Genetic variability and bottleneck studies in Zalawadi, Gohilwadi and Surti goat breeds of Gujarat (India) using microsatellites

Available online at www.sciencedirect.com

Small Ruminant Research 77 (2008) 58–64

Short communication

Genetic variability and bottleneck studies in Zalawadi, Gohilwadi and Surti goat breeds of Gujarat (India) using microsatellites Shadma Fatima a , C.D. Bhong a , D.N. Rank a , C.G. Joshi b,∗ a

Department of Animal Genetics and Breeding, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand 388001, Gujarat, India b Animal Biotechnology, College of Veterinary Sciences and Animal Husbandry, Anand Agricultural University, Anand 388001, Gujarat, India Received 5 October 2007; received in revised form 20 December 2007; accepted 29 January 2008

Abstract Indian goat breeds are recognized as an invaluable component of the world’s goat genetic resources. Microsatellite pairs were chosen from the list suggested by International Society for Animal Genetics (ISAG) and amplified in two multiplexes (Set-I: 7 microsatellites and Set-II: 11 microsatellites) for automated fluorescence genotyping to assess bottleneck and analyze genetic variability and genetic distances within and between three goat breeds viz. Zalawadi, Gohilwadi and Surti. The observed number of alleles ranged from 4 (Oar JMP-29) to 15 (ILSTS-030 and -034) with a total of 178 alleles and mean of 9.89 alleles across the three breeds. The overall heterozygosity, PIC and Shannon index values were 0.61, 0.60 and 1.50 indicating high genetic diversity. The maximum observed heterozygosity was found in Gohilwadi and minimum in Surti goat breed. The Nei’s standard genetic distance was minimum between Zalawadi and Gohilwadi, and maximum between Gohilwadi and Surti. Non-significant heterozygote excess on the basis of IAM, TPM and SMM models, as revealed from Wilcoxon sign-rank tests, along with a normal ‘L’-shaped distribution of mode-shift test, indicated no bottleneck in Zalawadi and Gohilwadi goat populations, whereas mild bottleneck in the recent past for Surti breed. This research on goat genetic diversity in Gujarat state provides valuable information on Zalawadi, Gohilwadi and Surti goat genetic resources, and will assist in developing a national plan for the conservation and utilization of indigenous goat breeds. © 2008 Elsevier B.V. All rights reserved. Keywords: Microsatellite; Zalawadi goat; Gohilwadi goat; Surti goat; Genetic diversity

1. Introduction The domestic goat (Capra hircus) is known for its ability to thrive on paltry fodder and to withstand harsh environments. From an agricultural standpoint, the ∗ Corresponding author. Tel.: +91 2692 261201; fax: +91 2692 261201. E-mail addresses: [email protected] (S. Fatima), [email protected] (C.D. Bhong), [email protected] (D.N. Rank), [email protected] (C.G. Joshi).

0921-4488/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2008.01.009

world’s 700 million goats provide reliable access to meat, milk, skin, and fibre for small farmers particularly in developing countries like India. India is bestowed with 17% of total world’s goat population comprised of 21 recognized and many non-descript breeds. Among them, Zalawadi, Gohilwadi and Surti are the three major goat breeds of Gujarat state and are known for meat quality and milk production. Molecular genetic characterization of these breeds has not been carried out. Hence, it is essential to genetically characterize and unfold the genetic diversity of indigenous breeds.

S. Fatima et al. / Small Ruminant Research 77 (2008) 58–64

Plate 1. Map of Gujarat showing breeding tract of Zalawadi, Gohilwadi and Surti breeds of goat.

