Mitochondrial DNA D-loop sequence variation among 5 maternal lines of the Zemaitukai horse breed
http://www.100md.com
《遗传学和分子生物学》
IDepartment of Veterinary Science, University of Kentucky, Lexington, KY, USA
IISiauliai University, Siauliai, Lithuania
ABSTRACT
Genetic variation in Zemaitukai horses was investigated using mitochondrial DNA (mtDNA) sequencing. The study was performed on 421 bp of the mitochondrial DNA control region, which is known to be more variable than other sections of the mitochondrial genome. Samples from each of the remaining maternal family lines of Zemaitukai horses and three random samples for other Lithuanian (Lithuanian Heavy Draught, Zemaitukai large type) and ten European horse breeds were sequenced. Five distinct haplotypes were obtained for the five Zemaitukai maternal families supporting the pedigree data. The minimal difference between two different sequence haplotypes was 6 and the maximal 11 nucleotides in Zemaitukai horse breed. A total of 20 nucleotide differences compared to the reference sequence were found in Lithuanian horse breeds. Genetic cluster analysis did not shown any clear pattern of relationship among breeds of different type.
Key words: D-loop, Equus caballus, phylogeny, polymorphism.
Introduction
Until recently conservation efforts have focused on wild species, but now domesticated animals are recognized as an important part of biodiversity and more efforts to save rare breeds are made. The Zemaitukai horse (Equus caballus) is a native breed of Lithuania. As a consequence of unfavourable historical and economic circumstancies, the number of Zemaitukai seriously declined and by 2003 the total population size was 147 animals. According to pedigree data, the Zemaitukai horse population now consists of two stallion (male) and five mare (female) family lines (Macijauskiene, 2002). Out of the five mare families the one of Kastanke is represented by significant number of individuals. The least numerous is the Mirta family. The other three are the Zibute, Arabe and Tulpe maternal families. Genetic relationship between Lithuanian and other horse breeds is a point of interest, as the origin of the breed is not exactly known.
Mitochodrial DNA (mtDNA) has strictly maternal inheritance (Hutchinson et al., 1974), which means mtDNA haplotypes should be shared by all individuals within a maternal family line. Mitochondrial DNA is useful for studying the evolution of closely related species and many studies have focused on the mitochondrial D-loop region, the most variable part of mtDNA (Ishida et al., 1994) due to a higher substitution rate than in the rest of the mtDNA genome (Cann et al., 1984). The entire horse mtDNA sequence has been reported (Xu & Arnason, 1994). Stability of maternal inheritance within documented horse pedigrees has been demonstrated in Lipizzan (Kavar et al., 1999) and Arabian horses (Bowling et al, 2000). Mitochondrial DNA sequence polymorphism has been used to examine genetic relationship within breeds (Hill et al., 2002; Luis et al 2002), among breeds (Kim et al., 1999; Mirol et al., 2002), between domestic and wild horse populations (Oakenfull & Ryder, 1998) and also to address questions of horse domestication (Lister et al., 1998; Vila et al., 2001).
Here we present a study designed to examine the validity of the breed pedigree, measure mtDNA diversity, and examine interbreed relationships, as this information could be useful in conservation and management of this rare horse breed.
Materials and Methods
Samples from Zemaitukai (ZO) mare families were: Arabes - Austeja ZRg59, Mirtos - Asveja Zrg173, Kastankes family - Kanarele ZRg40, Zibutes - Zemyna ZRg172 and Tulpes - Tola ZRg217, respectively. Three random samples from other two Lithuanian horse breeds Zemaitukai Heavy Type (ZH) and Lithuanian Heavy Draught (LH) were sequenced. In order to determine phylogenetic relationship three random samples from Finn horse (FH), Gotland (GT), Hanovarian (HA), Holstein (HO), Hucul (HU), Polish Heavy (PH), Wielkopolski (PI), Polish Primative (PO), Posavina (PV) and Trakehner (TK) breeds were sequenced. These are breeds that were available to us from the same general geographical area as Lithuania. DNA was extracted from fresh blood samples or frozen serum samples using the Puregene DNA extraction kit (Gentra Systems) following the manufacturer instructions. Primers from published horse mtDNA sequence (Xu and Arnason, 1994) were designed: Forward 5-CGCACA TT ACCCTGGTCTTG-3, Reverse 5-GAACCAGATGCCA GGTATAG-3.
