Whey protein polymorphisms in Sudanese goat breeds

Abstract

The aim of the present study was to assess genetic variation that is characteristic for Sudanese goat breeds in the milk whey protein genes (LALBA and BLG). Four Sudanese goat breeds were screened for variability in LALBA and BLG genes at the DNA level by comparative sequencing of five animals per breed. Sixteen SNPs were identified in LALBA: seven in the upstream region, six synonymous, and three in the 3´-UTR. Three novel synonymous SNPs in exon 2 (ss5197800003, ss5197800012, and ss5197800004) were found in Nubian, Desert, and Nilotic, but not in Taggar goats. One SNP in the promoter of LALBA (rs642745519) modifies a predicted transcription factor binding site for Tcfe2a. The SNPs in the 3'-UTR (rs657915405, rs641559728, and rs664225585) affect predicted miRNA target sites. With respect to haplotypes in the exonic region, haplotype LALBA-A is most frequent in Nubian, Desert, and Nilotic goats, while haplotype LALBA-D is prevalent in Taggar goats. In BLG, 30 SNPs were detected: eight in the upstream gene region, two synonymous, 17 intronic, and three in the 3'-UTR. Among the 30 identified SNPs, 15 were novel. Four of these novel SNPs were located in the upstream gene region, one was synonymous, and ten were intronic. The novel synonymous SNP (ss5197800017), located in exon 2, was only found in Nubian and Nilotic goats. The SNPs ss5197800010 and rs635615192 in the promoter are located in predicted binding sites of transcription factors (M6097, Elk3, Elf5, and GABPA). Among seven haplotypes detected in the coding region, haplotype BLG-A is most frequent in Nubian and Nilotic goats while haplotype BLG-B is most frequent in Desert and Taggar goats. The high variability in regulatory gene regions among Sudanese goats could potentially affect the quality and yield of whey proteins in goat milk and provide a wide resource for genetic improvement of milk production and milk technology characteristics.

Introduction

The protein fraction of goat milk, similar to that of other domestic ruminants, consists of about 80% caseins and 20% whey proteins (Fox et al. 2000). The two main whey proteins are alpha-lactalbumin (α-lactalbumin) and beta-lactoglobulin (β-lactoglobulin) which account for about 87% of total whey proteins (Acton 2013). The qualitative and quantitative content of each protein fraction depends on many factors, such as physiology (lactation stage, lactation order), environment, genetics, and nutrition (Cannas and Francescon 2008). Specific milk protein variants and bioactive peptides in milk are considered as targets for the production of health-promoting functional food due to their beneficial effects on human health and their potential prevention effects on human diseases (Vargas-Bello-Perez et al. 2019).

Whey proteins not only play an important role in nutrition but have also been found to exhibit antioxidant activity (Marshall 2004; Walzem et al. 2002). Alpha-lactalbumin is a calcium-binding metallo protein that catalyzes the final step of lactose synthesis in the lactating mammary gland (Brew 2011). Lactose is the most abundant milk sugar and an important osmotic regulator in the mammary gland. Lactose is produced from galactose moieties by lactose synthase, of which α-lactalbumin is the regulatory subunit. Lactose synthase is a heterodimer of α-lactalbumin and β-1, 4-galactosyltransferase (beta4Gal-T1). Alpha-lactalbumin regulates the catalytic activity of beta4Gal-T1, which is involved in the processing of galactose moieties in various secretory cells (Permyakov and Berliner 2000).

Alpha-lactalbumin plays an important role in regulating the volume of milk, and it has been considered a major gene for milk yield because lactose is the major osmotic active compound of milk and its production is associated with the movement of water into the secretory vesicle (Bleck and Bremel 1993). Furthermore, α-lactalbumin contains a relatively high proportion of essential amino acids and thus has physiological and nutritional significance for humans especially as a component of infant formulas (Layman et al. 2018). Alpha-lactalbumin may also be effective in preventing gastrointestinal infections among neonates (Ushida et al. 2007).

In goat, the α-lactalbumin gene (LALBA) is located on chromosome 5, consists of four exons and spans 2017 bp (NCBI, Accession NC_030812). LALBA knockout mice (LALBA-/-) produce milk lacking lactose but with an increased protein and fat content (Vilotte 2002).

