ISSN: 2469-2794 FRCIJ

Forensic Research & Criminology International Journal
Research Article
Volume 1 Issue 5 - 2015
Genetic Polymorphism of Eleven STR Loci in Rajput Population of Delhi, India
Tanya Chauhan*, KPS Kushwaha and Varsha Chauhan
LNJN National Institute of Criminology and Forensic Science, India
Received: October 28, 2015| Published: December 08, 2015
*Corresponding author: Tanya Chauhan, Assistant Professor, LNJN National Institute of Criminology and Forensic Science, 1, Institutional Area, Sector 3, Rohini, Delhi-110085, India, Tel: +919971180004; Email:
Citation: Chauhan T, Kushwaha KPS, Chauhan V (2015) Genetic Polymorphism of Eleven STR Loci in Rajput Population of Delhi, India. Forensic Res Criminol Int J 1(5): 00031. DOI: 10.15406/frcij.2015.01.00031

Abstract

This study is an attempt to generate genetic database for endogamous population of Rajputs of Delhi, India. Genetic polymorphism at eleven Short Tandem Repeat (STR) loci (F13A01, FESFPS, vWA, D16S539, D7S820, D13S317, HPRTB, F13B, CSF1PO, TH01 and TPOX) was examined in 87 unrelated Rajputs individuals to evaluate their significance in human identification. There was no evidence for departures from HWE (P>0.001) at all loci except locus HPRTB. All the loci showed high polymorphism with great power of exclusion. The combined matching probability (MP) of the eleven STR system was 1.965 x 10-11, indicating that the system has a much stronger inter-individual discriminating power. The eleven loci showed a combined power of discrimination (PD) of 0.999999. The data suggests that these loci are useful for identity testing, forensics and for solving paternity cases among the Rajput population in Delhi, India.

Keywords: DNA; STR loci; Genetic diversity; Population data; Rajputs; Forensics

Abbreviations

STR: Short Tandem Repeat; MP: Matching Probability; PD: Power of Discrimination; BDA: Bio Doc Analyze; PIC: Polymorphism Information Content; PE: Power of Exclusion; TPI: Typical Paternity Index

Introduction

Short Tandem Repeat (STR) markers are best choice for the genetic structure assessment of a population due to co-dominant inheritance, high polymorphism, mutation rate and ease of use [1,2]. Polymorphic STR loci have become useful tool for DNA analysis and typing for human identification and paternity testing for forensic purposes in most of the forensic laboratories in the world [3,4]. Determination of the allele frequencies and distribution of genotype are prerequisites for DNA typing of any population. India is a rich country in ethnic, cultural and linguistic variant groups. Human diversity in India is defined by 4693 different and documented population groups that include 2205 major communities, 589 segments and 1900 territorial units spread across the country. Like most other Indians, Rajput is endogamous population [5]. The Rajput clans emerged as a dominant community in northern, central, western India and current eastern Pakistan in the colonial period. They seem to have risen to prominence from the late 6th century C.E. and governed the majority of princely states in Rajasthan and Surashtra. The Rajput population and the former Rajput states are found spread through much of the subcontinent, particularly in north, west and central India. Rajput populations are found in Rajasthan, Gujarat, Delhi, Uttar Pradesh, Himachal Pradesh, Haryana, Jammu, Punjab, Sindh, Uttarakhand, Madhya Pradesh and Bihar. The state of Delhi comprises about 3.8% of the Rajput population [6]. A very few number of genetic studies have been carried out on Rajput population [7,8]. However, there is no published data on the allele frequency of STR loci in Rajput population of Delhi, India. Therefore, the present data would be used in the forensics and individual identification for this population group and this genetic data would enrich the genetic informational resource.

Materials and Methods

Sample collection

Blood samples were collected from 87 unrelated healthy donors resident in State of Delhi, India on FTA cards (Whatman technology, Sigma –Aldrich, USA).

STR markers

The selected STR markers are tetra-nucleotide ((F13A01, FESFPS, vWA, D16S539, D7S820, D13S317, HPRTB, F13B, CSF1PO, TH01, TPOX) located on 13 different chromosomes (Table 1).

