Brief Summary of Minutes of Annual Meeting

NRSP-8 2019 Annual Multispecies Report

Summarizing the 2019 accomplishments of National Animal Genome Research Program (NRSP-8) is a tall order, given the productivity and prolificacy of this group of researchers from land-grant universities and research institutions throughout the nation. The importance of animal agriculture to the U.S. agricultural economy should be kept in perspective. In 2017, U.S. cash receipts were almost evenly divided between plant (52%) and animal agriculture (48%).

The value of agricultural production in the United States rose over most of the past decade due to increases in production as well as higher prices. In 2017, the cattle industry had the highest value of livestock production at roughly $50.2 billion.  The poultry industries were the next largest commodity with production valued at around $42.7 billion, followed by hogs and pigs at $19.2 billion. The value of milk production was about $38.1 billion. Cumulatively, the 2017 value of U.S. animal agriculture was > $151 billion.

A major contributing factor to productivity increases (Figure 1) is the genetic improvement of animals through research. As outlined the newly published “Genome to phenome: improving animal health, production, and well-being–a new USDA blueprint for animal genome research 2018–2027[1]”, NRSP-8 scientists across the country were instrumental in creating many of the genomic tools and resources that enabled genetic improvements such as:

• The lifetime net merit (NM$) index, a gauge of dairy animal profitability, doubled over the past 10 years resulting in over $4 billion return (and counting) on a $100 million investment
• Poultry and swine and poultry breeding companies actively use genome information to accelerate genetic improvement by about 30%, resulting in considerable exports (Figure 2).

NRSP-8 is an umbrella organization of animal scientists who use genomics to provide solutions for the animal agriculture community. The membership of the NRSP-8 come from experiment stations throughout the nation AL, AR, AZ, CA, CO, DE, FL, IA, ID, IL, KY, MA, MD, MI, MN, MO, MS, NC, NE, NY, OK, PA, TX, UT, VA, VT, WA, WI, WY), non-land grant institutions (California State University, Fresno, City of Hope Beckman Research Institute, Middle Tennessee State University, National Research Institute of Animal Production, National University of La Plata, Norwegian University of Life Sciences, Racing Australia Equine Genetics Research Centre, Swedish University of Agricultural Sciences, The Laboratory of Racing Chemistry, Tufts University School of Veterinary Medicine, North Grafton, MA, University of Louisville, University of Sydney, USDA-ARS Beltsville Agricultural Research Center, USDA-ARS-Avian Disease & Oncology Laboratory, Western University of Health Sciences) and encompasses scientists from the dairy and beef cattle, poultry, equine, sheep, goat, swine, and aquaculture sectors. The impact of NRSP-8 on agriculture reaches every state and region of the U.S. The use of genomics to improve the genetics of US animal-based commodities has been adopted by nearly all the food and fiber animal-breeding industries for which tools have been developed.

The annual coordinator funds allocated to each NRSP-8 species group (including bioinformatics), help to provide critical infrastructure and tools for agri-animal genomic discoveries including; genomics and bioinformatics tools and databases, genetic resource populations with economically-important phenotypes, education and training of students, scientists, and outreach to the public.

Highlights and impacts summarized in this report for 2019 include:

• Leveraging of $65,000 coordinator funds to attract new grants e.g. the aquaculture group attracted $7,532,332 in new funding. This equates to over a 1:115 return on investment for the coordinator funds. Other species groups report leveraging seed funding to secure millions of dollars in matching industry, state and federal funds.
• The improved poultry genome assemblies have allowed for the identification of genes associated with heat stress, aflatoxin, and disease resistance in poultry. Validation of these regions for commercial breeding stocks will allow significant improvements in resistance to diseases will help with selection of animals that can tolerate extreme environmental fluctuations, and avoid diseases that cost the poultry industry >$100 million annually in the absence of antimicrobial therapies.
• The new horse genome assembly has been used in a multitude of research projects, ranging from identification of genetic defects to the origins of the modern horse, with the equine species group publishing an impressive 88 papers in 2019.
• Collectively the NRSP-8 group of researchers are prolific with over 200 basic and applied peer-review papers published in 2019, in addition to multiple industry collaborations, and outreach.
• Graduate students and postdocs are being trained in genomics and bioinformatics by all species groups, and coordinator funds are being used to bring students to conferences and training events, including a bioinformatics training in advance of the NRSP-8 Annual Meeting at PAG, these students/postdocs will be the future leaders in agriculture-oriented computational science.
• Over 4,685 users worldwide subscribe and are informed by the AnGenMap email list serve (https://www.animalgenome.org/community/angenmap/); and information about NRSP-8 is made publicly available through the https://www.animalgenome.org/ website maintained at Iowa State University.

Aquaculture NRSP-8 Report 2019

Coordinator:             Benjamin J. Reading, North Carolina State University

Co-coordinators:       Steven Roberts, Washington State University

Moh Salem, University of Maryland

Eric Peatman, Auburn University

Species Leaders:

Catfish:                       Sylvie Quiniou, ARS Stoneville, Mississippi,

Oyster/shellfish:          Dina Proestou, ARS University of Rhode Island, Rhode Island

Salmonids:                  Yniv Palti, ARS Leetown, West Virginia

Striped Bass:               Benjamin Reading, North Carolina State University, North Carolina

Workshop Chair 2019-2020: Louis Plough (lplough@umces.edu)

Workshop Chair-elect 2020-2021: Moh Salem (Mohamed.Salem@mtsu.edu)

Workshop Chair-elect 2021-2022: Rafet Al-Tobasei (Rafet.Al-tobasei@mtsu.edu)

Aquaculture Workshop

There were 17 oral presentations including presentations by 6 graduate students and postdocs who were presented with travel awards ($1000 each) and 3 invited plenary speakers.

Attendees

The number of attendees: 100 (80, 2019)

The number of institutes/organizations: 49 (43, 2019)

The number of countries: 13

Aquaculture dinner reception and poster session were sponsored in part by Illumina, National Breeding Program for the Hybrid Striped Bass Industry, and NC State University.

