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Transcript of M.sc. Nanoscience
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Studies on plant DNA isolation, PCR and sequencing of barcode
loci in tree species
A Project Report
SUBMITTED IN THE PARTIAL FULFILLMENT OF THE REQUREMENTS
FOR THE DEGREE
OF
Master of Science
In Nanosciences
Submitted by-
Nahid Akhtar
M.Sc. (H) Nanosciences
Roll No.: 0900142002
Integral University, Uttar Pradesh
Under the Guidance of
Dr. Sribash Roy
Plant molecular biology &Genetic Engineering Division
National Botanical Research Institute
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Lucknow
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ACKNOWLEDGEMENT
I take this privilege to express my thankfulness to all those people involved in this
endeavours.
I am bereft of words to find befitting and to show my deep sense of gratitude towards my
guide Dr. Sribash Roy. I shall remain ever indebted to him for his creative and expert
guidance, constant encouragement, incessant discussion, everlasting moral support,
constructive criticism, valuable suggestions, kindness and his trust on me. I learnt many
things from him during this course of study.
I am greatful to Prof.V.D.Gupta,Head of the department, Nanoscience, Integral University,
Lucknow for including me all those qualities, which make so meticulous, and for assisting
untiring at each and every stage during my project work.
My most sincere thanks are due to Mr.Abhinandan Mani Tripathi and Mr.Antariksh Tyagi for
their untiring help and suggestions throughout my studies by sacrificing their golden time
during the tenure of this project.
I wish to express my sincere gratitude to Dr.Samir Sawant , Dr.Hemant Yadav, Dr.S.N.Jena
gave their unstinted support in one way or the other.
I would also like to express my thanks to the members of our lab, Mr. Anukool srivastava,
Ms. Namrata Singh, Mr.Anuj Maurya Ms. Aastha Gupta ,Mr Ravi Shukla, Shipra and Parul
for their guidance, support cooperation, and suggestions throughout the work.
My vocabulary is not wide enough to reflect all my sense of regards and infinite gratitude to
my mother & my father for their love, affection, blessings, pivotal role in shaping my
academic career, guidance for the right path, tremendous moral boosting and sacrifice. I am
thankful to them for making me a self-reliant, positive and optimistic human being.
Nahid Akhtar
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CERTIFICATE
It is certified that the dissertation report entitled Studies on plant DNA isolation, PCR
and sequencing of barcode loci in tree species which is being submitted by Nahid
Akthar, Department of Nanosciences, Integral University carried out by her under my
supervision and guidance.
Signature of Co-Supervisor
Ms. Tarana Afreen Chandel
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Table of Contents-:Table of Contents-:
REFERENCES 35-40
5
CONTENT PAGE NO.
ABBREVIATIONS 4
INTRODUCTION 5-11
LITERATURE REVIEW 12-16
MATERIALS AND METHODS 17-28
RESULTS & DISCUSSION 29-34
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ABBREVIATIONS
bp Base pair
BME - mercapto ethanol
dNTPs Deoxy Nucleotide Tri Phosphates
DNA Deoxyribonucleic acid
EDTA Ethylene Diamine Tetraacetic Acid
HCl Hydrochloric acid
hr Hour
kDa Kilo Dalton
min Minute
Nm Nanometer
NaCl Sodium chloride
PCR Polymerase chain reaction
rpm Rotation per minute
TBE Tris-Borate-EDTA
UV Ultra violet
ITS Internal transcribed spacer
matK Maturase kinase
rbcL Ribulose Biophosphate Caboxylase Sub-unit L
CO1 CytochromeCoxidase1
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INTRODUCTION
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1. INTRODUCTION:
The number of living species on earth is unknown. There are approximately 1.7 million
named species and possibly another 10 million (not counting bacteria and archea) DNA based
identification could be vitally important in flagging specimens that represent undescribed
taxa .Comprehensive analysis of populations will help in identifying cryptic species ,which
may be for more prevalent than commonly realized, even among large animals.DNA
sequencing of the same gene (or set of gene )across diverse phyla will help unravel the
process that underlie speciation and reveal the diversity of life to an extent that is not now
possible .DNA sequences analysis is enormously useful in studies of evolutionary history and
origin of species on earth. Extensive sampling of DNA sequences has helped establish the
diversity of life and allowed researches to analyse be revolutionary relationships with in
groups in details.DNA sequencing has also been applied to identify specimens and resolve
species boundaries in populations apparently similar organisms.DNA sequence analysis of
uniform target gene to enable species identification has been termed DNA barcoding,by
analogy with Uniform product code barcodes on manufacturing goods. DNA bar-coding is
the use of a short DNA sequence or sequences from a standardised locus (or loci) as a species
identification tool. This method of using short orthologous DNA sequences called DNA
barcodes have been proposed and initiated to facilitate biodiversity studies, identify juvenilesassociate sexes ,enhanced forensic analyses and germplasm conservation.
With so much of human interference into environment and changing climatic
conditions many plant and animal species are onto the verge of extinction or some had
already faced extinction. Climate change and increasingly dramatic shifts in land threaten to
exacerbate the existing bio-diversity crisis. Meeting the imperative of environmental
stewardship requires support for the scientific endeavours of documenting, predicting and
managing ecological change on a global scale. This will require not only a one off description
of organism diversity (a formidable challenge in itself), but also the ability to monitor
biological communities year after year. Today, this task is carried out mainly by taxonomist
applying their specialist skills because the vast majority of other players in the field of
conservation and sustainable development are unable to identify any but the most familiar
species.
