Thesis report on Genetic study of Bhumij Tribe

Thesis Report On Genetic Study of Bhumij Tribe of Jharkhand using mitochondrial and Y chromosomal DNA markers

A thesis submitted in partial fulfillment of the requirements of the degree of Masters of Science in Biotechnology By: Smita Bernadet Kujur of Loyola College.

Work done at CCMB

ACKNOWLEDGEMENT I am heartily thankful to Dr. Ch Mohan Rao, Director of CCMB, Hyderabad, India for granting me his kind permission to work in CCMB. I am highly obliged to him for providing me with all the excellent facilities, rich source of knowledge and a healthy competitive environment.

With a deep sense of veneration and obligation to Dr. K.Thangaraj, Scientist , CCMB, Hyderabad, India, whose encouragement, guidance and support from the initial to the final level enabled me to develop an understanding of the subject.

I thank my co-guide Miss. Sakshi Singh [JRF], for her continuous support in my dissertation program. She was always there to listen and to give advice.She taught me how to express my ideas. She showed me different ways to approach a research problem and the need to be persistent to accomplish any goal. She guided me professionally as well as personally. She helped me day and night with my thesis preparation.May God bless her n may she come out with flying colours. I wish her all the success in her life ahead.Thank you maam.

I owe my sincere thanks to my Head of Department of Plant Biology and Biotechnology, Dr. Agastian. S. Theoder M.Sc., M.Phil., Ph.D. ( Head of the Department ), my department co-ordinator Mrs. Mary Dorothy Anitha Sebastian(SET) M.Sc. , my guide Ms. D. Jacintha Jasmine, staff of my department Ms. P. Margaret Sangeetha M.Sc., M.Phil, Dr. (Ms). Shirly George Panicker M.Sc. (Agri) Ph.D, Ms. Sally Gloriana M.Sc.,M.Phil, Ms. D. Jacintha Jasmine, M.Sc., M.Phil, Mr. Preetam, Mr. Victor.I am greatly indebted to them for their encouragement.

I thank Mr. A.Govardhan Reddy [Technical officer], Mr. Surya Narayan ,Mr. Rakesh Tamang [Proj. Asstt], Aditya Nath Jha [JRF], Sharath Anugula [Proj. Asstt], Mr. Nizam , Mr. Haneef. They were always available listen and talk about my ideas, to provide understanding, provide reagents and mark up my papers and chapters, and to ask me good questions to help me think through my problems (whether philosophical, analytical or computational).

Last but not the least, I owe special thanks to my respected father Mr. Srimanim Belkhas Kujur, my mother Mrs. Susheela Kujur, Mrs. Goretti, Mrs. Milaani Kullu, , Mr. Ananta Lal Tudu, , Mr. N.P.C Sardar, , Dr. Daisy , Mr. Surya Narayan ,Mr. Kuldip Khalkho, Mrs. Bina, Mr. Bhujang, they all helped me volunteerilly.

That’s not all. People with great personalities took out time from their busy schedule and helped me in blood sample collection. I am immense pleased to introduce to you a very honored person Shree. Dr Balram Singh Sardar (BISM), (village – Tirildih , Dist West Singbhum, Jharkhand) is a renowned

and poplar doctor from the Bhumij Community. He is holding the post of Secretary in the OYON AKHRA (in their language) which is the Central Executive Body of AADIM BHUMIJ MUNDA SAMAJ KAYYAN SAMITI. Shri. Subodh Singh Sardar, (village – Bhatin, Dist West Singbhum, Jharkhand) is popular Congress party leader. He contested the 2009 Assembly election of Jharkhand from Congress ticket. He is a Graduate.

Gunadhar Singh Sardar, (village – Gitilata, Dist West Singbhum, Jharkhand) is renowned social worker and community leader. He is one of the Trustee member of AADIM BHUMIJ MUNDA SAMAJ KALYAN SAMITI and Ex-Secretary. Presently he is one of the Advisor to the Samiti.

He is a Graduate.

Niranjan Singh Sardar (village – Tirildih, Dist West Singbhum, Jharkhand) is the NYA (in their language) that means Community Priest. He is also PRADHAN (village Head Man) of Tirildih village.

Amulya Singh Sardar (village – Bunudih, Dist West Singbhum, Jharkhand) is a renowned and veteran politician of Jharkhand Mukti Morcha (JMM).

He is Ex-MLA of Jharkhand Assembly. He is also the Secretary of Bunudih Branch of AADIM BHUMIJ MUNDA SAMAJ KALYAN SAMITI. Shatrudhan Singh Sardar (villge – Tentla, Dist West Singbhum, Jharkhand) is prominent distinguished member of Bhumij community. He holds the post of President of OYON AKHRA (in their language) which is the Central Executive Body of AADIM BHUMIJ MUNDA SAMAJ KALYAN SAMITI. He is just a matric but very active in social activities.

Miss. Mona Bhumij (village – Ghaghidih, Dist West Singbhum, Jharkhand) is the daughter of a retired TISCO employee Mr. Ghasiram Bhumij. She has all round qualitative skills. As a brilliant student she is doing her PG in Economics from Women’s Collge Jamshedpur. She is a talented sports woman and athlete participated at national level events of Hand Ball, Kabbaddi and Javelin Throw and won Medals. Her social and community life is also full of self service activities as she organizes classes for the children as well as grown ups under SARVSHIKSHA ABHIYAN ( a educational scheme of the govt.) from the Govt. Primary School, Ghaghidih as the centre. Miss Mona is presently the treasurer of the local Committee of this educational scheme. Besides this, she imparts tuitions to the local children free of cost. Last but not least, she is very much fond of gardening flowers, singing and listening music as extra curricular activities. She took lot of pain and cooperated to help me in collecting the blood samples by arranging meetings and convincing people to come forward for giving blood samples.

Mrs. Daisy is a local Senior Nurse working in the Govt. Health Centers located in the village habitats of majority Bhumij tribe. She is well known and gracefully respected in the Bhumij community. Another compounder, Mr. Surya Narayan also cooperated along with Mrs. Daisy. They also arranged the permission of the respected authorities an allowed to use the room of health Centre for collection of Blood samples.

Collection of blood was a very tedious job . People of this community are very superstitious and orthodox. The people named above helped me both directly and in directly in convincing the villagers to co-operate and gather at a arranged venue. All of them contributed in helping CCMB in there population genetic research. Thank you a lot for supporting me.

I offer my regards and blessings to all my friends of those who supported me in any respect during the completion of the project ,“ Joe Zacharias, Manju Kashyap, Vinee Khanna, Sapna Narvariya, Dheepa Kaliyaperumal, Devi.K ,Manimaran .M., Vidya, Anushah, Hema, Shobna, Pavitra, Richa, Satrupa,Upasna Saranji, Gayitri. They all are a blessing in my life.

Their presence made the CCMB Lab a wonderful workplace and home for the 6 months by indulging my ever expanding bookshelf space and computer equipment needs. Also thanks to the folks at the CCMB Lab for interesting discussions and being fun to be with.

Thanks, friends !

May God be there to support and guide you always in any form you as you were there for me. I express my heartiest gratitude to all of them for being a source of inexhaustible encouragement, unconditional love and inspiration to build up my educational career. Their influence is all over these pages and will stay all through my days to come.

Smita Bernadet Kujur

CONTENTS

Abstract Acknowledgement Chapter I: 1.1 1.2 1.3 1.4 1.5

Introduction to the Study Introduction Background Statement of Purpose Aims and Objectives of Study Hypothesis

Chapter II: Review of the Literature 2.1 Introduction of Bhumij Tribe Chapter III: 3.1 3.2 3.3 3.4 3.5 3.6

Methodology Sampling Materials and Method Purpose of Study Precautions The Research Site Data Collection

Chapter IV: Analysis of Results 4.1 Discussion of Results 4.2.1 Y-chromosomal Analysis 4.2.2 Mitochondrial DNA Analysis Chapter V : Summary and Conclusion 4.3 Summary and Conclusion

Terms Bibliography

LIST OF FIGURES Fig 1 : Human Mitochondrial DNA Fig 2 : Map of human haplotype migration, according to mitochondrial DNA Fig 3 : mt DNA haplogroup distribution of world Fig 4 : Structure of Y chromosome Fig 5: Major Haplogroup Frequencies Of the World Fig 6: The different transmission paths of genetic material. Y-chromosomes exclusively paternal, mitochondrial DNA entirely maternal.

Fig 7: Out-of-Africa model Fig 8: People who contributed Fig 9: Vacutainer Fig 10 : Transfering blood from syringe to vacutainer Fig11 : MJ Research PCR Fig 12 : PCR ( Eppendorf and Veriti) Fig 13: DNA Sequencer Fig 14: DNA sequencing analysis software Fig 15: Auto Assembler Software Fig 16 : aqueous layer, protein layer and solvent layer.

Fig 17 : 2 clear layers of DNA and Chloroform .

Fig 18 : DNA Extracted Fig 19 : Gel Check of Dilution Fig 20 : Gel Check of PCR products Fig 21 Map: Site of sample collection Fig 22 : Consent Form Table 1 Fig 23 : Frequency Chart of Y haplogroup

Table 2 Fig 24 : Frequency Distribution of mt DNA Figure 25. Worldwide frequency distribution of Haplogroup O. Figure 26. Relative frequency distribution of the four main subclades of Haplogroup O. Fig 27: Derived samples derived from M95 primer leads to O2a‐ Haplogroup On Y chromosome phylogenetic tree Fig 28: Derived samples derived from M82 primer leads to H1‐ Haplogroup On Y chromosome phylogenetic tree Fig 29 : A‐G Mutaion Fig 30: Insertion T Fig 31 : M82 primer haplogroup analysis giving Derived Fig 32 : M95 primer haplogroup analysis giving Ancestral Fig 33 : M95 primer haplogroup analysis Giving Derived

LIST OF ABBREVIATIONS

% C ATP bp cm cpm dATP dCTP DDW dGTP D-loop DNA dNTP ddNTP dTTP EDTA Et.Br Extn Figure g kb M mA mg min ml mm mM mtDNA mtRNA rRNA tRNA N nm NaOH ng OD

-

Percentage Degree Celsius Adenosine 5’-triphosphate base pair(s) centimeter counts per minute 2’-deoxyadenosine 5’-triphosphate 2’-deoxycytidine 5’-triphosphate Double distilled water 2’-deoxyguanosine 5’-triphosphate The displacement loop Deoxyribonucleic acid 2’-deoxynucleotide 5’-triphosphate 2’,3’-dideoxynucleotide 5’-triphosphate 2’-deoxythymidine 5’-triphosphate Ethylene diamine tetra acetic acid Ethidium bromide Extension Figure gram kilo base molarity milli ampere milligram minutes millilitre millimeter millimolar mitochondrial DNA mitochontrial RNA ribosomal RNA transfer RNA Normality nanometer sodium hydroxide nanogram Optical density

OH OL PCR pM RNA rpm.

