AABArchives Animal BreedingAABArch. Anim. Breed.2363-9822Copernicus PublicationsGöttingen, Germany10.5194/aab-65-1-2022Polymorphism detection of DGAT1 and Lep genes in Anatolian water buffalo (Bubalus bubalis)
populations in TurkeyPolymorphism detection of DGAT1 and Lep genes in Anatolian water buffaloIşıkRaziyerisik@nku.edu.trhttps://orcid.org/0000-0003-2982-6562Özkan ÜnalEmelozemel@nku.edu.trSoysalM. İhsanFaculty of Agriculture, Department of Agricultural Biotechnology,
Tekirdağ Namık Kemal University, Tekirdağ 59030, TurkeyFaculty of Agriculture, Department of Animal Science, Tekirdağ
Namık Kemal University, Tekirdağ 59030, Turkey
Acyl-CoA: diacylglycerol–acyltransferase 1 (DGAT1)
enzyme plays a key role in controlling the synthesis rate triglyceride from
diacylglycerol. Leptin (LP, OB, obese) is an important hormone that
synthesizes mostly from adipose tissue and regulates glucose metabolism and
homeostasis. DGAT1 and Lep genes are closely related to reproduction, growth, milk
yield and composition in water buffalo breeds. This study aimed to identify
genetic variation in the DGAT1 and Lep gene regions in 150 water buffalo individuals
from five different provinces of Turkey using DNA sequencing. A total of 38
nucleotide variations and indels have identified 761 bp long partial intron
2 and exon 3 and 5′ UTR regions of the Lep gene in Anatolian water buffalo
populations; 422 bp long partial exon 7–9 and exon 8 regions of DGAT1 gene were
amplified and two mutations were defined in the point of 155 and 275
nucleotide that is three genotypes for S allele and Y allele of DGAT1 gene in
intron 7 in Anatolian buffalo populations, respectively. These SNPs may have
an effect on reproduction, growth, milk yield and composition in water
buffalo populations and may prove to be useful for water buffalo breeding.
Introduction
The water buffalo (Bubalus bubalis) originated in tropical and subtropical regions such as
India, Southeast Asia, and China. Anatolian water buffalo that originated
from the Mediterranean subgroup of river buffalo, which is known as the Anatolian
water buffalo, adapted to harsh environmental conditions such as low-quality
pasture, parasites, and pathogens (Andreas et al., 2010; Soysal, 2013; Konca
and Akyüz, 2017).
In Turkey, generally farmers raise a very low number of buffaloes for family
consumption in small villages, while big farms with more than 100 heads
are located near the big cities. Turkey is composed of seven different
geographical regions (Marmara, Aegean, Black Sea, Central Anatolia, Eastern
Anatolia, Mediterranean and Southeastern Anatolia regions). Buffaloes are mainly
reared in the Marmara Region, Aegean Region, Black Sea Region, Central
Anatolia Region, Eastern Anatolia Region and Southeastern Anatolia Region of
Turkey (Atasever and Erdem, 2008). The provinces with the highest amount of
water buffalo presence are recorded as Samsun, Diyarbakır, Istanbul,
Tokat, Bitlis, Mus, Afyon, Kayseri, Sivas and Amasya (FAO, 2021). Buffaloes
are substantially raised for milk and meat production in Asian countries,
Italy, and Turkey. Buffalo milk that has a high percentage of fat is used
for Turkish desserts and making special yogurt. This fatty part of buffalo
milk is consumed in the traditional Turkish breakfast called “kaymak”.
Moreover, buffalo meat is used in the Turkish sausage-making industry.
The number of water buffaloes in Turkey has increased from 84 000 to 142 000
over the last 10 years (http://www.fao.org/faostat/en/#data/QA,, FAO, 2021) because Anatolian water buffalo are
included in a programme for the protection of gene resources by the Ministry of
Agriculture and Rural Affairs in Turkey. Also, The National Anatolian
Water Buffalo Breeding Project has been carried out with the cooperation of
the Republic of Turkey Ministry of Agriculture and Forestry and Anatolian
Water Buffalo Breeder Associations since 2013. Thus, some researchers have
been handled to identify genetic structure and variation of Anatolian
buffalo using ISSR markers (Aytekin et al., 2011), microsatellites
(Özkan Ünal et al., 2014; Gargani et al., 2009; Soysal et al., 2007)
DGAT1 gene (Özdil and İlhan, 2012) and leptin gene (Kaplan, 2018a).
