NANOTECH CONTROLLER
Nanotechnology will let us build fleets of computer controlled molecular tools and by
using this nano materials are created which these materials have very different properties than
materials at nano scale. They can be stronger, lighter, more electrically conductive, more
porus and less corressive than bulk materials. The nanomaterials have the potential to solve
unique biological challenges not currently possible.
The common examples of nanomaterials are found in scientific literature flurescence,
nanotubes, buckballs, quantum dots and nanoshells.
This was coined in1974 by Norio Taniguchi at the University of Tokyo.
Nanotechnology is actually a multitude of rapidly emerging technologies, most promising
potential of nanotechnology exists due to the laws of quantum physics.
Quantum physics laws take over at this scale enabling novel applications in optics,
electronics, magnetic storage, computing, catalysts and other areas.
It uses a basic unit of measure called a“nanometer” ( abbreviated nm) derived from
greek word for midget ,”nano” is a metric prefix and indicates a billionth part (10-9).There
are one million nm’s to ammeter
Each nm is only three to five atoms wide. They’re small. Really ~40,000 times smaller than
the width of an average human hair.
The invention of super small computers bacteria sized with todays MIPS capacity
processing power of billion laptops.
A nanometer is one thousandth of a micron and a thousandth of a millionth of meter
(a billionth of a meter). Imagine one billion nanometer in a meter perspective a nanometer. Is
about the width of six bounded carbon atoms, and approximately 40,000 are needed to equal
the width of an average human hair.
Another way to initialize nanometer 1 inch =25,400,000 nanometres.
A red blood cell is ~ 7,000 nm in diameter. And ~2,000 nm in height.
A virus is ~ 100 nm.
A buck ball is 1 nm.
A hydrogen atom is 1 nm.
For our purposes nanometers pertains to science, technology, manufacturing,
chemistry, health science, space programs, medical and engineering.
In engineering , it is used to reduce the size and increase the efficiency . But , in
medical it is used to overcome operations and severe risk taking disease. Think of your brain,
Now performing vastly superior levels Nano dots will become an as-needed addition to your
existing neurons extending your mental capabilities further than imagine .
The tablets, injections are used in nano technology for curing disease for living
organisms.
“But,in this tablets and injections are used for giving
instructions in brain ,and also we can be safe from any disease and we can cure any heart or
kidney problems without any surgery . During this treatment the human can continue his/her
regular work as usual. This can be taken as tablet, injection, food, cool drinks, oil applied to
hair etc”. for this construction a computer and the nano medicine is used.
Introduction
Background
The medical area of nano science application is one of the most potentially valuable, with
many projected benefits to humanity. Cells themselves are very complex and efficient nanomachines,
and chemists and biochemists have been working at the nano scale for some time
without using the nano label. Some areas of nano science aim to learn from biological nano
systems, while others are focusing on the integration of the organic and inorganic at the nano
scale. Many possible applications arising from this science are being researched.
Drug Delivery Using Nanoparticles and Molecular Carriers
Finally, drug delivery is likely to benefit from the development of nanotechnology.
With nanoparticles it is possible that drugs may be given better solubility, leading to better
absorption. Also, drugs may be contained within a molecular carrier, either to protect them
from stomach acids or to control the release of the drug to a specific targeted area, reducing
the likelihood of side effects. Such drugs are already beginning pre-clinical or clinical trials,
adhering to the strict regulatory requirements for new pharmaceuticals. Due to this,
development costs are often high and outcomes of research sometimes limited.
Lab on a Chip and Advanced Drug Delivery Systems
The ultimate combination of the laboratory-on-a-chip and advanced drug delivery
technologies would be a device that was implantable in the body, which would continuously
monitor the level of various biochemicals in the bloodstream and in response would release
appropriate drugs. For example, an insulin-dependent diabetic could use such a device to
continuously monitor and adjust insulin levels autonomously. There is no doubt that this is
the direction that current advances in which micro fluidics and drug delivery are heading.
Anesthesia
Anesthesia, or anaesthesia has traditionally meant the condition of having sensation
(including the feeling of pain) blocked. This allows patients to undergo surgery and other
procedures without the distress and pain they would otherwise experience. The word was
coined by Oliver Wendell Holmes, Sr. in 1846. Another definition is a "reversible lack of
awareness", whether this is a total lack of awareness (e.g. a general anaesthestic) or a lack of
awareness of a part of a the body such as a spinal anaesthetic or another nerve block would
cause. Anesthesia differs from analgesia in blocking all sensation, not only pain.
Today, the term general anesthesia in its most general form can include:
• Analgesia: blocking the conscious sensation of pain;
• Hypnosis: produces unconsciousness without analgesia;
• Amnesia: preventing memory formation;
• Relaxation: preventing unwanted movement or muscle tone;
• Obtundation of reflexes, preventing exaggerated autonomic reflexes.
Patients undergoing surgery usually undergo preoperative evaluation. It includes gathering
history of previous anesthetics, and any other medical problems, physical examination,
ordering required blood work and consultations prior to surgery.
There are several forms of anesthesia. The following forms refer to states achieved by
anesthetics working on the brain:
• General anesthesia: "Drug-induced loss of consciousness during which patients are
not arousable, even by painful stimulation." Patients undergoing general anesthesia
can often neither maintain their own airway nor breathe on their own. While usually
administered with inhalational agents, general anesthesia can be achieved with
intravenous agents, such as propofol.
• Deep sedation/analgesia: "Drug-induced depression of consciousness during which
patients cannot be easily aroused but respond purposefully following repeated or
painful stimulation." Patients may sometimes be unable to maintain their airway and
breathe on their own.
