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Genetic polymorphisms of milk genes (β- lactoglobulin and κ-casein) in

indigenous Awassi and improved Awassi sheep of Palestine

Article in Livestock Research for Rural Development · May 2020

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Livestock Research for Rural LRRD LRRD Guide for preparation LRRD Citation of this
Development 32 (5) 2020 Search Misssion of papers Newsletter paper

Genetic polymorphisms of milk genes (β-

lactoglobulin and κ-casein) in indigenous Awassi
and improved Awassi sheep of Palestine
F S Rashaydeh, N Sholi and R M Al-Atiyat 1
Palestine Technical University, Tulkarm, Palestine
[email protected]
1 Department of Animal Production, Mutah University, Jordan

Abstract

This study was investigating the genetic polymorphism and association of milk protein genes and yield in Awassi
sheep strains of Palestine. The polymorphism of β-lactoglobulin (β-LG) and Kappa casein (κ-CN) genes of the
two Awassi sheep strains in Palestine, known as indigenous (local) and improved Awassi strains, were
assessed on DNA level. The DNA of 54 individual sheep (23 local Awassi and 31 improved Awassi individuals
was extracted and then genotyped for ß-LG using Restriction Fragment Length Polymorphism (RFLP); RsaI
restriction enzyme technique, and for κ-CN using direct Sanger sequencing. The resulting amplified products for
ß-LG and κ-CN genes had the sizes of 471 bp and 670bp, respectively. The ß-LG gene revealed the presence of
three genotypes (AA, AB, and BB).While, results of the ß-LG sequence of exon 2 revealed a point mutation (C→
T), which is a single base nucleotide substitution of Thymine (T), instead of Cytosine (C) of the (X12817.1:c.112
T>C) locus in both local and improved Awassi strains. The mutation was responsible for the amino acid
exchange of Histidine to Tyrosine. The results of κ-CN sequence analysis showed the presence of three different
patterns; CC, TC and TT. In addition, they revealed a single base pair substitution (C→ T) in codon number 56
of exon 3. The significant effects of β-LG genotypes on fat content and density of milk in both strains were
reported, whereas certain κ-CN genotype showed effects on solid non-fat milk content in local Awassi only.
Furthermore, significant (P<0.05) differences were reported between local and improved Awassi of all tested
parameters in early season of milk and between fat, and lactose at the end of the milk season for both strains. It
is recommended to breed local Awassi sheep for outdoor rearing or extensive system in order to get more
content of milk components.

Keywords: gene Selection, β-lactoglobulin, κ-casein, RFLP, Sequencing

Introduction

Sheep breeds in Palestine are mainly indigenous (local) Awassi sheep, along with its derivatives; improved
Awassi and Assaf (Ahmed and Abdallah 2013). Awassi sheep are most commonly found in the Middle East in
which Palestine geographically located in its center. According to the Palestinian Center Bureau of Statistics
(PCBS 2013), Awassi, including local and improved strains, made up the majority of breeds (68%), while Assaf,
crossbreds, and others represented approximately 32%. Awassi sheep is a fat-tailed sheep with the advantages
of having great adaptability to tropical environmental conditions, nutritional fluctuations, and disease resistance
(Galal et al 2008). The intensive selection within Awassi breed produced an improved Awassi breed, which have
the highest milk production after the East Friesian breed (Galal et al 2008). The development process of the
improved dairy type of Awassi was throughout the 1930s and 1940s following within-breed selection as a dairy-
type Awassi strain until achieve a potential of producing over 500 liters of milk/ewe annually under an intensive
production system (Epstein 1985; Gootwine 2011).