Polymerase chain reaction (PCR) was carried out in 25 mL total reaction volumes, each containing 0.2 mM dNTPs, 0.5 mM of each primer, 2.5 mM MgCl2, 1XPCR buffer, 1 U of Taq polymerase (PE Applied Biosystems, MA), 1 U of ApliTaq Gold (PE Applied Biosystems, MA) and 50 ng of template DNA. The reaction mixture was heated to 95 °C for 5 min, followed by 30 cycles each consisting of 40 s denaturation at 94 °C, 45 s annealing at 55 °C, 45 s of extension at 72 °C and then a final 10 min extension at 72 °C.
Sequencing was carried out using BigDyeTM Terminator Cycle Sequencing Kit (PE Applied Biosystems, MA). Sequences were determined using the ABI Prism 377 DNA Sequencer. All sequences were confirmed by re-sequencing the same sample using a second independent PCR reaction. Sequence alignment was performed using reference equine mtDNA sequence (GeneBank X79547). Genetic clustering and molecular evolutionary analyses were conducted using MEGA version 2.1 (Kumar et al. 2001). In addition to the breeds listed above sequences belonging to other horse breeds from GeneBank (http://www.ncbi.nlm.nih.gov/GenBank) were incorporated into the analysis. The GeneBank accession numbers for selected sequencies are AY246174-AY246200, AY246209-AY246252 and AY246259-AY246271. The sequence of Equus prewalski was used as outgroup.
Results and Discussion
For other breeds, out of three randomly selected samples, two haplotypes for Finn horse, Holstein, Hucul, and Zemaitukai Heavy type and three haplotypes for rest of the breeds were observed. A total of 38 nucleotide differences compared to the reference sequence were found representing 9% of the total DNA sequence analyzed. Out of 38 detected mutations, there were 37 transitions and a single deletion in one individual of the Polish primitive horse breed. The deletion was located in stretch of five cytosines at position 15532.
For genetic cluster analysis, sequences of Arabian, Thoroughbred, Friesian, Akhal-Teke, Belgian, Caspian, Cleveland Bay, Clydasdales, Exmoor, Garrano, Haflinger, Lusitano, Noriker, Shetlands and Sorraia horse breeds were obtained from gene bank and included with the breeds sequenced in this study for analysis. The breeds represent a wide geographic area as well as the different horse types. Sequences were truncated to 353 bp for this analysis in order to maximize the number of breeds included. Both maximum parsimony and neighbour-joining analysis showed similar patterns. After truncating sequences for genetic cluster analysis, breeds that shared the same haplotype were pooled in to groups named C1, C2, C3 and C4. The C1 group contained the Holstein, Trakehner, Lithuanian Heavy Drought, Arabian, Sorraia and Hannovarian breeds. Hanovarian, Trakehner, Zemaitukai and Arabians were put in C2. Haplotypes of Akhal-Teke, Belgian, Haflinger, Zemaitukai, Polish Heavy, Garrano, Cleveland Bay, and Noriker were placed in C3. Hucul, Thoroughbred, Haflinger, Noriker and Lithuanian Heavy drought horse haplotypes formed the C4 group. Genetic cluster analysis did not shown any clear pattern of relationship among the domestic horse breeds whose relationships are well known historically (bootstrap analysis of 1000 replications, Figure 1). Haplotypes from the same breed frequently clustered in separate groups that included breeds of completely different horse breed types. This same pattern has been seen in other studies of horse mtDNA (Vila et al., 2001; Jansen et al., 2002), but are different from ones obtained using blood and protein typing data, were horse breeds clustered corresponding to their known ancestry (Juras et al., 2003). The lack of a pattern may be attributed the high rate of evolution in the control region, which may cause a site to mutate once, and then mutate later changing back to the original haplotype. The result would be the loss of an informative site. Also migration of individuals from one population to another before the establishment of the stud books or at the initial stage of breed formation. The mtDNA analysis provided no new information about the ancestry of the Lithuanian horse breeds.
