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Genetic characterization of the autochthonous sheep populations from Chiapas, Mexico
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Livestock Science 116 (2008) 156 – 161 www.elsevier.com/locate/livsci
Genetic characterization of the autochthonous sheep populations from Chiapas, Mexico J. Quiroz a , A.M. Martinez b , L. Zaragoza c , R. Perezgrovas c , J.L. Vega-Pla d,⁎, J.V. Delgado b a
Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, Carretera Huimanguillo Cárdenas Km. 1, Tabasco, México Departamento de Genética, Universidad de Córdoba, Edificio Gregor Mendel, Campus de Rabanales s/n, 14071-Córdoba, Spain c Universidad Autónoma de Chiapas-México d Laboratorio de Genética Molecular, Servicio de Cría Caballar y Remonta, Apartado Oficial Sucursal 2, 14071-Córdoba, Spain
b
Received 12 April 2007; received in revised form 19 September 2007; accepted 25 September 2007
Abstract The Tzotziles, a Mayan native group located in Chiapas, Southern Mexico, have sheep breeding as one of their principal means of subsistence. Sheep were introduced in the Americas by the Spaniards in the first half of the XVI Century. Three populations of sheep from Chiapas—Café, Chamula and Chiapas—were typed with 27 microsatellites. Genetic distances were calculated for three Iberian breeds, Spanish Merino, Churra, two Canary Islands breeds, Canaria and Palmera, and the French Mutton Merino (Precoce). In the case of Chiapas sheep most of the markers showed Hardy–Weinberg equilibrium and the fixation index of Fst (0.095) showed a moderate level of genetic differentiation. The three distinct sheep subpopulations (Café, Chamula and Chiapas) are genetically differentiated inside the Chiapas sheep population. These breeds could be historically related to Spanish sheep populations, but have diverged significantly as a result of genetic drift and selection. © 2007 Elsevier B.V. All rights reserved. Keywords: Sheep; Microsatellite; Genetic distance; Correspondence analysis; Population structure
1. Introduction The Tzotziles and other Maya native groups are located in Southern Mexico, particularly in the Chiapas region. Sheep and wool are economically important as well as a cultural patrimony of the native communities. Today sheep are considered as “animal companions”; their slaughtering is forbidden. Wool is manufactured to make distinctive ethnic clothes.
⁎ Corresponding author. Tel.: +34 957322344; fax: +34 957322493. E-mail address: [email protected] (J.L. Vega-Pla). 1871-1413/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.livsci.2007.09.022
The sheep from Chiapas are isolated within geographic areas that make the introduction and adaptation of exotic breeds very difficult. They are traditionally divided into three groups according to coat color: Café (dark brown), Chamula (black) and Chiapas (white). Microsatellite markers have been shown to be useful tools for the analysis of genetic differentiation among sheep populations as well as aid in conservation decisions for genetic resources (Gutiérrez-Espeleta et al., 2000; Saitbekova et al., 2001; Tomasco et al., 2002; Rendo et al., 2004; Peter et al., 2005; Paiva et al., 2005), Here we describe microsatellite marker analyses that were carried out to help to clarify the genetic structure of the native
J. Quiroz et al. / Livestock Science 116 (2008) 156–161
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Table 1 F statistics, probability of global equilibrium and number of alleles by locus Locus
Fis
Fit
Fst
p-val
Number of alleles
BM1818 BM1824 BM6506 BM6526 BM8125 CSRD247 CSSM66 D5S2 ETH10 ETH225 HSC ILSTS011 INRA35 INRA63 MAF209 MAF65 McM527 OarCP20 OarCP34 OarFCB11 OarFCB304 OarFCB48 RM006 SPS115 TGLA122 TGLA126 TGLA53
0.0777 0.0758 0.0787 0.0781 0.0789 0.0799 0.0777 0.0762 0.0767 0.0749 0.0784 0.0790 0.0753 0.0792 0.0776 0.0789 0.0753 0.0798 0.0791 0.0767 0.0790 0.0782 0.0791 0.0707 0.0680 0.0684 0.0754
0.1710 0.1644 0.1708 0.1677 0.1730 0.1732 0.1712 0.1679 0.1696 0.1691 0.1723 0.1715 0.1683 0.1711 0.1713 0.1720 0.1679 0.1705 0.1716 0.1709 0.1702 0.1717 0.1719 0.1646 0.1611 0.1632 0.1670
0.1012 0.0959 0.0999 0.0972 0.1022 0.1014 0.1015 0.0992 0.1006 0.1018 0.1018 0.1005 0.1006 0.0997 0.1017 0.1011 0.1001 0.0986 0.1004 0.0997 0.1014 0.1015 0.1008 0.1011 0.0999 0.1017 0.0991