Two α-lactalbumin protein variants, A and B, were found in goats, with A being dominant over variant B (Kumar et al. 2002). Polymorphisms in the LALBA gene have been most frequently studied in cattle, where five non-synonymous genetic variants were found (Caroli et al. 2009; Tetens et al. 2014).

Beta-lactoglobulin represents about 50% of the total whey protein in bovine, ovine, caprine, and buffalo milk (Sun 2012). The protein is absent in the milk of rodents, guinea pigs, camels, lamas, and humans (O'Mahony and Fox 2014). Beta-lactoglobulin is a member of the lipocalin super family of transporters for small hydrophobic molecules (Agudelo et al. 2013), also involved in other kind of biological process such as the synthesis of prostaglandins (Flower 1996). The protein can bind nonpolar hydrophobic molecules in multiple binding sites (Lozinsky et al. 2006) and is resistant to acid denaturation and proteolysis, a feature that is of pharmacological interest for the encapsulation and delivery of bioactive components (Barbiroli et al. 2008; Kontopidis et al. 2004). Beta-lactoglobulin can bind vitamin D, retinoic acid, cholesterol, fatty acids, and other aromatic compounds (Zimet and Livney 2009). Many biological functions have been proposed for β-lactoglobulin including improved lipid digestion (Perez et al. 1992), passive immune transfer (Ouwehand et al. 1997), and antimicrobial activity against mastitis pathogens (Chaneton et al. 2011). The role of β-lactoglobulin in milk appears to be primarily a nutritional one, and this protein poses a variety of functional and nutritional characteristics that have made this protein a versatile ingredient (Korhonen 2011; Oftedal 2012).

The β-lactoglobulin protein is encoded by the BLG gene, and it was the first milk protein in which a protein polymorphism was found (Aschaffenburg and Drewry 1955). In cattle, twelve protein variants are known (Caroli et al. 2009; Cui et al. 2012). The two most frequent protein variants A and B were associated with milk protein yield, quality, and cheese-making properties (Ng-Kwai-Hang and Grosclaude 1992; Yang et al. 2012).

In goat, the BLG gene is located in a GC-rich region on chromosome 11 (Hayes and Petit 1993). The gene consists of seven exons and spans 5132 bp (NCBI, Accession NC_030818). Several sequence variations have been identified in different goat breeds in the proximal promoter region and in the coding region , but no variation has been found causing an amino acid change (Ballester et al. 2005; Dettori et al. 2015b; Graziano et al. 2003; Pena et al. 2000; Sardina et al. 2012; Yahyaoui et al. 2000).

The main goat breeds in Sudan are Nubian, Desert, Nilotic, and Taggar goats (Wilson 1991). Nubian goats are the most productive dairy goats in Sudan (Steele 1996; Wilson 1991). Desert goats are dual purpose goats; they are very feed-efficient animals (Ismail et al. 2011). The Taggar goat is a dwarf, plump, meat-type goat that is well adapted to mountainous conditions (Bushara and Abu Nikhaila 2012). The Nilotic goat is a short statured meat-type goat known for its high fertility. Furthermore Nilotic goats are resistant to trypanosomiasis (Osman et al. 2008). More detailed information about Sudanese goat breeds can be found in Rahmatalla et al. (2017).

Genetic polymorphisms in milk protein genes have received considerable research interests since they can impact milk production traits, the properties of milk to produce certain dairy products (e.g., yogurt and cheese), and the nutritive value for human health (Bevilacqua et al. 2002; Ozdemir et al. 2018). Despite the importance of whey protein genes, these genes have not yet been genetically characterized in Sudanese goat breeds. The aim of the present study was to characterize the allelic variation of the two main whey protein genes LALBA and BLG in indigenous goat breeds in Sudan. The identification of such genetic variation is necessary for subsequent association studies, breed improvement, and conservation decisions.