SDDW SDS Sec. SNPs SRY SSC STR TAE TE Tris TMRCA

-

Origin of heavy chain replication The L-strand origin Polymerase chain reaction picomole ribonucleic acid Revolutions per minute Sterile Double Distilled water Sodium dodecyl sulphate Seconds Single Nucleotide Polymorphisms Sex-Determining Region On Y Chromosome Sodium saline citrate Short Tandem Repeat Tris-Acetate-EDTA Tris-EDTA Tris ( ecogniz methyl) amino methane Time to the most recent common ancestor

U

-

unit

UEP UV V v/v w/v g l MW YAP YCC

-

unique event polymorphism Ultraviolet Volts Volume/Volume Weight/Volume Microgram Microlitre Micro molar Watts Y-Alu polymorphism Y Chromosome Consortium

ABSTRACT

India is a conglomeration of various ethnicities with 4693 communities, 325 languages, 25 scripts and numerous endogamous groups. It is a home of several tribal pockets, which represents different genetic isolates and thus provides unique wealth to understand human evolution. These autochthonous tribal populations reveal striking diversities in terms of language, marriage practices as well as in their genetic architecture. The origin and settlement of the Indian people still remain intrigues for the scientist studying the impact of the past and modern migration of the genetic diversity and structure of contemporary populations.

Indian populations are stratified as tribe, caste and religious community.

Endogamy has probably been a major reason for genetic diversification among the people of this region. Taking geographical and ethnic diversity into account and to answer the question of origin and evolution of maternal and paternal lineages of Indian population .Above 400 base pairs of the HVR-1 region and selected coding regions of the mitochondrial DNA (mtDNA) and Y chromosome markers in 102 individuals of

Bhumij, an Austro-Asiatic tribe of

Jharkhand,

was

analyzed and compared with the data available from the Indian subcontinent.

Based on the mutations observed in the HVR -1 and selected coding region of mitochondrial DNA, haplogroups were assigned to each of the individual. It was observed that most of the individuals of Bhumij tribal population were falling in Indian specific macro haplogroup M, displaying the array of South Asian specific lineages. On the other hand, Y chromosomal analysis is showing 70% percentage of individuals falling into O2a-M95 haplogroup, found frequently among AustroAsiatic. Moreover, it is evident that our investigation of the small population is a snapshot with respect to the peopling of the Indian subcontinent. In future, detailed phylogeographic and phylogenetic analysis of more tribal population can reveal the detailed account of maternal and paternal lineages as well as genetic affinity of the Indian population.

Chapter 1 Introduction To The Study INTRODUCTION: Tracing about the origin and ancestral links of homo sapiens have been the subject of curiosity for various scientists. And a number of scholars have devoted themselves to disclose these hidden mysteries of Human origin and dispersal on earth.

Where did we come from, and how did we get here? This is the question which genetic anthropology field is seeking an answer for.

DNA studies indicate that all modern humans share a common female ancestor who lived in Africa about 140,000 years ago, and all men share a common male ancestor who lived in Africa about 60,000 years ago. These were not the only humans who lived in these eras, and the human genome still contains many genetic traits of their contemporaries. Humanity’s most recent common ancestors are identifiable because their lineages have survived by chance in the special pieces of DNA that are passed down the gender lines nearly unaltered from one generation to the next. These ancestors are part of a growing body of fossil and DNA evidence indicating that modern humans arose in sub-Saharan Africa and began migrating, starting about 65,000 years ago, to populate first southern Asia, China, Java, and later Europe. Each of us living today has DNA that contains the story of our ancient ancestors’ journeys.

When DNA is passed to our next generation, the processes that make each person unique from their parents is the combination of both their genomes. Some special pieces of DNA, however, remain virtually unaltered as they pass from parent to offsprings. One of these pieces are carried by Y chromosome. It is passed only from father to son.

Secondly, mitochondrial DNA (mtDNA), is passed (with few

exceptions) only from mother to child. Since the DNA in the Y chromosome does not undergo crossing over, it is like a genetic surname that allows scientists to trace back their paternal lineages.

Similarly, mtDNA allows both men and women to trace their maternal lineages. Both the Y chromosome DNA and mtDNA are subject to occasional harmless mutations that become inheritable genetic markers. After several generations, almost all male and female inhabitants of the region in which it arose carry a particular genetic marker. When people leave that region, they carry the marker with them. By studying the genes of many different indigenous populations, scientists can trace when and where a particular marker arose. Each marker contained in a person’s DNA represents a location and migration pattern of that person’s ancient ancestors. For example, roughly 70% of English men, 95% of Spanish men, and 95% of Irish men have a distinctive Y-chromosome mutation known as M173. The distribution of people with this mutation, in conjunction with other DNA analysis, indicates that they moved north out of Spain into England

and

Ireland

at

the

end

of

the

last

ice

age

(genomics.energy.gov).

Information about the history of our species comes from two main sources: the paleo-anthropological record and historical inferences based on current genetic differences observed in humans. Although both sources of information are fragmentary, they have been converging in recent years on the same general story (Underhill et; al.).

Since the 1990s, it has become common to use multilocus genotypes to distinguish different human groups and to allocate individuals to groups (Bamshad et al. 2004). These data have led to an examination of the biological validity of races as evolutionary lineages and the description of races in cladistic terms. The technique of multilocus genotyping has been used to determine patterns of human demographic history. Thus, the concept of “race” afforded by these

techniques is synonymous with ancestry broadly understood (Berg et al.,).

Y chromosome and mitochondrial DNA are transmitted uni-parentally through father and mother, respectively and don’t under go any recombination. Hence, markers present on both are useful to trace the paternal and maternal lineages. Haplotypes can be constructed by combining the allelic status of multiple markers, which would provide adequate information for establishing paternal lineages. The noncoding region (D-loop) of mtDNA, which harbors two hyper variable regions (HVR I and HVRII), shows variation between different populations. A large number of studies have been conducted on various populations using Y chromosome markers and mtDNA Dloop region to understand their origin, evolution and migration.

Indian populations reveal striking diversities in terms of language, marriage practices as well as in their genetic architecture. The social structure of the Indian population is governed by the hierarchical caste system. In India, there are nearly 5,000 well-defined endogamous populations. In addition to the native populations, there are a few migrant populations inhabiting various parts of India. Several important historical migrations into India caused amalgamation of migrant populations with the local population groups. Major demographic event like migrations, population bottlenecks and population expansion leave genetic imprints and alter gene frequencies. These imprints are passed onto successive generations, thus preserving the population’s history within the population.

Therefore, we have undertaken to disclose the genetic information about how different caste and tribal populations of India help to construct

ecognize

and help to construct the evolutionary tree

(Cavalli-Sforza et al.,).

Two major routes have been proposed for the initial peopling of East

Asia; one via Central Asia to Northeast Asia, which subsequently expanded towards Southeast Asia and beyond, and the other through

India to Southeast Asia and further to different regions of East Asia.[1] It is pertinent in this context that the Indian subcontinent has been considered as a major corridor for the migration of human populations to East Asia.[2-4] Given its unique geographic position, Northeast India is the only region which currently forms a land bridge between the Indian subcontinent and Southeast Asia, hence hypothesized as an important passage for the initial peopling of East Asia. This region is inhabited by populations belonging to IndoEuropean, Tibeto-Burman and Austro-Asiatic linguistic families. ‘‘BHUMIJ

TRIBE’’

come under austro-asiatic linguistic

population. Austro-Asiatic speakers, hypothesized as probably the earliest settlers in the Indian subcontinent ([5] and references their in), are also found in other parts of India as well as in East/Southeast Asia. Therefore, if Northeast India had served as an initial corridor, it is likely that the Austro-Asiatic tribes of this region should provide hitherto missing genetic link, which may reflect genetic continuity between Indian and East/Southeast Asian populations. Based on mitochondrial DNA (mtDNA) and Y-chromosome markers, Cordaux et al. [6] observed genetic discontinuity between the Indian and southeast Asian populations and inferred that Northeast India might have acted as a barrier rather than the facilitator of the movement of populations both into and out of India.

However, this study include only ‘‘BHUMIJ’’ Tribe of Jharkhand region from Jamshedpur district. Further evidence is needed by way of determining the mtDNA and Y-chromosome haplogroups/lineages of the Austro-Asiatic tribes of the northeastern region and their comparison with appropriate set of South and Southeast Asian populations.

Jharkhand

is

basically

an

agricultural

land.

Geographically it is covered by jungles, mountains, rivers and Chotanagpur plateau etc.

1.2 BACKGROUND :

HUMAN GENOME DIVERSITY PROJECT (HGDP) :­

The HGD Project was started internationally on mid-September

of 1993 and it has 13 countries participating in it. The Human Genome Diversity Project is an international project that seeks to understand the diversity and unity of the entire human species.

The Human Genome Diversity Project (HGDP) aims to collect biological samples from different population groups throughout the world, with the aim of building up a representative database of human genetic diversity. This seems a laudable aim, but the Project has been enmeshed in massive controversy since it was first proposed in 1991, with violent reactions from many of the indigenous people’s groups it proposes to study.

The eminent geneticist Luigi Luca Cavalli-Sforza of Stanford University first conceived by the HGDP. For many years, he and other geneticists and anthropologists have been visiting different ethnic groups around the world, collecting samples, and trying to build up a picture of how different human populations are related to each other.

The samples are seen as immensely valuable, but they are in laboratories spread around the world. In 1991, Cavalli-Sforza and a number of colleagues wrote a letter to the scientific journal, Genomics, pointing out the need for a systematic study of the whole range of human genetic diversity, within the context of the Human Genome Project. They pointed to a problem: ‘The populations that can tell us most about our evolutionary past are those that have been isolated for some time, are likely to be linguistically and culturally distinct and are often surrounded by geographic barriers. Such isolated populations are being rapidly merged with their neighbours, however, destroying irrevocably the information needed to reconstruct our evolutionary history. It would be tragically ironic if, during the same decade that

biological tools for understanding our species were created, major opportunities for applying them were squandered.

Major

demographic

events

like

migration,

population

bottlenecks and population expansion leave genetic imprints where gene frequency of the genome is altered (Thangaraj et, al., 1998).

These imprints are passed onto successive generations thus preserving the population history within the population. In general, human beings group themselves into units in such a way that members between units rarely exchange genes due to cultural and geographical barriers resulting in genetic divergence of population.

The Human Genome Diversity Project proposed in early nineties is a combined effort preceded by anthropologists, geneticists, doctors, linguists and other scholars from around the world aims at collecting the blood samples from different ethnic populations throughout the world aiming at building up a representative database of human genetic diversity.

The reason lying behind selecting only tribes for sampling is that they are believed to have been isolated during an evolutionary time, linguistically and culturally distinct and are often isolated by geographic barriers and thus prove to be best tools for study.

IN

THIS

P R O J E CT ,

THE

SUBJECT

OF

GENETIC

STUDY

IS

‘‘BHUMIJ TRIBE’’ F R O M JHARKHAND (CHOTANAGPUR PLATEAU), INDIA .

1.3 STATEMENT OF PURPOSE : How does DNA helps us to trace back?

Y chromosome and mitochondrial DNA are transmitted uni-parentally through father and mother respectively and do not undergo any recombination. Hence, markers present on both are useful to trace the paternal and maternal lineages. Haplotypes can be constructed by combining the allelic status of multiple markers, which would provide adequate information for establishing paternal lineages. The non-coding

region (D-loop) of mtDNA, which harbors two hyper variable regions (HVR I and HVRII), shows variation between different populations. A large number of studies have been conducted on various populations using Y chromosome markers and mtDNA D-loop region to understand their origin, evolution and migration.

Indian populations reveal striking diversities in terms of language, marriage practices as well as in their genetic architecture. The social structure of the Indian population is governed by the hierarchical caste system. In India, there are nearly 5,000 well-defined endogamous populations. In addition to the native populations, there are a few migrant populations inhabiting various parts of India Several important historical migrations into India caused amalgamation of migrant populations with the local population groups. Major demographic event like migrations, population bottlenecks and population expansion leave genetic imprints and alter gene frequencies. These imprints are passed onto successive generations, thus preserving the population’s history within the population. Therefore, we have undertaken to disclose the genetic information about caste and tribal populations of India to construct

ecognize

and to use the

ecognize

data to construct the

phylogenetic tree.

