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J. Appl. Genet. 45(4), 2004, pp.

445-452

New SSCP polymorphism within bovine STAT5A gene and its associations with milk performance traits in Black-and-White and Jersey cattle
Pawe Brym, Stanisaw Kamiski, Anna Ru
Department of Animal Genetics, University of Warmia and Mazury, Olsztyn, Poland

Abstract. Milk protein genes expression in cows mammary epithelial cells is regulated mostly by the action of prolactin mediated through the STAT5A transcription factor. The STAT5A gene is a potential quantitative trait locus (QTL) and genetic marker of production traits in dairy cattle. The sequence of the bovine STAT5A gene was analysed in this study to investigate if mutations in this sequence might be responsible for quantitative variations in milk yield and composition. Ten PCR fragments representing most important functional domains of STAT5A were screened for polymorphism. Using the SSCP method a new SNP (A/G) was found, located in intron 9 at position 9501 (GenBank AJ237937). The frequencies of alleles were estimated in 186 Black-and-White cows (0.52 and 0.48 for A and G, respectively) and in 138 Jersey cows (0.58 and 0.42 for A and G, respectively). For Black-and-White cows with different STAT5A genotypes no significant associations between STAT5A genotypes and milk performance traits were found. Statistically significant differences in the first and second lactations for milk yield, fat and protein content were found in Jersey cows. Cows with the GG genotype showed the highest milk yield, while cows with genotypes AA and AG showed higher protein contents when compared to cows with the GG genotype. Interestingly, cows with genotype AG showed significantly higher protein yields in comparison to cows with the AA genotype. For fat content, cows with genotype AA showed the highest level of this trait in the 1st and 2nd lactation. Further studies are necessary to evaluate an allele substitution effect in the population of sib-families of STAT5A heterozygous bulls.
Key words: bovine, polymorphism, SSCP, STAT5A.

Introduction
Signal Transducers and Activators of Transcription 5 (STAT5) are members of a family of transcription factors and are thought to play a central role in signal transmitting from prolactin to milk protein genes (Bole-Feysot et al. 1998). STAT5 dimers bind to GAS sequences located in promoters of milk protein genes and activate their transcription. Therefore they are suggested as candidate genes associated with milk protein yield and percentage in dairy cattle. STAT5 exists in two closely related forms, A and B, encoded by two separate genes (Darnell 1997). Gene disruption experiments proved their mandatory role for

mammary gland development and lactogenesis (Teglund et al. 1998). The bovine STAT5A and STAT5B genes were mapped on chromosome 19q17 (Goldammer et al. 1997) within 40 kb STAT locus containing also the STAT3 gene (Molenaar et al. 2000). STAT5A consists of 19 exons encoding 794 amino acid chains (Seyfert et al. 2000). Several mutations of the STAT5A gene (GenBank AJ242522 and AJ 237937) were reported. McCracken et al. (1997) found TG repeats of different length within intron 12. Antoniou et al. (1999), and Flisikowski and Zwierzchowski (2002) described SSCP variants of the STAT5A gene. The same group reported the deletion of CCT in intron 15 and the substitu-

Received: June 21, 2004. Revised: September 1, 2004. Accepted: September 20, 2004. Correspondence: S. Kamiski, Department of Animal Genetics, University of Warmia and Mazury, M. Oczapowskiego 5, 10-957 Olsztyn, Poland, email: stachel@uwm.edu.pl

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tion T/C at position 12743 within exon 16, which changed the amino acid sequence V686A (Flisikowski and Zwierzchowski 2003, Flisikowski et al. 2003a). Associations between STAT5A genotypes and beef production traits were found (Flisikowski et al. 2003b). The aim of this study was to screen most of the exons of the STAT5 gene in a sample of Jersey and Black-and-White cattle to find mutations, validate their frequency and evaluate them as potential markers of milk performance traits.

Materials and methods


Animals

Four groups of animals were included in the experiment: 1. inter-breed panel of individuals coming from several diverse breeds: Polish Black-and- White (n = 15), Jersey (n = 6), Polish Red (n = 6), Simmental (n = 3), Limousine (n = 3) and Charolaise (n = 3). 2. 13 Black-and-White bulls used in A. I. 3. 188 Black-and-White cows (one herd) 4. 138 Jersey cows (one herd)
DNA samples and PCR

