SNPs in the Stearoyl-CoA desaturase and their associations with the CLA
and MUFA content of milk fat from Canadian Holstein and Jersey cows.
Funded by DairyGen and NSERC
Submitted by Xin Zhao, McGill University
Introduction
Stearoyl-CoA desaturase (SCD) is an important lipogenic enzyme that catalyzes the
biosynthesis of monounsaturated fatty acids (MUFA) from their saturated counterparts as well
as the synthesis of conjugated linoleic acid (CLA) in the mammary gland and adipose tissues
of ruminant animals. CLA is a collective term for a series of conjugated dienoic positional and
geometrical isomers of linoleic acid (C18:2) found mainly in milk fat and to a lesser extent in
tissue fat. CLA has received wide attention in recent years because of its potential in protecting
against cancer, atherogenesis and diabetes (Dhiman et al., 2005). The bulk of the CLA in meat
and dairy products is synthesized endogenously in ruminant animals through the action of the
enzyme SCD or delta-9-desaturase on transvaccenic acid (TVA), an intermediate of rumen
biohydrogenation of linoleic and linolenic acids (Griinari et al., 2000). Endogenous synthesis
of CLA by SCD in the mammary gland is estimated to account for up to 78% of the CLA in
milk from cows on total mixed ration diets and for up to 91% of milk CLA from pasture-fed
cows [Griinari et al., 2000; Corl et al., 2001).
Breed differences in milk fat composition and a two to three fold variation in milk fat CLA has
been reported among animals on the same diet (Kelsey et al., 2003; Kelly et al., 1998a, b;
Lawless et al., 1999; Peterson et al., 2002). The variation among individuals in CLA would
primarily be due to differences in ruminal production of CLA and transvaccenic acid (TVA),
and the activity of delta-9 desaturase in the mammary gland. Peterson et al. (2002) reported a
range of approximately twofold for the individuals’ variation in delta-9 desaturase activity and
consistency in the individual hierarchy in desaturase ratios over time when cows were fed the
same diet or when they were switched between diets. It has also been reported that given the
same diet, Holsteins secrete significantly higher levels of CLA and MUFA (palmitoleic and
oleic acids) but lower levels of all the short and medium chain saturated fatty acids in their
milk than Jerseys (White et al., 2001; Lawless et al., 1999). White et al. (2001) reported CLA
levels of 0.41% vs. 0.32% of total fatty acids on total mixed rations (TMR) and 0.72% vs.
0.59% of total fatty acids on pasture in Holsteins and Jerseys, respectively. All these suggest
the existence of some genetic influence on SCD activities.
Hypothesis and objectives
Based on the above information we hypothesized that polymorphisms in delta-9-desaturase
gene are responsible for some of the variation in MUFA and CLA content of milk fat in dairy
cattle.
The specific objectives of the proposed work were
1) To determine the existence of polymorphisms in the SCD gene in Canadian Holsteins
and Jerseys
2) Based on the polymorphisms, to estimate allele and genotypic frequencies at the SCD
locus in Canadian Holsteins and Jerseys.
3) Based on the polymorphisms, to develop a strategy for quickly genotyping the
animals at the SCD locus to facilitate genetic selection for increased CLA content of
milk fat.
4) To test for associations between polymorphisms in the SCD gene and milk fat
percentage, milk fat yield, protein yield and percentage, somatic cell count and total
milk yield in Canadian Holsteins and Jerseys.
5) To test for associations between polymorphisms in the SCD gene and the CLA
content of milk in Canadian Holsteins and Jerseys.
6) To test for associations between polymorphisms in the SCD gene and the MUFA
content of milk fat in Canadian Holsteins and Jerseys.
Approach:
The study was divided into two phases: The pilot project to address the first three objectives
and the population study to address the last three objectives.
Progress on the Pilot Project
The pilot project was aimed at establishing the existence of polymorphisms in 50 randomly
selected Holsteins and 50 randomly selected Jerseys by direct DNA sequencing, determination
of different alleles at the SCD locus in the two breeds and development of a strategy for
quickly genotyping the animals at the SCD locus. To date the following have been done in
order to address the afore-mentioned objectives.
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Either blood or milk samples were collected from 50 randomly selected Holsteins and 50
randomly selected Jerseys for DNA isolation. Based on the SCD gene sequence deposited
in the gene bank (Accession No. AY241932), primers were designed for selective
amplification of the open reading frame region and the 5’ and the 3’ Untranslated regions
of the SCD gene using PCR. The PCR products were then sent to Genome-Quebec for
subsequent purification and sequencing.