In future the recorded data of mutated sites of a particular haplogroup can help the scientists to trace the cause and solution to many new diseases and help them to develop ne techniques of diagnosis and design new drugs.

1.4 AIMS AND OBJECTIVES OF THE STUDY :

GOALS OF HGD PROJECT:­ The Human Genome Diversity Project is a collaborative research project that is being developed on a global basis under the auspices of the Human Genome Organization (HUGO). ¾ The overall goal of the project is to arrive at a much more precise definition of the origins of different world populations by integrating genetic knowledge, derived by applying the new techniques for studying genes, with knowledge of history, anthropology and language. ¾ To investigate the variation occurring in the human genome by studying samples collected from populations that are representative of all of the world’s peoples. ¾ To create a resource for the benefit of all humanity and for the scientific community worldwide.

The resource will exist as a collection of biological samples that represents the genetic variation in human populations worldwide and also as an open, long-term, genetic and statistical database on variation in the human species that will accumulate as the biological samples are studied by scientists from around the world.

The major goals of HGDP:­ ¾ To identify all the approx 20,000-25,000 genes in human DNA, determination of the sequence of the 3 billion chemical base pair that make up human DNA. ¾ In silico storage of all DNA database. Improve tools for data analysis. ¾ Transfer related technologies to the private sector. Address the ethical, legal and social issues (ELSI) that may arise from the project. ¾ To provide information regarding human biological relationship among different groups and human history. ¾ To understand the cause and diagnostics of human diseases.

BENEFITS AND IMPLIFICATIONS OF HGDP:­ The project will reap fantastic benefits for human kind, some that we can anticipate and other that will surprise us. Generations of biologists and researchers have been provided with detailed DNA information that will be the key to understanding the structure, organization and function of DNA in chromosome. The information from HGDP provides information to clarify the origin and biological relationship of specific human populations and the evolution of human being in particular. The variations of frequencies in various populations can reveal how recently they shared a large pool of common ancestors.

HGDP IN INDIA:­ In India, Centre for Cellular and Molecular Biology [CCMB],

Hyderabad has pioneered the Human Genome Diversity Project in collaboration with several other institutes and universities. Around 6,200 different unrelated individuals have been sampled from various Indian populations & have been analyzed for their genetic diversity and phylogeny.

The origins of Indian tribes, who presently constitute about 8% of total population of India, have been subject to numerous genetic studies. India is a land of enormous human genetic, bio-geographic, socio-economic, cultural and linguistic diversity. More than 300 tribal groups are recognized in India and they are densest in the central and southern province. There are more than 800 dialects and a dozen major languages, grouped into those of Dravidian South India and Indo-Aryan North India. The resulting hypotheses range from referring to some tribes as the descendents of the original Paleolithic inhabitants of India while some are the recent immigrants. Hence, genetic diversity in India provides important clues to the evolutionary history of human beings.

TRACING GENETIC DIVERSITY:­ The past decade of advances in molecular genetic technology has

heralded a new era for all evolutionary studies, but especially the science of human evolution. Data on various kinds of DNA variation in human populations have rapidly accumulated. There is increasing recognition of the importance of this variation for medicine and developmental biology and for understanding the history of our species. Haploid markers from mitochondrial DNA and the Y chromosome have proven invaluable for generating a standard model for evolution of modern humans.

Conclusions from earlier research on protein polymorphisms have been generally supported by more sophisticated DNA analysis. Co-evolution of genes with language and some slowly evolving cultural traits, together with the genetic evolution of commensals and parasites that have accompanied modern humans in their expansion from Africa to the other continents, supports and supplements the standard model of genetic evolution. The advances in our understanding of the evolutionary history of humans attest to the advantages of multidisciplinary research.

Although molecular genetic evidence continues to accumulate that is consistent with a recent common African ancestry of modern humans, its ability to illuminate regional histories remains incomplete. A set of unique event polymorphisms associated with the non-recombining portion of the Y-chromosome (NRY) addresses this issue by providing evidence concerning successful migrations originating from Africa, which can be interpreted as subsequent colonization, differentiations and migrations overlaid upon previous population ranges. A total of 205 markers

identified

by

denaturing

high

performance

liquid

chromatography (DHPLC), together with 13 taken from the literature, are used to construct a parsimonious genealogy. Ancestral allelic states were deduced from orthologous great ape sequences. A total of 131 unique

ecognize

are defined which trace the micro evolutionary

trajectory of global modern human genetic diversification. The genealogy provides a detailed phylogeographic portrait of contemporary global population structure that is emblematic of human origins,

divergence and population history that is consistent with climatic, paleoanthropological and other genetic knowledge. The frequency of occurrence of different

ecognize

can be used to distinguish

populations and to shed light on the sub-structures within a population, also for inter and intra population variation studies. Population analyses have examined allele frequencies at autosomal genetic markers (CavalliSforza As in this project, when a significant number of individuals in a population et al., 1994). The incorporation of mitochondrial DNA during the 1980s added a powerful tool to the geneticists’ tool kit, since mtDNA does not recombine and is transmitted only through female germ line (Stoneking and Soodyall, 1996). The increasing number of polymorphic markers identified on the Y chromosome has allowed analyzing male lineages, (Hammer and Zegura, 1997). A set of highly polymorphic chromosome Y specific micro satellite became available for forensic, population genetic and evolutionary studies. However, the lack of a mutation frequency estimate for these loci prevents a reliable application.

MARKERS:­ The human genome comprise of actually two genomes: a complex nuclear genome, which account for 99.9995% of total genetic information and a simple mitochondrial genome, which accounts for the remaining 0.0005%.

During zygote formation, a sperm cell contributes its nuclear genome, but not its mitochondrial genome to the egg cell. Consequently, the mitochondrial genome of the zygote is determined exclusively by that originally found in the unfertilized egg. The mitochondrial genome is therefore maternally inherited. As a result, it does not undergo any genetic reshuffling and thus, is intact which makes it a unique tool for studying human origins. Thus, everyone carries with them a more or less exact copy of the mtDNA from their mother and their mother’s mother and so forth for countless generations. The term “more or less exact” is the key to scientist solving the mystery of human origins. That’s because like all DNA, mtDNA is subject to random mutations over the eons. As these mutations are passed on intact to next generation, they in effect become

“tracers” of family. A single type of circular double stranded molecule of 16,569 bases defines human mitochondrial genome.

MITOCHONDRIAL DNA (mtDNA) AS MARKER:­ The mtDNA (Fig:1) has no repetitive DNA, spacers or introns. The mtDNA contains 37 genes, all of which are involved in the production of energy and its storage in ATP. It encodes 13 mRNAs, 22 tRNAs and 2 rRNAs. mtDNA has two strands, a guanine rich heavy (H) strand and a cytosine rich light (L) strand. The heavy strand contains 12 of the 13polypeptide encoding genes, 14 of the 22 tRNA encoding genes and both rRNA encoding genes. The mtDNA is replicated from two origins. DNA replication is initiated at OH (Origin of heavy chain replication) using an RNA primer generated from the L-strand transcript. H-strand synthesis proceeds two-thirds of the way around the mtDNA, displacing the parental H-strand until it reaches the L-strand origin (OL), situated in a cluster of five tRNA genes. Once exposed on the displaced H-strand, OL folds a stem loop structure and L-strand synthesis is initiated and proceeds back along the H-strand template. Consequently, mtDNA replication is bi-directional but asynchronous (Clayton 1982).

The analysis of mitochondrial DNA (mtDNA) has been a potent tool in the understanding of human evolution, owing to its characteristics such as: •

High copy number 1000-10,000 copies per cell (Nass 1969; Bogenhagen et al.)

•

High substitution rate almost 10 times greater than nuclear DNA (Brown et al. 1979) and even higher in non-coding control region.

•

Maternal mode of inheritance (Giles et al., 1980). So the gene tree is an estimate of the maternal genealogy tells specifically about processes on the female side of the population history.

•

Semi-autonomously replicating molecule.

•

No repetitive DNA, spacers or introns.

•

Small size of the molecule and simple genome organization and hence easier to study.

•

They serve as “molecular clocks” as they can be used to calculate the divergence time elapsed.

However, almost all studies of human evolution based on mtDNA sequencing have been confined to the control region also called the D-loop or the displacement loop, which constitutes less than 7% of the mitochondrial genome.

Fig 1 : Human Mitochondrial DNA

Fig 2 : Map of human haplotype migration, according to mitochondrial DNA

MITOCHONDRIAL DNA CONTROL REGION:­ Mitochondrial DNA serves as a molecular clock, in that within its structure there is a 1200-base-pair non-coding segment, called the control region that carries the genetic signals needed for replication and transcription.

Since much of this DNA segment is not vital to the survival of the mitochondrion or of the host cell. (Other DNA segments are more vitalmutations could change the nature of the protein formed and gene expression, and therefore mutations could impact the survival of the organism that bears that gene.) By studying the number and variety of base changes within this control region, geneticists can determine the relatedness between individuals.

Using the mutation rate within the mitochondrial control region as a “molecular clock,” evolutionists can plot the course that hominid evolution has taken.

“The rate and pattern of sequence substitutions in the mitochondrial DNA (mtDNA) control region (CR) is of central importance to studies of human evolution”. The DNA sequence of the control region is termed hyper variable region because it accumulates point mutations at approximately 10 times the rate of nuclear DNA. In the human control region, the estimates of the rate of substitution were found to range between 2.8 (Cann et al. 1984) to

5 times (Aquadro & Greenberg 1983) the rate of the rest of the mtDNA. Most of the studies in which control region sequences have been used have focused on intraspecific patterns of variability and phylogenetic relationships of closely related species, a prominent example being the study of human population history. Polymorphic nucleotide sites within this loop are concentrated in two “Hyper variable segments”, HVRI (positions 1602416383) and HVRII (Wilkinson-Herbots et al. 1996). Hence HVSI and HVSII data can provide useful insights about inter and intra-specific population variations.

MITOCHONDRIAL DNA BASED HAPLOGROUPS: In human genetics, a human mitochondrial DNA haplogroup is a haplogroup defined by differences in human mitochondrial DNA. These haplogroups have led researchers to trace the matrilineal inheritance of modern humans back to human origins in Africa and the subsequent spread across the globe [e].

Known haplogroups are assigned the following letter codes: A, B, C, CZ, D, E, F, G, H, pre-HV, HV, I, J, pre-JT, JT, K, L0, L1, L2, L3, L4, L5, L6, L7, M, N, P, Q, R, S, T, U, UK, V, W, X, Y, and Z.

Fig 3 : mt DNA haplogroup distribution of world

Y CHROMOSOME AS A MARKER:­ Until recently, the Y chromosome seemed to fulfill the role of juvenile delinquent among human chromosomes-rich in junk, poor in useful attributes, reluctant to socialize with its neighbors and inescapable tendency to degenerate. The properties of Y chromosome read like a list of violations of the rulebook of human genetics.

However it is because of this disregard for the rules that Y chromosome proves to be such a superb tool for investigating human evolution. The availability of the near complete sequence and new polymorphisms, gives a highly resolved phylogeny and insights into its mutation processes throws further light on human evolution.