It is obvious that there are increment demands on sheep milk long time ago in the Mediterranean region
(Selvaggi et al 2015). Likewise, in Palestine, there is an increased demand for sheep’s milk, an essential
ingredient for making traditional and specific dairy products such as yogurt and white fermented cheese. The
dominant genetic improvement and breeding programs of sheep in Palestine mainly depended on selection
within the population considering phenotypic traits, and neglecting genes responsible for important economic
traits. However, there are several problems associated with phenotypic selection, including narrowing the
genetic base of a population and missing economic traits of interest. Moreover, most economic traits are
displayed only in large adult animals in which traits are recorded, so that only a few can be chosen for breeding.
Costs are very high in the case of progeny testing for milk yield and its quality, as the tested sires have to be
raised throughout testing their daughters. This is the primary reason why breeding programs are expensive
(Fleming et al 2018). However, selection for high milk content and yield is not as easy as it looks because milk is
a quantitative trait controlled by several genes and environmentally influenced. As a result, the estimated
breeding value from traditional selection methods alone is not sufficient enough for predicting the genetic merit.
In addition, milk production can only be measured in mature females; this makes it difficult to analyze the merit
of the males. However, with the advancements in DNA technology to identify genes utilizing gene assisted
selection (GAS) procedure, many of these problems can be resolved considering availability of performance and
parentage records. Recently, several genes found to be associated with milk quality and quantity, among them
are Beta lactoglobulin (β-LG) and Kappa casein (κ-CN) encouraging better utilization in GAS to select animals
for higher milk productivity (Raj, 2012). The gene encoding for β-LG has been found to be highly and specifically
expressed in the mammary gland during lactation (Mercier and Vilotte et al 1993). The β-LG is one of the major
whey proteins found in ruminant milk (Perez and Calvo et al 1995).On the other hand, the Κ-CN has a role in the
formation, stabilization and aggregation of the casein micelles, thus altering the manufacturing properties and
digestibility of milk (El-Shazly et al 2012). Selection efficiency, however, depends on allelic frequencies in the
breeds of choice and on the effect of these polymorphisms on selected traits. Therefore, to our knowledge, the
detection of genetic polymorphisms for both the β-LG and κ-CN genes in this study will be the first in Palestine,
specifically in west bank region. The main objective of this study was to detect genetic polymorphisms among
the ß-LG and κ-CN genes of the local and improved Awassi sheep strains to be utilized in GAS scheme for
breeding sheep with a great potential of milk yield and composition.

Materials and methods

The study involved 54 ewes belonging to local and improved Awassi. Each of these ewes had samples taken for
milk and blood in which 31 samples came from improved Awassi sheep located in Bait Quad Station in Jenin city
and 23 samples from the local Awassi sheep flocks from Nablus city in the West Bank region of Palestine. The
pedigree information for the 31 and 23 ewes of improved Awassi were reported in the Supplementary materials
(Table S1). The similar pedigree information of the 23 ewes of local Awassi was reported in Table S2. The
records show no close genetic relationship between the ewes of local Awassi and only a few ewes (two half sibs’
ewes of six sires) of improved Awassi breed.

Milk sample collection and analysis

The milk samples were taken in early morning and then transported into the ice-cooled box to the laboratory and
stored at -20°C for further analysis of milk composition (total protein, total fat, lactose, total solids and nonfat
solid contents) which was determined using Milk Analyzer (Milk scope –Model Julie C8-110-250V system).

Genotyping

Blood collection and DNA extraction

Blood samples were collected in 5 ml EDTA tubes from the jugular vein. Samples were stored at +4 °C during
transportation and then placed at +4 °C for DNA extraction in the next day. DNA was extracted from 100μl of
blood, using a commercial kit (Qiagen USA) following the manufacturer's protocol. The quality and quantity of
DNA were determined using NanoDrop spectrophotometer (2000, Thermo fisher, USA) and also by eye-
observation on 1.5% Agarose gel electrophoresis.

PCR and primers for the amplification of the ß-LG and-CN genes

Primers were designed according to available sheep gene sequences in the gene bank. We used the BLAST-N
program to ensure the specificity of forward and reverse primers for the two genes (β-LG and κ-CN). The
sequence of β-LG gene, its annealing temperature and expected product size (bp) were 5’-
CCCAAGATCCAAATGTTGCT-3’ / R 5’-CGCCGGGTACCAGTAAACTC-3’, 56.5 °C and 471 (bp). The sequence
of κ-CN Gene, its annealing temperature and expected product size were 5’-CTGGGTTCACTATTCCCAATG-3’ /
R 5’-TTGCTCATTTACCTGCGTTG-3’, 59 °C and 670 (bp). The DNA amplification of the β-LG gene was
achieved by PCR technique using forward primer and reverse primer (Table S3). The PCR amplification reaction
solution was performed in a total volume of 25 μl containing 2 μl (200 ng) DNA, 4.5 μl nuclease free water, 12.5
µl of GoTaq ® Green Master Mix (2x), 2 µl MgCl2 and 2 μl (0.8 µM) of each primer. The PCR cycling condition
was a preliminary denaturation at 95 ºC for 5 min, followed by 35 cycles, each cycle included denaturing at 95 ºC
for 30 seconds, annealing at 56.5 ºC for 40 seconds, and extension at 72 ºC for 40 seconds followed by 10 min
at 72 ºC as a final extension. The PCR reactions were performed on a LifePro Thermal Cycler. The PCR
products were visualized by resolving on 1.5% Agarose containing Ethidium bromide in parallel with 100-bp DNA
ladder. Gel electrophoresis was carried out at a constant voltage of 120 V for 45 min.

