SHORT COMMUNICATION: Mitochondrial DNA D-loop sequence diversity and origin of Chinese pony breeds (Equus caballus)

SHORT COMMUNICATION: Mitochondrial DNA D-loop sequence diversity and origin of Chinese pony breeds (Equus caballus) Sheng-lin Yang 1, Ai-Ping Li 2, Long-xing Xu 3, and Haibing Yang 1 1 Key Laboratory of Animal Genetics, Breeding and Reproduction of Educational Ministry in Guizhou Mountainous Area, Guizhou University, Guiyang, 550025, Guizhou Province, China (e-mail: shenglinyang@126.com); 2 Station of Animal Husbandry and Veterinarian of Kaili, Kaili, Guizhou 560000, China; and 3 Research Institute of Animal and Veterinary Science, Guiyang, Guizhou province. Received 6 December 2012, accepted 12 May 2013. Yang, S.-l., Li, A.-P., Xu, L.-x. and Yang, H. 2013. SHORT COMMUNICATION: Mitochondrial DNA D-loop sequence diversity and origin of Chinese pony breeds (Equus caballus). Can. J. Anim. Sci. 93: 313319. Previous studies based on mitochondrial DNA (mtdna) data have shown that Chinese horses have high genetic diversity. However, little is known about the genetic diversity of mtdna D-loop sequences and maternal origin of five Chinese pony breeds. In the present study, genetic diversity of 343-bp D-loop sequences for136 individuals representing five Chinese pony breeds was analyzed. To address the question of the single and multiple maternal origin of Chinese pony populations, 13 partial mtdna D-loop reference sequences from GenBank representing eight ancient and modern horse breeds (Connemara, Garrano, Sorraia, Pottok, Losino, Cheju, Tuva and a Swedish horse) were added to conduct the phylogenetic analyses. A total of 59 haplotypes and 50 polymorphic loci were detected, the haplotype diversity (h) ranged from 0.894 to 0.947 and nucleotide diversity (p) ranged from 0.0184 to 0.0229, suggesting relatively abundant genetic diversity in the Debao, Yunnan and the Guizhou breeds. The phylogenetic tree and median-joining network show multiple maternal origins of the five Chinese pony breeds. Key words: Pony, mitochondrial DNA, genetic diversity, origin Yang, S.-l., Li, A.-P., Xu, L.-x. et Yang, H. 2013. BRE` VE COMMUNICATION: La diversite de la se quence de la boucle D de l ADN mitochondrial et l origine des races de poney chinois (Equus caballus). Can. J. Anim. Sci. 93: 313319. Les e tudes pre ce dentes base es sur l ADN mitochondrial ont montre que les chevaux chinois posse` dent une grande diversite géne tique. Cependant, il y a peu de connaissances au sujet de la diversite ge nétique de la se quence de la boucle D de l ADN mitochondrial et au sujet de l origine maternelle de cinq races de poney chinois. Dans cette e tude, la diversite ge ne tique des se quences de 343 paires de bases de la boucle D de 136 individus repre sentant cinq races de poney chinois a e te analyse e. Pour traiter de la question de l origine maternelle unique ou multiple des populations de poney chinois, treize se quences partielles de boucle D d ADN mitochondrial de référence provenant de GenBank et repre sentant huit races anciennes et modernes de chevaux (Connemara, Garrano, Sorraia, Pottok, Losino, Cheju, Tuva et un cheval sue dois) ont été ajoute es pour mener l analyse phyloge ne tique. Un total de 59 haplotypes et 50 loci polymorphiques ont été de tectés. La diversite haplotypique (h) a varie de 0,894 a` 0,947 et la diversite nucle otidique (p) a varie de 0,0184 à 0,0229, ce qui sugge` re une diversite géne tique relativement grande des races Debao, Yunnan et Guizhou. L arbre phylogéne tique et le re seau de jonction des me dianes ont mis en e vidence des origines maternelles multiples des cinq races de poney chinois. Mots clés: Poney, ADN mitochondrial, diversite ge ne tique, origine Five native pony breeds are regarded as national treasures in China: the Ningqiang from Shanxi province in northwest China, the Debao from Guangxi province in southeast China, and the Yunnan, the Guizhou and the Sichuan from southwest China. These breeds, together with the Shetland pony in Great Britain, are known the two main pony sources in the world (Sun and Wang 2007), but there is no