STR Locus Location

Chromosomal Definition

GenBank Locus and Locus Definition

Repeat Sequence 5’→ 3’

Known Alleles

Allele Size Range (Bases)

CSF1PO

5q33.3-34

HUMCSF1PO, Human c-fms proto-oncogene for CSF-1 receptor gene

AGAT

6-15

295-323

TH01

11p15.5

HUMTH01, Human Tyrosine hydroxylase gene

AAGT

5-9, 9.3, 10, 11

183-199

TPOX

2p25.1-pter

HUMTH01, Human Thyroid peroxidase gene

AAGT

6-13

228-256

D16S539

16q24-qter

NA

AGAT

5, 8-15

276-396

D7S820

7q11.21-22

NA

AGAT

6-14

215-243

D13S317

13q22-q31

NA

AGAT

7-15

165-193

FESFPS

15q25-qter

HUMFESFPS, Human c-fes/fps proto-oncogene

AAAT

7-14

230-246

F13A01

6p24.3-p25.1

HUMF13A01, Human coagulation factor XIII a subunit gene

AAAG

3.2,4-16

281-331

vWA

12p12-pter

HUMVWA31, Human von Willebr and factor gene

AGAT

11, 13-21

143-171

F13B

1q31-q32.1

HUMBFXIII, Human factor XIII b subunit gene

AAAT

6-12

169-189

HPRTB

Xq26

HUMHPRTB, Human hypoxanthinephosphoribosyl-transferase gene

AGAT

11-15, 17

279-303

Table 1: Locus Specific Information.

DNA Extraction

A 1.2 mm punch from a dried sample spot on FTA paper was taken in a PCR tube. 200 μl FTA purification reagent ((Whatman technology, Sigma –Aldrich, USA) was added to PCR tube, incubated for 5 minutes at room temperature and then continuously agitated by using a pipette. This process was repeated thrice with FTA purification reagent and twice with 200 μl TE buffer (10mM Tris-HCl, 0.1mM EDTA, pH 8.0). Finally, the entire unspent TE buffer was removed and discarded by pipetting and the disc was allowed to dry at room temperature for overnight and was directly used for PCR amplification.

PCR Amplification

PCR amplification of the 11 STR loci viz. F13A01, FESFPS, vWA, D16S539, D7S820, D13S317, HPRTB, F13B, CSF1PO, TH01, TPOX was performed using Gene Print STR system kit (Promega Corporation, USA) in a 25 µl final reaction volume. The PCR reagents were used according to the manufacturer’s protocol (Promega Corporation, Madison, US) [9]. The samples were amplified on Biometra PCR thermal cycler. Thermal cycling parameters have been standardized in the laboratory for consistency of results (Table 2).

Program for Locus

Initial Incubation

Cycling for First 10 Cycles

Cycling for Last 20 Cycles

Extension Step

Hold Step

CTT Multiplex

96°C for 2 mins.

94°C for 1 min.

94°C for 1 min.

None

4°C

64°C for 1 min.

64°C for 1 min.

70°C for 1.5mins.

70°C for 1.5mins.

F13B

96°C for 2 mins.

94°C for 1 min.

90°C for 1 min.

None

4°C

60°C for 1 min.

60°C for 1 min.

70°C for 1.5mins.

70°C for 1.5 mins.

STRIII Multiplex FFV Multiplex HPRTB

96°C for 2 mins.

94°C for 1 min.

90°C for 1 min.

60°C for 30 mins.

4°C

60°C for 1 min.

60°C for 1 min.

70°C for 1.5 mins.

70°C for 1.5 mins.

Table 2: Amplification Programs for Different STR Loci.

Genotyping of amplified fragments

Amplified products (2.5 µl per sample mixed with an equal volume of STR 2X loading solution) were electrophoresed at 50 Watt for 2-4 h on a 40cmX20cm, 0.35mm thick 6% denaturing poly acrylamide gel by manual Geno Sequencer (Atto Corporation, Japan) with 0.5X TBE as gel running buffer. Various alleles were visualized by staining with silver stain (Promega Corporation, USA) described by Bassam et al. [10] and images were stored in the computer. The allelic ladders were run with the samples to determine the size of the amplified products. Direct comparison between the allelic ladders and amplified samples of the same locus allowed for determination of alleles. Alleles were designated by comparison with appropriate allelic ladders according to the standard nomenclature [11] and calculated the size relative to allelic ladder with Bio Doc Analyze (BDA) system (Biometra GmbH, Germany)

Statistical analysis

The genotype data of the 11 STR loci was compiled for eleven STR markers in Excel sheets to facilitate statistical treatment for generation of allele frequencies. Allele frequencies [12] heterozygosity (observed and expected) values were calculated using software Genetix 4.02 [13]. Tests for conformity to Hardy-Weinberg expectations (probability test) were performed through Gene pop 3.3d [14,15]. Statistical parameters of forensic importance like power of discrimination (PD), polymorphism information content (PIC)[16-18] matching probability (MP) and paternity indices viz. power of exclusion (PE) and typical paternity index (TPI) [16] were calculated using “Power Stats” Microsoft Excel workbook template provided by Promega Corporation [19,20].

