Leptin is mainly secreted by white adipose tissue in animals. Leptin acts by
stimulating or inhibiting the release of a neurotransmitter, which
eventually results in a decrease in food/feed intake and an increase in
energy expenditure. In this investigation, the polymerase chain reaction
(PCR) coupled with single-strand conformation polymorphism (SSCP) analysis
was used to reveal nucleotide sequence variations in bovine leptin gene
(LEP) in 338 cattle of a variety of breeds farmed in New Zealand (NZ) and
Nigeria. These included NZ Hereford, Angus, Shorthorn, and crossbred
Holstein-Friesian × Jersey cattle and the Nigerian Sokoto Gudali,
Red Bororo, White Fulani, and crossbred Holstein-Friesian × White
Fulani cattle. Sequence analysis of three regions of bovine LEP that
encompassed selected coding and non-coding regions, revealed a total of
12 nucleotide sequence variations (six in exons and six in introns). Of
these, three are reported here for the first time, whereas nine have been
previously described. Some of the variations identified were common in both
the NZ and Nigerian cattle breeds, while others were peculiar to particular
breeds from a specific region. The sharing of common variants across
different breeds irrespective of geography may indicate an evolutionary
relationship, just as the differences within a breed might be
attributable to either selective pressure for specific traits or random genetic
drift. The detection of both new and previously documented variations in
bovine LEP suggests that the gene is highly variable.
Introduction
One of the most important factors affecting animal productivity is feed
intake. Poor feed intake or inadequate nutrition can affect body weight,
growth, reproduction, and milk production, and it can also decrease
immunity. Accordingly, a desire to increase animal productivity may require
the adoption of measures aimed at increasing or maximizing feed intake. To
achieve this, having an understanding of the role of genetics in regulating
this important trait is important.
Leptin is a product of the gene that is now called LEP but was previously
known as OB, OBS, and LEPD. Bovine LEP has been mapped to chromosome 4 (Pomp et al.,
1997), and it consists of three exons separated by two introns. Exon 1 and
part of exon 2 (four nucleotides) are not translated, and only the remaining
part of exons 2 and 3 are translated into the functional 16 kDa leptin
protein of 146 amino acids in length.
Leptin is considered to be a protein hormone and is mainly secreted from
white adipose tissue. This protein is found to regulate feed intake, body
weight, immune function, and reproduction (Santos-Alvarez et al., 1999;
Kadokawa et al., 2000; Block et al., 2001). Leptin acts on receptors in the
lateral hypothalamus to inhibit hunger (Elias et al., 1999) and in the
medial hypothalamus to stimulate satiety (Elmquist et al., 1999). It
counteracts the effects of neuropeptide Y, a potent hunger promoter secreted
by cells in the gut and in the hypothalamus. The hypothalamic release of
neuropeptide Y can result in an increase in feed intake and an increase in
energy expenditure, among other things (Houseknecht et al., 1998).
Several nucleotide sequence variations have been reported in bovine LEP, with
these including microsatellite repeat number variation and nucleotide
sequence variation (Stone et al., 1996; Konfortov et al., 1999). Konfortov
et al. (1999) investigated a 1788 bp portion of LEP (comprised of exons 2 and 3 and parts of introns 1 and 2) in 13 different breeds of Bos taurus and Bos indicus cattle.
They reported 20 nucleotide variations, six of which occurred in the exons
and at a density of approximately 1 per 84 bp. The density of the intron
sequence variations was 1 per 92 bp, giving an overall density of
variation of one per 89 bp. This suggests the gene is polymorphic and that
if more cattle breeds are examined, more variation might be detected.
The aim of this study was therefore to investigate an extended region of
bovine LEP in a variety of cattle breeds from New Zealand (NZ) and Nigeria,
with the objective of identifying unique and novel variations specific to a
given breed or shared across breeds. This would enable improved
understanding of the gene and, subsequently, may allow identification of
potential gene markers associated with feed intake in cattle.
Materials and methodsCattle investigated
Our investigation was carried out in accordance with the Animal Welfare Act
1999 (NZ Government), whether blood was collected in NZ or Nigeria.
