92
Brief Notes Table 1 Allele frequencies for the novel MSTN mutation c.191T>C (p.L64P) and the MSTN:c.821del11 mutation for three different breeds.
Cattle breed
n
Allele frequency for the mutant allele MSTN:c. 191T>C (p.Leu64Pro)
German Gelbvieh Glanrind Limpurger
137 92 90
0.03 0.10 0.03
Allele frequency for the mutant allele MSTN:c. 821del11 0.07 0.00 0.01
double-muscled Gelbvieh cattle. In silico analysis of this mutation using POLYPHEN (http://genetics.bwh.harvard.edu/ pph2/) and SIFT (http://siftdna.org/www/Extended_SIFT_ chr_coords_submit.html) predicted a probably damaging effect on the protein. The amino acid leucin at position 64 of the MSTN protein is highly conserved among mammals (Fig. S1). The two double-muscled Gelbvieh cattle had one copy of the c.191T>C mutation and one copy of the 11-bp deletion. Genotyping of their parents confirmed compound heterozygosity of the c.191T>C and the 11-bp deletion mutation in these two animals (Fig. 1). All four parents were heterozygous for each one of the two mutations. In one case, the novel exon 1 mutation was transmitted by the sire and in the second case by the dam. The other two double-muscled Gelbvieh cattle were homozygous for the 11-bp deletion. Validation of the c.191T>C and c.821del11 mutations in 451 individuals revealed 19 Glanrind, seven German Gelbvieh and five Limpurger cattle as heterozygous for the c.191T>C mutation (Table S3). Of 29 double-muscled animals, 27 were homozygous for the 11-bp deletion and two for the mutation resulting in p.Cys313Tyr. Allele frequencies for c.191T>C were at 0.03 in German Gelbvieh and Limpurger, but at 0.10 in Glanrind (Table 1). Allele frequencies for the 11-bp deletion were 0.94 in Belgian Blue, 0.16 in German Angus, 0.07 in German Gelbvieh and 0.01 in Limpurger. The previously reported p.Phe94Leu variant from exon 1 of MSTN was associated with increased muscle mass in Limousin.6 In contrast to the novel detected mutation, the mutation resulting in the p.Phe94Leu substitution is located in a less conserved region (Fig. S1). The high frequency of 0.10 in Glanrind might be indicative of an origin of this mutation in the Glanrind and spreading through Glanrind sires into other breeds such as Gelbvieh and Limpurger. Our study showed a novel mutation with a probably damaging effect on the protein function of MSTN. We could validate this mutation in German Gelbvieh, Glanrind and Limpurger. Testing these breeds for the novel c.191T>C and 11-bp deletion mutations should be effective in preventing double-muscled animals.
Acknowledgements: We thank the cattle breeding associations and all cattle owners for providing samples and pedigree data. We acknowledge Mogens Drabert, Heike Klippert-Hasberg and Stefan Neander for their expert technical assistance. References 1 Allais S.. et al. (2010) J Anim Sci 88, 446–54. 2 Gill J.L. et al. (2009) Anim Genet 40, 97–100. 3 Grobet L. et al. (1998) Mamm Genome 9, 210–3. 4 Marchitelli C. et al. (2003) Mamm Genome 14, 392–5. 5 Quadros L. et al. (2008) Haemophilia 14, 628–9. 6 Sellick G.S. et al. (2007) Anim Genet 38, 440–6. Correspondence: O. Distl ([email protected])
Supporting information Additional supporting information may be found in the online version of this article. Figure S1 Alignment of the normal bovine MSTN protein (NP_001001525) and the p.Leu64Pro variant with known orthologous mammalian MSTN protein sequences. Table S1 Mutations in the bovine myostatin (MSTN) gene influencing muscle mass with polymorphism ID, location within MSTN, protein effect, resulting phenotype and cattle breeds where the mutation was observed as well as references. Table S2 Primer sequences with their product sizes (P) and annealing temperatures (AT) used for amplification of PCR-products, sequencing and genotyping within MSTN as well as the targeted regions within this gene. Table S3 Number of animals (n) per breed, their phenotypes and genotypes for the MSTN mutations p.Leu64Pro from exon 1 and the c.821del11 in exon 3. Mutated alleles are in bold.