The human Y chromosome (Figure.1.2) is approximately 60 Mb, linear molecule that determines maleness. It is an unusual segment of the human genome since, apart from two small regions in which pairing and exchange take place with the X chromosome, it is male specific and haploid and escapes from recombination.

These unique properties of the Y chromosome have important consequences for its mutation processes, its genes and in population genetics. Y chromosome pass down from father to son, largely unchanged, except by the gradual accumulation of mutations. Different populations often have characteristically different Y chromosome and these studies are likely to make a major contribution to our understanding of the origin of modern humans (Mark Jobling and Chris Tyler Smith, Trends in Genetics, 2000). By examining the difference between polymorphic Y-chromosomal markers one can attempt to reconstruct a history of human paternal lineages, population structure and history, genealogy, forensics and the investigation of selective influences in the Y chromosome. 95% of the Y chromosome has become a genetic junkyard because it does not recombine. In the Y-chromosome’s passage through the generations, changes occur randomly in its junk DNA and so the Y-chromosome of the contemporary populations retains a record of their passage through time. They can reveal the paternal genealogy of their owners and the relationships between different groups of individuals (Neil Bradman and Mark Thomas).

Properties of Y chromosome PAR1 SRY RPS4Y ZFY DYS7 (50f2/B) DYS7 (50f2/A)

KAL-Y YRRM1,2 STSP YRRM2 DYS7 (50f2/E)

AZFc

p Haploid

SMCY

AZFb

YRRM1,2 DAZ DYS7 (50f2/C) BPY2 CDY

sY160 102(d)2 pHY2.1

Heterochromatic region

AZFa

p arm Euchromatic region

AMELY YRRM1,2 TSPY DYS7 (50f2/D)

q arm

p Non-recombining region p Uniparental transmission

PAR2

Human Y Chromosome

Figure 4 : Structure of ‘Y’­Chromosome

FEATURES OF Y­CHROMOSOME:­ The Y chromosome has been a potent tool for studying human evolution owing to following characteristics:•

Paternal mode of inheritance as it passes from father to son and thus escapes meiotic recombination.

•

Only 3Mb of its length undergoes recombination and thus also referred as nonrecombining majority.

•

Haplotypes pass intact from generation to generations and change only by mutation.

•

Lower sequence diversity than elsewhere in nuclear genome.

•

Using binary polymorphism such as SNPs a unique phylogeny can thus be constructed.

•

More susceptible to genetic drift, a useful property for investigating past events.

•

Geographical clustering is further influenced by the behavior of men, bearers of Y-chromosome.

Y CHROMOSOMAL CHANGES:­ Changes that do occur from generation to generation are of four types: • INDELS: Insertions or deletions in the DNA at particular locations on the chromosome. One insertion particularly useful in population studies is the YAP, which stands for “Y chromosome Alu polymorphism. Alu is a sequence of approximately 300 letters (base pairs), which has inserted itself into a particular region of the DNA. There have been some half a million-Alu insertions in human DNA; YAP is one of the more recent.

• SNIPS: Are “single nucleotide polymorphisms” in which a particular nucleotide (an A for example) is changed (perhaps into a G). Stable indels and snips are relatively rare and, in the case of the latter, so infrequent that it is reasonable to assume they have occurred at any particular position in the genome only once in the course of human evolution. Snips and stable Alu’s have been termed “unique event polymorphisms” (UEPs).

Two other polymorphisms complete the marker set which can be used to unravel all Y chromosome history. ƒ

MICRO SATELLITES: Are short sequences of nucleotides (such as GATA) specific number of repeats in a particular variant (or allele) usually remains unchanged from generation to generation but changes do sometimes occur and the number may increase or decrease.

It is usually assumed that increases or decreases in the number of repeats take place in single steps, for instance from nine repeats to ten, but whether decreases in number are as common as increases has not been established. Changes in micro satellite lengths occur much more frequently than new UEP arise. What is more, while we can reasonably assume that a UEP has arisen only once, the number of repeat units in a micro satellite may have changed many times along a paternal lineage.

ƒ

MINISATELLITES: Extensively studied by Mark Jobling at the University of Leicester. Unlike micro satellites, in which the repeated sequences are short (often no more than 3 or 4 nucleotides), in minisatellites they are normally 10-60 base pairs long and the number of repeats often extends to several dozen. Changes during the copying process take place more frequently in minisatellites than in micro satellites and the mechanisms may be different in the two cases.

In using polymorphisms to study changes over time, we are fortunate in having markers, which change at different rates. Perhaps we can think of the UEPs as the hour hand, the micro satellite polymorphisms as the minute hand and the minisatellites as a sweep second hand of the evolutionary clock. Because most of the Y chromosome does not exchange DNA with a partner, a further benefit of using it to study evolution is that all the markers are joined one to another along its entire length. Such linkage of markers means that a haplotype constructed from a number of different markers records the

evolutionary history of the particular Y chromosome on which they are all located. Many polymorphic loci scattered over the entire nonrecombining part of the Y-chromosome can be identified. Among these polymorphisms, biallelic markers with a low mutation rate representing unique mutation events (UMEs) in human evolution, such as single base-pair substitutions (Underhill et al., 1997).

What is a Haplogroup?

The haplogroups are the major branches on Y chromosome tree, defined by single nucleotide polymorphism (SNPs), which have accumulated along different lineages as y chromosomes are passed from father to son over many generations . All haplogroups ultimately descend from a single Y chromosome carried by a male that lived in the distant past . The topology of the Y chromosome tree can be reconstructed by typing mutations in different human populations –as more SNPs are discovered (e.g., M254), the structure of the tree changes. Originally, the Y Chromosome Consortium (YCC) arbitrarily defined 18 haplogroups (A-R) , which represent the major divisions of human diversity based on Y chromosome SNPs. Currently , there are 20 haplogroups (A-T ). In turn , each of these major haplogroups has numbered subgroups or subclades, that are named with alternating letters and numbers.

Major Haplogroup Frequencies:­ The frequencies of 20 major NRY haplogroups are shown for each of 10 geographic regions. Each haplogroup is color-coded according to the tree figure ( also shown on the map legend ) .

The frequencies of each haplogroup are based on the following samples sizes for each region : •

Sub-Saharan = 229

North Africa = 131

•

Middle East = 180

Europe = 328

•

Central Asia = 264

South Asia = 195

•

North Asia = 496

East Asia = 461

•

The Pacific = 279

The Americas = 227

When haplogroup frequencies are close to zero , the corresponding pie slice is not readily visible .

Fig 5 : Major Haplogroup Frequencies Of the World

PATTERN OF INHERITANCE:­

Fig 6: The different transmission paths of genetic material. Y‐chromosomes exclusively paternal, mitochondrial DNA entirely maternal.

1.5 Hypothesis:­

R ECENT A FRICAN ORIGIN OF MODERN HUMANS [ C ] In paleoanthropology, the recent African origin of modern humans is the mainstream model describing the origin and early dispersal of anatomically modern humans. The theory is called the (Recent) Out-ofAfrica model in the popular press, and academically the recent singleorigin hypothesis (RSOH), Replacement Hypothesis, and Recent African Origin (RAO) model. The hypothesis that humans have a single origin (monogenesis) was published in Charles Darwin’s Descent of Man (1871).

The concept was speculative until the 1980s, when it was corroborated by a study of present-day mitochondrial DNA, combined with evidence based on physical anthropology of archaic specimens.

According to both genetic and fossil evidence, archaic Homo sapiens evolved to anatomically modern humans solely in Africa, between 200,000 and 100,000 years ago, with members of one branch leaving Africa by 60,000 years ago and over time replacing earlier human populations such as Neanderthals and Homo erectus. The recent single origin of modern humans in East Africa is the near-consensus position held within the scientific community.[19]The competing hypothesis is the multiregional origin of modern humans. Some push back the original “out of Africa” migration—in this case, by Homo erectus, not by Homo sapiens—to two million years ago.[20][21]

Fig 7: Out-of-Africa model

CHAPTER 2 REVIEW OF LITERATURE 2.1 I NTRODUCTION OF B HUMIJ T RIBE ( A )

Bhumij, a non-Aryan tribe of Manbhum, Singbhum, and Western Bengal, classed by Dalton and others, mainly on linguistic grounds, as Kolarian.

There can be no doubt that the Bhumij are closely, allied to, if not identical with, the Mundas; but there is little to show that they ever had a distinct language of their own. In 1850 Hodgson 2 published a short vocabulary prepared by Captain Haughton, then in political charge of Singbhum; but most of the words in this appear to be merely Ho. The most recent observer, 3 Herr Nottrott, of Gossner’s Mission, says that the Bhumij resemble the Mundas most closely in speech and manners, but gives no specimens of their language, and does not say whether it differs sufficiently from Mundâri to be regarded as a separate dialect.

Origin:­ I am inclined myself to believe that the Bhumij are nothing more than a branch of the Mundas, who have spread to the east, mingled with the Hindus, and thus for the most part severed their connection with the parent tribe. This hypothesis seems on the whole to be borne out by the facts observable at the present day. The Bhumij of Western Manbhum are beyond doubt pure Mundas. They inhabit the tract of the country which lies on both sides of the Subarnarakhâ river, bounded on the west by the edge of the Chotanagpur plateau, on the east by the hill range of which Ajodhyâ is the crowning peak, on the south by the Singbhum hills, and on the north by the hills forming the boundary between Lohardagâ, Hazaribagh, and Manbhum districts. This region contains an enormous number of Mundâri graveyards, and may fairly be considered one of the very earliest settlements of the Munda race. The present inhabitants use the Mundâri language, call themselves Mundas, and observe all the customs current among their brethren on the plateau of Chotanagpur proper. Thus, like all the Kolarians, they build no temples, but worship Buru in the form of a stone smeared with vermillion. A ecog is invariably composed of purely jungle trees, such as sâl and others, and can therefore be ecognize with certainly as a fragment of the primeval forest, left standing to form an abiding place for the aboriginal deities.

They observe the sarhul festival at the same time and in the same way as

their kindred in Lohardagâ and Singbhum, and the lâyâ or priest is a recognized village official. Marriages take place when both parties are of mature age, and the betrothal of children is unknown. Like the Mundas of the plateau, they first burn their dead and then bury the remains under gravestones, some of which are of enormous size. On certain feast days small supplies of food and money are placed under these big stones to regale the dead, and are extracted early the next morning by low-caste hindus. On the eastern side of the Ajodhya range, which forms a complete barrier to ordinary communication, all is changed. Both the Mundâri and the title of Munda have dropped out of use, and the aborigines of this eastern tract call themselves Bhumij or Sardâr, and talk Bengali. The physical characteristics of the race, however, remain the same; and although they have adopted Hindu customs and are fast becoming Hindus, there can be no doubt that they are the descendants of the Mundas who first settled in the country, and were given the name of Bhumij (autochthon) by the Hindu immigrants who found them in possession of the soil.

Internal Structure:­ The sub-tribes are numerous, and vary greatly in different districts. With the possible exception of the iron-smelting Shelo in Manbhum, the names of these groups seem to have reference to their supposed original settlements. It deserves notice that the tendency to form endogamous divisions seems to be stronger in outlying districts than it is at the recognized head-quarters of the tribe. Thus in Manbhum and Singbhum we find only one sub-tribe Shelo, which obviously got detached from the parent group by reason of its members adopting, or perhaps declining to abandon, the comparatively degraded occupation of iron-smelting. In Midnapur, or the other hand, the Bhumij settlements are of comparatively functional group of Shelo. The reason seems to be that when the stream of emigration is not absolutely continuous, successive sections of immigrants into distant parts of the country are affected in various degrees by the novel social influences to which they are exposed. Some groups become more rapidly hinduised than others, and thus there arise divergences of usage in matters of food and drink, which constitute a bar to intermarriage, and in time lead to the formation of sub-tribes. These divisions often outlast the differences of custom and ritual from which they took their origin, and in some cases the prohibition of intermarriage comes to be withdrawn, and the names alone remain to show that such a prohibition was once on force. The exogamous divisions of the tribe are totemistic, and closely resemble those met with among the Mundas. The rule of exogamy is simple.