A total of 338 cattle from NZ and Nigeria were investigated. The NZ cattle
included NZ Hereford (n=23), Angus (n=23), Shorthorn (n=18), and
crossbred Holstein-Friesian × Jersey (HF × J-cross) (n=166) breeds; while the Nigerian cattle included Sokoto Gudali (SG; n=18), Red Bororo (RB; n=34), White Fulani (WF; n=32), and crossbred
Holstein-Friesian × White Fulani (HF × WF-cross; n=24)
breeds. Of these eight breeds, the HF × J-cross and the HF × WF-cross breeds are bred for milk production, whereas the
Hereford, Angus, and Shorthorn breeds are farmed primarily for meat
production. The Nigerian Sokoto Gudali, Red Bororo, and White Fulani are
dual-purpose breeds. Unlike the NZ breeds, which are Bos taurus cattle, the Nigerian White Fulani, Red Bororo, and Sokoto Gudali are Zebu (Bos indicus) cattle and are
characterized by having a fatty thoracic hump on their shoulders, a large
dewlap, and being adapted to dry environmental conditions (Mattioli et
al., 2000).
Collection of blood samples
Blood samples from each animal were collected (either by piercing the ear or
from the tail) onto FTA cards (Whatman, Middlesex, UK) and air dried. DNA
from the dried blood was purified using a two-step procedure as described by
Zhou et al. (2006).
PCR primer design
Three pairs of primers were designed using DNAMAN Version 5.2.10 (Lynnon
BioSoft, Vaudreuil, Canada). These were designed to amplify three regions of
the bovine leptin gene (GenBank accession number U50365.1). The primer pair 1 (5′-gtctttgaggagatgatagcc-3′ and 5′-gctgtctttatgccagggg-3′) would amplify
a 443 bp fragment, consisting of exon 2 and parts of introns 1 and 2; the
primer pair 2 (5′-agctagtcaggttccacaag-3′ and 5′-ggttctgcaagggtattcag-3′)
would amplify a 400 bp fragment of intron 2; and the primer pair 3
(5′-ttgctctccccttcctcctg-3′ and 5′-ctcaggtttcttccctggac-3′) would amplify a
430 bp fragment, which consisted of exon 3 and part of intron 2.
Polymerase chain reaction (PCR)
The PCR reactions for all three regions were performed in 15 µL reactions
containing the genomic DNA on a 1.2 mm diameter disc of FTA paper, 0.25 µM
for each primer, 150 µM for each dNTP (Eppendorf, Hamburg, Germany), 3.0 mM Mg2+, 0.5 U of Taq DNA polymerase (Qiagen, Hilden, Germany), and
1× the reaction buffer supplied with the enzyme. For each of the
three regions, PCR amplification was undertaken in Bio-Rad S1000 thermal
cyclers (Bio-Rad, Hercules, CA, USA). The thermal cycling conditions
included an initial denaturation at 94 ∘C for 2 min,
followed by 35 cycles of 94 ∘C for 30 s, 60 ∘C
for 30 s, and 72 ∘C for 30 s, with a final extension step at
72 ∘C for 5 min.
The SSCP technique was used to detect genetic variation in the amplicons
obtained from the PCR reactions. A 0.7 µL aliquot of the amplicons was added
to 7 µL of loading dye containing 10 mM EDTA, 0.025 % bromophenol blue,
0.025 % xylene cyanol, and 98 % formamide. The samples were then
placed on a hotplate already set at 95 ∘C for 5 min
for denaturation, followed by snap-chilling on wet ice. Samples were then
loaded onto 16 cm × 18 cm, acrylamide : bisacrylamide (37.5 : 1)
(Bio-Rad) gels. Electrophoresis was carried out using Protean II xi cells
(Bio-Rad) for 18 h at 390 V, with a 12 % polyacrylamide gel
concentration, and run temperature of 13 ∘C for amplicon I (part of
intron 1, exon 2, and part of intron 2); for 16 h at 390 V, with 10 % polyacrylamide at 18 ∘C for amplicon II (part of intron 2); and
for 24 h at 390 V, with 10 % polyacrylamide plus 4 % glycerol
at 15 ∘C for amplicon III (part of intron 2 and exon 3). The silver
staining technique described by Byun et al. (2009) was used to reveal the
SSCP banding patterns.
Nucleotide sequencing
Based on the PCR-SSCP patterns identified, cattle that were homozygous with
unique banding patterns for amplicons I, II, and III were subjected to direct
sequencing at the Lincoln University DNA sequencing facility (Lincoln
University, NZ). For these cattle, the amplicons were purified using a
MiniElute™ PCR purification kit (Qiagen) and then directly sequenced
using the original PCR primers to prime the sequencing reactions in both
directions, using the forward and reverse primers.
For rare patterns that were only found in heterozygous form, the unique band
was excised from the wet gel and incubated at 69 ∘C for 1 h as per
Gong et al. (2011). A 1 µL aliquot of the incubated product was then mixed
with 14 µL of PCR pre-mixture and then amplified using identical conditions
to the original PCR reactions.