Materials and methods

Animals and sampling

Blood samples were collected from five unrelated goats per breed of the four major Sudanese goat breeds: Nubian, Desert, Taggar, and Nilotic. Does were chosen from different regions of Sudan in accordance with the recommendation of the International Society for Animal Genetics (ISAG) and the advisory group regarding animal genetic diversity of the Food and Agriculture Organization of the United Nations (FAO 2011) as described previously (Rahmatalla et al. 2017). In brief, Nubian goats were sampled from four locations in three states along the river Nile, Desert goat samples were collected from the Bara and Abu Zabad area in the North Kordofan state, Taggar goat samples were obtained from the Nuba Mountains and Dalang area in the South Kordofan state, and Nilotic goat samples were collected from the Kosti area in the White Nile state. Blood samples were collected from the jugular vein through the use of vacutainer tubes containing EDTA as anticoagulant (5 ml). DNA was extracted using the Puregen core kit A (Qiagen, Hilden, Germany).

Sequencing

Goat reference sequences were taken from the Capra hircus LWLT01 genome version (Bickhart et al. 2017) from the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/). Primers were designed using Primer3 (Untergasser et al. 2012) to amplify the promoter regions and all exons of the two genes including 5’- and 3’- exon flanking sequences (Additional file 1:Table S1). The amplified regions covered 1259 and 1574 bp before the transcription start sites of LALBA and BLG, respectively. The PCR products for sequencing the exonic regions had a length between 302 bp and 861 bp (Additional file 1: Table S1). PCR and sequencing were performed under standard conditions.

Sequence analysis

The obtained sequences were analyzed and compared with the reference sequences using the software DNA Baser v4.0 (Heracle BioSoft SRL) and multiple sequence alignment (http://www.ebi.ac.uk/Tools/msa/). The positions of identified sequence variants are presented in this paper relative to the LWLT01 genome (LWLT00000000.1, 2016/08/24). The identified sequence variants were confirmed by using the reverse primer. Transcription factor binding sites (TFBSs) were identified using the Protein–DNA Binding Sequence Motifs (MotifDb) (Shannon and Richards 2017), using binding sites from the JASPAR database (http://jaspar.genereg.net/). Position weight matrices (PWMs) encoding known TFBSs are not available for goats; as such Mus musculus PWMs were used for TFBS prediction. The predictions of miRNA target sites in 3'- and 5'-UTR of the LALBA and BLG genes were performed using miRanda (Betel et al. 2008) using known and predicted goat miRNA sequences from the miRBASE database (http://www.mirbase.org/index.shtml) with standard parameters.

Haplotypes at the LALBA promoter region, LALBA gene, BLG promoter region, and BLG gene were derived using the haplo.stats package (https://cran.r-project.org/web/packages/haplo.stats) available for the R language for statistical computing. The method assumes that all subjects are unrelated and that haplotypes are ambiguous (because of unknown linkage phase of the genetic markers). For deriving haplotypes, we used all SNPs identified in the upstream gene region and all SNPs located in all exons. Haplotypes were generated using 100 EM iterations and a minimum posterior probability of 1e-06. Haplotypes were generated per breeds (n = 5 per breed) and all breeds grouped together (n = 20).

Results

Sequence analysis of 3,415 bp of the caprine LALBA gene revealed sixteen SNPs, an average of 4.6 SNPs per 1,000 sequenced base pairs. Among the identified SNPs, seven were located in the upstream gene region, six were synonymous SNPs in exons 1, 2, and 3, and three SNPs were within the 3'-UTR. Surprisingly, no polymorphism was detected in the intronic regions of the LALBA gene (Table 1). SNPs in the promoter region of LALBA were detected in all Sudanese goat breeds used in this study, and the frequency of the alternative allele of the reference sequence ranged between 0.1 and 0.5. The synonymous SNPs rs660007102 and rs650426572 in exon 1 and rs663851286 in exon 3 segregated in all four goat breeds. The three novel synonymous SNPs in exon 2 (ss5197800003, ss5197800012, and ss5197800004) were identified in Nubian, Desert, and Nilotic goat breeds, but were not detected in Taggar goats. Three SNPs were found in the 3'-UTR of LALBA, two of them (rs657915405, rs641559728) were found in all breeds, while rs664225585 could only be detected in Taggar and Dessert goats. The SNP rs642745519, which was found in the promoter region of the LALBA gene, is located in a predicted binding site of the transcription factor Tcfe2a. The T allele leads to a predicted gain of the Tcfe2a-binding site. The three SNPs that are located in the 3'-UTR of the LALBA gene are predicted to affect the binding of several miRNAs (Table 2).

