The document discusses the study of paleogenomics, focusing on the evolutionary history of plant species through fossil records, genetic analysis, and living organisms' genetic structures. It highlights the challenges of analyzing ancient DNA and elaborates on methods of extracting and sequencing this DNA to understand ancient ecosystems and the evolution of various species. Key case studies, such as maize domestication, illustrate the complexities involved in plant evolution and adaptation over time.

1. Seminar-III
Maruthi Prasad B P
III PhD
PAMB 1066
Dept. of GPB, UASB

2. 2
Unravelling the evolutionary history
of species

3. 3
1. Studying direct evidence from the fossil records
Examining the preserved remains of ancient plant species
These fossils provide crucial insights into the morphological
changes, adaptations, and diversification of plants over geological
time
Radio carbon dating
Reconstruct the evolutionary history of various plant lineages
Limitations:
Preservation of soft plant tissues, such as flowers and fruits, is
rare in the fossil record, limiting our insights into reproductive
strategies and key aspects of plant biology
Bias in fossilization toward certain environmental conditions
Understanding How Plants Evolve: Different
Angles:

4. 2. Genetic analysis of germinated seeds or spores of
fossil material
Analyzing the fossil seeds or spores buried in the soil allows
researchers to uncover the dormant genetic information of past plant
populations
4
Understanding How Plants Evolve: Different
Angles:
Limitations:
Fossil seeds are having germination problem which poses a major
limitation to this method

5. 3. Studying the current genetic structure of
living organisms
Genetic Variation Analysis
Molecular Phylogenetics
Comparative Genomics
Population Genetics
Functional Genomics
5
Understanding How Plants Evolve: Different
Angles:
Limitation:
Living organisms are required for this approach, limiting the direct
study of extinct plant species and their genetic contributions to
evolutionary history

6. Paleogenomics:
Connecting the dots of
crop evolution from
Modern and Ancient
DNA
6

7. Case studies .
Introduction
Conclusion
01
Paleogenomics
02
03
04
05
06
Outline of Presentation
How is plaeogenome generated?
Applications of
Paleogenomics
Nature of Ancient DNA

8. Genomics in a Nutshell
Genomics is the study of an organism's complete set of DNA,
including genes and non-coding sequences
Involves sequencing, mapping, and analyze genomes to
understand genetic information
Mainstream genomics often focuses on living organisms
8

9. Overview of Paleontology
This interdisciplinary field combines elements of biology,
geology, and chemistry to unlock the mysteries of ancient life
 Insights into the processes that have shaped life over millions
of years, including extinction events, adaptations, and the
emergence of new species
9
Scientific discipline to study history of life on Earth
as revealed through the examination of fossils
Fossils are the preserved remains or traces of ancient
organisms

10. Paleontology
ancient organisms or
extinct organism
Genomics
study of an extinct
organism's
complete set of
DNA
PALEO-GENOMICS
Paleogenomics examines genomes of ancient
organisms, not just living ones
"Paleogenomics is a specialized field of study dedicated to unraveling
the genetic information of ancient organisms. Unlike traditional
genomics that focuses on living organisms, paleogenomics examines
the genomes of species that lived in the past, offering unique
insights into evolutionary history.”
What is Paleogenomics?
10

11. 11
Brief glossary of terms
Ancient DNA (aDNA): DNA extracted from ancient specimens
Archaeogenomics: paleogenomic research on samples excavated
from archaeological sites and analyzed in relation to their
archaeological (human) context
Molecular paleontologist: Scientist studying ancient organisms
DNA to understand their evolutionary relationships
Fossilized remains: Preserved remnants of ancient organisms which
provides valuable sources of ancient DNA
Paleobotany- Complementary field of studying ancient plant fossils

12. Key Emphasis of Paleogenomics
Studying ancient DNA: Paleogenomics centers on extracting and
analyzing DNA from ancient specimens, such as fossils and
preserved remains
Reconstructing ancient genomes: By piecing together fragments
of ancient DNA, researchers aim to reconstruct the genetic
makeup of organisms that are extinct.
Understanding evolutionary processes: To decipher how species
have evolved over time, providing a window into the dynamics of
ancient ecosystems
12

