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Study on genetic variation in yak breeds (populations) by RAPD Xiao Yuping∗, Jin Shuang, Deng Kangyun, Zhu Xiao, Chen Zhihua and Zhong Jincheng∗∗ College of Life Science and Technology, Southwest University for Nationalities, Chengdu 610041, Sichuan, P.R. China Summary The genetic diversity in Datong yak, Jiulong yak, Tianzhu yak, Maiwa yak (Hongyuan) and Maiwa yak (Ruoergai) was analyzed by RAPD. The results indicated that the indexes of the genetic diversity in these breeds are 0.207, 0.422, 0.178, 0.207 and 0.369 respectively. The genetic diversity in population is 0.293, and that of the whole population is 0.800. According to the results of the analysis, these five breeds can be divided into two groups, which is coincided with the results of Zhong Jincheng’s analysis based on the chromosome characteristics, which is also identical with the results of Zhao Sujun who applied the micro-satellite in the study, which is conformed to the actual conditions of the yak population’s geography distribution, the ecology conditions, the breeding of the breeds and differentiation. Keywords: Yak, RAPD, genetic diversity Introduction The yak (Bos grunniens) is a rare and valuable species, which is distributed on the Qinghai-Tibetan plateau and adjacent high mountain areas with the altitude above 3000m (Cai Li, 1992). They can make full use of rangeland resources in high mountains and have very strong ability to adapt the ecosystem environment of the high altitude and cold rangeland. They can live freely and multiply the offspring in the condition of low atmospheric oxygen, high solar radiation and short period of grass growth. They are almost the multipurpose livestock that can offer necessaries of production and life, such as milk, meat, down, hair, draught power and fuel. They are indispensable for the local stockbreeding economy. There are about 14 million heads of yak in the whole world, of which there are 13.4 million heads in China, occupy 95% of the total number. Inhabited and influenced by the ecosystem, the yak is a kind of primitive livestock, which is selected by nature rather than manpower. Therefore, analyzing the genetic diversity of the yak by virtue of theories and methods in modern molecular biology has great importance to protecting and making use of the genetic resources of the yak. RAPD（random amplified polymorphic DNA）was established by the two groups guided by Williams and Welsh respectively almost at the same time in 1990. It uses a series of single chains of nucleic acid with random arranged base pair (generally 10bp) as the primers, amplify the single primer of the studied genome DNA. The polymorphism of the amplified DNA fragments indicates the special fixed position for this fixed random primer for combining the model genome DNA when there are reverse-repeated sequences on the plate DNA which is complemented to the primer, the amplifying of the DNA fragments within certain scope is feasible. Materials and methods Materials Experiment animals are health yak that collected at random, breed (population), sample quantity and places etc. showed in Table 1. ∗

Xiao Yuping (1979-), Female, Master, Major Direction: Animal Genetic Breeding and Reproduction. Corresponding Author: Phone: 86-28-85522528, Fax: 86-28-85522799, E-mail: [email protected].

∗∗

Proceedings of the International Congress on Yak, Chengdu, Sichuan, P.R. China 2004

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YAK PRODUCTION IN CENTRAL ASIAN HIGHLANDS

Table 1. List of samples used for RAPD analyses.

Breeds(populations)