59

60

S. Fatima et al. / Small Ruminant Research 77 (2008) 58–64

Among available markers, microsatellites are the markers of choice for biodiversity evaluation owing to their unique characteristics and ease of applications. They are among most useful markers as they are easily transferred across ungulate taxa, being widely and successfully applied in conservation and diversity studies (Maudet et al., 2002). Fluorescent based automated fragment analysis along with multiplexing is a cost-effective way to increase the throughput for simultaneously typing of numerous microsatellite markers. Gohilwadi goats are large sized animals with black coat. They are mainly distributed in Bhavnagar, Amreli and Junagarh districts of Gujarat state. Zalawadi goats are distributed in Surendranagar and Rajkot districts of Gujarat state, also having black coat and long twisted horns pointed upward whereas Surti is a medium sized white coated animals mainly distributed in South Gujarat i.e. Vadodara, Bharuch, Surat, Navsari, and Valsad districts. They have small horns directed backward and well developed udder with long teats. Fig. 1 depicts the distribution areas of these three breeds. This study was undertaken to assess genetic variability, possible bottlenecks and to estimate genetic distances between Gohilwadi, Zalawadi and Surti goat breeds, using microsatellites recommended by ISAG/FAO. 2. Materials and methods Blood samples were randomly collected from 157 animals from different locations representing three indigenous goat

populations (50 Zalawadi; 50 Gohilwadi; and 57 Surti breeds). The population of Zalawadi, Gohilwadi and Surti goat breeds in the state was 0.46 millions, 0.59 millions and 0.155 millions, respectively (Anonymous, 2006). DNA was extracted from blood following John et al. (1991). To establish high throughput microsatellite analysis, 18 microsatellite markers were arranged by fragment size and fluorescent dye label, into two multiplexed PCR panels (multiplex Set-I: 7 markers and multiplex Set-II: 11 markers). Each multiplex PCR were carried out in 15 ␮l reaction containing 30–60 ng of DNA, 7.5 ␮l of 2× PCR mastermix (Fermentas) and 2 pmol of each primer (forward primer labelled at 5 with HEX, FAM, NED and PET fluorescent dyes). The PCR mix was subjected to an initial denaturation at 95 ◦ C for 5 min, followed by 35 cycles of 45 s at 95 ◦ C, 35 s at 60 ◦ C (for Set-I) or 58 ◦ C (for SetII) and 35 s at 72 ◦ C with final extension of 10 min at 72 ◦ C. One microliter of multiplex PCR products was mixed with 0.25 ␮l of Genescan-500 LIZ Size Standard (Applied Biosystems) and 8.75 ␮l of Hi-Diformamide (Applied Biosystems) and subjected to electrophoresis. Fragments were resolved on ABI prism® 310 Genetic Analyser (POP-4; run temperature 60 ◦ C) and collected data were analyzed by GeneMapper® software. The genotyped data were analyzed using Microsatellite Analyzer Software (MSA) version 4.0. The polymorphic information content (PIC) (Botstein et al., 1980) and Nei’s standard genetic distance (Nei, 1978) were calculated using PopGene version 1.31. The Wilcoxon sign-rank test (Luikart et al., 1998) for heterozyosity excess was applied to detect recent bottlenecks, under all three models i.e. infinite allele model (IAM), two phase model (TPM) and stepwise mutation model (SMM), using the program BOTTLENECK (Piry et al., 1999). The

Table 1 Variability parameters for all the loci pooled over three breeds Locus name

Observed alleles

Expected alleles

Minimum allele size

Maximum allele size

Ho

He

PIC

Shannon index

ETH-225 ILST-S002 ILSTS-005 ILSTS-008 ILSTS-019 ILSTS-029 ILSTS-030 ILSTS-033 ILSTS-034 ILSTS-049 ILSTS-065 ILSTS-082 ILSTS-087 Oar JMP-29 Oar HH-64 OMHC-1 RM-004 RM-088

9 12 12 8 8 9 13 15 15 8 6 11 11 4 7 9 7 14

4.47 4.80 5.07 4.55 7.82 4.57 5.46 5.01 7.05 5.82 3.15 6.27 8.79 3.03 6.97 3.82 5.98 6.81

138 106 164 166 145 144 146 144 150 166 110 108 136 112 122 182 106 114

156 130 188 184 159 164 172 176 180 179 130 128 158 118 134 206 118 140

0.68 0.93 0.47 0.48 0.61 0.49 0.38 0.58 0.69 0.73 0.30 0.60 0.81 0.24 0.76 0.57 0.65 0.97

0.60 0.67 0.61 0.55 0.82 0.53 0.64 0.62 0.71 0.73 0.37 0.72 0.84 0.35 0.79 0.50 0.72 0.70

0.56 0.62 0.60 0.51 0.78 0.47 0.62 0.60 0.68 0.68 0.32 0.67 0.82 0.29 0.75 0.47 0.67 0.68

1.35 1.38 1.62 1.34 1.86 1.21 1.74 1.60 1.89 1.55 0.72 1.75 2.02 0.65 1.74 1.14 1.58 1.84

0.61

0.64

0.60

1.50

Mean

9.89

5.52

Ho, heterozygosity observed; He, Nei’s heterozygosity expected.