(exon 16) forward 5 agctccaatcctcctcctgt 3, reverse 5 ggagtgtgttgggaggagtg 3; STAT5A 241 bp (exon 19) forward 5 gctccctctgatacccctct 3, reverse 5 acactggggattgtttccac 3. All the primers were tested for sequence similarities with the BLAST program (Altschul et al. 1990, www.ncbi.nih.nlm.gov). Primers were synthesized in MWG Biotech (www.mwgdna.com). PCR was carried out in 25 ml of mix containing: 1.25 ml 20x PCR buffer (400 mM (NH4)2SO4, 1.0 M Tris-HCl, pH 9.0), 1.3 ml dNTP (2 mM each), 70 pmol of each primer, MgCl2 in concentrations listed below, 0.8 U Tfl DNA Polymerase (Epicentre), 10x Enhancer (Epicentre) 100-600 ng of genomic DNA and H2O ad 25 ml. The PCR mix was placed on an Eppendorf Mastercycler 5330 thermocycler and subjected to thermal conditions, consisting of an initial DNA denaturation (94oC/2 min), 35 cycles of amplifications with constant temperatures of denaturation (94oC/30 s) and synthesis (72oC/30 s), but with different temperatures of primer annealing, and final synthesis (72oC/5 min) (indicated below).
Amplicon STAT5 240 bp STAT5 224 bp STAT5 219 bp STAT5 209 bp STAT5 251 bp STAT5 223 bp STAT5 235 bp STAT5 249 bp STAT5 241 bp Mg conc. (mMol) 2 1,5 2 3 2 2 1,5 1,5 1,5 Enhancer (ml/25m l) 3 2 4 5 2 3 2 4 4 Ta (oC) 58 61 62 58 58 59 60 61,5 61,5

Approximately 9 ml of blood were taken from animals. Genomic DNA was isolated from leukocytes with the MasterPure DNA Purification Kit (Epicentre). Based on the genomic sequences available in the GenBank (AJ237937), the following PCR primers were designed by PRIMER3 software (http://www-genome.wi.mit.edu.): STAT5A 240 bp (exon 9) forward 5 aagtgctcctgtcccttgtg 3, reverse 5 gggagggcagatgaagga 3; STAT5A 224 bp (exon 10) forward 5 ccagggtgcatacaggacag 3, reverse 5 gcaggttacgaggactcagg 3; STAT5A 219 bp (exon 11) forward 5 tcgtccccttttgatctcc 3, reverse 5 gattccagtctctggctttcc 3; STAT5A 209 bp (exon 12) forward 5 ccctggattcccgtagac 3, reverse 5 aagggtgggagaagaccact 3; STAT5A 251 bp (exon 13) forward 5 ttcccttttcttctctccctct 3, reverse 5 caggcaatgcgtcccttac 3; STAT5A 369 bp (exons 14 and 15) forward 5gggacacaagccctaacct 3, reverse 5 ggcaatgcagggaactca 3; STAT5A 223 bp (exon 14-intron 14-exon 15/1) forward 5 tgagatgatggggacacaag 3, reverse 5 gcatggagcacacagagtca 3; STAT5A 235 bp (exon 15/2) forward 5 ccagcagtgactctgtgtgct 3, reverse 5 tggagaatgggaatgaggaa 3; STAT5A 249 bp

The yield and specificity of PCR products were evaluated by electrophoresis in 2% agarose gel (Promega) with ethidium bromide. The results were observed, analysed and documented with the use of a Fluor-S MultiImager (Bio-Rad).
SSCP analysis

The SSCP technique (Orita et al. 1989) was optimised on the rules described by Hayashi and Yandell (1993) and the guidelines published by Electrophorese Technik GmbH (ETC) in a commercial bulletin attached to precast gels and buffers. Briefly, 0.5-2.0 ml of the PCR product were mixed with 5 ml of the denaturation solution (50 mmol NaOH, 1 mmol EDTA) and 1 ml of

New SSCP polymorphism within bovine STAT5A gene Table 1. Frequency of genotypes and alleles in locus STAT5A, SNP 9501 R
Number/frequency of genotypes Breed BW Jersey
A

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Allele frequency GG A
A A

N AA 186 138 47/0.253 41/0.297 AG 98/0.527 78/0.565 G 0.48 0.42 41/0.22 0.52 0.58

19/0.138

Within columns frequencies bearing the same superscript differ significantly at P 0.01.

