bhumij tribe of jharkhand, india
DESCRIPTION
College:Loyola College,Chennai.Duration: 6 months (2009-2010). Guide: Prof. K. Thangaraj.Lab: CCMB, Hyderabad.Work done independently.Done at CCMB- Hyderabad,India. My work on proving that Bhumij Tribe of Jharkhand belong to ancient race of Austro Asiatic Tribes which entered India before independance. This work is based on Field work and Biotechnological tools.TRANSCRIPT
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: Introduction to the Study 1.1 Introduction 1.2 Background 1.3 Statement of Purpose 1.4 Aims and Objectives of Study 1.5 Hypothesis Chapter II: Review of the Literature 2.1 Introduction of Bhumij Tribe Chapter III: Methodology 3.1 Sampling 3.2 Materials and Method 3.3 Purpose of Study 3.4 Precautions 3.5 The Research Site 3.6 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
% - Percentage �C - Degree Celsius ATP - Adenosine 5’-triphosphate bp - base pair(s) cm - centimeter cpm - counts per minute dATP - 2’-deoxyadenosine 5’-triphosphate dCTP - 2’-deoxycytidine 5’-triphosphate DDW - Double distilled water dGTP - 2’-deoxyguanosine 5’-triphosphate D-loop - The displacement loop DNA - Deoxyribonucleic acid dNTP - 2’-deoxynucleotide 5’-triphosphate ddNTP - 2’,3’-dideoxynucleotide 5’-triphosphate dTTP - 2’-deoxythymidine 5’-triphosphate EDTA - Ethylene diamine tetra acetic acid Et.Br - Ethidium bromide Extn - Extension Figure - Figure g - gram kb - kilo base M - molarity mA - milli ampere mg - milligram min - minutes ml - millilitre mm - millimeter mM - millimolar mtDNA - mitochondrial DNA mtRNA - mitochontrial RNA rRNA - ribosomal RNA tRNA - transfer RNA N - Normality nm - nanometer NaOH - sodium hydroxide ng - nanogram OD - Optical density
OH - Origin of heavy chain replication OL - The L-strand origin PCR - Polymerase chain reaction pM - picomole RNA - ribonucleic acid rpm. - Revolutions per minute SDDW - Sterile Double Distilled water SDS - Sodium dodecyl sulphate Sec. - Seconds SNPs - Single Nucleotide Polymorphisms SRY - Sex-Determining Region On Y Chromosome SSC - Sodium saline citrate STR - Short Tandem Repeat TAE - Tris-Acetate-EDTA TE - Tris-EDTA Tris - Tris (� ecogniz methyl) amino methane
TMRCA - Time to the most recent common ancestor
U - unit
UEP - unique event polymorphism UV - Ultraviolet V - Volts v/v - Volume/Volume w/v - Weight/Volume �g - Microgram �l - Microlitre �MW - Micro molar Watts YAP - Y-Alu polymorphism YCC - 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 Austro-
Asiatic. 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 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 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 Indo-
European, 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 PROJECT, THE SUBJECT OF GENETIC STUDY IS ‘‘BHUMIJ TRIBE’’ FROM 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).
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.
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.
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:
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.
In silico storage of all DNA database. Improve tools for data analysis.
Transfer related technologies to the private sector. Address the
ethical, legal and social issues (ELSI) that may arise from the project.
To provide information regarding human biological relationship
among different groups and human history.
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, paleo-
anthropological 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 (Cavalli-
Sforza 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 13-
polypeptide 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 vital-
mutations 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 16024-
16383) 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 chromosomeProperties of Y chromosome
p Haploid
p Non-recombining region
p Uniparental transmission
p Haploid
p Non-recombining region
p Uniparental transmission
SRYRPS4Y
ZFY
AMELYYRRM1,2
TSPYDYS7 (50f2/D)
KAL-YYRRM1,2
STSPYRRM2
DYS7 (50f2/E)
SMCY
YRRM1,2DAZ
DYS7 (50f2/C)BPY2CDY
sY160102(d)2pHY2.1
AZFa
AZFb
AZFc
DYS7 (50f2/B)DYS7 (50f2/A)
Human Y Chromosome
p arm
q arm
Hete
roch
rom
atic
regi
onEu
chro
mat
ic re
gion
PAR1
PAR2
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 non-recombining 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.