Code

heads

Sampling localities

Maiwa yak

M1

34

Ruoergai county of Sichuan Province

Maiwa yak

M2

34

Hongyuan county of Aba state of Sichuan Province

Jiulong yak

JL

32

Jiulong county of Sichuan Province

Datong yak

DT

45

Datong yak farm of Qinghai Province

Tianzhu white yak

TZ

28

Tianzhu white yak breeding farm of Gansu Provinc

Preparation of genomic DNA Extract the DNA by phenol: chloroform all DNA samples were diluted in 1×TE to a find concentration of 25~50ng/ul. The reaction conditions and processing of amplify of RAPD The total reaction system of PCR: in 25ul reaction volumes, 25ng plate DNA, 2.0ul 10×Buffer, dNTP 2.0mmol/L, MgCl2 2.0~2.5mmol/L, primer 20pmol, Taq DNA polymerase 1.5 U. PCR reaction procedure 94 5 min, 36 1 min, 72 2 min, forty cycling at 94 5 min, 36℃ 1 min, 72 2 min; final extension at 72 10 min. Amplified products were separated on 1.5% agarose gels, visualized by ethidium bromide staining and photographed. The selecting of polymorphic primers Comparing the amplifying results of 5 yak breeds (populations) with one primer in genome DNA, that is to say the appearance, amount, and spots of the brands of the amplifying. We will consider the ones as polymorphic primers that show differences in two populations of yak at least. The data statistics Record the bands Record the bands which is clear and can be repeated as “1”, otherwise “0”. Calculation of the hereditary distance between populations

1 r

Application of Rogers Formula: DR= [

1

( xi − yi ) 2 2 ∑ 2 ]

Among them the “r” is total bands in x, y community for all primers, xi, yi are the frequency. According to the hereditary distance, make use of the software pohylip (ver3.5) and adoption Neighbor-Joining method phylogenetic tree analysis. Calculation of the index of genetic diversity Adopt Shannon diversified index quantization formula: Genetic diversity index of species: H 0 = −

∑π

i

ln π i

General genetic diversity index of population: H sp = −

∑ π ln π

Proceedings of the International Congress on Yak, Chengdu, Sichuan, P.R. China 2004

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Average genetic diversity index in the population: H pop =

1 ∑ H0 n

Among above formula, π i expresses the frequency one amplified band to appearing in a population, π stands for a distribution frequency of one amplified band in the general population, n is counted for the species,

h pop / hsp is the hereditary proportion in the population of genetic diversity, (h pop − hsp ) / hsp is the genetic diversity in the proportions accounted for between the populations. Results The selecting results of the polymorphic primers There are 12 primers that have clear bands in the compound DNA of 20 primers, among which there are 5 primers having different extent of polymorphism, the remains are single RAPD band. The record number of the single primers is between 16-20, there are 91 RAPD fragments in record, 73 of them have polymorphism, the fragment is between 0.5-4.3kb (Table 2, Figure 1, 2, 3 and 4) Table 2. Amplification of the 5 random primers.

primers

Sequence (5，→3，)

CPZ356-04

Amplification

Polymorphism number (band)

number (band)

DT

JL

TZ

M1

M2

GGTGACGCAG

18

5

8

5

8

8

CPZ356-05

CCGCCTAGTC

20

5

8

2

11

12

CPZ356-15

ACGCATCGCA

20

12

11

10

6

8

CPZ356-17

CCGCCTAGTC

17

8

5

3

6

3

CPZ356-18

TGACGGCGGT

16

4

9

5

0

6

91

34

41

25

31

37

Total

M

1

2

3

4

5

6

7

8

M

1

2

3

4

5

6

7

8

Figure 1. The cpz356-04 random

Figure 2. The cpz356-04 random

amplified DNA products of Maiwa

amplified DNA products of Jiulong

yak (Ruoergai; M: 200bp DNA ladder).

(M: 200bp DNA ladder).

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YAK PRODUCTION IN CENTRAL ASIAN HIGHLANDS

M

1

2

3

4

5

6 7

8

M

1

2

3

4

5

6 7

8

Figure 3. The cpz356-17 random

Figure 4. The cpz356-17 random

amplified DNA products of Jiulong

amplified DNA products of Datong

yak (M: 200bp DNA ladder).

yak (M: 200bp DNA ladder).

The genetic variation of the five breeds (populations) According to the distribution of the 5 primers amplification RAPD bands in each population, apply the Shannon index to calculate the genetic variation index of the five yak populations (Table 3). Table 3. The distribution of the genetic diversity.