PCR product of ß-LG gene and κ-CN gene

The amplified product of 471 bp for β-LG was digested by the RsaI restriction enzyme in a digestion reaction
consisting of a 10 μl PCR product, 2 μl of 10 x buffer, and 2 μl of the restriction enzyme in a final volume of 32 μl.
The reaction mixtures were incubated for 3 hours at 37°C in temperature controlled water bath, and the digested
products were separated by electrophoresis on 3% Agarose gel stained with Ethidium bromide and then the
visualized bands were scored manually under UV light. The PCR amplified products were sequenced by Sanger
method using the same primers of amplification. The sequencing was done by Macrogen Incorporation (Seoul,
Korea).The results were visualized with FINICH TV program. The DNA amplification of the κ-CN gene was
achieved by PCR technique using forward and reverse primers. The PCR amplification reaction solution was
performed in the total volume of 25 μl containing 2 μl (200 ng) DNA, 4.5 μl nuclease free water, 12.5 µl of
GoTaq® Green Master Mix (2x), 2 µlMgCl2 and 2 μl (0.8 µM) of each primer. The PCR cycling condition was a
preliminary denaturizing at 95 ºC for 5 min, followed by 40 cycles, each cycle included denaturation at 95 ºC for
30 seconds, annealing at 59 ºC for 30 seconds, and extension at 72 ºC for 40 second followed by 10 min at 72
ºC as a final extension. The PCR product was separated on 1.5% Agarose containing Ethidium bromide in
parallel with 100 bp DNA ladder. Gel electrophoresis was carried out at a constant voltage of 120 V for 45 min.

The clean-up protocol was done when the successful gel electrophoresis and PCR product bands were seen on
the gel, PCR product " in order to chew up excess primers and remove excess unincorporated dNTPs, enzymes,
salts and small fragments from these products. In addition, this procedure was used to purify the amplified
fragments from agarose gel after electrophoresis or from the PCR reaction. So that, Antarctic Phosphatase (0.25
μl) and Exonuclease I (0.25 μl) enzymes were used to purify PCR products of 5 μl to form PCR reaction of 7 μl
as a total volume. Antarctic Phosphatase enzyme is responsible for removing the leftover nucleotides whilst the
role of Exonuclease I enzyme is to degrade the remaining primers. Further details of the cleaning procedures
and the PCR program used are shown in Supplementary files (Table S3-6). After PCR products were purified,
the next step was DNA sequencing. Sanger method of DNA sequencing was done depending on Big Dye
terminator. The Big Dye™ Terminators V1.1 Cycle Sequencing Reaction Kit (Applied Biosystems) included
ddNTPs (dideoxynucleotides) that inhibit chain-elongation by DNA polymerase. Samples were run on ABI
3130XL Genetic Analyzer (Applied Biosystem) at Bethlehem University. The results were visualized with FINICH
TV program.

Statistical analysis

Allele frequency and polymorphism under Hardy-Weinberg equilibrium (HWE) were analyzed using Cervus
software package (Marshall et al 1998). In order to evaluate the main effects of genotypes and their interactions
with the milk traits, General Linear Model (SAS 2008) for testing the hypothesized model assuming Hardy-
Weinberg equilibrium were used. The mixed model that describe the data was Yijknm = YRMOi + GENOj + Ak +
DLnm(k ) + Eijknm, where Yijknm is the milk composition variable in a test sample from each ewe, YRMOi is the
fixed effect due to ewes tested in the same time, GENO j is the fixed effect of the genotype evaluated for each
ewe, Ak is the random effect of ewes, DLnm(k ) is the random effect of test day and lactation nested within ewe
and Eijknm are the random residuals. Factors such as age and birth season were not includes because of all
ewes were considered of same age class (Supplementary Files- Table S1 and S2).