doi: 10.1111/age.12253
Haplotype diversity in autochthonous Balkan cattle breeds P. Hristov, D. Teofanova, B. Neov and G. Radoslavov Department of Animal Diversity and Resources, Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria Accepted for publication 9 October 2014
Description: The Bulgarian Grey cattle (BGC) and the Shorthorn Rhodopean cattle (SRC) are Balkan indigenous © 2014 Stichting International Foundation for Animal Genetics 46, 91–99
Frequency, %
© 2014 Stichting International Foundation for Animal Genetics, 46, 91–99
BGC-1 BGC-2 BGC-3 BGC-4.1 BGC-4.2 BGC-51 BGC-5.2 SRC-1 SRC-2 SRC-3.1 SRC-3.2 SRC-3.3 SRC-4.3 SRC-4.2 SRC-4.1 SRC-4.4 SRC-5.1 SRC-5.2 SRC-5.3
15974 . . . . . T . . . . . . . . . . . . T
C
15985 . . . . . . . . C . . . . . . . . . .
T . . . . . . . . . . . A . . . . . . .
G
16022
1
(.) Dot indicates the same sequence as the reference. Polymorphic sites characterized main haplogroups. 2 Bos taurus reference sequence GenBank Accession No. V00654.6
Shorthorn Rhodopean cattle
5.13 12.82 2.56 33.33 41.03 2.56 2.56 5.00 5.00 10.00 5.00 5.00 5.00 20.00 10.00 10.00 10.00 5.00 10.00
. . . . C . . . . . . . . . . C . . .
Frequency, number
2 5 1 13 16 1 1 1 1 2 1 1 1 4 2 2 2 1 2
T1 T2 T T3 T3 T3 T3 T1 T2 T3 T3 T3 T3 T3 T3 T3 T3 T3 T3
Haplogroup
Bulgarian Grey cattle
Haplotype
T
15965
Bovine reference sequence2
Breed
16042 . . . . . . . . . . . . C . . . . . .
T
16050 (T11) T . . . . . . . . . . . . . . . . . .
C
16057 (T21) . C . . . . . . C . . . . A . . . . .
G
16068 . . C . . . . . . . . . . . . . . . .
T
16074 . . . . . . . . C . . . . . . C . . .
T . . . . . . . . . . T . . . . . . . .
C
16084
Table 1 SNP variation detected in 59 D-loop sequence in two Balkan indigenous cattle breeds.
16085 . . C . . C . . . . . . . . . . . . .
T
16095 . . . . . . . G . . . . . . . . . . .
A
16101 . . . . . . . . C . . . . . . . . . .
T
16109 . . . . . . . . . C . . . . . . . . .
T
16113 (T11) C . . . . . . C . . . . . . . . . . .
T
16121 . . . . . . . . . . . . A . . . . . .
G
16133 . . . . . . . . . . . . . C . . . . .
T
16135 . . . . . . . . . . . . . . . . . C .
T
16141 . . . . . . . . . . . . C . . . . . .
T
16185 (T21) . A . . . . . . A . . . . . . . . . .
G
16228 . . . . . . . . . . . G . . . . . . .
A
16231 . . . . . T T . . . . . . . . . . . .
C
16255 (T1) C C C . . . . C C . . . . . . . . . .
T
16260 . . . . . . . . T . . . . . . . . . .
C
16264 . . . . . . . . . . . . . . . A . . .
G
16301 . T . . . . . . . . . . . . . . . . .
C
8 . . A . . . . . . . . . . . . . . . .
G
169 (T31) G G G G G G G G G . . . . . G G G G G
A
173 . . . . . . . . . . . . G G G G . . .
