Extensive reviews on identified candidate genes associated with different growth and reproduction traits in various sheep breeds published by [7] Xu & Li (2017) and [8] Gebreselassie et al. (2020) confirmed that these genes are distributed throughout the sheep genome. There was limited agreement between the previously reported studies in either the identified genes or the broader genomic regions where the genes were located. This could be ascribed to various factors, including breed differences, long-term selection practices followed in the breeds or flocks, sample sizes, models of analyses applied, levels of significance for identification of significant and suggestive SNPs and distance of significant or suggestive SNPs from the associated gene. For example, [9] Al-mamun et al. (2015) identified 39 SNPs associated with body weight in Australian Merino sheep, with a region on OAR6 containing 13 significant SNPs. Several SNPs related to growth and carcass traits in Scottish Blackface lambs were also reported on OAR6 ([10] Matika et al., 2016). However, no significant or suggestive SNPs were found for body weight or growth traits on OAR6 by [11] Zhang et al. (2013) or [12] Almasi et al. (2020), nor in the populations in the current study.
Many of the genes associated with reproduction in the current study were previously associated in literature with reproductive related traits, as well as with growth related traits in different farm animal species. Such genes associated with reproductive traits in the current study are MAP7D1, TRAPPC3, THRAP3, SPP2 and HDAC9 in the Cradock Merino population, ZFHX3 and PIK3C2A in the Grootfontein Merino population and SIX6 in the Afrino population.
Previous literature linked many of the genes associated with body weight in the current study to reproduction traits in farm animal species. Some of these genes were associated with both growth and reproductive related traits in literature. In the case of the Afrino population, all the genes associated with body weight in the current study (except for LDHD), were previously related to reproductive traits (RBP1, RBP2, PKP2, STK38L, IL1RN, ZNRF1, ZFP1, BSPH1, LIG1, CABP5, ELSPBP1). The genes TRPS1, ENKUR, EYA2 and RBMS3 in the Grootfontein Merino population and ZNF496 in the Cradock Merino population were also previously associated with reproductive traits.
Long-term selection in the flocks for both body weight and reproductive traits, especially the composite trait TWW, could have favoured genes with pleiotropic effects influencing both these traits, with the result that SNPs associated with such pleiotropic genes would be detected in the GWAS for the different traits. Furthermore, genes associated with BW and TWW in the Afrino and Grootfontein Merino populations featured in the same GO biological processes identified for these two populations. This confirms that the same underlying physiological processes are involved in these traits.
Previously published literature linked genes associated with either reproduction or body weight in this study to various reproductive processes from oocytogenesis through conception, implantation and pregnancy to milk production. Genes differentially expressed in or associated with bovine follicles or oocytes were MAP7D1 and ZNF496 [13] (Dickinson, 2016), TRAPPC3 [14] (Donnison & Pfeffer, 2004), RBP1 [15] (Mamo et al., 2011), PKP2 [16] (Franchi et al., 2016) and STK38L, ZFHX3, ZNRF1 [17] (Hatzirodos et al., 2014). In sheep, LIG1 and SPP2 [18] (Smith et al., 2019) were differentially expressed between fetal ovaries of fetuses whose dams were exposed to either maintenance or restricted nutrition. GRIK3, EYA2 and BSPH1 were part of a group of genes that were differentially expressed between a subset of Finnsheep and F1 crossbred ewes maintained on a flushing diet [19] (Pokharel et al., 2018). EYA2 was differentially expressed between uniparous and multiparous goat ovaries [20] (Ling et al., 2015). MAP7D1 and TRAPPC3 were associated with NLB and NLW in the Cradock Merino population, while SPP2 was also associated with NLB, supporting the above findings. In the Grootfontein Merino population, the ZFHX3 gene was linked to TWW. The gene SIX6 has been noted as a regulator of gonadotropin releasing hormone (GnRH) in cattle [21] (Cánovas et al., 2014) and sheep [22] (Mellon et al., 2018). In the Afrino population, SIX6 was linked to all three reproductive traits, NLB, NLW and TWW. According to [23] Grive et al. (2014) TAF4B is expressed in both somatic and germ cells in the ovary, and possibly play multiple roles in primordial follicle formation. It is also required for the initial establishment of the primordial follicle reserve at birth. Furthermore, infertility in TAF4B null female mice was associated with defects in early follicle formation and oocyte maturation [24] (Falender et al., 2005a). One of the gene ontology biological processes of TAF4B is “Positive regulation of stem cell proliferation” [25] (https://david.ncifcrf.gov/), which concurs with the associations found in literature. This gene was associated with TWW in the Afrino population in the current study.