Results

Beta lactoglobulin gene

The PCR-RFLP technique identified specific polymorphism of whey protein gene β-lactoglobulin. The resulted
PCR fragments all of 471-bp in size for 16 animals, as an example were presented in the supplementary
materials as in Figure S2. The digestion of PCR fragments (471bp) in size with RsaI enzyme revealed the
presence of two alleles, A and B, in the tested Awassi sheep (Figure 1). Three genotypes were observed; the
homozygous AA with two bands at 286 and 185 bp, the heterozygous AB genotype with four bands at 286, 220,
185 and 66 bp and the homozygous BB genotype with three bands at 220, 185 and 66 bp. Out of 54 tested
animals; 50 had AA genotype (27.8%), 35 had AB genotype (64.8%) whereas only 4 had BB genotype (7.41%).
In both Awassi breeds; AB genotype was recorded at the highest frequency compared with other two genotypes.
Improved Awassi sheep had the highest frequency of AB genotype (67.7%) with the lowest frequency of BB
genotype (3.23%). In tested local sheep; AB genotype frequency was lower than that found for tested improved
sheep but still with the highest frequency in this breed; it was 60.9%, while AA genotype frequency was 26.09%.
Although the frequency of BB genotype in local breed was the lowest (13.04%) but it was still higher than that in
the improved Awassi (3.23%). In all tested animals, the frequency of allele A (60.2%) was higher than the
frequency of allele B (39.8%). The results showed that alleles A and B were presented with different frequencies
in both tested Awassi sheep populations. Improved Awassi breed possessed the highest frequency of allele A
(62.9%) than that in local breed (56.5%) whereas the allele B frequency ranged from 37.1% in improved to
43.5% in local Awassi lines (Table 1). Hardy-Weinberg equilibrium test (HWE= 5.32; P<0.021) returned the
observed and expected genotypes (Table 1).

Figure 1. PCR-RFLP results for β -LG gene using RsaI restriction enzyme on 3% agarose
gel

The homozygous AA with two bands at 286- and 185-bp. The heterozygous AB genotype with four bands at
286-, 220-, 185- and 66-bp. The homozygous BB genotype with three bands at 220-, 185- and 66-bp. 3;’4lp
Table 1. Genotype and allele frequencies, Expected and Observed
Heterozygosity and HWE of β-LG gene in both Awassi breeds using Chi-
Square test
Local Awassi Improved Awassi Total
(No. 23) (No. 31) (No. 54)
AA 26.1 (6) 29.0 (9) 27.8 (15)
Genotype
AB 60.9 (14) 67.7 (21) 64.8 (35)
Frequencies (N)*
BB 13.0 (3) 3.2 (1) 7.4 (4)
Allele A 56.5 62.9 60.2
Frequencies B 43.5 37.1 39.8
Expected Heterozygosity Observed Heterozygosity PIC
0.48 0.65 0.36
* N : Number of animals

Sequencing result of the exon 2 in β-LG gene

Sequencing results revealed that there was a single base substitution of Thymine (T) nucleotide instead of
Cytosine nucleotide (C) at position 1617 bp of the (X12817.1: c.112T>C; chr3: 3,574,794) locus in both local
and improved Awassi (Figure 2) (Supplementary materials as in Figures S2 and 3). The mutation was a
missense mutation (Variant No.: rs430610497) where (CAC) codon change to (TAC) codon and as
consequence amino acid number 36 converted from Histidine to Tyrosine (p.Tyr36His).