0.0018 0.0001 0.0327 0.0118 0.0236 0.0982 0.0005 0.0026 0.0587 0.0000. 0.0026 0.0000 0.0000. 0.0010 0.0000. 0.4446 0.0001 0.0028 0.0021 0.2338 0.0001 0.0096 0.2870 0.0000 0.0000 0.0000 0.0006
16 7 10 13 9 18 25 13 7 9 16 15 16 17 16 13 13 13 10 18 24 14 12 10 15 21 13
sheep populations from Chiapas and its relationships with some Spanish breeds. 2. Materials and methods Blood or wool samples (number of samples in brackets) were obtained from a random sampling of Chamula (41), Café (42) and Chiapas (45) sheep from the state of Chiapas, Mexico. Other breeds sampled were the Palmera (47) and the Canary
Table 2 Population, sample size, N, mean number of alleles, MNA, expected, He, unbiased, Heu and observed, Ho heterozygosity levels and the values of Fis, confidence intervals of 95% are shown for each of the sheep populations in this study Population N
MNA He
He_u Ho
Fis
Chiapas Chamula Café Palmera Canaria Merino French Merino Churra
6.1 6.9 7.8 5.6 7.9 6.7 6.1
0.633 0.708 0.721 0.619 0.728 0.669 0.667
0.042, − 0.005–0.059 0.026, − 0.021–0.045 0.076, 0.033–0.096 0.091, 0.038–0.0143 0.058, − 0.004–0.076 0.074, 0.016–0.106 0.023, − 0.019–0.057
41 42 45 47 40 44 46
35 8.4
0.624 0.699 0.712 0.612 0.718 0.661 0.660
0.606 0.690 0.666 0.563 0.686 0.620 0.652
0.728 0.740 0.654 0.118, 0.078–0.132
Fig. 1. Factorial analysis of correspondence of three sheep populations from Chiapas, and other breeds.
(46) from the Canary Islands, Spanish Merino (44), Precoce French Mutton Merino (46), Churra (35). A panel of 27 microsatellite markers was used for amplification via Polymerase Chain Reaction (PCR) according to the method of Martinez et al. (2000). Fluorescently labeled PCR fragments were separated by electrophoresis in an automatic sequencer ABI377XL (Applied Biosystems, Foster City, CA, USA). Allelic frequencies, heterozygosity, number of migrant alleles, parameters of genetic differentiation (F statistics), and a Factorial Analysis of Correspondence, were calculated with the software Genetix v. 4.05 (Belkhir et al., 2000). Hardy– Weinberg equilibrium was accomplished by the method of Guo and Thompson (1992) using the Markov Chain of Monte Carlo algorithm in GENEPOP v. 3.1c software (Raymond and Rousset, 1995). Analysis of molecular variance (AMOVA) was performed with the software Arlequin 2.0 (Schneider et al., 1997) to evaluate the genetic structure of the populations. The
Table 3 DA genetic distances (below the diagonal) and number of migrants (above the diagonal) among the sheep populations Café Chamula Chiapas Merino French Merino Palmera Canaria Churra
0 10.26 0.093 0 0.115 0.096 0.199 0.217 0.228 0.233 0.255 0.198 0.182
4.26 4.94 0 0.241 0.256
2.55 2.39 1.76 0 0.207
2.00 1.95 1.46 2.08 0
1.70 1.53 1.23 1.15 1.23
2.95 2.64 1.77 2.09 1.88
4.13 3.17 2.02 2.60 3.06
0.278 0.281 0.315 0.307 0 2.39 1.97 0.219 0.247 0.227 0.252 0.206 0 3.99 0.205 0.238 0.211 0.192 0.242 0.170 0
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3. Results
Fig. 2. Genetic relationships among the sheep populations based on a neighbor-joining tree constructed with DA distances.
genetic structure of population is investigated here by an analysis of variance framework, as initially defined by Cockerham (1969, 1973),. The Analysis of Molecular Variance approach used (AMOVA, Excoffier et al., 1992) is essentially similar to other approaches based on analyses of variance of gene frequencies, but it takes into account the number of mutations between molecular haplotypes. A hierarchical analysis of variance partitions the total variance into covariance components due to intra-individual differences, inter-individual differences, and/or inter-population differences. The significance of the fixation indices is tested using a nonparametric permutation approach described in Excoffier et al. (1992). A large number of permutations is necessary to obtain some accuracy on the final probability. The DA genetic distance (Nei et al., 1983; Takezaki and Nei, 1996) was calculated using a Populations 1.2.28 software (Langella, 2002). A neighbor-joining tree was built with DA genetic distances and was graphically represented with TreeView software (Page, 1996). Analysis of individual assignment to populations was done using the Structure v. 2.0 software (Pritchard et al., 2000).