the loading buffer containing 0.25% bromophenol blue and 0.25% xylene cyanol, denatured for 13 min at 85oC, rapidly chilled on ice block and then loaded onto precast polyacrylamide (CleanGel DNA Analysis Kit, T = 10%, C = 2%, Amersham Biosciences). The samples were electrophoresed in the Multiphor II Electrophoresis System (Amersham Biosciences). A thermostatically controlled refrigerated circulator (MultiTemp III, Amersham Biosciences) was used to maintain constant temperature (4oC) of the gel. Electrophoresis was performed in DELECT gel buffers (ETC). The gel was run in the following conditions: 200 V, 20 mA, 20 min (preelectrophoresis) and 375 V, 30 mA, for 120 min or 170 min (for amplicons longer than 200 bp). The gels were stained by Silver Stain (Kucharczyk Techniki Elektroforetyczne). The patterns of DNA bands were observed and photographed with the GDS7500 System (UVP).
Sequencing of PCR products

(http://www.ualberta.ca/~fyeh). The significance of differences in allele and genotype frequencies was compared with the use of the 2 test. The analysis of associations between genotypes of STAT5A and milk performance traits was conducted with the use of the GLM procedure (STATISTICA 6, StatSoft Inc). The following model was used:
Yijk = + Gi + YSj + (GYS)ij + eijk

SSCP patterns were confirmed by dideoxy sequencing to determine the position and nature of polymorphism. 50 l of PCR products were purified with the PCR GenElute Kit (Sigma). PCR sequencing was conducted with the use of the ABI PRISMTM 377 DNA Sequencer (Applied Biosystems) and DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Biosciences). Both DNA strands were sequenced. The results were analysed with Chromas 1.45 (http://www. technelysium.com.au) and Blast 2.0 (Altschul et al. 1990) software. Differences between PCR products, as well as between PCR products and reference sequences available in the GenBank, were classified as experimental and computational SNPs, respectively.
Statistics

where: Yijk = analysed trait of cow k, = overall mean, Gi = effect of i-th genotype, YSj = effect of j-th season of calving , (GYS)ij = interaction between the main effects of the model, eijk = random error. The significance of differences between the means for each group was tested with the use of Duncan test (for significance levels: P 0.05 and P 0.01). The following milk performance traits were analysed: milk yield [kg], fat yield [kg], fat content [%], protein yield [kg], and protein content [%]. Data on milk performance traits was retrieved from breeding documentation made available by the owners of cows.

Results
Ten specific PCR products were obtained (Figure 1). The SSCP analysis was carried out in a 3-step strategy. First, samples from the inter-breed panel were screened to find any polymorphism. If such polymorphism was found, the SNP was approved as potentially polymorphic and subsequently sequenced. Approved SNPs occurring in Jersey and Black-and-White were validated in a population of these two breeds (Jersey, n = 138; Black-and-White, n = 188) and used for association studies. Inter-breed SSCP polymorphism within Black-and-White cattle was observed only for PCR product STAT5A 224 bp. Three SSCP patterns were observed: W1, W2 and W3 (Figure 2).

Genotype and allele frequencies, and the Hardy-Weinberg balance were calculated with the use of POPGENE ver. 1.31 software

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Association studies

Figure 1. Ten specific PCR products of STAT5A gene: 1 and 12 - FX 174/HaeIII molecular size marker; 2 240 bp; 3 - 224 bp; 4 - 219 bp; 5 - 209 bp; 6 - 251 bp; 7 - 369 bp; 8 - 223 bp; 9 -235 bp; 10 - 249 bp; 11 -241 bp

These SSCP patterns were sequenced. Sequence analysis revealed a single nucleotide substitution A into G in intron 9 (amplicon including the whole exon 10 and a part of exon 9), making possible a real genotype designation AA, GG and AG, for W1, W2 and W3, respectively (Figure 3). Thirteen Black-and-White bulls were screened to find het-

No significant associations between STAT genotypes and milk performance traits were found for Black-and-White cows. Statistically significant differences were found in the first and second lactations for milk yield, fat and protein content in Jersey cows. Cows with the GG genotype showed higher milk yields (+415.74 kg) in the 1st lactation in comparison to cows with the AA genotype (P 0.01) (Table 2). Jersey cows with genotypes AA and AG showed higher protein contents in comparison to cows with the GG genotype (p 0.05). For fat content, cows with genotype AA showed the highest level of this trait in the 1st and 2nd lactations. Interestingly, cows with genotype AG showed significantly higher protein yields (+12.3 kg) in comparison to cows with the AA genotype. Also, some minor associations in the 2 nd lactation were found at the significance level of 0.05 (Table 2).