Table 1 Sequence variation at the LALBA gene in Sudanese goat breeds
Table 2 Effect of single nucleotide polymorphisms (SNPs) within the upstream region and untranslated region (UTR) of the genes LALBA and BLG on micro-(mi) RNA and transcription factor binding sites (TFBS)

Using seven SNPs in the upstream gene region, the haplo.stats package distinguished seven haplotypes covering the LALBA promoter (Table 3). Haplotypes LALBA-A-up and LALBA-B-up were found in all four Sudanese goat breeds, whereas, haplotypes LALBA-C-up and LALBA-D-up were found only in Taggar and Nilotic goats. Using the nine synonymous SNPs found within the LALBA protein coding region, 16 haplotypes were detected when combining all animals together, 14 of these were found when analyzing haplotypes per breed (Table 4). We found that the haplotype LALBA-A is most frequent in Nubian, Desert, and Nilotic goats, while the haplotype LALBA-D is prevalent in Taggar goats. The haplotype LALBA-B was found in Nubian and Desert goats; all other haplotypes were found to be unique to a single breed.

Table 3 Haplotypes within haplotype blocks in the upstream region of the LALBA gene in four Sudanese goat breeds and their frequency
Table 4 Haplotypes within haplotype blocks in the exonic regions of the LALBA gene in the four Sudanese goat breeds and their frequency

The sequence analysis of 5,316 bp of the BLG gene detected 30 SNPs, with an average of 5.6 SNPs per 1,000 sequenced base pairs: eight in the upstream gene region, two synonymous SNPs, three in the 3'-UTR, and 17 in introns (Table 5). Four out of eight SNPs in the upstream gene region of the BLG gene were novel. Among these novel SNPs, ss5197800010 and ss5197800006 were detected in all Sudanese goat breeds, ss5197800013 was found only in Nubian and Nilotic goats, and ss5197800001 was detected only in Taggar goats. The novel SNP ss5197800010 and the known SNP rs635615192 in the promoter region reside in predicted transcription factor binding sites for M6097 for the novel SNP and Elk3, Elf5, and GABPA for the known SNP (Table 2, Sequence logos in Additional file 2). The novel synonymous SNP ss5197800017 in exon 2 was found in Nubian and Nilotic goats, but not in the other breeds. The known synonymous SNP rs655422073 in exon 3 was detected in all Sudanese goat breeds. Among the 17 SNPs in introns, ten were novel (Table 5). Three SNPs were detected in the 3'-UTR of the BLG gene; among them, rs654002978 and rs666423193 were found in all Sudanese goat breeds, while rs651951335 was only found in Nubian and Nilotic goats.

Table 5 Sequence variation at the BLG gene in Sudanese goat breeds

Haplotypes were constructed in the BLG promoter by using eight SNPs. When grouped all animals together, 15 haplotypes were detected in the Sudanese goat breeds (Table 6); only 13 haplotypes were detected when analyzing individual breeds. Haplotypes BLG-A-up and BLG-B-up were present in all four Sudanese goat breeds. Haplotype BLG-A-up is the most frequent haplotype in Desert and Nilotic goats, while haplotype BLG-B-up is most frequent in Nubian and Taggar goats. Haplotype BLG-E-up was observed in Nubian and Nilotic goats; all other haplotypes occurred in one breed only. Haplotypes in the protein-coding region of the BLG gene were constructed using the five identified synonymous SNPs. Seven haplotypes were derived (Table 7). Haplotype BLG-A was found in all Sudanese goats, while haplotype BLG-B was found in all but not in Nubian goats. The most frequent haplotype in Nubian and Nilotic goats is haplotype BLG-A; haplotype BLG-B is most frequent in Desert and Taggar goats. Haplotype BLG-C was found in Nubian and Nilotic goats, haplotype BLG-D in Desert and Nubian goats, and haplotype BLG-E in Desert and Taggar goats. The other haplotypes segregated in one breed only.