All microsatellites were polymorphic, except ETH10 which was monomorphic in some populations. A total of 383 alleles were detected in the 27 microsatellites analyzed showing a mean number of alleles of 14.19 with a maximum of 25 in CSSM66 and a minimum of 7 in ETH10 and BM1824. The Hardy–Weinberg disequilibrium tests were statistically significant only for six microsatellites in the Chiapas population and eight in Café and Chamula ones. None of the microsatellite loci were in disequilibrium in all of the investigated populations. However, TGLA122 and TGLA126 were in disequilibrium for 7 breeds. The Fis, Fit and Fst values (Table 1) were similar for all microsatellites. Twenty two loci deviated (p b 0.05) from the Hardy–Weinberg equilibrium in the pooled samples demonstrating genetic heterogeneity among breeds. The mean value of the F statistics and the confidence intervals at 95% after 1000 bootstraps were as follow: Fis = 0.076 (0.044–0.106), Fit = 0.169 (0.138– 0.198) and Fst = 0.100 (0.087–0.115). The mean value of Fis supports the general quality of the sampling and the presence of low levels of inbreeding in the majority of populations. The mean Fst indicates the existence of defined subpopulations. The AMOVA analysis showed similar results (Fis = 0.020, Fst = 0.095 and Fit = 0.113) with a highly significant p-value (P b 0.01). Geographic partitioning of variance breeds was tested grouping them in European (Churra Merino and French Merino), Canarian (Palmera and Canaria) and Mexican breeds. The AMOVA results were (Fis = 0.047, Fst = 0.0741 and Fit = 0.113) with a highly significant p-value (p b 0.01).
Fig. 3. Estimation of the population structure with k different values.
J. Quiroz et al. / Livestock Science 116 (2008) 156–161 Table 4 Proportion of animals of each population pre-defined in the 9 clusters Breed
Cluster 1
Café 0.013 Chamula 0.013 Chiapas 0.007 Canaria 0.013 Palmera 0.003 Merino 0.922 French 0.010 Merino Churra 0.012
2
3
4
5
6
7
8
0.008 0.019 0.009 0.010 0.007 0.006 0.004
0.658 0.083 0.025 0.012 0.004 0.012 0.008
0.012 0.019 0.010 0.034 0.004 0.008 0.006
0.009 0.007 0.006 0.011 0.708 0.008 0.003
0.014 0.012 0.005 0.894 0.008 0.014 0.006
0.245 0.819 0.924 0.010 0.003 0.010 0.008
0.032 0.017 0.009 0.011 0.261 0.008 0.006
0.015 0.010 0.904 0.009 0.017 0.007 0.012
There was more variability among populations than geographical groups. Table 2 shows the sample size, mean number of alleles, the heterozygosity and the values of Fis for each of the sheep populations. The factorial analysis of correspondence (Fig. 1) grouped the Chiapas populations in the left lower quadrant away from the other breeds. The information of the first two axes explains 42.31% of the variation among populations. There was a possible gene flow from the Churra and Canarian breeds based in the number of migrants. Also, close relationship between Cafe and Churra populations were detected in DA distances matrix (Table 3). Fig. 2 shows a neighbor-joining tree based on Nei's DA distances in which the clustering of the three populations from Chiapas is evident. The high values of bootstrapping supported the strength of the results in respect to the differentiation of breeds, although the main node obtained only 49% bootstrap. The branch length that separates the Chiapas nucleus from the others indicates the degree of differentiation that exists with the present Spanish breeds. The method of Falush et al. (2003) was used to assign individuals to inferred clusters based on the allele frequencies of each locus. The number of hypothetical clusters (K) was 2‑9 and 230,000 repetitions of the Markov Chain Monte Carlo algorithm were used. In Fig. 3, each individual is represented by a thin vertical line, which is divided in K colored segments that represent the fraction of each individual belonging to each one of the inferred clusters. The black lines separate the individuals of the different populations. When K = 2, a cluster was formed of the Mexico and Canary Islands populations (Canaria breed is divided in both clusters) and all remaining breeds were in another cluster. In the case of K = 3, the three Mexican
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populations clustered together. When K = 8, Café was separated from the Chiapas populations. Table 4 shows the proportion of individuals belonging to each population assigned for K = 9. Café, Chamula and Chiapas were grouped together in the same cluster 7 although a great proportion of Café individuals were assigned in a new cluster. 4. Discussion FAO defines a breed as a specific group of domestic animals with definable and identifiable external characteristics that permit to differentiate it from other similar groups of the same species. In this way, molecular biology techniques to characterize populations are very useful tools in order to obtain correct conclusions depending on the species. However, to establish credible hypotheses amenable to be tested by means of genetic analysis, it is necessary to have knowledge about both the old and recent history of the populations under research. This includes the reproductive management and their relationships with other populations, etc. The mean number of alleles was lower than the values showed by sheep from the North of the Iberian Peninsula (Rendo et al., 2004), but higher in general than those obtained for Finish and Russian sheep breeds (Tapio et al., 2003). The observed mean heterozygosities among sheep from Chiapas was lower than that in Swiss breeds (Saitbekova et al., 2001) with the exception of the mouflon; also it was lower than the Spanish Merino (Diez-Tascón et al., 2000). For breeds under conservation plans, the observed mean heterozygosity could be as low as 0.52 (Gutiérrez-Espeleta et al., 2000). The expected heterozygosities were similar to the Corriedale and Merino sheep from Uruguay (Tomasco et al., 2002). The level of genetic differentiation among domestic sheep breeds based on molecular genetic marker information is low in comparison with the reports in cattle (Fst = 0.186; Quiroz et al., 2004) or among subspecies of wild sheep (Ovis dalli) (Fst = 0.160) (Worley et al., 2004). The low differentiation among sheep breeds could be artificial and a consequence of the use of many microsatellites that are homologous with cattle located in highly conserved DNA regions. Alternatively, the domestication process and the ability of sheep to accompany human populations in their migrations probably contributed to the poor differentiation at a breed level due to continuous crossbreeding among different populations. This circumstance is also observed among domestic horses and goats (Ajmone-Marsan et al., 2001;