• Moderate sedation/analgesia or conscious sedation: "Drug-induced depression of
consciousness during which patients respond purposefully to verbal commands, either
alone or accompanied by light tactile stimulation." In this state, patients can breathe
on their own and need no help maintaining an airway.
• Minimal sedation or anxiolysis: "Drug-induced state during which patients respond
normally to verbal commands." Though concentration, memory, and coordination
may be impaired, patients need no help breathing or maintaining an airway.
The level of anesthesia achieved ranges on a continuum of depth of consciousness from
minimal sedation to general anesthesia. The depth of consciousness of a patient may change
from one minute to the next.
The following refer to the states achieved by anesthetics working outside of the brain:
• Regional anesthesia: Loss of pain sensation, with varying degrees of muscle
relaxation, in certain regions of the body. Administered with local anesthesia to
peripheral nerve bundles, such as the brachial plexus in the neck. Examples include
the interscalene block for shoulder surgery, axillary block for wrist surgery, and
femoral nerve block for leg surgery. While traditionally administered as a single
injection, newer techniques involve placement of indwelling catheters for continuous
or intermittent administration of local anesthetics.
o Spinal anesthesia: also known as subarachnoid block. Refers to a Regional
block resulting from a small volume of local anesthetics being injected into the
spinal canal. The spinal canal is covered by the dura mater, through which the
spinal needle enters. The spinal canal contains cerebrospinal fluid and the
spinal cord. The sub arachnoid block is usually injected between the 4th and
5th lumbar vertebrae, because the spinal cord usually stops at the 1st lumbar
vertebra, while the canal continues to the sacral vertebrae. It results in a loss of
pain sensation and muscle strength, usually up to the level of the chest (nipple
line or 4th thoracic dermatome).
o Epidural anesthesia: Regional block resulting from an injection of a large
volume of local anesthetic into the epidural space. The epidural space is a
potential space that lies underneath the ligamenta flava, and outside the dura
mater (outside layer of the spinal canal). This is basically an injection around
the spinal canal.
• Local anesthesia is similar to regional anesthesia, but exerts its effect on a smaller
area of the body.
History
Herbal derivatives
The first anesthesia (a herbal remedy) was administered in prehistory. Opium poppy capsules
were collected in 4200 BC, and opium poppies were farmed in Sumeria and succeeding
empires. The use of opium-like preparations in anaesthesia is recorded in the Ebers Papyrus
of 1500 BC
Non-pharmacological methods
Hypnotism and acupuncture have a long history of use as anesthetic techniques. In China,
Taoist medical practitioners developed anesthesia by means of acupuncture. Chilling tissue
(e.g. with ice) can temporarily cause nerve fibers (axons) to stop conducting sensation, while
hyperventilation can cause brief alteration in conscious perception of stimuli including pain
(see Lamaze).
Anesthetic agents
Local anesthetics
inhaled general anesthetic agents
intravenous anesthetic agents (non-opioid)
Current intravenous opioid analgesic agents
Current muscle relaxants
intravenous reversal agents
Anesthetic equipment
In modern anesthesia, a wide variety of medical equipment is desirable depending on the
necessity for portable field use, surgical operations or intensive care support. Anesthesia
practitioners must possess a comprehensive and intricate knowledge of the production and
use of various medical gases, anaesthetic agents and vapours, medical breathing circuits
and the variety of anaesthetic machines (including vaporizers, ventilators and pressure
gauges) and their corresponding safety features, hazards and limitations of each piece of
equipment, for the safe, clinical competence and practical application for day to day practice.
Anesthetic monitoring
Patients being treated under general anesthetics must be monitored continuously to ensure the
patient's safety.
Anesthesia record
The anesthesia record is the medical and legal documentation of events during an anesthetic.
It reflects a detailed and continuous account of drugs,
NANOTECHNOLOGY::
Nano is one billionth of one. Now we have the so-called microprocessors
and microarray technology that would reach the nano level within a few decades, we suppose.
Some call this technology to be nanotechnology and some others name it the molecular
nanotechnology, to be specific.
REASONS FOR APPLYIING NANOTECH TO BIIOLOGIICAL SYSTEMS::
Most animal cells are 10,000 to 20,000 nanometers in diameter. This means that
nanoscale devices (having at least one dimension less than 100 nanometers) can enter cells
and the organelles inside them to interact with DNA and proteins. Tools developed through
nanotechnology may be able to detect disease in a very small amount of cells or tissue. They
may also be able to enter and monitor cells within a living body. Miniaturization will allow
the tools for many different tests to be situated together on the same small device. This
means that nanotechnology could make it possible to run many diagnostic tests
simultaneously as well as with more sensitivity. In general, nanotechnology may offer a
faster and more efficient means for us to do much of what we do now.
NANOMEDICINE::
The emerging field of nanorobotics is aimed at overcoming the
shortcomings present in the traditional way of treatment of patients. Our bodies are filled
with intricate, active molecular structures. When those structures are damaged, health
suffers. Modern medicine can affect the work of the body in many ways, but from a
molecular viewpoint it remains crude. Molecular manufacturing can construct a range of
medical instruments and devices with greater abilities. The human body can be seen as a
workyard, construction site, and battleground form molecular machines. It works
remarkably well; using systems so complex that medical science still doesn’t understand
many of them.
BIOMEDICAL APPILICATIONS OF NANOROBOTS::
The enormous potential in the biomedical capabilities of nanorobots and
the imprecision and side effects of medical treatments today make nanorobots very
desirable. But today, in this revolutionary era we propose for nanomedical robots, since
they will have no difficulty in identifying the target site cells even at the very early stages
which cannot be done in the traditional treatment and will ultimately be able to track them
down and destroy them wherever they may be growing. By having these Robots, we can
refine the treatment of diseases by using biomedical, nanotechnological engineering.