The current study only identified one gene that was associated with conception rate in previous literature. PKP2 was one of the genes identified in a genome-wide association study that modulate conception rates in cattle [26] (Sugimoto et al., 2013). A range of genes have been identified that is associated with embryonic development in cattle (PKP2 and IER5L – [27] Killeen et al., 2014), sheep (EYA2 – [28] Ahbara et al., 2019) and mice (CUL7 – [29] Skaar et al., 2005; EYA2 – [30] Grifone et al., 2007; PTK7 – [31] Yen et al., 2009). In the current study, IER5L was linked to NLB in the Cradock Merino population, while PTK7 and CUL7 was associated with TWW in the Afrino population.
Several of the identified genes in the current study were previously associated with the uterus and placenta, as well as maintenance of pregnancy. CABP5 (sheep – [32] Burns et al., 2018), CAP1 (cattle – [33] Fortes et al., 2018), RBP1 and RPB2 (pig – [34] Ma et al., 2018) were associated with the uterus, while CUL7 was involved in mice placental development [35] (Tsunematsu et al., 2006). According to [25] DAVID (https://david.ncifcrf.gov/), one of the GO biological processes of CUL7 is “Placental development”. CABP5 and ELSPBP1 were part of a list of genes in genomic windows that explained more than 1% of the additive genetic variance for early pregnancy in Nelore heifers [36] (Oliveira Júnior et al., 2017). The IL1RN gene was involved in maternal recognition of pregnancy in cattle [37] (Mamo et al., 2012) and early pregnancy in sheep [38] (Pokharel et al., 2020). PKP2 was upregulated in the endometrium during the preimplantation stage of pregnancy in Finnsheep [38] (Pokharel et al., 2020) and, together with STK38L, was expressed in the endometrium of pigs at day 12 of pregnancy [39] (Kim et al., 2012).
Genes previously associated with milk production in cattle were ITFG1 [40] (Mei et al., 2018), ZNF496 [41] (Golik et al., 2011) and TRPS1 [42] (Do et al., 2017), while ZNRF1 was identified as a candidate gene for milk production in Valle del Belice dairy sheep [43] (Sutera, 2018).
Some of the identified genes in this study were also previously linked to spermatogenesis and male reproductive efficiency. TAF4B is important in spermatogonial stem cell maintenance, with high expression in spermatogonia, and some expression in spermatids [44, 45] (Falender et al., 2005b; Cooke et al., 2006). In mice lacking C14orf39, a complete arrest of spermatogenesis, as well as reduced testis size were observed [46] (Gómez-H et al., 2016). In the Afrino population, C14orf39 was associated with all three reproductive traits, NLB, NLW and TWW. RBMS3 was identified as one of the putative genes related to sperm fertility in Assaf sheep rams [47] (Serrano et al., 2019), while ELSPBP1 was differentially expressed in rams with high and low sperm motility [48] (Zhu et al., 2020). ELSPBP1 was associated with dead spermatozoa in cattle [49] (D’Amours et al., 2012) and consequently associated in various studies with spermatozoa in sub-fertile bulls [50, 51] (D’Amours et al., 2010; Kumar et al., 2016). The gene BSPH1 was indicated as a marker of sperm fertility in mice [52] (Heidari-Vala et al., 2020) and linked to sperm capacitation [53, 54] (Plante & Manjunath, 2015; Vala et al., 2018). Apart from being classified under the GO “Development processes” in the Afrino gene DAVID analysis, “Sperm capacitation” is another GO biological process of the BSPH1 gene [25] (https://david.ncifcrf.gov/). The HDAC9 gene was downregulated in 90-day old male goat kids and this was conducive to the vigorous development of spermatogenesis in this period [55] (Bo et al., 2020). TRPM8 is a testosterone receptor [56, 57] (Asuthkar et al., 2015a; Asuthkar et al., 2015b) and TRPM8 channels may be implicated in various physiological processes regulated by androgens [58] (Sutton et al., 2018). TRPM8 was also part of the genes classified under the molecular function “Channel activity” in the Cradock Merino DAVID gene analysis [25] (https://david.ncifcrf.gov/).
