International Journal of Legal Medicine

https://doi.org/10.1007/s00414-019-02198-8

POPULATION DATA

Evaluation of genetic polymorphism at 20 autosomal STR

loci in Rajput population of Himachal Pradesh, India
Vivek Sahajpal 1 & Abhishek Singh 2 & Mukesh Thakur 2 & Arun Sharma 1 & Kailash Chandra 2 & Shivkant Sharma 3

Received: 12 August 2019 / Accepted: 25 October 2019

# Springer-Verlag GmbH Germany, part of Springer Nature 2019

Abstract
In the present study, genetic diversity indices and forensic parameters of Rajput population of Himachal Pradesh, India, were
obtained at 20 autosomal short tandem repeat (STR) loci included in the PowerPlex® 21 System (Promega, USA). With 227
alleles and 11.35 ± 0.89 alleles per locus, none of the locus deviated from the Hardy–Weinberg equilibrium (HWE). The most
polymorphic and discriminative locus was Penta E. Phylogenetic tree showed high genetic affinity of Rajput population of
Himachal Pradesh with Rajputs of Madhya Pradesh and Yadav of Bihar. Overall, the tested 20 autosomal STRs were polymor-
phic in Rajput population and found suitable in forensic casework and population genetic studies.

Keywords PowerPlex® 21 System (Promega, USA) . Rajput population . Himachal Pradesh . Population genetic analysis . STRs .
DNATechnology (Use and Application) Regulation Bill, 2019

Situated in the core of Western Himalayas, Himachal Pradesh
is a state in northern India covering three Himalayan ranges,
i.e., The Outer Himalayan (Shivalik) range, Central
Himalayan range, and The Greater Himalayan range.
Accounting for 0.57% of India’s population, the total popula-
tion of the state is 6,864,602, among which 95.17% of indi-
viduals follow Hinduism. Brahmins, Rajputs, Kanets, Kolis,
and Rathis form the major sections of the Hindu society [1].
The Rajput populations residing in the state are the descen-
dants of downhill Rajputs immigrated to hill either due to
Muslim invaders or due to establishing a new princely state
Vivek Sahajpal and Abhishek Singh contributed equally to this work.
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00414-019-02198-8) contains supplementary
material, which is available to authorized users.
* Vivek Sahajpal
viveksahajpal@gmail.com
* Mukesh Thakur
thamukesh@gmail.com
1
State Forensic Science Laboratory, Directorate of Forensics Services,
Junga, Shimla, Himachal Pradesh 171218, India
2
Zoological Survey of India, New Alipore, Kolkata, West Bengal 700
053, India
3
Maharshi Dayanand University, Rohtak, India
[2]. Their distribution is throughout the northern, western, and
central India.
In the present study, a total of 179 genetically unrelated indi-
viduals (106 males and 73 females) were collected and brought
to the State Forensic Lab, Junga, Shimla, Himachal Pradesh. The
sample’s associated information was provided along with the
samples. Genomic DNA was extracted using DNeasy blood
and tissue kit (Qiagen, Germany). The 20 autosomal STR loci
and one sex identification marker were amplified in a total vol-
ume of 25 μl using the PowerPlex® 21 System (Promega, USA)
according to the manufacturer’s instruction. Both positive and
negative controls were used throughout the reactions. The sepa-
ration and detection of amplified products were carried out by
capillary electrophoresis using ABI 3130 Genetic Analyzer
(Applied Biosystems, USA). GeneMapper ID version 3.2
(Applied Biosystems, USA) was used for manual calling of al-
leles. Arlequin version 3.5 [3] was used to perform the exact test
for the Hardy–Weinberg equilibrium, and the STR analysis for
forensics (STRAF) online software [4] was used to generate the
allele frequency distribution table and forensic parameter values
for the 20 autosomal STR loci. GenAlEx 6.5 [5] was used to
generate genetic diversity characteristics such as observed het-
erozygosity (Ho), expected heterozygosity (He), and unbiased
heterozygosity (UHe). In addition, to assess the genetic distance
between the studied population and other previously reported 13
populations of India (Table S3), a UPGMA dendrogram was
constructed based on Dsw [6] distance obtained by the

POPTREE2 software [7]. Further to assess the clustering pattern
between the compared populations, principle component analy-
sis (PCA) was performed using allele frequency data in the
Paleontological Statistics (PAST) software version 3.02 [8].
The allele frequency distribution of Rajput population is
presented in Table S1. In total, 227 alleles with an average of
11.35 ± 0.89 alleles per locus were observed. After Bonferroni’s
correction [9] (α = 0.05, 0.05/20 = 0.0025), none of the locus
deviated from the Hardy–Weinberg equilibrium. No significant
variation in the mean heterozygosity estimates, i.e., observed
heterozygosity (Ho) (0.80 ± 0.01), expected heterozygosity
(He) (0.81 ± 0.01), and unbiased heterozygosity (UHe) (0.81
± 0.01), was observed. The evaluated forensic parameters such
as polymorphism information content (PIC), power of discrim-
ination (PD), power of exclusion (PE), paternity index (PI), and
probability of match (PM) are presented in Table S2. Locus
Penta E was found to be the most polymorphic and discrimina-
tive locus with 0.90 (PIC) and 0.98 (PD). The power of dis-
crimination ranged from 0.82 (TPOX) to 0.98 (Penta E), and
power of exclusion ranged from 0.37 (D5S818) to 0.83 (Penta
E). The combined power of discrimination and combined pow-
er of exclusion were 1 and 0.999999999, respectively. These
values suggest that the 20 autosomal STR loci were highly
polymorphic and can be used as a tool in forensic individual
identification and parentage testing. Further, the UPGMA den-
drogram based on Dsw genetic distance showed a close genetic
affinity of studied population with Rajputs of Madhya Pradesh
and Yadav of Bihar (Fig. S1). The PCA result of 14 populations
was consistent with the UPGMA dendrogram, showing the
genetic relatedness of Rajputs of Himachal Pradesh with other
populations of India (Fig. S2). In the context of the recent
introduced, “The DNA Technology (Use and Application)
Regulation Bill, 2019” passed in Lok Sabha by the Minister
of Science and Technology, Government of India, this study
aids in the formation of National DNA Data Bank and
Regional DNA Data Banks in each state. The Bill also assists
in the scientific upgradation of DNA testing laboratories in the
country. In conclusion, the 20 autosomal STR loci were poly-
morphic in Rajput population of Himachal Pradesh and can be
used in forensic analysis. This article follows the guidelines of
Int J Legal Med
the International Journal of Legal Medicine for the publication
of population data [10].
Acknowledgments We thank all individuals who generously donated
samples to undertake this study.
Compliance with ethical standards The study was conduct-
ed in compliance with the ethical standards. All the procedures prior to
their implementation were approved by the Ethical Committee of State
Forensic Science Laboratory, Directorate of Forensic Services, Shimla,
Himachal Pradesh.
Conflict of interest The authors declare that they have no conflict of
interest.
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