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Barker et al., 2001; Mirol et al., 2002; Xiang Long and Valentini, 2004). Regarding the variability detected by the selected microsatellites, ETH10 which was monomorphic in some populations, the remaining 26 markers showed a high mean number of alleles (14.19). The Hardy– Weinberg equilibrium of microsatellites was adequate for all populations except the Churra breed. The Fis, Fit and Fst values also demonstrated the near absence of uncontrolled biases such as great errors of sampling, inbreeding, etc. It is possible to consider that these three sheep populations from Chiapas have a relatively high degree of differentiation (Fst = 0.1) considering the Fst value of 0.07 obtained by Rendo et al. (2004) in a study across Spanish sheep breeds, or Fst = 0.03 in Merinos from different countries (Diez-Tascón et al., 2000). The assignment of individuals to populations showed a clear differentiation of Mexican sheep populations from the Merino, Churras and Canary Islands sheep. These results also indicate that the Café population must be reconsidered because several individuals previously assigned to this population showed a genetic divergence from the genetic profile estimated for this population. This finding must be supported by morphological, productive, functional and genealogical information of these discordant individuals. Genetic dispersion detected on the Café population is supported by the phenotypic studies of wool quality. Those studies showed the black and brown fibers that go from light brown to dark brown, resulting in different coat tones (Perezgrovas and Castro, 1998). The three different coat colors of the Mexican sheep populations have resulted in the reproductive isolation of the Chamula, Café and Chiapas, as a consequence of wool preferences by the native artisan industries throughout the centuries. The clear genetic differentiation between the Chamula and the Chiapas supports this conclusion, while the similarity observed between the Café and Chamula does not indicate a continuous reproductive isolation among these two populations. Our results agree with those obtained in previous works based on morphological characterization (Pedraza et al., 1992; Perezgrovas, 1998). Sheep breeding in Chiapas has survived in the present because of the animals' adaptation to the environment, their rusticity, and their integration in the rural economy of the Tzotzil natives who depend on them. Previously there were no formal conservation programs, only their own survival in the hard environment and their capacity to produce wool under these conditions. It is not possible to completely eliminate the
possibility of some influence from exotic breeds over these sheep populations; however, the adaptation of the local sheep to the challenging environment has always favored local genotypes. The influence of the Spanish breeds over the Chiapas populations is likely from both the morphological and historical point of view, but the results from our analyses did not support this assumption. This could be due to genetic drift over centuries in both Spanish as well as Chiapas sheep populations, as well as to other systematic effects (selection and migration) which separated these breeds from their Spanish ancestors although some common morphological traits were conserved. The introduction of the Spanish Merino to Mexico by Spanish colonizers is well documented (Rodero et al., 1992), although its influence cannot be appreciated from genetic distances. 5. Conclusion From a genetic perspective, it is possible to support the contention that the Café, Chamula and Chiapas are independent and lightly differentiated populations both among themselves as well as from other sheep breeds. This supports previous studies based on morphology (Pedraza et al., 1992, Perezgrovas, 1998) and productivity (Perezgrovas and Castro, 1998). Chiapas sheep populations are an important Mexican genetic heritage and must be immediately subjected to conservation programs linked to the indigenous culture where they have contributed over centuries. Acknowledgments We are particularly in debt to Cecilia Penedo and Phil Sponenberg for their valuable corrections and comments of this manuscript. This research was funded by the Andalucian Research Group “Mejora y Conservación de los Recursos Genéticos de los Animales Domésticos” (AGR218) sited at the University of Córdoba (Spain) as part of their efforts to improve the genetic diversity of the domestic animals. References Ajmone-Marsan, P., Negrini, R., Crepaldi, P., Milanesi, E., Gorni, C., Valentín, A., Cicogna, M., 2001. Assessing genetic diversity in Italian goat populations using AFLP markers. Anim. Genet. 32, 281–288. Barker, J.S.K., Tan, S.G., Moore, S.S., Muekherjee, T.K., Matheson, J.L., Selvaraj, O.S., 2001. Genetic variation within and relationships among populations of Asian goats, (Capra hircus. J. Anim. Breed. Genet. 118, 213–233.
