1a. Objectives (from AD-416):
ARS and the Cooperator (The International Water Buffalo Consortium, represented by Parco Tecnologico Padano [PTP] as coordinator and The Department of Animal Science University of Tuscia [UniTus] as administrative center) are interested in developing a first draft sequence assembly of the water buffalo genome based on the bovine species, Bubalis bubalis, which encompasses the prototypical beef and dairy animal in South Asian, Brazilian and Italian production environments. Such a resource is essential for developing informative SNP markers to intensify outputs in current production scenarios and enable selection of locally-adapted breeds in developing-world water buffalo populations. Our Project Plan has two objectives: 1) generate a 33X genome sequence coverage of DNA sequence from an Italian inbred female to use in building a genome sequence assembly and 2) generate additional sequence coverage from additional animals to identify SNP for development of a high density SNP assay that contains informative SNP for all breed types of water buffalo. The Cooperator has the funding and genomic DNA material to properly initiate this project, and the expertise and knowledge of the water buffalo production systems to extend application of the results to improve production. ARS has the expertise and facilities to generate the DNA sequence information on next-generation sequencing platforms and characterize the SNP that are potentially informative across all breeds of water buffalo.
1b. Approach (from AD-416):
The project will be managed jointly between ARS and the Cooperator. The Cooperator will evaluate the Water Buffalo herdbook in Italy to guide collection of tissues and/or DNA material from an appropriate buffalo for sequencing. The Cooperator will then provide genomic DNA templates from this animal along with funding to purchase reagents that will generate more than 33X genome equivalent coverage of DNA sequence. ARS will generate the DNA libraries, and provide next-generation DNA sequencing services to generate at least 33X genome coverage of sequence at cost with a small amount for the overhead costs of instrumentation. ARS will analyze, store, and distribute sequence information (as agreed upon with the Cooperator) that will be used for both genome assembly and SNP discovery. Identification of SNP for marker development will be carried out collaboratively by ARS and the Cooperator. Both ARS and the Cooperator will jointly aid development of the genome assembly led by outside parties that possess the necessary advanced expertise for this process. The Cooperator will provide some annotation expertise prepare the data and lead the development of the publication. All of these activities will be considered true collaborations and thus, by definition, each party will be considered to have provided a true intellectual contribution consistent with authorship.
3. Progress Report:
All ARS objectives were completed and a second-draft genome assembly of Buffalo (2.0) was produced using an improved short-read assembler (MASERCA). Sequencing the genomes (1.2 terabytes) of additional buffalos (N=68) of different breeds and aligning this sequence with the genome of the cow facilitated the identification of variations among buffalo genomes. Based on frequencies of variations within and among the breeds subsequently guided the selection of SNP to create a genotyping panel of 90,000 SNP that is being used to investigate the structure of buffalo population and to localize the genes having a major effect on phenotypic variation in the species. The 90,000 SNP genotyping panel was tested in buffalo populations from Italy and Brazil and used in a pilot association analysis to identify loci affecting milk production traits. Additionally, a draft genome assembly of the swamp buffalo was produced and is available at ftp://ftp.genome.umd.edu/pub/swamp_buffalo/1.0/. Completion of the manuscripts detailing all of these activities is in progress, and relies on non-funded partners from the University of Maryland for completion. It should be noted that some sequence production contribution and experimental planning was derived from ARS USMARC (Clay Center, NE). This research supported all three objectives of the in-house project to develop biological resources and computational tools to enhance characterization of ruminant genomes, utilize genotypic data to enhance genetic improvement of food animals across a spectrum of ruminant production systems, and characterize functional genetic variation for improved fertility and environmental sustainability of ruminants.