For each of the regions sequenced, the nucleotide sequences were aligned, and
other analyses were undertaken using DNAMAN Version 5.2.10 (Lynnon BioSoft).
Results
In this investigation, the PCR-SSCP technique revealed four banding patterns
(A1, B1, C1, and D1) in amplicon I (exon 2 and parts of introns 1 and 2), three patterns (A2, B2, and C2) in amplicon II (intron 2
region) and three patterns (A3, B3, and C3) in amplicon III (which
spans exon 3 and part of intron 2) (Fig. 1).
(a) Polymerase chain reaction–single strand conformation
polymorphism (PCR-SSCP) banding patterns obtained for the three amplicons
from the bovine leptin gene. (b) Nucleotide sequencing revealed the
different sequence variations identified in each of these regions. A dash
(–) represents a nucleotide deletion.
Of the four variants identified for amplicon I, the NZ HF × J
crossbred cattle had three variants (A1, B1, and C1), and the
Nigerian Red Bororo and crossbred HF × WF cattle had three
variants each (A1,B1 and D1), while two variants (A1 and B1)
were observed in the NZ Hereford, Angus, Shorthorn, Nigerian Sokoto Gudali,
and White Fulani cattle.
In the amplicon II region, all the eight breeds were observed to carry the
A2 and B2 variants, but only the Nigerian Red Bororo had the
C2 variant.
All eight breeds carried the A3, B3, and C3 variants in the
amplicon III region investigated. The frequency of each variant in each
region is presented in Table 1.
Variants and their respective frequencies identified in each of the
three amplicons of bovine leptin gene across the NZ dairy and beef and the Nigerian
dual-purpose cattle breeds investigated.
BreednExon 2 region Intron 2 region Exon 3 region variants and variants and variants and frequencies (%) frequencies (%) frequencies (%) A1B1C1D1A2B2C2A3B3C3NZ HF × J (dairy)16660.539.20.30.092.57.50.066.623.89.6NZ beef breeds6465.035.00.000.067.232.80.062.516.421.1Nigerian dual-purpose breeds10854.229.20.016.662.019.019.060.620.818.6
A total of 12 nucleotide variations were identified across the three
regions examined. Eleven were nucleotide substitutions: c.-107G/C and
c.-130C/T located in intron 1; c.73C/T (p.Arg25Cys) in exon 2;
c.144+808C/T, c.144+907C/T, and c.144+908T/C identified in intron 2; and
c.239C/T (p.Ala80Val), c.396C/T (p.Gly132=), c.399T/C (p.Val133=),
c.411T/C (p.Ala137=), and c.495C/T (p.Pro165=) located in exon 3. The
remaining nucleotide variation was a deletion (c.144+42delC), located in
intron 2.
Of them 12 variations identified across the three regions examined, the
deletion (c.144+42delC) and two substitutions (c.144+907C/T and
c.144+908T/C) were novel and are reported here for the first time (Fig. 2). The remaining nine variations, with their respective reference SNP (rs) numbers have
been previously reported to Ensembl (EMBL-EBI, Hinxton, United Kingdom,
release 96 – April 2019, ARS-UCD1.2 for the cow leptin gene).
(a) Sequence chromatograms illustrating the differences between
variants A2 and B2 and variant C2 of the bovine
LEP intron 2 region. The chromatogram of variant C2 indicates the
positions of the two nucleotide substitutions C>T and
T>C (nucleotide sequence variation c.144+907C/T and
c.144+908T/C respectively). (b) Chromatograms for A1, B1,
C1, and D1. The chromatogram of variant D1 illustrates the position of the deleted cytosine (c.144+42delC).
The alignment of the predicted amino acid sequence of leptin protein
obtained from the four variants (A1, B1, C1, and D1) in exon 2
and three variants (A3, B3, and C3) in exon 3 are given in Fig. 3.
(a) An alignment of the predicted amino acid sequence of leptin
protein obtained from the four variants (A1, B1, C1, and
D1) identified in exon 2. The grey highlighted areas mark the position
of the amino acid difference between the variant sequences and other
species. (b) Alignment of the putative amino acid sequence of leptin protein
from the three variants (A3, B3, and C3) in exon 3 and from
sequences in other species. The grey highlighted areas mark the position of
amino acids where the underlying nucleotide sequence is variable. The symbol
“–” represents the same amino acid as the top (reference sequence). The
amino acid sequence for cow was obtained from the coding sequences examined
in this study, whereas the others were obtained from GenBank JQ739233.1
(goat), MH716186.1 (sheep), and EU603265.1 (yak).