A
GenBank Accession Number KF373013 KF373014 KF373015 KF373016 KF373017 KF373018 KF373019 KF373020 KF373021 KF373022 KF373023 KF373024 KF373027 KF373028 KF373025 KF373026 KF373029 KF373031 KF373030
V00654
Brief Notes 93
94
Brief Notes breeds that are part of the Podolian and Brachicerous (Busha) cattle groups respectively. Concerning the mtDNA diversity of Balkan cattle breeds, previous studies focused on Busha cattle and Grey cattle populations.1 These investigations referred to the origin and worldwide migration of taurine breeds, and the Balkans were pointed out as an important route for European cattle dissemination. The aim of this study was to establish the mitochondrial D-loop genetic diversity of BGC and SRC and to reveal their state among other European Podolian and Brachicerous cattle breeds. Genotyping and sequence analysis: Two sets of 20 and 39 nasal swab samples respectively were collected from typical pure breed representatives and unrelated animals from different SRC and BGC herds. The D-loop region was amplified as described by Achilli et al.2 and directly sequenced. The sequences were deposited in GenBank under Accession No. KF373013–KF373031 (PopSet accession no. 530540029). On the basis of the polymorphic SNP positions, seven and 12 haplotypes for the BGC and SRC sets respectively were defined (Table 1). Comments: Both investigated breeds have mitotypes similar to other Brachicerous and Podolian cattle breeds. As expected there is a low frequency of the typical African T1 haplogroup in BGC and SRC populations (5%; Table 1). Its frequency is similar among the Italian peninsula cattle populations3 suggesting African genetic influence through a Mediterranean route. The T1 haplogroup was not found in Brachicerous (Busha) and Podolian type cattle (Fig. S1, Tables S1 and S2) due to the reduction of genetic diversity, receding from the center of origin. The T2 haplogroup, except among investigated population samples (Table 1), was found also in the Greek Brachicerous4 and Podolian cattle breeds3 with similar frequencies (Fig. S1, Tables S1 and S2). The data for T1 and T2 haplogroups can be explained by the later and limited livestock introduction from Anatolia.1 As expected, the T3 haplogroup has the highest frequency. Data shown in Fig. S1 revealed that T3 haplotypes are unequally disseminated from both Podolian and Busha cattle populations.1
Science, Bulgaria 28.12.2009).
(Grant
Number
YRG
02/23
References 1 Lenstra J.A. et al. (2014) Diversity 6, 178–87. 2 Achilli A. et al. (2008) Curr Biol 18, R157–8. 3 Bonfiglio S. et al. (2010) PLoS ONE 5, e15760. 4 Beja-Pereira A. et al. (2006) Proc Natl Acad Sci USA 103, 8113–8. 5 Ajmone-Marsan P. et al. (2010) Evol Anthropol 19, 148– 57. 6 Anderson S. et al. (1982) J Mol Biol 156, 683–717. Correspondence: P. Hristov ([email protected])
Supporting information Additional supporting information may be found in the online version of this article. Figure S1 A reduced median network of the Podolian and Busha mtDNA haplotypes. Table S1 D-loop mtDNA haplogroups and haplotypes observed in the Balkan Podolian cattle. Table S2 D-loop mtDNA haplogroups and haplotypes observed in the Balkan Brachicerous cattle
doi: 10.1111/age.12241
RegionPlot: an R package for regional plot association results for pigs F. Zhang1, Z. Y. Zhang1, Y. N. He, H. Chen, B. Yang, W. J. Deng and L. S. Huang Key Laboratory for Animal Biotechnology of Jiangxi Province and the Ministry of Agriculture of China, Jiangxi Agricultural University, Nanchang, Jiangxi 330045, China Accepted for publication 30 September 2014
Source/description: Genome-wide association studies (GWAS)
Conclusion: In conclusion, this study reveals high genetic diversity of the Balkan SRC and BGC breeds. SRC was presumed to be the earliest European cattle, evidenced by its mtDNA profile and supported by data for basic phenotypic characteristics of livestock from the Neolithic age to the period of the Roman Empire.5 On the contrary, BGC data confirm the later domestication and dissemination of Podolian cattle breeds.
have revealed hundreds of loci associated with economic important traits in pigs. To interpret and visualize these results, two visualization tools were developed in human genetics. However, the reference genome in LOCUSZOOM1 and an R script2 is human rather than other species, which precludes plotting a regional graph of association results for pigs. Moreover, the graphics produced by the R script lack important information, such as local linkage disequilibrium (LD) and genomic annotation (non-synonymous, synonymous, UTR, intron). Therefore, to develop a
Acknowledgements: This work was supported by National Science Fund of the Bulgarian Ministry of Education and
1 Both authors contribute equally to this study and should be considered as co-first authors.
© 2014 Stichting International Foundation for Animal Genetics 46, 91–99
Brief Notes Table 1 Allele frequencies for the novel MSTN mutation c.191T>C (p.L64P) and the MSTN:c.821del11 mutation for three different breeds.