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Livestock Science 116 (2008) 156 – 161 www.elsevier.com/locate/livsci
Genetic characterization of the autochthonous sheep populations from Chiapas, Mexico J. Quiroz a , A.M. Martinez b , L. Zaragoza c , R. Perezgrovas c , J.L. Vega-Pla d,⁎, J.V. Delgado b a
Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, Carretera Huimanguillo Cárdenas Km. 1, Tabasco, México Departamento de Genética, Universidad de Córdoba, Edificio Gregor Mendel, Campus de Rabanales s/n, 14071-Córdoba, Spain c Universidad Autónoma de Chiapas-México d Laboratorio de Genética Molecular, Servicio de Cría Caballar y Remonta, Apartado Oficial Sucursal 2, 14071-Córdoba, Spain
b
Received 12 April 2007; received in revised form 19 September 2007; accepted 25 September 2007
Abstract The Tzotziles, a Mayan native group located in Chiapas, Southern Mexico, have sheep breeding as one of their principal means of subsistence. Sheep were introduced in the Americas by the Spaniards in the first half of the XVI Century. Three populations of sheep from Chiapas—Café, Chamula and Chiapas—were typed with 27 microsatellites. Genetic distances were calculated for three Iberian breeds, Spanish Merino, Churra, two Canary Islands breeds, Canaria and Palmera, and the French Mutton Merino (Precoce). In the case of Chiapas sheep most of the markers showed Hardy–Weinberg equilibrium and the fixation index of Fst (0.095) showed a moderate level of genetic differentiation. The three distinct sheep subpopulations (Café, Chamula and Chiapas) are genetically differentiated inside the Chiapas sheep population. These breeds could be historically related to Spanish sheep populations, but have diverged significantly as a result of genetic drift and selection. © 2007 Elsevier B.V. All rights reserved. Keywords: Sheep; Microsatellite; Genetic distance; Correspondence analysis; Population structure
1. Introduction The Tzotziles and other Maya native groups are located in Southern Mexico, particularly in the Chiapas region. Sheep and wool are economically important as well as a cultural patrimony of the native communities. Today sheep are considered as “animal companions”; their slaughtering is forbidden. Wool is manufactured to make distinctive ethnic clothes.
⁎ Corresponding author. Tel.: +34 957322344; fax: +34 957322493. E-mail address: [email protected] (J.L. Vega-Pla). 1871-1413/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.livsci.2007.09.022
The sheep from Chiapas are isolated within geographic areas that make the introduction and adaptation of exotic breeds very difficult. They are traditionally divided into three groups according to coat color: Café (dark brown), Chamula (black) and Chiapas (white). Microsatellite markers have been shown to be useful tools for the analysis of genetic differentiation among sheep populations as well as aid in conservation decisions for genetic resources (Gutiérrez-Espeleta et al., 2000; Saitbekova et al., 2001; Tomasco et al., 2002; Rendo et al., 2004; Peter et al., 2005; Paiva et al., 2005), Here we describe microsatellite marker analyses that were carried out to help to clarify the genetic structure of the native
J. Quiroz et al. / Livestock Science 116 (2008) 156–161
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Table 1 F statistics, probability of global equilibrium and number of alleles by locus Locus
Fis
Fit
Fst
p-val
Number of alleles
BM1818 BM1824 BM6506 BM6526 BM8125 CSRD247 CSSM66 D5S2 ETH10 ETH225 HSC ILSTS011 INRA35 INRA63 MAF209 MAF65 McM527 OarCP20 OarCP34 OarFCB11 OarFCB304 OarFCB48 RM006 SPS115 TGLA122 TGLA126 TGLA53
0.0777 0.0758 0.0787 0.0781 0.0789 0.0799 0.0777 0.0762 0.0767 0.0749 0.0784 0.0790 0.0753 0.0792 0.0776 0.0789 0.0753 0.0798 0.0791 0.0767 0.0790 0.0782 0.0791 0.0707 0.0680 0.0684 0.0754
0.1710 0.1644 0.1708 0.1677 0.1730 0.1732 0.1712 0.1679 0.1696 0.1691 0.1723 0.1715 0.1683 0.1711 0.1713 0.1720 0.1679 0.1705 0.1716 0.1709 0.1702 0.1717 0.1719 0.1646 0.1611 0.1632 0.1670
0.1012 0.0959 0.0999 0.0972 0.1022 0.1014 0.1015 0.0992 0.1006 0.1018 0.1018 0.1005 0.1006 0.0997 0.1017 0.1011 0.1001 0.0986 0.1004 0.0997 0.1014 0.1015 0.1008 0.1011 0.0999 0.1017 0.0991
