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Livestock Science

Livestock Science 155 (2013) 17–22

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journal homepage: www.elsevier.com/locate/livsci

Assessment of genetic diversity and population structure ofVietnamese indigenous cattle populations by microsatellites

Lan Doan Phama,n, Duy Ngoc Do a,b, Nguyen Trong Binh a, Le Quang Nama,Nguyen Van Ba a, Tran Thi Thu Thuy a, Tran Xuan Hoan a, Vu Chi Cuong a,Haja N. Kadarmideen b

a Key laboratory of Animal Cell Technology, National Institute of Animal Sciences, Thuyphuong, Tuliem, Hanoi, Vietnamb University of Copenhagen, Faculty of Health and Medical Sciences, Department of Veterinary Clinical and Animal Sciences,Division of Genetics & Bioinformatics, Denmark

a r t i c l e i n f o

Article history:Received 27 February 2012Received in revised form27 March 2013Accepted 9 April 2013

Keywords:CattleGenetic diversityIndigenous populationMicrosatellitesVietnam

13/$ - see front matter & 2013 Elsevier B.V. Ax.doi.org/10.1016/j.livsci.2013.04.006

esponding author. Tel.: +84 432166147; fax:ail addresses: pdlanvn@yahoo.com (L.D. Phayfc@gmail.com (D.N. Do), binhniah@yahoo.cogma@yahoo.com (L.Q. Nam), nguyenba81@yBa), thuthuyniah@yahoo.com (T.T.T. Thuy),

X. Hoan), vn1899@gmail.comong), hajak@life.ku.dk (H.N. Kadarmideen).

a b s t r a c t

Cattle play a very important role in agriculture and food security in Vietnam. A high levelof cattle diversity exists and serves different needs of Vietnamese cattle keepers but hasnot yet been molecularly characterized. This study evaluates the genetic diversity andstructure of Vietnamese indigenous cattle populations, using microsatellite markers.A total of 410 individuals from six indigenous cattle populations and an exotic breedwas characterized using 27 microsatellite markers A total of 362 alleles was detected andthe number of alleles per locus ranged from 8 (INRA005 and ILSTS005) to 17 (ETH185). Thelevel of gene diversity was high indicated by a mean expected heterozygosity (He) acrosspopulations and loci of 0.73. Level of inbreeding (mean FIS¼0.05) and genetic differentia-tion (mean FST¼0.04) was moderate. The phylogenetic tree based on Reynolds geneticdistance reflected geographic distances. Structure analysis indicated five homogeneousclusters. The Brahman, Lang Son, Ha Giang and U Dau Riu cattle were assigned toindependent clusters while Nghe An, Thanh Hoa and Phu Yen cattle were grouped in asingle cluster. We conclude that Vietnamese indigenous cattle have high levels of geneticdiversity and distinct genetic structures. Based on these results, we recommend that forconservation homogenous populations (Nghe An, Thanh Hoa and Phu Yen) can be groupedto reduce costs and U Dau Riu, Lang Son and Ha Giang populations should be conservedseparately to avoid loss of genetic diversity.

& 2013 Elsevier B.V. All rights reserved.

1. Introduction

Besides milk and meat production, cattle have beentraditionally used for draught power in Vietnam. There are

ll rights reserved.

+84 43838068.m),m (N.T. Binh),ahoo.comhoantranvcn@yahoo.