S. Fatima et al. / Small Ruminant Research 77 (2008) 58–64

qualitative descriptor of the allele frequency distribution, “mode-shift” indicator was also used for assessing distortion in allele frequency, indicative of possible bottleneck.

3. Results and discussion The selected microsatellites were successfully amplified in two multiplex sets designed considering annealing temperature, product size and dye label. None of the microsatellite with the same range or dye overlapped leading to highly efficient protocol to estimate and analyze alleles of all the microsatellites present in the multiplex simultaneously. Similar high throughput multiplex system for goats was demonstrated by Glowatzki-Mullis et al. (2006) using 17 microsatellites simultaneously for parentage assessments. Iamartino et al. (2005) multiplexed 15 microsatellites to study genetic diversity in Italian and Alpine goat populations. The three (Zalawadi, Gohilwadi and Surti) goat populations exhibited high genetic diversity (Table 1) at all loci studied except Oar JMP-29 where only four alleles were seen. Barker (1994) suggested that microsatellite loci should have more than four alleles for studies of genetic distances to reduce the standard error of distance estimates. Total numbers of alleles observed per breed were 126, 141 and 127 alleles in Zalawadi, Gohilwadi and Surti breeds, respectively with 178 alleles across all the breeds. Loci ILSTS-034 in Zalawadi (Table 2) and Gohilwadi (Table 3) and ILSTS005 in Surti (Table 4) exhibited maximum of 12 alleles

61

among all 18 loci. The observed number of alleles exhibited per microsatellite was lowest for the microsatellite locus Oar JMP-29 (3) across the three breeds. The mean numbers of alleles observed were 7, 7.82 and 7 in Zalawadi, Gohilwadi and Surti, respectively. Private alleles were also found in all the three breeds but their frequencies were very less. For microsatellite loci, allelic diversity is more informative than heterozygosity to analyze possible genetic erosion in populations (Norris et al., 1999; Spencer et al., 2000). The mean allele number observed across the populations for all 18 loci was 9.89. The values were approximate with that of mean observed alleles (9.87) in 11 goat breeds of Switzerland studied by Glowatzki-Mullis et al. (2006) but higher than 6.90 observed in Chinese goat breeds (Li et al., 2002) and 5.37 in Indian goat breeds (Ganai and Yadav, 2001). It is also higher than the values reported for other African, Asian and French breeds (Martinez et al., 2004; Barker et al., 2001; Ouafi et al., 2002). However, the observed mean values were lower than those obtained for Mehsani (Aggarwal et al., 2007), Black Bengal and Changthangi goat breeds of India (Behl et al., 2003). The high number of alleles in the present study is indicative of adequate polymorphism and their appropriateness of selected loci for assessing genetic variation. The average observed heterozygosity across the populations was 0.61 with maximum heterozygosity observed for RM-088 (0.97) and minimum for locus Oar JMP-29 (0.24). The average heterozygosity observed

Table 2 Variability parameters in Zalawadi goat population Locus

Observed alleles

ETH-225 ILSTS-002 ILSTS-005 ILSTS-008 ILSTS-019 ILSTS-029 ILSTS-030 ILSTS-033 ILSTS-034 ILSTS-049 ILSTS065 ILSTS-082 ILSTS-087 Oar JMP-29 Oar HH-64 OMHC-1 RM-004 RM-088

8 8 9 5 8 7 5 11 12 8 3 6 8 3 6 6 7 6

7.47 10.40 4.49 3.38 13.00 8.50 2.68 6.12 11.11 12.58 2.13 6.60 15.47 2.11 10.28 3.72 11.94 7.55

