02G039501.1, encodes a potential DNA-binding ALOG protein involved in gene regulation.

A protein of unknown function encoded by gene Gohir.A02G039501.1 in Gossypium hirsutum , was studied using sequence and structure bioinformatic tools leading to its proposed function as a nuclear, DNA-binding ALOG protein involved in gene regulation during organ boundary specification and maintenance. The encoded protein contains a predicted nuclear localization sequence, an ALOG domain with conserved residues in the modeled DNA-binding regions and nearly identical sequence identity to Arabidopsis homologs involved in development of organ boundaries at the shoot apical meristem. The protein was modeled by AlphaFold2 to have a four-helix bundle that is structurally analogous to DNA-binding domains of XerC/D-like recombinases.

Gossypium hirsutum gene of unknown function Gohir.A03G0737001 encodes a potential Chaperone-like Protein of protochlorophyllide oxidoreductase (CPP1).

A gene of unknown function identified in Gossypium hirsutum , Gohir.A03G0737001.1, was studied using sequence and bioinformatic tools. The encoded protein (referred to here as GhCPP1-A0A1U8HKT6) was predicted to function as a Chaperone-like protein of protochlorophyllide oxidoreductase (CPP1), which is involved with initiation of photochemical reactions of chlorophyll biosynthesis. Sequence analysis indicates it is embedded in the chloroplast envelope membrane through four transmembrane regions and contains a J-like domain that is structurally similar to the J domain of DnaJ/Hsp40 "holdase" chaperone proteins.

A first look at the ability to use genomic prediction for improving the ratooning ability of sugarcane.

The sugarcane ratooning ability (RA) is the most important target trait for breeders seeking to enhance the profitability of sugarcane production by reducing the planting cost. Understanding the genetics governing the RA could help breeders by identifying molecular markers that could be used for genomics-assisted breeding (GAB). A replicated field trial was conducted for three crop cycles (plant cane, first ratoon, and second ratoon) using 432 sugarcane clones and used for conducting genome-wide association and genomic prediction of five sugar and yield component traits of the RA. The RA traits for economic index (EI), stalk population (SP), stalk weight (SW), tonns of cane per hectare (TCH), and tonns of sucrose per hectare (TSH) were estimated from the yield and sugar data. A total of six putative quantitative trait loci and eight nonredundant single-nucleotide polymorphism (SNP) markers were associated with all five tested RA traits and appear to be unique. Seven putative candidate genes were colocated with significant SNPs associated with the five RA traits. The genomic prediction accuracies for those tested traits were moderate and ranged from 0.21 to 0.36. However, the models fitting fixed effects for the most significant associated markers for each respective trait did not give any advantages over the standard models without fixed effects. As a result of this study, more robust markers could be used in the future for clone selection in sugarcane, potentially helping resolve the genetic control of the RA in sugarcane.

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.

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.

Two pathogen loci determine Blumeria graminis f. sp. tritici virulence to wheat resistance gene Pm1a.

Blumeria graminis f. sp. tritici (Bgt) is a globally important fungal pathogen of wheat that can rapidly evolve to defeat wheat powdery mildew (Pm) resistance genes. Despite periodic regional deployment of the Pm1a resistance gene in US wheat production, Bgt strains that overcome Pm1a have been notably nonpersistent in the United States, while on other continents, they are more widely established. A genome-wide association study (GWAS) was conducted to map sequence variants associated with Pm1a virulence in 216 Bgt isolates from six countries, including the United States. A virulence variant apparently unique to Bgt isolates from the United States was detected in the previously mapped gene AvrPm1a (BgtE-5612) on Bgt chromosome 6; an in vitro growth assay suggested no fitness reduction associated with this variant. A gene on Bgt chromosome 8, Bgt-51526, was shown to function as a second determinant of Pm1a virulence, and despite < 30% amino acid identity, BGT-51526 and BGTE-5612 were predicted to share > 85% of their secondary structure. A co-expression study in Nicotiana benthamiana showed that BGTE-5612 and BGT-51526 each produce a PM1A-dependent hypersensitive response. More than one member of a B. graminis effector family can be recognized by a single wheat immune receptor, and a two-gene model is necessary to explain virulence to Pm1a.

Gossypium hirsutum gene of unknown function Gohir.A03G007700.1 encodes a potential VAN3-binding protein with a phosphoinositide-binding site.

A gene of unknown function, Gohir.A03G007700.1 (gene ID: Gohir.A03G007700_UTX-TM1_v2.1; transcript ID: Gohir.A03G007700.1_UTX-TM1_v2.1), identified in Gossypium hirsutum was studied using bioinformatic analyses of the sequence and structure of its encoded protein. Results from domain prediction, conserved residues and structure comparison indicate the encoded plant-specific protein (UniProt A0A1U8N485) is part of the VAN3-binding protein family with a conserved phosphoinositide-binding site. Homology comparison suggests functional similarity with Arabidopsis FORKED-like FL5 and 6, which localize to the Golgi apparatus and are linked to vein development and leaf size phenotypes.