0.0018 0.0001 0.0327 0.0118 0.0236 0.0982 0.0005 0.0026 0.0587 0.0000. 0.0026 0.0000 0.0000. 0.0010 0.0000. 0.4446 0.0001 0.0028 0.0021 0.2338 0.0001 0.0096 0.2870 0.0000 0.0000 0.0000 0.0006
16 7 10 13 9 18 25 13 7 9 16 15 16 17 16 13 13 13 10 18 24 14 12 10 15 21 13
sheep populations from Chiapas and its relationships with some Spanish breeds. 2. Materials and methods Blood or wool samples (number of samples in brackets) were obtained from a random sampling of Chamula (41), Café (42) and Chiapas (45) sheep from the state of Chiapas, Mexico. Other breeds sampled were the Palmera (47) and the Canary
Table 2 Population, sample size, N, mean number of alleles, MNA, expected, He, unbiased, Heu and observed, Ho heterozygosity levels and the values of Fis, confidence intervals of 95% are shown for each of the sheep populations in this study Population N
MNA He
He_u Ho
Fis
Chiapas Chamula Café Palmera Canaria Merino French Merino Churra
6.1 6.9 7.8 5.6 7.9 6.7 6.1
0.633 0.708 0.721 0.619 0.728 0.669 0.667
0.042, − 0.005–0.059 0.026, − 0.021–0.045 0.076, 0.033–0.096 0.091, 0.038–0.0143 0.058, − 0.004–0.076 0.074, 0.016–0.106 0.023, − 0.019–0.057
41 42 45 47 40 44 46
35 8.4
0.624 0.699 0.712 0.612 0.718 0.661 0.660
0.606 0.690 0.666 0.563 0.686 0.620 0.652
0.728 0.740 0.654 0.118, 0.078–0.132
Fig. 1. Factorial analysis of correspondence of three sheep populations from Chiapas, and other breeds.
(46) from the Canary Islands, Spanish Merino (44), Precoce French Mutton Merino (46), Churra (35). A panel of 27 microsatellite markers was used for amplification via Polymerase Chain Reaction (PCR) according to the method of Martinez et al. (2000). Fluorescently labeled PCR fragments were separated by electrophoresis in an automatic sequencer ABI377XL (Applied Biosystems, Foster City, CA, USA). Allelic frequencies, heterozygosity, number of migrant alleles, parameters of genetic differentiation (F statistics), and a Factorial Analysis of Correspondence, were calculated with the software Genetix v. 4.05 (Belkhir et al., 2000). Hardy– Weinberg equilibrium was accomplished by the method of Guo and Thompson (1992) using the Markov Chain of Monte Carlo algorithm in GENEPOP v. 3.1c software (Raymond and Rousset, 1995). Analysis of molecular variance (AMOVA) was performed with the software Arlequin 2.0 (Schneider et al., 1997) to evaluate the genetic structure of the populations. The
Table 3 DA genetic distances (below the diagonal) and number of migrants (above the diagonal) among the sheep populations Café Chamula Chiapas Merino French Merino Palmera Canaria Churra
0 10.26 0.093 0 0.115 0.096 0.199 0.217 0.228 0.233 0.255 0.198 0.182
4.26 4.94 0 0.241 0.256
2.55 2.39 1.76 0 0.207
2.00 1.95 1.46 2.08 0
1.70 1.53 1.23 1.15 1.23
2.95 2.64 1.77 2.09 1.88
4.13 3.17 2.02 2.60 3.06
0.278 0.281 0.315 0.307 0 2.39 1.97 0.219 0.247 0.227 0.252 0.206 0 3.99 0.205 0.238 0.211 0.192 0.242 0.170 0
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3. Results
Fig. 2. Genetic relationships among the sheep populations based on a neighbor-joining tree constructed with DA distances.
genetic structure of population is investigated here by an analysis of variance framework, as initially defined by Cockerham (1969, 1973),. The Analysis of Molecular Variance approach used (AMOVA, Excoffier et al., 1992) is essentially similar to other approaches based on analyses of variance of gene frequencies, but it takes into account the number of mutations between molecular haplotypes. A hierarchical analysis of variance partitions the total variance into covariance components due to intra-individual differences, inter-individual differences, and/or inter-population differences. The significance of the fixation indices is tested using a nonparametric permutation approach described in Excoffier et al. (1992). A large number of permutations is necessary to obtain some accuracy on the final probability. The DA genetic distance (Nei et al., 1983; Takezaki and Nei, 1996) was calculated using a Populations 1.2.28 software (Langella, 2002). A neighbor-joining tree was built with DA genetic distances and was graphically represented with TreeView software (Page, 1996). Analysis of individual assignment to populations was done using the Structure v. 2.0 software (Pritchard et al., 2000).