13. Why to study ancient DNA?
13
1. Ancient DNA is a time capsule
2. Plant evolution and admixture
3. Ecology and evolution of life
4. Human migration
5. Development of agriculture
6. History of pathogens
7. Phenotypic traits

14. 14
Svante Pääbo
His research established the
scientific discipline of
PALEOGENOMICS
Nobel Prize in Physiology or
Medicine 2022
Paleogenomics: A Journey Through Time
Neanderthal Genome Sequencing
Identification of Denisovans human population
Developing and refining methods for extracting and
sequencing ancient DNA
First complete human paleogenome
(Denisovan Genomics) was
sequenced in 2010 from Denisova
Cave in Siberia
Paleogenomic studies reveal the
existence of the Denisovans group
of individuals, a sister group to
Neanderthals.

15. 15
 The paleogenome study conducted by Ragsdale et al.,
2023 using Africa, Britain and Neanderthal
 It was believed that humans are originated from single
group of African people
 Modern humans are came from 2 diverse groups that
merged in ancient time
Paleogenomics: A Journey Through Time

16. 16
1985 1990 2003 2003 2011
aDNA from mummified and
herbarium tissue (Rogers &
Bendish 1985)
aDNA analysis for sequence
diversity of Adh2 gene to infer
evolution of maize
(Goloubinoff et al., 1993)
First large-scale ancient DNA
metabarcoding study of plant diversity of
sediments (Willerslev et al., 2003)
First aDNA target capture to
overcome restrictions posed by
low endogenous and
contaminating DNA (Ávila-
Arcos et al., 2011)
Plant DNA of Miocene age
(Golenberg et al., 1990)
Ancient selection on maize
by humans revealed by
aDNA (Jaenicke-Despres et
al., 2003)
2005
aDNA from fossil pollen from
lake sediment in Sweden
(Parducci et al., 2005)
1993
Paleogenomics: A Journey Through Time

17. 17
2013 2016 2017
Complete ancient plant genomes of barley
and Maize (Mascher et al. 2016, Ramos-
Madrigal et al., 2016, Vallebueno-Estrada et
al., 2016)
Small plant RNA in Barley
(Smith et al., 2017)
Eukaryotic plant pathogens like
Phytophthora infestans that triggered the
Irish potato famine (Martin et al., 2013,
Yoshida et al., 2013)
Ancient plant genome
sequencing in Gossypium
(Palmer et al., 2012)
2012
Paleogenomics: A Journey Through Time

18. Ancient Genome Reconstruction
18
Synchronic Approach Allochronic Approach
Compares modern genomes to reconstruct ancestral genomes
over deep timescales of several millions of years
 Macro-evolution
 Direct sequencing of genomes from past plant fossil materials that
have been preserved over the past 10,000 years
 Micro-evolution

19. Ancient Genome Reconstruction using Synchronic
Approach
Ortholog
identification
→ pPGs
[Putative protogenes]
Synteny identification
→SBs
[Synteny blocks]
Ancestral chromosome
→ Core-pPGs
[Core protogenes]
Ancestral genome
→ oPGs
[Ordered protogenes]
(Pont et al., 2019) 19

20. Clustering or chaining collinear gene pairs
DRIMM-synteny,
ADHoRe,
DiagHunter,
DAGchainer,
SyMAP,
MCScanX
Reconstructing ancestral genomes
ANGES,
MRGA,
inferCARs
Tools for Synchronic approach
20

21. Paleogenomics work workflow- Allochronic Approach
(Pont et al., 2019) 21

22. 22
Sample collection
Sources of aDNA
(Estrada et al., 2018)

23. 23
2. Desiccated Leaves and stems
4. Seeds
Sample collection
1.Fossil Pollen grains
3. Fruits and nuts

24. Sample collection
24
1.Chaco Canyon (New Mexico, USA)
2.Jomon Period Sites (Japan)
3.Swiss Lake Dwellings
4.Mesoamerican Sites (e.g., Teotihuacan,
Mexico)
5.Neolithic Sites in the Near East (e.g.,
Jericho, Israel)
6.Ancient Egyptian Sites
7.Denisova Cave (Siberia, Russia)