evidence to show if this is correct or not. In China, an adult Equus caballus individual with a height at the withers less than or equal to 106 cm is classified as a pony, while the one with a height greater than 106 cm is called a common horse. The pony has long history of breeding, which has been traced back to the Han Dynasty of ancient China more than 2000 yr ago, because of its particular importance for riding, as a draft animal and for entertainment (Hou 1990). Until now, the origin of these pony populations has remained controversial. Historic, archaeological and biological evidence suggests that Chinese pony breeds might have a dual origin with an archaic breed from Hipparion and a possible arrival from north China, from where the ancient Qiang people migrated to the southern provinces with small horses (Bo 1998). Although some Chinese pony breeds have been individually involved in recent phylogenetic studies (Jiang et al. 2011), the genetic diversity and origin of five Can. J. Anim. Sci. (2013) 93: 313319 doi:10.4141/cjas2012-160 313

314 CANADIAN JOURNAL OF ANIMAL SCIENCE Table 1. Mitochondrial DNA D-loop sequence diversity indices of five Chinese pony breeds Breed Code z location Geographic Sample size (n) y Number of haplotype (k) y Haplotype diversity (h) y Nucleotide diversity (p) y Ningqiang NQ Shanxi 21 11 0.9190 0.0188 Debao DB Guangxi 30 20 0.9402 0.0224 Yunnan YN Yunan 30 15 0.8943 0.0229 Guizhou GZ Guzhou 25 19 0.9467 0.0194 Sichuan SC Sichuan 30 16 0.9287 0.0184 z NQ, DB, YN, GZ, and SC represent Debao, Guizhou, Ningqiang, Yunnan and Sichuan breed, respectively. y n is the sample size; k is the number of observed haplotypes; h is haplotype diversity; p is nucleotide diversity. Chinese pony breeds based on mitochondrial DNA (mtdna) information has not been systematically assessed. In this study, we investigated the genetic diversity and maternal origin of five Chinese pony breeds distributed in five different provinces of China (Shanxi, Guangxi, Sichuan, Yunan and Guizhou) using mtdna D-loop information. In total, 136 blood samples representing five unrelated Chinese pony breeds distributed in five different provinces were collected (Table 1). Additionally, to address the question of the single and multiple maternal origins of Chinese pony populations, 13 partial mtdna D-loop reference sequences from GenBank Table 2. Pony breeds for which the haplotype has been identified z Haplotype Pony breeds for which the haplotype has been identified representing eight ancient and modern horse breeds (Connemara, Garrano, Sorraia, Pottok, Losino, Cheju, Tuva and a Swedish horse) were added to conduct the phylogenetic analysis. To amplify the complete mtdna D-loop sequence, a pair of primers HA (5?-AGTCT- CACCATCAACCAAAGC-3?) and HB (5?-CCTGAAG TAGGAACCAGATG-3?) was used according to Xu and Arnason (1999), Sequences were edited using DNASTAR 5.0 software (DNASTAR, Madison, WI) and aligned using CLUSTALX software (Thompson et al. 1997). Haplotype diversity (h) and nucleotide diversity (p) for each breed were estimated using DNASP 4.0 (Rozas et al. 2003). A median-joining network was Haplotype Pony breeds for which the haplotype has been identified H1(KC968811) DB(1) H31 GZ(1) H2 DB(1),GZ(1),NQ(1),YN(1),SC(3) H32 GZ(1) H3 DB(7),GZ(6),YN(9),SC(7) H33(KC986938) NQ(1) H4 DB(1),NQ(2) H34 NQ(1) H5(KC968813) DB(1) H35(KC986939) NQ(5) H6 DB(1) H36 NQ(2),SC(1) H7 DB(1),GZ(1) H37 NQ(3) H8 DB(1) H38 NQ(2) H9 DB(3),GZ(1) H39 NQ(2) H10 DB(1) H40 NQ(1) H11 DB(1),GZ(1),SC(2) H41 YN(2) H12 DB(1),SC(3) H42 YN(4) H13 DB(1) H43(KF006236) YN(1) H14 DB(1) H44 YN(1) H15 DB(2) H45(KF006237) YN(1) H16 DB(1) H46 YN(1) H17 DB(2),GZ(1),SC(2) H47 YN(2) H18 DB(1),YN(1) H48 YN(2) H19 DB(1) H49 YN(1),SC(1) H20 DB(1),GZ(1) H50 YN(1),SC(1) H21(KF006234) GZ(1) H51 YN(1) H22 GZ(1) H52(KF006238) SC(1) H23(KF006235) GZ(1) H53 SC(2) H24 GZ(2),YN(2) H54(KF006233) SC(1) H25 GZ(1) H55 SC(2) H26 GZ(1),NQ(1) H56 SC(1) H27 GZ(1) H57 SC(1) H28 GZ(1) H58 SC(1) H29 GZ(1) H59 SC(1) H30 GZ(1) z Only two squences for each pony breed were submitted; the other squences have not been completed to submit.

