33 (2022) 201038

Contents lists available at ScienceDirect

Human Gene
journal homepage: www.journals.elsevier.com/human-gene

Revealing genetic structure in the paternal lineages and forensic profiling of

Bhotra tribe by the analysis of Y-STR multiplex platform
MuktikantaPanda a, c, 1, Awdhesh Narayan Sharma a, R.K. Kumawat b, Pankaj Shrivastava a, c, *, 1
a
Department of Anthropology, Dr. Harisingh Gour Vishwavidyalaya (A Central University), Sagar 470003, Madhya Pradesh, India
b
State Forensic Science Laboratory, Jaipur 302016, Rajasthan, India
c
DNA Fingerprinting Unit, State Forensic Science Laboratory, Department of Home (Police), Govt. of MP, Sagar 470001, Madhya Pradesh, India

A R T I C L E I N F O A B S T R A C T

Keywords: India has a diverse reservoir of genetic components, ethnicity and linguistic affiliations. Y-STR (Short Tandem
Y-STR Repeat) markers serve as a potential tool in the fields of anthropological genetics, ancestry exploration, as well as
Bhotra in forensic investigations. The present study was conducted to explore the molecular diversity in respect to male
Odisha
lineage, genomic heritage and forensic profiling of Bhotra tribe of Odisha, India, by using 23 Y-STR molecular
Forensic
Genetic diversity
markers. Blood samples were collected randomly from 133 unrelated healthy adult male individuals of Bhotra
population following the ethical standards.Out of 133 individuals, 109 unique haplotypes were observed.
Discrimination Capacity (DC) was observed at 0.8195; mean Gene Diversity (GD) was observed to be 0.615 along
with the Standard Error (SE) of 0.027. The locus DYS385a/b showed the highest genetic diversity (0.887). To
assess the genetic affinity of the Bhotra population with other Indian population that were reported to Y-STR
Haplotype Reference Database (YHRD) before, Multidimensional Scaling plot (MDS), Neighbor Joining (NJ) tree,
and Principal Coordinate Analysis (PCoA) were performed. The study population exhibited a high degree of
genetic diversity and indicated genetic affinity with the Majhi and Dorla tribes of Chhattisgarh. Study population
characterized by 12 different haplogroups and H1a1a (33.03%) was the most frequently observed haplogroup,
indicating the ancestral relationship of Bhotra with the European Romani population.To the best of our
knowledge, this is the first global report of the genetic diversity of the Bhotra tribe on 23 Y STR genetic markers,
which would be profoundly useful in forensic investigation, medical, and anthropological studies and would also
enrich the Indian Y-STR database.

1. Introduction
Microsatellites, also named as Y Chromosome Short Tandem Repeats
(Y-STRs), are the tandemly repeated tiny DNA sequences (2–6 bp)
positioned on the Y chromosome(Ellegren, 2004; Jobling and Gill, 2004;
Kayser and De Knijff, 2011). Structuring of the Y-STRs based on the
paternal inheritance and nature of the human population, makes them
suitable markers which could be extensively used for human evolution,
migration tracing, population history reconstruction, forensic in­
vestigations, genetic genealogy and male specific epidemiological
studies(Calafell and Larmuseau, 2017; Jobling and Tyler-Smith, 2003,
2017; Karafet et al., 2008; Kayser, 2007; Underhill and Kivisild, 2007).
The Y-Chromosomal study has also been implicated in the areas like the
sex specific migration between communal sets (social groups), corns
associated with different male behaviour (rules of marriage alliance and
male dominancy) of a specified stocks in the past context(Heyer et al.,
2012).
Complex geo-climatic setup of the Indian subcontinent is appropriate
forthe major diversity among the existing ethnic stocks, and hence there
exists a scope for the population geneticists as well as the anthropolo­
gists for the genomic investigations. Different anthropological and ge­
netic studies indicated toward the multifaceted historical context of the
population of the Indian subcontinent stimulated by the inward
genomicstream from varied ancestral worldwide populaces. Therefore,
the Indian landmass is considered the receiver and the donor of genetic
constituentsfromdifferentpools (Cavalli-Sforza et al., 1994; Chaubey
et al., 2007; Majumder, 2010; Thapar, 2002; Trivedi et al., 2008).
Various genomic investigations have been executed among the different