Recent research has shown that DGAT1 and LEP genes, where quantitative
trait loci are located, are related to reproduction (Demeter et al., 2009; Nasr et al.,
2016), growth (Nkrumah et al., 2004, 2005; Kulig and Kmieć, 2009), milk
yield and composition (Grisart et al., 2004; Tanpure et al., 2012; Meng et
al., 2013) in water buffalo and cattle (Banos et al., 2008). Acyl-CoA:
diacylglycerol–acyltransferase 1 (DGAT1) enzyme plays a key role in
controlling the synthesis rate triglyceride from diacylglycerol and FA-CoA
in adipocyte, which is important to supply the requirement of energy during
physiological phases such as pregnancy and lactation. Moreover, DGAT1 is a functional
candidate gene on quantitative trait locus (QTL), which is located on the
centromeric end of the bovine chromosome 14 (Bennewitz et al., 2004;
Coppieters et al., 1998; Grisart et al., 2002, 2004). In the eighth exon of
DGAT1, two single-nucleotide polymorphisms (SNPs) lead to the substitution of
lysine with alanine (K232A) that is closely related to variation in milk fat
content and milk performance (Winter et al., 2002). In some Anatolian,
Indian and Chinese water buffalo populations, it was seen that the eighth exon
of the DGAT1 gene has a fixed K allele (Tantia et al., 2006; Shi et al., 2012;
Özdil and İlhan, 2012).
Leptin (LP, OB, obese) is an important hormone that synthesizes mostly from
adipose tissue and regulates glucose metabolism and homeostasis (Houseknecht
et al., 1998; Orrù et al., 2007). It affects carcass traits and body
weight, feed intake (Nkrumah et al., 2005; Banos et al., 2008; Kulig and
Kmieć, 2009), fertility (Nasr et al., 2016), milk yield and composition
(Banos et al., 2008; Nasr et al., 2016), and immune function of cattle and
buffalo (Asiamah et al., 2009; Datta et al., 2013). Therefore, the leptin gene
is a crucial candidate gene for production traits in buffalo (Nasr et al.,
2016). Buffalo leptin gene located on chromosome 8 includes two exons and
introns with 167 amino acids (NC_058350) (https://www.ncbi.nlm.nih.gov/nuccore/MF679169, NCBI, 2021).
Nasr et al. (2016) revealed the associations with polymorphism in
the second exon region of leptin gene and fertility, as well as production traits in
Egyptian buffaloes.
The aim of this study was to identify genetic variation in the DGAT1 and Lep gene
regions in 150 water buffalo individuals from five different provinces of
Turkey using DNA sequencing.
Materials and methodsExperimental animals and blood collection
All procedures involving manipulation of the animals were conducted in
accordance with Turkish Guidelines on Animal Welfare. This research was
approved by the Ethics Committee on the Use of Animals, Republic of Turkey
Ministry of Agriculture and Forestry, Institute of Pendik Veterinary Control
Ethics Committee (case no. 2013/12). In this study, the 150 unrelated
Anatolian water buffalo blood samples used (0–6 months, 1–3 years; males,
n=46; females, n=104) were collected from East Anatolia (Bitlis,
Diyarbakır, Muş, n=40), Black Sea (Amasya-Merzifon, Samsun, Tokat,
Çorum, Giresun, Sinop, n=50), Central Anatolia (Kayseri, Sivas, n=17), and Aegean–Marmara (Afyon, Istanbul-Nakkaş/Danamandıra, Balıkesir, Bursa, Tekirdağ-Saray, n=43) regions of Turkey (Fig. 1).
Blood samples from animals were collected in 5 mL vacuum tubes, including
EDTA as an anticoagulant, and stored at -80 ∘C till the DNA extraction.
Genomic DNA was isolated by using a commercial DNA isolation kit (GeneJET
Whole Blood Genomic DNA Purification Mini Kit, Thermo Fisher Scientific)
according to the manufacturer's instructions.
Location of the Anatolian water buffalo samples collected
in this study.