All microsatellites were polymorphic, except ETH10 which was monomorphic in some populations. A total of 383 alleles were detected in the 27 microsatellites analyzed showing a mean number of alleles of 14.19 with a maximum of 25 in CSSM66 and a minimum of 7 in ETH10 and BM1824. The Hardy–Weinberg disequilibrium tests were statistically significant only for six microsatellites in the Chiapas population and eight in Café and Chamula ones. None of the microsatellite loci were in disequilibrium in all of the investigated populations. However, TGLA122 and TGLA126 were in disequilibrium for 7 breeds. The Fis, Fit and Fst values (Table 1) were similar for all microsatellites. Twenty two loci deviated (p b 0.05) from the Hardy–Weinberg equilibrium in the pooled samples demonstrating genetic heterogeneity among breeds. The mean value of the F statistics and the confidence intervals at 95% after 1000 bootstraps were as follow: Fis = 0.076 (0.044–0.106), Fit = 0.169 (0.138– 0.198) and Fst = 0.100 (0.087–0.115). The mean value of Fis supports the general quality of the sampling and the presence of low levels of inbreeding in the majority of populations. The mean Fst indicates the existence of defined subpopulations. The AMOVA analysis showed similar results (Fis = 0.020, Fst = 0.095 and Fit = 0.113) with a highly significant p-value (P b 0.01). Geographic partitioning of variance breeds was tested grouping them in European (Churra Merino and French Merino), Canarian (Palmera and Canaria) and Mexican breeds. The AMOVA results were (Fis = 0.047, Fst = 0.0741 and Fit = 0.113) with a highly significant p-value (p b 0.01).
Fig. 3. Estimation of the population structure with k different values.
J. Quiroz et al. / Livestock Science 116 (2008) 156–161 Table 4 Proportion of animals of each population pre-defined in the 9 clusters Breed
Cluster 1
Café 0.013 Chamula 0.013 Chiapas 0.007 Canaria 0.013 Palmera 0.003 Merino 0.922 French 0.010 Merino Churra 0.012
2
3
4
5
6
7
8
0.008 0.019 0.009 0.010 0.007 0.006 0.004
0.658 0.083 0.025 0.012 0.004 0.012 0.008
0.012 0.019 0.010 0.034 0.004 0.008 0.006
0.009 0.007 0.006 0.011 0.708 0.008 0.003
0.014 0.012 0.005 0.894 0.008 0.014 0.006
0.245 0.819 0.924 0.010 0.003 0.010 0.008
0.032 0.017 0.009 0.011 0.261 0.008 0.006
0.015 0.010 0.904 0.009 0.017 0.007 0.012
There was more variability among populations than geographical groups. Table 2 shows the sample size, mean number of alleles, the heterozygosity and the values of Fis for each of the sheep populations. The factorial analysis of correspondence (Fig. 1) grouped the Chiapas populations in the left lower quadrant away from the other breeds. The information of the first two axes explains 42.31% of the variation among populations. There was a possible gene flow from the Churra and Canarian breeds based in the number of migrants. Also, close relationship between Cafe and Churra populations were detected in DA distances matrix (Table 3). Fig. 2 shows a neighbor-joining tree based on Nei's DA distances in which the clustering of the three populations from Chiapas is evident. The high values of bootstrapping supported the strength of the results in respect to the differentiation of breeds, although the main node obtained only 49% bootstrap. The branch length that separates the Chiapas nucleus from the others indicates the degree of differentiation that exists with the present Spanish breeds. The method of Falush et al. (2003) was used to assign individuals to inferred clusters based on the allele frequencies of each locus. The number of hypothetical clusters (K) was 2‑9 and 230,000 repetitions of the Markov Chain Monte Carlo algorithm were used. In Fig. 3, each individual is represented by a thin vertical line, which is divided in K colored segments that represent the fraction of each individual belonging to each one of the inferred clusters. The black lines separate the individuals of the different populations. When K = 2, a cluster was formed of the Mexico and Canary Islands populations (Canaria breed is divided in both clusters) and all remaining breeds were in another cluster. In the case of K = 3, the three Mexican
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populations clustered together. When K = 8, Café was separated from the Chiapas populations. Table 4 shows the proportion of individuals belonging to each population assigned for K = 9. Café, Chamula and Chiapas were grouped together in the same cluster 7 although a great proportion of Café individuals were assigned in a new cluster. 4. Discussion FAO defines a breed as a specific group of domestic animals with definable and identifiable external characteristics that permit to differentiate it from other similar groups of the same species. In this way, molecular biology techniques to characterize populations are very useful tools in order to obtain correct conclusions depending on the species. However, to establish credible hypotheses amenable to be tested by means of genetic analysis, it is necessary to have knowledge about both the old and recent history of the populations under research. This includes the reproductive management and their relationships with other populations, etc. The mean number of alleles was lower than the values showed by sheep from the North of the Iberian Peninsula (Rendo et al., 2004), but higher in general than those obtained for Finish and Russian sheep breeds (Tapio et al., 2003). The observed mean heterozygosities among sheep from Chiapas was lower than that in Swiss breeds (Saitbekova et al., 2001) with the exception of the mouflon; also it was lower than the Spanish Merino (Diez-Tascón et al., 2000). For breeds under conservation plans, the observed mean heterozygosity could be as low as 0.52 (Gutiérrez-Espeleta et al., 2000). The expected heterozygosities were similar to the Corriedale and Merino sheep from Uruguay (Tomasco et al., 2002). The level of genetic differentiation among domestic sheep breeds based on molecular genetic marker information is low in comparison with the reports in cattle (Fst = 0.186; Quiroz et al., 2004) or among subspecies of wild sheep (Ovis dalli) (Fst = 0.160) (Worley et al., 2004). The low differentiation among sheep breeds could be artificial and a consequence of the use of many microsatellites that are homologous with cattle located in highly conserved DNA regions. Alternatively, the domestication process and the ability of sheep to accompany human populations in their migrations probably contributed to the poor differentiation at a breed level due to continuous crossbreeding among different populations. This circumstance is also observed among domestic horses and goats (Ajmone-Marsan et al., 2001;