Participants: 80

Posters: 15 (20, 2019)

Leveraged funds

4 small research projects were funded at $10,000 each for 2020 to provide preliminary data for grants: $40,000 (2019-2020); $30,000 (2018-2019).

Leveraged funds from diverse projects based on previously funded small research projects totaled more than seven million and a half dollars from federal sources in 2019.

Total Leveraged Funding in 2019: $7,532,332

This is over 1:100 return on investment for the aquaculture coordinator funds. Extramural funding agencies include NOAA, USDA NIFA, USDA AFRI, USDA Southern Regional Aquaculture Center and the Ratcliffe Foundation (non-profit). In particular the marine finfish and shellfish aquaculture initiatives of USDA and NOAA are to be thanked.

There were 29 publications in 2019 including one in the journal Nature.

Specific major activities include:

Catfish

Channel catfish genome assembly refined with optical mapping; blue catfish genome assembly released. DNA methylation profiles revealed differential methylation patterns between the two genders that underlie sex determination.

Shellfish and Crustaceans

Pacific white shrimp genome published; sequencing of whiteleg shrimp genome was initiated. Re-sequencing of wild and selected eastern oyster populations derived from multiple geographic regions along the US east Coast and Gulf of Mexico also were initiated. 600K and 50K SNP chips developed for eastern oyster were applied to different populations and RNA-seq analyses are ongoing to understand genomic basis for Dermo-resistance in Eastern Oyster. Three Eastern Oyster workshops were held: Epigenetics Workshop, Genome Workshop, Breeding Consortium Round Table.

Trout and Salmon

Genome assembly of doubled-haploid rainbow trout based on PacBio long read sequencing and scaffolding with Bionnano optical map and Hi-C contact map; genome assembly for Atlantic salmon is in progress using the tri-binning approach; Genome resequencing is underway in Chinook salmon and steelhead for broad representation of genomic variation across populations for each species. Genome-wide association studies identified genomic loci that affect fillet firmness, protein content, egg quality, fecundity, and egg size in rainbow trout. Allelic variation for candidate genes associated with migration timing/age at maturity was validated with markers in over 50,000 Chinook salmon and 20,000 steelhead. Contributions were made to development of FishGen.net database for storage of large-scale genotypes for genetic tagging and monitoring studies

Striped Bass

The second striped bass genome draft was uploaded to GenBank; transcriptome data is currently being processed by NCBI for annotation. A machine learning pipeline developed to analyze single nucleotide (SNP) markers (expressed quantitative trait loci, eQTL) related to growth in different strains of hybrid striped bass. Different and novel machine learning-based analytical platforms are focused on small molecule (metabolomics), gene expression (RNA-Seq), and protein (proteomics) profiling to better understand hybrid striped bass growth performance (heterosis effects) and reproductive success in several different wild stocks of striped bass in watersheds of the mid-Atlantic region. Different strains of white bass from the midwest were assembled to establish a base breeding population for familywise evaluations of growth and nutrient utilization on alternative, sustainable diets; genotyping-by-sequencing panel was developed from these white bass populations. Genetically improved striped bass and white bass transferred to industry from National Breeding Program for the Hybrid Striped Bass Industry.

Bovine NRSP-8 Report 2019

COOPERATING AGENCY AND PRINCIPAL LEADERS

University of California, Davis: Alison Van Eenennaam, alvaneenennaam@ucdavis.edu

University of Missouri-Columbia: Bob Schnabel, Co-coordinator, schnabelr@missouri.edu

Texas A&M University, Clare Gill, Co-coordinator, clare-gill@tamu.edu 

USDA ARS, Beltsville, Ben Rosen, Co-coordinator, Ben.Rosen@ars.usda.gov

Washington State University, Zhihua Jiang, Co-coordinator jiangz@wsu.edu

2020 Cattle Workshop Report

Workshop Chair 2019-2020: Ben Rosen (Ben.Rosen@ARS.USDA.GOV)

Co-Chair-elect 2020-2021:     Darren Hagen  (darren.hagen@okstate.edu)

Erdogan Memeli (em149@msstate.edu)

Co-Chair-elect 2021-2022      Cedric Gondro (gondroce@msu.edu).

International Plant and Animal Genome XXVIII Workshop Attendance:

Cattle/swine

Cattle/sheep/goat 1

Cattle/sheep/goat 2

Objective 1: “Advance the status of reference genomes for all species, including basic annotation of worldwide genetic variation, by broad sequencing among different lines and breeds of animals.”

We generated >1M pass filter CCS reads for 12 tissues (total CCS reads 14.6M), which produced an average of 50k polished high-quality isoforms per tissue using IsoSeq3 analysis protocol.  Tissues were from the 180d gestation Dominette fetus and included Abomasum, Adipose (SubQ), Adrenal Cortex, Cerebellum, Frontal Cortex, Kidney, Liver, Lung, Lymph Node, Skeletal Muscle, Thyroid, Tongue. We plan to do an analysis including all data to find total number of unique isoforms across all tissues, but it is computationally intensive so we have been waiting for the server to have a relatively idle period.  The data are being shared with Jim Reecy to perform analysis/comparison with adult tissues and across species (Tim Smith, US MARC).

We also generated 15 PromethION flow cells of data for the Angus_x_Brahman fetus, producing 301 Gb of data including the one flow cell run at UC Davis.  Ben Rosen has run an assembly of just the nanopore data to see how it would compare to the PacBio assembly. Initial contig (highly confident, uninterrupted sequence assembly) N50 for Angus and Brahman PacBio assemblies were 29.4 Mb and 23.4 Mb respectively. Preliminary results of the nanopore only assembly are encouraging, with these lengths more than doubling to 69.8 Mb and 71.8 Mb respectively..  Total data is >450x coverage, so we are hopeful that the assembly of sex chromosomes will be greatly improved.  We only got about 5x coverage of reads >100 kb, was hoping for more but DNA quality was a bit limiting due to age of the sample and shipping from Australia (Tim Smith, US MARC).

We also generated >60M reads (FAANG standard) RNAseq for each of the fetal tissues that had IsoSeq, for quantitation purposes (Tim Smith, US MARC).