The convention on biological diversity affirms that this so called taxonomistimpediment pose a serious threat to conservation and management of biological diversity.
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The urgent need for a rapid, accurate and web-accessible taxonomic resource will require the
full utilization of information technology and molecular biology (UK Government., 20004;
EPBRS., 2004). To reduce the risk of loss of genetic variability, there is urgent need to
conserve tree germplasm for future studies. The conservation needs and strategies can be
streamlined by resolving unambiguously between true genetic and phenotypic variability.
This has become possible due to recent developments in plant genomics which allow retrieval
of true genetic variation by directly examining sequence variation in DNA. Tree DNA
isolation, amplification and sequencing of most diversified genes are useful in several
applications, example. characterizing true genetic variation in the materials, understanding
genetic and evolutionary relationships.
Fortunately, a DNA based identification system( i.e.DNA barcoding)operable by
non-specialist to complement parallel development in taxonomic informatics is within reach.
As a uniform, practical method for species identification ,it appears to have broad scientific
applications. It will be great utility on conservation biology for example including
biodiversity surveys.DNA barcodes have been proposed as a shortcut that would provide
species identifications and as away to accelerate the discovery of new species. DNA Barcode
are the short segments of a gene sequence that evolve fast enough to differentiate species, but
have flanking regions that are sufficient conserved to enable the barcode region to be serviced
as universal primer. DNA bar-coding has already proved useful for identification (and in
some cases delimitation) of animal species, but plants (in a broad sense including land plants,
algae and lichens) are only beginning to attract the attention, importance of the DNA based
plant identification tool can be realized, however, several important scientific and
methodological questions must first be addressed. DNA Barcoding promises fast, accurate
species identification by focusing analysis on a short standardized segment of the genome.
DNA bar-coding involves sequencing a short stretch of DNA that will be universally presentin all species, ideally exhibits robust species-specific sequence variation. DNA bar-coding
involves sequencing a short stretch of DNA that will be universally present in all species,
ideally exhibits robust species-specific sequence variation. DNA bar coding is an exciting
new tool for taxonomic research.
DNA bar-coding has already proved useful for identification (and in some cases
delimitation) of animal species, but plants (in a broad sense including land plants, algae and
lichens) are only beginning to attract the attention, importance of the DNA based plant
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identification tool can be realized, however, several important scientific and methodological
questions must first be addressed.
The preferred gene for animal bar-coding, cytochrome oxidase1 (CO1 or cox1) is
not suitable for plant bar-coding because low rates of substitution in this gene have caused a
lack of sequence variation among plant species. Plants also pose unique challenges to bar-
coding: prevalent hybridization, polyploidy and apomixis may undermine the utility of even
an ideal bar-code locus. Thus, a pre requisite to a universal plant bar-coding procedure is a
period of rigorous experimentation to determine how indeed, if DNA bar-coding at a floristic
scale can succeed.
A desirable locus for DNA barcoding should be standardized (so that large databasesof sequences for that locus can be developed), present in most of the taxa of interest and
sequencable without species-specific PCR primersshort enough to be easily sequenced with
current technology and provide a large variation between species yet a relatively small
amount of variation within a species.
Although several loci have been suggested, a common set of choices are:
For animals and many other eukaryotes, the mitochrondrial CO1 gene
Forland plants, the concatenation of the rbcL , matKchloroplast genes, ITS ,trnH and
psbA.
Consortium for the Barcode of Life (CBOL) is an international collaborative effort which
aims to develop a mechanism capable of generating a unique genetic barcode for every
species of life on earth .The science behind the Consortium's initiative has met with
considerable controversy, with responses ranging from enthusiastic endorsement to strident
rejection. More work needs to be done to determine to what extent barcoding can
complement existing taxonomic methods in cataloguing the planet'sbiodiversity. In pursuing
this mission CBOL promotes-
1- The rapid compilation of DNA barcode record in a public library of DNA sequence.
2- The development of new instrument and processes that will make bar-coding cheaper,
faster and portable.
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http://en.wikipedia.org/wiki/PCR_primerhttp://en.wikipedia.org/wiki/PCR_primerhttp://en.wikipedia.org/wiki/Land_plantshttp://en.wikipedia.org/wiki/Land_plantshttp://en.wikipedia.org/wiki/RbcLhttp://en.wikipedia.org/w/index.php?title=MatK&action=edit&redlink=1http://en.wikipedia.org/wiki/DNA_barcodinghttp://en.wikipedia.org/wiki/Taxonomyhttp://en.wikipedia.org/wiki/Biodiversityhttp://en.wikipedia.org/wiki/Land_plantshttp://en.wikipedia.org/wiki/RbcLhttp://en.wikipedia.org/w/index.php?title=MatK&action=edit&redlink=1http://en.wikipedia.org/wiki/DNA_barcodinghttp://en.wikipedia.org/wiki/Taxonomyhttp://en.wikipedia.org/wiki/Biodiversityhttp://en.wikipedia.org/wiki/PCR_primer -
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3- The participation of taxonomist and taxonomic research organization in all regions and
countries.
4- The use of DNA bar-coding for the benefit of science and society.