Nanorobot designed to perform cell surgery
WHAT IS A MEDICINAL NANOROBOT ?
Nanorobots are theoretical microscopic devices measured on the scale of nanometers (1
nm equals one millionth of a millimeter). When fully realized from the hypothetical stage, they
would work at the atomic, molecular and cellular level to perform tasks in both the medical and
industrial fields that have heretofore been the stuff of science fiction Nanomedicine’s nanorobots
are so tiny that they can easily traverse the human body. Scientists report the exterior of a
nanorobot will likely be constructed of carbon atoms in a diamondoid structure because of its
inert properties and strength. Super-smooth surfaces will lessen the likelihood of triggering the
body’s immune system, allowing the nanorobots to go about their business unimpeded. Glucose
or natural body sugars and oxygen might be a source for propulsion, and the nanorobot will have
other biochemical or molecular parts depending on its task.
Nanorobot in Nanoscale
According to current theories, nanorobots will possess at least rudimentary two-way
communication; will respond to acoustic signals; and will be able to receive power or even reprogramming
instructions from an external source via sound waves. A network of special
stationary nanorobots might be strategically positioned throughout the body, logging each active
nanorobot as it passes, then reporting those results, allowing an interface to keep track of all of
the devices in the body. A doctor could not only monitor a patient’s progress but change the
instructions of the nanorobots in vivo to progress to another stage of healing. When the task is
completed, the nanorobots would be flushed from the body.
Nanorobot performing operations on blood cells
IMPLEMENTATION::
Anestthesiia USING NANOTECHNOLOGY::
Automated anesthesia delivery.
Automated delivery of inhalational anesthetics.
Automated I.V. anesthesia delivery system.
Nano assisted titration of I.V. agents with target controlled infusion.
CREATIION OF NANO DEVIICES::
The creation of the nano devices can be done using any of the two techniques that
are available. They are
• Top-down approach
• Bottom-up approach
CHALLENGES FACED BY NANOROBOTS::
While designing nonorobots in nanoscale dimensions there should be a better
understanding of how matter behaves on this small scale. Matter behaves differently on the
nanoscale than it does at larger levels. So the behaviour of the nanorobots must be taken
care so that the do not affect us both inside and outside the body.
Other challenges apply specifically to the use of nanostructures within biological
systems. Nanostructures can be so small that the body may clear them too rapidly for them
to be effective in detection or imaging. Larger nanoparticles may accumulate in vital
organs, creating a toxicity problem. So we need to consider these factors as they anticipate
how nanostructures will behave in the human body and attempt to create devices the body
will accept.
DESIIGN OF NANOROBOTS::
The nanorobots that we describe here will be floating freely inside the body
exploring and detect the various receptors eg GABA receptors in the brain, opioid
receptors, neuromuscular junction receptors. So, while designing such a nanorobot for
anesthesia, the main factors that are to be considered are given below.
TECHNIIQUE USED::
We use the bottom-up approach, which involves assembling structures atom-byatom
or molecule-by-molecule which will be useful in manufacturing devices used in
medicine.
SIIZE::
Nanorobots will typically be .5 to 3 microns large with 1-100 nm parts. Three microns
is the upper limit of any nanorobot because nanorobots of larger size will block capillary
flow.
STRUCTURE::
The nanorobot’s structure will have two spaces that are
Interior:
It will be a closed, vacuum environment into which liquids from the outside cannot
normally enter unless it is needed for chemical analysis.
Exterior: It will be subjected to various chemical liquids in our bodies.
CHEMIICAL ELEMENTS::
Carbon will likely be the principal element comprising the bulk of a
medical nanorobot, probably in the form of diamond or diamondoid/fullerene
nanocomposites largely because of the tremendous strength and chemicalinertness of
diamond. Many other light elements such as hydrogen, sulfur, oxygen,nitrogen, fluorine,
silicon, etc. may also be used
ABIILIITY TO DEFEND FROM IIMMUNE SYSTEM::
Immune system response is primarily a reaction to a "foreign" surface..
Passive diamond exteriors may turn out to be ideal. Several experimental studies hint that
the smoother and more flawless the diamond surface, the less leukocyte activity and the less
fibrinogen adsorption we will get. So it seems reasonable to hope that when diamond
coatings can be laid down with almost flawless atomic precision, making nanorobot exterior
surfaces with near-nanometer smoothness that these surfaces may have very low
bioactivity. Due to the extremely high surface energy of the passivated diamond surface and
the strong hydrophobicity of the diamond surface, the diamond exterior is almost
completely chemically inert and so opsonization should be minimized. If flawless diamond
surfaces alone do not prove fully bioinactive as hoped, active surface management of the
nanorobot exterior can be used to ensure complete nanodevice biocompatibility. Allergic
and shock reactions are similarly easily avoided.
ACQUIIRING POWER::
It could metabolize local glucose and oxygen for energy. Another possibility is
externally supplied acoustic power, which is probably most appropriate in a clinical setting.
There are literally dozens of useful power sources that are potentially available in the
human body.
COMMUNIICATON::
Having nanorobots inside the body it is very essential to know the actions done by it.
There are many different ways to do this. One of the simplest ways to send broadcast-type
messages into the body, to be received by nanorobots, is acoustic messaging. A device
similar to an ultrasound probe would encode messages on acoustic carrier waves at
frequencies between 1-10 MHz.
TRACKIING::
A navigational network may be installed in the body, with stationkeeping
navigational elements providing high positional accuracy to all passing nanorobots
that interrogate them, wanting to know their location. Physical positions can be
reported continuously using an in vivo communications network.
