Journal of Heredity Advance Access published online on November 5, 2008
Journal of Heredity, doi:10.1093/jhered/esn098
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Brief Communications |
Identification of Novel Single Nucleotide Polymorphisms in Promoter III of the Acetyl-CoA Carboxylase-
Gene in Goats Affecting Milk Production Traits
From Agricultural Research Council, Via Salaria 31, 00015 Monterotondo, Italy
Address correspondence to Moioli Bianca at the address above, or e-mail: bianca.moioli{at}entecra.it.
Acetyl-CoA carboxylase-
(ACACA) is the major regulatory enzyme of fatty acid biosynthesis. We have sequenced a fragment of Promoter III of the ovine ACACA gene in 211 goats of 5 breeds. The caprine sequence showed a high nucleotide identity (99%) with the ovine. We have identified 3 novel single nucleotide polymorphisms (SNPs) that fall in the core sequence of putative binding sites of transcription factors and have lower allele frequency than the wild type in all breeds. We evaluated the allele substitution effect of the SNPs on milk traits in the Saanen and the Local Grey breeds. Results from this study show that the mutations are associated with fat yield.
Key Words: acetyl-CoA carboxylase • goat • polymorphisms
Acetyl-CoA carboxylase is the flux-determining enzyme in the regulation of fatty acid synthesis in animal tissues; the expression of the mammary gland isoform of this enzyme, during lactation, is regulated by the acetyl-CoA carboxylase-
(ACACA) gene (Barber and Travers 1995, 1998). Badaoui et al. (2007) have sequenced 5.5 kb of the goat ACACA cDNA, identifying one silent single nucleotide polymorphism (SNP) at exon 45 (C5493T), which was associated with fat yield, although, because synonymous, it was not expected to produce a structural change in the ACACA enzyme. The same authors concluded that the found associations could be explained by the presence of linked mutations located in the ACACA gene or in a neighboring locus. The structural and functional features of the ovine ACACA gene were described by Travers and Barber (2001) who showed that gene transcription is initiated from multiple promoters in a tissue-specific fashion, so that differential promoter utilization results in ACACA mRNA with distinct 5' untranslated regions (UTR). In particular, mammary gland transcripts are regulated by promoter III (PIII). In the same region, Moioli et al. (2005) detected 3 novel SNPs in the ovine sequence and showed that the frequency of the variant alleles varied significantly among sheep breeds; however, the in silico analysis of the whole fragment indicated that no SNP occurred in any binding site of transcriptional factors (TF). Because no reference in literature was found for the analysis of this region in goats, the present study was performed in order to analyze the genetic variability of the 5' UTR, encoding PIII, of the ACACA gene, in goats of different breeds, recorded for milk production traits, through the search of SNPs.
 |
Materials and Methods |
|---|
 Top Materials and Methods ResultsDiscussionFundingReferences |
|---|
Goats for genotyping consisted of 211 animals: 104 Saanen (from 2 flocks), 51 Local Grey (6 flocks), 26 Syrian, 21 Maltese, and 9 Girgentana; the last 3 breeds were reared in the same experimental station. The Saanen goats are submitted to regular milk recording and selection activity for improvement of dairy traits, and genetic merit (GM) for the traits under selection are available (ASSONAPA 2008). Lactations of the goats of the other 4 breeds were recorded following the International regulation of milk recording activity.
An amplicon of 392 base pairs (bp) was obtained by polymerase chain reaction (PCR) amplification of the DNA of the 211 goats. The amplified region included the DNA fragment from 1179 to 1477 bp of the locus AJ292286 [GenBank] (Barber and Travers 1998) and from 1 to 128 bp of the locus AJ001056 [GenBank] (Barber and Travers 1995), the first fragment having the last 35 bp in common with the second fragment.
Primers used for the amplification and PCR conditions were as follows: forward primer: CGA CTG CTT TCC CTC TTG AC and reverse primer: ATT ACA ATG GGC TCC CAC AC; 35 PCR cycles at annealing temperature of 60 °C for 45 s were performed.