Efficient imaging and computer vision detection of two cell shapes in young cotton fibers.

Premise: The shape of young cotton (Gossypium) fibers varies within and between commercial cotton species, as revealed by previous detailed analyses of one cultivar of G. hirsutum and one of G. barbadense. Both narrow and wide fibers exist in G. hirsutum cv. Deltapine 90, which may impact the quality of our most abundant renewable textile material. More efficient cellular phenotyping methods are needed to empower future research efforts. Methods: We developed semi-automated imaging methods for young cotton fibers and a novel machine learning algorithm for the rapid detection of tapered (narrow) or hemisphere (wide) fibers in homogeneous or mixed populations. Results: The new methods were accurate for diverse accessions of G. hirsutum and G. barbadense and at least eight times more efficient than manual methods. Narrow fibers dominated in the three G. barbadense accessions analyzed, whereas the three G. hirsutum accessions showed a mixture of tapered and hemisphere fibers in varying proportions. Discussion: The use or adaptation of these improved methods will facilitate experiments with higher throughput to understand the biological factors controlling the variable shapes of young cotton fibers or other elongating single cells. This research also enables the exploration of links between early cell shape and mature cotton fiber quality in diverse field-grown cotton accessions.

Graph-based pangenomics maximizes genotyping density and reveals structural impacts on fungal resistance in melon.

The genomic sequences segregating in experimental populations are often highly divergent from the community reference and from one another. Such divergence is problematic under various short-read-based genotyping strategies. In addition, large structural differences are often invisible despite being strong candidates for causal variation. These issues are exacerbated in specialty crop breeding programs with fewer, lower-quality sequence resources. Here, we examine the benefits of complete genomic information, based on long-read assemblies, in a biparental mapping experiment segregating at numerous disease resistance loci in the non-model crop, melon (Cucumis melo). We find that a graph-based approach, which uses both parental genomes, results in 19% more variants callable across the population and raw allele calls with a 2 to 3-fold error-rate reduction, even relative to single reference approaches using a parent genome. We show that structural variation has played a substantial role in shaping two Fusarium wilt resistance loci with known causal genes. We also report on the genetics of powdery mildew resistance, where copy number variation and local recombination suppression are directly interpretable via parental genome alignments. Benefits observed, even in this low-resolution biparental experiment, will inevitably be amplified in more complex populations.

Gossypium hirsutum gene of unknown function, Gohir.A02G044702.1, encodes a potential B3 Transcription Factor of the REM subfamily.

A gene of unknown function, Gohir.A02G044702.1, identified in Gossypium hirsutum was studied using sequence and structure bioinformatic tools. The encoded protein (UniProt A0A1U8MGX4) was predicted to localize to the nucleus, was found to retain the B3 transcription factor domain with conserved DNA-binding residues and to most closely cluster with REM subfamily members of B3-domain containing proteins.

Outlook for Implementation of Genomics-Based Selection in Public Cotton Breeding Programs.

Researchers have used quantitative genetics to map cotton fiber quality and agronomic performance loci, but many alleles may be population or environment-specific, limiting their usefulness in a pedigree selection, inbreeding-based system. Here, we utilized genotypic and phenotypic data on a panel of 80 important historical Upland cotton (Gossypium hirsutum L.) lines to investigate the potential for genomics-based selection within a cotton breeding program's relatively closed gene pool. We performed a genome-wide association study (GWAS) to identify alleles correlated to 20 fiber quality, seed composition, and yield traits and looked for a consistent detection of GWAS hits across 14 individual field trials. We also explored the potential for genomic prediction to capture genotypic variation for these quantitative traits and tested the incorporation of GWAS hits into the prediction model. Overall, we found that genomic selection programs for fiber quality can begin immediately, and the prediction ability for most other traits is lower but commensurate with heritability. Stably detected GWAS hits can improve prediction accuracy, although a significance threshold must be carefully chosen to include a marker as a fixed effect. We place these results in the context of modern public cotton line-breeding and highlight the need for a community-based approach to amass the data and expertise necessary to launch US public-sector cotton breeders into the genomics-based selection era.

Leveraging National Germplasm Collections to Determine Significantly Associated Categorical Traits in Crops: Upland and Pima Cotton as a Case Study.

