A Research on Association between SCD1 and OLR1 Genes and Milk Production Traits in Iranian Holstein Dairy Cattle

Document Type: Research Articles


Department of Animal Science, Mashhad Branch, Islamic Azad University, Mashhad, Iran


The present study was carried out to investigate the association of C/T single nucleotide polymorphism(SNP)in exon 5 of stearoyl-CoA desaturase 1 (SCD1) gene and A/C SNP in the 3' untranslated region of oxidized low density lipoprotein receptor 1 (OLR1)gene with milk production traits in Iranian Holstein dairy Cattle. The blood samples of 153 (for OLR1) and 308 (for SCD1) dairy cattle from three different farms were used for genotyping. A 146 bp fragment of 3'UTR of OLR1 gene and a 400 bp fragment of exon 5 of SCD1 gene were amplified by standard PCR. Single nucleotide polymorphism of OLR1 and SCD1 gene was determined by polymerase chain reaction single-restriction fragment length polymorphism (PCR-RFLP) technique. The association between genotypes of OLR1 and SCD1 genes with milk production traits was studied by general linear models (GLM) procedure of SAS package and Duncan test was used for comparing means of traits. The frequency of AA, AV and VV genotype of SCD1 gene were 0.60, 0.32 and 0.08 respectively. The frequency of alleles A and V were 0.76 and 0.24. The genotype frequencies of AA, AC and CC in OLR1 gene were 0.22, 0.50 and 0.28 respectively. The frequency of allele A and C was 0.47 and 0.53. Thus, this population was in Hardy-Weinberg equilibrium for OLR1 but not for SCD1. The means of fat percentage for SCD1 genotypes were 3.43% and 3.33 for VV and AA respectively (P<0.05). The means of OLR1 genotypes were 8273 kg (CC), 8344 kg (AC) and 7178 kg (AA) for milk yield; 276.3 kg (CC), 277.6 kg (AC) and 239.7 kg (AA) for fat yield and for protein yield were 286.7 kg (CC), 290.5 kg (AC) and 253 kg (AA) (P<0.05). The results revealed these two SNP are appropriate for marker assisted selection.



Quantitative traits are controlled by large numbers of genes and also influenced by environmental factors. Recent researches with farm animals have quantified the effect of candidate genes on economically important traits. Identification of genes with large effects on milk production traits would be useful for genetic improvement programs in dairy cattle (Wang et al. 2016; Cecchinato et al. 2015; Hosseinpour et al. 2013). Stearoyl-CoA desaturase (SCD) is an enzyme that plays an important role in the biosynthesis of fatty acids. This enzyme belongs to a large family of enzymes that are involved in the synthesis of saturated fatty acids and they are found in both animals and plants. Stearoyl-CoA desaturase (SCD) is the enzyme responsible for conversion of saturated fatty acids into 9-monounsaturated fatty acids in mammalian adiposities (Campbell et al. 2001; Taniguchi et al. 2004). Two SCD isoforms are known in cattle, SCD5 is expressed in brain and is located on chromosome 6 and SCD1 is located on chromosome 26 and expressed in adipose and mammary tissue (Schennink et al. 2008). The cytosine to thymine substitution causes alanine change to valine amino acids on protein (A293V). Several researches have shown significant effects of genotypes SCD1A293V in exon 5 on the composition of fatty acids in milk and milk production traits (Moioli et al. 2007; Kgwatalala et al. 2009; Clark et al. 2010). The major protein oxidized low density lipoprotein receptor1 (OLR1), was initially identified in bovine aortic endothelial cells, this protein binds, internalizes, and degrades oxidized low-density lipoprotein (Sawamura et al. 1997). The oxidized form of the low-density lipoprotein (oxLDL) is involved in endothelial cell injury, dysfunction, and activation, all of which are implicated in the development of atherosclerosis (Mehta and Li, 1998).The oxLDL and its lipid constituents have numerous damaging effects on secretory activities of the endothelium, including induction of apoptosis (Imanishi et al. 2002). The OLR1 gene encodes a vascular endothelial cell-surface receptor that binds and degrades the oxidized forms of low-density lipoproteins (oxLDL) (Mehta and Li,  2002). The genomic sequence of bovine OLR1, released by Baylor College of Medicine, contains five exons and located on chromosome 5. The length of this gene is 11373 base pairs (GenBank accession no. NW_215807). The bovine OLR1 gene encodes 270 AA that has a 72% identity to the human protein (Sawamura et al. 1997). Many studies have been conducted on QTL in bovine chromosome 5 near OLR1 affecting milk production traits between 1999 and 2004 (Heyen et al. 1999; Olsen et al. 2002; Ashwell et al. 2004; De Koning et al. 2001; Viitala et al. 2003; Rodriguez-Zas et al. 2002). Direct cDNA and genomic sequencing of OLR1 revealed 2 single nucleotide polymorphisms (SNP) inexon 4, 5 SNP in intron 4 and 1 in the 3' untranslated region (3'UTR) (Khatib et al. 2006). Some researchers reported that allele C of SNP in the 3'UTR had significant effects on fat yield and fat percentage. Khatib et al. (2006) reported significant effects of A/C SNP in the 3′-untranslated region of OLR1on milk fat yield and fat percentage in a granddaughter-design Holstein bull population. Association between OLR1 haplotypes and milk production traits was further confirmed in a daughter-design study of Holstein cows and in an Italian Brown Swiss population (Khatib et al. 2007). Schennink et al. (2009) reported a significant association between OLR1 and milk fat percentage in Dutch Holstein-Friesian cattle. Because of the role of OLR1 in lipid metabolism and degradation of Ox-LDL, the OLR1 gene has been regarded as a candidate gene affecting milk production traits in dairy cattle (Khatib et al. 2006). Thus, the objective of this research was to study the association between genes OLR1 and SCD1 and milk production traits in Iranian Holstein dairy cattle.