STRUCTURE OF NANOROBOT::
The nanorobot consists of three main parts like the receptor sensor, CPU, effector and the
power system. The purpose of receptor sensor is to identify the different anesthesia receptors on
the cell. The effector is used to produce the post receptor event.The CPU controls all the activities
.The power system provides the necessary energy for the working of the nanorobot.
GP120
layer GABA
layer
REQIREMENTS OF THE NANOROBOT:
1. It should e very small so that the blood capillary flow is not affected.
2. It should not be affected by the WBC.
3. It should be capable of attaching to anesthesia receptors only.
4. It should make its operations in the brain with GABA receptors, in the muscles with
neuromuscular junction and in the spinal cord with the opioid receptors .
5. It should be made of cheaper rates, so that the patient can afford it easily.
OPERATION::
The designed anesthesia nanorobots are injected into the blood stream. These nanorobots attaches
to the various receptor in the different parts of the body and produces the effects.
GABA receptors produces the loss of consciousness and amesia
Neuromuscular junction produces the full muscle relaxation
These two gives the good intubation conditions for securing the airway.
Opioid receptors produce the good analgesia.
In Spinal cord attaches to the sodium channel receptor and produces the spinal anesthesia
Arrangement
to produce
the effect
Recept
or
Power System
Central
Processin
g Unit
effector
Site for
receptor
attachment
ADVANTAGES::
1. More than million people in this world are undergoing surgery where anesthesia is essential.
Currently an anesthesiologist is required to give the anesthesia and carefull titration of the
drugs is essential to prevent the side effects like hypotension, desaturation, preventing the
intubation response.
2. As the nanorobot do not generate any harmful activities there is no side effect. It operates at
specific site only.
3. The initial cost of development is only high but the manufacturing by batch processing
reduces the cost.
4. Can be used in both general as well as spinal anesthesia.
5. Reduces the mortality and morbidity associated with anesthesia.
6. Patient satisfaction.
7. Less drug consumption and hence less side effects.
8. No peaks and downs in pain relief.
9. Labour analgesia; can provide pain relief for the mother at the time of delivery.
DISADVANTAGES::
1.The nanorobot should be very accurate, otherwise harmful effects may occur.
2.The initial design cost is very high.
3.The design of this nanorobot is a very complicated one
CONCLUSION::
The paper is just a recent advancement in the field of nanotechnology gives the hope of
the effective use of this technology in medical field. This paper gives an idea of giving exhalent
pain relief to millions of patients who undergo various types of surgery and also pain relief to
terminally ill cancer patients. Using this technology we can conduct safe and painless delivery
and provide exhalent care to the mother and baby.
Thursday, September 3, 2009
Use of DNA barcodes to identify flowering plants
Methods for identifying species by using short orthologous DNA sequences, known as “DNA barcodes,” have been proposed and initiated to facilitate biodiversity studies, identify juveniles, associate sexes, and enhance forensic analyses. The cytochrome c oxidase 1 sequence, which has been found to be widely applicable in animal barcoding, 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. We therefore propose the nuclear internal transcribed spacer region and the plastid trnH-psbA intergenic spacer as potentially usable DNA regions for applying barcoding 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 interspecific divergence. The trnH-psbA spacer, although short (≈450-bp), is the most variable plastid region in angiosperms and 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 barcoding purposes.
angiosperm internal transcribed spacer Plummers Island species identification trnH-psbA
The identification of animal biological diversity by using molecular markers has recently been proposed and demonstrated on a large scale through the use of a short DNA sequence in the cytochrome c oxidase 1 (CO1) gene (1-5). These “DNA barcodes” show promise in providing a practical, standardized, species-level identification tool that can be used for biodiversity assessment, life history and ecological studies, and forensic analysis. Engineered DNA sequences also have been suggested as exact identifiers and intellectual property tags for transgenic organisms (6). A Consortium for the Barcode of Life (http://www.barcoding.si.edu/) has been established to stimulate the creation of a database of documented and vouchered reference sequences to serve as a universal library to which comparisons of unidentified taxa can be made. Here, we propose two DNA regions for barcoding plants and provide an initial test of their utility.
DNA barcoding follows the same principle as does the basic taxonomic practice of associating a name with a specific reference collection in conjunction with a functional understanding of species concepts (i.e., interpreting discontinuities in interspecific variation). Presently, some controversy exists over the value of DNA barcoding (7), largely because of the perception that this new identification method would diminish rather than enhance traditional morphology-based taxonomy, that species determinations based solely on the amount of genetic divergence could result in incorrect species recognition, and that DNA barcoding is a means to reconstruct phylogenies when it is actually a tool to be used largely for identification purposes (8-10). In support of barcoding as a species identification process, Besansky et al. (11), Janzen (12, 13), Hebert et al. (1-4), and Kress (14) have offered arguments for the utility of DNA barcoding as a powerful framework for identifying specimens. Our objective in this paper is not to debate the validity of using barcodes for plant identification, but rather to determine appropriate DNA regions for use in flowering plants.
A portion of the mitochondrial CO1 gene was deliberately chosen for use in animal identification when DNA barcoding was proposed (1), and its broad utility in animal systems has been demonstrated in subsequent pilot studies (1-5). The taxonomic limits of CO1 barcoding in animals are not fully known, but it has proven useful to discriminate among species in most groups tested (2). The choice of a DNA region usable for barcoding has been little investigated in other eukaryotes, whereas in prokaryotes, rRNA genes are favored for identifications (e.g., ref. 15). Among plants, especially angiosperms, DNA-based identifications, although not strictly through the use of DNA barcodes, have been creatively used to reconstruct extinct herbivore diets (16, 17), to identify species of wood (18), to correlate roots growing in Texas caves with the surface flora (19), and to determine species used in herbal supplements (20). However, some of these identifications have not been entirely successful at the species level, and DNA barcoding per se has not yet been applied to plants. The primary reason that barcoding has not been applied to plants by the emerging initiative is that plant mitochondrial genes, because of their low rate of sequence change, are poor candidates for species-level discrimination. The divergence of CO1 coding regions among families of flowering plants has been documented to be only a few base pairs across 1.4 kb of sequence (21, 22). Furthermore, plants rapidly change their mitochondrial genome structure (23), thereby precluding the existence of universal intergenic spacers that otherwise would be appropriately variable unique identifiers at the species level (e.g., ref. 24).