25. 25
Ancient DNA Extraction Methods
1.Preparation:
 Clean the fossil samples to remove surface contaminants
 If necessary, grind or pulverize the sample to increase surface
area and facilitate DNA extraction
2.Decalcification (if applicable):
 If the fossil contains mineralized material (e.g., calcium
carbonate), decalcify the sample using a weak acid (e.g., EDTA)
 To dissolve the minerals and expose the organic material
containing DNA
3.DNA Extraction:
 Perform DNA extraction using specialized protocols optimized
for ancient DNA recovery
 Utilize methods such as phenol-chloroform extraction, silica
column-based purification, or magnetic bead-based extraction to
isolate DNA from the fossil material

26. 26
Ancient DNA Extraction Methods
(Donato et al., 2018)

27. 27
Nature of Ancient DNA
1. Degradation or Damage of DNA in
deceased tissues
A. Short DNA fragment lengths
B. Low endogenous DNA content
C. High rates of nucleotide damage
2. Exogenous DNA contamination from
modern DNA

28. 28
A. Short DNA fragment lengths
 Shorter fragment lengths compared to fresh DNA
 Environmental factors and enzymatic activity
contribute to DNA fragmentation over time
 Challenges in PCR amplification and sequencing
 Adapt extraction and amplification techniques for
successful analysis of short aDNA fragments
1. Degradation or Damage of DNA in
deceased tissues

29. 29
B. Low endogenous DNA content
 Endogenous DNA refers to the authentic genetic material
originating from the fossil
 Ancient tissues often yield low endogenous DNA content due to
degradation
 Contaminating environmental DNA may contribute to the overall
DNA content but is non-endogenous.
 Rigorous contamination controls and selective amplification
strategies are essential for accurate analysis of low-endogenous
DNA samples
1. Degradation or Damage of DNA in deceased
tissues

30. 30
1. Degradation or Damage of DNA in deceased
tissues
Ancient DNA (aDNA) in deceased tissues experiences chemical and
environmental degradation
Deamination, a prevalent hydrolytic DNA damage, is notably
swift for cytosine, leading to the conversion of cytosine to uracil in
the DNA molecule
This chemical transformation occurs naturally in dead organisms and
has significant consequences for subsequent DNA analyses
 During PCR, deoxyuridine residues in the template strand prompt
DNA polymerases to incorporate deoxyadenosine at positions
where deoxyguanosine would typically be added
 Deamination induces C→T and G→A transitions in the DNA
sequence, creating mismatches during replication
Purine loss results in the formation
of overhanging ends

31. 31
2. Exogenous DNA
contamination
 aDNA often coexists with abundant environmental DNA
mostly from microbes, fungi, and plants that colonized the
remains, resulting in extremely small proportion of endogenous
DNA in an extract
 For Ex, the first Neanderthal genome was reconstructed from
DNA samples with only 1-5% endogenous DNA (Green et al.,
2010)
Control Measures:
1. Laboratory Controls: Enforce strict separation and employ
dedicated equipment for aDNA analysis to minimize cross-
contamination
2. Authentication Markers: To differentiate between endogenous
and exogenous DNA
3. Protective Measures while working with aDNA
4. Negative Controls: Implement mock extractions and PCR
controls without ancient DNA to monitor and detect
contamination at every stage of the process
5. Unique Barcoding: Employ unique barcoding for aDNA
libraries to identify and discard contaminated sequences
 Exogenous DNA can distort the genetic information derived from
ancient samples, leading to misinterpretations
 Sources of Exogenous DNA
 Modern DNA
 Researcher's DNA
 Environmental DNA

32. Aspect Paleogenetics Paleogenomics
Focus
Primarily involves
studying genes
Involves the study of entire
genomes
Data
Sources
Analyzes specific
genetic markers
Utilizes high-throughput
sequencing tech
Time
Scale
Typically spans shorter
genetic regions
Encompasses entire genomic
sequences
Applicatio
ns
Investigates specific
genetic traits
Offers a more comprehensive
evolutionary view
32
Paleogenetics vs Paleogenomics

33. Before introduction of NGS technologies, conventional
PCR followed by Sanger sequencing was used for aDNA
sequencing
It is not feasible for large-scale genomic studies due to its
limited throughput and high cost
Paleogenomics using Next-Generation
Sequencing and Library Preparation
33
 NGS technologies can generate millions to billions of
DNA sequence reads
 Provides whole-genome-scale data within a short period
and at much lower cost per base