Samples and phenotypic data

Blood samples of 153 (for OLR1) and 308 (for SCD1) of Iranian Holstein dairy cattle were randomly selected for genotyping from three different farms in the Khorasan Razavi province of Iran. Records of 136 (OLR1) and 274 (SCD1) cows were used for the association study. The Studied traits were 305-day milk, fat and protein yield and fat and protein percentage.


Genotyping of SNP

Approximately 5 mL of blood were collected from the jugular vein of each animal in EDTA tubes. The aliquots of whole blood were stored at -20 ˚C. The genomic DNA from blood samples was extracted using the GuSCN-Silica Gel method and standard protocol with commercial kit of DIAtom DNA Prep (Biokom). The quality of DNA was examined by agarose gel electrophoresis. Genotyping was carried out using the PCR-RFLP technique. The 146 bp fragment of OLR1 gene from 3'UTR (GenBank accession no. NC_007303.4; 107080852; 107092157 described in Khatib et al. 2006)was amplified by standard PCR by Biometra thermo cycler (Germany). The sequences of the primers were (F 5´-TCCCTAACTTGTTCCAAGTCCT-3´) and (R 5´-CTCTACAATGCCTAGAAGAAAGC-3'), respectively (Komisarek and Dorynek, 2009). The total volume of reaction was 25 µL that contained one unit (0.2 µL) of Taq polymerase, 200 µM (0.5 µL) of dNTP, 2 mM MgCl2, 10 pM (3 µL) primer mix and 2.5 µL standard buffer in 13.8 µL dH2O. Fifty nano grams (5 µL) DNA were added to the reaction mix. The thermal cycling conditions for the PCR were as follows: initial denaturation at 94 ˚C for 5 min, cyclic denaturation at 94 ˚C for 30 s, cyclic annealing of primers at 62 ˚C for 30 s, cyclic elongation at 72 ˚C for 45 s (for 30 cycles) and final elongation at 72 ˚C for 5 min (Komisarek and Dorynek, 2009). The PCR products were separated by electrophoresis on 2% agarose gel and visualized on gel documentation system (UVP, USA). The PCR product was digested with PstI restriction enzyme (Fermentas). The 5 µL of PCR product was mixed with 2 µL 10X buffer, 5 µL dH2O and 2 units of PstI enzyme and digested over 5 hours at 37 ˚C. Polyacrylamide gel electrophoresis methods were used to identify genotypes. The 400bp fragment of SCD1 gene that included exon 5 was amplified with standard PCR based on GenBank accession no. AY241932. The primers were as follow: F (5´-CCC ATT CGC TCT TGT TCT GT-3´) and R (5´-CCC ATT CGC TCT TGT TCT GT-3´). The total volume of PCR reaction was 25 µL similar to OLR1 fragment. The thermal program of PCR reaction was initial denaturation at 94 ˚C for 3 min, cyclic denaturation at 94 ˚C for 45 s, cyclic annealing of primers at 54 ˚C for 30 s , cyclic elongation at 72 ˚C for 90 s (for 34 cycles) and final elongation at 72 ˚C for 10 min (Kgwatalala et al. 2009). The PCR products were separated by electrophoresis on 2% agarose gel and visualized on UVP gel documentation system. The PCR product was digested with restriction enzyme NcoI (Fermentas). The 3 µL of PCR product was mixed with 2 µL 10X buffer, 4.5 µL dH2O and 2 units of NcoI and digested over 5 hours at 37 ˚C . The digested fragment was loaded in 2% agarose gel and visualized on UVP gel documentation system.