The analysis of mitochondrial DNA of Zemaitukai horses adds useful information for the effective management and conservation of this rare breed. The mtDNA results confirm at least five remaining maternal lineages in Zemaitukai horse breed, which are useful for development of breeding strategies, aimed at evening the genetic contribution of different maternal founding lineages. The mtDNA data also provides additional insights into the genetic diversity of the breed, which, in combination with data from nuclear genes, can be used to maximize the maintenance of genetic diversity within the Zemaitukai horse.
Accession numbers
GenBank accession numbers AY575103-AY575139 for sequences obtained in this study are as follows: FH1-2, GT1-3, HA1-3, HO1-2, HU1-2, LH1-3, PH1-3, PI1-3, PO1-3, PV1-3, TK1-3, ZH1-2 and Z01-5.
Acknowledgments
The authors appreciate greatly the help of Dr. K. Graves for designing primers.
References
Bowling A, Del Valle A and Bowling M (2000) A pedigree-based study of mitochondrial D-loop DNA sequence variation among Arabian horses. Animal Genetics 31:1-7.
Cann PL, Brown WM and Wilson AC (1984) Polymorphic sites and the mechanism of evolution in human mitochondrial DNA. Genetics 106:479-499.
Hill EW, Bradley DG, Al-Barody M, Ertugul O, Splan RL, Zakharov I and Cunningam EP (2002) History and integrity of thoroughbred dam lines revealed in the equine mtDNA variation. Animal Genetics 33:287-294.
Hutchinson CA, Newbold JE, Potter SS and Hall EM (1974) Maternal inheritance of mammalian mitochondrial DNA. Nature 251:536-8.
Ishida N, Hasegawa T, Takeda K, Sakagami M, Onishi A, Inumaru S, Kamtsu M and Mukoyama H (1994) Polymorphic sequence in the D-loop region of the equine mitochondrial DNA. Animal Genetics 25:215-221.
Jansen T, Forster P, Levine MA, Oelke H, Hurles M, Renfrew C, Weber J and Olek K (2002) Mitochndrial DNA and the origin of the domestic horse. Proceedings National Academy of Science 99:10905-10910.
Juras R, Cothran EG and Klimas R (2003) Genetic analysis of three Lithuanian native horse breeds. Acta Agriculturae Scandinavica, Section A 53:180-185.
Kavar T, Habe F, Brem G and Dovc P (1999) Mitochondrial D-loop sequence variation among the 16 maternal lines of the Lipizzan horse breed. Animal Genetics 30:423-430.
Kim KI, Yang YH, Lee SS, Park C, Ma R, Bouzat JL and Lewin HA (1999) Phylogenetic relationship of Cheju horses to other horse breeds as determinated by mtDNA D-loop sequence polymorphism. Animal Genetics 30:102-108.
Kumar S, Tamura K, Jakobsen IB and Nei M (2001) MEGA2: Molecular Evolutionary Genetics Analysis software. Bioinformatics 12:1244-1245.
Lister AM, Kadwell M, Kaagan LM, Jordan WC, Richards MB and Stanley HF (1998) Ancient and modern DNA in study of horse domestication. Ancient Biomolecules 2:267-280.
Luis C, Bastos-Silveira C, Cothran EG and Oom MM (2002) Variation in the control region sequence between the two maternal lines of the Sorraia horse breed. Genetics and Molecular Biology 25:309-311.
Macijauskiene V (2002) Monitoring of the Zemaitukai horse breed. Animal Husbandry 40:3-12.
Marklund S, Chaudhary R, Marklund L, Sandberg K and Andersson L (1994) Extensive mtDNA diversity in horses revealed by PCR-SSCP analysis. Animal Genetics 26:193-196.
Mirol PM, Peral Garcia P, Vega-Pla JL and Dulout FN (2002) Phylogenetic relationship of Argentinean Creole horses and other South American and Spanish breeds inferred from mitochondrial DNA sequences. Animal Genetics 33:356-363.
Oakenfull EA and Ryder OA (1998) Mitochondrial control region and 12S rRNR variation in Przewalski horse (Equus przewalski). Animal Genetics 29:456-459.
Vila C, Leonard JA, Gotherstrom A, Marklund S, Sandberg K, Linden K, Wayne RK and Ellegren H (2001) Widespread origins of domestic horse lineages. Science 291:474-477.