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Analysis of the ORF sequences of the SCD gene revealed SNPs: A702G, T762C and
C878T in both breeds and an additional SNP, G435A, unique only to Holsteins. The SNPs
were linked resulting in 4 different genetic variants (alleles) in Holsteins containing: A
(G435 A702 T762 C878), A1 (A435 A702 T762 C878), B (G435 G702 C762 T878) and B1 (A435 G702
C762 T878) and only variants A and B in Jerseys. The A variant was by far the most
prevalent in both breeds suggesting that this may indeed be the wild or dominant allele at
the SCD locus, followed by the B variant. The A1 and B1 variants occur at relatively low
frequencies in Holsteins. One SNP C878T resulted in a non-synonymous codon change
while the rest resulted in synonymous codon changes giving rise to only two protein
variants (type A containing alanine and type V containing valine).
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A paper entitled ‘Single nucleotide polymorphisms in the open reading frame of the
Stearoyl-CoA desaturase gene and the resulting genetic variants in Canadian
Holstein and Jersey Cows’ has been published in DNA Sequence. 18 (5):357-362.
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Since only SNP C878T resulted in a non-synonymous codon change that might affect the
functionality of the resulting SCD enzyme, we have already developed a strategy for
distinguishing between the A (A1) and B (B1) variants based on restriction fragment
length polymorphism and this is currently used for quickly genotyping the cows at the
SCD locus in the population study .
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The 5’ and 3’ UTR of most genes are increasingly recognized as containing some
sequence motifs with regulatory roles in gene expression. In view of this, we also screened
the 5’ and 3’ UTRs of the SCD gene for SNPs. No SNPs were identified in the 5’UTR and
fifteen SNPs (G1571T, G1644C, C1763A, T2053C, A2584G, T2668C, A3007G, C3107T,
G3208A, T3290C, G3497A, G3682A, A4399T, C4533T and G4881A) were identified in
the 3´UTR region of the SCD gene. The SNPs linked together resulting in 3 different
haplotypes or regulatory variants in Holsteins: H1 (G1571G1644C1763C2053A2584C2668
A3007C3107G3208T3290G3497G3682A4399C4533G4881),H2 (G1571G1644A1763C2053A2584C2668G3007C3107
G3208T3290 G3497G3682A4399C4533G4881) and H3 (T1571C1644A1763T2053 G2584 C2668 G3007 T3107 A3208
C3290 A3497A3682T4399T4533A4881) and only H1 and H3 haplotypes or regulatory variants in
Jerseys. The search for functional motifs in the 3´UTR region of the SCD gene revealed the
presence of an internal ribosome entry site (IRES) motif only in the H1 regulatory variant.
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Previous studies reported the existence of the IRES motif only in the 5´UTRs of most
genes but we hereby report its presence also in the 3´UTR of the Bovine SCD gene and are
currently establishing the role of this motif and the different versions of IRES motif found
in the H1 and H3 regulatory variants on expression of the reporter gene in both prokaryotic
and eukaryotic cells. Once this is established, we will prepare the second manuscript on
the SNPs in the 3´UTR of the SCD gene in Canadian Holsteins and Jerseys
Progress on the Population study
The population study seeks to establish associations between the genotype at the SCD locus
and milk composition traits, as well as associations between the genotype at the SCD locus
and the CLA and MUFA content of milk fat of 1000 Holsteins and 1000 Jerseys enrolled in
PATLQ (VALACTA) program in Quebec. To date the following have been done in the
population study.
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Milk composition data (Milk fat yield and content, Milk protein yield and content, Somatic
cell count and Total milk yield) have been collected in 1000 Holsteins and 700 Jerseys.
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A total of 500 Holsteins and 400 Jerseys have already been genotyped at the SCD locus
and the genotyping work is still in progress.
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We have determined the CLA and MUFA content of 700 Holsteins and 500 Jerseys using
gas chromatography. Determination of the CLA and MUFA work is also in progress.
Description and Justification of the deviation from the original objectives
In the initial proposal we intended to genotype and measure the CLA and MUFA of a 1000
Jerseys in the population study but may be forced to scale down the sample size due to fewer
animals registered with PATLQ (VALACTA). To date we have acquired 700 Jersey milk
samples from PATLQ (VALACTA) and hope to get more samples before the end of the year
depending on availability.
References
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