Statistical analysis

The Hardy-Weinberg equilibrium for allele and genotype frequencies was analyzed with Chi-square test using POPGENE software (Yeh et al. 1999). The association between genotypes and traits was assessed with the (GLM) procedure of SAS (2004) according to the following general linear model:

Yijnl= µ + Gi + HYSj + Lk + Sl + eijkl


Yijkl: value for each milk-related trait.

μ: overall mean.

Gi: fixed effect of the ith genotype (3 genotypes for each gene: AA, AV and VV for SCD1 or AA, AC and CC for OLR1).

HYSj: fixed effect of herd (1, 2, 3), year and season of parturition.

Lk: kth lactation (1 and 2).

Sl: random effect of sire (1,…,128).

eijkl: residual effects.

Due to use the records of traits, the fixed effects and the sire effect (as random genetic effect) were considered in the model. Genotype means were compared with the Duncan test (P˂0.05).



Gene and genotypic frequencies

Genotype and allele frequencies are shown in Table 1. The SCD1 genotypic frequencies for AA, AV and VV were 0.60, 0.32 and 0.08, respectively. The frequencies of alleles A and V of SCD1 were 0.76 and 0.24. The population was not in Hardy Weinberg equilibrium for gene SCD1. Because of population was under selection and probably other dispersive factor such as population size was affected. The higher frequency of the A allele is in agreement with results reported by other studies. Schennink et al. (2008) reported the frequency of allele A and V as 0.73 and 0.27. Kgwatalala et al. (2009) genotyped 525 Canadian Jersey cows for the SCD1 gene and reported genotypic frequencies as 0.686, 0.244 and 0.07 for the AA, AV and VV genotypes, respectively and frequencies of alleles A and V of 0.808 and 0.192. Genotype frequencies were in Hardy-Weinberg equilibrium. Cows of 3 breeds of northern Italy, Jersey, Valdostana and Piedmontese were genotyped at exon 5 of the SCD gene; the frequency of A allele was 0.94 for Jersey, 0.65 for Valdostana and 0.42 for Piedmontese (Moioli et al. 2007). Clark et al. (2010) determined the polymorphism of 143 and 215 cows in two studies. The distribution of genotypes among 143 dairy cows was 72 AA, 60 AV, and 11 VV animals. Therefore, allele frequencies were 0.71 (A) and 0.29 (V). In study 2, the distribution of genotypes for 215 dairy cows was 111 (AA), 85 (AV) and 19 (VV) animals. Allele frequencies were 0.71 and 0.29 for the A and V alleles. Frequencies of SCD genotypes in the sample of Italian Friesian cows were 0.27, 0.6 and 0.13 for AA, AV and VV genotypes, respectively (Mele et al. 2007). The higher frequency of the A allele (0.57) compared to allele V (0.43) agreed with the result of this study. The genotypic frequencies of gene OLR1 (AA, AC and CC) were 0.22, 0.5, and 0.28 respectively. Frequencies of A and C alleles were 0.47 and 0.53. The Chi-square test revealed that the population was in Hardy-Weinberg equilibrium, the two alleles frequencies were near to 0.5 so the population was in equilibrium and probably the selection and other factors were not effective. Khatib et al. (2006) and Komisarek and Dorynek (2009) reported the same frequency of 0.46 and 0.43 for allele A and 0.54 and 0.57 for allele C in the US, Polish and Holstein cattle populations respectively. Soltani-Ghombavan et al. (2013) estimated the frequency of allele C and A to be 0.483 and 0.517 in Holstein dairy cattle from 5 farms in the Esfahan province of Iran. Schennink et al. (2009) reported frequencies of 0.29 and 0.71 for alleles A and C in a Dutch Holstein population.