Primers, total DNA extraction and PCR
Primer sequences of DGAT1 and Lep genes that are used for amplification and sequencing
are shown in Table 1. Reverse primer of Lep gene is designed using Primer3web
online software (version 4.1.0) (https://primer3.ut.ee/, last access: 12 March 2021) based
on NCBI Reference Sequence NC_058350 (91132528-91149368). For
PCR amplification reactions, the 35 µL PCR volume contained 20 ng genomic DNA, 0.5 µM of each primer, 1× PCR buffer
((NH4)2SO4), 200 µM dNTP, 1.5 mM MgCl2 and 1 U
of Taq DNA polymerase (Taq DNA Polymerase, Thermo Scientific, USA). The
cycling protocol was 4 min at 94 ∘C for initial denaturation, 35 cycles of amplification; 94 ∘C for 30 s, 56.1 ∘C
annealing for 30 s, 72 ∘C for 2 min and 15 min at 72 ∘C
for final extension. Afterward, the PCR products were checked on 1 %
agarose gel using horizontal electrophoresis and the gels were stained using
SafeView™ Classic (Applied Biological Material Inc. Canada).
Primer sequences of DGAT1 and Lep genes.
Gene regionsPrimersLength (bp)ReferencesDGAT1F: 5′-CAC CAT CCT CTT CCT CAA G-3′ R: 5′-GGA AGC GCT TTC GGA TG-3′412Shi et al. (2012)LepF: 5′-GTC TGG AGG CAA AGG GCA GAT-3′ R: 5′-GAT GGT CCA AAG GCT CTG AA-3′758Lien et al. (1997) (forward primer)Sequencing and data analysis
A total of 412 bp of DGAT1 gene and 558 bp of Lep genes were sequenced on an Applied Biosystems
3500XL Genetic Analyzer System (Applied Biosystems, USA) to determine those
gene sequences. DGAT1 and Lep fragments were blasted with the NCBI BLAST tool
(https://blast.ncbi.nlm.nih.gov/Blast.cgi, last access: 12 March 2021) to defined
orthologue sequences on the NCBI GeneBank database. Homolog sequences were
downloaded by NCBI GenBank database and aligned with ChromasPro 2.4.3
program (Technelysium Pty Ltd) and BioEdit (v7.0.9) Sequence Alignment
Editor with Clustal W multiple alignment modules (Hall, 2011). A heatmap was
performed for the differential nucleotide (SNP) of the selected sequences of
Lep gene region using an R script with the R function “heatmap” of the package
“ggplot2” based on the genetic distance of sequences analysed with the
MEGA7 software (Molecular Evolutionary Genetics Analysis, version 6.0). The
3D tertiary structure of the proteins based on the studied Lep gene region was
predicted using the I-TASSER server (Yang et al., 2015). The genotypic and
allelic frequencies of the Lep gene and the Hardy–Weinberg equilibrium of
the populations were calculated using the PopGene program (Yeh et al.,
2000).
Results
In the studied samples, the partially second intron and third exon regions
of the leptin gene were amplified. A total of 758 and 761 bp length samples were sequenced (AH013754). These variations were deposited in the
NCBI GenBank database with the accession numbers MZ230555–MZ230565. As 18
nucleotide changes in the third exon and 20 nucleotide changes in the intron
regions, a total of 38 point nucleotide variations have been identified in
the studied regions of the Lep gene (Table S1 in the Supplement). Also, 3 bp insertion/deletions
(indels) have defined position 47–49 (position 3998 at AH013754) in the
second intron region of the Lep gene for the first time. In this study, we have
detected three nonsynonymous amino acid changes and 13 silent
mutations in third exon sequence of Lep amino acid residues in Anatolian
buffalo breeds (Table 2). The amino acids change as T39A and V47I have been
defined for the first time in Anatolian buffalo breeds. According to Fig. 2, the heatmap revealed the differential nucleotide (SNP) of the Lep gene
region based on the genetic distance of sequences. While Lep A, C, and H alleles
were close to MF490270 LEP-CGGA allele, Lep-I allele was close to HE605297 sequence,
which was retrieved from the NCBI GenBank database. The Lep-B allele of
Anatolian buffalo was the same clade as the LEP-TAGA allele (MF490273).
Heatmap showing the differential nucleotide (SNP) of the
selected sequences of Lep gene region.
The nucleotide variations and amino acid changes in Lep amino
acid residues.
* Location is the point of mutations that occurred in the studied Lep gene amino acid
residues.
The predicted 3D tertiary structure of the Anatolian water buffalo leptin
peptide and the effect of the detected mutations on its 3D tertiary
structure are shown in Fig. 3. While the C-score of native 3D tertiary
structure of leptin peptide was -0.74 (estimated TM-score =0.62±0.14, estimated RMSD =5.8±3.6 Å), the C-score of leptin peptide
with the three mutations was -0.69 (estimated TM-score =0.63±0.14,
estimated RMSD =5.7±3.6 Å).