Table 6 Haplotypes within haplotype blocks in the upstream region of the BLG gene in the four Sudanese goat breeds and their frequency
Table 7 Haplotypes within haplotype blocks in the exonic regions of the BLG gene in the four Sudanese goat breeds and their frequency

Discussion

The sequence analysis of the LALBA and BLG genes in four Sudanese goat breeds identified a high genetic variability in and between breeds. With respect to allele frequencies reported in this study, we have to point to the limited sample size of only five animals per breed that were sequenced, which might not represent the whole populations under study and could introduce a bias toward the most common alleles found in the population. Despite the small sample size, a high number of novel SNPs were identified compared to other goat breeds. Nevertheless, none of them changed the encoded protein sequences. Since many SNPs are located in regulatory sequence motives, an influence of single SNPs or haplotypes on the transcriptional activity can be assumed.

The sequence analysis of the LALBA gene identified sixteen SNPs, seven in the upstream gene region, six synonymous, and three within the 3´UTR of exon 4. Many sequence variants that we found in the examined Sudanese goat breeds had been identified before in other goat breeds. With respect to the LALBA gene, for example, six out of eight SNPs that were identified in the promoter region of the Sudanese goats were also found in the indigenous Spanish breed Murciano–Granadina (Zidi et al. 2014).

The T allele of SNP rs646115281 and the G allele of SNP rs671985793 have been associated with higher lactose content in Italian Sarda goats (Dettori et al. 2015a). In addition, they found that CT heterozygotes at rs646115281 and AG heterozygotes at rs671985793 were characterized by delayed rennet coagulation time, curd firming time, and low curd firmness. In our Sudanese breeds, we found that these two alleles (T at rs646115281 and G at rs671985793) are the most common alleles. Although no information exists about the lactose content in goat milk of the Sudanese breeds, we assume that the high frequency of those alleles might indicate selection for higher lactose content also in our Sudanese breeds.

The sequence analysis of the coding regions of the LALBA gene in our Sudanese goats revealed six synonymous SNPs in exons 1 (rs660007102, rs650426572), exon 2 (ss5197800003, ss5197800012, and ss5197800004), and exon 3 (rs663851286), and additional three SNPs within the 3´UTR. The SNP rs650426572 in exon 1 which was found in all Sudanese goats and segregates also in Xinong Saanen and Guanzhong goat breeds in China (Mahmood and Usman 2010). In those breeds, this SNP (rs650426572) was associated with body size; individuals carrying the CT genotype were significantly bigger in terms of chest circumference than those with the CC reference genotype (Mahmood and Usman 2010). Although we did not have individual phenotypic data of our Sudanese goats, we found that the alternative allele T was enriched in Nilotic and Taggar goats, which are meat producing breeds, while the C allele was more frequent in Nubian and Desert goats, which are dairy and dual purpose breeds, respectively. Nevertheless, we cannot confirm the association with body size or chest circumference unless we have individual phenotypic data. The alternative allele T of the SNP in exon 3 which was found in Sudanese goat breeds occurs also widespread, for example, in Red Syrian, Girgentana, and a local goat population in Naples in Italy (Cosenza et al. 2003), in different breeds across Turkey (Ağaoğlu et al. 2014), in a native breed in Saudi Arabia (El-Hanafy et al. 2016), and non-described local breeds in Sri Lanka (Ariyarathne et al. 2017) . The only known non-synonymous SNP in exon 3 of the LALBA gene that leads to leucine to phenylalanine substitution was not found in the examined Sudanese goats. So far, this mutation has been identified in Mongolian White Cashmere and Chinese goat breeds where the T-allele carriers had higher cashmere yield (Lan et al. 2008; Lan et al. 2007).

With respect to the BLG gene, so far, no genetic variations that could modify the amino acid sequence of the β-lactoglobulin protein has been detected neither in Sudanese nor other goat breeds (Ballester et al. 2005) although different protein variants were reported through protein electrophoresis (Chianese et al. 2000; Macha 1970; Moioli et al. 1998).