7

7.75

Mean

Expected alleles

Minimum allele size

Maximum allele size

Ho

He

PIC

Shannon index

138 106 166 166 145 144 154 144 152 166 110 114 136 112 124 182 106 116

156 122 184 180 159 158 166 170 180 179 118 124 152 118 134 198 118 136

0.77 0.96 0.40 0.26 0.79 0.91 0.19 0.55 0.76 0.75 0.16 0.45 0.87 0.16 0.83 0.41 0.67 0.98

0.64 0.74 0.45 0.35 0.81 0.69 0.28 0.57 0.77 0.79 0.20 0.63 0.84 0.19 0.76 0.39 0.78 0.64

0.61 0.74 0.48 0.32 0.77 0.65 0.30 0.57 0.73 0.77 0.20 0.57 0.82 0.16 0.71 0.37 0.70 0.63

1.36 1.60 1.10 0.74 1.78 1.34 0.62 1.39 1.80 1.70 0.40 1.16 1.88 0.40 1.55 0.83 1.66 1.21

0.60

0.58

0.56

1.25

Ho, heterozygosity observed; He, Nei’s heterozygosity expected.

62

S. Fatima et al. / Small Ruminant Research 77 (2008) 58–64

Table 3 Variability parameters in Gohilwadi goat population Locus

Observed alleles

Expected alleles

Minimum allele size

Maximum allele size

Ho

He

PIC

Shannon index

ETH-225 ILSTS-002 ILSTS-005 ILSTS-008 ILSTS-019 ILSTS-029 ILSTS-030 ILSTS-033 ILSTS-034 ILSTS-049 ILSTS-065 ILSTS-082 ILSTS-087 Oar JMP-29 Oar HH-64 OMHC-1 RM-004 RM-088 Mean

9 11 7 7 7 4 11 9 12 6 5 8 9 3 7 9 7 9 7.82

6.77 9.67 6.36 5.96 12.88 3.79 16.96 10.68 7.87 13.12 4.98 10.72 16.68 4.08 11.83 7.98 9.85 7.15 9.15

138 106 166 166 147 148 146 154 150 167 114 108 136 114 122 182 106 114

156 130 180 180 159 160 172 176 174 177 130 122 154 118 134 206 118 136

0.56 0.93 0.43 0.48 0.75 0.37 0.39 0.78 0.63 0.75 0.45 0.78 0.83 0.31 0.76 0.75 0.60 1.00 0.63

0.61 0.72 0.59 0.56 0.80 0.40 0.90 0.76 0.66 0.80 0.50 0.75 0.85 0.43 0.78 0.66 0.73 0.62 0.67

0.60 0.71 0.56 0.53 0.77 0.36 0.86 0.72 0.63 0.77 0.41 0.72 0.83 0.35 0.75 0.62 0.70 0.59 0.64

1.41 1.62 1.25 1.21 1.73 0.79 2.25 1.66 1.50 1.67 0.85 1.66 1.98 0.70 1.64 1.39 1.52 1.24 1.44

Ho, heterozygosity observed, He, Nei’s heterozygosity expected.

was 0.60, 0.63 and 0.58 for Zalawadi, Gohilwadi and Surti goats, respectively. Tables 2–4 shows that Gohilwadi goats had the highest heterozygosity whereas Surti goats had the lowest. The observed mean genetic heterozygosity (0.61) in this study was lower than that reported for Black Bengal goats (0.69) and Chegu goats (0.66) (Behl et al., 2003) but higher than that reported for Swiss goat breeds (0.51–0.58) (Saitbekova et al., 1999).

The mean values for Shannon index pooled over the populations was 1.50, with 1.25, 1.44 and 1.33 individually for Zalawadi, Gohilwadi and Surti goats. Among all the three populations Gohilwadi showed highest gene diversity on the basis of Shannon index. The statistical assessment of informativeness of a marker is denoted by PIC values which varied between 0.29 (Oar JMP-29) and 0.81 (ILSTS-087) with mean PIC of 0.60 across the pop-

Table 4 Variability parameters in Surti goat population Locus

Observed Alleles

Expected Alleles

Minimum Allele size

Maximum Allele size

Ho

He

PIC

Shannon Index

ETH-225 ILSTS-002 ILSTS-005 ILSTS-008 ILSTS-019 ILSTS-029 ILSTS-030 ILSTS-033 ILSTS-034 ILSTS-049 ILSTS-065 ILSTS-082 ILSTS-087 Oar JMP-29 Oar HH-64 OMHC-1 RM-004 RM-088

