Genetic Variations of 13 Indigenous Chinese Goat ... - Springer Link

C 2006) Biochemical Genetics, Vol. 44, Nos. 3/4, April 2006 ( DOI: 10.1007/s10528-006-9013-6

Genetic Variations of 13 Indigenous Chinese Goat Breeds Based on Cytochrome b Gene Sequences Shilin Chen,1 Bin Fan,1 Bang Liu,1 Mei Yu,1 Shuhong Zhao,1 Mengjin Zhu,1 Tongan Xiong,1 and Kui Li1,2 Received 16 August 2004—Final 16 August 2005 Published online: 12 May 2006

Phylogenetic relationships among and genetic variability within 13 Chinese indigenous goat breeds and Boer goat were analyzed using cytochrome b gene sequences. There were 44 variable sites found in a 642 bp sequence, and 46 Cyt b haplotypes were subsequently defined. The phylogeny analysis of haplotypes in combination with goat Cyt b sequences from GenBank shows that Chinese goats are obviously separated from wild goats and might come from Capra aegagrus. Further analysis indicated that indigenous Chinese goats might descend from at least two lineages; most of the individuals analyzed could be classified into lineage A as defined by Luikart, but five other goats were of uncertain lineage. The Tibet plateau is a possible place of origin for Chinese goats. The neighbor-joining tree based on pairwise differences among populations shows that most Tibetan goats, except the Middle Tibet type, cluster closely with North China goats, and then with South China goats. This result confirms that differences in genetic structure exist among goats in different geographic locations. Nucleotide diversity varied among populations. Tibet and North China goats had higher genetic diversity than South China goats. The fixation index (Fst = 87.72%) suggested that most of the total genetic variation was due to variation within populations. In addition, the results indicate that Cyt b gene sequence information alone might not be enough for phylogeny analysis among breeds within species, as shown by fewer polymorphic sites and lower bootstrap values on the neighbor-joining tree. KEY WORDS: phylogeny relationship; genetic variability; Cyt b gene; Chinese goat.

1 Laboratory of Molecular Biology and Animal Breeding, College of Animal Science and Technology,

Huazhong Agricultural University, Wuhan, 430070, P.R. China. whom correspondence should be addressed; e-mail: [email protected], likuihau@ yahoo.com.

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89 C 2006 Springer Science+Business Media, Inc. 0006-2928/06/0400-0089/1

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Chen, Fan, Liu, Yu, Zhao, Zhu, Xiong, and Li

INTRODUCTION The domestic goat (Capra hircus) is classified as Capra, Caprovinae, Bovidae, Ruminatia, Artiodactyle. China raises the largest number of goats in the world and has an abundance of goat breeds. There are 34 breeds of indigenous goats listed in the Domestic Animal Diversity Information System (DAD-IS) of FAO, and 20 are described in detail in Sheep and Goat Breeds in China (Tu et al., 1989). The goat is one of the most important livestock species in animal husbandry, offering a variety of agricultural products such as wool, milk, mutton, and kid leather. The goat also has a strong fitness and foraging capability, which makes it adaptable to numerous areas throughout China, from the frigid and warm zones of North China to the subtropical zone of South China. It is generally recognized that the domestic goat originated in west Asia about 12,000 years ago (Maijala, 1998; Zeder and Hesse, 2000). Modern goat breeds are found in the boundary region of the Tibet plateau in China and the adjacent mid-Asian countries, and evidence obtained from archeological material and historical records indicates that goat farming has been practiced in China for about 7000–10,000 years (Li, 1993; Tu et al., 1989). In the last two decades, various molecular markers have been explored and commonly utilized in the studies of genetic diversity and phylogenetics of domestic goats in China. Based on studies of RFLP and the mtDNA genome, Li et al. (1999) deduced that the origin and evolution of modern Chinese goat breeds were independent of those of exotic goats and that the indigenous goats could be grouped into two main types, the North type and the South type. Chang et al. (2000) thought that the Chinese goat might have been subject to a period of tameness after its emergence in the Tibet plateau region and then spread toward east and south China. Our previous studies using microsatellite markers also revealed that the genetic relationships of indigenous goats were in accordance with their geographical distribution, and that they could be further classified into two systematic branches (Li et al., 2002). There is less information, however, on phylogenetic studies of Chinese goats by means of mtDNA sequences. The cytochrome b (Cyt b) gene is one of the important coding genes in mtDNA; it is about 1.2 kb in length. Because of its maternal inheritance, its well-known gene structure and sequence, the occurrence of low recombination, and other characteristics, the Cyt b gene has been widely used for phylogenetic studies of various species (Giuffra et al., 2000; Hiendleder et al., 1998; Irwin et al., 1991; Luikart et al., 2001). Here we carried out Cyt b gene sequencing of 13 Chinese indigenous goat breeds and the Boer goat to demonstrate the genetic variations and possible origins of these breeds.