Marriage:­ The aboriginal usage of adult-marriage still holds its ground among the

Bhumij, though the wealthier members of the tribe prefer to marry their daughters as infants. The extreme view of the urgent necessity of early marriage is unknown among them, and it is thought no shame for a man to have a grown-up daughter unmarried in his house. The Bhumij ecognize polygamy, and in theory at least impose no limitation on the number of wives a man may have.

Widow­marriage: Widow‐marriage is freely permitted by the sanga ritual. It is deemed right for a widow to marry her late husband’s younger brother or cousin, if such an arrangement be feasible; and in the event of her marrying an outsider, she forfeits all claim to a share in her late husband’s property and to the custody of any children she had with the first husband Divorce: The Bhumij of Manbhum allow divorce only when a woman has been guilty of adultery.

Religion:­ The religion of the Bhumij is flexible within certain limits, according to the social position and territorial status of the individuals concerned.Zamindars and well-to-do tenure-holders employ Brahmans as their family priests, and offer sacrifices to Kali or Mahâmâyâ. The mass of the people revere the sun under the names of Sing-Bonga and Dharm, as the giver of harvests to men and the cause of all changes of seasons affecting their agricultural fortunes. They also worship a host of minor gods like Jâhir-Buru, Kârâkâtâ, (Kârâ = ‘buffalo,’ and Kâtâ = ‘to cut’), Bâghut, Kudra and Bisaychandi etc.

Occupation:­ The original occupation of the Manbhum Bhumij is believed by themselves to have been military serviceFor many years agriculture has been the sole profession of all the sub tribes except the iron-smelting Shelo. A few have engaged in petty trade, and some have emigrated to the tea districts of Assam. Their relations to the land are various. The great bulk of the Bhumij, who are simple cultivators and labourers, stand on a far lower social level that the landholding members of the tribe.

Language:­ Their language is almost identical with Mundârí is also spoken by the Bhumij tribe of Singbhum and neighbourhood. Santhâlí language is

spoken in the west of the district. In Manbhum they are found in the west, and, according to Mr. Risley, speak Mundârí language. The Bhumij on the eastern side of the Ajodhya range speak Bengali. The Tamariâs are a subtribe of the Bhumij, who were originally settled in Pargana Tamar of Ranchi. Their dialect does not differ from that of the Bhumij proper.

Other Tamariâs speak a dialect of Magahí.(6)

List of People who contributed in blood sample for research : NAME 1) 2) 3) 4) 5) 6) 7) 8) 9) 10) 11) 12) 13) 14) 15) 16) 17) 18) 19) 20) 21) 22) 23) 24) 25) 26) 27) 28) 29) 30) 31) 32) 33) 34) 35) 36) 37) 38) 39) 40) 41) 42) 43) 44) 45) 46)

BHUJANG PRASAD SINGH [M] MANOJ SARDAR [M] KANDI BHUMIJ [F] SUNDARI BHUMIJ [F] GURVA BHUMIJ [M] PURENDAR BHUMIJ [M] PARSURAM SARDAR [M] GITA SARDAR [F] Mr. SARDAR [M] SUKLAL SARDAR [M] KUNI SARDAR [M] MEENA SARDAR [F] RUP SINGH [M] ARUN SINGH [M] SRIKANT SINGH [M] SOHAN SINGH [M] GULAB SINGH [M] INDRA SINGH [M] DEVA SINGH [M] MOHAN SINGH [M] PADMAWATI SINGH[F] LAKHAN SINGH [M] SRIKANT SARDAR [M] NARAYAN SARDAR [M] KANHAI SARDAR [M] BIRBAL SARDAR [M] ARUN SARDAR [M] SRIKANT SARDAR [M] MANGAL SINGH HANSDA [M] DHURMU SARDAR [M] BHUDRAI SARDAR [M] RUPCHAND SARDAR [M] Mrs. HULSAI SARDAR [F] NARDE SARDAR [M] SUDARSAN SARDAR [M] Mr. GUNADHAR SARDAR [M] SHEFALI SINGH [F] SATRUGHON SARDAR [M] HARISHCHANDRA SINGH [M] KAMALANI SINGH [F] ARJUN SINGH [M] SUDARSAN BHUMIJ [M] GANESH SINGH [M] NARAYAN SINGH [M] RATAN SARDAR [M] H. SARDAR [M]

VILLAGE - CHOLAGORA - TILKAGARH - GHAGIDIH - GHAGIDIH - GHAGIDIH - GHAGIDIH - GHAGIDIH - GHAGIDIH - GHAGIDIH - GHAGIDIH - GHAGIDIH - GHAGIDIH - DOMJURI - DOMJURI - DOMJURI - DOMJURI - DOMJURI - DOMJURI - DOMJURI - DOMJURI - DOMJURI – DOMJURI – GOMIASAI – KITADIH – GOMIASAL – BHELAIDIH – VELAIDIH – VELAIDIH – JONRAGARA – TILKAGARH – TILKAGARH – TILKAGARH – TILKAGARH – TILKAGARH – TILKAGARH – GITILATA – GITILATA – TETLA – CHANDPUR - TIRILDIH – GITILATA – BALIDIH – GITILATA – GITILATA – TIRILDIH – TIRILDIH

47) 48) 49) 50) 51) 52) 53) 54) 55) 56) 57) 58) 59) 60) 61) 62) 63) 64) 65) 66) 67) 68) 69) 70) 71) 72) 73) 74) 75) 76) 77) 78) 79) 80) 81) 82) 83) 84) 85) 86) 87) 88) 89) 90) 91) 92) 93) 94) 95) 96)

MOSO SARDAR [M] BHAWARI SARDAR [M] HARISHCHANDRA SINGH [M] DHIRENDAR [M] SURAJ PRABHASH SINGH [M] AJIT SINGH [M] BALRAM SARDAR [M] BASANTI SARDAR [F] SOBINAY SINGH [M] R.SINGH [M] RAMKADA SARDAR [M] NIRMAL SARDAR [M] LAKHI RAM HANSDA [M] ARUN SARDAR [M] TUNU SINGH [M] MADHUSUDAN SINGH [M] LAKHAN SINGH [M] BELBATI SINGH [F] RAGHU BHUMIJ [M] NAND SINGH [M] Mr. GURUCHARAN [M] BABLU SARDAR [M] Miss. SABITA SARDAR [F] Miss. KUNI SARDAR [F] BALARAM SARDAR [M] Mr. ASIT SARDAR [M] Mr. GUNADHAR BHUMIJ [M] Mrs. ANJALI SINGH [F] Mr. BADAL SARDAR [M] Mr. VIDYADHAR SINGH [M] SANTOSH SINGH [M] UTAM KUMAR SINGH [M] KARTIK SARDAR [M] BHIRANJAN SARDAR [M] MIRJA SARDAR [M] JANTA SADAR [M] PUSHPLATA SINGH[M] DARA SINGH [M] Miss. BASANTI SARDAR [F] VIJAY SARDAR [M] LALA SINGH [M] SITARAM BHUMIJ [M] BISHEKHAR SARDAR [M] KARTIK SARDAR [M] SHASHI CHARAN SINGH [M] NANDLAL BHUMIJ [M] Miss. SUMITRA SINGH [F] JANVI SINGH [F] AJAY SINGH [M] RAJU SINGH [M]

– CHIRING – TILKAGARH – GITILATA – KUDADA – KHADADERA – TIRILDIH – TUDI – BADEDIH – TIRILDIH – TIRILDIH – BALIDIH – DEGPA – SANKARPUR – DEGAM – HATNABEDA – HATNABERA – CHANDPUR – CHARGIRA – GHAGIDIH – CHAIDIH – KHADADERA – KAWALI – PAURU – RANIDIH – TIRILDIH – PICHALI – CHAIGARA – GUTKA – BAHARDARI – BHUNTKA – CHANDPUR – PICHALI – BHURIDIH – BALIDIH – BALIDIH – BAHARDADIH – GITILATA – TIRILDIH -- PAURU – KARANDIH – BAHARDADIH – CHAIGARA – PICHALI – JANUMDIH – PICHALI – CHANGIRA – TIRILDIH – TIRILDIH – RAJABDSH – HATNADERA

97) 98) 99) 100)

Mr. AMULYA SARDAR [M] Mr. SUBODH SARDAR [M] Mr. RAJMOHAN [M] Mr. NIRANJAN SINGH [M]

– KUDRUKOCHA – JHARIA – SARDAR – TIRILDIH

Fig 8: People who contributed: ­

CHAPTER 3 METHODOLOGY 3.1 Sampling :­

Intravenous blood samples were collected from a total of 100 healthy unrelated individuals belonging to Bhumij tribe , which are Austro-Asiatic groups.Vacutainers were used to store blood with ice gels to maintain the cold temperature. Vacutainers contains EDTA (the potassium salt, or K2EDTA). This is a strong anticoagulant and these tubes are usually used for full blood counts (CBC) and blood films.Blood can be stored in it for 45 days from the date of collection.

3.2 MATERIALS & METHODS:­ Blood Collection Kit DISPOVAN® 10ml sterile syringes and VACUETTE® tubes were used for blood collection. The interior of the tube wall is coated with EDTA K3.

The tube is also available with an 8% liquid EDTA solution. The EDTA binds calcium ions thus blocking the coagulation cascade. Erythrocytes, leucocytes and thrombocytes are stable in EDTA anticoagulated blood for up to 24 hours at 4o C.

Fig 9 : Vacutainer

Fig 10 : Transfering blood from syringe to vacutainer

Materials required :­

•

Gloves

•

Apron

•

50 ml Falcon tubes

•

14 ml Falcon tubes

•

Tube stand

•

Gel Tray

•

Combs

•

Electrophoresis tank

•

Marker

•

Tissue roll

•

Autoclave Tape

•

Sequencing plates

•

Plate flap

•

Pipettes

•

1.5 ml eppendorf

•

PCR vials

•

Ice

•

Pippete Tips

•

Aluminium foil

•

Agrose

•

10 ml Disposable syringe

•

Torniquet

•

Cotton

•

Petriplate

•

Flask 250 ml

•

Measuring cylinder

•

Water bath

Reagents Required:REAGENT A (TRITONATE BUFFER ):

Tris HCl (pH 8.0)

-

10ml (pH8)

Sucrose

-

109.54 gm(320mM) for osmoregulation

MgCl2

-

5 ml(5mM) for creation of pore on cell surface

Triton X 100

-

10 ml to lyse the RBCs

DDW

-

1000ml (autoclaved)

Reag ent B (Lysis Buffer II):

Tris HCl (pH 8.0)

-

40ml(400mM)

NA-EDTA

-

12ml(60mM)

NaCl

-

15ml(150mM)

SDS

-

5ml

REAGENT C:

Sodium per chlorate -

35.115 gm

Milli Q water

50 ml

-

TRIS SATURATED ALCOHOL:

Phenol

-

Distilled

8-Hydroxy Quinoline -

0.1%

Tris HCl (pH 8.0)

-

0.5 M

Tris HCl (pH 8.0)

-

0.1 M

CHLOROFORM : ISOAMYL ALCOHOL (24:1):

Chloroform

-

24ml

Isoamyl Alcohol

-

1ml

T. E. BUFFER Tris HCl (pH 7.5)

-

1ml (10mM)

EDTA (pH 8.0)

-

0.2 ml (1mM)

Make it upto 100ml with DDW

70% ALCOHOL: Absolute Alcohol

-

70ml

DDW

-

30ml

REAGENTS USED FOR GEL ELECTROPHORESIS:1 X TAE BUFFER: 10 x TAE Buffer - 50 ml DDW

- 950 ml

6 X LOADING DYE:

Bromophenol Blue

-

0.125g

Xylene Cyanol FF

-

0.125g

Glycerol

-

15ml

(Diluted with DDW to make up volume to 50ml) ETHIDIUM BROMIDE:

Ethidium Bromide

-

10mg

DDW

-.