24 cattle breeds currently present in the country includingseven locally defined populations named by their place oforigin (Nghe An, Thanh Hoa, Ba Ria and Tuy Hoa or PhuYen cattle); by their typical characteristics (U Dau Riu andYellow) or by ethnic people who keep them (H'mongcattle) (http://dad.fao.org/). These populations or breedsare adapted to local conditions with distinct characteristicsvaluable for local farmers or cattle keepers and maypresent rare and valuable genetic resources for livelihood.In the absence of herd book registration for local popula-tions, breeding decisions are based on phenotypic perfor-mance and culturally desired phenotypes. Therefore,

Fig. 1. Geographic localizations of the seven cattle populations analyzedin this study. Legends: (I) Brahman in Ho Chi Minh City, (II) Ha Giangprovince, (III) Lang Son province, (IV) Xuan Thanh district, Nghe Anprovince, (V) Phu Yen province, (VI): Thanh Hoa province, and (VII) NamDan district, Nghe An province. The different colors indicate distincteconomic zones. (For interpretation of the references to color in thisfigure legend, the reader is referred to the web version of this article.)

L.D. Pham et al. / Livestock Science 155 (2013) 17–2218

Vietnamese cattle are probably mainly differentiated accord-ing to their local environments. Genetic exchange betweenpopulations is probably limited due to the large geo-graphic distances and may have led to further divergence.On the other hand, Vietnamese cattle are raised insmall holder farms and do not suffer from intensiveselection, so that more genetic diversity within breeds isexpected and genetic differentiation between breeds maybe relatively less than in breeds from the industrializedparts of the world.

In addition, because of recent demands to increasecattle performance, exotic breeds are introduced eitherfor crossing with indigenous cattle or even for replacement.Consequently, indigenous populations decline quickly.Conservation of this indigenous genetic resource is veryimportant for sustainable development of the agriculturalsector and livelihood of poor farmers in Vietnam. Nationalprograms for conservation of Vietnamese agriculturaldiversity have been initiated since 1990 to prevent theextinction of local breeds. However, up to now, conserva-tion decisions are mainly based on population size andpopulation trends of the perceived breeds. Prioritization ofpopulations for conservation and deciding whether breedsshould be conserved separately or with some populationsmerged is difficult without knowledge of their geneticdiversity and differentiation. Understanding the geneticdiversity and genetic relationships of indigenous popula-tions will enable a better conservation strategy for Viet-namese cattle.

Microsatellite markers are the most common tool forstudying or establishing population structure, populationdifferentiation and reconstruction of phylogenic relation-ships among populations. In the last 15 years, the useful-ness of microsatellite markers for the estimation of geneticdiversity and relationships among cattle breeds has beendocumented in numerous studies (MacHugh et al., 1998;Kantanen et al., 2000; Chikhi et al., 2004; Gautier et al.,2007; Li et al., 2007; Zhang et al., 2007; Flury et al., 2009;Sodhi et al., 2011). Some studies have reported a linkbetween cattle genetic diversity and resistance to infec-tious diseases such as bovine tuberculosis (Kadarmideenet al., 2011) underlining the importance of cattle diversityin adaptation. The first study that estimated geneticdiversity of cattle breeds in Vietnam was by Berthoulyet al. (2010) who found high diversity of H'mong cattle andpossible genetic admixture from Yellow cattle with H'mongcattle. The authors recommended studying other indigenouscattle populations for better conservation strategies inVietnam. The present study was performed to evaluatethe genetic diversity and genetic structure of six indigen-ous cattle populations, to provide guidelines for conserva-tion strategies of Vietnamese cattle.

2. Materials and methods

2.1. Sampling collection

The study population consisted of 410 individuals fromseven regions representing six different indigenous cattlepopulations. They were Ha Giang (HG; n¼60), Lang Son(LS; n¼62), Nghe An (NA; n¼53), Phu Yen (PY; n¼62),

Thanh Hoa (TH; n¼64); and U Dau Riu (UR; n¼49).An exotic breed, Brahman (BR; n¼60) was analyzed tocompare with indigenous populations. The Brahman waschosen for this study because it was recently imported andhas not mixed with indigenous populations yet. The sexratio was approximately 50% in all populations except UR(ratio 75%), because farmers give a higher priority to maleUR cattle for draught power. Samples were taken from 3 to5 different villages for each population depending onavailability of cattle. Ear tissue samples were collectedfrom various geographical locations including four eco-nomical zones in Vietnam (Fig. 1). To ensure that theanimals were unrelated and had characteristics typical ofthe populations, we used both the pedigree informationand the knowledge of local herdsmen via questionnaireinterviews whenever possible.

