Genetic diversity, population structure, and taxonomic confirmation in annual medic (Medicago spp.) collections from Crimea, Ukraine.

Annual medic (Medicago spp.) germplasm was collected from the Crimean Peninsula of Ukraine in 2008 to fill gaps in geographic coverage in the United States department of Agriculture, Agricultural Research Service, National Plant Germplasm System (NPGS) temperate-adapted forage legume collection. A total of 102 accessions across 10 Medicago species were collected. To assess genetic diversity, population structure, and to confirm taxonomic identities, the collections were phenotypically and genetically characterized. Phenotyping included the use of 24 descriptor traits while genetic characterization was accomplished using a 3K Diversity Array Technologies (DArTag) panel developed for alfalfa (Medicago sativa L.). For both field and molecular characterizations, a reference set of 92 geographically diverse and species-representative accessions were obtained from the NPGS collection. Phenotypic descriptors showed consistency among replicated plants within accessions, some variation across accessions within species, and evident distinctions between species. Because the DArTag panel was developed for cultivated alfalfa, the transferability of markers to the species being evaluated was limited, resulting in an average of ~1,500 marker loci detected per species. From these loci, 448 markers were present in 95% of the samples. Principal component and phylogenetic analysis based on a larger set of 2,396 selected markers clustered accessions by species and predicted evolutionary relationships among species. Additionally, the markers aided in the taxonomic identity of a few accessions that were likely mislabeled. The genotyping results also showed that sampling individual plants for these mostly self-pollinating species is sufficient due to high reproducibility between single (n=3) and pooled (n=7) biological replicate leaf samples. The phenotyping and the 2,396 Single Nucleotide Polymorphism (SNP) marker set were useful in estimating population structure in the Crimean and reference accessions, highlighting novel and unique genetic diversity captured in the Crimean accessions. This research not only demonstrated the utility of the DArTag marker panel in evaluating the Crimean germplasm but also highlighted its broader application in assessing genetic resources within the Medicago genus. Furthermore, we anticipate that our findings will underscore the importance of leveraging genetic resources and advanced genotyping tools for sustainable crop improvement and biodiversity conservation in annual medic species.

Current challenges and future of agricultural genomes to phenomes in the USA.

Dramatic improvements in measuring genetic variation across agriculturally relevant populations (genomics) must be matched by improvements in identifying and measuring relevant trait variation in such populations across many environments (phenomics). Identifying the most critical opportunities and challenges in genome to phenome (G2P) research is the focus of this paper. Previously (Genome Biol, 23(1):1-11, 2022), we laid out how Agricultural Genome to Phenome Initiative (AG2PI) will coordinate activities with USA federal government agencies expand public-private partnerships, and engage with external stakeholders to achieve a shared vision of future the AG2PI. Acting on this latter step, AG2PI organized the "Thinking Big: Visualizing the Future of AG2PI" two-day workshop held September 9-10, 2022, in Ames, Iowa, co-hosted with the United State Department of Agriculture's National Institute of Food and Agriculture (USDA NIFA). During the meeting, attendees were asked to use their experience and curiosity to review the current status of agricultural genome to phenome (AG2P) work and envision the future of the AG2P field. The topic summaries composing this paper are distilled from two 1.5-h small group discussions. Challenges and solutions identified across multiple topics at the workshop were explored. We end our discussion with a vision for the future of agricultural progress, identifying two areas of innovation needed: (1) innovate in genetic improvement methods development and evaluation and (2) innovate in agricultural research processes to solve societal problems. To address these needs, we then provide six specific goals that we recommend be implemented immediately in support of advancing AG2P research.

Mitochondrial genome datasets for the sweetpotato weevil, Cylas formicarius elegantulus (Coleoptera: Brentidae), collected in the United States.

The sweetpotato weevil, Cylas formicarius elegantulus (Summers) (Coleoptera: Brentidae), is one of the most destructive pests of sweetpotato worldwide. Genomic analyses of sweetpotato weevils can provide insights into their genetic diversity, population structure, and dispersal as well as provide information to support management strategies. Adult sweetpotato weevils were collected by various methods from Ipomoea batatas L. (sweetpotato) or I. coccinea L. (red morning glory) in the U.S. states of Georgia, Hawaii, South Carolina, and Texas. Genomic DNA was extracted from individual weevil specimens and sequenced using Illumina NovaSeq. A total of 181 GB of 150 base pair (bp) paired-end reads were generated for 40 specimens. Mitochondrial genomes were assembled for each specimen via reference mapping and annotated using Geneious Prime. Full mitochondrial genome sequences range from 17,141 to 17,152 bp with an average GC content of 21.8% and average coverage of 3307 × . A maximum likelihood phylogenetic analysis considering the mitochondrial protein coding genes is provided. Mitochondrial genomes and assembled reads are deposited in NCBI GenBank, providing 40 mitogenomes of C. formicarius elegantulus collected in the U.S.

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.

Live imaging of chromosome dynamics.