Several genes identified in the current study were previously linked with body weight and various growth related traits in sheep, cattle and pigs. The HDAC9 and EYA2 genes were reported to be involved with myogenesis in these species. HDAC9 was associated with myogenesis and muscle development in sheep [59] (Cheng et al., 2020), cattle [60] (De Vos, 2018) and pigs [61] (Zhang et al., 2014). The EYA2 gene was linked to muscle differentiation and development in cattle [62, 63] (Heanue et al., 1999; Hudson et al., 2013), goats [64] (Ling et al., 2019) and pigs [65] (Pérez-Montarelo et al., 2012). EYA2 was also associated with BW in the Grootfontein Merino population. Downregulated expression of the HDAC9 gene has been observed in callipyge animals relative to non-callipyge sheep [66] (Vuocolo et al., 2007). Furthermore, EYA2 was reported to be associated with muscle hypertrophy in goats [64] (Ling et al., 2019).
Some genes were specifically associated with muscle or fat in the live animal. For example, MAP7D1 (cattle – [67] Sweeney et al., 2016), SPP2 (sheep – [68, 69] Trukhachev et al., 2016a; Trukhachev et al., 2016b) and LIG1 (pigs – [70] Da Costa et al., 2004) were associated with or expressed in muscle, while TRAPPC3 (sheep – [71] González-Calvo et al., 2017), THRAP3 (cattle – [72] Perez et al., 2010), PIK3C2A (pigs – [73] Kim et al., 2015) and STK38L (cattle – [74] Lim et al., 2013) were associated with intramuscular or subcutaneous fat. From the genes mentioned here, LIG1 and STK38L were associated with BW in the Afrino population, while XIRP2 was linked to BW and TWW in the Grootfontein Merino population.
Genes identified in the literature to be generally associated with growth are ZFHX3 (cattle – [75] Xu et al., 2017; goats – [76, 77] Zhang et al., 2015; Wei et al., 2018), SIX6 (cattle – [78] Huai et al., 2011; goats – [79] Pan et al., 2011), EYA2 (cattle – [80] Somavilla, 2015) and RBMS3 (cattle – [81] Widmann et al., 2013). Genes associated with body weight at specific ages were the TRPS1 gene which was linked to fetal [82] (Xu et al., 2014) and post weaning weight in sheep [11] (Zhang et al., 2013) and the HDAC9 and LIG1 genes which were associated with birth weight in pigs [61] (Zhang et al., 2014) and cattle [83] (Cole et al., 2014) respectively. Most of these genes were associated with BW in the Afrino or Grootfontein Merino populations. SPP2 was differentially expressed in the loin muscle of Merino sheep with high and low body weight [68, 69] (Trukhachev et al., 2016a; Trukhachev et al., 2016b), while PIK3C2A was associated with body weight in pigs [84] (Bovo et al., 2020). IL1RN was associated with carcass weight in cattle [85] (Daetwyler et al., 2012). Two of the GO biological processes of IL1RN are “Lipid metabolic process” and “Response to glucocorticoid” [25]. This gene was categorised under the “Response to lipid” biological process in the Afrino DAVID gene analysis.