2.2. DNA extraction, PCR amplification and microsatellitegenotyping

Genomic DNA was extracted from ear tissue samplesusing AccuPREP Genomic DNA extraction kit (Cat.No.K-3032) (Bioneer, Korea). The quality of genomic DNA

L.D. Pham et al. / Livestock Science 155 (2013) 17–22 19

was checked by NanoDrop2000 (Thermo Scientific, USA)before any further application. The multiplex PCR ampli-fication of 27 FAO/ISAG microsatellite loci was carried outin 20 ml reaction volume. The PCR products were labeledwith fluorescent dyes and genotyped using a capillarysequencer (Beckman Coulter CEQ8000, Fullerton, CA, USA).

2.3. Statistical analysis

A pre-analysis check for data errors and removal ofduplicate samples or genetically identical individuals wasperformed with the Excel add-in MS_tools.xla (Park, 2001).Number of alleles, allele frequency and genetic diversitywere calculated using Genetix 4.4 (Belkhir et al., 2004).Allelic richness by rarefaction and F-statistics were esti-mated using Fstat 2.9.3 (Goudet, 1995). The Genepop 1.2software (Raymond and Rousset, 1995) program was usedto perform exact Hardy–Weinberg equilibrium test.Based on an assumption that Vietnamese cattle has shortdivergence time, the matrix of Reynolds et al.'s (1983)distance was computed with 1000 bootstraps on loci tobuild Neighbor Joining (NJ) tree in Population v.1.2.28(Langella, 1999) and the dendrogram was depicted usingthe software package TreeView version 1.6.6 (Page, 1996).To help setting up conservation priorities, the Molkin 2.0package (Gutierrez et al., 2005) was used for quantificationof the contribution of each indigenous population to thediversity of the whole dataset using the method proposedby Caballero and Toro (2002). The genetic structure andindividual assignments using the Bayesian clustering withthe admixture model was implemented in Structure 2.1(Pritchard et al., 2000). Assuming that the dataset could berepresented by K separate genetic clusters (K¼1–10), 10runs for each K value with 40,000 iterations following aburn-in period of 60,000 and without any prior informa-tion was performed. To find optimal alignments of 10independent runs, the computer program Clumpp 1.1(Jakobsson and Rosenberg, 2007) was used; the outputobtained was used directly as input by the cluster visua-lization program Distruct (Rosenberg, 2004).

3. Results

3.1. Loci polymorphism and population genetic variation

Loci polymorphism, evaluated by the number of allelesper locus, allelic richness, the mean of total allele number,heterozygosity, as well as the polymorphism informationcontent (PIC) are described in the Table 1. A total of 362alleles was detected for the 27 microsatellites in this study.Number of alleles per locus ranged from 8 (INRA005 andILSTS005) to 17 (ETH185) with a mean value of 12.15. Mostof the loci were highly informative (PIC40.5), except ETH3and TGLA227 which had moderate values (PIC¼0.48 and0.46, respectively). Expected heterozygosity, ranged from0.48 (TLGA227) to 0.85 (CSRM60). Of the 27 loci, four locihad negative inbreeding coefficients and the mean FIS wasmoderate (0.07). Nineteen of 27 loci deviated from HardyWeinberg equilibrium (Po0.05) (Table 1).

Across the populations, the total number of allelesranged from 225 in BR to 265 in PY population (Table 2).