Cattle breed
n
Allele frequency for the mutant allele MSTN:c. 191T>C (p.Leu64Pro)
German Gelbvieh Glanrind Limpurger
137 92 90
0.03 0.10 0.03
Allele frequency for the mutant allele MSTN:c. 821del11 0.07 0.00 0.01
double-muscled Gelbvieh cattle. In silico analysis of this mutation using POLYPHEN (http://genetics.bwh.harvard.edu/ pph2/) and SIFT (http://siftdna.org/www/Extended_SIFT_ chr_coords_submit.html) predicted a probably damaging effect on the protein. The amino acid leucin at position 64 of the MSTN protein is highly conserved among mammals (Fig. S1). The two double-muscled Gelbvieh cattle had one copy of the c.191T>C mutation and one copy of the 11-bp deletion. Genotyping of their parents confirmed compound heterozygosity of the c.191T>C and the 11-bp deletion mutation in these two animals (Fig. 1). All four parents were heterozygous for each one of the two mutations. In one case, the novel exon 1 mutation was transmitted by the sire and in the second case by the dam. The other two double-muscled Gelbvieh cattle were homozygous for the 11-bp deletion. Validation of the c.191T>C and c.821del11 mutations in 451 individuals revealed 19 Glanrind, seven German Gelbvieh and five Limpurger cattle as heterozygous for the c.191T>C mutation (Table S3). Of 29 double-muscled animals, 27 were homozygous for the 11-bp deletion and two for the mutation resulting in p.Cys313Tyr. Allele frequencies for c.191T>C were at 0.03 in German Gelbvieh and Limpurger, but at 0.10 in Glanrind (Table 1). Allele frequencies for the 11-bp deletion were 0.94 in Belgian Blue, 0.16 in German Angus, 0.07 in German Gelbvieh and 0.01 in Limpurger. The previously reported p.Phe94Leu variant from exon 1 of MSTN was associated with increased muscle mass in Limousin.6 In contrast to the novel detected mutation, the mutation resulting in the p.Phe94Leu substitution is located in a less conserved region (Fig. S1). The high frequency of 0.10 in Glanrind might be indicative of an origin of this mutation in the Glanrind and spreading through Glanrind sires into other breeds such as Gelbvieh and Limpurger. Our study showed a novel mutation with a probably damaging effect on the protein function of MSTN. We could validate this mutation in German Gelbvieh, Glanrind and Limpurger. Testing these breeds for the novel c.191T>C and 11-bp deletion mutations should be effective in preventing double-muscled animals.
Acknowledgements: We thank the cattle breeding associations and all cattle owners for providing samples and pedigree data. We acknowledge Mogens Drabert, Heike Klippert-Hasberg and Stefan Neander for their expert technical assistance. References 1 Allais S.. et al. (2010) J Anim Sci 88, 446–54. 2 Gill J.L. et al. (2009) Anim Genet 40, 97–100. 3 Grobet L. et al. (1998) Mamm Genome 9, 210–3. 4 Marchitelli C. et al. (2003) Mamm Genome 14, 392–5. 5 Quadros L. et al. (2008) Haemophilia 14, 628–9. 6 Sellick G.S. et al. (2007) Anim Genet 38, 440–6. Correspondence: O. Distl ([email protected])
Supporting information Additional supporting information may be found in the online version of this article. Figure S1 Alignment of the normal bovine MSTN protein (NP_001001525) and the p.Leu64Pro variant with known orthologous mammalian MSTN protein sequences. Table S1 Mutations in the bovine myostatin (MSTN) gene influencing muscle mass with polymorphism ID, location within MSTN, protein effect, resulting phenotype and cattle breeds where the mutation was observed as well as references. Table S2 Primer sequences with their product sizes (P) and annealing temperatures (AT) used for amplification of PCR-products, sequencing and genotyping within MSTN as well as the targeted regions within this gene. Table S3 Number of animals (n) per breed, their phenotypes and genotypes for the MSTN mutations p.Leu64Pro from exon 1 and the c.821del11 in exon 3. Mutated alleles are in bold.