Progression of meiosis has been traditionally reconstructed from microscopic images collected from fixed cells. However, studies conducted in a number of species, including plants, indicate that this approach has clear shortcomings in accurately portraying the dynamic nature of meiotic processes. Here, we describe two methods to study chromosome dynamics in live meiocytes in maize, a protocol to observe chromosomes during meiotic prophase I and a technique to monitor chromosome segregation in anaphase I and anaphase II. The first method relies on culturing intact maize anthers and observing meiocytes embedded in the anthers with multiphoton excitation (MPE) microscopy. This approach circumvents difficulties in culturing isolated prophase I meiocytes in plants. The second technique uses culturing isolated meiocytes, which is possible with anaphase cells. Both methods can be fairly easily adapted for use in other plant species. We also detail the kinds of time-lapse movies that can be captured and analyzed using this technique, and describe software that can be utilized for analysis of movies chromosome dynamic in live meiocytes.

SNP discovery with EST and NextGen sequencing in switchgrass (Panicum virgatum L.).

Although yield trials for switchgrass (Panicum virgatum L.), a potentially high value biofuel feedstock crop, are currently underway throughout North America, the genetic tools for crop improvement in this species are still in the early stages of development. Identification of high-density molecular markers, such as single nucleotide polymorphisms (SNPs), that are amenable to high-throughput genotyping approaches, is the first step in a quantitative genetics study of this model biofuel crop species. We generated and sequenced expressed sequence tag (EST) libraries from thirteen diverse switchgrass cultivars representing both upland and lowland ecotypes, as well as tetraploid and octoploid genomes. We followed this with reduced genomic library preparation and massively parallel sequencing of the same samples using the Illumina Genome Analyzer technology platform. EST libraries were used to generate unigene clusters and establish a gene-space reference sequence, thus providing a framework for assembly of the short sequence reads. SNPs were identified utilizing these scaffolds. We used a custom software program for alignment and SNP detection and identified over 149,000 SNPs across the 13 short-read sequencing libraries (SRSLs). Approximately 25,000 additional SNPs were identified from the entire EST collection available for the species. This sequencing effort generated data that are suitable for marker development and for estimation of population genetic parameters, such as nucleotide diversity and linkage disequilibrium. Based on these data, we assessed the feasibility of genome wide association mapping and genomic selection applications in switchgrass. Overall, the SNP markers discovered in this study will help facilitate quantitative genetics experiments and greatly enhance breeding efforts that target improvement of key biofuel traits and development of new switchgrass cultivars.

Using information technology to improve adult immunization delivery in an integrated urban health system.

BACKGROUND: Adult immunizations prevent morbidity and mortality yet coverage remains suboptimal, in part due to missed opportunities. Clinical decision support systems (CDSSs) can improve immunization rates when integrated into routine work flow, implemented wherever care is delivered, and used by staff who can act on the recommendation. METHODS: An adult immunization improvement project was undertaken in a large integrated, safety-net health care system. A CDSS was developed to query patient records and identify patients eligible for pneumococcal, influenza, or tetanus immunization and then generate a statement that recommends immunization or indicates a previous refusal. A new agency policy authorized medical assistants and nurses in clinics, and nurses in the hospital, to use the CDSS as a standing order. Immunization delivery work flow was standardized, and staff received feedback on immunization rates. RESULTS: The CDSS identified more patients than a typical paper standing order and can be easily modified to incorporate changes in vaccine indications. The intervention led to a 10% improvement in immunization rates in adults 65 years of age or older and in younger adults with diabetes or chronic obstructive pulmonary disease. Overall, the improvements were sustained beyond the project period. The CDSS was expanded to encompass additional vaccines. CONCLUSIONS: Interdepartmental collaboration was critical to identify needs, challenges, and solutions. Implementing the standing order policy in clinics and the hospital usually allowed immunizations to be taken out of the hands of clinicians. As an on-demand tool, CDSS must be used at each patient encounter to avoid missed opportunities. Staff retraining accompanied by ongoing assessment of immunization rates, work flow, and missed opportunities to immunize patients are critical to sustain and enhance improvements.

Imaging chromosome dynamics in meiosis in plants.

Progression of meiosis has been traditionally reconstructed from microscopic images collected from fixed cells. This approach has clear shortcomings in accurately portraying the dynamics of meiotic processes. Studies conducted in recent years mostly in unicellular fungi have shown that chromosomes in meiotic prophase exhibit dynamic motility that cannot be accurately examined using fixed cell imaging. However, in contrast to yeast, research on meiotic chromosome dynamics in multicellular eukaryotes has been lagging. This was in part because meiocytes in multicellular eukaryotes reside deep within reproductive organs and are often refractory to culturing. Here, we describe a method in which intact, live-plant reproductive organs (anthers) are cultured to enable monitoring chromosome dynamics of meiocytes using multiphoton excitation (MPE) microscopy. The method was developed for use in maize but can be applied to other plant species and adapted for use in other taxa in which meiocytes are embedded in multicellular reproductive structures. MPE microscopy allows visualization of meiocytes embedded within native tissue in planta and thus meiocytes remain intact for the entire imaging procedure. We detail the kinds of time-lapse movies that can be captured and analyzed using this technique and also highlight software packages that can be utilized for analysis of movies chromosome dynamic in live meiocytes.

