Participants and Sample collection
This study was conducted in compliance with ethical standards and was approved by the Institutional Ethical Committee of the Jaipur National University, Jaipur, Rajasthan, India, vide letter no. JNUMSRC/IEC/2018/45 dated 20.07.2018. The peripheral blood samples were collected from randomly selected 669 unrelated healthy adult individuals from the various populations of Rajasthan, namely; Mina tribe (n=202), Gujjar (n=150) and mixed population of Rajasthan (n=317) after obtaining written informed consent following the declaration of Helsinki. The samples of Mina and Gujjar population were collected from the central and south-eastern parts of Rajasthan. The mixed population of Rajasthan was sampled from almost the whole state [Fig.1].
DNA Extraction and Genotyping
The samples were directly amplified without DNA extraction using PowerPlex® 21 multiplex system (Promega, CA, USA) as per recommendations of the manufacturer's except that the 10 µl reaction volume was used. The direct amplification protocol was initially standardized and the profiles generated by the direct method were compared with the DNA profiles obtained from the extracted DNA using standard organic extraction method Phenol Chloroform Isoamyl Alcohol (PCIA) as described earlier . The Amplified DNA fragments were analyzed by using ABI 3500XL Genetic Analyzer using 36 cm capillary and POPTM - 4 (Thermo Fisher Scientific, USA-Thermo). Allelic ladder provided along with kit was used to obtain the DNA profiles. Data was analyzed using GeneMapperTM ID-X Software Version 1.5 (Thermo). The peak height threshold was 50 and 200 Relative Fluorescence Unit (RFU) for the heterozygous and homozygous allele respectively.
Obtained data was statistically analyzed. Allele frequency and observed heterozygosity were calculated by using GenAlEx 6.5 software . Arlequin v3.5 software was used for calculation of Observed Heterozygosity (Hobs), Expected Heterozygosity (Hexp) and Hardy Weinberg Equilibrium (HWE) and AMOVA. Further, Arlequin v3.5 software was also used for comparison of allele frequencies of the studied population data with the previously published population data by Fst pairwise distance. The previously reported populations, namely; Kahar Population (Uttar Pradesh) , Populations of Uttar Pradesh, Khatri (Punjab) , Baniya (Punjab), Jatt Sikh (Punjab), Population of Jharkhand, Oraon (Chhattishgarh) , Gond-1 (Madhya Pradesh) , Gond-2 (Madhya Pradesh), Gond (Madhya Pradesh) , Central Indian Populations , Bhil (Gujrat) , Bhil (Madhya Pradesh) , Maheli (Bengal) , Kora (Bengal) , Lodha (Bengal) , Oraon (Chotanagpur) , Santhal (Chotanagpur) , Munda (Chotanagpur) , Yerukula (Andhra Pradesh) , Naikpod Gond (Andhra Pradesh) , Lambadi (Andhra Pradesh) , Chenchu (Andhra Pradesh) , Munda (Jharkhand) , Kurmans (Tamilnadu), Iyengar (Tamilnadu) , Mahadev Koli (Maharashtra) , Konkanastha Brahmin (Maharashtra) , Sakaldwipi Brahmin (Jharkhand) , Balmiki (Punjab) , Population of Madhya Pradesh , Indian Population , Populations of Bangladesh , Han population of Southern China, Tibetan population (Nepal) , Royal Kingdom of Bhutan Population , Kashmiri Muslim population living in Pakistan , Pakistani population , Pakistani groups (Balochi, Brahui, Sindhi, Pathan, and Barusho), were used to explore the genetic diversity and affinity with the studied Mina, Gujjar and mixed population of Rajasthan. Based on Nei’s genetic distances , Neighbour-joining (NJ) dendrogram was drawn using by using POPTREE2 software with bootstrap value of 1000 replications over loci and viewed in MEGA v6 software . Graphical representation of genetic distances (Dst) of studied populations with other reported populations was also performed based on Principal Component Analysis (PCA) plot generated by PAST 3.02a software. Our studied population data and the data set of previously reported Pakistani population, East Asian population and Indian populations were used for STRUCTURE software, from k=2 to k=10 performed by 20 iterations after a burn-in of 100000 and selected the k (k=5) by evaluation of the ∆k and estimated log-probability data statistics.
Quality control standards for this study were followed. The authors have passed the proficiency test of GITAD, Spain (http://gitad.ugr.es). This research article follows the guidelines formulated by the BMC Genetics.