34. NGS platforms require construction of a sequencing library,
where all DNA fragments in a sample are normally end-repaired
and ligated to universal sequencing adapters
Enables the sequencing of all the fragments simultaneously
For an aDNA sample, three major limiting factors:
Extremely low copy number of endogenous DNA
The high levels of PCR inhibitors
Abundance of damaged bases
34
Paleogenomics using Next-Generation
Sequencing and Library Preparation
Lead to less efficient conversion of DNA fragments into
an adapter-ligated form and substantial loss of endogenous
DNA due to several rounds of purification and inflation of
exogenous DNA content during amplification

35. Library Construction Methods
1. Double-stranded Library
construction method
2. Single-stranded Library
construction method
35

36. 36
Blunt-end repair
Double-stranded
methods
A. 454-type Meyer
and Kircher, 2010
B. Illumina-type
Bentley et al., 2008
Adapter fill-in
Indexing oligo
A-tailing
Indexing-PCR Indexing oligo
Indexing-PCR

37. 1. Compromised Sensitivity to Single-Stranded Fragments:
Fragments of surviving aDNA may be single-stranded,
only double-stranded fragments of DNA become part of the
sequencing library in this method
2. Higher Template Requirement
The preparation of double-stranded libraries often requires a
higher amount of starting material, which can be a
limitation when working with limited or highly degraded
ancient DNA samples
37
Limitations of Double-stranded Library

38. Single-stranded
methods
38
Denaturation
A. Gansauge and Meyer, 2013 B. ssDNA2.0 Gansauge et al., 2017
Biotinylated
adapter
Biotinylated
adapter

39. Advantages:
DNA molecules are tightly bound to streptavidin-coated beads,
which avoids the loss of molecules in purification step.
Single stranded molecules can be recovered
Recovery of more short reads
39
Single-stranded Library
Disadvantages:
It is more costly and time consuming
Conversion efficiency drops for molecules longer than 120bp

40. 40
Targeted Enrichment
Target enrichment is increasing the proportion of endogenous DNA
in an aDNA library
Reduces the amount of DNA sequenced and therefore cost of
sequencing required.
1.Selective Amplification:
 Target enrichment involves selectively amplifying specific
genomic regions of interest from ancient DNA sample
(organellar genomes, exomes, single chromosome or even
whole nuclear genome)
 So we can enrich for selected genomic regions
2.Focused Sequencing:
 This method allows researchers to concentrate sequencing
efforts on specific genetic markers or regions of the genome

41. 41
Targeted
Enrichment
In-solution beads capture Microarray-based capture
Hybridization
Sequencing library
Baits library- Sequence
is similar to existing
organism
Sequencing targeted
molecules only
(Lindqvist et al., 2019)

42. A
general
pipeline
for
bioinformatic
analyses
in
paleogenomic
studies
42

43. Authentication of aDNA
43
Deamination as Authentication Marker:
 Deamination, a common postmortem DNA damage,
increases with age of fossil
 Elevated levels of deamination serve as an indicator of
ancient DNA, aiding in authentication

44. A
general
pipeline
for
bioinformatic
analyses
in
paleogenomic
studies
44

45. 45
Applications
of
Plaeogenomics
Evolutionary
Insights
Genetic
Diversity
Historical &
Cultural
Insights
Nutritional
quality
and Stress
tolerance

46. Bridging Time Gaps: Paleogenomics in Plant
Research
It is estimated that approximately 2,500 species of plants have been
subject to domestication since the last glacial period, over 12,000
years ago
However, the domestication history of most crop species remains
unclear, with several fundamental questions regarding wild progenitor
species, timing of domestication and geographic regions of
domestication, yet to be answered
Paleogenomics helps us time-travel through plant DNA, solving
mysteries about how ancient plants became the crops we rely on
today, answering questions about their origins, timelines, and where
they first grew
46

47. 47
Case study 1
Maize (Zea mays ssp. mays) evolved from its wild ancestor, Balsas
teosinte (Z. mays ssp. parviglumis), in modern-day lowland
Mexico around 9000 years ago
Maize's journey from wild teosinte to a dominant food crop is a
fascinating tale of adaptation and human cultivation