Genetic Variation of Cytochrome b in Chinese Goats

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MATERIALS AND METHODS Sample Collection Blood samples of 84 individuals belonging to 14 goat populations were collected. Four populations of the Tibetan goat were sampled in different geographic districts of Tibet (North Tibet, NT; Middle Tibet, MT; East Tibet, ET; Southeast Tibet, SET). The Inner Mongolia Cashmere goat (NM), the Liaoning Cashmere goat (LN), the Wu’an goat (WA), and the Matou goat (MAT) were sampled from their locations of origin. The individuals studied were unrelated or they had the least relationships in three generations. Genomic DNA was extracted according to a modified phenol and chloroform method. DNA samples of five other goats (Chuandong White, CDW; Wu, WU; Black, BL, Nanjiang Brown, NJB; Small Xiang, SX) were kindly provided by Prof. Wei Hong (Third Military Medical University of China, Chongqing). Additionally, the Boer goat (BE) was used as the outgroup population. Detailed sampling information is shown in Table I. Mitochondrial DNA Cyt b Sequencing Six individuals from each goat population were used for Cyt b gene sequencing. The primers were designed as follows: the first pair of primers (Primer 1-F: 5 -CCTCCTGCTCGCAACAA TAG-3 and Primer 1-R: 5 GGGATGTTCGACTGGCTGTC-3 ) was used to amplify a 684 bp PCR product.

Table I. Sampling Information for 14 Goat Populations Population East Tibetan goat Southeast Tibetan goat Middle Tibetan goat North Tibetan goat Mongolia Cashmere goat Liaoning Cashmere goat Wu’an goat Matou goat Wu goat Chuandong White goat Small Xiang goat Black goat Nanjiang Brown goat Boer goat

Abbreviation ET SET MT NT NM LN WA MAT WU CDW SX BL NJB BE

Sample collection Basu, Tibet Langkazhi, Tibet Linzhi, Tibet Naqu, Tibet Yikezhao State, Mongolia Gaizhou City, Liaoning Wu’an County, Hebei Yunxi County, Hubei Three Gorges, Sichuan Three Gorges, Sichuan Leishan County, Guizhou Three Gorges, Sichuan Three Gorges, Sichuan Yichang City, Hubei

Geographic types Tibet Tibet Tibet Tibet North China North China North China South China South China South China South China South China South China South China

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The PCR conditions were: 0.4 µm of each primer, 160 µm of each dNTP, 2.5 mM MgCl2 , 1.5 unit Taq DNA polymerase enzyme, 2.5 µL buffer, 1.5 µL DNA template, and ddH2 O in a total of 25 µL volume. The PCR amplifications were conducted using a PTC-100 Thermal Controller (MJ Research) according to the program, 95◦ C, 2 min; 35 cycles of 95◦ C, 30 s, 50◦ C, 30 s, 72◦ C, 1 min; and final extension of 72◦ C, 10 min. PCR target fragments were recovered in low-melting agarose gel and purified using Promega’s Wizard PCR Preps DNA purification kit (A7170). The purified PCR products were sequenced directly by the Genomic and Bioinformatic Center, Chinese Academy of Science (Beijing). The second pair of primers (Primer 2-F: 5 -AGCCCTCGCCCATAGTCCA-3 and Primer 2R: 5 -TGTAGTAAGGGTGAA A TGG-3 ) were used as “internal primers” for sequencing a 642 bp fragment from the Cyt b gene.

Statistical Analysis The quality of the 642 bp Cyt b gene sequence for individuals was firstly evaluated on the basis of sequencing peak value, and then these sequences were entered into the DNAstar (ver. 5.01) software package to obtain sequence order and alignment by the Megalign program. The basic statistical estimators, including nucleotide composition, the ratio of transitions and transversions, and haplotype analyses, were computed with the software MEGA2 (http://mep.bio.psu.edu). Neighbor-joining trees were constructed under Kimura 2-parameter distance, Tamura 3-parameter distance, and Tamura-Nei distance, with α = 0.29 of gamma distribution referenced to that of Luikart et al. (2001). Bootstraps of 1000 replications were carried out in order to test the robustness of the phylogenetic tree. The diversity indices including haplotype diversity (H), the average number of differences among pairs of haplotypes (K), the proportion of polymorphic sites (pS ), nucleotide diversity (π ), and the average number of pairwise differences between populations (Dxy ), were calculated by Arlequin software (Schneider et al., 2000). The neighbor-joining tree based on pairwise differences among populations was implemented with the neighbor procedure of the Phylip software package (Felsenstein, 1993). The hierarchical analysis of variance partition and exact test of population differentiation were estimated with the AMOVA procedure of the Arlequin software (Schneider et al., 2000). In addition, the reported Cyt b sequences of domestic and wild goats were downloaded from GenBank and used for a combined analysis with sequences of this study. These sequences were: Capra hircus (GenBank AB00407075, AB110594-97), C. aegagrus (AB004069, AB110592-93, AF034739), C. pyrenaica (AJ010047, AJ010050, AJ010054, AJ010057), C. cylindricornis (AF034737), and C. caucasica (AF034738).