Pimer

H0

Hsp

Hpop

Hpop/Hpop

(Hsp-Hpop)/Hsp

0.406

0.629

0.307

0.488

0.512

0.471

0.357

0.571

0.381

0.667

0.333

0.217

0.440

0.224

1.365

0.197

0.144

0.856

0.235

0.185

0.242

0.093

0.637

0.287

0.451

0.549

0.202

0.418

0.180

0.369

0.270

0.797

0.295

0.370

0.630

0.207

0.422

0.178

0.369

0.207

0.800

0.293

0.424

0.576

DT

JL

TZ

M1

M2

CPZ356-04

0.186

0.371

0.194

0.324

CPZ356-05

0.099

0.474

0.115

CPZ356-15

0.409

0.614

CPZ356-17

0.228

CPZ356-18 average

From Table 3 we can see that the genetic variation index of Datong yak is 0.207, Jiulong yak is 0.422, the Tianzhu white yak is 0.178, Maiwa yak is 0.369 (Ruoergai), 0.207 (Hong Yuan), the genetic variation index in population is 0.293, and that of the whole population is 0.800. The genetic variation index amounts to 42.4%, 57.6% in population and within population respectively. The results indicate that the heredity and variation is primarily found in the breed. The genetic differentiation and classification among the breeds of the yak According to Rogers genetics distances formula, we calculated five breeds (populations) of yak genetic distances DR (Table 4). Table 4. Genetics distances between the five breeds.

Breeds

DT

JL

TZ

JL

0.159

TZ

0.145

0.148

M1

0.180

0.172

0.179

M2

0.178

0.143

0.217

M1

0.140

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Analyzed by the neighbor-Joining, the results show that: the five populations yak can be classified as two groups. Jiulong yak can be a single group, the rest can be a groups. The two kinds yak of Maiwa first forms a group at a near level, Datong yak and Tianzhu yak forms a group at a far level, then these two groups form a group at a more far level (Figure 5). M1 M2 DT TZ JL Figure 5. The neighbor -Joining tree for the five yak breeds.

Discussions The genetic diversity of the yak breed (population) The study analyses the genetic variation of the yak by virtue of Shannon’s informative diversity index. The results indicate that: the heredity and variation were found mainly among the breed in the population. The Tianzhu yak’s hereditary index to lower than the other four populations in the five populations, which indicates the genetic variation of the Tianzhu yak is shorter than the other populations, the variation level is relatively lower in the community breeding, has high quality of even breed, has higher purity of heredity, while the highest index of genetic variation belong to the Jiulong yak, its number is 0.422, which indicates that Jiulong yak has more abundance in the genetic diversity, this breed has higher level of population heredity, and lower level of purity of heredity. The differentiation and classification of the yak breed (population) The reasonable classification of hereditary resources of yak is of great actual importance in terms of reasonable conservation and utilization of the yak hereditary resources and developing the economy of livestock in high and cold areas. The study adopts the RAPD technique to study the population genetic diversity in five breeds (population) of yak, calculating the genetics distances DR among five breeds (populations) according to Roger’s formula. Analyzing the results, we can found that the five populations can be classified into two breeds, the results is identical with the results of Zhong Jincheng (2001) who adopted the chromosome analysis and the results of Zhao Sujun (2004) who applied the micro-satellite, and also identical with actual situations such as geographic distribution, ecosystem conditions, the cultivation of the breed and differentiation of the yak population. References Cai Li. 1992. China yak. Beijing, China Agriculture Press. Zhao Sujun. 2004. Study on Genetic Diversity of Microsatellite Loci in Yak. Master Dissertation of Southwest University for Nationalities. Zhong Jincheng, Chen Zhihua et al. 2001. Cluster analysis of the yak breed. Journal of Southwest University for Nationalities (Natural Science Edition). 27 (1): 92-94.

_________________________________________________________________________________ This manuscript is reproduced in the IVIS website with the permission of International Livestock Research Institute (www.ilri.cgiar.org) Proceedings of the International Congress on Yak, Chengdu, Sichuan, P.R. China 2004