1ml

(Stored in dark bottles)

PCR COMPONENTS :

Master Mix (6µl) : + 4 µl DNA Milli Q

– Make the master mix upto 6µl

PCR Buffer - 1 × (no. of samples)n µl MgCl2

– 0.8 × n µl

Forward Primer : M23 primer – 0.05 × n µl M12 primer – 0.1 × n µl M15 primer – 0.14 × n µl M95 primer – 0.2 × n µl M82 primer – 0.2 × n µl Reverse Primer: M23 primer – 0.05 × n µl M12 primer – 0.1 × n µl M15 primer – 0.14 × n µl M95 primer – 0.2 × n µl M82 primer – 0.2 × n µl Dntps

– 0.6 × n µl

Taq Polymerase - 1 × n µl REAGENTS FOR PCR SEQUENCING AND PROCESSING:

Big Dye – 25µl Sequencing Buffer – 175µl Formamide – 500 µl Milli Q - 500µl 80% Ethanol – 9600 µl 3M Sodium Acetate – 120µl Absolute alcohol – 3 ml

INSTRUMENTS USED:

•

Centrifuge (Eppendorff 5810R, Biofuge, Remi R8C)

•

PCR Thermo Cyclers (MJ Research PTC 200, Gene Amp 9700,

Eppendorff, Veriti)

Fig11 : MJ Research PCR

Fig 12 : PCR ( Eppendorf and Veriti) •

Electrophoresis Apparatus (Pharmacia Biotech EPS600, Hoefer power

pack) •

Trans illuminator (Syngene)

•

Vortex

•

ABI PRISM® 3730xl DNA Analyzer

The ABI PRISM® 3730xl DNA Sequencer automatically analyzes DNA molecules labeled with multiple fluorescent dyes. It consists of a charge couple device (CCD) camera and a power Macintosh® computer that includes software for data collection and data analysis. After samples are loaded onto

the system’s vertical gel, they undergo electrophoresis, laser detection, and computer analysis. Electrophoretic separation can be viewed on-screen in realtime.

Fig 13: DNA Sequencer

SEQUENCE ANALYSIS SOFTWARES:

Sequencing Analysis Software™ Ver. 5.2 Two software packages automatically process gel files or raw sample files to analyze sample files with base calls matching sequence peaks.

Sequencing Analysis Software™ Ver. 5.2 is used for analysis of data for 3730 and 3730xl genetic analyzers running on a Mac® OS platform.

Sequencing Analysis Software™ is powered by multiple base caller algorithms to perform signal processing and classification of peaks from raw data collected from ABI PRISM® Genetic Analyzers. The result yields accurate sequence data with electropherograms that can be viewed by Sequencing Analysis Software™ or Edit View software. If the KB basecaller is used. It defines and displays mixed bases along with calculated quality values. It calculates clear range and sample score. It creates output files in ABI (.seq), FASTA (.seq), Phred (.phd.1), and standard chromatogram format (.scf) formats. It also generates an analysis report containing sample analysis statistics.

Fig 14: DNA sequencing analysis software

Auto Assembler Version 3.1.2

This is a sequence assembly program and can handle at least 1000 sequences of 500 bp. It allows on-screen alignment of chromatograms. The manufacturer claims that the software has no known limitations or bugs. It certainly has the nice feature of lining up all the electropherograms under each other making analysis easier. Moreover, it is

user-friendly for editing process.

Fig 15: Auto Assembler Software

Protocol : Isolation of DNA from blood: DNA was isolated by Phenol-Chloroform method modified by Dr. Thangaraj (17)(b)(18)

To 9ml of blood sample, 36ml of Reagent-A was added in a 50ml polypropylene tube. The solution was mixed gently till the solution became clear.

The above solution was centrifuged at 2,700 rpm for 7min to obtain a pellet free from RBCs. The supernatant containing lysed RBCs were discarded carefully.

The pellet was disturbed thoroughly and half the volume as that of blood sample (roughly 2ml) of Reagent B was added. The solution was mixed thoroughly by inverting very gently for 3-4min till the solution became viscous.

To the above solution, 500µl of Reagent C was added and mixed gently for 3-4min. It precipitates protein molecules which may inhibit PCR.

2ml each of phenol and chloroform was added to the above mixture.It is a deproteinising reagent.

It was mixed well and centrifuged at 3,500 rpm for 8min to separate 3 layers viz. aqueous layer, protein layer and solvent layer.

Fig 16 : aqueous layer, protein layer and solvent layer.

The aqueous layer containing DNA was carefully transferred into a 15ml polypropylene centrifuge tube using a broad mouth tip.

3ml of chloroform was added to the supernatant and mixed gently for 1 min. It was then centrifuged at 2,000 rpm for 5min. Chloroform removes left over phenol and protein which hinders PCR. Centrifugation resulted to give 2 clear layers of DNA and Chloroform .

Fig 17 : 2 clear layers of DNA and Chloroform .

The aqueous phase having DNA obtained was transferred to a fresh polypropylene centrifuge tube.

To this phase, 2ml of chilled isopropyl alcohol was added. It was mixed gently to precipitate the DNA.

Fig 18 : DNA Extracted

The DNA thread was spooled out and transferred to a fresh Eppendorf tube.

The DNA was washed twice with 70% alcohol and vortexed for 10 seconds.

The pellet was dried properly for 10-15 min to ensure that whole alcohol had dried off.

The pellet was dissolved in 100 ml of TE Buffer and incubated in water bath at 55ºC for 45 min to enhance the dissolution.

The DNA samples were stored at 4ºC. Dilution of DNA: 5 µl of DNA was mixed in 450 µl of TE buffer in a fresh eppendorf.

It was incubated in 4oC overnight.

Gel Electrophoresis : 0.96 gm of agrose was added to 120 ml of 1 x TAE buffer.

It was boiled and cooled to room temperature.

1 drop of EtBr was added and gel was casted in the gel tray.

5µl of diluted DNA was loaded with loading dye in the gel.

Bands were analysed under UV transilluminator to confirm proper dilution.

Fig 19 : Gel Check of Dilution

PCR : 4µl diluted DNA was added to labeled autoclaved PCR vials.

Master mix of 6µl was added to each vial.

Master mix for 23rd primer: 1. Milli Q – 2.5 × n (no. of samples) 2. PCR Buffer – 1 × n (no. of samples) 3. MgCl2 – 0.8 × n (no. of samples) 4. Forward primer – 0.05 × n (no. of samples) 5. Reverse primer – 0.05 × n (no. of samples) 6. Dntps – 0.6 × n (no. of samples) 7. Taq polymerase – 1 × n (no. of samples) Master mix for 15th primer: 1. Milli Q – 2.32 × n (no. of samples) 2. PCR Buffer – 1 × n (no. of samples) 3. MgCl2 – 0.8 × n (no. of samples) 4. Forward primer – 0.14 × n (no. of samples) 5. Reverse primer – 0.14 × n (no. of samples) 6. Dntps – 0.6 × n (no. of samples) 7. Taq polymerase – 1 × n (no. of samples) Master mix for 12th primer: 1. Milli Q – 2.4 × n (no. of samples) 2. PCR Buffer – 1 × n (no. of samples) 3. MgCl2 – 0.8 × n (no. of samples) 4. Forward primer – 0.1 × n (no. of samples) 5. Reverse primer – 0.1 × n (no. of samples) 6. Dntps – 0.6 × n (no. of samples) 7. Taq polymerase – 1 × n (no. of samples)

Master mix for 95th primer: 1. Milli Q – 2.2 × n (no. of samples) 2. PCR Buffer – 1 × n (no. of samples) 3. MgCl2 – 0.8 × n (no. of samples) 4. Forward primer – 0.2 × n (no. of samples) 5. Reverse primer – 0.2 × n (no. of samples) 6. Dntps – 0.6 × n (no. of samples) 7. Taq polymerase – 1 × n (no. of samples)

Master mix for 82nd primer: 1. Milli Q – 2.2 × n (no. of samples) 2. PCR Buffer – 1 × n (no. of samples) 3. MgCl2 – 0.8 × n (no. of samples) 4. Forward primer – 0.2 × n (no. of samples) 5. Reverse primer – 0.2 × n (no. of samples) 6. Dntps – 0.6 × n (no. of samples) 7. Taq polymerase – 1 × n (no. of samples)

PCR conditions :Mt DNA primer:- [ M23, M15, M12 ] •

95o C– 5 min.

•

95o C– 30 sec.

•

58o C– 30 sec.

•

72o C– 3 min.

•

72o C– 7 min.

•

4o C- ∞

•

35 cycles

Y DNA primer:- [ M95, M82] •

96o C– 5 min.

•

94o C– 1 min.

•

54o C– 1 min.

•

72o C– 1 min.

•

72o C– 5 min.

•

4o C- ∞

•

35 cycles

Gel Electrophoresis of PCR products : Gel check was done for each amplified PCR product.

Fig 20 : Gel Check of PCR products These amplified products were stored at 4oC .

Sequencing of PCR products : 1µl of PCR product was added to each well of sequencing plate.

1/3rd of the primer concentration [a] used for PCR × no. of samples was calculated as [b].

Any one of the primer either forward or reverse was used.

Milli Q = ( 2.2 – [a] ) × no. of samples was added to [b].

The resulting mixture of primer and milli Q was added 2.2 µl to each well.

175 µl of Sequencing buffer + 25µl of Big Dye was prepared into a mixture.

1.8 µl of the Dye Buffer mix was added to each well.

The plate was centrifuged at 12000 rpm for few seconds.

The plate was covered properly with alcohol washed flap and kept for sequence PCR.

Conditions for sequence PCR:•

96o C– 10 sec.

•

55o C– 5 sec.

•

60o C– 4 min.

•

4o C- ∞

•

30 cycles Processing :-

120µl of Sodium Acetate + 3 ml of absolute alcohol was made into a mixture.

25µl of the above mixture was added to all the wells of sequencing plate.

It was incubated at room temperature for 10 min.

The plate was wrapped in tissue roll.

It was centrifuged at 4000 rpm at 16oC for 16 min.

The plate was gently inverted to discard sodium acetate mixture.

100 µl of 80% alcohol [ 8ml alcohol + 2 ml Milli Q] was added to precipitate DNA. It was centrifuged at 4000 rmp at 16oC for 11 min.

The plate was gently inverted to discard alcohol.

An inverse spin of < 300 rpm was given to discard excess alcohol.

The plate was incubated in dark for 10 min. to vapourize remaining alcohol.