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RESULTS Cytochrome b Polymorphism and Haplotype Analysis There were 44 variable sites found in the sequenced Cyt b gene fragments, and 46 Cyt b haplotypes could be defined (Fig. 1). All 44 variable sites were single nucleotide substitutions, and no insertions and deletions were detected. Among these polymorphic sites, there were 14 singleton polymorphic sites of two variants, and 30 parsimony informative polymorphic sites of two variants. Three major haplotypes were shared by 20, five, and four individuals. Assuming a gamma distribution parameter of α = 0.29, a neighbor-joining tree of goat haplotypes was constructed from the Kimura 2-parameter model, in which the total goat haplotypes could be roughly divided into three clades (Fig. 2). The indigenous Chinese goats and Boer, Pakistani goats (Sultana et al., 2003), and four wild goats formed a major clade. Within this clade two main branches could be roughly identified. Most indigenous goats clustered closely with a wild goat C. aegagrus (AB004069), and only five indigenous goats clustered with another wild goat C. aegagrus (AF034739). Additionally, four wild goats of C. pyrenaica were classified into one clade, and two wild goats of C. aegagrus were in another clade.

Genetic Variability and Genetic Structure To demonstrate genetic variability within Chinese goat populations, the basic demographic parameters, including haplotype number (A) and diversity (E), the average number of differences among pairs of haplotypes (K), the proportion of polymorphic sites (pS ), and nucleotide diversity (π ), based on Cyt b gene sequence information for 13 indigenous goat populations were estimated (Table II). Haplotype diversity estimates were between 0.6 and 1.0, and nucleotide diversity varied widely among populations, ranging from 0.15% in WU goats to 1.57% in NM goats. Comparing these five estimators, NM goats had the higher genetic variability, followed by MAT, ET, LN, SET, SX, NT, CDW, BL, MT, NJB, WA, and WU goats. As far as the geographic locations are concerned, intrapopulation genetic variation within Tibet goats was the highest, followed by North China goats and South China goats. The second neighbor-joining tree shows that these 13 indigenous populations could be roughly divided into two major groups (Fig. 3). Seven populations of Tibetan and North China goats, except MT goats, clustered into one group, and the other six populations of South China goats clustered into another group. The cluster patterns were concordant with their geographic localities and were also in agreement with our previous studies using microsatellite markers (Li et al., 2002).

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Fig. 1. Distribution of 44 observed variable sites and 46 Cyt b haplotypes found in goats from 14 localities.


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Fig. 2. Phylogenetic tree constructed from the neighbor-joining method using the Kimura 2parameter model and assuming α = 0.29. The whole topology tree could be classified into three clades, A, B, and C. The number at the branch node indicates the percentage occurrence in 1000 bootstrap replications. The bar scale indicates the genetic distance among Cyt b haplotypes.

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Table II. Genetic Variability Statistics for Cyt b in 13 Populations of Chinese Goats Populations

A K H pS π

MT

NT

ET

SET

LN

NM

WA

WU

SX

NJB

CDW BL

MAT

4 2.40 0.80 0.009 0.37

3 4.67 0.83 0.014 0.72

6 8.33 1.00 0.026 1.29

5 6.20 1.00 0.022 0.95

6 6.86 1.00 0.028 1.07

6 10.06 1.00 0.037 1.57

4 1.87 0.87 0.006 0.29

3 1.00 0.60 0.005 0.15

4 4.80 0.87 0.017 0.75

5 1.93 0.93 0.006 0.30

3 4.00 0.73 0.015 0.62

5 8.80 1.00 0.028 1.37

4 3.80 0.80 0.014 0.59

Note. A, number of haplotypes in each population. K, mean number of pairwise differences within population. H, haplotype diversity. pS, proportion of polymorphic sites. π , nucleotide diversity (in %).

The AMOVA results of the hierarchical analysis of molecular variance of population structure showed that a large percentage (87.72%) of the total Cyt b gene variation was within populations, and a much smaller percentage was among populations (12.28%). Thus, the phylogeographic structure of Chinese goats is weak and similar to other goats in other continents (Luikart et al., 2001). DISCUSSION Polymorphism of Cyt b Gene Sequence Because of the well-known gene structure and lack of recombination, the Cyt b gene has been generally used alone or in combination with other mtDNA encoding genes and hypervariable regions for phylogenetic studies between species. Generally speaking, the AT content is always higher than the GC content in Cyt b. Our

Fig. 3. The neighbor-joining tree based on pairwise differences (Dxy ), showing the genetic relationships among indigenous Chinese goat populations. The bar scale indicates the pairwise differences among populations.