Xu X and Arnason U (1994) The complete mitochondrial DNA sequence of the horse, Equus caballus: Extensive heteroplasmy of the control region. Gene 148:657-662.(E. Gus Cothran; Rytis Jur)
IISiauliai University, Siauliai, Lithuania
ABSTRACT
Genetic variation in Zemaitukai horses was investigated using mitochondrial DNA (mtDNA) sequencing. The study was performed on 421 bp of the mitochondrial DNA control region, which is known to be more variable than other sections of the mitochondrial genome. Samples from each of the remaining maternal family lines of Zemaitukai horses and three random samples for other Lithuanian (Lithuanian Heavy Draught, Zemaitukai large type) and ten European horse breeds were sequenced. Five distinct haplotypes were obtained for the five Zemaitukai maternal families supporting the pedigree data. The minimal difference between two different sequence haplotypes was 6 and the maximal 11 nucleotides in Zemaitukai horse breed. A total of 20 nucleotide differences compared to the reference sequence were found in Lithuanian horse breeds. Genetic cluster analysis did not shown any clear pattern of relationship among breeds of different type.
Key words: D-loop, Equus caballus, phylogeny, polymorphism.
Introduction
Until recently conservation efforts have focused on wild species, but now domesticated animals are recognized as an important part of biodiversity and more efforts to save rare breeds are made. The Zemaitukai horse (Equus caballus) is a native breed of Lithuania. As a consequence of unfavourable historical and economic circumstancies, the number of Zemaitukai seriously declined and by 2003 the total population size was 147 animals. According to pedigree data, the Zemaitukai horse population now consists of two stallion (male) and five mare (female) family lines (Macijauskiene, 2002). Out of the five mare families the one of Kastanke is represented by significant number of individuals. The least numerous is the Mirta family. The other three are the Zibute, Arabe and Tulpe maternal families. Genetic relationship between Lithuanian and other horse breeds is a point of interest, as the origin of the breed is not exactly known.
Mitochodrial DNA (mtDNA) has strictly maternal inheritance (Hutchinson et al., 1974), which means mtDNA haplotypes should be shared by all individuals within a maternal family line. Mitochondrial DNA is useful for studying the evolution of closely related species and many studies have focused on the mitochondrial D-loop region, the most variable part of mtDNA (Ishida et al., 1994) due to a higher substitution rate than in the rest of the mtDNA genome (Cann et al., 1984). The entire horse mtDNA sequence has been reported (Xu & Arnason, 1994). Stability of maternal inheritance within documented horse pedigrees has been demonstrated in Lipizzan (Kavar et al., 1999) and Arabian horses (Bowling et al, 2000). Mitochondrial DNA sequence polymorphism has been used to examine genetic relationship within breeds (Hill et al., 2002; Luis et al 2002), among breeds (Kim et al., 1999; Mirol et al., 2002), between domestic and wild horse populations (Oakenfull & Ryder, 1998) and also to address questions of horse domestication (Lister et al., 1998; Vila et al., 2001).
Here we present a study designed to examine the validity of the breed pedigree, measure mtDNA diversity, and examine interbreed relationships, as this information could be useful in conservation and management of this rare horse breed.
Materials and Methods
Samples from Zemaitukai (ZO) mare families were: Arabes - Austeja ZRg59, Mirtos - Asveja Zrg173, Kastankes family - Kanarele ZRg40, Zibutes - Zemyna ZRg172 and Tulpes - Tola ZRg217, respectively. Three random samples from other two Lithuanian horse breeds Zemaitukai Heavy Type (ZH) and Lithuanian Heavy Draught (LH) were sequenced. In order to determine phylogenetic relationship three random samples from Finn horse (FH), Gotland (GT), Hanovarian (HA), Holstein (HO), Hucul (HU), Polish Heavy (PH), Wielkopolski (PI), Polish Primative (PO), Posavina (PV) and Trakehner (TK) breeds were sequenced. These are breeds that were available to us from the same general geographical area as Lithuania. DNA was extracted from fresh blood samples or frozen serum samples using the Puregene DNA extraction kit (Gentra Systems) following the manufacturer instructions. Primers from published horse mtDNA sequence (Xu and Arnason, 1994) were designed: Forward 5-CGCACA TT ACCCTGGTCTTG-3, Reverse 5-GAACCAGATGCCA GGTATAG-3.