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.
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 non-
recombining 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:
RECENT AFRICAN 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-of-Africa model in the popular press, and academically the recent single-origin 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 INTRODUCTION OF BHUMIJ TRIBE(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 inter-marriage, 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 sub-tribe 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 VILLAGE 1) BHUJANG PRASAD SINGH [M] - CHOLAGORA 2) MANOJ SARDAR [M] - TILKAGARH 3) KANDI BHUMIJ [F] - GHAGIDIH 4) SUNDARI BHUMIJ [F] - GHAGIDIH 5) GURVA BHUMIJ [M] - GHAGIDIH 6) PURENDAR BHUMIJ [M] - GHAGIDIH 7) PARSURAM SARDAR [M] - GHAGIDIH 8) GITA SARDAR [F] - GHAGIDIH 9) Mr. SARDAR [M] - GHAGIDIH 10) SUKLAL SARDAR [M] - GHAGIDIH 11) KUNI SARDAR [M] - GHAGIDIH 12) MEENA SARDAR [F] - GHAGIDIH 13) RUP SINGH [M] - DOMJURI 14) ARUN SINGH [M] - DOMJURI 15) SRIKANT SINGH [M] - DOMJURI 16) SOHAN SINGH [M] - DOMJURI 17) GULAB SINGH [M] - DOMJURI 18) INDRA SINGH [M] - DOMJURI 19) DEVA SINGH [M] - DOMJURI 20) MOHAN SINGH [M] - DOMJURI 21) PADMAWATI SINGH[F] - DOMJURI 22) LAKHAN SINGH [M] – DOMJURI 23) SRIKANT SARDAR [M] – GOMIASAI 24) NARAYAN SARDAR [M] – KITADIH 25) KANHAI SARDAR [M] – GOMIASAL 26) BIRBAL SARDAR [M] – BHELAIDIH 27) ARUN SARDAR [M] – VELAIDIH 28) SRIKANT SARDAR [M] – VELAIDIH 29) MANGAL SINGH HANSDA [M] – JONRAGARA 30) DHURMU SARDAR [M] – TILKAGARH 31) BHUDRAI SARDAR [M] – TILKAGARH 32) RUPCHAND SARDAR [M] – TILKAGARH 33) Mrs. HULSAI SARDAR [F] – TILKAGARH 34) NARDE SARDAR [M] – TILKAGARH 35) SUDARSAN SARDAR [M] – TILKAGARH 36) Mr. GUNADHAR SARDAR [M] – GITILATA 37) SHEFALI SINGH [F] – GITILATA 38) SATRUGHON SARDAR [M] – TETLA 39) HARISHCHANDRA SINGH [M] – CHANDPUR 40) KAMALANI SINGH [F] - TIRILDIH 41) ARJUN SINGH [M] – GITILATA 42) SUDARSAN BHUMIJ [M] – BALIDIH 43) GANESH SINGH [M] – GITILATA 44) NARAYAN SINGH [M] – GITILATA 45) RATAN SARDAR [M] – TIRILDIH 46) H. SARDAR [M] – TIRILDIH
47) MOSO SARDAR [M] – CHIRING 48) BHAWARI SARDAR [M] – TILKAGARH 49) HARISHCHANDRA SINGH [M] – GITILATA 50) DHIRENDAR [M] – KUDADA 51) SURAJ PRABHASH SINGH [M] – KHADADERA 52) AJIT SINGH [M] – TIRILDIH 53) BALRAM SARDAR [M] – TUDI 54) BASANTI SARDAR [F] – BADEDIH 55) SOBINAY SINGH [M] – TIRILDIH 56) R.SINGH [M] – TIRILDIH 57) RAMKADA SARDAR [M] – BALIDIH 58) NIRMAL SARDAR [M] – DEGPA 59) LAKHI RAM HANSDA [M] – SANKARPUR 60) ARUN SARDAR [M] – DEGAM 61) TUNU