Figure 2. The homozygous TT of B-LG gene detected in Awassi sheep strains

(Mutant)

κ-CN gene

The fragment of 670-bp was amplified by polymerase chain reaction (Supplementary materials- Figure S4).
Sequencing results recorded the presence of three different genotypes; CC, TC and TT in the 54 tested sheep
animals. The sequence analysis showed the nucleotide C in wild type (Supplementary materials- Figure S5)
converted into T (Supplementary materials- Figure S6). This mutation was synonymous variant where no change
to the encoded amino acid. The sequencing results of κ-CN gene showed nucleotide C in local Awassi was
converted to T in improved Awassi (Supplementary materials- Figures S6, 7 and 8). In addition, the results
showed that the genotype (CC) was present only in one individual of local Awassi sheep (4.35%) whereas the
genotype (TT) was found in more tested animals (higher frequencies). The highest frequency (83.87%) was
recorded in improved Awassi for genotype (TT) with the absence of genotype (CC). The frequencies of patterns
(TC) and (TT) in local Awassi were 39.13 and 56.52%, respectively (Table 2).

Table 2. The Genotypic pattern of κ-CN gene in both local and

improved Awassi breeds
Pattern frequencies
Genotype I (TT) Genotype II (CT) Genotype III (CC)
Breeds
No. of No. of No. of
Freq. Freq. Freq.
animals animals animals
Local 13 56.5% 9 39.1% 1 4.40%
Improved 26 83.9% 5 16.1% 0 0.00%
Total 39 140% 14 55.3% 1 4.40%

Effect of β-LG and κ-CN gene on milk composition

First, in the Local Awassi, the associations between β-LG genotype and milk composition were shown in Table
3. In general, associations were examined between β-LG polymorphisms and milk composition in which
significant differences noticed for fat and density percentage. Accordingly, greater association was observed
between fat ratio in milk analysis at the end of the season and genotype AA in comparison to genotype AB and
BB. In addition, β-LG genotype AA was associated with the higher density ratio in milk analysis at the end of the
season compared to genotype BB (Table 4). The other milk traits remained unaffected. On the other hand, the κ-
CN gene was significantly associated only with higher SNF of early milk stage. In more details, it was observed
also a significant effect of κ-CN genotype TT compared to genotype TC (Table 4). Other genotypes ofκ-CN gene
showed no associations with other milk composition (See supplementary files; Table S7 for more details).
Second, in the improved Awassi, the associations between β-LG and κ-CN genotypes with milk composition are
shown in Table 5. Unexpectedly, the statistical analysis within breed showed that, for the tested animals, there
were no significant effects of the genotypes on milk composition of improved Awassi ewes.
Table 3. Genotype effect of β-lactoglobulin and κ-Casein on milk
composition in Local Awassi sheep breeds, based on with F-test
and its probability values

Variable Std B-LG F-Value κ-CN F-Value

N Mean
(%) Dev F-Value Pr> F F-Value Pr> F
Early of the season
Fat 23 8.99 2.04 0.65 0.54 0.79 0.47
SNF 23 10.4 0.81 0.84 0.45 4.02 0.03
Density 23 30.9 3.96 1.65 0.22 1.82 0.19
Protein 23 3.82 0.30 0.87 0.44 2.20 0.14
Lactose 23 5.74 0.47 1.17 0.33 2.24 0.14
Solids 23 0.72 0.11 1.04 0.38 1.56 0.24
End of the season
Fat 23 6.82 1.34 4.66 0.02 0.63 0.55
SNF 23 7.69 0.95 0.09 0.91 0.56 0.58
Density 23 24.9 2.69 2.62 0.01 0.30 0.75
Protein 23 3.28 0.18 0.88 0.43 0.24 0.79
Lactose 23 4.81 0.47 0.06 0.99 1.80 0.20
Solids 23 0.73 0.11 1.02 0.34 1.57 0.24

Table 4. Significant differences of β-lactoglobulin genotypes on fat

percentage and density fat in final stage and significant differences of κ-
casein genotypes on solid Non Fat (SNF) in first stage in local Awassi
sheep breed based on Least Significant differences test (LSD)
Difference Significant
BLG 95% Confidence
Variable Between (P value)
Comparison Limits
Means level
Fat 2 AA – AB 1.43 0.22 2.65 <0.001
Fat2 AA – BB 2.18 0.42 3.94 <0.001
Density2 AA – BB 2.76 0.09 5.433 <0.001
SNF 1 TT – TC 0.796 0.137 1.46 <0.001
1: Early of the season, 2: End of the season,

Table 5. Genotype effects of β-lactoglobulin and κ-Casein on

milk composition in improved Awassi sheep breeds with F-test
and its probability values
Std B-LG F-Value κ-CN F-Value
Variable N Mean
Dev F-Value Pr> F F-Value Pr> F