For plant molecular systematic investigations at the species level, the internal transcribed spacer (ITS) region of the nuclear ribosomal cistron (18S-5.8S-26S) is the most commonly sequenced locus (25). This region has shown broad utility across photosynthetic eukaryotes (with the exception of ferns) and fungi and has been suggested as a possible plant barcode locus (26). Species-level discrimination and technical ease have been validated in most phylogenetic studies that employ ITS, and a large body of sequence data already exists for this region (>36,000 angiosperm sequences were available in GenBank in December 2004, although these sequences have not been filtered for taxa, so it is not certain how many species are represented). However, the limitations of this nuclear region in some taxa are well established. ITS has reduced species-level variability in certain groups (especially recently diverged taxa on islands), divergent paralogues that require cloning of multiple copies, and secondary structure problems resulting in poor-quality sequence data (25, 27). In some cases, the preferential amplification of endophytic or contaminating fungi may occur, although this can be eliminated with plant-specific primer design (28, 29).
An advantage of the ITS region is that it can be amplified in two smaller fragments (ITS1 and ITS2) adjoining the 5.8S locus, which has proven especially useful for degraded samples. The quite conserved 5.8S region in fact contains enough phylogenetic signal for discrimination at the level of orders and phyla (29), although identification at this taxonomic level is not the concern of barcoding. Alignments are trivial to optimize for 5.8S due to the few indels found in plants and fungi (30). In contrast for phylogenetic reconstruction, ITS or any rapidly evolving noncoding region can require complex sequence alignment for homology assessments. Thus, the 5.8S locus can serve as a critical alignment-free anchor point for search algorithms that make sequence comparisons for both phylogenetic and barcoding purposes. The utility of conserved regions such as 5.8S to generate a pool of nearest neighbors for refined comparisons will be critical for effective database searches, especially when comparing a sequence that has no identical match in a sequence library. GenBank blast searches with our ITS data (see below) returned correct matches for the sequences in GenBank. This success suggests that despite alignment concerns, current search algorithms will be fast and effective at using ITS for species-level identifications, given an adequate database for comparison. For all of these reasons, ITS, even with its recognized limitations, is a prime candidate as an effective locus for DNA barcoding in plants.
However, the recognition that ITS has certain functional limitations for DNA barcoding of plants is a compelling argument that a search for additional loci is warranted. For phylogenetic investigations, the plastid genome has been more readily exploited than the nuclear genome and may offer for plant barcoding what the mitochondrial genome does for animals. It is a uniparentally inherited, nonrecombining, and, in general, structurally stable genome. Universal primers are available for a number of loci and intergenic spacers that are evolving at a variety of rates. The plastid locus most commonly sequenced by plant systematists for phylogenetic purposes is rbcL, followed by the trnL-F intergenic spacer, matK, ndhF, and atpB (e.g., refs. 31-33). rbcL has been suggested as a candidate for plant barcoding (34), even though it has generally been used to determine evolutionary relationships at the generic level and above. Besides rbcL and atpB, all of the latter plastid loci have been used at the species level with various degrees of success. Most of them (except the trnL-F spacer) require full-length sequences of >1 kb to yield enough sequence length to discriminate species. Most relevant to plant barcoding, no region of the plastid genome has been found to have the high level of variation seen in most animal CO1 barcodes, although a few intergenic spacers have shown more promise than any plastid locus now in general use (33).
When evaluating other genetic loci appropriate for plant DNA barcoding, three criteria must be satisfied: (i) significant species-level genetic variability and divergence, (ii) an appropriately short sequence length so as to facilitate DNA extraction and amplification, and (iii) the presence of conserved flanking sites for developing universal primers. With regard to sequence length, we note that in CO1 barcoding systems, the 600- to 700-bp length fortuitously matches high-quality sequence data from average capillary sequencer reads, although it is expected that routine read length will improve with new technology. An important rationale for using short sequences also resides in the need to obtain useful data from potentially degraded samples found in museum specimens. Amplicon size and gene copy number have been shown to account for much of the variability of amplification success: smaller sizes and increased copy number promote greater success with PCR, presumably by increasing the likelihood that a desired sequence has been preserved (18).
Previous SectionNext SectionMaterials and Methods
Determining Suitable Regions of the Genome. To screen for appropriate levels of sequence divergence in the plastid genome, we chose two closely related flowering plant species for comparison, Atropa belladonna and Nicotiana tabacum (Solanaceae). Both species have complete sequence data available for their plastid genomes (35-37). Twenty-nine additional complete plastid genomes spread across a wide range of plant groups are also available for comparison: algae (five genera in various families), mosses and liverworts (three genera in different families), ferns and relatives (three genera in different families), gymnosperms (two species in the genus Pinus), and angiosperms (eight genera in eight different families, two genera in the Fabaceae, and four genera and several cultivars in the Poaceae). We selected Nicotiana and Atropa, even though they belong to different subfamilies of Solanaceae (38), because they represent the most closely related taxa among the genomes available in the angiosperms. The complete plastid genomes of the taxa in the Fabaceae and the Poaceae include cultivars, hybrids, and more distantly related genera. We aligned the Nicotiana and Atropa genomes, and raw divergence levels (i.e., number of base-pair discordances divided by length of sequence under consideration) were individually estimated across all genes, introns, and intergenic spacers (Fig. 1). Plastid regions with raw sequence differences ≥2% (Table 1) were categorized as the most variable segments, and therefore the most promising of the plastid genome for DNA barcoding when normalized for length. The nuclear ITS region and plastid rbcL gene were used as baseline comparisons for these chloroplast test regions (Table 1). To further narrow down the number of remaining regions usable for barcoding purposes, we applied a sequence criterion of 300-800 bp and a stable presence across multiple plastid genomes of both monocots and dicots.