Observable qualitative traits are relatively stable across environments and are commonly used to evaluate crop genetic diversity. Recently, molecular markers have largely superseded describing phenotypes in diversity surveys. However, qualitative descriptors are useful in cataloging germplasm collections and for describing new germplasm in patents, publications, and/or the Plant Variety Protection (PVP) system. This research focused on the comparative analysis of standardized cotton traits as represented within the National Cotton Germplasm Collection (NCGC). The cotton traits are named by 'descriptors' that have non-numerical sub-categories (descriptor states) reflecting the details of how each trait manifests or is absent in the plant. We statistically assessed selected accessions from three major groups of Gossypium as defined by the NCGC curator: (1) "Stoneville accessions (SA)," containing mainly Upland cotton (Gossypium hirsutum) cultivars; (2) "Texas accessions (TEX)," containing mainly G. hirsutum landraces; and (3) Gossypium barbadense (Gb), containing cultivars or landraces of Pima cotton (Gossypium barbadense). For 33 cotton descriptors we: (a) revealed distributions of character states for each descriptor within each group; (b) analyzed bivariate associations between paired descriptors; and (c) clustered accessions based on their descriptors. The fewest significant associations between descriptors occurred in the SA dataset, likely reflecting extensive breeding for cultivar development. In contrast, the TEX and Gb datasets showed a higher number of significant associations between descriptors, likely correlating with less impact from breeding efforts. Three significant bivariate associations were identified for all three groups, bract nectaries:boll nectaries, leaf hair:stem hair, and lint color:seed fuzz color. Unsupervised clustering analysis recapitulated the species labels for about 97% of the accessions. Unexpected clustering results indicated accessions that may benefit from potential further investigation. In the future, the significant associations between standardized descriptors can be used by curators to determine whether new exotic/unusual accessions most closely resemble Upland or Pima cotton. In addition, the study shows how existing descriptors for large germplasm datasets can be useful to inform downstream goals in breeding and research, such as identifying rare individuals with specific trait combinations and targeting breakdown of remaining trait associations through breeding, thus demonstrating the utility of the analytical methods employed in categorizing germplasm diversity within the collection.

There and back again; historical perspective and future directions for Vaccinium breeding and research studies.

The genus Vaccinium L. (Ericaceae) contains a wide diversity of culturally and economically important berry crop species. Consumer demand and scientific research in blueberry (Vaccinium spp.) and cranberry (Vaccinium macrocarpon) have increased worldwide over the crops' relatively short domestication history (~100 years). Other species, including bilberry (Vaccinium myrtillus), lingonberry (Vaccinium vitis-idaea), and ohelo berry (Vaccinium reticulatum) are largely still harvested from the wild but with crop improvement efforts underway. Here, we present a review article on these Vaccinium berry crops on topics that span taxonomy to genetics and genomics to breeding. We highlight the accomplishments made thus far for each of these crops, along their journey from the wild, and propose research areas and questions that will require investments by the community over the coming decades to guide future crop improvement efforts. New tools and resources are needed to underpin the development of superior cultivars that are not only more resilient to various environmental stresses and higher yielding, but also produce fruit that continue to meet a variety of consumer preferences, including fruit quality and health related traits.

The spinach YY genome reveals sex chromosome evolution, domestication, and introgression history of the species.

BACKGROUND: Spinach (Spinacia oleracea L.) is a dioecious species with an XY sex chromosome system, but its Y chromosome has not been fully characterized. Our knowledge about the history of its domestication and improvement remains limited. RESULTS: A high-quality YY genome of spinach is assembled into 952 Mb in six pseudo-chromosomes. By a combination of genetic mapping, Genome-Wide Association Studies, and genomic analysis, we characterize a 17.42-Mb sex determination region (SDR) on chromosome 1. The sex chromosomes of spinach evolved when an insertion containing sex determination genes occurred, followed by a large genomic inversion about 1.98 Mya. A subsequent burst of SDR-specific repeats (0.1-0.15 Mya) explains the large size of this SDR. We identify a Y-specific gene, NRT1/PTR 6.4 which resides in this insertion, as a strong candidate for the sex determination or differentiation factor. Resequencing of 112 spinach genomes reveals a severe domestication bottleneck approximately 10.87 Kya, which dates the domestication of spinach 7000 years earlier than the archeological record. We demonstrate that a strong selection signal associated with internode elongation and leaf area expansion is associated with domestication of edibility traits in spinach. We find that several strong genomic introgressions from the wild species Spinacia turkestanica and Spinacia tetrandra harbor desirable alleles of genes related to downy mildew resistance, frost resistance, leaf morphology, and flowering-time shift, which likely contribute to spinach improvement. CONCLUSIONS: Analysis of the YY genome uncovers evolutionary forces shaping nascent sex chromosome evolution in spinach. Our findings provide novel insights about the domestication and improvement of spinach.

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.