Polymerase chain reaction (PCR) was carried out in 25 mL total reaction volumes, each containing 0.2 mM dNTPs, 0.5 mM of each primer, 2.5 mM MgCl2, 1XPCR buffer, 1 U of Taq polymerase (PE Applied Biosystems, MA), 1 U of ApliTaq Gold (PE Applied Biosystems, MA) and 50 ng of template DNA. The reaction mixture was heated to 95 °C for 5 min, followed by 30 cycles each consisting of 40 s denaturation at 94 °C, 45 s annealing at 55 °C, 45 s of extension at 72 °C and then a final 10 min extension at 72 °C.
Sequencing was carried out using BigDyeTM Terminator Cycle Sequencing Kit (PE Applied Biosystems, MA). Sequences were determined using the ABI Prism 377 DNA Sequencer. All sequences were confirmed by re-sequencing the same sample using a second independent PCR reaction. Sequence alignment was performed using reference equine mtDNA sequence (GeneBank X79547). Genetic clustering and molecular evolutionary analyses were conducted using MEGA version 2.1 (Kumar et al. 2001). In addition to the breeds listed above sequences belonging to other horse breeds from GeneBank (http://www.ncbi.nlm.nih.gov/GenBank) were incorporated into the analysis. The GeneBank accession numbers for selected sequencies are AY246174-AY246200, AY246209-AY246252 and AY246259-AY246271. The sequence of Equus prewalski was used as outgroup.
Results and Discussion
For other breeds, out of three randomly selected samples, two haplotypes for Finn horse, Holstein, Hucul, and Zemaitukai Heavy type and three haplotypes for rest of the breeds were observed. A total of 38 nucleotide differences compared to the reference sequence were found representing 9% of the total DNA sequence analyzed. Out of 38 detected mutations, there were 37 transitions and a single deletion in one individual of the Polish primitive horse breed. The deletion was located in stretch of five cytosines at position 15532.
For genetic cluster analysis, sequences of Arabian, Thoroughbred, Friesian, Akhal-Teke, Belgian, Caspian, Cleveland Bay, Clydasdales, Exmoor, Garrano, Haflinger, Lusitano, Noriker, Shetlands and Sorraia horse breeds were obtained from gene bank and included with the breeds sequenced in this study for analysis. The breeds represent a wide geographic area as well as the different horse types. Sequences were truncated to 353 bp for this analysis in order to maximize the number of breeds included. Both maximum parsimony and neighbour-joining analysis showed similar patterns. After truncating sequences for genetic cluster analysis, breeds that shared the same haplotype were pooled in to groups named C1, C2, C3 and C4. The C1 group contained the Holstein, Trakehner, Lithuanian Heavy Drought, Arabian, Sorraia and Hannovarian breeds. Hanovarian, Trakehner, Zemaitukai and Arabians were put in C2. Haplotypes of Akhal-Teke, Belgian, Haflinger, Zemaitukai, Polish Heavy, Garrano, Cleveland Bay, and Noriker were placed in C3. Hucul, Thoroughbred, Haflinger, Noriker and Lithuanian Heavy drought horse haplotypes formed the C4 group. Genetic cluster analysis did not shown any clear pattern of relationship among the domestic horse breeds whose relationships are well known historically (bootstrap analysis of 1000 replications, Figure 1). Haplotypes from the same breed frequently clustered in separate groups that included breeds of completely different horse breed types. This same pattern has been seen in other studies of horse mtDNA (Vila et al., 2001; Jansen et al., 2002), but are different from ones obtained using blood and protein typing data, were horse breeds clustered corresponding to their known ancestry (Juras et al., 2003). The lack of a pattern may be attributed the high rate of evolution in the control region, which may cause a site to mutate once, and then mutate later changing back to the original haplotype. The result would be the loss of an informative site. Also migration of individuals from one population to another before the establishment of the stud books or at the initial stage of breed formation. The mtDNA analysis provided no new information about the ancestry of the Lithuanian horse breeds.
The analysis of mitochondrial DNA of Zemaitukai horses adds useful information for the effective management and conservation of this rare breed. The mtDNA results confirm at least five remaining maternal lineages in Zemaitukai horse breed, which are useful for development of breeding strategies, aimed at evening the genetic contribution of different maternal founding lineages. The mtDNA data also provides additional insights into the genetic diversity of the breed, which, in combination with data from nuclear genes, can be used to maximize the maintenance of genetic diversity within the Zemaitukai horse.
