We performed genome-wide association studies to associate QTL with udder and teat conformation traits, with special focus on previously identified mastitis-risk traits of fore udder attachment, rear teat end shape, rear teat width, and udder height (15). These studies provide insight into the genetic regulation of teat and udder confirmation and mastitis susceptibility as well as novel traits and specific markers that may be used in genetic selection. A comprehensive list of candidate genes may be found in Supplementary Table 1; here we discuss those most biologically relevant to mastitis risk associated traits.
Our goal in performing PCA on these risk traits was to identify a single measure which may describe mastitis risk based on udder and teat conformation. Risk PC1 described this risk (excepting rear teat end shape), and GWA for this new composite measure significantly associated novel QTL (15:7287030-7311314) not identified by individual assessment of those risk traits (Figure 1). Candidate genes identified in this region were related to both cell division (Centrosomal Protein 126 (CEP126)) and immune cell progenitor differentiation (Angiopoietin Like 5 (ANGPTL5)), suggesting that Risk PC1 does indeed reflect both mastitis and udder and teat morphology (16, 17).
In this study cohort, we previously associated loose fore udder attachment with high odds of elevated milk somatic cell count and clinical mastitis diagnosis, making fore udder attachment among these cows a relevant criterion on which to base culling and management decisions for mastitis control (Miles et al., 2019). A case-control GWA for extremes in fore udder attachment (loose versus tight) identified a number of genes related to both immune function and cell proliferation near associated markers at 2:126359098-126364670 (Figure 2). Wiskott-Aldrich Syndrome Protein Family 2 (WASF2) is a cytoplasmic protein implicated in cell migration, phagocytosis, and immune synapse formation (18). Similarly, Nuclear Distribution C (NUDC) is critical to cytokinesis, Stratifin (SFN) may regulate cell cycle progression, and Keratinocyte Differentiation Factor 1 (KDF1) serves as an essential regulator of epidermis formation (19-21). In contrast, this same genomic region houses genes related to immune function, including Ficolin 3 (FCN3) which is an essential component of the lectin complement pathway, Ribosomal Protein S6 Kinase A1 (RPS6KA1) which has been implicated in activated Toll Like Receptor 4 signaling, High Mobility Group Nucleosomal Binding Domain 2 (HMGN2) which is known to have antimicrobial activity against bacteria, viruses, and fungi, as well as Zinc Finger 683 (ZNF683), a tissue-resident T-cell transcription regulator (22-25). The variety in function among candidate genes associated with fore udder attachment, relating to both the physical trait and immune function, reinforce the role of this udder type trait as a major risk factor for mastitis.
Similarly, we previously associated rear teat end shape with increased odds of both elevated somatic cell count and clinical mastitis in this cohort of cows, and in this study investigated the potential genetic regulation of this trait (Miles et al., 2019). Upon examining rear teat end shape (Figure 5), a similar candidate gene pattern emerged with gene functions related to both cell division (explaining teat morphology variation) and immune response regulation (reinforcing teat end shape as an appropriate indicator of mastitis risk). Significantly associated SNP BovineHD2600014687 positioned at 26:50630351 resides within Kinase Non-Catalytic C-Lobe Domain Containing 1 (KNDC1), which has been implicated in the regulation of cellular senescence and cell cycle progression (26). In addition, Adhesion G Protein-Coupled Receptor A1 (ADGRA1) was identified within a 1 Mb window of this SNP and belongs to a family of receptors known to regulate immune signaling (27). A pattern of candidate gene function emerged like that of fore udder attachment, reinforcing that it may be appropriate to select for mastitis-resilient cows based on udder and teat conformational traits.