500µl of formamide + 500 µl of Milli Q is made into a mixture. 10µl of this mix was added to each well.

The sequencing plate was kept for DNA analysis.

Note: Sodium acetate precipitates DNA. Alcohol removes unnecessary molecules and washes the DNA. Formamide is toxic. It is used to convert double stranded DNA into single strands which helps in sequencing of either forward or reverse strands.

DNA analysis: Analysis was done by the Sequencing Analysis Software™ Ver. 5.2.

Blue peaks indicate good sequences. Red and yellow peak gives noisy sequences.

Auto Assembler Version 3.1.2 helps to auto assemble the sequences matching it with the mitochondrial map. It makes assembling easier. We note down the mutations , site of mutation and sample number.

3.3 PURPOSE OF STUDY : Since the completion of the human genome sequencing project, the discovery and characterization of human genetic variation is a principal focus for future research. Comparative studies across ethnically diverse human populations and across human and nonhuman primate species is important for reconstructing human evolutionary history and for understanding the genetic basis of human disease.

3.4 PRECAUTIONS:­

Gloves should be worn.

Use autoclave tips, eppendorf , falcon tubes and PCR vials.

Store the samples at 4oC and work in an aseptic environment . The exhaust

fan should be on while working.

Protein contamination should be avoided while pipetting out supernatant as it hinders PCR. EtBr is light sensitive and carcinogenic. It should be wrapped in foil and handled wearing gloves.

Big Dye is light sensitive. It should me added to the mixture in dim light.

Pipetting error should be avoided.

PCR reagents should be fresh and always be kept in deep freezer.

Gel should be handled with gloves as it contains EtBr. Analysis of haplogroup should be perfect, avoid noise conditions.

3.5 THE RESEARCH SITE :­

Fig 21 Map: Site of sample collection (state:Jharkhand, district: East and West Singhbhum)

Country : India State : Jharkhand District : East and West Singhbhum Village: CHOLAGORA, TILKAGARH, GHAGIDIH, DOMJURI, GOMIASAI, KITADIH, BHELAIDIH, VELAIDIH, JONRAGARA, TILKAGARH, GITILATA, TETLA, CHANDPUR, TIRILDIH, BALIDIH etc.

3.6 DATA COLLECTION

Blood samples were collected by the consent of the people . They were given a consent form to fill .

Fig 22 : Consent Form

CHAPTER IV: ANALYSIS OF RESULTS 4.1 Results :Bhumij Tribe populations show O-M95 as the most common haplogroup. This haplogroup is also found in a relatively high frequency in the Khasi and Nicobarese. This may underscore that the Mundari, Khasi-Khmuic and MonKhmer groups of India are not only linguistically related but also genetically linked, probably with a single but relatively broad paternal genetic source.

This haplogroup has been reported to be absent or present in low frequency in other linguistic groups of India [7,8,9,10,11,12], suggesting a distinct genetic identity of the Indian Austro-Asiatic populations. Thus the predominance of this haplogroup both in Austro-Asiatic populations of India and Southeast Asia and its absence/negligible presence in other Asian populations suggests a common genetic heritage of the people of this linguistic family.

Table 1 TRIBE NAME BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ

SAMPLE No. 1 2 5 6 7 9 10 13 14 15 16 17 18 19 20 22 23 24 25

GENDER M M M M M M M M M M M M M M M M M M M

M95 Primer D D D

D D D

D D D D D D

M82 Primer

A

A D A

HG O2a O2a O2a UC UC UC O2a O2a O2a UC UC H1 UC O2a O2a O2a O2a O2a O2a

BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ

26 27 28 29 30 31 32 34 35 36 38 39 41 42 43 44 45 46 47 49 50 51 52 53 55 56 57 58 59 60 61 63 65 66 67 68 71 72 73 75 76 77 78 79 80 81 82 84 86 87 88 89

M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M M

D A D D A A D A D D D D

A D D D D D A

D D D D D A A D D

D D D D A

D

D D D D D D D D D D D D

A

D D

D

A

D

A

O2a UC O2a O2a H1 H1 O2a H1 O2a O2a O2a O2a UC UC O2a O2a O2a O2a O2a UC UC O2a O2a O2a O2a O2a H1 UC O2a O2a UC O2a O2a O2a O2a UC UC O2a UC O2a O2a O2a O2a O2a O2a O2a O2a O2a O2a O2a O2a UC

BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ BHUMIJ

90 91 92 95 96 97 98 99 100

M M M M M M M M M

D D D D D

D A D

A

D

O2a O2a O2a O2a O2a UC O2a H1 O2a

Fig 23 : Frequency Chart of Y haplogroup X axis : Haplogroup distribution Y axis : No. of Samples

Table 2 Haplo‐ Group

SAMPLE NO.

M23 Primer

M12

M15 Primer

1

10398G 10400T

M

2

16231C 16223T 16291T 16319A 16362C 16519C 16360G

10398G 10400T

M

3 4

16231C 16223T 16291T 16319A 16362C 16519C 16545[DEL]T 16239T 16245G 16302G 16330G 16345G 16372A 16373A 16384A

UC UC

5

10025[DEL]A 10398G 10400T

6

16223T 16291T 16519C 16545[DEL]T 16330G 16387T

10398G 10400T

M M

7 8

15938T 16102C 16111T 16232T 16330G 16353T 16373A 16545[DEL]T 16551[DEL]T

8557A

UC M39a2

9

10

16519C

UC UC

11 12

16223T 16325C 16223T 16295T 16318T[TR] 16325C 16395[DEL]C 16417T[TR] 16426A 16451A 16460T 16499C[TR] 16503A

13

16183C 16189C 16223T

8047C

14

15

16223T 16274A 16319A 16471A 16233C 16234A 16265C 16294A 16323A

8064A

16

10398G 10400T 10025[DEL]A 10398G 10400T

M

M

M38b 10398G 10400T

M UC UC

17

16275 [DEL]A 16438A 16073G[TR]

10025[DEL]A 10398G 10400T 10750G

18

16051G 16075C 16399G

10109T

19

20

21

16223T 16309G

22

16145A 16223T 16240G 16261T 16311C 16319A 16519C

M

UC

UC UC 10398G 10400T 10025[DEL]A 10398G 10400T

M

M4a

23

16189[DEL]T 16201A 16225T 16245T 16246T 16247T 16248T 16270G

8047C

24

25

16189C 16223T 16325C 16468C

26 27

16129A 16182C 16183C 16189C 16223T 16325C 16468C

28 29 30 31

32

16290T 16519C 15924G 16126C 16183C 16223T 16232A 16245T 16092C 16145A 16185T 16239T 16325C 16266T 16304C 16311C 16357[DEL]T 16362C 16092C 16145A 16185T 16239T 16325C

36

16318T 16318T[TR]

37

38

16209C 16223T 16275G 16438A

8047C

40 41

16294T 16319A 16356C 16463G

43

16129A 16266T 16290T 16318G 16320T

47

10398G 10400T 10398A 10454C

10398G 10400T 10398G 10400T 10398G 10400T 10398G 10400T

M UC

M

M M

M UC

10398G 10400T 10398G 10400T

M M R6a1

M

UC 10398G 10400T

16362C 16115A 16146G 16519C 16170C 16183C 16189C 16225T 16226T 16227T 16230T 16239T 16240G 16245T 16246T 16247T 16248T 16319A 16519C 16223T 16274A

UC UC

45 46

UC

UC

42

44

UC 10398A 10400T

8149G

35

39

UC UC

8149G

UC

UC UC

8047C 8392A

16111T 16092C 16145A 16185T 16239T 16325C 16189C 16223T 16261T 16269T+ 16274A 16311C 16319A 16319A 16352C 16086C 16223T 16234A 16274A 16382T

33 34

10398G 10400T

10143A 10289G 10398G 10400T

R7a1a

M

10398G 10400T

M

48 49

50 51

16183C 16189C 16223T 16086C 16111T 16223T 16399G 16223T 16270T 16274A 16319A 16352C 16086C 16269T 16279A 16353T 15938T 16102C 16111T 16216T[TR] 16228G[TR] 16230T[TR] 16260A 16275G

8047C

10398G 10400T

UC

UC

10398G 10400T M

52

53

16223T 16362C 16343G 16355T

54

55

16223T 16318T[TR] 16325C 16075C 16093C 16260T 16261T 16262T 16319A 16362C

10143A 10400T 10289G 10398G 10400T 10398G 10400T

10143A 10289G

56

8149G

57

M38b

16223T 16284G 16327T 16398A

58

59

60

61

62

16017C 16093C 16126C 16145A 16223T

63

64

65 66

16183C [TR] 16189C 16194C+ 16223T 16256T 16274A 16319A 16390A

67

16189C 16194C+ 16195C+ 16223T 16325C 16468C

68

69

70

16179T 16223T 16289G 16294T 16319A 16463G 15954G

71

72

16179T 16223T 16289G 16294T 16319A 16428C[TR]

M M

R7

UC 10118C 10325A 10370C 10398G 10400T

10398G 10400T 10398G 10400T 10398G 10400T 10531G

M UC UC M M M31a2

UC 10143A 10289G

10398G 10400T 10398G 8047C 10400T 10025[DEL]A 10398G

10400T

UC

UC UC UC

UC UC UC M40a1a M

M40a1a

73

74

75

76

77

16189C 16223T 16275G

78

79

80

8047C

81 82

16188T 16223T 16231C 16233C 16234A 16362C 16170C[TR] 16172C+ 16183C [TR] 16189C 16194C+ 16223T 16274A 16319A 16320T

83

84

85

86

87

16180C[TR] 16189C 16194C+ 16195C+ 16223T 16232T+ 16519C

88

89

90

91 92 93 94 95

96

10398G 10400T 10398G 10400T 10398G 10400T 10398G 10400T 10143A 10289G 10398G 10400T 10398G 10400T

8110C

10398G 10400T 10398G 10400T

M M

UC M M

UC

M M

UC UC 10398G 10400T

M

UC

8047C

UC

16194C+ 16195C+ 16189C 16223T 16274A 16319A 16320T 16519C 16170C 16172C+ 16183C 16221A 16225T 16228A 16230T 16233T 16239T 16242A

10398G 10400T

16170C[TR] 16182C 16183C 16189C 16213A 16214A 16223T 16228A 16234A 16236A 16238A 16239A 16242T 16319A 16409[DEL]T 15954G 16214G[TR] 16231G 16239T

M

UC 10398G 10400T

10025[DEL]A 10398G 10400T

16179T 16223T 16289G 16294T 16319A 16356C 16463G 16189C 16194T+ 16196+ 16223T 16300G

M

UC

M UC

M

M40a1a

UC

UC

10398G 10400T

M

16245T 16246T 16247T 16248T

97

98

16319A 16320T 16129A 16266T 16290T 16318G 16320T 16362C 16179T 16223T 16289G 16294T 16319A 16356C

99 100

10398G 10400T 10398G 10400T 10398A 10400T 10398G 10400T

M M

UC M40a1a

Fig 24 : Frequency Distribution of mt DNA

4.2 DISCUSSION : 4.2.1 Y-chromosomal Analysis: In present study Bhumij, an astroasiatic tribal population of Jharkhand is showing Haplogroup M95-O2a as the most abundant(70%) Y-DNA haplogroup.O2a subclade of haplogroup O is already known to be the most abundant Y-DNA haplogroup of all in austroasiatic tribes of India.