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Download as PowerPoint SlideFig. 1. Plastid genome variation between deadly nightshade (A. belladonna; shown) and tobacco (N. tabacum), adapted from Shinozaki et al. (35). Shown are a complete genome (A), loci with ≥1% sequence difference between species (B), and loci with ≥2% sequence difference between species (C). The letters in C correspond to spacer regions listed in Table 1.
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In this window In a new windowTable 1. Sampled loci in plastid genomes of Atropa belladonna and Nicotiana tabacum that were found to have base-pair sequence divergences ≥2%
Selecting Taxa for Testing. To empirically test the regions identified as most appropriate for barcoding in our comparison of the plastid genomes of Atropa and Nicotiana (Table 1), we selected two sets of flowering plant taxa. The first taxon set consisted of 2 or 3 species in each of eight genera spread across seven families of plants for a total of 19 species (Table 2 and Table 3, which is published as supporting information on the PNAS web site). The second taxon set included a geographically circumscribed flora comprised of taxa that are not closely related but represent a broad range of angiosperms in 50 plant families, including 83 species in 72 genera (Table 3). The selection of the two taxon sets was made so as to test each locus for appropriate sequence length and divergence, primer success across a wide taxonomic spectrum, and the viability of routinely extracting DNA from dried herbarium specimens, compared with fresh or silica-dried tissue. The species in the first taxon set were selected because they represent a diverse set of species pairs across the angiosperms (including monocots and dicots) with various levels of phylogenetic distance as previously shown in research by the authors using other genetic markers (W.J.K. and K.J.W., unpublished data). In addition, high-quality DNA extractions from living plants, silica-dried tissue, and/or herbarium specimens were readily available for these taxa. The genera were not selected randomly and were not biased a priori toward low or high levels of interspecific divergence. The second taxon set was selected to represent a floristic sample that would be used in a typical plant DNA barcoding project. The samples were taken from Plummers Island, MD, a National Park Service habitat reserve in the Potomac River that has been studied and inventoried by biologists in the Washington, DC, area for >100 years, making it an appropriate test site for barcoding trials. For the Plummers Island taxa, tissue samples were taken from dried leaves only on herbarium specimens located in the U.S. National Herbarium (Smithsonian Institution) collected between 1960 and 2000 (Table 3). These samples were used to compare ITS and rbcL as standards to the best plastid regions identified in the tests of taxon set one. A smaller set of older herbarium collections of Erysimum cheiranthoides (Brassicaceae) prepared as early as 1897 were compared with more recent collections made as recently as 1997 from the same populations to empirically test the relationship between specimen preservation status, age, and DNA quality (see Fig. 2, which is published as supporting information on the PNAS web site).
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In this window In a new windowTable 2. Sequence length and percent sequence divergence for nine plastid regions sampled for eight genera from taxon set one
DNA Analysis. New DNA extractions were performed with the DNeasy Plant Mini kit (Qiagen, Valencia, CA) after tissue disruption of 0.5-1 cm2 of leaf tissue in a FastPrep FP-120 bead mill (Qbiogene, Carlsbad, CA). DNA extractions followed manufacturer's protocols with the modification of buffer AP1 lysis conditions by the addition of 0.4 mg of proteinase, 15 mg of DTT, and incubation at 42°C for 12 h on a rocking platform. This method can easily be scaled up to a 96-well format for large-scale (high-throughput) barcoding purposes. Amplification by PCR used puReTaq Ready-To-Go PCR beads (Amersham Pharmacia Biosciences) and direct sequencing of purified PCR products used bigdye 3.1 software on a 3100 sequencer, both from Applied Biosystems. Universal primers for selected genes and intergenic spacers were taken from investigations described in refs. 39-41 and Table 4, which is published as supporting information on the PNAS web site. Comparative rbcL data were generated for the Plummers Island flora by splitting the gene into two overlapping fragments (1f-724r and 636f-1368r), because test amplifications on a portion of the samples netted only 31% success as a full-length fragment vs. 94% as two pieces.
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In our comparison of the Atropa and Nicotiana plastid genomes, the most variable regions that tentatively met the barcode criteria were nine intergenic spacers: trnK-rps16, trnH-psbA, rp136-rps8, atpB-rbcL, ycf6-psbM, trnV-atpE, trnC-ycf6, psbM-trnD, and trnL-F (listed in order of decreasing variability; Table 1 and Fig. 1). By comparison, ITS had a much higher divergence value (13.6%) than any of the plastid regions, and rbcL was by far the lowest in divergence (0.83%). Although three spacers (atpB-rbcL, ycf6-psbM, and psbM-trnD) were slightly to moderately longer than our 800-bp cutoff, we included them in our further analysis because of their high interspecific variability.