* Corresponding author at: Department of Anthropology, Dr. Harisingh Gour Vishwavidyalaya (A Central University), Sagar 470003, Madhya Pradesh, India.
E-mail addresses: pankaj.shrivastava@rediffmail.com, ecsdsjbp@gmail.com (P. Shrivastava).
1
Author with equal credential.

https://doi.org/10.1016/j.humgen.2022.201038
Received 11 October 2021; Received in revised form 26 December 2021; Accepted 27 April 2022
Available online 10 May 2022
2773-0441/© 2022 Elsevier B.V. All rights reserved.
MuktikantaPanda et al.
Fig. 1. Geographical location of study area selected for this study.
Indian populations at diverse geography in reference to the variety of
parentage (patrilineal and matrilineal) and the autosomal molecular
markers(Kumawat et al., 2020; Perera et al., 2021; Shrivastava et al.,
2017; Sreekumar et al., 2020).
Herein, Bhotra, a distinct endogamous tribe, restricted to the KBK
region of the southern Odisha and the Bastar region of Chhattisgarh
states of the Indian republic(Bhottada, 2014) was selected for the pre­
sent study. The area is known for the homeland of the differ­
entindigenous populations, enriched by the river Indravati and its
tributaries. Historically, the regioncame under the earliestfamous Indian
kingdoms like Atavika Rajya, part of Kalinga during 3rd-4th century B.C.
and afterwards was ruled by the Satabahan empires, Nala Dynasty,
Kakatiya and Suryavansi rulers in the Christian era till the British rule
(NIC-Bastar, 2021; NIC-Nabarangpur, 2021). Bhotra tribe is character­
ized as a major indigenous tribe in Odisha state, having a population of
4,50,771 and the existing social hierarchy is represented in three sec­
tions, viz. Sana, Madhya and Bada (Bhottada, 2014; Tandon, 1959).
Traditionally, the males enjoy domination over females, and the nuclear
family system exists in this tribe. Occupationally, they depend on settled
agriculture with seasonal forest assemblages. Bharti, a semi-autonomous
languageaffiliated to the southern branch of Indo-Aryan language (Indo-
European language family), is spoken by the people of this tribe, which
is deprived of any script (Bhottada, 2014; Das et al., 1996). Originally,
the Bhotra were the native of Warangal region of undivided Andhra
Pradesh state of the Indian republic, and migrated and steadily settled
over Bastar region of Chhattisgarh and undivided Koraput region of
Southern Odisha(Das et al., 1996; Russell and Hiralal, 1916).
2. Materials and methods
2.1. Selection of study area
We selected Nabarangpur district (Fig. 1) located in the South part of
Odisha, India, for this study. Of the total Bhotra population in the Odisha
and Chhattisgarh states, 48.96% lives in the Nabarangpur district of
Odisha alone. The selected district is approximately located inthe Cen­
tral part of the Bhotra dominated area of Odisha and Chhattisgarh states.
It is considered as the epicentre for the interstate migration between
Odisha and Chhattisgarh in the context of the marriage alliances, hence
preferably considered in the present study.
2
Human Gene 33 (2022) 201038
2.2. Sample collection, DNA extraction and quantification
In this study, 133 unrelated healthy adult male individuals of Bhotra
tribe were randomly selected. The blood samples of the selected in­
dividuals were collected after obtaining written informed consent and
following the ethical guidelines and Declaration of Helsinki(Rickham,
1964).DNA was extracted using Phenol Chloroform Isoamyl Alcohol
(PCIA) organic extraction method from the collected blood samples
(Sambrook et al., 1989).For each sample, 500 μl of blood from stored
EDTA vile was taken for DNA extraction, achieved by steps like cell lysis
and protein digestion, extraction with organic solvents and concen­
trating the DNA. The extracted DNA was dissolved in TE buffer and
stored at − 800 for subsequent processing.
Using the PowerQuant® system (Promega, CA, USA-Promega), the
quality and quantity of isolated DNA wereassessedon the RT-PCR 7500
(Thermo Fisher Scientific, CA, USA-Thermo) system by following the
manufacturer's protocol.The PowerQuant® System is a 4-target probe-
based and 5-coloured, qPCR assay that quantifies the entire quantity
of amplifiable Y-chromosomal and autosomal DNA in a single assay.
DNA samples were diluted to a concentration of 1 ng/1 μl according to
the qPCR results for subsequent PCR amplification.
2.3. Y-STR marker's amplification and genotyping
23 Y-STR markers (DYS391,DYS576, DYS389I, DYS448, DYS389II,
DYS458, DYS19, DYS481, DYS549, DYS533, DYS438, DYS437, DYS570,
DYS635, DYS439, DYS392, DYS643, DYS390,DYS393, DYS456,
DYS385a/b and Y-GATA-H4) of each individual sample were amplified
using 5 dye-based PowerPlex® Y-23 System kit (Promega, Madison, WI,
USA) on Verity™ fast thermal cycler (Thermo) according to the rec­
ommendations of the manufacturer. Amplified DNA fragments were
alienated through the capillary electrophoresis (CE) in Genetic Analyzer
ABI 3500 XL (Thermo). The acquired data was analyzed in Gene­
Mapper®HID software version 1.6 (Thermo Fisher Scientific, Waltham,
MA, USA). The control DNA (2800 M) was used as the positive control.
The Y- STR haplotype data of this study was submitted to the YHRD
marked as Bhotra, Population, Odisha, India, and the accession number
obtained was YA004674. The submitted data hasbeen released in R63
series of YHRD (http://www.yhrd.org).
 MuktikantaPanda et al.
Table 1
Allele frequencies and gene diversity values for the 23 Y-STR loci in Bhotra population of Odisha, India (n = 133).
DYS576 DYS389I DYS448 DYS389II DYS19 DYS391 DYS481 DYS549 DYS385a/b