4 4 12 7 8 5 7 8 11 6 3 10 10 3 7 5 6 11

6.16 6.07 13.50 11.09 17.21 5.09 11.01 6.13 10.05 6.85 4.15 12.65 16.56 4.29 15.00 4.77 8.67 17.82

138 110 164 166 145 146 148 156 152 167 114 108 136 114 122 184 106 116

146 122 188 184 159 164 170 172 180 179 118 128 158 118 134 194 116 140

0.71 0.90 0.60 0.71 0.29 0.19 0.54 0.42 0.80 0.58 0.30 0.57 0.74 0.26 0.67 0.54 0.67 0.93

0.54 0.53 0.78 0.72 0.84 0.48 0.74 0.54 0.70 0.58 0.40 0.77 0.82 0.41 0.81 0.45 0.65 0.84

0.47 0.42 0.75 0.68 0.81 0.41 0.69 0.51 0.67 0.50 0.35 0.73 0.80 0.36 0.78 0.42 0.61 0.81

0.95 0.84 1.80 1.49 1.90 0.86 1.53 1.17 1.54 1.01 0.69 1.72 1.90 0.70 1.75 0.91 1.33 1.93

Mean

7.06

9.84

0.58

0.64

0.60

1.33

Ho, heterozygosity observed; He, Nei’s heterozygosity expected.

S. Fatima et al. / Small Ruminant Research 77 (2008) 58–64

ulations. The mean PIC values were 0.56, 0.64 and 0.60 for Zalawadi, Gohilwadi and Surti populations, respectively which is quite comparable with 0.65 PIC values observed in Mehsani goats (Aggarwal et al., 2007). Genetic markers exhibiting PIC values more than 0.5 are considered as informative in genetic population analysis (Botstein et al., 1980). The Nei’s standard genetic distance between Zalawadi and Gohilwadi, Zalawadi and Surti and Gohilwadi and Surti goat breeds were 0.128, 0.151 and 0.195, respectively. This suggested that Zalawadi and Gohilwadi are more closely related in comparison to Zalawadi and Surti and Gohilwadi and Surti which is also reflected by the phenotypic similarity and close geographic distribution of Zalawadi and Gohilwadi goats. A comparative low rate of inbreeding was depicted from mean values (−0.058, 0.057 and 0.070) of withinpopulation inbreeding coefficient (FIS ) for Zalawadi, Gohilwadi and in Surti, respectively. Among the three breeds the FIS values are highest for Surti breeds, possibly because of small population size of Surti breed. These values showed low rate of inbreeding within the populations as compared to 45 rare breeds of 15 European and Middle Eastern countries reported by Canon et al. (2006) (FIS = 0.10). Significant heterozygote deficiency has also been reported in Marwari (FIS = 0.264) (Kumar et al., 2005) and Mehsani (FIS = 0.16) goat breeds (Aggarwal et al., 2007). The stepwise mutation model, the infinite allele model and two phase mutation model under Wilcoxon sign-rank test and shift mode test were used to find out recent bottleneck (heterozygosity excess) in the three goat populations. In a population at mutation-drift equilibrium (i.e. effective size of which has remained constant in the recent past) there is approximately an equal probability that a locus shows genetic diversity excess or deficit. The Wilcoxon sign-rank test were used for assessing bottleneck because of its relatively high statistical power (Luikart and Cornuet, 1998) as it can be used for as few as four polymorphic loci and any number of individuals (15–40 individuals and 10–15 polymorphic loci is recommended to achieve high power). This condition is easily fulfilled with 18 loci and 50 individual each of three breeds. The heterozygosity excess obtained (Table 5) were non significant (P < 0.5) under all the models in the Zalawadi and Gohilwadi populations, but Surti showed mild bottleneck only with IAM. These results were consistent with the normal L-shaped distribution of allele frequency in Zalawadi and Gohilwadi (graph not shown). However, the allele frequency distribution in the Surti breed displayed a mode shift to the right, indicating a putative bottleneck that might have