From the results of this study, as well as evidence from other studies performed on sheep and cattle available in literature, some suggestive SNPs and genes with pleiotropic effects were identified that warrant further investigation. The following SNPs were either linked to more than one trait or gene, or the genes associated with these SNPs were previously associated with both reproductive and body weight traits in sheep and cattle. Such a SNP in the Cradock Merino population is OAR1_10554666.1, which is associated with both NLB and NLW and the genes MAP7D1, TRAPPC3 and THRAP3. MAP7D1 is expressed in bovine oocytes [13] (Dickinson, 2016) and bovine muscle [67] (Sweeney et al., 2016), TRAPPC3 in bovine oocytes [14] (Donnison & Pfeffer, 2004) and subcutaneous fat of lambs [71] (González-Calvo et al., 2017), while THRAP3 is involved in the fat profile of bovine muscle [72] (Perez et al., 2010). SNP s20120.1 on OAR1 is associated with NLB and the genes TRMP8, a testosterone receptor [58] (Sutton et al., 2018), and SPP2, previously linked with body weight and expressed in ovine muscle [68, 69] (Trukhachev et al., 2016a; Trukhachev et al., 2016b) and sheep fetal ovaries [18] (Smith et al., 2019). The HDAC9 gene (SNP OAR4_28811142.1), associated with NLB, NLW and TWW in the Cradock Merino population, was previously linked to spermatogenesis in goats [55] (Bo et al., 2020). This gene is involved in myogenesis and muscle development in sheep, cattle and pigs, was observed in callipyge sheep [66] (Vuocolo et al., 2007) and was associated with birth weight in pigs [61] (Zhang et al., 2014).
ZFHX3 linked to OAR14_39202046.1 and associated with TWW, was expressed in bovine follicles [17] (Hatzirodos et al., 2014) and previously linked to growth in cattle [75] (Xu et al., 2017) and goats [77] (Wei et al., 2018). SNP OAR7_76295917.1 in the Afrino population was associated with NLB, NLW and TWW, as well as with two genes, SIX6 and C14orf39. SIX6 is a regulator of GnRH in cattle and sheep [21, 22] (Cánovas et al., 2014; Mellon et al., 2018) and involved in puberty [86] (Fortes et al., 2016) and growth [78] (Huai et al., 2011) in cattle. C14orf39 was previously linked to spermatogenesis [46] (Gómez-H et al., 2016). The TAF4B (SNP OAR23_32551191.1) gene warrants further investigation due to its association with TWW in the Afrino population and evidence from literature linking it to primordial follicle formation [23] (Grive et al., 2014) and sperm stem cell maintenance [45] (Cooke et al., 2006).
SNPs associated with body weight in the current study are OAR9_64654880.1, OAR13_80729511.1 and OAR19_4811675.1 in the Grootfontein Merino population and OAR3_201667351.1 and OAR14_56900862.1 in the Afrino population. The gene TRSP1 (SNP OAR9_64654880.1) was previously associated with milk production in cattle [42] (Do et al., 2017) and fetal and post weaning weight in sheep [11, 82] (Zhang et al., 2013; Xu et al., 2014). SNP OAR13_80729511.1 might be an important marker, due to its associated gene, EYA2 being involved with various reproductive and growth traits. EYA2 is expressed in the ovaries of sheep [19] (Pokharel et al., 2018) and goats [20] (Ling et al., 2015) and involved in embryonic development in sheep [28] (Ahbara et al., 2019). It is also linked to myogenesis [62] (Heanue et al., 1999) and growth [80] (Somavilla, 2015) in cattle. Another gene associated with body weight in the Grootfontein Merino population, RBMS3 (SNP OAR19_4811675.1), was also previously linked to growth in cattle [81] (Widmann et al., 2013) and to sperm fertility in sheep [47] (Serrano et al., 2019). STL38L (SNP OAR3_201667351.1) in the Afrino population was associated with marbling score in cattle [74] (Lim et al., 2013) and expressed in bovine follicles [17] (Hatzirodos et al., 2014) and the porcine endometrium [39] (Kim et al., 2012). The most important SNP associated with body weight in the Afrino population was OAR14_56900862.1, linked to four genes (BSPH1, LIG1, CABP5, ELSPBP1). These genes were associated with sperm fertility in sheep and cattle and pregnancy in sheep and cattle, expressed in sheep ovaries and muscle and involved in birth weight of pigs.