Live imaging of rapid chromosome movements in meiotic prophase I in maize.

The ability of chromosomes to move across the nuclear space is essential for the reorganization of the nucleus that takes place in early meiotic prophase. Chromosome dynamics of prophase I have been studied in budding and fission yeasts, but little is known about this process in higher eukaryotes, where genomes and chromosomes are much larger and meiosis takes a longer time to complete. This knowledge gap has been mainly caused by difficulties in culturing isolated live meiocytes of multicellular eukaryotes. To study the nuclear dynamics during meiotic prophase in maize, we established a system to observe live meiocytes inside intact anthers. We found that maize chromosomes exhibited extremely dynamic and complex motility in zygonema and pachynema. The movement patterns differed dramatically between the two stages. Chromosome movements included rotations of the entire chromatin and movements of individual chromosome segments, which were mostly telomere-led. Chromosome motility was coincident with dynamic deformations of the nuclear envelope. Both, chromosome and nuclear envelope motility depended on actin microfilaments as well as tubulin. The complexity of the nuclear movements implies that several different mechanisms affect chromosome motility in early meiotic prophase in maize. We propose that the vigorous nuclear motility provides a mechanism for homologous loci to find each other during zygonema.

In the beginning: the initiation of meiosis.

The most-critical point of reproductive development in all sexually reproducing species is the transition from mitotic to meiotic cell cycle. Studies in unicellular fungi have indicated that the decision to enter meiosis must be made before the beginning of the premeiotic S phase. Recent data from the mouse suggest that this timing of meiosis initiation is a universal feature shared also by multicellular eukaryotes. In contrast, the signaling cascade that leads to meiosis initiation shows great diversity among species.

Subfunctionalization of PhyB1 and PhyB2 in the control of seedling and mature plant traits in maize.

Phytochromes are the primary red/far-red photoreceptors of higher plants, mediating numerous developmental processes throughout the life cycle, from germination to flowering. In seed plants, phytochromes are encoded by a small gene family with each member performing both distinct and redundant roles in mediating physiological responses to light cues. Studies in both eudicot and monocot species have defined a central role for phytochrome B in mediating responses to light in the control of several agronomically important traits, including plant height, transitions to flowering and axillary branch meristem development. Here we characterize Mutator-induced alleles of PhyB1 and a naturally occurring deletion allele of PhyB2 in Zea mays (maize). Using single and double mutants, we show that the highly similar PhyB1 and PhyB2 genes encode proteins with both overlapping and non-redundant functions that control seedling and mature plant traits. PHYB1 and PHYB2 regulate elongation of sheath and stem tissues of mature plants and contribute to the light-mediated regulation of PhyA and Cab gene transcripts. However, PHYB1 and not PHYB2 contributes significantly to the inhibition of mesocotyl elongation under red light, whereas PHYB2 and to a lesser extent PHYB1 mediate the photoperiod-dependent floral transition. This sub functionalization of PHYB activities in maize has probably occurred since the tetraploidization of maize, and may contribute to flowering time variation in modern-day varieties.

Cereal phytochromes: targets of selection, targets for manipulation?

Plants respond to shading through an adaptive syndrome termed shade avoidance. In high-density crop plantings, shade avoidance generally increases extension growth at the expense of yield and can be at odds with the agronomic performance of the crop as a whole. Studies in Arabidopsis are beginning to reveal the essential role phytochromes play in regulating this process and to identify genes underlying the response. In this article, we focus on how phytochrome signaling networks have been targeted in cereal breeding programs in the past and discuss the potential to alter these pathways through breeding and transgenic manipulation to develop crops that perform better under typical high density conditions.

Structure and expression of maize phytochrome family homeologs.

To begin the study of phytochrome signaling in maize, we have cloned and characterized the phytochrome gene family from the inbred B73. Through DNA gel blot analysis of maize genomic DNA and BAC library screens, we show that the PhyA, PhyB, and PhyC genes are each duplicated once in the genome of maize. Each gene pair was positioned to homeologous regions of the genome using recombinant inbred mapping populations. These results strongly suggest that the duplication of the phytochrome gene family in maize arose as a consequence of an ancient tetraploidization in the maize ancestral lineage. Furthermore, sequencing of Phy genes directly from BAC clones indicates that there are six functional phytochrome genes in maize. Through Northern gel blot analysis and a semiquantitative reverse transcriptase polymerase chain reaction assay, we determined that all six phytochrome genes are transcribed in several seedling tissues. However, expression from PhyA1, PhyB1, and PhyC1 predominate in all seedling tissues examined. Dark-grown seedlings express higher levels of PhyA and PhyB than do light-grown plants but PhyC genes are expressed at similar levels under light and dark growth conditions. These results are discussed in relation to phytochrome gene regulation in model eudicots and monocots and in light of current genome sequencing efforts in maize.