Fifteen population-specific alleles were found in sevenpopulations. Gene diversity was found to be the highestin PY (He¼0.75) and the lowest in BR (He¼0.71). Nopopulation showed heterozygote deficiency (Po0.05),however there were slight differences of inbreeding coef-ficients amongst them (Table 2). The genetic variationamong populations (mean FST) was moderate (4%). Notethat positive contributions to diversity from a givenpopulation using the Caballero and Toro's (2002) methodmean that the remaining dataset after removing of thepopulations shows increased overall diversity, i.e. such apopulation does not contribute to the overall diversity, andtheir diversity is contained in other populations. Among theindigenous populations, NA, TH and UR did not contribute tothe total genetic diversity while HG, LS and PY increasedthe total diversity of Vietnamese cattle.

3.2. Genetic distance and phylogenic tree

The highest genetic distance was found between BRand LS (0.098) and the lowest distance was found betweenTH and PY (0.017) (Table 3). As expected, BR had a uniquebranch in the phylogenic tree. Among indigenous popula-tions, UR formed a new branch, TH, NA, PY formed a sub-branch with HG, and LS grouped in another sub-branch.The genetic distances reflected geographic distances (Fig. 2).

3.3. Population structure analysis

The estimated genotype membership coefficient orassigned probability (q) for each individual is shown inFig. 3. Similar to genetic distance, two homogenous clus-ters were separated at K¼2: an exotic population (q¼0.92)and indigenous populations (q¼0.74–0.95). However, thebest possible genetic clustering was found at K¼5, indivi-duals in HG (q¼0.67), LS (q¼0.81) and UR (q¼0.68)tended to assign into their own cluster while the threeremaining populations (NA, PY and TH) were assigned intoa single cluster (q¼0.48 to 0.71).

4. Discussion

4.1. Genetic diversity among populations

According to Barker (1994), the standard is thatselected microsatellite loci ought to have at least fouralleles to be considered useful for the evaluation of geneticdiversity. Based on this criterion, the 27 microsatellite locithat were used in the present study are suitable forpopulation genetic analyses. The mean number of allelesper population per locus found in this study is higher thanthose found in European breeds (MNA¼6.5) (Canon et al.,2001) or Southeastern European breeds (MNA¼6.5) (Beja-Pereira et al., 2003) but lower than those found in Chineseyellow cattle (MNA¼16.55) (Zhang et al., 2007). The highmean value of expected heterozygosity (He¼0.73) indicatesthat Vietnamese cattle populations have a high level of genediversity. This result is in agreement with studies of indigen-ous populations from developing countries such as He¼0.73in Chinese yellow cattle (Zhang et al., 2007) or Ethiopian cattle(Dadi et al., 2008) while He in breeds of developed countries

Table 2Genetic variation among seven Vietnamese cattle populations.

Populationa Size NAb Allelerichness

Privatealleles

Hoc Hed FISe CDf

BR 60 225 7.97 1 0.63 0.71 0.12 −0.42HG 60 261 9.02 3 0.69 0.74 0.08 −0.27LS 62 240 8.34 3 0.67 0.73 0.08 −0.37NA 53 251 8.82 3 0.71 0.74 0.04 0.04PY 62 265 9.09 3 0.72 0.75 0.05 −0.47TH 64 261 8.96 1 0.68 0.72 0.06 0.40UR 49 248 8.86 1 0.68 0.72 0.06 0.28Mean – 328 8.72 2 0.68 0.73 0.07 –

a See abbreviation in Table 1.b See abbreviation in Table 1.c See abbreviation in Table 1.d See abbreviation in Table 1.e See abbreviation in Table 1.f Contributions to Diversity according to Caballero and Toro's (2002)

(negative values of Loss/Gain increased total diversity while positivevalues decreased total diversity).

Table 3The Reynolds et al.'s (1983) genetic distance among seven populations.

BR HG LS UR PY TH NA

BR 0.000 0.057 0.098 0.055 0.058 0.071 0.046HG 0.000 0.032 0.027 0.026 0.028 0.024LS 0.000 0.044 0.033 0.042 0.037UR 0.000 0.033 0.029 0.022PY 0.000 0.020 0.017TH 0.000 0.020NA 0.000

Table 1The polymorphism of 27 loci across seven populations.