The results of our intrageneric tests across eight genera in the first taxon set demonstrated conspicuous differences between the nine plastid regions with respect to our three barcoding criteria: amplification success, sequence length, and sequence divergence. Only three regions (trnH-psbA, rp136-rpf8, and trnL-F) were successfully amplified for all eight genera and 19 species; the other regions, including ITS, could not be amplified in one or more taxa (Table 2). Sequence length in the nine plastid regions ranged from 204 to 1,240 bp, with mean length in all but two (ycf6-psbM and psbM-trnD) falling within our 300- to 800-bp optimum length criterion (Table 2). ITS had the highest between-species sequence divergence values in four of the five genera successfully amplified (Table 2), with a mean sequence divergence of 2.81% across the five genera. trnH-psbA ranked first in divergence value in six of the eight genera and in 11 of the 14 species pairs, compared with the other eight plastid regions; trnV-atpE and trnC-ycf6 ranked highest for the remaining two genera and three species pairs (Table 2). trnH-psbA ranked highest (1.24%) in mean percent sequence divergence across all genera, whereas trnV-atpE (0.29%) and ycf6-psbM (0.30%) ranked lowest (Table 2).
In our broader taxonomic sampling of the Plummers Island flora in which only herbarium material was used, none of the loci could be successfully amplified for all of the 83 species tested, which we suggest may be related to primer design or to more fundamental changes in gene structure during herbarium specimen preparation and storage (see ref. 33). Amplification success was highest for trnH-psbA (100%), followed by rbcL (5′ half; 95%), and ITS (88%, although high-quality sequence data were not obtained from all ITS amplifications). We could not detect any general correlation between specimen age and amplification success, indicating that herbarium specimens in apparently good condition and as old as 20 years can be successfully used to establish DNA-sequence reference libraries. Moreover, amplification of full-length ITS was possible (results not shown) for the five specimens of Erysimum cheiranthoides collected between 1897 and 1997 (Fig. 2), indicating that significantly older specimens also may be used.
Because of the high sequence divergence value in the majority of genera in our taxon set one and the high amplification success of the trnH-psbA spacer in all of our test samples, this region became the focus of our examination of the plastid genome for further analyses of barcoding potential. The trnH-psbA amplicon ranged from 247 to 1,221 bp, whereas the intergenic spacer alone (excluding primer-binding regions and small regions of flanking exon) ranged from 119 to 1,094 bp across 53 families of flowering plants, including both the Plummers Island species and the taxonomic groups (extremes were Thalictrum and Trillium, respectively; see Table 2 and Table 5, which is published as supporting information on the PNAS web site). Most taxa (92%) had amplicons falling between 340 and 660 bp, which is within our suggested length criterion for successful barcoding. All species in our sampling had unique trnH-psbA spacer sequences, which is very relevant to the question of using this gene for barcoding plants.
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The ITS and rbcL loci provide a baseline against which to compare other genes and intergenic spacers in our directed search for sequences to use in plant DNA barcoding. Besides ITS, those single-copy nuclear genes or their introns that are gaining prominence in species-level molecular systematics studies (e.g., leafy, waxy, pistillata, and RPB2), also were considered. However, because of the lack of universal primers (either published or with potential development by using current information) and poor success by using existing primers, these loci have been eliminated as potential barcode loci. The poor success by using existing primers is probably due to the difficulty of amplifying genes with low numbers of copies in degraded samples and the frequent need to clone PCR products before sequencing. We, therefore, turned our attention to the plastid genome in search of the most variable sequences that would also meet the criteria needed for maximum utility (i.e., variability, universal primers, and short length) and that could be used in place of or in addition to the ITS region. The significantly greater length of rbcL (usually 1,428 bp; Tables 1 and 2) causes problems because it is necessary to use four primers for double-stranded sequencing of the entire gene. Although this number of primers is equivalent to that needed if a two-loci system is used for barcoding purposes, the level of interspecific variation we observed in rbcL is less than the variation detected in either ITS or trnH-psbA alone (Table 2). Furthermore, this gene has been previously discounted for discrimination at the species level (e.g., refs. 31, 42, and 43).
We suggest that the trnH-psbA intergenic spacer is the best plastid option for a DNA barcode sequence that has good priming sites, length, and interspecific variation. In our trials across a diverse set of genera in seven plant families, three plastid regions (trnH-psbA, rp136-rpf8, and trnL-F) ranked highest with respect to amplification success and appropriate sequence length, but trnH-psbA demonstrated nearly 3 times the percentage sequence divergence of these other two regions (1.24% in trnH-psbA vs. 0.44% in both rp136-rpf8 and trnL-F; Table 2). The two spacers with the next highest mean sequence divergence after trnH-psbA (atpB-rbcL at 0.63% and trnC-ycf6 at 0.55%) could not be amplified in one or more of the test genera. In only one genus (Solidago; Asteraceae), exceptionally low sequence divergence in trnH-psbA prevented discrimination among the three species tested, although insertion/deletion differences still allowed us to distinguish among the species. This lack of sequence divergence between taxa was true for one or more species pairs in ITS and all other plastid spacers, except atpB-rbcL, in our test sample. In only 2% of our samples did homopolymer regions adversely affect sequence quality in trnH-psbA.
For a number of reasons, we refrained from a statistical test of differences among mean sequence divergences of the nine spacer regions. First, the sample size in our survey was too restricted to provide a meaningful statistical test (although the standard error of the mean of trnH-psbA does not directly overlap with the means of any of the other spacers). More importantly, as pointed out by Shaw et al. (33), genera within and between families of plants are phylogenetically nonequivalent, i.e., lineages recognized as genera may have quite different divergence rates depending on the various life history traits of the included species. Therefore, statistical comparisons between genera with respect to genetic distance are not valid or warranted at this time. Our intent in calculating these mean percent divergences across loci is to provide a qualitative evaluation of each spacer region for barcoding purposes. In this respect, we consider the high divergence value of trnH-psbA, which permits species discrimination in the largest number of taxa we tested (six of the eight genera and 11 of the 14 species pairs), as strong support for its use as a plant barcode.