The detection of heterozygous samples was performed through the denaturing high-performance liquid chromatography (DHPLC) Transgenomic WAVE system for nucleic acid fragment analysis, which allows the resolution of DNA fragments on the basis of differential retention of double-stranded versus single-stranded DNA (Underhill et al. 1997). All heterozygous samples detected in this way were submitted to direct sequencing, in both directions, on a PerkinElmer ABI Prism 310 DNA sequencer. Genotyping of the homozygous samples was performed by mixing a homozygous sample of known sequence with each of the unknown homozygous, as proposed by Moioli et al. (2005). More than one-third of the homozygous samples was also submitted to direct sequencing, in both directions, to confirm the genotyping results.
Statistical Analysis
Allelic substitution effect on milk production traits was estimated by regressing the phenotype on the number of copies of one allele of each SNP (Sherman et al. 2008) using a general linear model in SAS. Two analyses were performed.
- (1) On the Saanen goats only, using as dependent variable the GM, as issued by the Breed Society (ASSONAPA 2008), at the following traits:
- - total merit (= GM kg fat + 4 x GM kg protein)
- - GM kg fat
- - GM kg protein
- - GM percentage fat
- - GM percentage protein.
- - GM kg fat
- (2) Because no GM were available for the local breeds (Grey goats, Maltese, Syrian, and Girgentana) that have no Herdbook, a further analysis was performed on the average daily milk parameters of the lactation performed in 2006 by the Local Grey goats (51 animals) and the Saanen goats of one flock (27 animals), considering the following dependent variables:
- - g fat
- - g protein
- - percentage fat
- - percentage protein.
- - g protein
- - total merit (= GM kg fat + 4 x GM kg protein)
Also in this analysis, which included the breed as fixed effect, the allelic substitution effect on milk production traits was estimated by regressing the phenotype on the number of copies of one allele of each SNP.
|
Results |
|---|
|
TopMaterials and MethodsResultsDiscussionFundingReferences |
|---|
Direct sequencing of the goat fragment showed a high nucleotide identity (99%) with the ovine sequence. Three novel variants were identified in the analyzed goats at locus AJ292286 [GenBank] —1206 bp: C/T; 1255 bp: A/G; and 1322 bp: T/C. At the sites where SNPs had been found in sheep (Moioli et al. 2005), the analyzed goats were all homozygous.
Three types of profile, diagnostic for the presence of a heteroduplex, were evident, through DHPLC analysis, at the partial denaturating temperature of 60 °C (Figure 1).
|
In Table 1, allele frequencies of the variants are reported; because the SNP at position 1255 bp was found only in one goat of the Saanen breed, this variant was omitted in the table.
|
The occurrence of the mutated allele at 1206 and 1322 bp is 13% and 4%, respectively. The Saanen and the Local Grey goats show frequency or the mutated allele slightly higher than the average (Table 1).
The in silico analysis of the fragment was performed with the Genomatix software (Genomatix 1998–2008). The variant at 1206 bp falls in the core sequence of the zinc-binding protein factors, which belong to the group of TF known to regulate housekeeping genes and was identified within the most abundant conserved TF-binding sites of promoters for the oxidative phosphorylation system genes (van Waveren and Morales 2008). The variant at 1255 bp falls in the core sequence of the nuclear receptor (NR) subfamily 2 factors: NRs are transcription factors capable of exerting regulation of gene expression in the nucleus in response to various extra- and intracellular signals (Zhang et al. 2004). The variant at 1322 bp falls at the end of the core sequence of the basonuclin rDNA transcription factor, which is a nuclear zinc finger protein considered to be a transcriptional regulator (Andersen et al. 1997).
Due to the low occurrence of the rare allele in 3 breeds and because of the low numbers of available milk recorded goats in the same, we performed the statistical analysis for the association of the SNP with milk traits only on the Saanen and Local Grey goats. Moreover, variant 1255 A/G was not considered in the association analysis because it occurred only in one sample of Saanen.