Locus NAa Alleles richness Effective allele PICb Hoc Hed FISe FST

f HW testg

CSRM60 12 10.28 4.00 0.83 0.69 0.85 0.16 0.04 n

CSSM66 11 8.11 6.48 0.69 0.57 0.73 0.18 0.07 n

HEL1T 12 7.73 3.68 0.57 0.56 0.61 0.02 0.07 n

INRA063 10 7.74 2.54 0.75 0.60 0.78 0.14 0.10 n

INRAO37 10 7.89 4.48 0.72 0.73 0.74 -0.03 0.06 NSHEL13 11 8.66 3.83 0.72 0.64 0.75 0.09 0.06 n

SPS115 14 9.92 4.02 0.80 0.68 0.83 0.15 0.03 n

TGLA126 9 6.99 5.70 0.70 0.60 0.73 0.17 0.03 n

INRA032 14 9.48 3.73 0.79 0.70 0.81 0.14 0.01 n

ETH3 12 7.31 5.34 0.48 0.42 0.50 0.13 0.03 n

ILSTS006 12 9.42 2.01 0.78 0.64 0.80 0.20 0.02 n

BM1818 10 7.75 5.04 0.76 0.76 0.78 0.01 0.02 NSHEL9 16 10.83 4.55 0.84 0.79 0.86 0.05 0.04 n

ETH152 12 8.72 6.90 0.72 0.79 0.75 -0.06 0.02 NSINRA005 8 6.68 4.05 0.76 0.78 0.79 -0.02 0.04 NSHAUT27 13 9.35 4.77 0.79 0.85 0.81 -0.07 0.02 n

TGLA227 13 7.01 5.18 0.46 0.44 0.48 0.05 0.04 n

BM 1824 10 7.44 1.92 0.68 0.63 0.73 0.10 0.04 n

INRA035 16 9.62 3.65 0.83 0.77 0.84 0.05 0.05 n

ILSTS005 8 6.43 6.36 0.58 0.53 0.60 0.09 0.03 n

MM12 15 9.69 2.51 0.76 0.76 0.78 0.02 0.03 NSETH185 17 13.62 4.61 0.86 0.75 0.87 0.11 0.04 n

BM2113 11 9.05 7.62 0.79 0.75 0.81 0.02 0.06 n

ETH10 9 6.20 5.23 0.70 0.71 0.75 0.03 0.02 NSETH225 15 9.88 3.93 0.77 0.77 0.79 0.02 0.03 NSTGLA122 16 10.77 4.84 0.82 0.79 0.83 0.01 0.04 NSINRA023 12 8.90 6.00 0.78 0.72 0.80 0.08 0.04 n

Mean 12.15 8.72 4.55 0.73 0.68 0.76 0.07 0.04

a Number of alleles.b Polymorphism information content.c Observed heterozygote.d Expected heterozygote.e Inbreeding coefficient index.f Genetic differentiation index.g Hardy–Weinberg equilibrium test.n Significant deviation from Hardy–Weinberg equilibrium (Po0.05); NS: not significant.

L.D. Pham et al. / Livestock Science 155 (2013) 17–2220

ranged from 0.61 to 0.71 in 18 European local breeds (Canonet al., 2001) or He¼0.41 in Japanese Black cattle (Kim et al.,2002). Nineteen of 27 loci were found to deviate from Hardy–Weinberg equilibrium. Several factors can cause deviation

such as inbreeding, genetic drift, selection, population sub-division (Wahlund effect), the presence of null alleles, andselection in favor of homozygotes. All loci are in the ISAGstandard panel and they are specially selected for absence ofnull alleles. All populations showed slight, although notstatistically significant, deviations from Hardy–Weinberg equi-librium indicating that inbreeding might be a cause. Someindigenous populations (UR, LS and HG) were selected fordraught power; it is possible that some alleles are in favor forthis selection. Finally, population subdivision mentioned instructure analysis among seven Vietnamese populations mayalso have contributed to this result (Fig. 3).