Figure 2. A) Polymorphism of STAT5A 224 bp PCR product in Black-and-White cows. Three SSCP patterns were observed. B) SSCP patterns: W1 (bands c, d),W2 (bands a, b, c, d), W3 (bands a, b).

erozygous ones for the SNP inheritance analysis in the sub-family. Only one such sib-family was found confirming the Mendelian segregation of STAT5 alleles (data not shown). The frequency of A and G alleles in both breeds was almost equal. However, a relatively low frequency of the GG genotype in Jersey cows was observed (0.138) (Table 1). Allele frequencies within the breed group were in accordance with the Hardy-Weinberg balance. The difference in the frequency of the GG genotype between Jersey and Black-and-White was highly significant (P 0.01).

Discussion
In the Marker Assisted Selection in dairy cattle some genes are proposed as potential candidates associated with dairy performance traits. Among different candidates, STAT5 seems to be promising because it plays a crucial role in transmitting signals from lactogenic hormones to milk protein gene promoters. It is believed that SNPs occurring within such genes may influence the chemical content of milk or at least be an effective DNA marker of a subregion of the dairy cattle genome.

New SSCP polymorphism within bovine STAT5A gene

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Table 2. Means and their standard deviations (in brackets) of milk performance traits in Jersey cows with different STAT5A genotypes, SNP 9501 R
Milk Lactation Genotype 9501 AA N [kg] [kg]
A

Fat [%] 5.76


A

Protein [kg] 149.80 (22.28) 153.22 (18.73) 156.84 16.28 152.70 20.52 163.48
a a.b A

[%] 3.89A (0.21) 3.90B (0.23) 3.68A.B (0.21) 3.87 (0.23) 3.93a (0.20) 3.98A (0.20) 3.82a.A (0.25) 3.94 (0.21)

41

3857.85

221.56 (28.28) 216.99 (30.89) 229.79 (28.46) 220.10 (29.92) 248.32 (30.15) 244.21 (33.90) 248.21 (27.82) 246.17 (31.50)

(549.97) 9501 AG 78 3914.44


a

(0.46) 5.58 (0.55) 5.42

(613.37) 9501GG 19 4272.74


Aa

(553.88) Total 138 3946.96 (598.14) II 9501 AA 25 4167.88


a.b

(0.64) 5.61 (0.54) 5.98

(542.09) 9501 AG 42 4419.14


b

(0.49) 5.56
a

(18.85) 175.78
a

(536.34) 9501 GG 14 4594.86


a

(0.62) 5.45
b

(20.81) 174.50 (18.09) 171.76 (20.32)

(613.99) Total 81 4371.96 (565.44)

(0.65) 5.67 (0.62)

Within columns means marked by the same superscripts differ significantly; small letter at the level P 0.05, capital letters at the level P 0.01.

Taking that into account, several exons coding most important functional domains of STAT5 (DNA binding domain, SH2 domain) were selected for the screening of single nucleotide polymorphism. Using the PCR-SSCP method only one SNP was found, located in intron 9. The SNP was deposited in the GenBank database under acc. no AY484401 for Jersey and AY339394 for Black-and-White cattle. Because the detected SNP is a silent mutation, it cannot be causative for any phenotypic variance and can be suggested as a new marker for the genome region in which it resides. STAT5 is a relatively complex gene (19 exons), therefore the space of searching for polymorphism was limited to exons which might have the most significant function in transmitting signals from prolactin and the activation of transcription of milk protein genes. On the basis of literature data (Ihle 1996, Darnell 1997, Becker et al. 1998, Seyfert et al. 2000) 3 functional domains of STAT5A were selected to be screened for polymorphisms, namely the DNA binding domain, SH2 domain and C-terminal transactivating domain. Those three domains are coded by exons 9-19 (Figure 4). Among the ten PCR products, the SSCP polymorphism was detected in one, STAT5A 224 bp. A polymorphism was also found

in STAT5A 223bp amplicon (data not shown). However, because this point mutation occurred only in Limousine cattle, no further analysis was conducted. The sequencing of amplicon STAT5A 224 showed a SNP in position 9501 R (A/G), located in intron 9, in reference to the GenBank sequence (AJ237937). Almost equal frequencies of alleles in both breeds suggest that STAT5A SNP is linked with different causative alleles of an unknown locus affecting milk performance traits. Also, similar frequencies of alleles A and G show a high informative content of the SNP and its perspective application as a genetic marker. The polymorphism of the bovine STAT5A gene has been lately studied intensively by the Zwierzchowski team (Institute of Animal Genetics and Breeding, Jastrzbiec, Poland). In a review paper, Flisikowski and Zwierzchowski (2003b) reported 17 SNPs and STRs. However, only several ones were published in original papers and only those will be discussed in this study. These authors identified 2 non-synonymous SNPs 2 K256N and A316S located in exons 7 and 8, as well as 7 polymorphisms within the STAT5A promoter. For 3 SNPs within the STAT5A promoter, they determined their significance for gene expression. Computer-aided analysis (TESS program) revealed that these nucleotide substitutions