63

Table 5 Bottleneck analysis for Zalawadi, Gohilwadi and Surti populations using Wilcoxon rank test under infinite allele, stepwise mutation and two phase model Models

Zalawadi (P)

Gohilwadi (P)

Surti (P)

IAM TPM SMM

0.6952 0.98658 0.99997

0.24754 0.94065 0.99832

0.00237** 0.58414 0.99883

Parameters for TPM: variance = 30.00, proportion of SMM = 70.00%, estimation based on 1000 replications. P, probability; IAM, infinite allele model; TPM, two phase model; SMM, stepwise mutation model. ** Indicate highly significant deviation from equilibrium as value less than 0.05.

occurred in this population. Though, the Surti population is not apparently faced with an immediate extinction risk, but demographic and genetic monitoring coupled with the survey of the environmental conditions is recommended. Tantia et al. (2004) reported heterozygosity excess and genetic bottleneck in the Indian goat breeds (Chegu and Black Bengal). Li et al. (2002) studied the genetic equilibrium of 12 Chinese goat populations using 17 microsatellite loci, all except three Tibetan populations showed deviation from the equilibrium. Deviation from mutation-drift equilibrium has been reported in several populations, however they were mainly associated with heterozygosity deficiency viz., in Mehsani goats (Aggarwal et al., 2007), California big horn sheep (Whittaker et al., 2004) and in Xalda Spanish breeds (Alvarez et al., 2004). Among all the seven population of Baltic sheep only Estonian Ruhnu population showed a slight distortion in distribution of allelic frequency (Grigaliunaite et al., 2003). Severely bottlenecked populations are important to identify for conservation, as they are likely to suffer from inbreeding depression, loss of genetic variation, fixation of deleterious alleles as well as increased demographic stochasticity, any of which can ultimately reduce adaptive potential and the probability of population persistence (Frankham, 1995). The data reported here provides a valuable insight into genetic diversity and genetic relationships between Zalawadi, Gohilwadi and Surti breeds, which can be used to improvise the breeding strategies. Acknowledgements The present study was conducted under Core laboratory project, Network Projects on Animal Genetic Resources, National Bureau of Animal Genetic Res-

64

S. Fatima et al. / Small Ruminant Research 77 (2008) 58–64

ources (NBAGR), Indian Council of Agriculture Research (ICAR), India. References Aggarwal, R.A.K., Dixit, S.P., Verma, N.K., Ahlawat, S.P.S., Kumar, Y., Kumar, S., Chander, R., Singh, K.P., 2007. Population genetics analysis of Mehsana goat based on microsatellite markers. Curr. Sc. (92), 1133–1137. Alvarez, I., Royo, J., Fernandez, I., Gutierrez, J.P., Gomez, E., Goyache, F., 2004. Genetic relationships and admixture among sheep breeds from Northern Spain assessed using microsatellites. J. Anim. Sci. (82), 2246–2252. Anonymous, 2006. Summary report on 17th Livestock Census 2003, Directorate of Animal Husbandry, Krishi Bhavan, Gujarat. Barker, J.S.F., 1994. A global protocol for determining genetic distances among domestic livestock breeds. In: Proceeding of the 5th World Congress on genetics Applied to livestock production, 7–12 August, vol. 21. University of Guelph, Ontario, pp. 501–508. Barker, J.S.F., Tan, S.G., Moore, S.S., Mukherjee, 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. Behl, R., Sheoran, N., Be hl, J., Vijh, R.K., Tantia, M.S., 2003. Analysis of 22 heterologous microsatellite markers for genetic variability in Indian goats. Anim. Biotechnol. (14), 167–175. Botstein, D., White, R.L., Skolnick, M., Davis, R.W., 1980. Construction of a genetic linkage map in man using restriction fragment length polymorphism. Am. J. Hum. Genet. (32), 324– 331. Canon, J., Garcia, D., Garc´ıa-Atance, M.A., Obexer-Ruff, G., Lenstra, J.A., Ajmone-Marsan, P., Dunner, S., 2006. Geographical partitioning of goat diversity in Europe and the Middle East. Anim. Genet. (37), 327–334. Frankham, R., 1995. Effective population size/adult population size ratios in wildlife: a review. Genet. Res. (66), 95–107. Ganai, N.A., Yadav, B.R., 2001. Genetic variation within and among three Indian breeds of goat using heterologous microsatellite markers. Anim. Biotechnol. (12), 121–136. Glowatzki-Mullis, M.L., Muntwyler, J., Gaillard, C., 2006. Costeffective parentage verification with 17-plex PCR for goats and 19-plex PCR for sheep. Anim. Genet. (38), 81–91. Grigaliunaite, I., Haldja, M.T., Grislis, V.Z., Kantanen, J., Miceikien, I., 2003. Microsatellite variation in the Baltic Sheep breeds. Veterinarija Ir Zootechnika. T. (21), 1–8. Iamartino, D., Bruzzone, A., Lanza, A., Blasi, M., Pilla, F., 2005. Genetic diversity of Southern Italian goat populations assessed by microsatellite markers. Small Rumin. Res. (57), 249–255.