Bovine Y-Chromosome Since January of 2019 Brenda Murdoch from the University of Idaho ran 25 cycles of cells for chromosome isolation and fluorescence-activated cell sorting (FACS) preparation. Thirteen of these trails have been done with cultured white blood cells isolated from whole blood, and twelve have been done with a fibroblast cell line. Chromosome samples were initially sent for fluorescence-activated cell sorting (FACS) at Stanford’s FACS facility; however, several FACS trials did not return an adequately isolated sample, and the project has begun implementation of a magnetic streptavidin-bead capture method as an alternative to FACS. DNA has been extracted from isolated chromosomes and sent to Tim Smith for validation of enrichment.  This project is ongoing (Brenda Murdoch, University of Idaho). 

Wansheng Liu at Penn State reported in previous years that two of the lost Holstein Y lineages, namely ZIMMERMAN ALSTAR PILOT (born in 1954) and ROSAFE CALIBAN (born in 1953), were recovered and produced 3 and 5 male offspring, respectively. We have completed the evaluation of these young bulls and submitted a manuscript to J. Dairy Sci. Here is the abstract of this paper: More than 99 percent of all known Holstein artificial insemination bulls in the United States can be traced through their male lineage to just two bulls born in the 1950s and all Holstein bulls can be traced back to two bulls born in the late 1800s. As the Y-chromosome is passed exclusively from sire to son, this suggests there is limited variation for much of the Y-chromosome and that there has been significant loss of genetic diversity in the artificial insemination era. Two additional male lineages that are separate from modern lineages prior to 1890 were present at the start of the artificial insemination era and had semen available from the USDA–National Animal Germplasm Program; semen from representatives of those lineages were used for in vitro  embryo production by mating to elite modern genetic females, resulting in the birth of seven bulls and eight heifers. Genomic evaluation of the bulls suggested that lineages from the beginning of the AI era could be reconstituted to breed average for total economic merit in one generation when mated to elite females due to high genetic merit for fertility, near average genetic merit for fat and protein yield, and below average genetic merit for udder and physical conformation. Semen from the bulls is commercially available to facilitate Y-chromosome research and efforts to restore lost genetic diversity.

Sequence and Assembly of the Holstein Y Chromosome. The objective of this study was to sequence and assemble the Holstein Y by using multiple types of whole genome sequencing data from a single bull. The sequence data sets include the illumina paired-end (PE), illuminia mate pair (MP), PacBio long reads (PB), and Dovetail Chicago reads (Hi-C). The initial contigs was assembled from the PE reads. The total length of the contigs was 17.3M. Then the contigs were scaffolded with MP reads, and the total length was 19.2 M with the largest contig increased from 36K to 123K. The gaps of the scaffolds were filled with PB reads, and the total scaffold length was improved into 24.2M. At last the scaffolds were improved by Hi-C reads, and the final assembly was 24.3Mb with 7208 contigs, and the N50 is 7618 bp. Gene annotation indicated that all 12 known X-degenerate (Xd) and 5 known Y-ampliconic (Ya) genes are present in the draft Holstein Y assembly, and the copy number of these Y-linked genes were estimated. Differing from the Hereford Y, we found that RBMY and UBE1Y genes are multicopy in the Holstein Xd region.

Functional Annotation of Animal Genomes (FAANG) initiative

Brenda Murdoch from the University of Idaho completed the collection of fetal cattle tissues from four (3 males and 1 females) 14 month old Line 1 Hereford animals. The samples from eight tissues (skeletal muscle, liver, adipose, spleen, hypothalamus, brain cortex/whole, cerebellum, and lung) from the animals have been collected for RNA-Seq, DNase-Seq, ATAC-Seq, ChIP-Seq, DNA-methylation, Hi-C and other assays. Data are being integrated to functionally annotate regulatory elements within the bovine genome. We expect that the FAANG cattle initiative will be significantly expanded with added collaborations and assays supported by the recent FAANG Program Area NIFA funding (Brenda Murdoch, University of Idaho).

Wansheng Liu at Penn State oversees the bovine tissue RNA extraction and RNA-seq and small RNA-seq library construction and sequencing (at Zoetis). The accomplishments for the cattle FAANG project in 2019 are summarized below. This project seeks to generate high quality transcript and chromatin status datasets from a comprehensive set of assays in tissues collected from animals closely related to Dominette L1, the individual from which the reference genome was sequenced. Moreover, multiple developmental stages, mammary gland tissue from Holstein cows, and cultured cells are being evaluated for a wide-ranging list of agriculturally important tissues/cell types. So far, transcriptomic data, including RNA-seq, small RNA-seq, and ATTS-seq has been collected from two biological replicates of 28 adult tissues and 10 fetal tissues. Current datasets include 65 ATTS libraries with a total of 388M sequenced reads, 123 RNA-seq libraries, and 123 small RNA-seq libraries. Analysis of these data will provide a comprehensive characterization of the expressed regions of the genome as well as accurate comparisons of differential gene expression across multiple tissues that will be harnessed for the identification of regulatory elements active in the bovine genome (Wansheng Liu, Pennsylvania State University).

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Identify relationships between sensor data and feed intake in Holstein dairy cattle.  Associations were identified between adjusted daily dry mater intake (i.e. feed intake) and health events with rumen bolus pH, temperature and activity and ear tag temperature, activity and rumination measurements from two ear tag technologies (P < 0.05).  Daily changes in feed intake were estimated in response to animal health events to determine the daily impact on feed intake.  The impact of ambient temperature on feed intake and health events was also evaluated.  Results indicate automated sensor traits act as indicators of feed intake. Health events appear to have long lasting influence on sensor trait and feed intake phenotypes.  Objective 1b: Participation in national effort to develop a feed efficiency genetic evaluation in US dairy cattle.  Feed intake was collected on 48 lactating cows in 2019 towards the goals and objectives of the FFAR/ CDCB funded project:  Improving dairy feed efficiency, sustainability and profitability by impacting farmers’ breeding and culling decisions, headed by lead PI Dr. Mike Vandehaar at Michigan State University.  This research program links the use of molecular and quantitative genetics data as well as new high-throughput phenotyping technologies for use in applied animal breeding by AI companies and breeders (James Koltes, Iowa State University).