Association analysis

In general, the used regression model was significant for all studied traits with OLR1 or SCD1 genotypes included in model. The coefficient of determination (R2) was higher than 0.8 and 0.9 for most of traits that showed the fixed and random effects in general linear model justify the variation in traits. Trait means, standard errors, and coefficients of variation are also shown in Table 2.


Table 1 Genotype and allele frequencies


SCD1: stearoyl-CoA desaturase 1 and OLR1: oxidized low density lipoprotein receptor 1.

HWE: Hardy-Weinberg equilibrium.

** (P˂0.01).

NS: non significant.


Table 2 Analysis of general linear model for studied traits including SCD1 or OLR1 genotypes


SCD1: stearoyl-CoA desaturase 1 and OLR1: oxidized low density lipoprotein receptor 1.

MY: milk yield; FY: fat yield; PY: protein yield; FP: fat percentage and PP: protein percentage.

SE: standard error.

CV: coefficient of variation.


The coefficient of variation range for 305-d milk, fat and protein yield were between 14.7 (for fat yield) to 18.8 (for protein yield), this value for fat and protein percentage were lower and the range was between 6.25 (for fat percentage) and 7.3 (for protein percentage). The results of comparing traits means with different SCD1 and OLR1 genotype by Duncan multiple rang test are shown in Table 3.



The SCD1 genotypes showed significant effects on fat percentage but no effect on the other traits. The means of fat percentage for VV and AA genotype were 3.43% and 3.33% (P<0.05), this result was similar to Komisark and Dorynek (2009), that showed a positive effect of VV genotype on fat percentage. We found 305-d milk and protein yield means of cows with genotype AA to be higher than those of cows with the VV genotype. Kgwatalala et al. (2009) reported that allele A was positively associated with increased 305-d milk and protein yields. Effects of SCD genotype were not observed for milk yield or composition (Clark et al. 2010). The SCD1 genotype did not significantly affect fat or protein percentage and fat, protein, or milk yield (Schennink et al. 2008).



The comparison of traits mean for OLR1 genotypes are shown in Table 3. The milk yield mean of cows with genotype AA (7178 kg) was significantly higher (P<0.05) than those of cows with genotypes AC (8344 kg) and CC (8273 kg). The means of fat and protein yield for cows with the AC and CC genotypes were higher than the corresponding values for cows with the AA genotype (P<0.05). The means for fat and protein percentage in AA cows were somewhat higher than those for AC and CC cows, but differences were not significant (P˃0.05). Although other SNP were identified in OLR1, only the 3′-UTR SNP was found to be associated with milk traits in the Holstein population. Khatib et al. (2006) reported the positive effect of allele C on fat and protein percentage and fat yield (P<0.05). Soltani-Ghombavani et al. (2013) revealed a significant effect of OLR1 gene on fat and protein percentage and breeding value of fat yield in Iranian Holstein cattle, they found the value of milk yield for CC and AC genotype more than AA genotype but there was no significant association. Interestingly, quantitative real-time PCR analysis revealed that the expression level of OLR1 was higher in individuals bearing the CC genotype compared with the AA genotype of the 3′-UTR SNP, suggesting that C is the nucleotide causing increased expression of OLR1 or is in strong linkage disequilibrium with the causative SNP (Khatib et al. 2006). Komisarek and Dorynek (2009) reported a significant association between C allele and fat yield and fat percentage. The significant effect of allele C on fat yield in this study was similar to those reported by Khatib et al. (2006); Komisarek and Dorynek (2009) and Soltani-Ghombavan et al. (2013). The association of CC genotype with milk yield shown in this study disagreed with the findings of Khatib et al. (2006) and Komisarek and Dorynek (2009).


Table 3 Compare genotypes mean for milk production traits with different genotypes of SCD1 or OLR1 gene


SCD1: stearoyl-CoA desaturase 1 and OLR1: oxidized low density lipoprotein receptor 1.

MY: milk yield; FY: fat yield; PY: protein yield; FP: fat percentage and PP: protein percentage.

The means within the same column with at least one common letter, do not have significant difference (P>0.05).

SE: standard error.