Discussion
Recently, there has been some interest in leptin gene variations in
livestock, mainly as a consequence of their potential for use as
gene markers for improving the efficiency of selection for quantitative
traits.
Of the 12 variations identified in this study, three are novel, and nine
have been described previously. Of the nine reported previously, two would
if expressed, change the amino acid sequence of the protein. These are
c.73C/T (p.Arg25Cys) in exon 2 and c.239C/T (p.Ala80Val) in exon 3. The
p.Arg25Cys substitution would result from the C/T substitution located 73 bp
from the start of exon 2 in a region that is proximal to the signal sequence
of leptin and that has been described previously (Konfortov et al., 1999;
Buchanan et al., 2002; Lagonigro et al., 2003). The signal sequence contains
21 amino acids and it is cleaved off before leptin is excreted from adipose
tissue (Zhang et al., 1997). The predicted cleavage site of the signal
sequence is C terminal to an alanine at position 21 (Zhang et al., 1997).
The p.Ala80Val would result from a C/T substitution located 95 bp from the
start of exon 3. It has also been described previously (Konfortov et al.,
1999; Haegeman et al., 2000).
Zhang et al. (1997) suggested that the p.Arg25Cys change is situated in the
first of the four α helices of the leptin protein and at a location that varies considerably between species. They reported glutamine in
primates and mice, arginine in dogs, tryptophan in pigs, and histidine in
rats. Therefore, it is likely that the p.Arg25Cys change is allowed because
it does not have functional significance. This view was also shared by
Konfortov et al. (1999). It is also conceivable that this position may be
polymorphic in other species. This is supported by the amino acid alignment
of leptin protein from different ruminant species (Fig. 3), with position
25 of the protein in goats being an arginine residue, while sheep have
glutamine, and yak have serine. Variation at this position therefore appears
to be tolerated. Since arginine and cysteine have alkaline and neutral side
chains respectively, this variation could be considered a nonconservative
substitution.
However, Buchanan et al. (2002) hypothesized that the p.Arg25Cys change has
a functional effect on the leptin molecule. The paper suggested that the presence
of cysteine in the α helix of the leptin molecule may disrupt the
binding of leptin to its receptor. This is supported by the observation that
the leptin receptor contains a conserved trough typical of haemopoietic
cytokine receptors, into which the A and D helixes of haemopoietic cytokines
dock (Sprang and Bazan, 1993). They argued that a change between two very
different amino acids, such as arginine and cysteine at this location, may
have deleterious effect on this process. Also, their report suggests that
the presence of another unpaired cysteine in the leptin molecule could
destabilize the disulfide bridge found between the two existing cysteines,
thus, affecting the structure of the leptin molecule and, consequently, its
function (Rock et al., 1996). Studies have shown that this disulfide bridge is
critical for biological function (Rock et al., 1996; Zhang et al., 1997).
In comparison, the p.Ala80Val polymorphism is located in a conserved region
of the leptin protein (Komisarek and Dorynek, 2005). The c.396C/T (p.Gly132=),
c.399T/C (p.Val133=), c.411T/C (p.Ala137=), and c.495C/T (p.Pro165=)
nucleotide sequence differences in exon 3 are all synonymous and occur in a
conserved region of leptin.
Three nucleotide changes, which included a deletion (c.144+42delC) and two
substitutions (c.144+907C/T and c.144+908T/C) occurred in the intron 2
region. They are reported here for the first time. In amplicon I, the
D1 variant, carrying the deletion c.144+42delC was identified only in
the Nigerian Red Bororo and the crossbred Holstein-Friesian × White Fulani cattle breeds. In amplicon II, the B2 variant, with the
haplotype c.144+808C, c.144+907T, and c.144+908C was found in all the
NZ and Nigerian breeds investigated, whereas the C2 variant for amplicon II, which carries the substitutions c.144+808T, c.144+907C, and
c.144+908T was only detected in the Red Bororo breed. The occurrence of
variant B2 with GenBank accession number MN082389 in all the nine breeds
(NZ and Nigerian cattle breeds) investigated suggests that this variation
is shared across cattle of Bos taurus and Bos indicus (African Zebu cattle) origin. This result
is not surprising, given the close relationship between Bos taurus and Bos indicus in terms of
origin. There are suggestions that these two species originated from a
common ancestor, and the wild auroch, Bos primigenius, is the progenitor of all taurine and
zebu (African Bos indicus) cattle (Edwards et al., 2007). Estimates of divergence times for Bos taurus and Bos indicus from a common ancestor are all pre-Neolithic and range from
approximately 2 million to 330 000 years ago, depending on the genetic
markers and the calibration of the evolutionary molecular clock (Hiendleder
et al., 2008). There are also some reports that suggest the Bos indicus populations may
have been produced at a later date through breeding and selection from Bos taurus
cattle (Epstein, 1971; Epstein and Mason, 1984). Either way, each of these
aforementioned studies highlighted the evolutionary link between Bos taurus and Bos indicus
cattle. The sharing of common haplotypes suggest that cattle studied here
might be more closely related, although it does not provide definitive
proof. The suggestion is supported by Dunner et al. (2003) who suggested
that the pattern of haplotype sharing is an indicator of the history of the
different bovine breeds, and thus the distribution of shared haplotypes is
useful in describing population relationships.