Haplogroup O was one of eight haplogroups detected in an Indian population at frequencies > 5% (overall, 22.9% with 14.6% Subclade O2a and 8.0% Subclade O3a3c; Sengupta et al. 2006). A relatively high proportion of Haplogroup O was detected across all tribal linguistic classes (Austroasiatic, Dravidian, Indo-European, and Tibeto-Burman) but the haplogroup was rare within caste populations, supporting theories that caste and tribal populations within India had separate origins (Cordaux et al. 2004). The Austroasiatic language family has a high prevalence in Southeast Asia, and it is thought to be one of the oldest language families in India. These two observations suggest that there may be a linkage between Indian and Southwest Asian Austroasiatics. Based on current distributions of Haplogroup O, Austroasiatic speakers in India likely originated from Southeast Asia, but other results indicate that the demographic history may not be this simple. More recent studies argue that Austroasiatic populations originated in India, and then migrated to Southeast Asia via the Northeast Indian corridor (Kumar et al.

2007).

Figure 25. Worldwide frequency distribution of Haplogroup O. The red area within each pie chart indicates the frequency of Haplogroup O within that location. The labels and associated pie charts also indicate the average frequency of Haplogroup O within different language families of China. It is clear from this frequency distribution map that Haplogroup O is most prevalent within East and Southeast Asia, with moderate frequencies detected in men from Central Asia and Oceania.

Figure 26. Relative frequency distribution of the four main subclades of Haplogroup O.

There is a wide discrepancy in the time and place of origin of Subclade O2a.

The SNP mutation M95 that defines Subclade O2a is currently thought to have orginated in Indian Austroasiatic populations approximately 65,000 years ago (Kumar et al. 2007), although previous studies have argued for a Southeast Asian origin approximately 8,800 years ago (Kayser et al. 2003, Karafet et al.

2005). Yet another study estimated the age of O2a to be 11,700 Âą 1,600, which provides support for the previous age estimate of 8,800 (versus 65,000; Sengupta et al. 2006). This subclade is detected mostly in Southeast Asia, in south Asian tribal populations, in populations of India (Sengupta et al. 2006, Kumar et al. 2007) and at a low frequency in Japan (1.9%; Hammer et al.

2006). O2a shows an interesting pattern in India as it occurs a high frequencies within all tribal language classes (Austroasiatic, 53.1%; Dravidian, 26.7%; Tibeto-Burman, 18.4%; Indo-European, 28.6%) but is virtually absent in caste populations (Sengupta et al. 2006). Recent data indicates that, on average, there seems to be a decreasing frequency of O2a from India to Southeast Asia (but see Karafet et al. 2005 and references therein that found highest frequencies of O2a to occur in Southeast Asia). For example, the average frequency of Subclade O2a in Austroasiatic populations is estimated at 54%, whereas the same study found O2a in 38% of Austroasiatic men in Southeast Asia and only 14.7% of non-Austroasiatic Southeast Asians (Kumar et al. 2007). So far only two men in Oceania have been found to carry M95 (Sue et al. 2000, Capelli et al. 2001). A study on the Andaman and Nicobar Islands found that of the 30% (n = 10) of Andamanese men that were Haplogroup O, 10% (1 of 3) were in Subclade O2a, and all of the Nicobarese were in Subclade O2a (11 men were tested).

H1-M82 haplogroup is found at a high frequency in Indian Subcontinent. It is generally rare outside of the Indian subcontinent but is common among the Romani people, particularly the H-M82 subgroup. It is a branch of Haplogroup F, and is believed to have arisen in India between 20,000 and 30,000 years ago. Its probable site of introduction is India because it is high concentrated here. It seems to represent the main Y-haplogroup of the indigenous paleolithic inhabitants of India, because it is the most frequent Yhaplogroup of tribal populations (25-35%). On the other hand, its presence in upper castes is quite rare (ca. 10%).So, low percentage (7.5%) of H1a-M82 haplogroup is quite explainable in Bhumij population of Jharkhand.

Fig 27: Derived samples derived from M95 primer leads to O2a‐ Haplogroup On Y chromosome phylogenetic tree

Fig 28: Derived samples derived from M82 primer leads to H1‐ Haplogroup On Y chromosome phylogenetic tree

4.2.2 Mitochondrial DNA Analysis: In present study, macrohaplogroup M shows highest occurrence of about 50 %. M is the single most common mtDNA haplogroup in Asia, and peaks in Bangladesh where it represents two thirds of the maternal lineages, and is ubiquitous in India where it has a 60% frequency. Due to its great age, haplogroup M is an mtDNA lineage which does not correspond well to present-day ethnic groups, as it spans Siberian, Native American, East Asian, Southeast Asian, Central Asian, South Asian, Melanesian populations at a considerable frequency . Among the descendants of M are C, D, E, G, Q, and Z, with Z and G being observed in North Eurasian populations, C and D being shared between North Eurasian and Native American populations, E being observed in Southeast Asian populations, and Q being observed in Melanesian populations. The lineages M31, M38, M39, M4 and M40 are specific to South Asia. Haplogroup M4 is found mainly in South Asia but some sequences in Eastern Saudi Arabia. M4a has been reported in Gujarat, India.

Haplogroup R is a very extended mitochondrial DNA (mtDNA) haplogroup and is the most common macro-haplogroup in West Eurasia. The most recent study dates the origin of haplogroup R to 66.6kya. South Asia lies on the way of earliest dispersals from Africa and is therefore a valuable well of knowledge on early human migration. The analysis of the indigenous haplogroup R lineages in India points to a common first spread of the root haplotypes of M, N, and R along the southern route some 60â&#x20AC;&#x201C;70 kya.

Haplogroup R has wide diversity and antiquity among varied ethnic status and different linguistic families in South Asia. In indian western region among the castes and southern region among the tribes show higher haplogroup diversity than the other regions, possibly suggesting their autochthonous status. R6'7 (16362) shows the most important presence is among Austro-Asiatic languages speakers from India (10%). Small frequencies in India and Pakistan.R7 subclades in india, R7a mainly found in East India, specially in Santals from Bihar and R7b in Dravidian tribes of East India.

Fig 29 : A‐G Mutaion

Fig 30: Insertion T

Fig 31 : M82 primer haplogroup analysis giving Derived

Fig 32 :M95 primer haplogroup analysis giving Ancestral

Fig 33 : M95 primer haplogroup analysis Giving Derived M82 primer : CATTTTCAT_AT gives Ancestral CCTGAAA_C gives Derived M95 primer: TTAGTG_T_TGG gives Derived TTAGTG_C_TGG gives Ancestral

4.3 SUMMARY AND CONCLUSION

The objective of this project was to infer about the genetic diversity of

Bhumij tribal population of Jharkhand with other populations of India. In the overall analysis, it was observed that most of the individuals of Bhumij tribe population were falling in Indian specific macro haplogroup M displaying the array of South Asian specific lineages.

In addition Y chromosomal analysis is showing 70% percentage of individuals falling into O2a-M95 haplogroup, found frequently among Austro-Asiatic peoples.

Also it is evident that our investigation of the small population can offer no more than snapshot of Indian pre history from the genetic perspective.

In future detailed phylogeographic and phylogenetic analyses of more tribal population can reveal some interesting patterns of maternal as well as paternal lineages and genetic footprints of India population.

Recent studies by Dr. Thangaraj et.al. 2005 a, b opens new insights to many unique studies that can be made to found unique patterns of genetic [g]

foot prints of different maternal and paternal lineages in India.

TERMS

ALLELE: The specific nucleotide (A, T, G, C) found at a location on the chromosome.

CAMBRIDGE REFERENCE SEQUENCE (CRS): The reference sequence to which all-human mtDNA sequences are compared. The CRS was the first complete human mtDNA sequence, published in 1981.

CHROMOSOME: Condensed DNA. This “compact packaging” allows DNA to fit in the nucleus of a cell. The human genome contains 23 pairs of chromosomes for a total of 46. We receive 23 from our mother and 23 from our father. Each chromosome is a single strand of DNA containing genes.

Genes provide information for the structure and function of proteins, the building blocks of life. 23 Chromosome Pairs

ELECTROPHEROGRAM: The output of an automated genetic analyzer that shows the sequence of a sample through fluorescent detection.

EUKARYOTE: An organism whose cells have a nucleus and other membrane bound organelles. All organisms except viruses, bacteria and blue-green algae are eukaryotes.

GENETIC MARKERS: exact locations on the Y-chromosome that scientists use to look for specific information.

HAPLOGROUP: A group of lineages defined by linked diagnostic mutations.

Human mtDNA haplogroups are labeled A-Z and are often regionally specific.

Human Y-chromosome haplogroups are grouped by letter (A-R). The relative frequency of these haplogroups varies from population to population.

HAPLOTYPE: A more specific subgroup of a haplogroup. For example, your mtDNA sequence and the sequences of other individuals whose mtDNA exactly matches your own, are considered a haplotype. Many different haplotypes are grouped together to form a more generalized unit, called a haplogroup.

LOCUS: The position of a gene on a chromosome.

MITOCHONDRION: An extra-nuclear (outside the nucleus) organelle responsible for energy production within the cell.

MITOCHONDRIAL DNA (mtDNA): A circular genome located in the mitochondrion that contains different information than DNA found in the nucleus. It is approximately 16,569 base pairs in length.

MUTATION: The process of a change in the genome through a mistake in the cellular machinery that copies DNA.

NUCLEUS: The membrane bound organelle containing the genome of humans organized into chromosomes. Note that mtDNA is located in the mitochondrion, outside of the nucleus.

NUCLEOTIDE: Informational sub-units, when strung together in a specific sequence make-up DNA. There are four different sub-units: Adenine (A), Guanine (G), Thymine (T), and Cytosine(C). Adenine and Thymine normally pair together and Guanine and Cytosine normally pair together. Nucleotides are also referred to as bases.

NUCLEOTIDE POSITION (np): The position of each nucleotide in a genome is called the Nucleotide position (np).

POINT MUTATION: one nucleotide is exchanged for another nucleotide by mistake at a specific location.

POLYMERASE CHAIN REACTION (PCR): A powerful method that exploits certain features of DNA replication for amplifying specific DNA segments. The method amplifies specific DNA segments by cycles of template denaturation; primer addition; primer annealing and replication using thermo stable DNA polymerase. The degree of amplification achieved is set at a theoretical maximum of 2N, where N is the number of cycles, e.g. 20 cycles gives theoretical1048576 fold amplification.

POLYMORPHISM: A difference in the DNA sequence among individuals or groups.

PROTEINASE: An enzyme that digests or breaks apart proteins.

PURINE: A type of nucleotide or base, the information subunits of DNA. Adenine (A) and guanine (G) are purines.

PYRIMIDINE: A type of nucleotide or base, the information subunits of DNA. Thymine (T) and cytosine (C) are pyrimidines.

SNP: Single Nucleotide Polymorphism, a specific type of point mutation.

TRANSITION: A type of nucleotide-pair mutation involving the replacement of a purine with another purine, or of a pyrimidine with another pyrimidine

(e.g. GC with AT. This type of mutation is much more common than a transversion.

TRANSVERSION: A type of nucleotide-pair mutation involving the replacement of a purine with a pyrimidine, or vice versa for example GC with TA. This type of mutation is much less common than a transition.

•

Transition

Transversion

C to T

G to T

G to A

C to A

Y-CHROMOSOME: Humans each have one pair of sex chromosomes. The Y-chromosome is associated with male characteristics in mammals. Females normally do not have a Y-chromosome, but instead have two X-chromosomes (XX). Males have one X-chromosome and one Y-chromosome (XY).

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