The GM for the considered parameters, used in the statistical analysis (1), was estimated from all the lactations of the 104 Saanen goats (228 lactations) and is expressed as deviation from the corresponding milk trait averages of more than 32 000 standard lactations (210 days) performed by 15 615 does, from 1999 to 2006.
Because no GM were available for the Local Grey goats, descriptive statistics of the average daily milk parameters of the lactation of the Saanen and Local Grey goats, used in the statistical analysis (2), are reported in Table 2.
|
The estimates of the allele substitution effect on the GM, for the Saanen goats and for the various milk traits, obtained from the statistical analysis (1) are presented in Table 3. The mutated allele at 1206 bp contributes to increase fat yield (+0.35 kg; P = 0.18), protein yield (+0.36 kg; P = 0.12), and consequently the total merit (+18 kg; P = 0.12). The mutated allele at 1322 bp contributes to increase fat yield (+0.78 kg; P = 0.05), protein yield (+0.58 kg; P = 0.10), and consequently the total merit (+32 kg; P = 0.08). Estimate for fat percentage is also positive.
|
In Table 4, we reported the estimates of the allele substitution effect on the daily milk production parameters of one lactation, obtained from the statistical analysis (2), for the Saanen and Local Grey goats. A positive effect of the mutated allele at 1206 bp was found on fat percentage and protein percentage (+0.17; +0.08), whereas the mutated allele at 1322 bp positively affects fat percentage (+0.25) and fat and protein yield (+10.31 g; +4.62 g), although the value is statistically significant only for fat yield.
|
|
Discussion |
|---|
|
TopMaterials and MethodsResultsDiscussionFundingReferences |
|---|
ACACA gene is known to influence the synthesis of fatty acids; in this work, we have analyzed a portion of the 5' UTR, encoding Promoter III, which regulates mammary gland transcripts. We expected, therefore, that the novel SNPs influenced, particularly, fat content. The statistical analysis allows to draw only preliminary conclusions, because of the feeble statistical significance of the allele substitution effects, due to the low frequency of the mutations. Globally, from the results of the association analysis on the GM of the Saanen goats, it appears that fat and protein yield are affected by the variant alleles at both loci; in the analysis performed on the day test of one lactation, both variants show a positive effect on fat content, whereas variant at 1322 bp positively affect fat yield.
Different expression of this gene in the lactating mammary gland (Veltri 2000) of 2 Italian sheep breeds with different milk fat content suggested a direct involvement of this gene in the fatty acid synthesis during lactation, and Badaoui et al. (2007) suggested that the presence of mutations in the Promoter could be associated with milk fat content. The 3 identified novel SNPs in Promoter III of the ovine ACACA gene fall in the core sequence of putative binding sites of transcription factors, therefore they might explain the difference in milk fat content of goats of different genotypes. The question arises why the SNPs have such low frequency. The Saanen breed had the highest frequency of the variant alleles (Table 1), and it is the only breed where sound selection programs have been performed in Italy. However, only recently the selection objective was directed to the increase of protein and fat yield; in the past, only milk yield was taken into account in the selection schemes. For the other breeds, which are all local and nonselected breeds, milk fat, and protein content was seldom recorded, and shepherds were obviously prone to select the breeding stock among the higher milk producers.
Our results need to be followed by further association studies using larger, diverse populations, in order to achieve a reliable picture of the variability of this locus and its influence on milk fat and other traits related to milk composition. The 3 novel SNPs can be easily detected through DHPLC analysis (Figure 1) so avoiding expensive and time-consuming direct sequencing.