L.D. Pham et al. / Livestock Science 155 (2013) 17–22 21

4.2. Genetic relationship and population structure

Vietnamese yellow cattle are believed to originate fromZebu cattle from India that were crossed with Taurine cattlefrom China (Berthouly et al., 2010). During the adaptationprocess, variation in environmental conditions, agriculturalhabits, and ethnic cultures caused distinct characteristics ofcattle in Vietnam. Our measurement of genetic distance wasin agreement with geographic distance. Moreover, indivi-dual assignment at K¼5 revealed four homogenous clustersin Vietnamese cattle. HG and LS tended to form an inde-pendent genetic cluster, probably due to isolation by geo-graphic distance. HG and LS are both raised in northernmountainous regions, so they are isolated from the otherpopulations, while the structure analysis also revealed clear

Fig. 2. Genetic relationship among the seven cattle populations analyzedin this study. Legends: Neighbor Joining dendrogram based on Reynoldset al.'s (1983) genetic distances estimated with 27 microsatellites.Numbers on the nodes in NJ dendrogram are bootstrap values of 1000replications.

Fig. 3. Clustering assignment of the seven cattle populations obtained by Strucvertical line that is divided into segments whose size and color correspond to thcluster. Breeds are separated by thin black lines. (For interpretation of the referenof this article.)

differentiation between HG and LS. Conservation of HG andLS cattle should thus be done separately and in situ tomaintain their local diversity as well as to avoid transfercosts. UR cattle can also be considered as an independentcluster (q¼0.68), although it had a small genetic distance toNA and shared some common genes (as shown by whitelanes in Fig. 3) with NA cattle. UR cattle is naturally adaptedto the micro-ecology of the region of Nam Dan in Nghe Anprovince; this region has a shortage of water for plantsespecially during the dry season, and has poor soil andperhaps the worst climate in Vietnam. According to Xuanet al., (2006), it has always been impossible to introducevehicles or any machinery into Nam Dan village because ofits gravel ground, infertile soil and landscape full of rocks.Therefore, UR plays a critical role in the whole agriculturalsystem by providing power for plowing and transport,fertilizer for land, meat for local farmers and it is consideredas a member of the farmer's family. Interestingly, we foundthat UR cattle contribute less to overall genetic diversity(Table 2). This may be due to the relative low population sizeof UR in this analysis compared to the other populations.Leroy et al. (2009) also found that in the approach ofCabalero and Toro populations contributing a lot to the totaldiversity were mostly non-endangered breeds. Further ana-lysis is needed to clarify this issue of biodiversity contribu-tion; however, it is necessary to conserve UR because oftheir small population size, cultural value and importancefor local agricultural systems.

5. Conclusion

Our study is the first to provide insight into diversity ofindigenous cattle populations in Vietnam at the molecularlevel. Molecular diversity is relatively high compared to

ture analyses. Legends: Each of the 410 animals is represented by a thine relative proportion of the animal genome corresponding to a particularces to color in this figure legend, the reader is referred to the web version

L.D. Pham et al. / Livestock Science 155 (2013) 17–2222

cattle from industrialized countries. This study contributesto the knowledge of genetic structure and molecularcharacterization of these indigenous populations. Basedon traditional classification and these results, we sug-gested to conserve the U Dau Riu cattle at the highestpriority. HG and LS should be conserved separately toavoid loss of genetic diversity. Moreover, the results alsoindicate that conservation of NA, TH and PY cattle can becombined in a group to reduce costs.

Conflict of Interest Statement

No actual or potential conflict of interest in relation tothis article exists.

Acknowledgment

This study was financed by the Vietnam NationalProgram of Development of Biotechnology in Agriculture(2009–2011), Ministry of Agriculture and Rural Develop-ment. The authors would like to thank local farmers fortheir help during sampling process.

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