Results and Discussion

Allelic variation at each locus in Rajput population is described in Table 3. The combined allele distribution for the eleven STR loci in the present study ranged from 5 to 9 at different loci. All STR loci exhibited considerable variation in this population. The distribution of observed allele frequencies, heterozygosity (Observed and expected), P values at eleven STR loci and common forensic efficiency parameters useful for forensic and paternity tests have been tabulated in Table 4.

STR Locus

No. of Alleles Observed at Each Locus (no. of  Samples Studied=87)

Alleles Observed in Present Study

Alleles Observed in Bihar Population [7]

Alleles Observed   in Haryana Population  [8]

CSF1PO

8

7-14

8-14

14-Aug

THO1

6

6- 9, 9.3, 10

Same

Same

TPOX

7

7-12, 14

8-13, 15

12-Jul

D16S539

6

8-13

8-14

Not Studied

D7S820

8

6-13

7-14

Not Studied

D13S317

8

7-14

Same

Not Studied

FESFPS

5

9-13

Not Studied

14-Aug

F13A01

9

3.2,4-8, 14-16

Not Studied

Not Studied

vWA

8

14-21

13-19

Not Studied

F13B

5

6,8-11

Not Studied

Not Studied

HPRTB

6

11-15, 17

Not Studied

Not Studied

Table 3: Variation at Each STR Locus in Rajput Population.    

Alleles

CSF1PO

D7S820

D13S317

D16S539

F13A01

F13B

FESFPS

HPRTB

THO1

TPOX

vWA

3.2

0.133

4

0.084

5

0.398

6

0.010

0.169

0.111

0.316

7

0.006

0.042

0.010

0.139

0.184

0.006

8

0.006

0.250

0.200

0.053

0.006

0.210

0.109

0.425

9

0.023

0.104

0.100

0.171

0.309

0.006

0.253

0.155

9.3

0.121

10

0.218

0.156

0.150

0.132

0.364

0.217

0.017

0.057

11

0.339

0.198

0.260

0.276

0.006

0.367

0.110

0.293

12

0.351

0.167

0.210

0.250

0.319

0.234

0.052

13

0.046

0.073

0.060

0.118

0.090

0.351

14

0.011

0.010

0.024

0.221

0.011

0.107

15

0.036

0.078

0.065

16

0.012

0.202

17

0.006

0.315

18

0.220

19

0.065

20

0.018

21

0.006

MP

0.137

0.061

0.076

0.096

0.093

0.149

0.145

0.117

0.093

0.162

0.091

Combined MP

1.965 x 10-11

Expressed as 1 in    7.3

16.5

13.2

10.5

10.7

6.7

6.9

8.5

10.7

6.2

110

PD

0.863

0.939

0.924

0.904

0.907

0.851

0.855

0.883

0.907

0.838

0.909

PIC

0.66

0.81

0.79

0.77

0.74

0.66

0.65

0.72

0.74

0.66

0.76

PE

0.395

0.745

0.637

0.533

0.59

0.581

0.484

0.161

0.586

0.525

0.781

TPI

1.55

4

2.78

2.11

2.44

2.38

1.89

0.94

2.42

2.07

4.64

Ho

0.6782

0.875

0.8333

0.7632

0.7952

0.7901

0.7349

0.4675

0.7931

0.7586

0.8916

He

0.7116

0.8281

0.8121

0.7978

0.7675

0.7157

0.7078

0.7554

0.7755

0.703

0.7882

PHW

0.238

0.9913

0.5795

0.0545

0.2916

0.569

0.9391

0.0000

0.8621

0.4132

0.4446

Total Alleles

174

96

100

76

166

162

166

154

174

174

168

Table 4: Allele Frequency and other Forensic Efficiency Parameters of Rajput Population.
MP: Matching Probability, PD: Power of Discrimination, PE: Power of exclusion; TPI: Typical Paternity index; Ho: Homozygotes (%); He: Heterozygotes (%) PHW: Probability value of significant deviation from Hardy Weinberg Equilibrium

None of the alleles in eleven STR loci exceeded 50% frequency reflecting the usefulness and validity of these loci in calculated paternity indices and discriminating individuals [12]. The high level of heterozygosity observed (range 0.468-0.892) for eleven STR system is an indication of that the Rajput population has a high level of genetic variation and there would be successfully utilized in discriminating between individuals. The results indicate that for the analysed population, all the STR loci met Hardy-Weinberg expectations except locus HPRTB. Significant deviation from HW expectations at HPRTB locus was depicted in probability test. The results revealed deficiencies of heterozygotes at this locus.