GD = 0.743 GD = 0.642 GD = 0.590 GD = 0.734 GD = 0.523 GD = 0.521 GD = 0.596 GD = 0.503 GD = 0.887

Allele Frequency Allele Frequency Allele Frequency Allele Frequency Allele Frequency Allele Frequency Allele Frequency Allele Frequency Allele Frequency

15 0.008 11 0.008 18 0.526 26 0.008 13 0.075 9 0.008 20 0.008 11 0.053 10,14 0.008
16 0.098 12 0.203 19 0.361 27 0.015 14 0.030 10 0.549 21 0.023 12 0.662 11,14 0.098
17 0.241 13 0.444 20 0.113 28 0.173 15 0.639 11 0.421 22 0.038 13 0.233 13,15 0.015
18 0.383 14 0.346 29 0.301 16 0.248 13 0.023 23 0.594 14 0.038 13,16 0.008
19 0.195 30 0.353 17 0.008 24 0.203 15 0.015 13,17 0.038
20 0.068 31 0.143 25 0.068 13,18 0.068
21 0.008 32 0.008 26 0.008 13,19 0.015
27 0.060 14,16 0.090
14,17 0.015
DYS533 DYS438 DYS437 DYS570 DYS635 DYS390 DYS439 DYS392 15,15 0.008
GD = 0.576 GD = 0.548 GD = 0.249 GD = 0.810 GD = 0.749 GD = 0.698 GD = 0.731 GD = 0.487 15,16 0.248
Allele Frequency Allele Frequency Allele Frequency Allele Frequency Allele Frequency Allele Frequency Allele Frequency Allele Frequency 15,17 0.068
10 0.045 9 0.278 13 0.008 15 0.045 19 0.008 21 0.338 10 0.135 10 0.023 15,18 0.015
3