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Figure 3. Sequence analysis of polymorphic region within intron 9 of the bovine STAT5A gene representing A/G transversion at position 9501 (GenBank AJ237937) or at position 40 (GenBank AY484401 and AY339394). SNP is indicated by an arrow. A) Sequence of PCR product STAT5A 224 bp showing SSCP pattern W1. B) Sequence of PCR product STAT5A 224 bp showing SSCP pattern W3. C) Sequence of PCR product STAT5A 224 bp showing SSCP pattern W2

may potentially change the binding sites of transcription factors: HNF3, E2F and AP2 (Flisikowski and Zwierzchowski unpublished). In exon 7 (6852 C/T) a silent SNP was detected (Flisikowski and Zwierzchowski 2002). That polymorphism has been used as a genetic marker of meat performance traits in beef cattle. It was shown that cattle with the CC genotype have a higher body mass, growth rate, carcass yield, and other productive and technological traits at the age of 9 and 15 months than cattle with the CT genotype. Homozygotes TT were found only in Polish local breeds: Polish Red and Biaogrzbietka (Flisikowski et al. 2003b). In intron 15 (position 12549), the deletion of CCT has been reported (Flisikowski and Zwierzchowski 2003a). This polymorphism is located

within an amplicon analysed earlier by Antoniou et al. (1999), who described two SSCP patterns. In exon 16 Flisikowski et al. (2003a) showed point mutation in position 12743 (T/C) which causes amino acid substitution (V686A) and is located very close to tyrosine 694, which plays a key role in the phosphorylation, activation and dimerisation of STAT5. All the polymorphisms described in original papers and identified in this study are presented in Figure 4. It is commonly known that the SSCP method has some limitations in SNP detection even when careful optimisation is carried out. Although all the steps in SNP discovery with the SSCP method was standardized (the same apparatus and electrophoresis conditions, precast gels and commercial buffers, a uniform quality of PCR products, etc.), it was not possible to confirm polymorphism 12743 (C/T), reported by Flisikowski et al. (2003a). There are 3 possible explanations for that difference: 1) different SSCP conditions (temperature, electrophoresis parameters); 2) amplicon screened by Flisikowski was longer (281 bp), and encompassed deletion CCT in position 12549 (Flisikowski and Zwierzchowski 2003a). In effect, after SSCP genotyping, the observed differenced might be due to 4 nucleotides. Unfortunately, in our investigations the CTT deletion was placed within the forward primer sequence of STAT5A 249 bp amplicon (primers were designed and used before the report by Flisikowski and Zwierzchowski was published). So, in the subsequent cycles of PCR, the primer could eliminate a potential polymorphism in position 12549, limiting detection only to SNP 12743 Y; 3) different populations under study and rather low numbers of animals genotyped. In conclusion, in the analysed herds of Polish Black-and-White cows and Jersey cows, a new silent mutation was identified, located within intron 9. Statistical analysis revealed associations between STAT5A genotypes and some milk performance traits in Jersey cows, but not in Black-and-White. The SNP may be used in the near future to construct the bovine haplotype map applied in the new strategy of QTL mapping. The validation of the described SNP in the population of two dairy breeds has a special value, because it facilitates the introduction of the SNP into a set of SNPs markers of a certain region of the chromosome

New SSCP polymorphism within bovine STAT5A gene

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Figure 4. Mutation map of the bovine STAT5A gene. The figure shows the scheme of the STAT5A gene organization with position of PCR products analysed in the present study. Protein structure with most important functional domains, tyrosine 694, and missense mutation V686A were also indicated. All identified mutations were placed in appropriate sites of STAT5A gene according to the numbering used by Seyfert et al. 2000 (GenBank AJ242522, AJ237937) and the reference in which the detection of mutation was for the first time reported is indicated next to the position of mutation. The type of mutation was marked by one letter symbols. Y = (C or T), R = (A or G), (TG)12 = TG repeat polymorphism, CCTdel = deletion of CCT, Ex = exon

and also as effective marker in paternity testing and phylogenetic studies. Further studies are necessary to evaluate an allele substitution effect in a population of sib-families of STAT5A heterozygous bulls.
Acknowledgments. This study was financially supported by the State Committee of Scientific Research (Grant No. 6 P06D 006 20). The authors are grateful to Professor Krzysztof Walawski for a critical review of the manuscript and for his help in collecting some blood samples. We also would like to thank Elbieta Wjcik for excellent technical assistance.
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