Four barcoding primer selected for this work viz rbcl, trnH-psbA, matK and ITS in which
first three are taken from chloroplast genome and ITS were taken from Internal transcribed
spacer (ITS) region of nuclear region of genome. Like mitochondria cytochrome oxidase 1
gene (CO1) of animal kingdom there were no any single locus reported in plant which act
completely as barcode. Due to this region we were taken four loci for our study. Keeping
above problems in mind we have taken following objective-
I) DNA isolation of plants
II) PCR amplification with all four loci
III) Sequencing of these amplified product
IV) Phylogenetic analysis
Until now the biological specimens were identified using morphological keys but in most
cases an experienced professional taxonomist is needed. If a specimen is damaged or is in animmature stage of development, even specialist may be unable to make identification. Bar-
coding solves these problems because non-specialist can obtain bar-code from tiny amount of
tissues. This is not to say that traditional taxonomy has become less important but rather that
DNA bar-coding can serve a dual purpose as a new tool in the taxonomist tool box
supplementing his/her knowledge well as being an innovative device for non experts who
need to make a quick identification.
1.2 GENES USED FOR BAR-CODING:
In animals a portion of mitochondrial gene cox 1(CO1) is standardarised as potential bar-
code.In land plants this mitochondrial gene cox 1 will not succeed to low levels of variability
in the mitochondrial DNA of land plants. The closest equivalent source of a plant bar-coding
region is the Plastid Genome. This genome shares many of the desirable attributes of animal
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mitochondrial DNA for bar-coding such as conserved gene order and high copy number in
each cell enabling easy retrieval DNA for PCR and Sequencing.
One problem with plastid DNA however, is its generally slow rate of evolution and challenge
has been to find a plastid region that is sufficiently variable for DNA bar-coding. A suitable
region should ideally show enough variation within it to discriminate among species, yet to
be conserved enough to be present and routinely retrievable across the >400 million years of
evolutionary divergence represented by extent land plant diversity. This is a non- trival
problem; finding a marker or perhaps set of marker for which primer binding sites are
conserved but which shows high levels of variability across all groups of land plants
represent a set of contradictory targets. If markers have highly conserved primer binding sites
they tend to also be internally more conservative, whereas for the most variable regions it is
difficult to identify sites for reasonably conserved primers.An additional desirable trait for
potential bar-coding region is to have a reading frame so that the presence of non- sense
substitutions could be used as a criterion to evaluate how good sequencing reactions/ editing
have been. For a non- coding region (introns or intergenic spacer) to represent a viable
altenative it is necessary for it to have-
1- Universal primers and standard PCR protocols.
2- Consistently higher variation than coding region.
3- A non- coding complicated pattern of molecular evolution.
Plastid genes used for barcoding are-
a) rbcL- Ribulose Biophosphate Caboxylase Sub-unit L
b) psbA- trnH spacer
c) matk- Maturation Kinase
a) rbc L - Ribulose Biophosphate Caboxylase Sub-unit L it is a protein encoding plastid
gene. It has been proposed as a potential barcode by several sets of researchers ( Chase et
al., 2005; Newmaster et al., 2006 ). This region has shown a fair degree of success in
discriminating species and is regarded as benchmark locus in phylogenetic investigation
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by providing a reliable placement of taxon into a plant family and /or genus (W. John
Kress et al., 2007).
b) trnH- psbA spacer- is a non-coding intergenic spacer region found in plastid DNA (Kress
et al., 2005; Shaw et al., 2007). This region is one of the most variable non-coding
regions of the plastid genome in angiosperms in terms of having the highest percentage of
variable site (Shaw et al., 2007). This variation means that this inter-genic spacer can
offer high levels of species discrimination (Kress etal., 2005; Shaw et al., 2007).
These are many problems with alignment for this locus caused by high rates of insertion/
deletions; alignment of the trnH- psb A spacer across larger families of angiosperms is highly
ambiguous. It appears that even within closely related taxa, great length differences exist,
such that at greater taxonomic distance no shared sequence remains. Further more in some
groups of plants, the trnH -psbA spacer is exceedingly short (less than 300bp; Kress et al.,
2006) and in some groups of plans it is much longer. Despite of these problems trn H-psb A
spacer is considered as suitable for barcoding.
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REVIEW OF LITERATURE
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22.. Review of Literature
DNA barcoding the recently proposed DNA based project for specie identification ,has
attracted much attention and controversy. Proponents envision that a short fragment of DNA
can be used to diagnose taxa , increasing the speed ,objectivity and efficiency of specie
identification. Initial tests of genetic barcoding using mitochondrial markers on animal
reported near 100% accuracy , indicating that the method can be highly accurate under
certain condition.