11 0.353 10 0.602 14 0.857 16 0.188 20 0.241 22 0.316 11 0.301 11 0.662 15,19 0.023
12 0.541 11 0.113 15 0.128 17 0.188 21 0.353 23 0.023 12 0.331 12 0.015 15,20 0.128
13 0.060 14 0.008 16 0.008 18 0.233 22 0.083 24 0.030 13 0.226 13 0.271 15,21 0.038
19 0.211 23 0.233 25 0.293 14 0.008 14 0.030 16,16 0.023
20 0.135 24 0.083 16,17 0.045
16,18 0.023
DYS643 DYS393 DYS458 DYS456 YGATA H4 17,17 0.015
GD = 0.587 GD = 0.673 GD = 0.692 GD = 0.485 GD = 0.546 17,18 0.008
Allele Frequency Allele Frequency Allele Frequency Allele Frequency Allele Frequency 18,21 0.008
9 0.113 11 0.030 15 0.038 13 0.015 10 0.015
10 0.301 12 0.218 16 0.173 14 0.038 11 0.338
11 0.023 13 0.286 17 0.406 15 0.677 12 0.579
12 0.556 14 0.444 18 0.331 16 0.233 13 0.068
13 0.008 15 0.023 19 0.053 17 0.023
18 0.008
20 0.008

The table shows allele frequencies for each investigated locus except for DYS385a/b, for which genotype frequencies were calculated for the combination of the two alleles. GD = gene Diversity, Major allele frequencies
per locus are in bold.

Human Gene 33 (2022) 201038

MuktikantaPanda et al.
Fig. 2. Gene Diversity (GD) at 23 Y-STR lociin Bhotra population of Odisha, India.
2.4. Statistical analyses
The quality of the DNA profile was estimated by observing param­
eters like the interlocus peak height ratio and balanced DNA profile.
Genomic data was analyzed for different descriptive statistics like the
Hardy-Weinberg Equilibrium (HWE), Gene diversity (GD)and allele
frequencies of the studied population. Genetic Diversity (GD) is thede­
∑
gree of polymorphism at a certain locus calculated as GD = 1- pi2, (pi is
the allele frequency)(Dogan et al., 2014). GenAlEx 6.0 software was
used for allele and haplotype frequency calculation(Peakall and Smouse,
2006). Y- Chromosome STR Haplotype Reference Database (YHRD), an
online tool (http://www.yhrd.org) was used for the analysis of Multi­
dimensional Scaling (MDS) to infer the genetic affinity among different
populations; Molecular Variance (AMOVA) based on Rst and associated
probability values (p-values) among the studied and other compared
populations with 10,000 permutations.An online tool NevGen hap­
logroup predictor (https://www.nevgen.org) was used to allocate the
halpogroups for each respective Y-STR profile. Principal Component
Analysis (PCA) plot and construction Neighboring-Joining (NJ) tree
were performed using PAST software(Hammer et al., 2001). The
discrimination capacity (DC) was calculated by the formula: DC = h/n
(h = different haplotypes observed in the population and n = sample
size).
3. Results and discussion
3.1. Genetic diversity
The allele frequency distribution and locus wise Gene Diversity (GD)
of the studied population at 23 Y- STR loci have been shown in Table 1
and Fig. 2. In the studied population, among 23 Y STR loci, a total of 124
alleles were observed, with the mean allele number of 5.39 per locus.
Out of 133 individuals, one hundred nine unique haplotypes were
observed (Table S1). The Discrimination capacity (DC) was calculated
using the formula DC = h/n, where ‘h’ is the number of observed hap­
lotypes and ‘n’ is the number of samples. The DC for the studied popu­
lation was found to be 0.8195.
3.2. Genomic affinity of the study population