Characterization of the bovine PRAMEY protein in testis, epididymis and spermatozoa.

We reported last year the characterization of PRAMEY protein dynamic in spermatozoa, fluid and tissues from testis and caput and cauda segments of the epididymis by western blot with a PRAMEY-specific antibody. We continued to work on this project in 2019 to further study the potential role of PRAMEY during sperm capacitation and acrosome reaction, and we hope to complete this work in 2020 (Wansheng Liu, Pennsylvania State University).

Determine the functional role of PRAME in spermiogenesis using a Prame-knockout (KO) mouse model. We continued to work on the KO mouse models to study the functional role of PRAME during spermiogenesis. We have finished the characterization of the Prame cKO mice and found that the cKO mice are fertile with a distinctively reproductive phenotype,  i.e. the testis size (P<0.01) and sperm count (P<0.05) are significantly reduced by 12% at 4 months of age when compared to the Prame floxed mice. Histological, immunofluorescence with germ cell-specific markers and TUNEL analyses of testis cross-sections at postnatal day 7 (P7), P14, P21, P35, P120, and P365 indicated a significant increase in apoptotic germ cells at P7 and P14, and an increase in abnormal seminiferous tubules at P21 and P35. Germ cells were gradually lost resulting in two different phenotypes in the Prame cKO testes: Sertoli-cell-only (SCO) for some of the affected tubules in young mice (at P35) and germ cell arrest at spermatogonia stage for other affected tubules in mature mice. Both phenotypes were a consequence of disruption in RAR signal pathway by the depletion of Prame at a different time point during the first and subsequent rounds of spermatogenesis. The results suggest that Prame plays a minor, but important role in spermatogenesis and different paralogs in the Prame gene family may be functionally and partially redundant (Wansheng Liu, Pennsylvania State University).

Generated high –throughput RNA sequence of microRNAs from 74 longissimus lumborum biopsies from F3 Bos indicus x Bos taurus steers.  These sequence data are coupled with extensive meat and carcass phenotypes (Penny Riggs, Texas A&M).

Objective 2: “Develop strategies to identify and exploit genes and allelic variation that contribute to economically relevant phenotypes and traits, in part through improving functional annotation of the genomes of our species.”

Genetic associations with antral follicle count in beef cattle

Antral follicle number was counted on 216 heifers from the University of Idaho’s Nancy M. Cummings Research, Education and Extension Center. The heifers used were between 10 and 14 months at the time of sampling and samples were taken during two different years, 70 from year one and 146 from year two. The herd is a crossbred herd with sires from Angus, Hereford, Simmental, and SimAngus breeds. Antral Follicle Count (AFC) was determined using an ultrasound probe with follicles greater than 3mm in diameter being recorded. The Bovine 50K GGP Single Nucleotide Polymorphism (SNP) marker chip and then was imputed up to 850K SNP markers (with Bob Schnabel). The most significant SNPs are located on chromosome 23 and 2 and are located in regions with genes of biological significance to AFC as they affect transcription, anti-apoptosis, intracellular calcium levels, angiogenesis, energy production/metabolism, and cell proliferation (Brenda Murdoch, University of Idaho).

Single cell RNA-sequencing

Single cell RNA-sequencing was performed in fetal gonads at 35 and 49 days of gestation.  Gonads were collected from fetuses recovered from timed-pregnant heifers, two male fetuses were used for day 35 collection and one male fetus for day 49. The genital ridge/gonad was dissected and dissociated to achieve a single cell suspension, which was then cryopreserved until single-cell library preparation. On September 10th, 4,000 and 12,000 cells corresponding to day 35 and 49 samples, respectively, were submitted to the UC Davis Genome Center for 10X Genomics Chromium single Cell RNA-seq preparation. The libraries were sequenced in a single HiSeq4000 lane. Sequencing data is being processed to assess library quality. Another sequencing lane is expected to be necessary to achieve sufficient read coverage. (Pablo Ross, UC Davis).

Ovaries were harvested immediately after slaughter of heifers at days 35 and 49 of gestation and transported to the laboratory.  Small fragments of ovarian cortex from the first heifer (slaughtered on 8/14/19) were cryopreserved at -80 °C in medium composed of 90% fetal bovine serum and 10% DMSO immediately after slaughter, and the cell isolation procedure was performed in thawed tissue. For the second heifer (slaughtered on 8/27/19), fresh ovarian tissue was processed into a single cell suspension, and the cells were then cryopreserved in the same freezing medium and temperature until sequencing. Preparations of cell suspension: ovarian cortex was cut into fragments of approximately 5 mm and processed into a single cell suspension. The cell suspension was then filtered through a 100 mm and 40 mm cell strainers, centrifuged at 300g for 5 minutes and resuspended in 1mL of HBSS with 0.04% (v/v) BSA. Trypan blue cell viability test was used to ensure at least 80% cell viability before and after thawing. Cell suspensions were transported in ice to the Genome Center for 10X Genomics Chromium single Cell RNA-seq preparation on 9/6/2019 and 9/10/2019. Libraries were sequenced in a single HiSeq4000 lane. (Anna Denicol, UC Davis)

Single-cell transcriptome analysis of germ cells at postnatal day 7 from Prame cKO and floxed mice. Cells from the entire testis of 3 Prame cKO and 1 WT mice at P7 were subjected to single-cell RNA-seq using 10X Genomics. Approximately 4000 cells from each sample and 130K reads (pair-end, 100 bp per read) per cell were obtained and analyzed. All cells in the P7 testis were clustered into 5 clusters based upon the transcriptome profile. Cell type in each cluster was identified by germ cell-specific markers, such as Nanos3 and c-Kit. Sertoli and other non-germ cells were clustered in groups II and V. All germ cells were clustered in three groups: undifferentiated (III) and differentiated spermatogonia (I) and an undefined cluster of germ cells (IV). Interestingly, Cluster IV shows substantial difference between the cKO and WT mice. The proportion of Cluster IV cells was 5% in the WT testis, while 8.3% in average (6-10%) in cKOs. (Wansheng Liu, Pennsylvania State University).