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Barker et al., 2001; Mirol et al., 2002; Xiang Long and Valentini, 2004). Regarding the variability detected by the selected microsatellites, ETH10 which was monomorphic in some populations, the remaining 26 markers showed a high mean number of alleles (14.19). The Hardy– Weinberg equilibrium of microsatellites was adequate for all populations except the Churra breed. The Fis, Fit and Fst values also demonstrated the near absence of uncontrolled biases such as great errors of sampling, inbreeding, etc. It is possible to consider that these three sheep populations from Chiapas have a relatively high degree of differentiation (Fst = 0.1) considering the Fst value of 0.07 obtained by Rendo et al. (2004) in a study across Spanish sheep breeds, or Fst = 0.03 in Merinos from different countries (Diez-Tascón et al., 2000). The assignment of individuals to populations showed a clear differentiation of Mexican sheep populations from the Merino, Churras and Canary Islands sheep. These results also indicate that the Café population must be reconsidered because several individuals previously assigned to this population showed a genetic divergence from the genetic profile estimated for this population. This finding must be supported by morphological, productive, functional and genealogical information of these discordant individuals. Genetic dispersion detected on the Café population is supported by the phenotypic studies of wool quality. Those studies showed the black and brown fibers that go from light brown to dark brown, resulting in different coat tones (Perezgrovas and Castro, 1998). The three different coat colors of the Mexican sheep populations have resulted in the reproductive isolation of the Chamula, Café and Chiapas, as a consequence of wool preferences by the native artisan industries throughout the centuries. The clear genetic differentiation between the Chamula and the Chiapas supports this conclusion, while the similarity observed between the Café and Chamula does not indicate a continuous reproductive isolation among these two populations. Our results agree with those obtained in previous works based on morphological characterization (Pedraza et al., 1992; Perezgrovas, 1998). Sheep breeding in Chiapas has survived in the present because of the animals' adaptation to the environment, their rusticity, and their integration in the rural economy of the Tzotzil natives who depend on them. Previously there were no formal conservation programs, only their own survival in the hard environment and their capacity to produce wool under these conditions. It is not possible to completely eliminate the
possibility of some influence from exotic breeds over these sheep populations; however, the adaptation of the local sheep to the challenging environment has always favored local genotypes. The influence of the Spanish breeds over the Chiapas populations is likely from both the morphological and historical point of view, but the results from our analyses did not support this assumption. This could be due to genetic drift over centuries in both Spanish as well as Chiapas sheep populations, as well as to other systematic effects (selection and migration) which separated these breeds from their Spanish ancestors although some common morphological traits were conserved. The introduction of the Spanish Merino to Mexico by Spanish colonizers is well documented (Rodero et al., 1992), although its influence cannot be appreciated from genetic distances. 5. Conclusion From a genetic perspective, it is possible to support the contention that the Café, Chamula and Chiapas are independent and lightly differentiated populations both among themselves as well as from other sheep breeds. This supports previous studies based on morphology (Pedraza et al., 1992, Perezgrovas, 1998) and productivity (Perezgrovas and Castro, 1998). Chiapas sheep populations are an important Mexican genetic heritage and must be immediately subjected to conservation programs linked to the indigenous culture where they have contributed over centuries. Acknowledgments We are particularly in debt to Cecilia Penedo and Phil Sponenberg for their valuable corrections and comments of this manuscript. This research was funded by the Andalucian Research Group “Mejora y Conservación de los Recursos Genéticos de los Animales Domésticos” (AGR218) sited at the University of Córdoba (Spain) as part of their efforts to improve the genetic diversity of the domestic animals. References Ajmone-Marsan, P., Negrini, R., Crepaldi, P., Milanesi, E., Gorni, C., Valentín, A., Cicogna, M., 2001. Assessing genetic diversity in Italian goat populations using AFLP markers. Anim. Genet. 32, 281–288. Barker, J.S.K., Tan, S.G., Moore, S.S., Muekherjee, T.K., Matheson, J.L., Selvaraj, O.S., 2001. Genetic variation within and relationships among populations of Asian goats, (Capra hircus. J. Anim. Breed. Genet. 118, 213–233.
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