The Analysis of the molecular variance (AMOVA) pairwise distances
4
Human Gene 33 (2022) 201038
based on Rst values between the studied population and selected pop­
ulations used for comparison from YHRD hasbeen shown in Table 2.To
reveal the genetic affinity of the studied population with the Indian
population reported previously in Y Chromosome Haplotype Reference
Database, Multidimensional Scaling plot (MDS) on pairwise Rst based
genetic distance was performed(Fig. 3.). Principal Coordinate Analysis
(PCoA) (Fig. 4.) and pairwise Rst genetic distance based Neighbour­
Joining tree (Fig. 5.) were performed, and findings of these genetic
analyses showed consistency to each other. All these analyses indicated
that the overall Bhotra population showed ahigh degree of genetic di­
versity, and it showed the genetic affinity with the Majhi and Dorla
population of Chhattisgarh state. The Indian population previously re­
ported from Uttar Pradesh, India has a very distant affinity with the
presently studied Bhotra population.
3.3. Haplogroup diversity
Out of the 109 unique haplotypes, 107 were assigned to different
haplogroups, whereas 2 haplotypes had not been assigned to any hap­
logroups. The Bhotra population was characterized by total 12 hap­
logroups viz. C1, D1b1b, E1b1a, E1b1b, G2a2a-Major, H1a1a, J2b2a,
L1a, O1b1, R1a, R2 and T (Fig. 6.1 and 6.2). The 4most frequently
observed haplogroupswere H1a1a (33.03%), O1b1 (23.85%), R1a
(9.17%) and T (8.26%), those together contributed ~75%.
HaplogroupH1a1a is commonly found in the Indian castes and tribes
as well in Romanian populations(Rai et al., 2012). This results indicated
a past hereditary relationship of Bhotra with Romanians and other
inland populations of Indian subcontinent.A previous study concluded
about the Indian origin of the European Romani populations(Rai et al.,
2012). This previous data was found in coherence withour present re­
sults.The haplogroup R1a has been seen as the most frequently repre­
sented haplogroup associated withEurasia (Karafet et al., 2008; Trivedi
et al., 2008), whereas in the Bhotra population, it is represented as the
3rd most frequent haplogroup. A previous study reported that the hap­
logroup frequency of R1a decreased from the North to South direction in
the Indian continent (Singh et al., 2018). Findings of Sharma et al.
indicated that subclades of haplogroup R1a, i.e. R1a1 is present as a
founder lineage among Brahmin caste group as well as directed toward
the tribal link to Indian Brahmins(Sharma et al., 2009). Overall the
haplogroup analysis indicated that the Bhotra male lineage is associated
to multi-ethnic groups during its origin and evolution.
 MuktikantaPanda et al.
Table 2
Analysis of molecular variance pairwise distances based on Rst values between studied population (Bhotra) and selected populations used for comparison from YHRD.
Population Odisha, Chhattisgarh, Andhra Assam, Jharkhand, Madhya Maharashtra, Rajasthan, Uttar West Uttar Chhattisgarh, Chhattisgarh, Uttar
India India [Dorla] Pradesh, India India Pradesh, India [Indian] India Pradesh, Bengal, Pradesh, India [Majhi] India [Muria] Pradesh,
[Bhotra] India [Indian] [Indian] India [Indian] India India India India
[Indian] [Indian] [Indian] [Indian] [Kahar] [Tharu]

