Genetic Diversity of Autosomal STR Markers in the Brahmin Population of Rajasthan and Haryana: Significance in Population and Forensic Genetics

The aim of the study is to evaluate the suitability of STRs for molecular characterization and forensic applications in unrelated Brahmins of Rajasthan and Haryana states, India. Materials and Methods A total of 203 male DNA samples from various districts of Haryana (n=104) and Rajasthan (n=99) were genotyped using the GlobalFiler® PCR Amplification Kit. Allelic frequencies and different forensic parameters like PD, PE, PIC, PM, Ho, He, UHe, and TPI were calculated with different software. Results More than 200 alleles were present in both populations, ranging from 6.0 to 35.2 and SE33 was the most polymorphic marker. The combined power of discrimination was 1. To know the relatedness with other Indian Brahmin populations, the UPGMA dendrogram and principal component analysis plot were visualized to show that both populations are close to each other and in nearby Saraswat Brahmins of Himachal Pradesh. This study showed a genetic relationship and forensic examination in the Haryana and Rajasthan Brahmin populations and various ethno-linguistically diverse populations of India. Conclusion The results imply that the highly polymorphic 21 autosomal STR loci might be applied for individuals’ forensic identification and parentage testing. This study also suggests that the kit having both autosomal and Y-STR markers is appropriate for a better understanding of the genetic and forensic examination in the Brahmin population of Haryana and Rajasthan.


Introduction
Genetic diversity is essential for the evolution of a species, and it offers the genetic make-up of a species based on the total number of gene distinctiveness features. Diversity reasonably determines population or human social organization in language, ethnicity, culture, geography, social aspect, and communities worldwide. These characteristics are common in the alpine environment, where people frequently live in separate communities far from developed areas [1]. Some traditions emerged due to the assimilation of varied cultures and others died over a period of time. On the other hand, certain cultures have remained steadfast and persistent in demonstrating their presence over time [2]. India has always welcomed people of many racial groups and ethnicities. According to various pieces of evidence, it is possible that modern people left Africa via the southern coastal route and India emerged as a vital human mobility corridor by the initial wave of migration along the south coast route [3]. The interaction and conversion of ideas may 64 СТМ ∫ 2023 ∫ vol. 15 ∫ No.1 clinical supplements have resulted in hybridization between populations of various ethnolinguistic categories in India that further resulted in cultural, linguistic, and genetic variation [4]. In 1919, Ludwik and Hanka Hirszfeld, pioneers in blood typing, first reported the genetic variation among human populations [5]. However, the variations studied in blood groups were insufficient to identify a particular individual from a gene pool. Thus, this area of interest was revolutionized by detecting variations in the human gene pool at the DNA level [6]. In the early days, restriction fragment length polymorphism analysis was employed for DNA analysis. Later on PCR-based assays (with increased sensitivity and power of discrimination) were used along with SNPs, VNTRs, and STRs. In 1990, Alec Jeffrey, with the Human Genome Project, created a wide range of STR sequences [7].
By the mid-1990s, multiple STR markers were added in single multiplexed reactions in forensic DNA testing. Several studies have been used with autosomal STR markers to investigate genetic relationships among Indian groups. The caste system in India is supported by four primary groups or varna: Brahmin, Kshatriya, Vaishya, and Sudra. The castes are further divided into sub-castes and individuals belonging to these sub-castes practice endogamy, a system in which an individual marries inside a specific group [8].
The aim of the study was to investigate the molecular characterization and forensic applications of Brahmins who practice endogamy i.e. they marry within the same caste, practice gotras (clan) system, and do not marry in the same gotras. . Twenty-one autosomal and three sex-determining markers were amplified in a total volume of 25 μl using the GlobalFiler ® PCR Amplification Kit (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. Throughout the reactions, positive and negative controls were employed. Capillary electrophoresis was used to separate and detect amplified products using an ABI 3500xL Genetic Analyzer (Applied Biosystem, USA). GeneMapper IDX version 1.6 (Applied Biosystem, USA) was utilized for manual allele calling.

Statistical data processing.
To calculate the allelic frequency distribution, as well as forensic parameters like polymorphism information content (PIC), probability of match (PM), typical paternity index (TPI), observed heterozygosity (Ho), expected heterozygosity (He), and unbiased heterozygosity (UHe) for 21 autosomal STR loci, STRAF (STR analysis for forensics) online software [11] and GenAlEx 6.5 were used [12]. Additionally, a UPGMA dendrogram [13] was generated based on the D sw distance determined by POPTREE2 software [14] to examine the genetic distance between the present study population and formerly observed 10 Brahmin groups of India. The principal component analysis (PCA) plot was also created with the help of allelic frequency data through Paleontological Statistics (PAST) software version 3.02 [15] to evaluate the grouping pattern between these populations.
Quality assurance. A positive control DNA template (2800 M), provided along with the kit, and negative controls were used throughout the study. The experimental work was carried out in an accredited laboratory conforming to ISO/IEC 17025 standard. Further, the authors have also qualified International DNA Proficiency Test (http://gitad.ugr.es/principal.htm).

Results and Discussion
In the Haryana Brahmin population, a total of 61 distinct alleles were detected in the Haryana Brahmin community, ranging from 6.0 to 34.2 and 229 alleles with an average of 10.409. Table 1 shows the allele frequency distribution among Haryana Brahmins. The observed allele frequency ranged from 0.005 to 0.452, showing allele 11 (0.452) as the most common in the study population at the D2S441 locus. The researchers discovered Ho as 0.760±0.040, He as 0.789±0.022, and UHe as 0.793±0.022. Table 2 lists the forensic parameters that were evaluated, including PIC, power of exclusion (PE), power of discrimination (PD), PM and TPI as well as number of alleles (Na), number of effective alleles (Ne), Ho, UHe, and He.  These results imply that 21 autosomal STR loci were more polymorphic to a greater extent and might be utilized for individuals' forensic identification and parentage testing.

Conclusion
This has been a pilot study to bring the genetic characteristics of the Haryana and Rajasthan Brahmin population together and demonstrating the utility of a panel of specific markers in the disputed forensic cases. The genetic data from the present study could be helpful in population genetics and forensics. We suggest further extending this study encompassing a greater more number of samples. It can be conducted by using the representative samples belonging to varied castes/ ethnic groups while keeping the immigration routes of the human population in Haryana and Rajasthan states under consideration. In addition, the fine scale understanding of population assignment can be aided by application of genomic data using next generation sequencing. In addition, based on the DNA technology, Regulation bill was enacted by the Ministry of Science and Technology of the Indian Government in Lok Sabha (2019). The presently conducted study would aid in the development of DNA Data Banks at national, as well as state, levels.