48. 48
H0- Single Domestication Event
H1- Stratified Domestication Model
Objective:

49. 49
Sample Type Number of
Samples
Source
Modern Landraces 40 Indigenous maize landraces from
South America
Archaeological
Samples
9 Excavated maize samples from
archaeological sites
Modern Genomes 68 Published modern maize
genomes
Ancient Genomes 2 Published ancient maize genomes
Teosinte Genomes 2 Published teosinte genomes
Material and Method:

50. 50
Archaeogenomic evidence
Maize was Partially
domesticated in Mexico by
~5300 yr B.P.
Carrying a mixture of wild-
type and maize-like alleles
at loci involved in the
domestication syndrome
This partially domesticated
maize was grown in
Mexico
This state persisted even after maize had become established in other
regions

51. 51
f4 Statistics
 f4 statistics demonstrated excess allele sharing between the Pan-
American lineage and wild parviglumis compared with other maize
 Revealing nonuniform crop-wild gene flow after initial
domestication

52. 52
Output of study:
Complex Domestication Process: Maize domestication is not
a singular event; it involves a complex, stratified process
South American Maize Dynamics: South American maize
exhibited partial domestication and divergence during early
domestication stages
Multiple Dispersal Waves: Dispersal of maize occurred in
multiple waves, challenging the notion of a single migration
event

53. 53
Herbarium Paleogenomics: Plant
Archival DNA Explored
herbaria contains preserved plants, seeds, fungi, and algae
collected at different phenological stages
Herbarium specimens are commonly preserved on paper sheets or
stored in folded packets or small boxes, which can be treated with
solvents or pesticides to protect the specimens

54. 54
Herbarium Paleogenomics: Plant Archival
DNA Explored
 The history of modern herbaria began in
the 16th century
 Luca Ghini, Professor of medical botany,
at the University of Bologna
Luca Ghini (1490–1556)
Carl Linnaeus
In the 18th century, Carl Linnaeus
developed his herbarium “cabinet”
collection
 Includes approximately 14,000 sheets
of plant specimens and several
zoological specimens
 Developed a method to make plant specimens transportable and
preservable over time
 He created the first herbarium (Hortus siccus) in the year
1544
Linnaeus provided innovative instruction on how to
establish an herbarium collection

55. Herbarium Paleogenomics: Plant
Archival DNA Explored
55
Global herbarium collections:
 In 1935, the International Association for Plant Taxonomy (IAPT)
established the “Index Herbarium” (http://sweetgum.nybg.org/science/ih/)
 Comprehensive resource that serves as a global directory
of herbaria.
~400 million
specimens
~3500
active herbaria
(Papalini et al., 2023)

56. 56
01
02
03
04
Muséum National d’Histoire
Naturelle, France
New York Botanical Garden, USA
Komarov Botanical Institute,
Russia
Royal Botanic Gardens, UK
Global herbarium collections:
Herbarium Paleogenomics: Plant
Archival DNA Explored
(Papalini et al., 2023)

57. 57
Herbarium Paleogenomics: Plant
Archival DNA Explored
 DNA decay is fast (6x
faster than bone)
 Damage accumulates
faster
 High endogenous DNA
content

58. 58
Herbarium Paleogenomics: Plant Archival DNA
Explored
(Papalini et al., 2023)

59. 59
 Objective: To analyze the genomic evolution of
Phytophthora infestans, the causal agent of potato late
blight which caused Irish potato famine in 19th century

60. Materials and Methods
60
 Historic herbarium specimens
from the initial 1845 outbreaks in
Belgium and later outbreaks in
Europe
 Modern isolates from North
America
 High-throughput Sequencing:
Shotgun sequencing of DNA
extracts from blight-infected
potato leaves
Phylogenetic Analysis: To assess evolutionary relationships

61. 61
Results
The diversity within historic samples highlights the complexity of P.
infestans evolution
Phylogenetic Analysis:

62. Output of study:
62
Phylogenetic analysis provide insights into the evolutionary
history of P. infestans
Identified distinct nuclear genotypes in historical European
samples
Understanding the multiple introductions is crucial for
tracing the pathogen's migratory history and its impact on
global potato late blight outbreaks