SINGH [M] – HATNABEDA 62) MADHUSUDAN SINGH [M] – HATNABERA 63) LAKHAN SINGH [M] – CHANDPUR 64) BELBATI SINGH [F] – CHARGIRA 65) RAGHU BHUMIJ [M] – GHAGIDIH 66) NAND SINGH [M] – CHAIDIH 67) Mr. GURUCHARAN [M] – KHADADERA 68) BABLU SARDAR [M] – KAWALI 69) Miss. SABITA SARDAR [F] – PAURU 70) Miss. KUNI SARDAR [F] – RANIDIH 71) BALARAM SARDAR [M] – TIRILDIH 72) Mr. ASIT SARDAR [M] – PICHALI 73) Mr. GUNADHAR BHUMIJ [M] – CHAIGARA 74) Mrs. ANJALI SINGH [F] – GUTKA 75) Mr. BADAL SARDAR [M] – BAHARDARI 76) Mr. VIDYADHAR SINGH [M] – BHUNTKA 77) SANTOSH SINGH [M] – CHANDPUR 78) UTAM KUMAR SINGH [M] – PICHALI 79) KARTIK SARDAR [M] – BHURIDIH 80) BHIRANJAN SARDAR [M] – BALIDIH 81) MIRJA SARDAR [M] – BALIDIH 82) JANTA SADAR [M] – BAHARDADIH 83) PUSHPLATA SINGH[M] – GITILATA 84) DARA SINGH [M] – TIRILDIH 85) Miss. BASANTI SARDAR [F] -- PAURU 86) VIJAY SARDAR [M] – KARANDIH 87) LALA SINGH [M] – BAHARDADIH 88) SITARAM BHUMIJ [M] – CHAIGARA 89) BISHEKHAR SARDAR [M] – PICHALI 90) KARTIK SARDAR [M] – JANUMDIH 91) SHASHI CHARAN SINGH [M] – PICHALI 92) NANDLAL BHUMIJ [M] – CHANGIRA 93) Miss. SUMITRA SINGH [F] – TIRILDIH 94) JANVI SINGH [F] – TIRILDIH 95) AJAY SINGH [M] – RAJABDSH 96) RAJU SINGH [M] – HATNADERA
97) Mr. AMULYA SARDAR [M] – KUDRUKOCHA 98) Mr. SUBODH SARDAR [M] – JHARIA 99) Mr. RAJMOHAN [M] – SARDAR 100) Mr. NIRANJAN SINGH [M] – 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)
Reagent 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 real-
time.
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.
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 Mon-
Khmer 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
SAMPLE No. GENDER
M95 Primer
M82 Primer HG
BHUMIJ 1 M D O2a BHUMIJ 2 M D O2a BHUMIJ 5 M D O2a BHUMIJ 6 M UC BHUMIJ 7 M A UC BHUMIJ 9 M UC BHUMIJ 10 M D O2a BHUMIJ 13 M D O2a BHUMIJ 14 M D O2a BHUMIJ 15 M UC BHUMIJ 16 M A UC BHUMIJ 17 M D H1 BHUMIJ 18 M A UC BHUMIJ 19 M D O2a BHUMIJ 20 M D O2a BHUMIJ 22 M D O2a BHUMIJ 23 M D O2a BHUMIJ 24 M D O2a BHUMIJ 25 M D O2a
BHUMIJ 26 M D O2a BHUMIJ 27 M A A UC BHUMIJ 28 M D O2a BHUMIJ 29 M D O2a BHUMIJ 30 M A D H1 BHUMIJ 31 M A D H1 BHUMIJ 32 M D O2a BHUMIJ 34 M A D H1 BHUMIJ 35 M D O2a BHUMIJ 36 M D O2a BHUMIJ 38 M D O2a BHUMIJ 39 M D O2a BHUMIJ 41 M UC BHUMIJ 42 M A UC BHUMIJ 43 M D O2a BHUMIJ 44 M D O2a BHUMIJ 45 M D O2a BHUMIJ 46 M D O2a BHUMIJ 47 M D O2a BHUMIJ 49 M A UC BHUMIJ 50 M A UC BHUMIJ 51 M D O2a BHUMIJ 52 M D O2a BHUMIJ 53 M D O2a BHUMIJ 55 M D O2a BHUMIJ 56 M D O2a BHUMIJ 57 M A D H1 BHUMIJ 58 M A UC BHUMIJ 59 M D O2a BHUMIJ 60 M D O2a BHUMIJ 61 M UC BHUMIJ 63 M D O2a BHUMIJ 65 M D O2a BHUMIJ 66 M D O2a BHUMIJ 67 M D O2a BHUMIJ 68 M A UC BHUMIJ 71 M UC BHUMIJ 72 M D O2a BHUMIJ 73 M UC BHUMIJ 75 M D O2a BHUMIJ 76 M D O2a BHUMIJ 77 M D O2a BHUMIJ 78 M D O2a BHUMIJ 79 M D O2a BHUMIJ 80 M D O2a BHUMIJ 81 M D O2a BHUMIJ 82 M D O2a BHUMIJ 84 M D O2a BHUMIJ 86 M D O2a BHUMIJ 87 M D O2a BHUMIJ 88 M D O2a BHUMIJ 89 M A UC