DNA barcoding promises fast ,accurate species identifications by focusing analysis on a short
standardized segment of the genome (Herbert et al 2003).Several studies have now
established that sequence diversity in a approx.650 bp region near the 5 end of mitochondrial
cytochrome oxidase subunit I(cox I, also referred to as COI)gene provides strong species
level resolution for varied animal groups including birds(Herbert et al ,2004),fishes (Ward et
al ,2005),springtails(Hogg and Hebert,2005),spiders(Barrett and Hebert,2005 )and moths
(Hebert et al ,2003,Janzen et al 2005).These early results have provoked larger scale
barcoding efforts and global projects for fishes and birds have now been initiated
(Marshall,2005).These projects represent the first way in series of initiatives which will
demand the capability to assemble barcode rapidly and cost effectively .As one looks further
to the future the need for substantial analytical capacity looms. For e.g. an effort to barcode
the 1.7 millions described species (Hawksworth,1995) would require the assembly of some
20 million barcodes, given a target of about 10 barcodes per species. This total will rise 5 fold
if barcode coverage is desired for all 10 million eukaryote species(eg.Hammond,1992)
producing a sequence library of 65 billion base pairs ,Approximately twice current size of
genebank (april,2005).This task could be completed within decade by establishing 50 core
laboratories, each producing 200000 barcode record per year .When viewed from the
perspective of major genomic facilities, some of which generate more than 50 million
sequence a year, the production goals for barcode facilities may seem modest.
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This technique of species identification have recently been proposed as
solutions to the crisis of taxonomy and received significant attention from scientific journals,
grant agencies , natural history museums, and mainstream media. Meieret al, tested two key
claims of molecular taxonomy using 1333 mitochondrial COI sequences for 449 species of
Dipterans and investigated whether sequences can be used for species identification (DNA
bar-coding) and find a relatively low success rate (
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populations belonging to three species. Thus, for the first time, DNA barcodes have been
found to identify entities below the species level that may constitute separate conservation
units or even species units. Rach et al (2007) findings suggest that character-based DNA
barcoding can be a rapid and reliable means for (i) the assignment of unknown specimens to a
taxonomic group, (ii) the exploration of diagnosability of conservation units, and (iii)
complementing taxonomic identification systems. (Rach et al., 2007)
Cornman et al (2007) investigated the phylogeography of Iris missouriensis (Iridaceae),
which is widely distributed in western North America. R. Scott and Cornman et alutilized
transposon display and DNA sequencing to quantify nuclear and chloroplast genetic
structure. Their objectives were (i) to characterize the geographic structure of genetic
variation throughout the species range, (ii) to test whether both margins of the range show
reduced genetic diversity as predicted by north south expansion and contraction associated
with climate change, and (iii) to determine whether the subspecies Iris missouriensis ssp.
longipetala is genetically distinct. Cornman et al (2007) found that genetic diversity was
significantly lower in the northern part of the range but was not significantly different
between the central and southern regions, indicating greater stability of the southern margin
vs. the northern. Among-population differentiation was high (PT = 0.52). The largest
divisions in each marker set were concordant and separated the southern Rocky Mountainsand Basin and Range provinces from the remainder of the range. The boundaries of this
phylogeographic break do not coincide with gaps in present-day distributions or
phylogeographic breaks identified in other species, and may indicate a measure of
reproductive isolation. Consistent with current treatments, Cornman et al(2007) did not find
support for the taxonomic placement I. missourienis ssp. longipetala as a distinct species.
Although transposon display has been used to investigate relationships among crop
accessions and their wild relatives, to their knowledge, this is the first use of these markers
for population-level phylogeography of a nonmodel species and further demonstrates their
utility in species recalcitrant to amplified fragment length polymorphism protocols.
(Cornman et al., 2007)
The evolution rates of mtDNA in early metazoans hold important implications for
DNA bar-coding. Huan et al (2007) present a comprehensive analysis of intra- and inter
specific COI variabilities in Porifera and Cnidaria (separately as Anthozoa, Hydrozoa, and
Scyphozoa) using a data set of 619 sequences from 224 species. Huan et al (2007) found
variation within and between species to be much lower in Porifera and Anthozoa compared to
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Medusozoa (Hydrozoa and Scyphozoa), which has divergences similar to typical metazoans.
Given that recent evidence has shown that fungi also exhibit limited COI divergence, slow-
evolving mtDNA is likely to be plesiomorphic for the Metazoa. Higher rates of evolution
could have originated independently in Medusozoa and Bilateria or been acquired in the
Cnidaria + Bilateria clade and lost in the Anthozoa. Low identification success and
substantial overlap between intra- and interspecific COI distances render the Anthozoa
unsuitable for DNA bar-coding. Caution is also advised for Porifera and Hydrozoa because of
relatively low identification success rates as even threshold divergence that maximizes the
bar-coding gap does not improve identification success. (Huang et al., 2008)
The cytochrome c oxidase 1 sequence, which has been found to be widely applicable
in animal bar-coding, is not appropriate for most species of plants because of a much slower
rate of cytochrome c oxidase 1 gene evolution in higher plants than in animals. Kress et al
(2005) therefore propose the nuclear internal transcribed spacer region and the plastid trnH-
psbA intergenic spacer as potentially usable DNA regions for applying bar-coding to
flowering plants. The internal transcribed spacer is the most commonly sequenced locus used
in plant phylogenetic investigations at the species level and shows high levels of inter specific
divergence. The trnH-psbA spacer, although short (450-bp), is the most variable plastid
region in angiosperms and it is easily amplified across a broad range of land plants.Comparison of the total plastid genomes of tobacco and deadly nightshade enhanced with
trials on widely divergent angiosperm taxa, including closely related species in seven plant
families and a group of species sampled from a local flora encompassing 50 plant families
(for a total of 99 species, 80 genera, and 53 families), suggest that the sequences in this pair
of loci have the potential to discriminate among the largest number of plant species for bar-
coding purpose. (Kress et al., 2005)
Newmaster et al (2007) determined the relative utility of six coding (Universal
Plastid Amplicon UPA, rpoB, rpoc1, accD, rbcL, matK) and one noncoding (trnH-psbA)
chloroplast loci for bar-coding in the genus Compsoneura using both single region and
multiregion approaches. Five of the regions we tested were predominantly invariant across
species (UPA, rpoB, rpoC1, accD, and rbcL). Two of the regions (matK and trnH-psbA) had
significant variation and show promise for bar-coding in nutmegs. Newmasteret al(2007)
demonstrate that a two-gene approach utilizing a moderately variable region (matK) and a
more variable region (trnH-psbA) provides resolution among all the Compsonuera species.