Work towards this objective involves studies characterizing a major gene for bovine ovulation rate in cattle.  Current work involves creating a de novo sequence assembly of a homozygote for the allele which will be used to identify the probably variant.  The variant will be validated by creating a cell line containing the putative causative polymorphism using gene editing and verifying the mutations effect on gene expression (Brian Kirkpatrick, University of Wisconsin)

Objective 3: “Facilitate analysis, curation, storage, distribution and application of the enormous datasets now being generated by next-generation sequencing and related "omics" technologies with regard to animal species of agricultural interest.”

NRSP8 Bioinformatics Training Workshop @PAG supported by Bovine Coordinator Funds

Provided $3,000 support to the Workshop as well as covered registration for 6 students, and travel costs for 5 graduate students to attend PAG.

A pilot study was done to better understand what aspects of the maternal environment influence the developing rumen microbiome in calves. Currently, samples from this study have been collected and 16S amplicon sequencing of the microbial DNA is complete. Bioinformatic analysis is currently ongoing where I am training 2 M.S. students and 1 Ph.D. to utilize these tools of metagenomic sequencing to characterize the microbiome. (Hannah C. Cunningham-Hollinger, University of Wyoming)

The effect of the rumen microbiome and the effects on feed efficiency is a major theme in our lab. Utilizing Next Generation Sequencing of the rumen microbiome, we are able to characterize not only what microbes are present but also their functional role. Recent studies investigating the maternal factors that impact early colonization of the rumen microbiome have been underway.  The long-term potential impact is discovering management, feeding, or other intervention strategies that may allow for programming of the microbiome to improve efficiency in offspring later in life.  This project was funded by the Wyoming INBRE ($10,000) (Hannah C. Cunningham-Hollinger, University of Wyoming)

While the DNA samples we collect are not necessarily from the host but rather used to identify microbial communities at various locations (rumen, vagina, placenta, etc.) these samples are coupled with phenotypic information and will be used to develop selection tools based on the microbiome to select for improved efficiency. Host genetics plays a critical, however, the rumen microbiome has a strong connection to feed efficiency and as such should also be considered when making selection decisions (Hannah C. Cunningham-Hollinger, University of Wyoming)

Recent work has been published utilizing key microbial taxa to predict feed efficiency in sheep. There is an effort in our lab to utilize various data sets that have GrowSafe feed intake data and rumen microbiome data, to evaluate the use of this strategy in beef cattle. This has the potential to predict efficiency based on the rumen microbiome and/or to understand which microbes are most critical in animals with desirable feed efficiency. There are various new sequencing techniques that make the possibility of chute-side testing a reality (MinION, iSeq, etc.). It is imperative that a host of candidate microbial populations be well established so this chute-side testing can be effective and beneficial (Hannah C. Cunningham-Hollinger, University of Wyoming)

Impacts

• There are now several high quality “reference” genomes for major cattle breeds, which has open up the possibility of developing a pangenome. Several groups have recently used this data to develop successful grants to develop tools to develop a bovine pangenome. This will be a more generally useful resource for industry as compared to a single reference genome.
• This has also revealed New insights into mammalian sex chromosome structure and evolution using high-quality sequences from bovine X and Y chromosomes
• Genomic data is being used widely in both the beef and dairy industry for genomic prediction

Funding Secured:

• 2019-2020 University of Wyoming Agricultural Experiment Station Competitive Grant Program. Late gestation maternal nutrition influence on the developing rumen microbiome in cattle. Funded. $9,866.64. (Hannah C. Cunningham-Hollinger, University of Wyoming)
• 2019-2020 Private industry funding. Investigations of RumaCell on post-weaned calf performance, efficiency, rumen microbiome, and coccidia prevalence. Funded by Pacer Technology. ($20,000) (Hannah C. Cunningham-Hollinger, University of Wyoming)
• 2018-2019 Wyoming INBRE Sequencing and Bioinformatic Analysis Program. Survey of the maternal/reproductive microbiomes influence on the neonatal rumen microbiome in cattle. Funded. $8,594.72 (Hannah C. Cunningham-Hollinger, University of Wyoming)
• Prenatal Stress Modulation of the Hypothalamic-Pituitary- Adrenal Axis and Telomere Length, Funded by USDA NIFA (May 1, 2019 - April 30, 2021), awarded May 1, 2019 (Funded - In Progress, Spring 2019, PI Thomas Welsh with CoPI Charles Long, CoPI David Riley, CoPI Penny Riggs, CoPI Rodolfo De Carvalho Cardoso, CoPI Ronald Randel
• IPA at the US Department of the Interior (October 1, 2018 - August 31, 2019), awarded October 2, 2018 ($191,030.00), Completed, Summer 2019, PI Penny Riggs
• Pilot Project to Develop Feasibility Data for Bovine Germline Complementation. Rustici Rangeland and Cattle Research Endowment Grant. $50,000 A.L. Van Eenennaam (PD) 1/1/2019 – 12/31/2019
• Functional Annotation of the Bovine Genome; 2018-67015-27500; P. Ross (PI), H. Zhou, J. Medrano et al; 1/15/2018-1/14/2022;. $2,500,000.

Poultry NRSP-8 Report 2019

NRSP-8 Poultry Annual Report October 1, 2018 – September 30, 2019

Poultry Genome Coordinators: Huaijun Zhou (UC Davis); Hans Cheng (USDA-ARS)

Chair: Kent Reed (University of Minnesota)

Secretary: Bindu Nanduri (Mississippi State University)

The NRSP-8 Poultry Workshop held January 11-12, 2020 in conjunction with NC1170 Poultry Workshop at the Plant & Animal Genome Conference, San Diego CA, and attendance overview:

• Attendance during the 1.5 day workshop averaged n=45 with peak attendance in excess of 90.
• Representatives of 16 agricultural experiment stations attended from across the US including the membership of NRSP-8 Poultry group: Iowa State, Michigan State, University of Arizona, University of Arkansas, Western University of Health Sciences, Mississippi State University, Univ of Delaware, Univ of Georgia, University of California Davis, University of Minnesota, Beckman Research Institute.
• Attendees also included members of the poultry layer and broiler breeding companies, and scientists from the United Kingdom, Germany, Canada, Sweden, Netherlands, Bangladesh, Australia and China.