Odisha, India * 0.001 0.0006 0.0001 0 0 0 0 0 0 0 0.0005 0.0091 0

[Bhotra]
Chhattisgarh, 0.0423 * 0 0 0 0 0 0 0 0 0 0 0.0011 0
India [Dorla]
Andhra 0.0569 0.1173 * 0.0563 0.2001 0.0469 0.0779 0.0035 0 0.044 0.0954 0.0022 0.0661 0.0905
Pradesh,
India
[Indian]
Assam,India 0.1018 0.1543 0.0329 * 0.1146 0.044 0.0539 0.0288 0 0.0613 0.0687 0.0003 0.0134 0.6489
[Indian]
Jharkhand, 0.1253 0.1981 0.0107 0.0266 * 0.3953 0.4126 0.0306 0.0001 0.3041 0.5406 0 0.0126 0.0728
India
[Indian]
Madhya 0.1058 0.1311 0.0179 0.0255 − 0.0002 * 0.6297 0 0 0.0822 0.0327 0 0.0003 0.0035
Pradesh,
India
[Indian]
Maharashtra, 0.1633 0.2023 0.0238 0.0382 − 0.0013 − 0.0042 * 0.0401 0.001 0.1354 0.0453 0 0.0001 0.0316
5

India
[Indian]
Rajasthan, 0.1359 0.1892 0.0414 0.0314 0.0251 0.0208 0.0192 * 0 0.5776 0 0 0 0.0027
India
[Indian]
Uttar Pradesh, 0.2505 0.2417 0.1357 0.1612 0.0949 0.0751 0.0504 0.0949 * 0 0 0 0 0
India
[Indian]
West Bengal, 0.1665 0.2468 0.0368 0.0419 0.0049 0.0187 0.0201 − 0.0051 0.1051 * 0.0857 0 0.0013 0.0334
India
[Indian]
Uttar Pradesh, 0.0927 0.1359 0.0142 0.0251 − 0.0043 0.0105 0.022 0.0443 0.1333 0.0227 * 0 0.008 0.0169
India
[Kahar]
Chhattisgarh, 0.0302 0.1107 0.0515 0.1006 0.102 0.1197 0.1608 0.1515 0.2759 0.1458 0.0783 * 0.0005 0
India
[Majhi]
Chhattisgarh, 0.0215 0.0503 0.0187 0.0449 0.0423 0.0385 0.0709 0.0752 0.1646 0.0761 0.0252 0.0376 * 0.0004
India
[Muria]

Human Gene 33 (2022) 201038

Uttar Pradesh, 0.0923 0.1405 0.0162 − 0.0085 0.0213 0.0234 0.0286 0.026 0.14 0.0362 0.0214 0.0959 0.0485 *
India
[Tharu]
*
p values are shown above the diagonal and Rst values below it.
MuktikantaPanda et al.
Fig. 3. Multi-dimensional scaling (MDS) plot showing Y-STR based relationship between studied (Bhotra) and compared populations based on pairwise Rst values.
Fig. 4. Principal Coordinate Analysis (PCoA) showing Y-STR based relationship between studied (Bhotra) and compared populations based on pairwise Rst values.
3.4. Forensic application
The studied 23 Y–STR markers hold significance in forensic in­
vestigations. The discrimination capacity (DC)with a high value of
0.8195 makes it ideal for forensic genetic application. Details of the
haplotype allele frequency of the Bhotra population have beenmen­
tioned in Table-1. From this study, it could be predicted that the dis­
tribution of the alleles of 23Y-STR markers merged in the PowerPlex® Y-
23 kit is highly helpful and useful for various forensic application
perspectives.
4. Conclusion
Here we studied the Bhotra population hoped to fill the gap in the
genetic survey in the Indian subcontinent with reference to Y-STR
markers. Overall, the Bhotra population showed ahigh degree of genetic
diversity with the Indian population of Uttar Pradesh and showed the
genetic affinity with the Majhi and Dorla population of Chhattisgarh.But
linguistically, the Majhi tribe and Dorla tribe are affiliated with the Indo-
European and Dravidian language groups, respectively. Here the genetic
6
Human Gene 33 (2022) 201038
affinity doesn't unequivocally trail the linguistic affiliation. The hap­
logroup analysis predicts that the Bhotra population shares its ancient
ancestry closely with the European Romani population. Interestingly,
the Bhatri language of the Bhotra tribe and Romani language of the
Romani population belongs to the Indo-Aryan branch of Indo-Europian
language family, depicting the linguistic connection among them in the
past.The genetic data of the studied population will add to the potential
tools to uncover the studied population's genetic ancestry and potential
use in forensic investigations and may enrich the existing Indian popu­
lation Y-STR database.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.humgen.2022.201038.
Ethical statement
The present study protocol was approved by the Institutional Ethics
Committee, Dr. H. S. Gour Vishwavidyalaya, Sagar-470003, (M.P.) vide
letter No. DHSGV/IEC/2021/19/1 of Dated 10/08/2021.
MuktikantaPanda et al. Human Gene 33 (2022) 201038

Fig. 5. Rectangular Neighbor-Joining tree showing relationship between studied and compared population based on a distance matrix of Rst.

Fig. 6.1. Haplotype Distribution in the Study Population.

Author's contribution. agencies in the public, commercial, or not-for-profit sectors.

MKP and PS designed the study;MKP collected the samples;MKP and Informed consent
PS did analytical work;MKP and RK did statistical analysis; MKP pre­
pared the manuscript; PS and ANS reviewed the manuscript. All authors This study was conducted with written informed consent following
read and approved the final manuscript. the declaration of Helsinki.

Funding sources
Declaration of Competing Interest
This research did not receive any specific grant from funding
The authors declare that they have no conflict of interest in the

7
MuktikantaPanda et al. Human Gene 33 (2022) 201038

Fig. 6.2. Subclades Wise Distribution of Major Haplogroups in the Study Population.

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