Accession numbers
GenBank accession numbers AY575103-AY575139 for sequences obtained in this study are as follows: FH1-2, GT1-3, HA1-3, HO1-2, HU1-2, LH1-3, PH1-3, PI1-3, PO1-3, PV1-3, TK1-3, ZH1-2 and Z01-5.
Acknowledgments
The authors appreciate greatly the help of Dr. K. Graves for designing primers.
References
Bowling A, Del Valle A and Bowling M (2000) A pedigree-based study of mitochondrial D-loop DNA sequence variation among Arabian horses. Animal Genetics 31:1-7.
Cann PL, Brown WM and Wilson AC (1984) Polymorphic sites and the mechanism of evolution in human mitochondrial DNA. Genetics 106:479-499.
Hill EW, Bradley DG, Al-Barody M, Ertugul O, Splan RL, Zakharov I and Cunningam EP (2002) History and integrity of thoroughbred dam lines revealed in the equine mtDNA variation. Animal Genetics 33:287-294.
Hutchinson CA, Newbold JE, Potter SS and Hall EM (1974) Maternal inheritance of mammalian mitochondrial DNA. Nature 251:536-8.
Ishida N, Hasegawa T, Takeda K, Sakagami M, Onishi A, Inumaru S, Kamtsu M and Mukoyama H (1994) Polymorphic sequence in the D-loop region of the equine mitochondrial DNA. Animal Genetics 25:215-221.
Jansen T, Forster P, Levine MA, Oelke H, Hurles M, Renfrew C, Weber J and Olek K (2002) Mitochndrial DNA and the origin of the domestic horse. Proceedings National Academy of Science 99:10905-10910.
Juras R, Cothran EG and Klimas R (2003) Genetic analysis of three Lithuanian native horse breeds. Acta Agriculturae Scandinavica, Section A 53:180-185.
Kavar T, Habe F, Brem G and Dovc P (1999) Mitochondrial D-loop sequence variation among the 16 maternal lines of the Lipizzan horse breed. Animal Genetics 30:423-430.
Kim KI, Yang YH, Lee SS, Park C, Ma R, Bouzat JL and Lewin HA (1999) Phylogenetic relationship of Cheju horses to other horse breeds as determinated by mtDNA D-loop sequence polymorphism. Animal Genetics 30:102-108.
Kumar S, Tamura K, Jakobsen IB and Nei M (2001) MEGA2: Molecular Evolutionary Genetics Analysis software. Bioinformatics 12:1244-1245.
Lister AM, Kadwell M, Kaagan LM, Jordan WC, Richards MB and Stanley HF (1998) Ancient and modern DNA in study of horse domestication. Ancient Biomolecules 2:267-280.
Luis C, Bastos-Silveira C, Cothran EG and Oom MM (2002) Variation in the control region sequence between the two maternal lines of the Sorraia horse breed. Genetics and Molecular Biology 25:309-311.
Macijauskiene V (2002) Monitoring of the Zemaitukai horse breed. Animal Husbandry 40:3-12.
Marklund S, Chaudhary R, Marklund L, Sandberg K and Andersson L (1994) Extensive mtDNA diversity in horses revealed by PCR-SSCP analysis. Animal Genetics 26:193-196.
Mirol PM, Peral Garcia P, Vega-Pla JL and Dulout FN (2002) Phylogenetic relationship of Argentinean Creole horses and other South American and Spanish breeds inferred from mitochondrial DNA sequences. Animal Genetics 33:356-363.
Oakenfull EA and Ryder OA (1998) Mitochondrial control region and 12S rRNR variation in Przewalski horse (Equus przewalski). Animal Genetics 29:456-459.
Vila C, Leonard JA, Gotherstrom A, Marklund S, Sandberg K, Linden K, Wayne RK and Ellegren H (2001) Widespread origins of domestic horse lineages. Science 291:474-477.
Xu X and Arnason U (1994) The complete mitochondrial DNA sequence of the horse, Equus caballus: Extensive heteroplasmy of the control region. Gene 148:657-662.(E. Gus Cothran; Rytis Jur)