In this cohort of cows, we previously associated increasing rear teat width with increased odds of clinical mastitis (Miles et al., 2019). We first assessed this trait quantitatively to account for continuous variation in rear teat width, and a linear GWA for rear teat width also revealed candidate genes related to both cell division and immune function at a number of different QTL spanning the genome (Figure 6). Near a significantly associated SNP at BTA 11:104129366, we identified Caspase Recruitment Domain-Containing Protein 9 (CARD9), a key modulator of immune response related to TLR and NOD2 signaling pathways as well as NF-kB activation (28, 29). Furthermore, genes related to cell differentiation and survival were identified including Notch Receptor 1 (NOTCH1), a highly conserved protein with an extracellular domain containing many epidermal growth factor repeats and whose signaling is heavily involved in cell fate specification, and Epidermal Growth Factor- Like 7 (EGFL7) which is involved in Notch binding (30). In addition, a Myomaker Myoblast Fusion Factor (MYMK) resides in this region and has been associated with muscle hypertrophy, making it a strong candidate for impacting variation in teat morphology (31). Genes were also investigated in various regions on BTA 16, including Centrosomal Protein 350 (CEP350) which plays a critical role in microtubule binding and spindle integrity during cell replication (32). This region is also home to Laminin Subunit Gamma 1 and 2 (LAMC1/2), which are thought to regulate cell organization into tissues, potentially contributing to variation in teat width (33). In regards to immune function, nearby genes were investigated including Major Histocompatibility Complex Class I-Related (MR1) which is critical to adaptive immune response, Ribonuclease L (RNASEL) is involved in interferon regulation, and DExH-Box Helicase 9 (DHX9), which has been found to control TLR-stimulated immune responses (34-36). A significantly associated QTL at BTA 19:29058547-29063744 is located within the Growth Arrest Specific 7 (GAS7) gene, which while previously understood to influence neuron differentiation, was recently found to be abundantly expressed in murine alveolar macrophages, though its exact roles in immune responses are still unknown (37). Furthermore, HIC ZBTB Transcriptional Repressor (HIC1) and Tyrosine 3-Monooxygenase Activation Protein (YWHAE) have both been associated with the regulation of cell proliferation, and in the case of HIC1 its function as a transcriptional regulator has been specifically tied to immune homeostasis (38, 39). A significantly associated QTL on BTA 25:40126743-40190566 resides within the Sidekick Cell Adhesion Molecule 1 (SDK1) gene, an adhesion molecule isoform in the immunoglobulin superfamily primarily known for synapse formation in the retina, though Sidekicks in general are expressed in many different tissue types (40). A significantly associated SNP at 25:35208040 was interrogated for candidate genes in the surrounding area, which included Cut Like Homeobox 1 (CUX1), known for its role in morphogenesis as well as regulation of antigen presenting cells (41, 42). Myosin Light Chain 10 (MYL10), implicated in immune cell transmigration, and Tripartite Motif Containing 56 (TRIM56), an ubiquitin-ligase with a role in antiviral innate immunity, were also found in this region (43, 44). A case-control GWA study was also performed to identify genes which may potentially drive extremes in morphology, and significantly associated a single SNP at BTA 25:38568564. Candidate genes near this SNP included Polypeptide N-Acetylgalactosaminyltransferase 2 (GALNT2) which is believed to be involved in O-linked glycosylation of the immunoglobulin A1 hinge region and PiggyBac Transposable Element Derived 5 (PGBD5), a transposase suspected to mediate genomic rearrangements (45, 46). The large number of QTL identified spanning the genome suggests rear teat width may be highly polygenic.
We previously associated low rear udder height with increased odds of clinical mastitis (Miles et al., 2019), and a case-control comparison of low udder height versus all other udder height types also identified several significantly associated QTL spanning the genome (Figure 4). A significantly associated SNP at 14:27024015 lay within the Aspartate Beta-Hydroxylase (ASPH) gene, known for hydroxylating epidermal growth factors and contributing to dysmorphic features (47). Furthermore, a significantly associated QTL on BTA 18 was near Troponin T1, Slow Skeletal Type (TNNT1), a sarcomere regulatory complex associated with muscle weakness, which could feasibly contribute to weak rear udder attachments, and consequently lower udder height (48). In addition, NLR Family Pyrin Domain Containing 2 (NLRP2), a member of the NLR family known to be regulators of immune responses, was in this same region (49). While we considered low rear udder height as a risk factor for clinical mastitis, the majority of candidate genes identified in this area seem more related to the physical trait itself rather than the indirect trait of “mastitis-risk”, making rear udder height a less compelling trait by which to base breeding and culling decisions for mastitis control.
We hypothesized that the genotype by environment interaction may be greater in multiparous cows who have had greater mastitis exposure and mechanical manipulation of the udder and teats via milking, and thus evaluated a primiparous-only subset of cows (n = 144) for each trait. Only linear GWAs assessing continuous variation in the trait resulted in significant associations; the lack of success with the case-control approach is likely explained by few risk phenotypes (< 20%) observed in primiparous cows. In these linear GWA, QTL were only significantly associated with two traits (front teat placement and udder depth) likely due to low power from a smaller sample size. For udder depth, different inheritance patterns and QTL were identified for each population (Figure 3), which is likely explained by farm culling resulting in different genotypic frequencies among primiparous and multiparous cows. The total cohort GWA associated a single SNP at BTA 5:113268242 located within Transcription Factor 20 (TCF20) which is primarily associated with human neurodevelopmental disorders (50). The primiparous only subset significantly associated one QTL on BTA 17. Antisense genes Nudix Hydrolase 6 (NUDT6) and Fibroblast Growth Factor 2 (FGF2) were in linkage with this QTL, and are hypothesized to regulate cell proliferation (51). Also in linkage with this QTL lay Sprouty RTK Signaling Antagonist 1 (SPRY1), which has been shown to influence mammary epithelial morphogenesis during post-natal development by negatively regulating epidermal growth factor signaling in the murine mammary gland (52). We hypothesize that this QTL was masked in our analysis of the total cohort due to the differing genotypic frequencies in multiparous versus primiparous populations, suggesting to truly elucidate the genetic mechanism underlying these morphological characteristics primiparous populations with minimal exposure to selective pressures must be evaluated.