Early of the season

Fat 31 6.05 1.78 1.80 0.19 3.35 0.08
SNF 31 9.18 0.60 1.02 0.38 1.07 0.31
Density 31 28.81 2.55 1.06 0.36 0.73 0.40
Protein 31 3.37 0.27 1.15 0.33 0.31 0.58
Lactose 31 5.05 0.33 0.03 0.97 0.73 0.40
Solids 31 0.71 0.05 0.95 0.40 0.01 0.93
End of the season
Fat 31 5.01 0.71 0.71 0.500 2.98 0.10
SNF 31 7.28 0.74 0.03 0.972 0.03 0.88
Density 31 24.7 4.92 1.30 0.289 1.64 0.21
Protein 31 3.09 0.45 1.30 0.288 0.30 0.59
Lactose 31 4.38 0.43 2.39 0.110 0.00 0.93
Solids 31 0.71 0.06 1.02 0.399 0.04 0.94

Milk composition for the local and improved Awassi

The mean differences between different parameters of milk composition between local and improved Awassi are
shown in Table 6. There were significant (p<0.05) differences between local and improved Awassi of all tested
parameters in the early milk season whereas there were only significant differences between fat and lactose at
the end of the milk season between both strains as shown in Table 6.

Table 6. Significant mean differences (P<0.05) between milk composition in local and improved Awassi on first and
final season based on Least Significant differences test (LSD)
Variable
Breed
Fat SNF Density Protein Lactose Solids1 Fat2 SNF2 Density2 Protein2 Lactose2 Solids2
1 1 1 1 1
Local Awassi 8.99a 10.4a 30.9a 3.8a 5.74a 0.86a 6.83a 7.69a 24.9a 3.28a 4.81a 0.72a
Improved Awassi 6.05b 9.18b 28.8b 3.4b 5.05b 0.76b 5.10b 7.28a 24.7a 3.09a 4.38b 0.71a
1: Early of the season, 2: End of the season

Discussion

Dairy sheep are considered as one of the most important livestock species, even more so than dairy cow in rural
and developing areas. In fact, they have a potential role as a traditional small-scale dairy sector for supporting
food security, human health, and poverty alleviation (Al-Atiyat 2014). The sheep milk is an excellent raw material
for the milk processing industry especially in cheese production (Boyazoglu and Morand-Fehr 2001). Therefore,
the target of the sheep breeding has shifted from meat to milk and wool (Gürsoy et al 2006). Similarly,
Palestinian farmers and breeders are breeding local Awassi for better milk quantity and quality because of its
better adaptation to local harsh environment over improved Awassi. Previously, several investigations proved
that sheep genetic polymorphisms affect quantity and quality of milk (El-Shazly et al 2012). The following
discussion details our findings and results of the genetic polymorphisms of both β-LG and κ-CN genes in Awassi
sheep strains and the opportunities for selection of high potential milk ewes on this basis.

Genetic Polymorphisms of β-LG

Beta-Lactoglobulin is a major milk protein gene in ruminants representing 60 to 65% of total whey protein in milk.
Ovine β-LG gene has been assigned to chromosome 3 and its complete sequence consists of 7379 nucleotides
arranged in seven small exons and six introns (El-Shazly et al 2012). In our study, PCR-RFLP results identified
three genotypes: AA, AB and BB in both breeds. The AB genotype had appeared with high frequencies in both
improved (67.74%) and local (60.87%) Awassi Sheep. The genotype AA had appeared with moderate
frequencies, fewer than AB genotype and greater than BB genotype. Allele A was more frequent than allele B in
both Awassi sheep breeds. The results were similar to other findings such as with the Egyptian sheep breeds;
Barki, Rahmani and Ossimi, in which the allele A was more frequent than allele B (Othman et al 2013), Noami,
Sawakni, Harry and Nagdi (El-Shazly et al 2012); Turkish sheep breeds; Kıvırcık, Gökçeada, and Sakız (Elmaci
et al 2006); Karagouniko breed (Triantaphyllopoulos et al., 2017); Racka and East Friesian sheep (Kawecka and
Radko 2011). Other researchers have also reported that allele A was dominant in sheep breeds (Dario et al
2008, Corral et al 2010). In contrary, higher frequency of dominant B allele had been reported in Awassi and
other breeds (Kusza et al 2015; Triantaphyllopoulos et al 2017). In addition, Yousefi et al (2013) and Staiger et
al (2010) reported that allele B was dominant in different sheep breeds. The presence of B allele was mainly
encountered in sheep breeds from India, with its ancestral origin (Selvaggi et al 2015). According to Rozbicka-
Wieczorek et al (2015), the high frequency of B allele had been also observed in British dairy sheep, Hungarian
Merinos, Racka and Polish Lowland breeds. On the other hand, no evidence was found of C allele in our study,
which was considered a rare variant detected only in few breeds such as Merinoland, Lacha, Carranzana,
Spanish Merino, Serra da Estrela and White and Black Merino originated from the Spanish Merinos (Selvaggi et
al 2015).