doi: 10.1111/age.12253
Haplotype diversity in autochthonous Balkan cattle breeds P. Hristov, D. Teofanova, B. Neov and G. Radoslavov Department of Animal Diversity and Resources, Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria Accepted for publication 9 October 2014
Description: The Bulgarian Grey cattle (BGC) and the Shorthorn Rhodopean cattle (SRC) are Balkan indigenous © 2014 Stichting International Foundation for Animal Genetics 46, 91–99
Frequency, %
© 2014 Stichting International Foundation for Animal Genetics, 46, 91–99
BGC-1 BGC-2 BGC-3 BGC-4.1 BGC-4.2 BGC-51 BGC-5.2 SRC-1 SRC-2 SRC-3.1 SRC-3.2 SRC-3.3 SRC-4.3 SRC-4.2 SRC-4.1 SRC-4.4 SRC-5.1 SRC-5.2 SRC-5.3
15974 . . . . . T . . . . . . . . . . . . T
C
15985 . . . . . . . . C . . . . . . . . . .
T . . . . . . . . . . . A . . . . . . .
G
16022
1
(.) Dot indicates the same sequence as the reference. Polymorphic sites characterized main haplogroups. 2 Bos taurus reference sequence GenBank Accession No. V00654.6
Shorthorn Rhodopean cattle
5.13 12.82 2.56 33.33 41.03 2.56 2.56 5.00 5.00 10.00 5.00 5.00 5.00 20.00 10.00 10.00 10.00 5.00 10.00
. . . . C . . . . . . . . . . C . . .
Frequency, number
2 5 1 13 16 1 1 1 1 2 1 1 1 4 2 2 2 1 2
T1 T2 T T3 T3 T3 T3 T1 T2 T3 T3 T3 T3 T3 T3 T3 T3 T3 T3
Haplogroup
Bulgarian Grey cattle
Haplotype
T
15965
Bovine reference sequence2
Breed
16042 . . . . . . . . . . . . C . . . . . .
T
16050 (T11) T . . . . . . . . . . . . . . . . . .
C
16057 (T21) . C . . . . . . C . . . . A . . . . .
G
16068 . . C . . . . . . . . . . . . . . . .
T
16074 . . . . . . . . C . . . . . . C . . .
T . . . . . . . . . . T . . . . . . . .
C
16084
Table 1 SNP variation detected in 59 D-loop sequence in two Balkan indigenous cattle breeds.
16085 . . C . . C . . . . . . . . . . . . .
T
16095 . . . . . . . G . . . . . . . . . . .
A
16101 . . . . . . . . C . . . . . . . . . .
T
16109 . . . . . . . . . C . . . . . . . . .
T
16113 (T11) C . . . . . . C . . . . . . . . . . .
T
16121 . . . . . . . . . . . . A . . . . . .
G
16133 . . . . . . . . . . . . . C . . . . .
T
16135 . . . . . . . . . . . . . . . . . C .
T
16141 . . . . . . . . . . . . C . . . . . .
T
16185 (T21) . A . . . . . . A . . . . . . . . . .
G
16228 . . . . . . . . . . . G . . . . . . .
A
16231 . . . . . T T . . . . . . . . . . . .
C
16255 (T1) C C C . . . . C C . . . . . . . . . .
T
16260 . . . . . . . . T . . . . . . . . . .
C
16264 . . . . . . . . . . . . . . . A . . .
G
16301 . T . . . . . . . . . . . . . . . . .
C
8 . . A . . . . . . . . . . . . . . . .
G
169 (T31) G G G G G G G G G . . . . . G G G G G
A
173 . . . . . . . . . . . . G G G G . . .