Table 3. Variable sites of partial of D-loop region (343 bp) in Chinese five pony breeds Variable nucleotide position 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 4 4 4 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 7 7 7 7 7 7 7 7 7 7 7 8 8 8 8 8 8 8 8 8 8 9 9 9 2 3 3 4 4 8 8 9 9 9 0 0 0 0 0 1 1 1 3 3 4 5 5 6 6 7 0 2 2 2 2 4 7 7 7 7 7 0 0 0 1 1 1 1 1 1 1 Haplotype 4 5 6 6 2 4 0 2 4 5 5 7 8 0 1 2 3 4 5 6 7 3 5 9 0 9 6 7 2 3 0 5 6 8 0 0 1 5 6 7 6 7 9 0 1 3 5 6 7 8 X79547 T T A T C C A C C G A A T G T C T G A A T A C A A T G A G T G T G T A C C C T A C C A A C T G G G G H1 C C G.. T.. T A...... C...... G...... A..... T............. H2. C................................................ H3. C............................................... T H4. C......... G... T. A......... G. C A..... T.. G.. G....... H5. C............. T. A........... C A... G. T....... T..... H6 C C G.. T... A..... T C A..... G...... A..... T....... T..... H7. C.. -.... A..... T..... G........ A..... T.... T....... T H8 C C G.. T... A..... T C...... G...... A..... T........... T. H9 C C G.. T... A...... C...... G...... A..... T............ T H10 C C G.. T... A...... C...... G...... A..... T............. H11. C............. T.... C.... C.... A..... T... -........ T H12 C C G.. T... A..... T C A..... G...... A..... T............. H13. C....... A..... T. A....... C... C A. A. G. T.. G.... T.... T H14. C....... A. G... T. A........... C A... G. T.. G.... T.... T H15. C........ G.... T. A........... C A... G. T....... T.... T H16. C........... A................................... T H17. C............. T........ G..... A.. C............... T H18. C.. -.... A..... T.............. A..... T.... T....... T H19. C....... A....................................... T H20. C.......... C.. T.. G G..... C... C A.... T. T C. -........ T H21. C....... A.... C T.............. A..... T... T......... H22. C.......... C.. T.. G G..... C... C A.... T. T C. T......... H23 C C G.. T... A..... T C...... G...... A..... T......... T T. T H24 C C G.. T... A..... T C...... G...... A..... T............ T H25. C............................ A.................. T H26. C.. -.... A..... T.............. A..... T.... T........ H27. C.............................................. A. H28. C.. -.... A. G... T.............. A..... T.... T....... T H29. C.. -.... A..... T..... G........ A..... T.... T........ H30. C............................. C................. T H31. C..... T... G... T...... T. G. A.. C A.................. T H32. C................................. T............. T H33. C..... T... G... T...... T. G. A.. C A................... H34. C......... G... T. A........... C A... G. T.. G.... T..... H35. C............. T.............. A.. C................ H36.................................................. H37. C........... A.................................... H38. C......... G... T........ G..... A................... H39. C............. T. A....... C... C A. A. G. T.. G.... T..... H40 C C G.. T... A..... T C A..... G...... A..... T........ C.... H41. C............. T. A.......... A C A. A. G. T....... T..... H42. C............. T. A.......... A C A. A. G. T....... T.... T H43. C..... T....... T...... T. G. A.. C A................ A. T YANG ET AL. * ORIGIN OF CHINESE PONY BREEDS 315

316 CANADIAN JOURNAL OF ANIMAL SCIENCE Table 3 (Continued) Variable nucleotide position 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 4 4 4 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 7 7 7 7 7 7 7 7 7 7 7 8 8 8 8 8 8 8 8 8 8 9 9 9 2 3 3 4 4 8 8 9 9 9 0 0 0 0 0 1 1 1 3 3 4 5 5 6 6 7 0 2 2 2 2 4 7 7 7 7 7 0 0 0 1 1 1 1 1 1 1 Haplotype 4 5 6 6 2 4 0 2 4 5 5 7 8 0 1 2 3 4 5 6 7 3 5 9 0 9 6 7 2 3 0 5 6 8 0 0 1 5 6 7 6 7 9 0 1 3 5 6 7 8 H44. C..... T....... T...... T. G. A.. C A.................. T H45. C..... T....... T...... T. G. A.. C A................... H46. C..... T. A. G... T...... T. G. A.. C A.................. T H47 C C G.. T... A..... T C...... G...... A..... T.......... A. T H48 C C G.. T... A..... T C...... G...... A..... T.......... T. T H49 C C G.. T... A..... T C A..... G...... A..... T............ T H50. C............. T.... C.... C.... A..... T... -......... H51. C. C.. G.. A..... T....... G...... A..... T............ T H52. C............. T........ G..... A.. C................ H53 C C G.. T... A..... T C............. A..... T.......... T. T H54. C............................................. T. T H55 C C G.. T......... T C...... G...... A..... T............ T H56................................................. T H57 C C G.. T... A..... T C A..... G...... A..... T...... G...... H58. C............. T. A........... C A... G. T.. G.... T..... H59. C.............................................. T. generated using the program NETWORK 4.1 (Bandelt et al. 1999). A 343-bp (1547615818) of mtdna D-loop sequence from a Swedish horse (accession no. X79547) was used as a reference to examine the polymorphic sites among 136 Chinese pony sequences. Fifty-nine haplotypes were identified and defined by 50 polymorphic nucleotide sites including 12 singleton variable sites and 38 parsimony informative sites: 46 transitions, 1 transversions, 2 deletions and one site with both transition and transvertion, suggesting higher polymorphisms and a strong bias towards transitions (Table 3). Among the 59 haplotypes, 15 haplotypes (25.4%) were detected more than once and were thus shared by individuals among breeds or within breeds. The largest haplotype, H3, consisted of 29 individuals, the second-largest haplotype, H2, contained seven individuals, and there were two haplotypes (H17, H35) with five individuals, four haplotypes (H9, H11, H12, H42) with four individuals, and the other haplotypes possessed one to three individuals each (Table 3). Thirty-four haplotypes (57.6%) were singletons: 10 (29.4%) from the Guizhou breed (accession numbers: KF006234, KF006235), 10 (29.4%) from the Debao (KC968811, KC968813), 6 (17.6%) from the Sichuan (KF006233, KF006238), 5 (14.7%) from the Yunnan (KF006236, KF006237), and 3 (8.9%) from the Ninqiang (KC986938, KC986939). The number of haplotypes identified in each breed ranged from 11 to 20, and haplotype diversity values (h) ranged from 0.894 in the Yunnan breed to 0.947 in the Guzhou breed. The Yunnan breed showed the highest nucleotide diversity value (p 0.0229), whereas the Ningqiang and Sichuan breeds displayed lower nucleotide diversity values (p0.0188 and 0.0184, respectively), indicating relatively abundant genetic diversity in the Debao, Yunnan and the Guizhou breeds, whereas the Ningqiang and Sichuan breeds displayed the lower genetic diversity (Table 1). This may be partly because the Ningqiang breed has recently undergone a severe bottleneck; the Ningqiang breed is reduced to 300 individuals (Jiang et al. 2011). In this study only 21 adult pony samples could be collected. Additionally, all of Ningqiang ponies were raised on one Genetic Resource Conservation farm and probably had close genetic relationships among them. Accordingly, it has been reported that the numbers of Yunnan and Guizhou ponies are reduced to 1184 and 1421, respectively (Kong et al. 2010; Huang et al. 2012), these ponies are raised in farmers courtyards and no selective pressure occurred among them. These results are in accordance with those reported by Lei et al. (2009) and Yue et al. (2011). Previous research grouped equine mtdna haplotypes into seven major haplogroups (AG) (Vila et al. 2001; Jansen et al. 2002; McGahern et al. 2006; Lei et al. 2009), but few pony breeds were used in their studies. In the present study, 13 partial mtdna D-loop sequences representing seven pony breeds including Connemara (C1: AF481246; C2: AF481247) from Ireland, Garrano