Sequencing analysis of B-LG detected a substitution from Cytosine (allele C) to Thymine (allele T) at the
nucleotide number 192, which leads to amino acid exchange from Histidine (CAC) to tyrosine (TAT), The
mutation genomic number is chr3:3,574,794 according to GenBank: X12817.1. It is missense mutation convert
amino acid number 36 from Histidine to Tyrosin and from (CAC) codon to (TAC) codon. In other words, β-LGA
has Tyrosine whereas β-LG B has Histidine. These two amino acid variants are corresponding to a single
nucleotide substitution (T→C) in theβ-LG gene. Other researchers detected the rare variant of β-LG C as a
subtype of ovine β-LG A with a single exchange of amino acid from Arginine to Glutamic acid at position 148
(Picariello et al 2012).

Genetic Polymorphism of κ-CN Gene

Casein is a milk protein secreted by mammary gland cells (Akers et al 2016). It constitutes about 78-82% of
ovine milk protein, and it is divided into four main groups: αS1casien, αS2 casein, β-casein, and κ-CN (Doosti et
al., 2011). The κ-CN plays an important role in the formation, stabilization and aggregation of the casein micelles
thus altering the manufacturing properties and digestibility of milk (El-Shazly et al 2012). Although κ-CN is widely
polymorphic in cattle with six variants (Atamer et al 2017), it is considered to be monomorphic in sheep (Othman
et al 2013). In our study, the sequence results of a 670 bp fragment of κ-CN reported the presence of three
different patterns: CC, TC and TT in the 54 tested animals . The sequence analysis showed a SNP C→T
chr6:85,316,422 in codon number 56 of exon three. This mutation was a synonymous variant -a sequence
variant where there is no resulting change to the encoded amino acid-. The results showed the presence of
genotype III (CC) in rare frequency whereas the genotype I (TT) was found in high frequencies. Nevertheless,
the highest (83.8%) frequencies were recorded in improved Awassi for patterns TT and absence of CC. In local
Awassi, the frequencies of the three recorded patterns were 4.3%, 39% and 56.5% for patterns CC, TC and TT,
respectively. It is worth mentioning that the dominance of pattern T would be considered monomorphic in sheep
considering the very low frequency of C. Similarly, Othman et al (2013) reported the monomorphism of κ-CN in
Egyptian sheep breeds; Rahmani, Barki and Ossimi. Furthermore, many studies reported the monomorphic in κ-
CN in different sheep breeds; for example, in Harry and Nagdi (El-Shazly et al 2012), in Ossimi sheep (Othman
et al 2013), in Czech Sumava and Valachian breeds (Sztankoova et al 2011), in Churra da Terra Quente
(Selvaggi et al 2015), in Indian sheep (Singh et al 2015).