John, S.W., Weitzner, G., Rozen, R., Scriver, C.R., 1991. A rapid procedure for extracting genomic DNA from leukocytes. Nucleic Acids Res. (19), 408. Kumar, D., Dixit, S.P., Sharma, R., Pandey, A.K., Sirohi, G., Patel, A.K., Aggarwal, R.A.K., Verma, N.K., Gour, D.S., Ahlawat, S.P.S., 2005. Population structure, genetic variation and management of Marwari goats. Small Rumin. Res. 59, 41–48. Li, M.H., Zhao, S.H., Bian, C., Wang, H.S., Weid, H., Liu, B., Yu, M., Fan, B., Chen, S.L., Zhu, M.J., Lia, S.J., Xiong, T.A., Li, K., 2002. Genetic relationships among twelve Chinese indigenous goat populations based on microsatellite analysis. Genet. Sel. Evol. (34), 729–744. Luikart, G., Cornuet, J.M., 1998. Empirical evaluation of a test for identifying recently bottlenecked populations from allele frequency data. Conserv. Biol. (12), 228–237. Luikart, G., Sherwin, W.B., Steele, B.M., Allendorf, F.W., 1998. Usefulness of molecular markers for detecting population bottlenecks via monitoring genetic change. Mol. Ecol. (7), 963–974. Martinez, A.M., Carrera, M.P., Acosta, J.M., Rodriguez-Gallardo, P.P., Cabello, A., Camacho, E., Delgado, J.V., 2004. Genetic characterisation of the Blanca Andaluza goat based on microsatellite markers. S. Afr. J. Anim. Sci. (34), 17–19. Maudet, C., Luikart, G., Taberlet, P., 2002. Genetic diversity and assignment tests among seven French cattle breeds based on microsatellite DNA analysis. J. Anim. Sci. (80), 942–950. Nei, M., 1978. Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics (89), 583–590. Norris, A.T., Bradley, D.G., Cunningham, E.P., 1999. Microsatellite genetic variation between and within farmed and wild Atlantic salmon (Salmo salar) populations. Aquaculture (180), 247–264. Ouafi, A.T., Babilliot, J.M., Leroux, C., Martin, P., 2002. Genetic diversity of the two main Moroccan goat breeds: polygenetic relationships with four breeds reared in France. Small Rumin. Res. (45), 225–233. Piry, S.G., Luikart, G., Cornuet, J.M., 1999. BOTTLENECK: a computer program for detecting recent reductions in the effective population size using allele frequency data. J. Hered. (90), 502–503. Saitbekova, N., Gaillard, C., Obexer-Ruff, G., Dolf, G., 1999. Genetic diversity in Swiss goat breeds on microsatellite analysis. Anim. Genet. (30), 36–41. Spencer, C.C., Neigel, J.E., Leberg, P.L., 2000. Experimental evaluation of the usefulness of microsatellite DNA for detecting demographic bottlenecks. Mol. Ecol. (9), 1517–1528. Tantia, M.S., Behl, R., Sheoran, N., Singh, R., Vijh, R., 2004. Microsatellite data analysis for conservation of two goat breeds. Indian J. Anim. Sci. (74), 761–767. Whittaker, D.G., Ostermannb, S.D., Boycec, W.M., 2004. Genetic variability of reintroduced California big sheep in Oregon. J. Wildlife Manage. (68), 850–859.