Performing Department
Animal Sciences
Non Technical Summary
A major portion of predicted population growth will occur in developing nations where small ruminants such as goats and sheep are important livestock species for improving food security. The callipyge trait in sheep shows that increasing muscle mass by 35% without increasing feed consumption is biologically possible (Jackson et al. 1997; Freking et al. 1998b). This type of large phenotypic gain is what is needed to meet the demands of growing populations. Genetically the trait is 100% penetrant, meaning that the allele causes hypertrophy regardless of the genetic background of the local flocks. The callipyge allele has been tested in several breeds in the Middle East and Asia and increases carcass weights and yields in all breeds tested so far (Gootwine et al. 2003). The high penetrance suggests that callipyge alters fundamental rate limiting genes for muscle growth.The knowledge gained from studying the callipyge trait will be implemented through genetic improvement of meat animal species. The association of specific genes with traits such as muscle mass help to improve the selection of genetic markers from quantitative trait loci which may contain hundreds of genes. Furthermore, the functional DNA sequences for the callipyge mutation and polar overdominance are short and amenable to gene editing. It is possible to create new alleles such as a microRNA-resistant DLK1 allele that would alter the inheritance from polar overdominance to polar dominance. Polar overdominance also makes integrating the trait into a flock more difficult without DNA testing because a callipyge allele inherited from the dam prevents hypertrophy. The paternal microRNA-resistant DLK1 allele would not be negatively regulated by the maternal microRNA and homozygous animals would be expected to have muscle hypertrophy. A polar dominant trait can be easily integrated into small flocks using selection based on visible phenotypes without DNA testing.The PD has an existing collaboration with Drs. Cockett and Polejaeva at Utah State University that has been successful at moving the callipyge mutation into goats. In 2017, we have two healthy founder Spanish goat clones that are homozygous for the callipyge mutation (Yang et al. 2016). These founders do not have hypertrophy but that is expected for homozygous animals (C/C). They will be used as sires to determine if the mutation causes muscle hypertrophy in paternal heterozygous (+/CP) goats. If the callipyge mutations functions the same in goats, to would also be possible to create a microRNA resistant DLK1 allele in callipyge goats as well.This project will produce an in depth understanding of the genes that drive muscle hypertrophy in callipyge sheep. Creating the callipyge allele in goats and changing the mode of inheritance for the trait will be an important proof of principle of using gene editing to modify genetic regulation and inheritance of agriculturally important phenotype. Creating new alleles in sheep and goats based on the callipyge trait has potential to substantially increase efficient meat production in parts of the world where the population is growing.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The long term goal of this research is to increase meat production using genetic tools based on the callipyge trait. The current project will identify genetic regulators of muscle growth and test the interactions of RNA's from the DLK1-DOI3 locus needed to simplify the inheritance of callipyge to polar dominance.Determine the effect of the four callipyge genotypes on transcriptome changes in hypertrophied and non-hypertrophied muscle using RNA sequencing.Identify the miR329a target sequence in the ovine DLK1 3'UTR, and determine sequence changes that make the ovine DLK1 mRNA resistant to miR329a repression.
Project Methods
Objective 1. The muscle RNA samples and phenotypic data for this objective have already been collected and no further animal sampling will be necessary. RNA-sequencing data was obtained for semimembranosus muscle in all 4 genotypes with 5 biological replicates. Five replicates were determined to be sufficient by power analysis of previous data (Bidwell et al. 2014).The Project Director has gained experience with analysis of RNA-sequencing data and has 1 node on the Snyder Bioinformatics computing cluster for running analysis (Bidwell et al. 2014; Bi et al. 2016; Kelly et al. 2017) . Genome-guided transcriptome analysis methods using the Tuxedo suite of programs will be used for this analysis (Trapnell et al. 2010; Trapnell et al. 2012a). The basic workflow for the Tuxedo suite has been well documented (Trapnell et al. 2012b). The PD is aware of the newer suite of tools (HISAT, Stringtie and Ballgown) from the Center for Computational Biology, Johns Hopkins University (Frazee et al. 2015; Kim et al. 2015; Pertea et al. 2016). Other open source tools such as EdgeR and DEGseq (Robinson et al. 2010; Wang et al. 2010) are available through Bioconductor/R as well. These programs are favored by some as they do not use the extensive normalization employed in Cufflinks. The expression values (FPKM or read count) generated by these programs will be analyzed for transcriptional responses to the callipyge mutation under several modes of gene action. This will identify genes that respond to the paternally expressed genes or the maternally expressed ncRNA.The expression values for transcriptome features (genes, transcripts, etc.) will be analyzed using a series of three orthogonal contrasts (Freking et al. 1998a). The PD has previously used this analysis with qPCR data for genes from the DLK1-DIO3 region (Bidwell et al. 2004). The contrast values for each genotype under the specific genetic models are shown