Conversely, Schennink et al. (2009) also found no association between OLR1 gene and fat percentage in Dutch Holstein Friesian cows agreeing on results here. Similarly, no association was observed between polymorphism in OLR1 and either milk production traits or reproduction traits in a Czech Fleckvieh population (Rychtarova et al. 2014).



The frequency of allele A of SCD1 was 0.76 in the sample of Iranian Holstein dairy cattle studied here, and the association of genotype with fat percentage was significant. The population was in linkage disequilibrium for SCD1, perhaps due to selection for fat percentage in this population. Although the frequency of the C allele of gene OLR1 was somewhat higher than that of the A allele, the population was in linkage equilibrium. However, the C allele had significant effects on milk, fat and protein yields, it could be concluded the favorable allele for SCD1 and OLR1 gene are A and C allele, thus these alleles could be used in a marker-assisted selection program to increase favorable alleles frequencies in the population and also these SNPs are proper for having on commercial SNP panels.

Ashwell M.S., Heyen D.W., Sonstegard T.S., Van Tassell C.P., Da Y., VanRaden P.M., Ron M., Weller J.I. and Lewin H.A. (2004). Detection of quantitative trait loci affecting milk production, health, and reproductive traits in Holstein cattle. J. Dairy Sci. 87, 468-475.

Campbell E.M.G., Gallagher D.S., Davis S.K., Taylor J.F. and Smith S.B. (2001). Mapping of the bovine stearoyl coenzyme A desaturase (SCD) gene to BTA26. J. Anim. Sci. 79, 1954-1955.

Cecchinato A., Chessa S., Ribeca C., Cipolat-Gotet C., Bobbo T., Casellas J. and Bittante G. (2015). Genetic variation and effects of candidate-gene polymorphisms on coagulation properties, curd firmness modeling and acidity in milk from Brown Swiss cows. Animal. 9(7), 1104-1112.

Clark L.A., Thomson J.M., Moore S.S. and Oba M. (2010). The effect of Ala293Val single nucleotide polymorphism in the stearoyl-CoA desaturase gene on conjugated linoleic acid concentration in milk fat of dairy cows. Canadian J. Anim. Sci. 90, 575-584.

De Koning D.J., Schulmant N.F., Elo K., Moisio S., Kinos R., Vilkki J. and Maki-Tanila A. (2001). Mapping of multiple quantitative trait loci by simple regression in half-sib designs. J. Anim. Sci. 79, 616-622.

Heyen D.W., Weller J.I., Ron M., Band M., Beever J.E., Feldmesser E., Da Y., Wiggans G.R., VanRaden P.M. and Lewin H.A. (1999). A genome scans for QTL influencing milk production and health traits in dairy cattle. Physiol. Genomics. 1, 165-175.

Hosseinpour Mashhadi H., Nassiri M.R., Emam Jome Kashan N. and Vaez Torshizi R. (2013). Associationbetween DGAT1 genotype and breeding value of milk production traits in Iranian Holstein bulls. Iranian J.Appl. Anim. Sci. 3, 811-815.

Imanishi T., Hano T., Sawamura T., Takarada S. and Nishio I. (2002). Oxidized low density lipoprotein potentiation of Fas-induced apoptosis through lectin-like oxidized-low density lipoprotein receptor-1 in human umbilical vascular endothelial cells. Circ. J. 66, 1060-1064.

Kgwatalala P.M., Ibeagha-Awemu E.M., Mustafa A.F. and Zhao X. (2009). Influence of stearoyl-coenzyme a desaturase 1genotype and stage of lactation on fatty acid composition of Canadian Jersey cows. J. Dairy Sci. 92, 1220-1228.

Khatib H., Leonard S.D., Schutzkus V., Luo W. and Chang Y.M. (2006). Association of the OLR1 gene with milk composition in Holstein dairy cattle. J. Dairy Sci. 89, 1753-1760.

Khatib H., Rosa G.J., Weigel K., Schiavini F., Santus E. and Bagnato A. (2007). Additional support for an association between OLR1 and milk fat traits in cattle. Anim. Genet. 38, 308-310.

Komisarek J. and Dorynek Z. (2009). Effect of ABCG2, PPARGC1A, OLR1 and SCD1 gene polymorphism on estimated breeding values for functional and production traits in Polish Holstein-Friesian bulls. J. Appl. Genet. 50, 125-132.