Finally, ACACA is certainly not the only candidate gene affecting milk fat content. In goats, several studies are in progress (Moioli et al. 2007), aiming to identify SNPs in candidate genes that have proved to influence milk fat content in cattle, like stearoyl-CoA desaturase, lipoprotein lipase, acyl CoA-diacylglycerol-acyltransferase 1; it is therefore expected that further field studies will be carried on, to demonstrate the association between genetic variants and milk quality.
|
Funding |
|---|
|
TopMaterials and MethodsResultsDiscussionFundingReferences |
|---|
This study is part of the "GENZOOT" research programme, funded by the Italian Ministry of Agriculture (132/7303/2006) to D.M.G.
|
Acknowledgments |
|---|
We thank Dr Salvatore Murru, Director of the Italian Sheep and Goats Breeders Association, for making the genetic evaluations results available for this study and Prof. Bruno Ronchi of the University of Viterbo for providing part of the DNA samples used for this study.
|
Footnotes |
|---|
Corresponding Editor: James Womack
Received May 20, 2008
Revised July 21, 2008
Accepted October 15, 2008
|
References |
|---|
|
TopMaterials and MethodsResultsDiscussionFundingReferences |
|---|
-
Andersen B, Hariri A, Pittelkow MR, Rosenfeld MG. Characterization of Skn-1a/i POU Domain factors and linkage to papillomavirus gene expression. J Biol Chem. (1997) 272(25):15905–15913.
ASSONAPA. Internal statistics [Internet] (2008) Rome (Italy): Italian Association of Sheep and Goats Breeders. Available from www.assonapa.it.
Badaoui B, Serradilla JM, Tomas A, Urrutia B, Ares JL, Carrizosa JA, Sanchez A, Jordana J, Amills M. Goat acetyl-coenzyme A carboxylase : molecular characterization, polymorphism, and association with milk traits. J Dairy Sci. (2007) 90:1039–1043.
Barber M, Travers M. Cloning and characterization of multiple acetyl-CoA carboxylase transcripts in ovine adipose tissues. Gene (1995) 154(2):271–275.[CrossRef][Web of Science][Medline]
Barber M, Travers M. Elucidation of a promoter activity that directs the expression of acetyl-CoA carboxylase with an alternative N-terminus in a tissue-restricted fashion. Biochem J (1998) 333:17–25.[Web of Science][Medline]
Genomatix. Genomatix Software GmbH, München, Germany (1998–2008) Available from www.genomatix.de.
Moioli B, D'Andrea M, Pilla F. Candidate genes affecting sheep and goat milk quality. Small Rumin Res. (2007) 68:179–192.[CrossRef]
Moioli B, Napolitano F, Orrù L, Catillo G. Single nucleotide polymorphism detection in Promoter III of the Acetyl-CoA carboxylase- gene in sheep. J Anim Breed Genet. (2005) 122:418–420.[CrossRef][Web of Science][Medline]
Sherman EL, Nkrumah JD, Murdoch BM, Li C, Wang Z, Fu A, Moore SS. Polymorphisms and haplotypes in the bovine NPY, GHR, GHRL, IGF2, UCP2, and UCP3 genes and their associations with measures of growth, performance, feed efficiency and carcass merit in beef cattle. J Anim Sci. (2008) 86:1–16.
Travers M, Barber M. Acetyl-CoA carboxylase- : gene structure-function relationships. J Anim Sci. (2001) 79(E. Suppl):136–143.
Underhill PA, Jin L, Lin AL, Mehdi SQ, Jenkins T, Vollrath D, Davis R, Cavalli-Sforza LL, Oefner PJ. Detection of numerous Y chromosome biallelic polymorphisms by denaturing high performance liquid chromatography. Genome Res. (1997) 7:996–1005.
van Waveren C, Moraes CT. Transcriptional co-expression and co-regulation of genes coding for components of the oxidative phosphorylation system. BMC Genomics (2008) 9:18.[CrossRef][Medline]
Veltri C. Structure and expression variability of genes involved in milk production in small ruminants. (2000) [Campobasso (Italy)]: Università del Molise. [PhD thesis].
Zhang Z, Burch PE, Cooney AJ, Lanz RB, Pereira FA, Wu J, Gibbs RA, Weinstock G, Wheeler DA. Genomic analysis of the nuclear receptor family: new insights into structure, regulation, and evolution from the rat genome. Genome Res. (2004) 14:580–590.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||