Newmasteret al(2007) sampled including the recently evolved C. sprucei and C. mexicana.
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Newmasteret al(2007) classification analyses based on nonmetric multidimensional scaling
ordination, suggest that the use of two regions results in a decreased range of intraspecific
variation relative to the distribution of interspecific divergence with 95% of the samples
correctly identified in a sequence identification analysis. (Newmasteret al, .2007)
MATERIALS
AND
METHODS
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3. Material and Methods-:
3.1. Source of experimental materials
Green leaves were collected and identified in their flowering stage on the basis of in their leaf
morphology and floral structure from different location of Uttar Pradesh (Table-1). These
samples were stored either in silica gel or in -80c. These leaves were used for isolation, PCR
amplification and for sequencing.
3.2. Chemicals used
All the chemicals used in the present investigations were of analytical grade. Where as all the
chemicals of molecular biological grade were supplied by Bangalore Genie and Applied
Bioscience.
3.2.A. Buffer and Stock solution for DNA isolation-:1. Extraction Buffer 100 ml
CTAB (4%)
Tris (100mM)
EDTA (20 mM)
NaCl (1.4M)
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2. M Tris buffer (pH 8.0) 100ml
Weighed 12.11 g Tris base and dissolved in 80 ml Milli -Q water. Adjusted the pH to 8.0 with
0.1 N HCl and made up volume to 100 ml. Autoclaved and stored at room temperature.
3. 0.5 M EDTA (pH 8.0) 100ml
Weighed 18.61g Na2EDTA and dissolved in 80 ml Milli-Q water. Adjusted the pH to 8.0
with 1N NaOH. Shaken vigorously on a magnetic stirrer for some time to ensure that all the
solutes have dissolved. Made up to the volume 100 ml. Autoclaved and stored at room
temperature.
4. 5 M NaCl 100 ml
Weighed 29.2 g of NaCl and dissolved in 80 ml distilled water. Made up the volume 100 ml.
Autoclaved and stored at room temperature.
5. CTAB extraction buffer (pH 8.0) 100 ml
Weighed CTAB powder 2.5 g, added 1M Tris buffer (10 ml), 5M NaCl (30 ml) and 0.5
M EDTA (5 ml). pH was adjusted to 8.0 with 0.1N HCl. Made up the volume 100 ml. Stored
at room temperature.
6. 70% Ethanol 100 ml
Mixed 70 ml absolute ethyl alcohol in 30 ml distilled water. Stored at 0C.
7. Isopropanol 100ml
Stored at -20C in dark colored bottle.
8. TE buffer 100ml
Added 1 M Tris (pH 8.0) (1 ml), 0.5 M EDTA (0.1 ml), and distilled water (98.90 ml).
Adjusted pH to 8.0. Autoclaved and stored at room temperature.
3.2. B. Solution for DNA purification
1. Polyvinylpyrolidone(PVP)
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2. Chloroform: Isoamyl alcohol (24:1) 50 ml
Added 2 ml of Isoamyl alcohol into 48 ml of Chloroform. Stored it in a brown colored
bottle at RT.
Note: Mixing chloroform with other solvents may cause serious hazard. Do not mixes
chloroform with acetone and strong base.Isoamyl alcohol should be handled carefully.
Vapours are poisonous.
3. RNase 1 ml
Took 10 mg of RNase A and dissolved in 1 ml of distilled water. Dispensed into aliquots and
stored at -200C.
3.2.C Solutions for gel electrophoresis
1. DNA loading dye (6X) 10ml
Weighed bromophenol blue (0.25% w/v) (0.0025 g), xylene cyanol FF (0.25% w/v) (0.025g) and added 30% glycerol (3 ml) and double distilled water (8 ml). Made up the volume to
10 ml in distilled H2O and set pH 8.0 with 1 N NaOH. The dye is aliquoted into eppendorf
tubes and stored at 4C. We also used the Red Taq Polymerase which contains red dye which
do not required any more dye from outside.
2. Electrophoresis buffer (10X TBE) pH 8.0 1 litre
Weighed 108 g Tris base, 55 g boric acid and 9.5g EDTA, disodium salt. All the constituents
were dissolved in 750 ml double Milli -Q water. Adjusted pH to 8.0 with sodium hydroxide.
Filtered and adjusted final volume to 1 L. Autoclaved and kept at room temperature.
3. Ethidium bromide (10X) 10 ml
Weighed 10 mg Ethidium bromide (EtBr) and dissolved in 10ml Milli-Q water. Stored at RT.