Poultry Coordination funds partially supported a total 11 scientists attending the workshop. The 2020 poultry Jorgensen Travel award winner Nnamdi Ekesi was then introduced and each gave a lightning talk on their area of research. Finally, we had 4 junior scientists given 2 minutes lightning talks about their research.

Grants

University of Arkansas:

• Empowering US broiler production for transformation and sustainability; USDA-NIFA Sustainable Agriculture Systems; 9/2019 - 8/2024; $9,919,300; PD Bottje CoPIs Dridi, Rochell, Hargis, Erf, Kong, Kidd, Kuenzel, Owens, Kwon, Sun, Rhoads, Alrubaye, Tabler (MS), and others.
• Global expression pathway analysis training: target obesity. Chancellor’s Discovery, Creativity, Innovation, and Collaboration Fund. 9/2017-7/2019; $76,500; PI: W. Bottje CoPI: D. Rhoads, B. Kong.

Douglas Rhoads, Univ of Arkansas:

• Validation of a SNP panel for breeding against ascites in broilers. NIFA-AFRI; 3/2018-2/2021; $500,000; PI: Rhoads
• Evaluation of Zinpro micronutrients for protection against BCO lameness and improving bone health for broilers raised on litter flooring with bacterial challenge. Zinpro; 2/2018-12/2018; $47,797; PI: Rhoads
• Whole Genome Resequencing to Identify Genetic Determinants of Resistance to Bacterial Chondronecrosis with Osteomyelitis Leading to Lameness. Cobb-Vantress, Inc; 9/2018-8/2019; $55,846; PI: Rhoads

Wayne Kuenzel, Univ of Arkansas:

• Antistress compounds as effective tools for addressing chronic stress. Arkansas Biosciences Institute. 7/2018 to 6/2019; $50,000; PI: W. Kuenzel, Co-PIs: S. Krishnaswamy, S.W. Kang.

Byung-Whi Kong, Univ of Arkansas:

• Gene editing and transgenic poultry production. Chancellor’s Discovery, Creativity, Innovation, and Collaboration Fund. 7/01/2019-6/30/2021; $115,665; PI: B. Kong CoPI: W. Kuenzel.
• Determination of roles of mitochondrial small RNAs in metabolic disease phenotypes using isocitrate dehydrogenase 2 (IDH2) knock out mouse and genetically selected chicken models. Arkansas Bioscience Institute. 7/2017-6/2020; $148,500; PI: B. Kong.

Huaijun Zhou, University of California, Davis:

• Genomics for improving animal production; USDA NIFA National Need Training Grant 2014-38420-21796; H. Zhou, J. Murray, P. Ross;$238,000.
• Genomic Editing for Enhanced Animal Production; USDA NIFA National Need Training Grant; P. Ross, H. Zhou., J. Murray; $238,000.
• Improving food security in Africa by enhancing resistance to disease and heat in chickens; Feed the future innovation lab for genomics to improve poultry; USAID AID-OAA-A-13-00080; H. Zhou, S. J. Lamont, J. Dekkers etc; $5,000,000.
• Functional Annotation of the Swine Genome;2018-67015-27501;C. Tuggle (PI), H. Zhou, et al; 1/15/2018-1/14/2022; $2,500,000.
• High throughput characterization of gene transcript variants by full-length single-molecule sequencing to improve farm animal genome annotation; P. Ross(PI), H. Zhou, J. Mendaro; 1/1/2017-12/31/2019; $460,000.
• Genome wide identification and annotation of functional regulatory regions in livestock species; 2015-67015-22940;H. Zhou (PI), P. Ross, I. Korf; 1/1/2015 – 12/31/2019; $500,000.

Susan Lamont, Iowa State University:

• US-UK Collaborative Research: Host Resistance to Avian Pathogenic E. coli. USDA-NIFA-AFRI/BBSRC; $499,999.
• Industry funding: Aviagen Limited, EW Group, Hy-Line, International.

Jack Dekkers, Iowa State University:

• Industry Funding: Iowa Egg Industry Center

Marcia Miller; Beckman Research Institute, CA:

• MHC-Y-Directed Immune Responses during colonization of Chickens by Campylobacter; USDA NIFA; Grant No. 2016-10247; 06/01/2017-05/31/2020; $387,518.00.

Kent Reed, Univ of Minnesota:

• USDA National needs fellowship for enhancing animal production: Addressing national need in poultry production; USDA-NIFA-NNF; 2016-2021; $241,000; PI: Reed.

Hans. H. Cheng, USDA-ARS:

• ARS CRIS Project, Employing Genomics, Epigenetics, and Immunogenetics to Control Diseases Induced by Avian Tumor Viruses.
• ARS CRIS Project, Genetic and Biological Determinants of Avian Herpesviruses Pathogenicity, Transmission, and Evolution to Inform the Development of Effective Control Strategies.
• USDA, AFRI, award no. 2017-05741, Genomic screens to identify regulatory elements with causative polymorphisms accounting for Marek’s disease genetic resistance in chicken. PI, Cheng; co-PIs, Erez Lieberman Aiden (Baylor) and Bill Muir (Geneysis Bioinformatic Services). $498,116.