The predicted 3D tertiary structure of the Anatolian water
buffalo leptin peptide; (a) native structure and (b) the effect of the detected
amino acid changes on its 3D tertiary structure.
The genotype and allele frequencies belong to some SNPs of Lep gene are listed
in Table 3. Lep-G allele frequencies were 0.85, 0.83 and 0.82 for the point
of 164, 287 and 403 SNPs, respectively. While Lep-C allele frequency was 0.67,
Lep-T allele frequency was 0.33 for the point of 562 SNP. In this study, the
genotype distributions in SNPs of Lep gene were not found in Hardy–Weinberg
equilibrium (p<0.05) except for SNP in the point of 287
(p>0.05).
The genotype and allele frequency belong to some SNPs of
the Lep gene in Anatolian water buffaloes.
Mediterranean water buffalo DGAT1 gene consists of 17 exons located on chromosome
15, and it is 10.224 bp long in the NCBI reference sequence
(NC_037559). A total of 422 bp long
partial exon 7–9 and exon 8 regions of DGAT1 gene in studied Anatolian buffalo
populations (NC_037559) are amplified. A mutation is detected in the
point of 155 nucleotide that is homozygote C and G and heterozygote S allele of
DGAT1 gene in intron 7. The nucleotide changes from C to G in the DGAT1 gene and from
A to G of Lep gene as shown in Fig. 4. Also, we identified a variation at the
point of 275 nucleotide that is homozygote C and T and heterozygote Y allele of
DGAT1 gene in intron 7. These variations are deposited in the NCBI GenBank
database with the accession numbers MZ230551-MZ230554. Table 4 shows
the nucleotide differences of reference bovine and buffalo sequences that
deposited in the NCBI GenBank and studied sample sequences in the current
research.
Nucleotide changes from C to G in intron 7 region of
DGAT1 gene (point of 155 nucleotide, a) and from A to G in point of 403
nucleotide Lep gene (point of 111 amino acid, b).
The nucleotide variations between the studied sequences
and reference sequences of DGAT1 gene.
* The reference sequences were deposited by previous studies to the NCBI GenBank.
Discussions
In this study, the partially second intron and third exon regions of the
leptin gene and the partial exon 7, 9 and exon 8 regions of DGAT1 gene were
amplified in Anatolian buffalo populations. A total of 38 nucleotide
variations in the Lep gene and 4 nucleotide variations in DGAT1 gene have been
identified in the studied populations.
Nasr et al. (2016) reported three genotypes (AA, AG and GG) of leptin locus,
and the animals that have AA genotype had the highest milk yield (2332.34 kg, p=0.04) and fat yield (155.75 kg, p=0.06) in Egyptian buffaloes.
But they revealed that the GG genotype (43.67 kg, p=0.02) had higher
birthweight compared with AA genotype (39.35 kg, p=0.02). Orrù et al. (2007) reported that the G allele (0.640) was predominantly in the T1131G
locus of leptin gene in Italian and Egyptian buffaloes. Kaplan (2018b)
studied the same region of the Lep gene and reported three genotypes as TT, GT
and GG in a small population of Anatolian buffaloes. In Italian and Egyptian
buffalo, Orrù et al. (2007) reported 11 SNPs: G3333A,
C1221T, G3195A, C1221T, G3434A, T1015C, C1071T, G1072A, T1081C, T1143C and
T1145G loci of Lep gene. Jhala et al. (2009) identified three variations in exon
3 of leptin gene as A42G, A44G and A250G in Mehsana buffalo. In another
research on this buffalo breed carried out by Tanpure et al. (2012), five
SNPs were determined at positions 98, 111, 172, 209 and 266 in the first
intron of leptin gene. In Mediterranean water buffaloes, Scatà et al. (2012) revealed eight SNPs (A83G, A90G, A121G, G256T, A283G, G959T, A1010C,
G1254A) in the 5′ flanking and first exon regions of leptin gene. The
non-synonymous SNPs at positions A114G (EU888289.1), C163A (EU825672.1),
A211G (JQ045625.1), G288A (AY177609.1), A310G (KP864436.1), A322G
(JN689387.1), G330C (JQ045625.1), C348T (JQ045625.1), T360C (HE605297.1)
and G379A (Orru et al., 2007) caused amino acid changes (S71G, T87N,
N103S, E129K, E136G, Y140C, E143Q, R149W, S153P, and R159Q, respectively) in
LEP polypeptide. The T27C mutation (KP897166.1), which is close to the
3′ splicing site of the second intron region of the Lep gene, dislocates
a splicing silencer and creates a new intron splicing silencer, while A114G
and A310G SNPs of the third exon dislocate two splicing enhancers and create
new exonic splicing enhancers that may cause alternative splicing and intron
retention (Braunschweig et al., 2014; Mahrous et al., 2020).