BHUMIJ 90 M D O2a BHUMIJ 91 M D O2a BHUMIJ 92 M D O2a BHUMIJ 95 M D O2a BHUMIJ 96 M D O2a BHUMIJ 97 M A UC BHUMIJ 98 M D O2a BHUMIJ 99 M A D H1 BHUMIJ 100 M D O2a
Fig 23 : Frequency Chart of Y haplogroup
X axis : Haplogroup distribution
Y axis : No. of Samples
Table 2
SAMPLE NO.
M23 Primer M12 M15 Primer Haplo‐ Group
1 10398G 10400T M
2 16231C 16223T 16291T 16319A
16362C 16519C 16360G 10398G 10400T M
3 UC 4 16231C 16223T 16291T 16319A
16362C 16519C 16545[DEL]T 16239T 16245G 16302G 16330G 16345G 16372A
16373A 16384A
UC
5
10025[DEL]A 10398G 10400T M
6 16223T 16291T 16519C
16545[DEL]T 16330G 16387T 10398G 10400T M
7 8557A UC 8 15938T 16102C 16111T 16232T
16330G 16353T 16373A 16545[DEL]T 16551[DEL]T
M39a2
9 UC
10 16519C UC
11 16223T 16325C 10398G 10400T M
12 16223T 16295T 16318T[TR] 16325C 16395[DEL]C
16417T[TR] 16426A 16451A 16460T 16499C[TR] 16503A
10025[DEL]A 10398G 10400T
M
13 16183C 16189C 16223T 8047C M38b
14 10398G 10400T M
15 8064A UC
16
16223T 16274A 16319A 16471A 16233C 16234A 16265C 16294A 16323A
UC
17 16275 [DEL]A 16438A
16073G[TR]
10025[DEL]A 10398G 10400T 10750G
M
18 16051G 16075C 16399G 10109T UC
19 UC
20 UC
21 16223T 16309G 10398G 10400T M
22 16145A 16223T 16240G
16261T 16311C 16319A 16519C
10025[DEL]A 10398G 10400T M4a
23
16189[DEL]T 16201A 16225T 16245T 16246T 16247T 16248T
16270G 8047C 10398G 10400T UC
24 UC
25 16189C 16223T 16325C 16468C UC
26 8047C 8392A UC
27 16129A 16182C 16183C 16189C 16223T 16325C 16468C
UC
28 16290T 16519C 15924G 16126C 16183C 16223T 16232A 16245T UC
29 16092C 16145A 16185T 16239T
16325C 8149G 10398A 10400T UC
30 16266T 16304C 16311C 16357[DEL]T 16362C UC
31 16092C 16145A 16185T 16239T 16325C
8149G UC
32 16111T 10398G 10400T M
33 16092C 16145A 16185T 16239T
16325C 10398A 10454C UC
34 16189C 16223T 16261T 16269T+ 16274A 16311C
16319A
UC
35
16319A 16352C 16086C 16223T 16234A 16274A
16382T 10398G 10400T M
36 16318T 16318T[TR] 10398G 10400T M
37 10398G 10400T M
38 8047C 10398G 10400T M
39 16209C 16223T 16275G
16438A UC
40 10398G 10400T M
41 16294T 16319A 16356C
16463G UC
42 10398G 10400T M
43 10398G 10400T M
44 16129A 16266T 16290T
16318G 16320T R6a1
45 16362C 16115A 16146G 10143A 10289G R7a1a
46 16519C 16170C 16183C 16189C 16225T 16226T 16227T 16230T 16239T 16240G 16245T