Mehta J.L. and Li D. (2002). Identification, regulation and function of a novel lectin–like oxidized low–density lipoprotein receptor. J. Am. Coll. Cardiol. 39, 1429-1435.

Mehta J.L. and Li D. (1998). Identification and autoregulation of receptor for OX-LDL in cultured human coronary artery endothelial cells. Biochem. Biophys. Res. Commun. 248, 511-514.

Mele M., Conte G., Castiglioni B., Chessa S., Macciotta N.P., Serra A., Buccioni A., Pagnacco G. and Secchiari P. (2007). Stearoyl-CoA desaturase gene polymorphism and milk fatty acid composition in Italian Holsteins. J. Dairy Sci. 90, 4458-4465.

Moioli B., Contarini G., Avalli A., Catillo G., Orru L., De Matteis G., Masoero G. and Napolitano F. (2007). Short communication: effect of stearoyl-coenzyme a desaturase polymorphism on fatty acid composition of milk. J. Dairy Sci. 90, 3553-3558.

Olsen H.G., Gomez-Raya L., Vage D.I., Olsaker I., Klungland H., Svendsen M., Adnoy T., Sabry A., Klemetsdal G., Schulman N., Kramer W., Thaller G., Ronningen K. and Lien S. (2002). A genome scan for quantitative trait loci affecting milk production in Norwegian dairy cattle. J. Dairy Sci. 85, 3124-3130.

Rodriguez-Zas S.L., Southey B.R., Heyen D.W. and Lewin H.A. (2002). Interval and composite interval mapping of somatic cell score, yield, and components of milk in dairy cattle. J. Dairy Sci. 85, 3081-3091.

Rychtarova J., Sztankoova Z., Kyselova J., Zink V., Stipkova M., Vacek M. and Stolc L. (2014). Effect of DGAT1, BTN1A1, OLR1 and STAT1 genes on milk production and reproduction traits in the Czech Fleckvieh breed. Czech J. Anim. Sci. 59 (2), 45-53.

SAS Institute. (2004). SAS®/STAT Software, Release 9.1. SAS Institute, Inc., Cary, NC. USA.

Sawamura T., Kume N., Aoyama T., Moriwaki H., Hoshikawa H., Aiba Y., Tanaka T., Miwa S., Katsura Y., Kita T. and Masaki T. (1997). An endothelial receptor for oxidized low-density lipoprotein. Nature. 386, 73-77.

Schennink A., Heck J.M., Bovenhuis H., Visker M.H., Van Valenberg H.J. and Van Arendonk J.A. (2008). Milk fatty acid unsaturation: genetic parameters and effects of stearoyl-CoA desaturase (SCD1) and acyl-CoA: diacylglycerol acyltransferase 1 (DGAT1). J. Dairy Sci. 91, 2135-2143.

Schennink A., Bovenhuis H., Le´on-Kloosterziel K.M., van Arendonk J.A.M. and Visker M.H.P.W. (2009). Effect of polymorphisms in the FASN, OLR1, PPARGC1A, PRL and STAT5A genes on bovine milk-fat composition. Anim. Genet. 40, 909-916.

Soltani-Ghombavan M., Ansari-Mahyari S., Ghorbani G.R. and Edriss M.A. (2013). Association of a polymorphism in 3' untranslated region of OLR1 gene with milk fat and protein in dairy cows. Arch. Tierz. 56, 32-40.

Taniguchi M., Utsugi T., Oyama K., Mannen H., Kobayashi M., Tanabe Y., Ogino A. and Tsuji S. (2004). Genotype of stearoyl-CoA desaturase is associated with fatty acid composition in Japanese Black cattle. Mamm. Genome. 14, 142-148.

Viitala S.M., Schulman N.F., de Koning D.J., Elo K., Kinos R., Virta A., Virta J., Maki-Tanila A. and Vilkki J.H. (2003). Quantitative trait loci affecting milk production traits in Finnish Ayrshire dairy cattle. J. Dairy Sci. 86, 1828-1836.

Wang Q., Hulzebosch A. and Bovenhuis H. (2016). Genetic and environmental variation in bovine milk infrared spectra. J. Dairy Sci. 99(8), 6793-6803.

Yeh F.C., Yang R.C. and Boyle T. (1999). POPGENE. Microsoft Windows-Based Freeware for Population Genetic Analysis. Release 1.31. Univ Alberta, Edmonton, Canada.