Lep-G allele frequencies were high for the point of 164, 287 and 403 SNPs,
respectively. The genotype distributions in SNPs of Lep gene were not found in
Hardy–Weinberg equilibrium mostly (p<0.05). This can be because of
adaptation or fitness, which is a result of random effects of genetic drift
in evolutionary process (Andrews, 2010).
In this study, the C-score of the native 3D tertiary structure of leptin
peptide was -0.74, the C-score with the three amino acid changes T39A, V47I
and R111Q of leptin peptide was -0.69. Mahrous et al. (2020) revealed that
while the S71G improved the stability of the leptin protein, R159Q
decreased the stability of mature leptin peptide tertiary structure -0.25 kcal/mol. Also, Mahrous et al. (2020) reported that the amino acid N103S in
leptin polypeptide is conserved in humans and river buffalo, but this amino
acid variation does not exist in Anatolian water buffalo populations.
Li et al. (2018) revealed an SNP (g.9046T>C) caused a
nonsynonymous amino acid change from arginine to histidine in exon 17 of
DGAT1 gene, which was significantly associated with the fat percentage that TT
genotype had a significantly higher fat percentage than the CC genotype in
Riverine buffalo and Swamp buffalo. Similar to Li et al. (2018), de Freitas et al. (2016) carried out the nonsynonymous SNP g.11785
T>C that causes the alteration of alanine to valine (Ala484Val)
in exon 17 of the DGAT1 gene that was significantly related to fat and protein
percentage of milk in Murrah buffaloes in Brazil (P<0.05) (de Freitas et al., 2016). Contrary to de Freitas et al. (2016), Meng et al. (2013) found that the SNP c.1350C>G in exon 17 was not related
to milk yield and compositions in Dehong and hybrid (F1: Mora buffalo × Dehong buffalo) buffaloes. In this study, the amino acid change
from alanine to valine was not detected in exon 17 of the DGAT1 gene, since
strict breeding programmes were not implemented for milk yield and composition
in Anatolian buffaloes. In Murrah buffalo breed, Cardoso et al. (2015)
reported that DGAT1-VNTR (variable number of tandem repeats) in the promoter
region was significant for the fat percentage of milk because of a located
binding site of Sp1 (specificity protein 1), which is a transcription factor
for the mRNA synthesis.
Cesarani et al. (2021) revealed that WssGBLUP is a weighted version of
ssGBLUP, which is some major genes or SNPs, as DGAT1 for fat content has more
weights that could increase the accuracy of breeding values for growth, milk
traits.
Conclusion
Many studies have been carried out about relations between DGAT1 and Lep genes and
economic production traits. In this study, 38 polymorphisms, indels in the
Lep gene region and several variations in the DGAT1 gene region were identified
which will improve reproduction, growth, milk yield and composition traits
in water buffalo based on marker-assisted selection. For this reason, in
future studies, these relationships should be defined in water buffalo
populations.
Ethical statement
The experimental protocol was approved by the Republic of Turkey Ministry of
Agriculture and Forestry Pendik Veterinary Control Institute Local Ethics
Committee (AEC approval number: 12/2013).
Data availability
The data are available from the corresponding author upon request.
The supplement related to this article is available online at: https://doi.org/10.5194/aab-65-1-2022-supplement.
Author contributions
RI and EÖÜ designed the study. RI, EÖÜ and MİS performed the statistical analysis and wrote the paper.
Competing interests
The contact author has declared that neither they nor their co-authors have any competing interests.
Disclaimer
Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Financial support
This research was supported by the Scientific Research Projects Coordination
Unit of Namık Kemal University, under project number
NKUBAP.00.24.AR.14.32, and the Republic of Turkey Ministry of Agriculture
and Forestry General Directorate of Agricultural Research and Policies,
under project number TAGEM 13/AR-GE/29.
Review statement
This paper was edited by Steffen Maak and reviewed by Levent Mercan and one anonymous referee.
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