The number of alleles at many loci viz. CSF1PO, TH01, TPOX, FESFPS, vWA, D7S820 and D16S539 are different those reported for Rajput population from other states (Table 3). However, the heterozygosity is generally within the range of those reported in Rajput population from other states [7,8] PIC values > 0.5 for all STR loci (range 0.65-0.81) indicating that the analysed system is informative and useful for identification purpose. The combined matching probability (MP) of the eleven STR systems was 1.965 x 10-11, indicating that the system has a much stronger inter-individual discriminating power. The eleven loci showed a combined power of discrimination (PD) of 0.999999. Practically this degree of MP and PD means that no other individual with the same profile for all eleven loci could exist in Rajput population, as these two parameters express the probability of two random DNA profile matching at the loci tested: MP expressed as 1 individual for eleven STR loci is ~ one Trillion is a population that is over 1.24 billion population in country.

Conclusion

All eleven loci were found to be informative and useful for forensic identity testing. The expected heterozygosity and the power of discrimination calculated from the gene frequencies obtained in the population reveal that the combination of 11 STR loci has a high forensic efficiency. In addition, the present study involves the development of forensic databases for indigenous population of Delhi, India.

Acknowledgement

Thanks are accorded to Ministry of Homes, Government of India for financial support. Excellent technical assistance provided by Mr. Pawan Kumar and Mr. Bhagat Singh is duly acknowledged. The work was carried out under the XI FYP project, MHA GOI.

References

  1. Tautz D, Renz M (1984) Simple sequences are ubiquitous repetitive components of eukaryotic genomes. Nucleic Acids Res 12(10): 4127-4138.
  2. Weber JL, May PE (1989) Abundant class of Human DNA polymorphisms which can be typed using the polymerase chain reaction. Amer J Human Genet 44(3): 388-396.
  3. Chakraborty R, Stivers DN, Su B, Zhang Y, Budowle B (1999) The utility of short tandem repeat loci beyond human identification: implication for development of new DNA typing system. Electrophoresis 20(8): 1682-1696.
  4. Butler JM (2001) Forensic DNA typing: Biology and technology behind STR markers. (2nd edn) Academic Press, UK, pp. 688.
  5. Singh KS (1998) India's Communities. People of India National Series Volume IV. Oxford University Press, India, pp. 1230.
  6. Census of Delhi (1901) Census posts.
  7. Ashma R, Kashyap VK (2002) Genetic study of fifteen important STR loci among four major ethnic groups of Bihar, India. J Forensic Sci 47(5): 1139-1142.
  8. Kushwaha KPS, Rao MB, Chauhan K (2007) Population Genetics of 12 PCR based DNA markers among people of Haryana. J of Crimino and criminalistics 28(2): 45-51.
  9. Promega Technical Manual (2006) GenePrint® STR Systems (Silver Stain Detection), Promega Corporation, Madison, USA.
  10. Bassam BJ, Caetaro-Anolles G, Gresshoff PM (1991) Fast and sensitive silver staining of DNA in polyacrylamide gel. Anal Biochem 196(1): 80-83.
  11. Bär W, Brinkmann B, Budowle B, Carracedo A, Gill P, et al. (1997) DNA recommendations: further report of the DNA Commission of the ISFH regarding the use of short tandem repeat system. International Society for Forensic Haemogenetics. Int J Legal Med 110(4): 175-176.
  12. Weir BS, Cockerham CC (1984) Estimating F-statistics for the analysis of population structure. Evolution 38(6): 1358-1370.
  13. Belkhir K, Borsa P, Goudet J, Chikhi L, Bonhomme F (1997) Genetix ver. 4.02, Genetics logicielsous windows pour Iage ’ne’ Tiquedes populations.
  14. Raymond M, Rouseet F (1995) An exact test for population differentiation. Evolution 49(6): 1280-1283.
  15. Raymond M, Rousset F (1998) Genepop version 3.3d Population genetics software for exact test and ecumenicism.
  16. Brenner C, Morris JW (1989) Paternity index calculations in single locus hyper-variable DNA probes validation and other studies. Proceedings from International Symposium of Human Identification. Promega Corp, USA, pp. 23-53.
  17. Jones DA (1972) Blood samples: probabilities of discrimination. J Forensic Sci Soc 12(2): 355-359.
  18. Botstein D, White RL, Skolnick M, Davies RW (1980) Construction of a genetic linkage map in man using restriction fragment length polymorphism. Am J Hum Genet 32(3): 314-331.
  19. Tereba A (1999) Tools for analysis of population statistics. Profiles DNA 2: 14-16.
  20. Power Stats (2015) Microsoft Excel Workbook template from Promega: Genetic Identity.  
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