We confirmed the SNP rs659299918 in the BLG promoter region that segregates in all Sudanese breeds and that also occurs in Alpine, Saanen, Girgentana, Garganica, Montefalcone, Sarda (Cosenza et al. 2003; Dettori et al. 2015b), and Derivata di Siria goats (Sardina et al. 2012). Dettori et al. (2015b) found in Sarda goats that rs659299918 was associated with the milk, fat, and protein yields, but not with the total protein content. The SNPs rs659299918 and rs635615192 which occurred in Sudanese goats were found also in Italian Sarda goats (Dettori et al. 2015b). In Sarda goats, these two SNPs affected the milk pH value with rs659299918 being highly significant. Goats homozygous for the T allele showed lower pH values, shorter rennet coagulation times (RCT), and lower curd firming rates (k20) than heterozygous CT goats (Dettori et al. 2015b). We found that the T allele that is beneficial in Sarda goats was fixed in Nubian and Taggar goats and was most frequent in Desert and Nilotic goats.

The novel SNP ss5197800010 and the known SNP rs635615192 change TFBS in the promoter region of the BLG gene. Therefore, these two SNPs could affect the binding of transcription factors and thereby influence the transcriptional rate and finally the milk protein content and ratio of different proteins in the milk. The polymorphisms in the promoter region of the BLG gene could have a functional role associated with milk composition as reported for cattle by Braunschweig and Leeb (2006) and for goats by Dettori et al. (2015b).

Our study also detected the SNP rs666423193 that is located within the 3´UTR of the BLG gene in all Sudanese goats. Previously, this SNP has been detected in the Spanish Payoyas, Malaguen and Mucriano-Giranadinas goats, French Saanen (Pena et al. 2000), Indian (Kumar et al. 2006), Barki, Damascus (El-Hanafy et al. 2010), and native Saudi Arabian goat breeds (Hanafy et al. 2015).

Although several studies were performed to characterize whey protein genes in diverse goat breeds in Asia and Europe, only a few studies have been carried out in the context of Africa. This study highlights the high genetic variation of whey protein genes in Sudanese goats, which belong to the largest populations in Africa and which contribute to other breeds in the world. Although the number of animals sequenced from each breed was low, we could gain an idea regarding the segregation of the alternative alleles in each breed. The pattern of distribution of haplotypes occurring in the genes in Sudanese goat breeds could result from natural selection for milk or meat traits and adaptation to the different geographical conditions. The new genetic variants found in this study and the observed haplotypes need further investigation to assess their functional effects on milk production traits before they can be used as selection markers for milk yield or quality. This requires reliable phenotypic measurements that can be used in association analyses between whey protein gene variants and milk yield and composition.

As Sudan is located in northeastern Africa, it shares borders with seven other countries: Egypt, South Sudan, Central African Republic, Chad, Eritrea, Ethiopia, and Libya. We expected that the four breeds investigated in this paper share a genetic background with breeds across those countries. This shared genetic background might have contributed to an increased genetic diversity especially in animals from the border regions. This might be due to the cross-border movements of pastoralist for grazing resources and water. Therefore, it will be interesting to investigate the relationship of the breeds investigated in this study with breeds not only in the neighboring countries but also in the African and world-wide context.

This is the first study to present information about genetic variants found in whey protein genes of Sudanese goat breeds. The high genetic variability detected within these whey protein genes provides a genetic basis for the genetic improvement of milk production and milk processing characteristics. However, before this improvement can be realized, association studies are necessary to evaluate the functional effects of these variants on milk production traits.

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Acknowledgments

The authors thank the goat owners in Sudan, the management staff of the Goat Research Stations Wad Medani, Kuku and Dongola, as well as the farms of Bahri and the Sudan University for providing goat samples.

Funding

SR and this study were funded by Georg Foster Research Fellowship provided by Alexander von Humboldt Foundation, Germany.

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Correspondence to Siham A. Rahmatalla or Gudrun A. Brockmann.

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Rahmatalla, S.A., Arends, D., Ahmed, A.S. et al. Whey protein polymorphisms in Sudanese goat breeds. Trop Anim Health Prod 52, 1211–1222 (2020). https://doi.org/10.1007/s11250-019-02119-2

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Keywords

  • Whey protein
  • Sudanese goat breeds
  • Single nucleotide polymorphism
  • Genetic diversity