Effect of B-LG and κ-CN Genotypes on Milk Composition

Local Awassi sheep

The β-LG showed a unique protein fraction profile considering lactoglobulin protein content depends on breed
and lactation stage, in sheep and goat. The polymorphic genes of β-LG, were proven to be associated with traits
of interest in sheep milk, thus to be considered in a future selection for milk composition and rheological
properties. In more details, the association of β-LG polymorphism with milk yield and composition has been
reported in sheep (El-Shazly et al 2012), cows (Ng-Kwai-Hang et al 1998) and goats (Kahilo et al 2014). The
physicochemical characteristics of milk are important for the efficient development of milk industry and
marketing. Therefore, β-LG locus has been extensively studied as one of the genes that may affect the
economically important traits. Some studies observed that β-LG polymorphism significantly affects milk yield,
milk fat, and protein content (Bolla et al 1989). However, other studies failed to detect any effect of its genetic
polymorphism on milk production traits. The present study confirmed the significant effect of β-LG genotypes
(genotype AA compared to genotype AB and BB) on fat content and density of milk, observed greater fat ratio in
milk analysis at the end of the milk production season. Our results are in agreement with previous results
reported by Özmen and Kul (2016) who found that the genotype AA showed high fat content in Awassi sheep
breed. In similar, the AA genotype produced a higher fat percentage in Merino Sheep (Corral et al 2010).
Contrary results were reported for significant associations between AB genotype of β-LG gene and higher
percentages of fat (Selvaggi et al 2015;Yousefi et al 2013). On the other hand, Çelık and Özdemir (2006)
reported that AB genotype is associated with higher lactose content in Awassi Sheep. Our results, of within
breed, showed that homozygotes AA in local Awassi breed have a higher density in milk composition compared
to homozygotes BB. These results are similar to the findings of Sumantri et al (2008) who also reported no
significant effect of β-LG genotypes on other milk composition parameters in improved and local Awassi. In
addition, Kawecka and Radko (2011) found no statistical associations between β-LG genotypes and milk
composition in Polish Mountain, East Friesian, and Polish Merino and Austrian Bergschaf sheep.

It is well known that the performance of cheese production and processing depends on the structure of κ-CN
protein (Bonfatti et al 2010). In this study, no significant difference was reported between the genotypes of κ-CN
gene and milk composition in local Awassi except for genotype TT compared to genotype TC with higher SNF in
early milk production stage. However, the result is in agreement with previous findings which reported that BB
genotype of κ-CN had more influence on the milk fat, and protein yield of the Sahiwal cattle and genotype TT
showed higher fat and protein contents in Chinese Holstein Cattle (Hamza et al 2011). According to Othman et
al (2013), cheese production can be increased by 10 percent if milk is from a cow of the BB genotype when
compared to AA genotype. Therefore, it proposed to select in favor of BBgenotype in breeding programs
preferring more milk produced.

Improved Awassi sheep

The improved Awassi was a result of intensive selection within the local Awassi sheep for highest milk
production. In this study, the associations between β-LG and κ-CN genotypes and milk composition were not
significantly different and thus were in agreement with previous reports by Selvaggi et al (2015). However,
significant differences between local and improved Awassi of all tested parameters in the early stage of milk
production of first lactation were reported. On the other hand, there was only significant difference between fat
and lactose percentages in the late milking stage of both strains. Other researchers such as Triantaphyllopoulos
et al (2017) showed non-significant differences in fat and protein content between two breeds; Karagouniko and
Chios sheep breeds. They explained the result as a consequence of combinatorial effect between breed and
environment and to the breed itself. The later was reported recently for Tsigai and Improved Valachian ewes
(Orancova et al 2015).

Conclusion

Both strains of Awassi have shown genetic polymorphisms for B-LG and κ-CN genes. The Palestinian local
Awassi breed proved to be significantly higher in milk composition than improved Awassi.
 The associations between certain genotypes of both breeds with milk composition parameters would be
useful in implementation plan for gene selection scheme by Palestinian farmers and breeders. Therefore, it
is advisable to breed local Awassi sheep as the main breed for extensive and semi-intensive production
system, to get an increase content of milk components.

It is recommended to shift the national breeding strategy from disseminating improved Awassi to
disseminating elite local Awassi for the farmers and breeders. The establishment of nuclei for the pure elite
local Awassi breed to be used for future breeding programs is recommended in cooperation with the
private sector and industry.

Investigation on the polymorphism of genes like prolactin and other casein genes for those breeds is
recommended.

Acknowledgment

The authors would like to thank the staff members of Palestinian Center for Livestock Improvement (PCLI) and
members of Bait Qad Station for help in samples collection and providing animals.

Conflict of interest

The authors declare that no conflicts of interests exist.

Supplementary information

Supplementary Materials - Genetic polymorphisms of milk genes in indigenous Awassi and improved Awassi
sheep of Palestine

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Received 22 February 2020; Accepted 8 April 2020; Published 1 May 2020

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