Kompetitive Allele Specific PCR (KASP™) genotyping of 48 polymorphisms at different caprine loci in French Alpine and Saanen goat breeds and their association with milk composition

Introduction

The goat sector within the European Union accounts for 12.3 million head (EUROSTAT, 2016), concentrated geographically in Greece, Spain, Romania, France and Italy (10.68 million altogether), with the farmers essentially focused on dairy production (milk and cheese production). According to Eurostat reports for the year 2016, Romania alone holds a flock of 1.48 million breeding goats, representing approximately 12% of the goats reared in the EU. As in other European countries, the main focus of the sector is milk and cheese production, with goat meat being considered a marginal product. Milk production traits are of fundamental importance in livestock production and are strongly related to the efficiency and economy of the farm (Erhardt et al., 2010; An et al., 2011). However, there are significant differences in the average milk production of goats between regions, mostly due to management and feeding systems and to genetic background (breed, selection). In Romania, goats are reared predominantly in extensive low-input production systems (Raducuta, Calin & Purdoiu, 2012), and the unimproved indigenous Carpatina breed represents over 90% of the population. The breed has modest production outputs, with milk yields estimated at 220 to 350 kg/lactation (Padeanu, 2001, Pascal et al., 2011).

Consequently, farmers started to import Saanen and French Alpine goats, the main specialized dairy goat breeds reared worldwide. The milk yield estimated for the two breeds ranges between 800 to 1100 kg/lactation when they are reared in intensive production systems (Palhiere et al., 2014). Both the Saanen and Alpine breeds are reared as purebreds where large commercial farms are concerned, while on small- and medium-sized farms, which practice more extensive rearing, the bucks are used for crossbreeding with the indigenous Carpatina breed in order to improve their milk yield and udder conformation.

The ongoing growth and increasing accessibility of genetic and genome sequencing data has given new perspectives for research in domestic ruminant species (Dong et al., 2013; Elsik et al., 2009). Polymorphism of milk proteins, which has been linked to the chemical composition and biological characteristics of milk, as well as the processing properties of cheese, is well studied and documented worldwide (Martin et al., 2002; Vinesh et al., 2013). Selection can be carried out to increase the frequency of alleles with a positive effect on a given production trait (Dekkers, 2004). Variations in these genes, which show strong associations with economically important production traits, such as traits related to milk production, are useful tools for marker-assisted selection (Parmentier et al., 2011). In the case of dairy goats, the effect of caseins—especially that of α_s1 casein (CSN1S1)—on milk composition (mainly protein content) has received significant attention from both geneticists and animal scientists in the last decade. Moreover, the state of knowledge on goat genetics has been significantly improved from the year 2010 onward, subsequent to the sequencing of the goat genome and the release of the goat SNP50, which allowed several genome-wide association studies (GWAS) (Martin et al., 2016). The aim of the current pilot research was to investigate the polymorphism of 48 SNPs of 40 genes on dairy production and major milk components using genotyping methods in French Alpine and Saanen goats reared in Romania. Furthermore, association study was performed between milk traits and polymorphic SNPs. The ultimate purpose was to find and describe polymorphisms that could prove useful for the design of future genetic improvement schemes in the two dairy breeds.

Materials & Methods

Results

For all investigated SNPs the KASP assays produced 2,230 identified allele calls and 122 unidentified allele calls with an allele call rate of 94,81% and a mean of unidentified allele calls of 5.19%. A total of 13 SNPs (27.08%) among the selected 48 SNPs were polymorphic across the two populations and further used for the association study (Table 2). The polymorphic SNPs were found in the following genes: CAST, CLEC4E, DES, GHRHR, HSP90AA1, IL15RA, IL1RN, IL8, MITF, PPRC1, SOCS3, TNF and TNFSF13. Although the set of SNPs was selected from dbSNP, a large number of SNPs (n = 35, 72.92%) were not polymorphic and were therefore excluded from the association study. Thirty-five SNPs were not polymorphic (Table 1).