Note:Ethidium bromide is highly carcinogenic. Use gloves
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Genomic DNA isolation-:
For genomic DNA isolation CTAB method (Doyle and Doyle, 1987) with slight
modification was used for the present study. In this method cetyl trimethyl ammonium
bromide buffer (CTAB) is used in the extraction of DNA, which serves as a detergent to
lyase the wall of cells for release of DNA.
Principle-:
The efficiency of isolation of genomic DNA depends upon the lysis of cells to release cells
contents. In order to prepare a cell extract, Tris buffer is the actual buffering component. -
mercaptoethanol acts as reducing agent and reduces disulphides and thus disrupts the tertiary
structure of proteins and denatures them. EDTA chelates Mg++ ions that are essential for
overall structure of cell envelope and for DNase activity.
Cetyl trimethyl ammonium bromide (CTAB) forms insoluble CTAB-DNA complexwhen added in cell extract. DNA-CTAB complex get precipitated leaving other component in
supernatant. Sample in Solution is then neutralized with potassium acetate solution,
centrifuged to remove this insoluble cell debris. Isopropanol precipitates nucleic acids (both
DNA and RNA). RNAcontamination can be removed by RNase treatment. Ethanol
precipitates nucleic acids and proteins along with other components in solution
Protocol-:
1gm of fresh leaves was ground to fine powder in liquid nitrogen by using mortar and
pestle.
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The powder was suspended in 12 ml of ice cold CTAB extraction buffer (2%, w/v
CTAB; 50 mM Tris-base, 20 mM EDTA, 1.4 NaCl and 0.1% v/v, ME) and
homogenized.
The mixture was kept at 65oC in water-bath for 3 hrs for lysis.
After incubation, the temperature of lysate was brought down to the room
temperature.
The lysate was extracted with 0.7 volume chloroform: isoamyl alcohol (24:1) and
gently mixed.
The mixture was centrifuged at 10,000 rpm for 10 min at room temp.
The aqueous layer was collected in a fresh polycarbonate tube and the supernatant
was discarded after centrifugation.
Then 0.7 volume of chilled isopropanol was added gently mixed and keep it at -20C
for overnight.
Then centrifuge at 8000 rpm for 10 min at 4C.
Supernatant was decanted by inverting the tube on paper towel to remove any residual
liquid.
The pellet was washed with 70% ethanol and dried at room temperature.
The nucleic acids containing genomic DNA were dissolved in 1ml TE buffer
containing DNase free RNase and incubate for 30 min at RT for RNA degradation.
The genomic DNA was extracted once with phenol: chloroform (1:1) and twice with
chloroform: isoamyl alcohol (24:1).
DNA was precipitated with 0.7 volume isopropanol, keep it at-20C for 1 hr.
Then centrifuge at 8,000 rpm for 10 min and supernatant was decanted by inverting
the tube, dried at room temp.
Dissolved in Milli- Q water and quantified on Nano drop spectrophotometer.
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Quantification of genomic DNA-:
The genomic DNA dissolved in Milli-Q water was taken for quantification by UV
absorbance at 260 nm. To measure the concentration, Eppendorf Biophotometer was used.
Reference was set against sterile Milli -Q water and then after absorbance of the sample was
measured at 260 nm and 280 nm. The concentration in ng/ l was recorded on the screen
along with the optical density at 260 nm, 280 nm and ratio of OD 260/280. An O.D. of 1.0 at
260 nm is equivalent to 50 g/l of double stranded DNA. The ratio of OD 260/280 gave an
indication of the amount of RNA or protein contamination in the preparation. A value of 1.8
is optimum for best DNA preparation. A value of the ratio below 1.8 indicates the presence of
protein in the preparation and a value above 1.8 indicates that our sample has RNA
contamination.
Agarose gel electrophoresis of the genomic DNA-:
Agarose gel electrophoresis of the isolated genomic DNA was performed to know
about the quality and quantity of DNA. Larger molecules migrate slower because of greater
frictional drag and they form their way through the pores of the gel less efficiently than
smaller molecules. As the size of genomic DNA is quite big, a 0.8% gel was used to visualize
the genomic DNA. A 0.8% gel resolves DNA molecules in the range of 0.7-8.5 kb (Fig-1).
3.3. PCR amplification
Following primers were selected for PCR amplification and Sequencing
Gene or spacer region Primer sequence
psbA-trnH
psbA3'f 5GTTATGCATGAACGTAATGCTC3
TrnHf 5CGCGCATGGTGGATTCACAATCC 3
rbcL
1.1F 5ATGTCACCACAAACAG3
724.1R 5ATGTACCTGCAGTAGC3
matK
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matK NBRI F1 5TCCCCATCCATCTGGAA3
matK 3.2r 5CTTCCTCTGTAAAGAATTC3
ITS
ITS5A(F) 5CCTTATCATTTAGAGGAAGGAG3
ITS4(R) 5TCCTCCGCTTATTGATATGC3
Genomic DNA isolated from the leaf sample was subjected to PCR amplification using the
forward and reverse primers of rbcL, matK, trnH- psbA and ITS. PCR amplification was
performed in 50 l volume in a thermocycler (Gene Amp 9700, Perkin Elmer, and USA).