16246T 16247T 16248T
10398G 10400T
M
47 16319A 16519C 16223T
16274A 10398G 10400T M
48 16183C 16189C 16223T 8047C 10398G 10400T M38b
49 16086C 16111T 16223T
16399G UC
50
16223T 16270T 16274A 16319A 16352C 16086C 16269T
16279A
UC
51 16353T 15938T 16102C 16111T 16216T[TR] 16228G[TR]
16230T[TR] 16260A 16275G
10398G 10400T
M
52
10143A 10400T 10289G UC
53 16223T 16362C 16343G 16355T 10398G 10400T M
54 16223T 16318T[TR] 16325C 10398G 10400T M
55
16075C 16093C 16260T 16261T 16262T 16319A
16362C 10143A 10289G R7
56 8149G UC
57 16223T 16284G 16327T
16398A
10118C 10325A 10370C 10398G 10400T M
58 UC
59 UC
60 10398G 10400T M
61 10398G 10400T M
62 16017C 16093C 16126C 16145A
16223T
10398G 10400T 10531G M31a2
63 UC
64 10143A 10289G UC
65 UC 66 16183C [TR] 16189C 16194C+
16223T 16256T 16274A 16319A 16390A
UC
67 16189C 16194C+ 16195C+ 16223T 16325C 16468C UC
68 UC
69 UC
70 16179T 16223T 16289G 16294T
16319A 16463G 15954G 10398G 10400T M40a1a
71 8047C 10398G 10400T M
72 16179T 16223T 16289G
16294T 16319A 16428C[TR]
10025[DEL]A 10398G 10400T M40a1a
73 10398G 10400T M
74 UC
75 10398G 10400T M
76 10398G 10400T M
77 16189C 16223T 16275G 10398G 10400T M
78 10143A 10289G UC
79 10398G 10400T M
80 8047C 10398G 10400T M
81 16188T 16223T 16231C 16233C
16234A 16362C 8110C UC 82 16170C[TR] 16172C+ 16183C
[TR] 16189C 16194C+ 16223T 16274A 16319A 16320T
10398G 10400T
UC
83 10398G 10400T M
84 10398G 10400T M
85 UC
86 UC
87 10398G 10400T M
88 16180C[TR] 16189C 16194C+ 16195C+ 16223T 16232T+
16519C 8047C
UC
UC
89
10025[DEL]A 10398G 10400T M
90
16194C+ 16195C+ 16189C 16223T 16274A 16319A 16320T
16519C 16170C 16172C+ 16183C 16221A 16225T
16228A 16230T 16233T 16239T 16242A
UC
91 10398G 10400T M
92 16179T 16223T 16289G 16294T
16319A 16356C 16463G M40a1a
93 16189C 16194T+ 16196+
16223T 16300G UC
94 95 16170C[TR] 16182C 16183C
16189C 16213A 16214A 16223T 16228A 16234A 16236A 16238A 16239A
16242T 16319A
UC
96 16409[DEL]T 15954G
16214G[TR] 16231G 16239T 10398G 10400T M
16245T 16246T 16247T 16248T
97 10398G 10400T M
98 16319A 16320T 10398G 10400T M
99 16129A 16266T 16290T 16318G 16320T 16362C
10398A 10400T
UC
100 16179T 16223T 16289G 16294T
16319A 16356C 10398G 10400T 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 ± 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 Y-haplogroup 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 60–70 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
foot prints of different maternal and paternal lineages in India.[g]
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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