Impacts

• Our members are highly focused on fundamental, translational and applied research to benefit U.S. Agriculture and through genomics improve poultry health and contribute to the productivity of the relevant industries. Below are listed some of the highlights from 2018-19 research. Many of the efforts are focused on projects that directly impact poultry health and production.
• We have been using the existing assemblies for high resolution (10 kb regions) QTL mapping through whole genome resequencing. We have mapped 42 regions for ascites and 11 regions for bacterial resistance.  We are working with primary breeding companies to validate these regions and to expand the analysis to commercial products.  Validation of these regions for commercial breeding stocks will allow significant improvements in resistance to two diseases that cost the industry >$100 million annually.
• Additional data have been published to support our proposed suggestion that the nucleus of the hippocampal commissure (NHpC) be added to the classical hypothalamo-pituitary-adrenal (HPA) axis in avian species due to its early activation of corticotropin releasing hormone gene expression within that structure following an imposed stressor.
• Providing new molecules and additional key mechanisms into the cellular pathways for muscle growth and muscle mass development in breast muscle of broilers will improve production efficiency and hopefully prevent metabolic myopathy such as ‘woody breast’.
• Identification of genes that are associated with resistance to heat stress and Newcastle disease virus and can be used to genetic enhancement of disease resistance of chicken in adaption to hot climate.
• Knowledge of genes associated with enhanced immune response may inform further information on vaccine efficacy in poultry production.
• ChIP-seq and ATAC-seq assays developed and other omic data generated for regulatory elements annotation will be important for animal genome community.
• Genetic variation was characterized in commercial, research and indigenous lines of chickens.
• Genes, pathways and genomic regions associated with important biological traits in chickens were identified.
• The feasibility of applying molecular genetics and genomics to analysis of variation in structure, function and gene expression within the chicken genome was demonstrated.
• The improved typing method makes it feasible to expand efforts to understand the impact of MHC-Y genetic variability on immunity and disease resistance in chickens.
• Evidence continues to accumulate supporting the likelihood that MHC-Y contributes to the genetics of immune responses in chickens.
• Identification of molecular mechanisms associated with altered muscle development will result in development of mitigation strategies based on improved genetic selection, nutritional intervention, and other strategies to improve poultry muscle food quality and quantity.
• AFB1 causes annual industry losses estimated in excess of $500 M. Increasing innate resistance to AFB1 could result in numerous health benefits. Transformational improvements in AFB1 resistance require a multidisciplinary approach to identify protective alleles with potential to reduce disease.
• Genetic markers to improve AFB1-resistance have a potentially high commercial value and positive economic impact to industry, owing to improvements in health and well-being, productivity, and a safer product for consumers.
• The gastrointestinal health of an animal is key to its successful growth and development. Elimination of sub therapeutic antibiotics for growth promotion and health in poultry will leave a critical void. This project will improve our mechanistic understanding of host-microbiome interactions in the avian host, and identify feasible approaches towards modulating the turkey intestinal microbiome resulting in enhanced health and performance.
• Determining the purity of tumor samples has aided our efforts to identify Ikaros and other candidates as the first driver genes for MD. This supports our hypothesis that somatic mutations are required in addition to MDV infection to get tumors in susceptible birds.
• The TCR genes and usage play a role in response to MDV infection. As the TCR interactions with the MHC, this makes sense as the MHC has a major influence on MD genetic resistance.
• Advancement in understanding the underlying genetic and epigenetic factors that modulate vaccine efficacy would greatly improve the development of strategy in design of new vaccines, and therefore better control of the disease. The findings that MD vaccines-induced differentially expressed microRNAs in primary lymphoid organ, bursae, suggest the epigenetic factors are highly likely involved in modulating vaccine protective efficacy in chicken.
• Functionally annotating the chicken genome will benefit research in agricultural animals. Epigenetic modifications such as histone tail modifications and DNA methylation are not only key to the regulation of unique transcriptome patterns, these modifications are indispensable as genome annotators to uncover cell- and tissue-specific regulatory elements. Uncovering the location of regulatory elements and determining their interactions will provide the necessary framework to understand how regulatory networks govern gene expression and how genetic and environmental influences alter these networks to impact animal growth, health and disease susceptibility or resistance.
• HPIDB (https://hpidb.igbb.msstate.edu/) allows agricultural researchers to predict host-pathogen protein-protein interaction (HPI) data for proteins of their interest, where none exist in the literature. This in turn allows researchers to interrogate high through put datasets (RNA-Seq and proteomics) in the context of HPI to better understand infectious diseases of relevance to US agriculture.
• We provided information about poultry gene functions (via the AgBase database) and developed and deployed analysis tools on CyVerse. This includes the development of iMicrobe (http://openwetware.org/wiki/Imicrobe), a resource that makes large scale microbial data accessible and queryable for all researchers, and Chickspress, a chicken gene expression resource (http://geneatlas.arl.arizona.edu). Another resource is VERVE Net (http://vervenet.us), a virus ecology research and virtual exchange network.
• We improved standardized naming across chicken genes, enabling researchers to consistently and unambiguously report their scientific findings. These efforts enable researchers to have equal access to tools that help them analyze their genomics data sets, and data to support these anlayses. Analyzing and understanding genomics data sets in turn ensures that poultry researchers can translate their finding into gains for the industry.

NRSP-8 Equine 2019 Annual Report (Coordinator and Workshop)

Leadership:

Coordinators:

Ernest Bailey, University of Kentucky

Samantha Brooks, University of Florida

Molly McCue, University of Minnesota

NRSP8 Workshop:

Chair:   Annette McCoy, University of Illinois

Co-chair: Mike Mienaltowski, University of California, Davis

2019 Equine Workshop Report

The workshop met Saturday afternoon (Jan 11, 2020) and Sunday morning (Jan 12, 2020) at the Plant and Animal Genome Conference in San Diego, CA.

Attendees:

January 12: 80

January 13: 55

Station Reports were provided by scientists from 20 laboratories including those at Cornell University, University of Florida, University of Kentucky, University of Louisville, University of Minnesota, Michigan State University, Illinois State University, University of Nebraska, Texas A&M University, University of California-Davis, Argentina, Brazil, Sweden, Denmark, South Korea and China.

PAG 2019 Workshop Presentations

Invited Speaker: Dr. Marcio Costa, from the University of Montreal, who presented his perspective on what is known about the equine intestinal microbiota.

There were eleven abstract presentations, including two sharing results from the Equine FAANG initiative. Brief station reports were provided by 15 lab groups in the U.S. and three international lab groups during which they shared their ongoing research efforts and made requests for future collaborations and students/post-docs. There was also an update for the upcoming Havemeyer equine genetics/genomics workshop to be hosted by Cornell University July 26-29, 2020.