PCR reaction was set up as following in a 0.2 ml PCR tube-:
:
PCRProgramme -:
Cycle for amplification of rbcL, matK, trnH-psbA & ITS
26
Template DNA (20-100ng) : 1 l
DNA Taq buffer (10X) : 5.0 l (1X)
dNTPs mixture (4mM) : 2.5 l (.2mM)
Primer F (10 pmol) : 2.0 l (10-20 pmol)
Primer R (10 pmol) : 2.0 l (10-20 pmol)
Taq DNA Polymerase (5U/l) : .5l(2.5U)
Milli-Q water : 37 l
Total : 50.0 l
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Cycle Denaturation Annealing Polymerization
First cycle 94C 4 min - - - -
34 Cycle 94C 40
second
48-58 C 45
second
72C 1 min
Last cycle - - - - 72C 7 min
Annealing temperature was variable according to the melting temperature of the primers.
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3.3.1 Agarose gel electrophoresis of the PCR Amplicons-:
The horizontal electrophoresis system was used to separate, identify and purify DNA
fragments using different concentration of agarose gel matrix and appropriate electrophoresis
buffer (Sam brook et al., 1989).
The edges of the gel-casting tray supplied with the electrophoresis assembly (Bangalore
Genei, India) were sealed with adhesive tape to form an enclosed chamber and the comb of
appropriate thickness was adjusted on sides of the chamber.
Desired amount of agarose (Sigma, USA) was dissolved in desired volume of 0.5x TBE
buffer (90 mM Tris base, 90 mM boric acid, 2 mM EDTA, pH 8.0) to get required
concentration of agarose gel matrix by heating in microwave oven, until the agarose was
completely dissolved.
The solution was allowed to cool at 50-55oC .
Ethidium bromide solution (0.5g/ml) was added and poured into casting tray and allowed to
solidify.
The comb and side tape were removed from the casting tray and gel was transferred to
electrophoretic chamber filled with sufficient amount 0.5x TBE buffer.
The DNA samples were mixed with gel loading dye (0.25% bromophenol blue in 4% w/v
sucrose) in 4:1 ratio and loaded in wells.
Electrophoresis was performed at desired constant voltage by connecting the chamber with
electric power supply instrument (Pharmacia EPS 500, Bio-Rad 200/2.0) to allow the DNA to
migrate from the cathode to anode. The gel was allowed to run for about 80% of its length.
The gels was examined on UV transilluminator (Fotodyne) and documented on gel
documentation system (Bio-Rad, USA).
Amplified DNA was then eluted by PCR clean up kit or by Gel Extraction.
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3.3.2. PCR Clean up from Kit (Nucleospin extract II, MACHEREY-
NAGEL)
Adjust DNA binding condition. Volume of PCR reaction is 50 ml. Make it upto
100ml by adding distilled water.
Mix 1 volume of sample with 2 volumes of buffer NT (Binding Buffer).
Load the sample on the Qiagen quick spin column, placed in a 2ml collection tube
and centrifuge at 1000 rpm for 1 min discard flow through.
Wash silica membrane.
Add 600l Buffer NT3 (Wash Buffer), centrifuge at 11000 rpm for 1 min discard
flow through.
Give the blank spin at 13,000 rpm for 1 min.
Dry the column (silica membrane) at room temperature for 5-10 min.
For DNA elution, place the column in clean 1.5 ml eppendrof tube.
Add 25lof MQ. Keep at room temperature for 5 min. centrifuge at 10,000 rpm for 1
min.
DNA is been collected in the tube.
3.4 Sequencing PCR and Clean up-:
Samples are prepared for sequencing after clean up 200- 300ng DNA is required for
sequencing. Template was lyophilized.
PCR Reaction- Total reaction volume- 5l
Template (100-200ng) : 2 l
Buffer : 1.75
Primer (10pmol/ l) : 2l
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Big Dye Reaction Mixture : .5l
Milli-Q water : 5.75
Total : 10
3.4. Sample Sequencing and Analysis:
Samples are prepared for sequencing after clean up. 200- 300ng DNA is required for
sequencing.
Sequencing PCR set up reaction- Total reaction volume- 10l
Master Mix-:
Template (100-200ng) 1 l
Dilution buffer 1.75l
Primer (10pmol/ l) 0 .5 l
Ready Reaction Mix (2.5x) 0 .5 l
Milli-Q water 6.25
Total 10 l
PCR Programme set up :
Step 1:
Rapid thermal ramp to 96Cfor 1 min.
Initial denaturation at 96C for 10sec
Step2:
Repeat the following for 25 cycle:
Rapid thermal ramp to 96C
Denaturation at 96C for 10 sec
Rapid thermal ramp to 50C
Primer annealing at 50C for 5 secs.
Rapid thermal ramp to 60C.
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Extension for 5mins.
Step:3
Rapid thermal ramp 4C and hold until ready to purify.
Protocol for purification of Sequencing PCR Product: Ethanol/EDTA Plate
Precipitation method:
Add 12 l of Sol-I having 125mM EDTA to pcr reaction product and mixed.
Add 52 l of Sol-II containing 200 l of 3M sodium acetate (4.8pH) and 5 ml
absolute ethanol mixed it by inversion.
Incubate at RT for 15 min.
Spin at a speed 3,850 rpm for 30 min at RTC.
Decant the supernatant and add 100 l 70% ethanol. Spin at 3,850 rpm for 30 min at
RT.
Dry the pellet by centrifuging it in invert condition and then kept it at 37C for 10
minutes.