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study was consistent with that, showing proportions of 56:44. As an encoding gene of mtDNA, the occurrence of mutation of the Cyt b gene is medium compared to mutation in the D-loop and other encoding genes. There were 44 polymorphic sites found in a 642 bp sequence; most of these mutations (30 sites) were nonsense mutations, and only a few (14 sites) were shift mutations. The genetic variations detected in this study were much fewer than those in mtDNA D-loop, with 160 variable sites in a 481 bp sequence (Luikart et al., 2001).

Phylogenetic Relationships of Chinese Goat Populations According to phylogenetic analysis using mtDNA D-loop and Cyt b gene sequencing, Luikart et al. (2001) deduced that domestic goats throughout the world might have three maternal origins, and lineage A is the most important of the three. Mannen et al. (2001) further concluded that domestic goats came from two kinds of C. aegagrus. Recently, Sultana et al. (2003) classified Pakistani goats into four lineages, in addition to lineages A, B, and C as defined by Luikart et al. (2001). On the basis of the phylogenetic patterns of Cyt b gene haplotypes in this study, Chinese indigenous goats were clustered with C. aegagrus, though they were obviously separated from other wild goats. Further analysis indicated that these indigenous goats could roughly be classified into two lineages. One comprising most of the individuals undoubtedly corresponded to lineage A of Luikart et al. (2001) and Sultana et al. (2003). It is uncertain whether the other lineage, comprising five individuals, corresponds to lineage B or C, since not enough Cyt b gene sequence information could be obtained from GenBank. It is accepted that domestic goats in China descended from the Tibet plateau and the adjacent areas of Middle Asia. There are two kinds of wild goats (Capra aegagrus aegagrus and Capra a. ibex) that possibly contributed to the origin and evolution of domestic goats, and both of them can still be found in Tibet, Qinghai, and Inner Mongolia. As shown in Fig. 2, Tibet goats mingled into these two lineages, which might confirm that the Tibet area is an important region for goat origin and domestication.

Population Genetic Diversity of Chinese Goat Breeds Based on analyses of morphology and performance traits and molecular markers such as RAPD and mtDNA-RFLP, there are great differences in genetic structure between goats in North and South China (Gao and Kong, 2000; Li et al., 1999, 2000). As far as the breed cluster in this study is concerned, goat breeds in Tibet, except MT, and North China goats were classified into one branch, and other goats in South China were grouped into a second one. This clustering further showed the existence of differences between goats in South and North China.

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The phylogenetic relationships between Cyt b haplotypes were relatively loose, as shown by the low bootstrap values on the cluster branches. The fixation index (Fst = 87.72%) indicated that the majority of the total genetic variation was due to differences within populations, and only a small amount was due to variation among populations. The extent of gene differentiation of these goat breeds was in accordance with that obtained from microsatellites (Li et al., 2002). From three statistical indices (H, K, π ), the population variability of the North type and Plateau type was higher than that of the South type. The π value further indicated that the North type had the highest extent of nucleotide variability, followed by the Plateau type and the South type. The higher variation of the North type may be due to the larger population size and extensive farming system. But π estimates showed that the genetic diversity of these indigenous goat breeds was the same as that obtained from mtDNA RFLP detection and was also lower compared with that obtained from other molecular markers such as RAPD and microsatellites (Barker et al., 2001; Chen et al., 2001; Kim et al., 2002; Saitbekova et al., 1999). Our previous studies using microsatellite loci showed that the average gene heterozygosity of these goat breeds was between 0.611 and 0.784 (Li et al., 2002; Yang et al., 1999). The differing results might be caused by differences between intranucleic and internucleic gene materials with respect to the mode of inheritance, selection pressure, and mutation rate. Meanwhile, it also revealed that the Cyt b gene sequence is less polymorphic than the D-loop and might have lower advantages for phylogenetic analysis among breeds within species. In this study, on the basis of mtDNA Cyt b sequence analysis, Chinese indigenous goats have at least two origins and a loose genetic structure. The population genetic diversity within North China was higher than that of South China.

ACKNOWLEDGMENTS This research was supported by the Key Project of the National Basic Research and Developmental Plan of China (G2000016103), the Key Project of the National Natural Science Foundation of China (30330440), National High Science and Technology Foundation of China (2004AA222170), and National Natural Science Foundation of China (30270951). Authors Shilin Chen and Bin Fan contributed equally to this work.

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