A portion of the Saturday workshop was dedicated to a community discussion, as part of the larger animal genomics community discussion regarding the future of NRSP8. There were four questions posed for discussion:

1. What are the next steps we are taking as a community in terms of discovery science? What will we be investigating over the next 5-10 years? What tools do we need to build?
2. What would help us the most? Where do investments need to be made that will do the most good in driving discovery forward?
3. How will we get our results out to the industry? Who are the stakeholders that we need to engage as our champions?
4. What are our options for community funding moving forward once NRSP8 ends?

A white paper will be generated summarizing the results of this discussion. Briefly, the following key points were brought forth:

• We are poised to make the jump from genome to phenotype, and from there to clinical management including the development of informative biomarkers. Phenotype can be defined at many levels (cell, tissue, organism, herd). This work will allow us to work towards the goal of personalized medicine for horses, which is something that owners and breeders are looking for.
• We need to consider moving away from a single reference genome. Can we move to data-driven rather than reference driven discovery? This involves taking the machinery for making genomes and turning it to annotating genomes.
• Tools that need to be developed include shareable repositories for data of all types (SNP, long-read data, functional annotation etc.) and a shared biobank.
• We need to invest in translating our discoveries to our stakeholders (veterinarians and owners/breeders) so that they will provide support for infrastructure development/maintenance and further discovery research. We should leverage existing extension frameworks through the agricultural experiment station directors to accomplish this, as well as use industry media contacts. We need to effectively communicate what tools are available, how to use them, and what the underlying science means. This communication will also allow us to get feedback about what is important to our stakeholders.
• The horse is well-poised to be used as a model for sports medicine/performance-related traits and diseases. We can propose a comparative biology/genomics angle to funding agencies (e.g. NIH). There is also a new NSF call with a specific line for investigation of complex traits, and IOS is focusing on “reunifying biology” with an interest in cross-disciplinary collaboration.
• The best bet for the horse to be a part of a new multispecies NRSP would be if it was focused on phenotype prediction (and development of bioinformatics tools to facilitate this).
• There may be an opportunity to tap industry for money (pharma, feed/supplement companies, breed organizations), but we need to include them in our meetings so we know where their interests lie.

At the conclusion of the workshop, Felipe Avila of the University of California, Davis was elected co-chair for the 2020 workshop (meeting at PAG in January 2021).  Mike Mienaltowski will assume major responsibilities as chair of the workshop meeting.

Travel support: The Jorgenson Travel Award was won by Sian Durward-Akhurset of the University of Minnesota for the presentation entitled, “The frequency of loss of function alleles in the equine population”.   Additional Travel Awards were also made to 24 other students using the NRSP8 Coordinator Funds. 

Progress on the Workshop Objectives:

1: Advance the status of reference genomes for all species, including basic annotation of worldwide genetic variation, by broad sequencing among different lines and breeds of animals.

The new assembly of the horse genome, Ecab 3.0, was published in 2018 and made available on NCBI and ENSEMBL genome browsers.  The assembly has been used extensively in research during the current year with exceptions and problems being reported to the team that organized the new assembly. The assembly was based on the existing Sanger sequence data along with Illumina HiSeq short reads, CHiCago and Hi-C long-insert libraries, Gap-filling with PacBio and a 10x Chromium library to identify and phase variants. The final assembly has 4.5Mb contig N50, 85Mb scaffold N50, and 70Mb more sequence assigned to chromosomes.

2: Develop strategies to identify and exploit genes and allelic variation that contribute to economically relevant phenotypes and traits, in part through improving functional annotation of the genomes of our species.

For the Functional Annotation of Animal Genomes (FAANG) initiative, sampling and preservation of 86 tissues, 2 cell lines, and 5 fluids from two Thoroughbred mares was completed in 2016 (Burns, et. al 2018) and data continues to be added to the community databases.  During 2019, work began to add two stallions to this project and tissues will be available for testing during 2020.   This biobank has been instrumental in the development of epigenetic assays and data collection for the horse, including RNA-seq, ChIP-seq, and CTCF-binding assays. This sequencing was completed in 2018 and uploaded to EMBL-ENA (https://www.ebi.ac.uk/ena/data/view/PRJEB26698).

Because of the “adopt-a-tissue” effort, we have also identified a set of tissues for which functional annotation will have the greatest impact on immediate research endeavors being conducted by members of the community. Additionally, USDA Species Coordinator Funds were appropriated for ChIP-seq analysis of additional tissues.

3: Facilitate analysis, curation, storage, distribution and application of the enormous datasets now being generated by next-generation sequencing and related "omics" technologies with regard to animal species of agricultural interest.

Datasets from whole-genome sequencing of the two mares (https://www.ebi.ac.uk/ena/data/view/PRJEB26698), mRNA-seq (https://www.ebi.ac.uk/ena/data/view/ERA1487553) and smRNA-seq (in submission) across 47 tissues from the two mares and reduced read bisulfate sequencing (RRBS) across 8 tissues (in submission) on the two mares continue to be publicly available at EMBL-ENA.

Communication:  The coordinators maintain an email list and use it to broadcast information for USDA-NRSP8, the USDA, the Havemeyer Foundation and other information relevant to the workshop.  In addition to the PAG conference, workshops are held once every two years at a Dorothy Russell Havemeyer Workshop and at a conference of the International Society for Animal Genetics.  Many of the NRSP8 members also participant in the biennial Equine Science Society Conferences.

International Society for Animal Genetics (ISAG) Conference 2019: A workshop meeting for the horse genome project was held at the ISAG meeting in Lleida, Spain, July 7-12, 2019.  More information about that meeting can be found at the conference website: https://www.isag.us/2019/

Website:  A new website for the International Horse Genome program was set up including reports from the different meetings, identification of participants and tools.  The website can be found at:  https://horsegenomeworkshop.com/

September 2020 Havemeyer International Equine Genome Workshop

A workshop meeting will be held in connection with this program. Details can be found at the following website:   https://havemeyergenome2020.com/

Funding in 2019 Reported by 9 US Stations and 7 International Stations

Internal                      Industry                     Federal

$311,400                    

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