Denaturation with HI-Dye
1Add 10 l Hi-Dye formamide in each tube then kept in PCR at 98 C
And keep it on ice
Kept it for sequencing in ABI- capillary sequencer. Sequencing was performedbidirectionally.
After 1hr sequence is obtained.
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RESULTS AND DISCUSSION
.
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.
Results and Discussion-:
Genomic DNA Isolation:
Genomic DNA of tree species: Genomic DNA was isolated from leaves of tree
species collected from different parts of UP following CTAB method as described in
materials and methods. Quantity of genomic DNA was measured using nanodrop
spectrophotometer and quality was checked on ethidium bromide stained 0.8% agarose gel
electrophoresis. Quality of genomic DNA was quiet good as revealed in Fig. 1.
Figure 1: Ethidium stained agarose gel showing gDNA from tree samples
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The quality and the yield of gDNA varied depending on tree species. Some tree
species have high phenolic substances which hindered isolation of good quality DNA.
Quality of DNA also depends on age of the leaf materials used for DNA extraction. In
general we observed that fresh and young leaves yielded good quality DNA as compared to
preserved and old leaves. Genomic DNA could not be isolated following standard CTAB
method because of very high content of phenolic substances. We need to modify the protocol
to isolate gDNA from these species.
PCR amplification of genomic DNA with selected barcode loci
Four different barcode loci viz. ITS, matK, rbcL and trnH-psbA were tested in
different tree species to check the PCR amplification efficiency. The PCR success rate of
different loci are given in table 1. The highest PCR success was observed with rbcL (97%)
followed by ITS (92%), trnH-psbA ( 85%) and matK (66%). Other studies have also reported
that rbcL is a good barcode locus as far as PCR amplification rate is concerned. Similarly ourresults also support the finding of others that matK is the least successful barcode locus as far
as PCR amplification is concerned. Quality of PCR amplification of trees genomic DNA was
quiet good. A representative agarose gel of PCR product with rbcL, ITS and trnH-psba and
matK primers are shown in Fig 2, 3, 4 and 5 respectively
Figure 2: Agarose gel electrophoresis of PCR products using rbcL primers
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Figure 3: Agarose gel electrophoresis of PCR products using ITS primers
Figure 4: Agarose gel electrophoresis of PCR products using trh-psbA primers
Figure 5: Agarose gel electrophoresis of PCR products using matK primers
PCR clean up and Sequencing of PCR products-:
The samples which showed positive PCR reactions were cleaned up using commerciallyavailable kits. This step removes the unused dNTPS and other chemicals. The cleaned up
PCR product was again quantified using nanodrop spectrophotometer and checked on
agarose gel. The quality and quantity of the cleaned up products were very good. This
ensures that the sequencing of the PCR products will be off good quality.
After cleaned up, 20 -30 ng of template DNA was used for sequencing reaction
following manufacturers protocol as described in materials and methods. The representative
eltrophorogram of sequences is shown in Fig 7. The sequencing success rate of different loci
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is predicted in Table 1. Sequencing success ranged from 85% for rbcL to 50% for matK
(Table 1). The alignment of sequences was straight forward except in case of trnH-psbA,due
to high variation in sequence length. The mean sequence lengths of ITS (ITS1+5.8S+ITS2),
matK, rbcL and trnH-psbA were 602.2, 488.1, 479.0 and 410.0bp, respectively (Table 1).
This lower success rate of PCR using matKmay be due to the instability and the uniqueness
of the primer 3'-end in matKsequences of as reported in other cases.
Figure 7: The representative electropherogram of sequences
Table 1: Results for four loci tested in species of the representatives species-:
36
Locus
# species Mean
sequence
length
%PCR
success
%
sequencing
success
Mean
interspecific
distances
(k2p)
Mean
intraspecific
distances(k2p)
ITS 60 602.2 92 71 0.0100.001 0.0020.001
matK 60 478.1 66 50 0.0030.001 0.0010.001
Rbcl 60 469.0 97 85 0.0010.0004 0.00010.0002
trnH-
psbA
60 410 85 72 0.0070.002 0.00070.001
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In most of the recent plant barcoding studies, the coding regions ofmatKand rbcL and the
non-coding plastid intergenic spacer of trnH-psbA have been suggested as prime candidates
for barcoding. Following the first suggestion by Kress et al. (2005) several subsequent
reports projected trnH-psbA as a strong candidate for plant barcoding. However, Consortium
for the Barcoding of Life (CBOL) disregarded trnH-psbA as it does not consistently provide
bidirectional unambiguous sequences reads Erstwhile studies have focused predominantly on
plastid regions for barcoding. Chase et al. (2005) and Kress et al. (2005) recovered highest
mean percentage sequence divergence (2.81 and 5.7% respectively) for nrITS region for plant
barcoding. However, the use of ITS region as barcode locus has often been considered
unfavourable because of the presence of paralogues in several plant taxa. Yet, in other
studies, ITS has been used successfully as barcode locus. More recently, ITS2 has been
projected as an important plant barcode locus.
In the present study, we attempted to extract good quality gDNA from diverse group
of tree species. Following gDNA isolation, PCR and sequencing success rate were evaluated
using different barcode loci. The sequences were corrected and aligned. However, detailed
analysis for barcoding purposes remains to be worked out.
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