Among the polymorphic SNPs, two were located in introns (rs660778229-HSP90AA1 and rs635583012-SOCS3), two in the 3′ UTRs (rs671256096-DES and rs664283580-MITF), and one was an upstream variant (rs669986850-CLEC4E). In addition, eight SNPs were missense substitutions (rs666806435-CAST, rs671391101-GHRHR, rs635969404-IL15RA, rs640582069-IL1RN, rs665173888-IL8, rs671509714-PPRC1, rs661165283-TNF and rs669561078-TNFSF13).

The different genotype frequencies are shown in Table 2. The results of our study outlined that homozygous genotypes were more frequent than heterozygous in most cases. Only the Alpine goats were in Hardy-Weinberg disequilibrium at the g.62894878A>G locus (rs671391101) (P < 0.05).

The studied individuals’ genotypes were analysed for association with milk yield and composition in the Saanen and Alpine breeds and in both populations together. The four SNPs—rs671391101 (GHRHR), rs640582069 (IL1RN) rs635583012 (SOCS3) and rs635969404 (IL15RA)—had significant effects on milk production, protein, fat and lactose in the Alpine breed and in both populations collectively (Table 3). No significant effects on investigated traits were found for any other polymorphic SNP (results not presented). No significant effects were recorded in the Saanen population regarding milk production, protein, fat or lactose content. Milk yield and fat percentage did not show any significant association with genotype in either the Saanen or the Alpine breed. In the case of total population, the g.46353777T>G SNP located in the IL1RN gene was associated with milk production; the g.62894878A>G SNP in the GHRHR gene, the g.10354813C>T SNP in the IL15RA gene and the g.46353777T>G SNP in the IL1RN gene were associated with fat percentage (P < 0.05); the g.46353777T>G SNP in the IL1RN gene with protein percentage (P < 0.05); and the g.52626440T>G SNP in the SOCS3 gene with lactose percentage. Out of these associations, the g.52626440T>G (rs635583012) SNP located in the intron of the SOCS3 gene and the g.46353777T>G (rs640582069) SNP located in the IL1RN gene were associated with lactose percentage; the g.62894878A>G SNP in the GHRHR gene and the g.46353777T>G SNP located in the IL1RN gene were associated with protein percentage in the Alpine population (P < 0.05). The current findings also underline the significant differences found between the Saanen and Alpine goat breeds based on all 13 markers for milk yield (P ≤ 0.05).

Discussion

The Kompetitive Allele Specific PCR genotyping assay has been successfully used in order to genotype different organisms (He, Holme & Anthony, 2014; Semagn et al., 2014; Landoulsi et al., 2017). In our work KASP technology was used in Saanen and Alpine goats reared in Romania in order to investigate the polymorphism of 48 SNPs of 40 genes that were previously reported to be associated with the production traits or that potentially contributed to important metabolic conditions. The KASP genotyping system used in the present study generated a high assay success rate (94,81%) and offered a highly locus specificity and accurate genotyping solution. Ours results are in accordance with previously reported KASP assay success rate (Semagn et al., 2014). Furthermore, according to previous studies the KASP genotyping system has low-labour and economic advantages over other genotyping assays (Landoulsi et al., 2017).

The results of this study showed that only 13 of the studied SNPs were polymorphic. The genetic variants CC of rs669986850, TT of rs671256096, AG of rs671391101, TT of rs664283580, AA of rs671509714 and GG of rs669561078 were not observed in the Saanen or the Alpine. The missing genotypes may be attributable to the relatively small number of animals studied.

