Conservation Genetics

, Volume 11, Issue 3, pp 671–673 | Cite as

Ten polymorphic microsatellite loci in Tibetan chicken, Gallus gallus domesticus

  • Kong Yang
  • Xia Luo
  • Yong Wang
  • Ying Yu
  • Zhihua Chen
Research Article

Abstract

Through an improved enrichment protocol, a genomic library for (AC)12 repeats was constructed and 34 microsatellite loci were isolated and characterized in an endangered animal, Tibetan chicken, Gallus gallus domesticus. In the 34 loci, ten loci showed a distinct allelic variation ranging from 4 to 14 alleles in 54 individuals tested. Polymorphism information content (PIC) ranged from 0.590 to 0.869 with an average of 0.713. Average observed and expected heterozygosities were 0.7988 (ranged from 0.310 to 1.000) and 0.7495 (ranged from 0.609 to 0.897), respectively. These ten microsatellites loci would be the valuable genetic markers for further investigation of Tibetan chicken.

Keywords

Tibetan chicken Microsatellite (AC)n 

Tibetan chicken (Gallus gallus domesticus) is an endemic species to China and mainly distributed in Qinghai Province and Tibetan Plateau (Ji et al. 2001). It was estimated that the purebred Tibetan chicken was reduced from 750,000 to <100,000 individuals in the last 20 years, largely because of the introgression of other chicken species into purebred Tibetan chicken population. Moreover, the area of purebred Tibetan chicken decreased dramatically. Therefore, in order to protect this species, agriculture ministry of China listed Tibetan chicken in the first class “key” species of animals in 2000 (Ji et al. 2001). In addition, in order to protect this species, a study on genetic diversity of the population is clearly needed.

Microsatellite marker should be one of the most suitable DNA markers for this purpose (Zhang et al. 2006). Although microsatellites have been isolated in many chicken species (Wu et al. 2004), no report was found in the Tibetan chicken. In this research, we reported the isolation and characterization of ten polymorphic microsatellite loci in the Tibetan chicken.

A genomic library for (AC)12 repeats was constructed following the method of Bloor et al. (2001) and Zhang et al. (2008). The DNA of the genomic library was extracted from a male Tibetan chicken. Out of 63 recombinant clones screened by PCR, 47 (74.6%) positive clones were found and insured by sequencing.

About 34 primers were obtained from the 47 microsatellite sequences based on the program primer3.0 (Rozen and Skaletsky 2000). Each primer was labeled with a fluorescent dye (6-FMA or HEX) in order to detect the polymorphism and heterozygosity in 54 Tibetan chickens housed at Nyingtri county of Tibet Autonomous Region, P. R. China. The DNAs from the 54 Tibetan chickens were all extracted from blood using a standard proteinase K digestion and phenol–chloroform extraction procedure (Sambrook et al. 1989). PCR amplifications were performed in 25 μl reactions containing 50 ng genomic DNA, 1.0 μM of each primer, 1.5 mM MgCl2, 200 μM of each dNTP, 1× PCR buffer, and 0.5 U of Taq DNA polymerase (TaKaRa, Japan). Amplifications were conducted in an iCycler PCR System (Bio-rad, USA) with an initial denaturation at 95°C for 5 min, 35 cycles consisting of 95°C for 30 s, a primer-specific annealing temperature (Table 1) for 30 s, 72°C for 45 s and ending with a single extension of 72°C for 10 min. The amplified products were separated on an ABI 377 PRISM™ DNA sequencer. Fragment lengths were assigned using GeneScan software (ABI) and a GeneScan-500 [ROX] size standard. Allele heterozygosity and polymorphism information content (PIC) were calculated using the program Cervus 2.0 (Marshall et al. 1998). Deviations from Hardy–Weinberg equilibrium (HWE) and linkage disequilibrium (LD) were analyzed using GENEPOP 3.4 (Raymond and Rousset 2002).
Table 1

Characterization of ten microsatellite loci in Tibetan chicken, including repeat motif, number of genotypes (Ng), number of alleles observed (Na), allele size range, annealing temperature (Ta), observed heterozygosity (Ho), expected heterozygosity (He), polymorphic information content (PIC), and GenBank accession numbers