in Table 1. The analysis occurs in three stages with correction for multiple testing at each stage. The first stage determines an overall effect of genotype on the FPKM/count value for the selected feature. For features with a significant effect of genotype, the first series of orthogonal contrasts (additive,Table 1. Contrast Values by GenotypeGenetic Model+/+CM/++/CPC/CAdditive-100-1Dominance-111-1Reciprocal Het0-110Maternal Het-120-1Polar Overdom-1-13-1dominance, and reciprocal heterozygote) will identify changes in gene expression that correspond to those basic genetic models. Features that have a significant reciprocal heterozygote effect are analyzed in a second set of orthogonal contrasts (additive, maternal heterozygote, polar overdominance) to refine the heterozygous genetic model. Gene ontology (GO) analysis will provide insight into function of the differentially expressed genes relative to metabolism, cell cycle, cell signaling, and structural proteins affected by the callipyge mutation.The primary tools will be AgBase (www.agbase.msstate.edu) as a source of gene association files. AgBase also provides tools to retrieve GO terms based on genome co-ordinates generated from RNA-seq data (Genome2Seq) as well as retrieving annotation from highly similar genes in other species (GOanna). Complete gene set list and the subset of differentially expressed genes for different gene models (Gene Ids, log 2 fold change and GO terms) will be analyzed for gene set enrichment using BioConductor/R GAGE (Luo et al. 2009).The exon/intron organization of the maternal ncRNA are not well known but northern blotting indicates that there are numerous splice variants (Bidwell et al. 2001). RNA-sequencing can readily identify splice junctions, including rarely used junctions. Determining specific association of exons with RNA-seq is currently a likelihood estimate relative to the most dominant isoforms (Trapnell et al. 2010). Characterizing the maternal ncRNA will require isolation of cDNA to identify specific exon association of the splice variants. Solution hybrid selection to will be used to isolate cDNA for the maternal ncRNA. Oligo nucleotides (~100 nt) will be synthesized using exon sequences from the lncRNA with an added T7 RNA polymerase binding site. RNA will be synthesized by in vitro transcription with biotinylated rUTP to generate hybrid selection probes. After solution hybridization to single stranded cDNA, the sequences for the lncRNA cDNA will be captured by streptavidin coated paramagnetic beads. The cDNA will be converted to double stranded DNA and subjected to long read sequencing technologies.The transcriptome analysis will produce candidate genes that respond to DLK1 as well as those that are regulated by the maternal ncRNA and associated microRNA. The PD will collaborate with Dr. Shihuan Kuang to examine the function of the candidate genes. We have previously collaborated to examine the role of PARK7, that was identified in our microarray data (Fleming-Waddell et al. 2009; Yu et al. 2014). Transcription factors and components of signal transduction pathways will be priority candidate genes. Collaboration with Dr. Kuang will enable a range of transgenic and cell culture models to be used to determine the role of candidate genes in muscle growth or protein accretion.Objective 2. Long term goal is to create a new callipyge allele that will have a polar dominant inheritance pattern (see Literature Review) for muscle hypertrophy. The most direct approach to make the DLK1 transcript resistant to miR329a repression will be to alter the target sequence in the DLK1 3'UTR. The target sequence is in the untranslated region so there should be no effect on DLK1 biological activity. One could also delete a portion of the miR329a sequence but it is not known what other genes may be regulated by miR329a so this approach risks unknown secondary effects. The objectives for current proposal will use sheep cell culture models to determine the miR329a target sequences in the ovine DLK1 3'UTR, and determine a minimal sequence change needed to make the ovine DLK1 mRNA resistant to miR329a repression.Dr. Kola Ajuwon will be a collaborator on this project and the cell culture work will be conducted in his laboratory. He has an established program working with adipocytes. Dr. Ajuwon has previously isolated sheep stromovascular cells using his established methods (Qu et al. 2015). These sheep primary cells express endogenous DLK1 (Figure 1), whereas muscle satellite cells from callipyge sheep rapidly down-regulate DLK1 expression in cell culture. The down-regulation of endogenous sheep DLK1 will be determined using transfection of commercial miRNA mimics. A second microRNA (miR15) that is known to regulate DLK1 in adipocytes will be used as a positive control and a sequence scrambled miRNA mimic will be used as a negative control (Andersen et al. 2010).Experiments to test a resistant DLK1 allele will use easily transfected CHO cells in a co-transfection assay. Sheep DLK1 expression plasmids will be made with an intact target/seed sequence as control and three test constructs with alteration of 2, 4 or all 6 bases of the target/seed sequence. The cells will be transfected with the DLK1 constructs by electroporation and plated in cell culture. The cells will be subsequently transfected with the RNA mimics and controls described above in a dose titration design. In both objectives, the ability of the miRNA mimics to decrease DLK1 protein expression will be determined by plate based spectrofluorometry of fixed and stained cells as in Figure 1, as well as western blot analysis using the anti-bovine DLK1 antibody. Quantitative PCR will be used to determine the effect of the treatments on mRNA expression in the sheep adipocytes.