The results of the current study showed that the homozygous genotypes were more frequent than heterozygous in most cases. All results were tested for deviations from HWE that was used as a means of quality control. Only the Alpine breed was in Hardy-Weinberg disequilibrium at the g.62894878A>G locus (rs671391101) (P < 0.05) and therefore this locus raises question for further study on large number of samples. The Hardy-Weinberg disequilibrium for this SNP in French Alpine may be attributable to either selection or the genotyping error of KASP assays that produced 18 identified allele calls and 6 unidentified allele calls. Among the polymorphic SNPs, two were located in introns (rs660778229, rs635583012), and those variants have recently been demonstrated to be significantly associated with quantitative traits in different species, despite the fact that they do not change amino acids (Clark et al., 2015; Maitra et al., 2014; Sun et al., 2015; Zhao et al., 2013; Zhou et al., 2016). The results obtained here on single SNP analyses confirmed the previous findings, since it indicated that the goat SOCS3 g.52626440T>G beside GHRHR g.62894878A>G, IL1RN g.46353777T>G and IL15RA g.10354813C>T might play an important role in affecting milk production traits and could be linked to major genes that affect milk production traits in goats. Current findings revealed the significant association and the effects of those loci and the investigated milk traits. It was surprising since those genes are not milk protein genes or closely related to them.

SOCS3 is a member of the suppressors of cytokine signalling (SOCS) family of proteins that has a negative effect on cytokine signalling (Krebs & Hilton, 2001). However, polymorphism of SOCS3 gene was associated with somatic cell score trait in cattle (Strillacci et al., 2014; Wang et al., 2015), mammary development pathway (Raven et al., 2014) and is expressed in goat milk fat globules in response to experimental intramammary infection with Staphylococcus aureus (Strillacci et al., 2014; Wang et al., 2015).

Growth hormone (GH) was found to have wide range effect on a variety of physiological parameters such as lactation, reproduction, growth traits and metabolism (Mousavizadeh et al., 2009). Marques et al. (2006) detected high levels of polymorphisms at the five exons and the 5′-UTR and 3′-UTR regions of growth hormone gene in Serra da Estrela ewes and found that the two copies of the ovine GH gene (namely, GH2-N and GH2-Z) genotypes significantly affect milk yield in the breed. In contrast, significant differences in milk traits included milk yield, fat, protein, casein and lactose percentage were detected among Sarda sheep genotypes at polymorphic loci only for the GH2-Z gene (Dettori et al., 2015). However, there are no information about polymorphism and its associations with milk production traits in goats. Polymorphism and association study of exon 2 and exon 3 of the growth hormone gene with growth traits in goats from two Indian breeds did not find any significant association between genotypes and body development traits (Singh et al., 2015).

In the current study, two less-studied interleukins in goats were found to have a significant association with milk related traits. The IL1RN had also significant SNP for milk yield and located on chromosome 11 like a whey protein, β-lactoglobulin gene in goats. However, genetic variants of BLG gene involved this study (rs646939931) was not polymorph. Members of the IL-1 family are pleiotropic cytokines that have important rules in inflammation, immunoregulation and other homeostatic functions in the body (Mora & Weigert, 2016). Marcos-Carcavilla et al. (2007) studied the IL-1 family members as candidate genes for scrapie susceptibility detection in sheep. However, no studies on goats were published up-to-date. IL-15γ, IL-15Rβ and IL-15Rα are composing the chains of the interleukin-15 receptor (IL-15R). In human interleukin-15 receptor alpha is a high affinity IL-15 binding protein that is crucial for mediating IL-15 functions such as memory CD8 T, cell proliferation and NK, NK/T cell, and intestinal intraepithelial lymphocyte development (Schluns, Stoklasek & Lefrançois, 2005). Moreover, no previous study was performed for polymorphism or association studies with production traits in farm animals.

Conclusion

In summary, this study validated a KASP genotyping assays in goat that can replace the different genotyping assays developed for marker-assisted selection. The validated KASP genotyping assay used for the 48 SNPs will be helpful for diverse applications in goat breeding programs. Furthermore, preliminary results of the current study shows evidence of significant associations between four SNPs located on four genes, which were poorly studied before within the genetic variants and milk production traits in Alpine goats, indicating the potential role of SOCS3, GHRHR, IL1RN and IL15RA variants on relevant traits in dairy production.

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