Locus

Primer sequence (5′–3′)

Repeat motif

Ng

Na

Size (bp)

Ta (°C)

Ho

He

PIC

GenBank accession number

MberH7-2

F:5′-CTGTGACTTGGCATTTCT-3′

R:5′-CACCATTTTATAGCGTGAC-3′

(TG)17

54

10

110–144

53.5

0.833

0.778

0.734

EU939313

MberC8-1

F:5′-CAGGTGGCAATCACAACG-3′

R:5′-AAGGCACCCAAAGCAAGG-3′

(AC)6GC(AC)7

51

13

174–226

50

0.963

0.745

0.693

EU939314

MberH5-4

F:5′-TCCTTCTTGCCTTCTACC-3′

R:5′CTTCCACTCCTCCCTTACT-3′

(TG)8

53

4

138–146

50

1.000

0.743

0.681

EU939315

MberA4-10

F:5′-TGGGTCAGACGGGCTTTG-3′

R:5′-GTCCTTGCCAGAGGCTTC-3′

(TG)15

51

11

159–188

56

0.889

0.897

0.869

EU939316

MberB12-6

F:5′-GGAAGCCCTACAGAAACC-3′

R:5′-GCCAGATGTCTTTAATGC-3′

(AC)11

53

9

300–320

53.5

0.875

0.846

0.809

EU939317

MberC11-10

F:5′-CGGTCAGTCAGGCAGCAA-3′

R:5′-AAGGCACCCAACGCAAGG-3′

(AC)8GC(AC)5

54

14

200–238

50

0.964

0.857

0.828

EU939318

MberD11-5

F:5′-AGCTACGTCTGGCCCTAA-3′

R:5′-AATGCTCCTGCTGTCCTT-3′

(TG)5GC(TG)6

52

7

186–218

55.8

0.964

0.732

0.679

EU939320

MberD8

F:5′-AAACAGAGGCCCAAACCGA-3′

R:5′-TTGGTGATAGGTGGACGGTTG-3′

(AC)14

54

14

185–247

56

0.310

0.623

0.590

FJ198069

MberH11

F:5′-TGAGGAAGCAGCGGGTAA-3′

R:5′-GTTGGCCCTTGCTTGTTTG-3′

(TG)20

54

7

184–206

52

0.627

0.665

0.604

FJ198068

MberF12

F:5′-TGAGGAAGCAGCGGGTAA-3′

R:5′-AAAAGCGATGATGTGAATAAGG-3′

(TG)11

54

6

102–128

54

0.563

0.609

0.643

FJ198067

About 10 loci of the 34 primers showed a distinct allelic variation ranging from 4 to 14 alleles in the individuals examined (Table 1). Their polymorphism information content (PIC) ranged from 0.590 to 0.869 with an average of 0.713. Average observed and expected heterozygosities were 0.7988 (range 0.310–1.000) and 0.7495 (range 0.609–0.897), respectively. No loci showed significant deviations from HWE (P > 0.01). Following Bonferroni correction, highly significant LD tests were not shown for any pair of loci (P > 0.01 for all comparisons). In addition, the remaining 24 primers were mono-morphism in the individuals tested.

The results indicated that these ten microsatellite markers would be useful in genetic diversity analyses and population management of Tibetan chicken population.

Notes

Acknowledgments

We thank Dr. Wenping Zhang and Xiuyue Zhang for giving us a lot of help in data analysis and advices in experiments. This research was funded by the Sichuan Youth Science and Technology Foundation (08ZQ026-027) and Southwest University for Nationalities Foundation of P. R. China.

References

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Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Kong Yang
    • 1
    • 2
  • Xia Luo
    • 3
  • Yong Wang
    • 2
  • Ying Yu
    • 1
  • Zhihua Chen
    • 1
  1. 1.College of Life Sciences and TechnologySouthwest University for NationalitiesChengduPeople’s Republic of China
  2. 2.Key Laboratory for The Qing-Tibet Plateau Animal Genetic Resource Conservation and Exploitation of Sichuan ProvinceChengduPeople’s Republic of China
  3. 3.College of ManagementSouthwest University for NationalitiesChengduPeople’s Republic of China

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