Polymorphism Identification , RH Mapping and Association of α-Lactalbumin Gene with Milk Performance Traits in Chinese Holstein *

Lactose synthase catalyses the formation of lactose which is the major osmole of bovine milk and regulates the milk volume. Alpha-lactalbumin (α-LA) is involved in the synthesis of lactose synthase in the mammary gland. Therefore α-LA is regarded as a plausible candidate gene for the milk yield trait. To determine whether α-LA is associated with milk performance traits, 1,028 Chinese Holstein cows were used to detect polymorphisms in the α-LA by means of single-strand conformation polymorphism (SSCP). Two nucleotide transitions were identified in the 5′ flanking region and intron 3 of α-LA. Associations of such polymorphisms with five milk performance traits were analyzed using a general linear model procedure. No significant associations were observed between these polymorphisms and the five milk performance traits (p>0.05). RH mapping placed α-LA on BTA5q21, linked most closely to markers U63110, CC537786 and L10347 (LOD>8.3), which is far distant from the region of the quantitative trait locus (QTL) on bovine chromosome 5 for variation in the milk yield trait. In summary, based on our findings, we eliminated these SNPs from having an effect on milk performance traits. (


INTRODUCTION
Lactose, produced in the mammary epithelial cell and packaged into secretory vesicles, is the major osmotic component of milk (Tucker, 1981).Because lactose cannot diffuse out of the secretory vesicles (Larson, 1985), water is drawn into the vesicles to balance the osmotic pressure.Alpha-lactalbumin (α-LA) is a major whey protein of bovine milk and is essential for the biosynthesis of lactose in the mammary gland.Within the mammary epithelial cells, α-LA forms a complex with the membrane bound enzyme β-1, 4-galactosyltransferase to form lactose synthase (Kuhn et al., 1980).Lactose synthase then synthesizes lactose in the Golgi apparatus, and α-LA and the lactose produced by the complex are secreted into the milk.So the concentration of α-LA is hypothesized to regulate milk volume through this mechanism by increasing or decreasing the amount of lactose secreted into the milk.
Dairy cow produces milk that contains approximately 1.2 g/L of α-LA and 5% lactose (Tucker, 1981).Previous study showed that both mRNA and protein levels of α-LA increase greatly at parturition, remain elevated throughout lactation, and then decrease within 2 d after the start of involution (Goodman and Schanbacher, 1991).The bovine α-LA has been localized on BTA5q21 (Threadgill and Womack, 1990;Hayes et al., 1993).Molecular genetic studies indicate that α-LA spans approximately 3,090 bp and is composed of four exons and three introns (Vilotte et al., 1987;Bleck and Bremel, 1993a).Polymorphisms have been reported at positions +15, +21, +54 and -1,689, -1,466 within the 5′ flanking region of α-LA (Bleck and Bremel, 1993a;Voelker et al., 1997), most of them, however, were focused on the 5′ flanking regions of the α-LA gene.Till now, only the polymorphism at the position +15 (α-LA (+15)) in the 5′ flanking region of α-LA gene has been reported to be associated with increased milk yield in Holstein cattle.The cows with α-LA (+15) AA (an adenine on both alleles at position +15) genotype had statistically higher PTA for milk, kilograms of protein, protein dollars, U63110, CC537786 and L10347 (LOD>8.3),which is far distant from the region of the quantitative trait locus (QTL) on bovine chromosome 5 for variation in the milk yield trait.In summary, based on our findings, we eliminated these SNPs from having an effect on milk performance traits.(Key Words : α-Lactalbumin, SNP, Milk Performance Traits, RH Mapping, Chinese Holstein) kilograms of fat, and fat dollars than the cows with α-LA (+15) BB.While, the α-LA (+15) BB animals had higher protein and fat percentages than α-LA (+15) AA animals (Bleck and Bremel, 1993b).In yak monomorphic pattern of α-LA was observed by Mao et al. (2004) and some polymorphism loci had been identified in Water Buffalo (Dayal et al., 2006).
As for candidate genes for milk product traits, prolactin was confirmed to be associated with milk yield, fat yield, protein yield and protein percentage in Chinese Holstein (He et al., 2006).Considering α-LA is an important candidate gene for milk performance traits, the aims of the present study were to screen the complete nucleotide sequence of the α-LA to identify its polymorphisms and determine whether there exists association between the polymorphisms and milk performance traits in Chinese Holstein.