63. Multiple Episodes of Domestication
63
 Paleogenomics is changing our understanding of domestication
 It gives us the opportunity to witness the process as it unfolds
instead of being limited to studying the end product

64. 64
The earliest Domestic horse specimen
from Botai culture site dated to around
5500 years ago
Przewalski’s horses have long been considered to
be the only living wild horse
Modern day horses
Extinct wild Horses specimens
Objective of the study:

65. 65
Przewalski’s horse
Modern horse
Whether Przewalski
is a wild species ?
Is it a wild progenitor of
Modern day horses ?
Objective of the study:

66. Sample Type
Number of
Samples Description
Botai Culture Horse
Genomes
20
Genomes from horses
associated with the Botai
culture sites over the past 5000
years
Other European and
Central Asian Horse
Genomes
20
Genomes from horses
excavated from locations in
Europe and Central Asia (past
5000 years)
Previously Published
Ancient and Modern
Domestic Horse Genomes
N/A
Comparison with existing
ancient and modern domestic
horse genomes
19th Century Przewalski’s
Horse Genome
1
Genome from a Przewalski’s
horse dating back to the 19th
century
66
Material and Method:

67. 67
Results

68. Domestication De-domestication/
Feralization
Feralization/De-domestication
Przewalski’s horse
Przewalski’s horses are a feral population
descended from the horses that were first
domesticated at sites like Botai
Modern domestic horses likely evolved from
a from a different lineage of wild horses.
68

69. Output of study:
69
The surprising findings of this study reveal that domestication can be a
complex process, characterized by feralization, population turnover
events, and interbreeding between wild and domestic stocks
These findings underscore the point that past population dynamics
cannot always be inferred from present-day DNA variation
The conventional view of crop evolution
includes domestication to landrace from
wild plants and improvement of modern
cultivars from the landrace
Feral plants (de-domesticates) form the
fourth node and, therefore, extend crop
evolutionary complexity

70. Domestication, as an extensive selection process, can also lead to
a reduction in the genetic diversity of cultivated species
Hence, investigating new sources of genetic variation is critical
for crop improvement
70
Paleogenomics opens a new path to directly access the genetic
information that has changed at various stages of plant
domestication
Utilizing Paleogenomics for Enhancing Crop Genetic Diversity
and Trait Improvement
Enabling the identification of lost genetic diversity and the potential
to reintroduce extinct loci related to desired traits

71. Studies of modern plant DNA have already detected
several genetic loci associated with tolerance to abiotic and
biotic factors
DNA capture-enrichment methods on aDNA can be used
to investigate such genes of ancient plants, and identify
allelic variants for these traits
71
Unlocking Ancient Genetic Variation for Modern Crop
Resilience

72. 72
(Donato et al., 2018)
Unlocking Ancient Genetic Variation for Modern Crop
Resilience

73. 73
Structural Variants in Ancient Genomes
(Pont et al., 2019)

74. 74
Paleogenomic study on the Black Death revealed
the genomic makeup of the ancient pathogen
Yersinia pestis, shedding light on its evolutionary
history and dynamics of historical pandemics
Ancient Pathogens Through Human History:
The Black Death, a devastating pandemic in the
14th century
Ancient bacterial and viral genomes provide a
window into evolutionary processes of pathogens
that have accompanied humans throughout history
Most fatal pandemics in human history, as many as
50 million people perished, 50% of Europe’s
population

75. 75
FUTURE PROSPECTS
There is a need for development of techniques
to study Paleoproteomics, Ancient
Epigenomics and Ancient RNA, will offer a
multidimensional understanding of ancient
organisms' biology
There is a need to study how plant species
have evolved and adapted to environmental
changes through time to adopt climate
change and habitat modification
Best practices for ethical sampling of
remains for aDNA research, as these are finite
resources
Build collaborations between genetics and
other disciplines, such as archaeology and
environmental studies to ensure
paleogenomic results are interpreted within
their appropriate cultural, historical, and
environmental contexts
Develop bioinformatics tools specifically
tailored for analyzing ancient DNA (aDNA)
data

76. Conclusion
76

77. 77
Thank you