In the amplicon I region investigated, the D1 variant carrying the novel
deletion c.144+42delC was proximal to the putative splice-donor site. The
deletion variant was only identified in the Nigerian Red Bororo cattle and
the crossbred Holstein-Friesian × White Fulani-cross cattle. This
suggests that this variation may only be found in the African Zebu cattle,
since it was not observed in the NZ HF × J-cross cattle or the
other NZ beef breeds. The occurrence of this variation around the
exon/intron boundary may possibly have functional implications for gene
expression and protein assembly. To illustrate this point, Sjakste et al. (2011) investigated variation in MSTN in Latvian Darkhead sheep and reported
nucleotide sequence variations (c.373+18G/T and c.373+101C/T) at the
splice-donor site and showed that the G nucleotide in the c.373+18
position, initiated single-strandedness in the first CUG repeat and G
triplet location. They further suggested that hairpin-loop development in
that region could be followed by rearrangement of the spatial topology
between the two other CUG repeats. Such perturbation of the pre-mRNA
secondary structure could potentially influence sequence interaction with
different regulatory proteins and the efficiency of lariat formation, and
this could then potentially affect transcription and splicing efficiency
(Chasin, 2007; Hiller et al., 2007; Aznarez et al., 2008). There is also
evidence suggesting that introns are functionally active participants in
gene and genome functionality, as they can encode regulatory elements
(Yutzey et al., 1989) that participate in splicing, transcription, and
recombination events. It will therefore be interesting to further
investigate the effect of this deletion in the Red Bororo and Holstein-Friesian × White Fulani-cross cattle.
In the amplicon I region examined, the C1 variant (carrying the
nucleotide sequence variations c.-107C and c.-130T) had the lowest
frequency, which was also reflected at the genotype level. Again, this is
not surprising, especially considering the fact that this variant was only
identified in one heterozygous sample (A1C1) out of the 166 samples
of NZ HF × J-cross cows investigated. Since this variant had very
low frequency in the cattle studied here, it may in future be difficult to
ascertain how it affects the function of the gene. The investigation of more
cows and more breeds may also resolve this issue, especially as in the
Ensembl report on Iranian Bos taurus from the NextGen Project (NextGen Consortium, 2020) suggests the G and C
nucleotides in c.-107G/C (rs29004485) had a frequency of 69 % and 31 %
respectively (remapped to ARS-UCD1.2; release 150), and the C and T alleles
in the nucleotide variation c.-130C/T (rs29004484) had a frequency of 69 %
and 31 % respectively.
Conclusion
The detection of new as well as previously documented variations in bovine
leptin gene indicates a possibility of identifying potential gene marker(s)
for the selection of specific traits for increased efficiency in animal
production. Some variants were common across the Bos taurus and Bosindicus species, which
supports the theory of a possible descent from a common ancestor.
Data availability
Data are available upon request to the corresponding author.
Author contributions
ILH collected data, designed primers, carried out the experiment, and wrote the paper. JGHH and HZ designed and supervised the experiment, helped
with results interpretation, and edited the paper. SAH, OJO, and GM
optimized PCR-SSCP conditions and edited the paper. All authors
reviewed and approved the final paper.
Competing interests
The authors declare that they have no conflict of interest.
Acknowledgements
The authors thank Chika Nwosuh at the National Veterinary Research
Institute Jos, Nigeria, for his support in sample collection and the staff
of the Gene-Marker Laboratory at Lincoln University for technical support
and facilities provided.
Review statement
This paper was edited by Steffen Maak and reviewed by two anonymous referees.
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