Animals
Blood samples of 1,028 Chinese Holstein cows were collected randomly from 8 Chinese Holstein cattle farms in Beijing, in which regular and standard performance testing (DHI) have been carried out since 1999.The phenotypes for five milk performances traits, i.e. milk yield (MY), fat yield (FY), protein yield (PY), fat percentage (FP), and protein percentage (PP), over 305-d were obtained from the Beijing Dairy Cattle Center.With animal model and the additive genetic relationship matrix, the breeding value of five traits were evaluated simultaneously using the PEST computer programs (Groeneveld, 1990), which is a software package for multivariate prediction and estimation.Pedigrees of animals detected in the present study were traced back three generations to create the numerator relationship matrix, so that the A -1 matrix included 2,604 animals.The animal model is as follow: Where Y is the phenotype for each milk performance traits; hys is herd-year-season effect; T is the effect of the fetal number; M(T) is the month effect of calving nesting within the fetal number; α is the random polygenic component account for all known pedigree relationships ("animal model"; (Lynch and Walsh, 1997), including ungenotyped individual whose phenotypes were ignored), p is permanent environmental effect, and e is a random residual.Maximum likelihood solutions for σ a 2 , σ p 2 and σ e 2 were obtained using the REMLF90 program.

PCR-SSCP and sequencing analysis
Genomic DNA was isolated from blood samples by the phenol-chloroform method.Using oligo 6.0 program, 11 pairs of primers were designed according to the genomic sequence of α-LA (GenBank accession no.X06366) to amplify eleven fragments (designated LA1, LA2, LA3, LA4, LA5, LA6, LA7, LA8, LA9, LA10, and LA11).The gene from -707 bp upstream of the first exon to 548 bp downstream of the last exon, except a 331 bp fragment from position 1,435 to 1,765 was analyzed for each cow.The primer sequences of the amplified fragments were shown in Table 1.PCR reactions were carried out in a total of 20 µl volume including 50 ng of genomic DNA, 0.2 mM each primer, and 0.5 U of Taq DNA polymerase.The PCR protocol was 94°C for 7 min, followed by 35 cycles of 94°C for 30 s, annealing for 45 s and 72°C for 30 s, with a final extension at 72°C for 10 min.Two µl of PCR product of each sample was mixed with 8 µl of denaturing buffer (98% formamide, 0.09% xylene cyanole FF, and 0.09% bromophenol blue) and then denatured at 98°C for 10 min, followed by a rapid chill on ice for 10 min.The denatured PCR products were electrophoresed on 12% acrylamide gels for 18 h at 8 V/cm and stained by 0.2% AgNO 3 for 20 min, and then 3% Na 2 CO 3 for about 5 min (Qu et al., 2005).Genotypes were recorded according to band patterns.

Association analysis
Associations between SNPs within α-LA and 5 milk performance traits were performed with GLM procedure of SAS 8.02 software, respectively.The significance of differences among various genotype effects of each SNP on estimated breeding values (EBVs) for MY, FY, PY, PP, and FP were calculated using Duncan's multiple-range test.
Because the estimated breeding values are the best available estimates of the additive genotype of the cow, no environmental effects were included in the model.The model used was as follows: Where Y ij is EBV for each milk performance trait; µ is overall mean; S i is sire effect; G j is genotype effect; e ij is random residual effect.

Radiation hybrid mapping
Radiation hybrid mapping was carried out using the forward and reverse primers: 5´-CCATAAAGCACTCTG TTC-3´ and 5´-GAGCAAGGGTCAAAAGTC-3´, and a 5,000-rad RH panel consisting of 90 cell lines (Womack et al., 1997).The PCR protocol was 94°C for 7min, followed by 35 cycles of 94°C for 30 s, 58°C for 45 s, and 72°C for 30 s, and a final extension at 72°C for 10 min.The amplification of each cell line was assessed by electrophoresis in 2% agarose gel.The hybrids were scored for presence/absence of the amplified product.Each marker was repeated twice to minimize possible experimental bias.
Map positions of the α-LA will be given relative to markers in the third generation whole genome comparative map of cattle and humans (Everts-van der Wind et al., 2005).RH analysis was done using RHMAPPER-1.22 (Slonim et al., 1997).