YANG ET AL. * ORIGIN OF CHINESE PONY BREEDS 317 1 6 4 5 5 1 5 8 4 8 4 2 1 8 2 1 2 4 5 6 1 5 1 1 4 1 7 4 7 7 8 4 1 1 4 4 6 1 2 1 4 0 5 2 2 7 2 7 4 6 4 3 1 7 5 4 2 5 1 7 1 3 3 1 6 1 1 3 3 5 3 6 0 2 1 7 8 1 1 3 9 1 4 3 7 5 5 4 6 1 7 1 7 5 3 2 5 5 7 6 4 5 6 3 4 7 0 9 9 3 0 5 7 7 0 1 5 5 3 6 9 5 1 3 8 5 0 3 5 7 0 H 6 H 4 0 H 1 2 H 5 7 H 4 9 H 2 4 H 4 7 H 2 3 H 4 8 H 8 A F 4 8 1 2 4 7 H 9 H 1 H 1 0 H 5 5 H 5 3 A F 4 8 1 2 4 6 H 5 1 H 2 1 H Q 8 2 7 1 2 9 A F 0 1 4 4 0 5 H 2 6 H 7 H 2 9 H 1 8 H 2 8 H 5 0 A Y 2 4 6 2 3 2 H 1 1 H 2 0 H 2 2 H 4 H 1 5 H 5 H 1 4 H 3 4 H 5 8 H 1 3 H 3 9 H 4 1 H 4 2 H Q 8 2 7 1 2 7 H 2 5 H 1 9 H 3 0 H 5 4 H 3 H 3 2 H 1 6 H 3 7 H 2 7 H 5 9 H 2 H 5 6 H 3 6 X 7 9 5 4 7 H 1 7 H 5 2 H 3 5 A F 4 8 1 3 2 4 H 3 8 A Y 2 4 6 2 3 1 H 4 5 H 4 3 H 4 4 H 3 1 H 4 6 A Y 2 4 6 2 6 4 H 3 3 A Y 2 4 6 2 6 0 A F 4 6 6 0 1 1 A F 4 6 6 0 1 2 A B C D E F Fig. 1. Neighbour-joining tree based on pairwise genetic distances using 343 bp of the D-loop region among 59 haplotypes representing five Chinese pony breeds and 13 haplotypes representing eight ancient and modern horse breeds. The digits at the nodes are the bootstrap support based on 1000 replicates.

318 CANADIAN JOURNAL OF ANIMAL SCIENCE Fig. 2. Median joining phlogenetic network constructed for horse mitochondrial DNA sequences using 343 bp of the control region. Letters AF shows six main haplogroups. Circles represent sequence haplotypes, the area being proportional to the frequency of the corresponding haplotypes. The colour indicates which of 13 horse breeds are represented by all haplotypes in this study: red Ningqiang, yellowdebao, greenyunnan, blueguizhou, blacksichuan, brownpotok, bottle greengarrano, purple Losino, orangeconnemara, pinkcheju, dark browntuva, whitereference horse (Swedish), cyansorraia. Points are theoretical intermediate nodes introduced by the median algorithm branches between haplotypes represent mutations. (G1: AY246231; G2: AY246232) from Portugal, Pottok (P1: AF466011; P2: AF466012), Sorraia (S1: AY246260; S2: AY246264) and Losina (L1: HQ827127; L2: HQ8 27129) from Spain, Cheju (AF014405) from Korea, Tuva (AF481324) from Russia, and a reference breed (Swedish horse: X79547), together with 136 D-loop partial sequences of five Chinese pony breeds were applied to conduct the phylogenetic analysis (Kim et al. 1999; Hill et al. 2002). Five Chinese pony breeds, plus ancient and modern breeds, were grouped into six different lineages (Fig. 1). Accordingly, a total of 72 haplotypes were assigned to six haplogroups, AF, while ancient and modern breeds were assigned to haplotypes A, B, C, E, and F (Fig. 2). G1, P1, P2 and TUVA were grouped into the same lineages, F and L1, L2 and the reference sequence were clustered into the lineage E (Fig. 1). The most frequent haplotype, H3, and the second most frequent, H2, appear in five Chinese pony breeds (Fig. 2), which suggests that H3 and H2 may be the main haplotypes of Chinese ponies. The phylogenetic network shows that all five Chinese pony breeds appear to be distributed in the majority of haplogroups, suggesting that extensive gene flow has existed among different pony breeds. The mtdna D-loop data provide further evidence for Chinese ponies having multiple maternal origins. Overall, the current study provides a new understanding of the mtdna genetic diversity and origins of Chinese ponies. The results show that haplotype diversity (h)and nucleotide diversity (p) are not far from each population; all of the five pony breeds display relatively abundant genetic diversity except for the Ningqiang and the Sichuan breeds. The median-joining network shows that extensive gene flow occurred among these pony breeds. This work was supported by the Natural Science Foundation of China (NSFC: 30960162). The authors acknowledge the assistance with sample collection of five Animal Husbandry Departments in China. Bandelt, H. J., Forster, P. and Rohl, A. 1999. Median-joining network for inferring intraspecific phylogenetics. Mol. Biol. Evol. 16: 3748. Bo, W. C. 1998. Analysis of the origin of Chinese primitive pony. J. Anim. Sci. Vet. 4: 3335. Hill, E. W., Bradley, D. G., Al-Barody, M., Ertugrul, O., Splan, R. K., Zakharov, I. and Cunningham, E. P. 2002. History and

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