A
GenBank Accession Number KF373013 KF373014 KF373015 KF373016 KF373017 KF373018 KF373019 KF373020 KF373021 KF373022 KF373023 KF373024 KF373027 KF373028 KF373025 KF373026 KF373029 KF373031 KF373030
V00654
Brief Notes 93
94
Brief Notes breeds that are part of the Podolian and Brachicerous (Busha) cattle groups respectively. Concerning the mtDNA diversity of Balkan cattle breeds, previous studies focused on Busha cattle and Grey cattle populations.1 These investigations referred to the origin and worldwide migration of taurine breeds, and the Balkans were pointed out as an important route for European cattle dissemination. The aim of this study was to establish the mitochondrial D-loop genetic diversity of BGC and SRC and to reveal their state among other European Podolian and Brachicerous cattle breeds. Genotyping and sequence analysis: Two sets of 20 and 39 nasal swab samples respectively were collected from typical pure breed representatives and unrelated animals from different SRC and BGC herds. The D-loop region was amplified as described by Achilli et al.2 and directly sequenced. The sequences were deposited in GenBank under Accession No. KF373013–KF373031 (PopSet accession no. 530540029). On the basis of the polymorphic SNP positions, seven and 12 haplotypes for the BGC and SRC sets respectively were defined (Table 1). Comments: Both investigated breeds have mitotypes similar to other Brachicerous and Podolian cattle breeds. As expected there is a low frequency of the typical African T1 haplogroup in BGC and SRC populations (5%; Table 1). Its frequency is similar among the Italian peninsula cattle populations3 suggesting African genetic influence through a Mediterranean route. The T1 haplogroup was not found in Brachicerous (Busha) and Podolian type cattle (Fig. S1, Tables S1 and S2) due to the reduction of genetic diversity, receding from the center of origin. The T2 haplogroup, except among investigated population samples (Table 1), was found also in the Greek Brachicerous4 and Podolian cattle breeds3 with similar frequencies (Fig. S1, Tables S1 and S2). The data for T1 and T2 haplogroups can be explained by the later and limited livestock introduction from Anatolia.1 As expected, the T3 haplogroup has the highest frequency. Data shown in Fig. S1 revealed that T3 haplotypes are unequally disseminated from both Podolian and Busha cattle populations.1
Science, Bulgaria 28.12.2009).
(Grant
Number
YRG
02/23
References 1 Lenstra J.A. et al. (2014) Diversity 6, 178–87. 2 Achilli A. et al. (2008) Curr Biol 18, R157–8. 3 Bonfiglio S. et al. (2010) PLoS ONE 5, e15760. 4 Beja-Pereira A. et al. (2006) Proc Natl Acad Sci USA 103, 8113–8. 5 Ajmone-Marsan P. et al. (2010) Evol Anthropol 19, 148– 57. 6 Anderson S. et al. (1982) J Mol Biol 156, 683–717. Correspondence: P. Hristov ([email protected])
Supporting information Additional supporting information may be found in the online version of this article. Figure S1 A reduced median network of the Podolian and Busha mtDNA haplotypes. Table S1 D-loop mtDNA haplogroups and haplotypes observed in the Balkan Podolian cattle. Table S2 D-loop mtDNA haplogroups and haplotypes observed in the Balkan Brachicerous cattle
doi: 10.1111/age.12241
RegionPlot: an R package for regional plot association results for pigs F. Zhang1, Z. Y. Zhang1, Y. N. He, H. Chen, B. Yang, W. J. Deng and L. S. Huang Key Laboratory for Animal Biotechnology of Jiangxi Province and the Ministry of Agriculture of China, Jiangxi Agricultural University, Nanchang, Jiangxi 330045, China Accepted for publication 30 September 2014
Source/description: Genome-wide association studies (GWAS)
Conclusion: In conclusion, this study reveals high genetic diversity of the Balkan SRC and BGC breeds. SRC was presumed to be the earliest European cattle, evidenced by its mtDNA profile and supported by data for basic phenotypic characteristics of livestock from the Neolithic age to the period of the Roman Empire.5 On the contrary, BGC data confirm the later domestication and dissemination of Podolian cattle breeds.
have revealed hundreds of loci associated with economic important traits in pigs. To interpret and visualize these results, two visualization tools were developed in human genetics. However, the reference genome in LOCUSZOOM1 and an R script2 is human rather than other species, which precludes plotting a regional graph of association results for pigs. Moreover, the graphics produced by the R script lack important information, such as local linkage disequilibrium (LD) and genomic annotation (non-synonymous, synonymous, UTR, intron). Therefore, to develop a
Acknowledgements: This work was supported by National Science Fund of the Bulgarian Ministry of Education and
1 Both authors contribute equally to this study and should be considered as co-first authors.
© 2014 Stichting International Foundation for Animal Genetics 46, 91–99