Identification of SNPs within α-LA
Eleven α-LA fragments (LA1 to LA11), which cover almost the entire length of the α-LA gene, were amplified.The positions of the fragments were shown in Figure 1.
With the eleven sets of primers, all of fragments of α-LA were successfully amplified.Two single nucleotide polymorphisms (SNPs), α-LA (+15) (a single base variation 15 bp 3´ of the transcription start point) and α-LA (I3) (a single base variation in intron 3), were identified (Figure 2), in which three kinds of band patterns were observed, and named as genotypes AA, BB and AB, respectively.As shown in Table 2, the frequency for allele B (0.6841) at α-LA (+15) locus was higher than that for allele A (0.3159).

Association between α-LA and milk performance traits
With PEST program, the EBVs of milk yield, fat yield, protein yield, fat percentage, and protein percentage over 305 days were estimated on 1,028 Chinese Holstein cows, respectively.Means, standard deviations, and minimum and maximum values of the EBVs for all 5 traits are summarized in Table 3.
Based on the EBVs, associations were tested of α-LA (+15) and α-LA (I3) for five milk performance traits.No significant associations with five milk performance traits were observed for these two SNPs (p>0.05)(Tables 4 and  5).

RH mapping of α-LA
Statistical analysis of the RH mapping vectors indicated that the α-LA gene was linked most closely to markers U63110, CC537786 and L10347, situated on BTA5.Two-  point LOD scores between α-LA and the three markers are summarized in the Table 6.These data demonstrated that α-LA is significantly linked to the three markers.Figure 4 represents the position of α-LA on cytogenetic, radiation hybrid and linkage maps.

DISCUSSION
Initially, screening for variation was performed on α-LA in 1028 Chinese Holstein cows.Only two polymorphisms were identified in the LA4 and LA9 fragments, namely α-LA (+15) and α-LA (I3) polymorphism loci.However, we did not find the polymorphisms the positions +21 and +54 (Bleck and Bremel, 1993a).The α-LA (I3) polymorphism locus, T2413C, was found for the first time in this study.The genotypes and alleles frequencies of α-LA (+15) were in good agreement with the result previously published (Bleck and Bremel, 1993b).A allele within the population is about 31.59%(Table 2).In addition, independent association studies were performed based on breeding value for five milk performance traits by general linear model.No significant association between the two SNPs and milk production traits was observed in present research.The result of α-LA (+15) polymorphism loci having no association with milk  performance traits was inconsistent with previous research result (Bleck and Bremel, 1993b).These results may have arisen from differences in the number of animals analyzed and statistical approaches taken.
Finally, we mapped α-LA in a bovine hamster radiation hybrid panel.α-LA was significantly linked to markers U63110, CC537786 and L10347 with lod scores more than 8.3, located on BTA5q21.Quantitative trait locus (QTL) affecting milk yield (MY) has been identified on BTA5.A chromosome-wise significant QTL for MY with large effect was detected between the marker interval BM1819-BM2830 using a granddaughter design (Viitala et al., 2003).Probably the same QTL was detected by De Koning et al. (2001) and Bennewitz et al. (2003).As showed in Figure 4, the three marks were assigned to a region on bovine chromosome 5 between CSSM34 (315 cR and 45 cM) and RM500 (398 cR and 55.6 cM).So the α-LA gene was located to the marker interval CSSM34-RM500 and was more than 22 cM away from the QTL for milk yield along the BTA5, because the interval with two boundary markers, RM500 (398 cR and 55.6 cM) and BM1819 (77.60 cM), is 22 cM.
In conclusion, two polymorphism loci were identified in 5´ flanking region and intron 3 respectively.The association studies results indicated that neither of them was associated with milk performance trait.α-LA was assigned to BTA5q21, closely linked to three marks, far away from the QTL region for milk yield along the BTA5.Based on the association studies and RH mapping, we excluded these SNPs having effect on milk performance traits.

Figure 1 .Figure 2 .
Figure 1.Map of the positions of the eleven amplified fragments within α-LA gene.Open arrowheads indicate the locations of the polymorphic sites.

Table 1 .
Primer sequences of the 11 amplified fragments Fragments 1

Table 2 .
Genotypes 1The animal numbers for each genotype are difference due to the missing data during the genotyping.

Table 3 .
Means, standard deviations, and minima and maxima of the EBV for the 5 milk production traits

Table 4 .
p values for associations between each of SNPs in bovine α-LA gene and milk performance traits

Table 5 .
Least square means of breeding value for 5 milk performance traits(LS Means±SE)

Table 6 .
Pairwise two-point lod scores between α-LA and three