The universality of trnH-psbA for differentiating among all flowering plant species clearly needs further investigation (see below), especially in taxa with extremely short spacers that may not contain enough sequence variation for species-level discrimination (e.g., Thalictrum and Solidago in our study and Minuartia in ref. 33). This spacer region also is present in other nonflowering land plants. In a search of GenBank, we found that the trnH-psbA spacer has been successfully amplified in angiosperms, gymnosperms, ferns, mosses, and liverworts, although we do not know at this time the degree of between-species divergence. Further study is needed to determine whether this plastid region is as variable in the nonflowering plants as we have shown for our test angiosperms, and therefore whether it is of broad utility as a barcode across the total spectrum of land plants.
Our findings on the properties of trnH-psbA agree with Shaw et al. (33) in their extensive survey of noncoding plastid DNA for phylogenetic purposes. By applying our barcode criteria (i.e., length considerations and universality) to the framework of their study, we conclude that trnH-psbA has greater potential for species-level discrimination than any other locus they analyzed. Similar to our results, they demonstrated that trnH-psbA amplified and sequenced easily with an average length of 465 bp across the 30 taxa they surveyed. Although this region was the second most variable of the 21 spacers they tested in terms of potentially informative characters, they ranked its utility for phylogenetic purposes as low (tier 3) because of its short length. Our analysis of the number of nucleotide substitutions within genera across all taxa in the 21 plastid regions presented by Shaw et al. (33) indicates that the trnH-psbA spacer has the highest percentage nucleotide difference (0.0135 difference per base pair), even though at least 8 of the 21 other regions showed a greater total number of nucleotide substitutions because of their longer length. The interspecific nucleotide differences in trnH-psbA ranged from 18% to 105% higher than that of the other eight most variable plastid regions. Because short sequence length is an important criterion for barcoding, the high frequency of nucleotide differences of trnH-psbA, in combination with its relatively short length, is a significant advantage. Other studies also have shown a high percentage of interspecific divergence for trnH-psbA, and in most cases, the highest in all plastid regions tested (e.g., refs. 44-48).
Despite this high level of interspecific variation, trnH-psbA has found only limited use in species-level phylogenetic reconstruction because of the short length as well as the difficulty of alignments resulting from a high number of indels (e.g., refs. 49-51). In contrast with the problems of indels for phylogenetic construction, we suspect that indels will ultimately enhance the information needed for species identifications, once the appropriate informatics tools for barcoding are developed. In the set of species we sampled, sequences were alignable within genera, but problematic above that rank. In the one case (Solidago) where sequence divergence was not sufficient to separate species, the presence of unique indels allowed easy discrimination among the taxa. Blaxter (34) advocates ease of alignment as a criterion when evaluating the utility of barcode loci. We do not consider difficulty of alignment to be a major obstacle to the applicability of either ITS or trnH-psbA for the primary purpose of DNA barcoding, i.e., identification. Although ease of alignment is desirable, it is not necessary for barcoding. Searches in GenBank by using our data from both loci with a blast search returned correct identities at both the gene and species level. blast searches are anchored and canalized by conserved regions in both loci, 5.8S in ITS and the small region of flanking exon for trnH-psbA. Intraspecific variation in both ITS and trnH-psbA is known to be relatively low, compared with interspecific variation (27, 52), although in the present study, our intraspecific sampling was insufficient to address this issue.
The extraction of DNA from specimens in herbarium collections was highly successful. This success may be due to the specimens having been air-dried and in a good state of preservation as evidenced by the generally green appearance of the leaves selected for extraction (Fig. 2). Plant voucher specimens vary in how and when they are dried after being pressed. If specimen-drying facilities are not immediately available, especially in humid tropical climates, botanists often treat pressed specimens with ethanol to temporarily preserve them against fungal attack and degradation. Alcohol has been shown to be detrimental to recovering high-quality DNA (53), although how it will affect the short sequences needed for barcoding is unknown. We are encouraged by the fact that museum specimens of insects dried from ethanol storage readily yield CO1 sequences. A more thorough investigation and optimization of methods to extract high-quality barcode DNA from herbarium collections in a high-throughput format will be critical to efficiently build a sequence-database library for plant DNA barcodes. Our positive results by using well preserved specimens indicate that the a priori selection of apparently undegraded plant samples will be an important determinant of success. Fortunately, herbaria often have more than one specimen per species among which to select for successful DNA barcoding.
We have shown here that there are gene sequences suitable for DNA barcoding of flowering plants. It may be necessary to employ more than one locus to attain species-level discrimination across all flowering plant species. Algorithms for combining barcoding sequences from two or more DNA regions to yield species-level unique identifiers are now needed. We believe that ITS and trnH-psbA serve as good starting points for large-scale testing of DNA barcoding across a large sample of angiosperms. A good test would be to expand taxon sampling through the application of both ITS and trnH-psbA to barcode the estimated 8,000 species of flowering plants of Costa Rica (54).
angiosperm internal transcribed spacer Plummers Island species identification trnH-psbA
The identification of animal biological diversity by using molecular markers has recently been proposed and demonstrated on a large scale through the use of a short DNA sequence in the cytochrome c oxidase 1 (CO1) gene (1-5). These “DNA barcodes” show promise in providing a practical, standardized, species-level identification tool that can be used for biodiversity assessment, life history and ecological studies, and forensic analysis. Engineered DNA sequences also have been suggested as exact identifiers and intellectual property tags for transgenic organisms (6). A Consortium for the Barcode of Life (http://www.barcoding.si.edu/) has been established to stimulate the creation of a database of documented and vouchered reference sequences to serve as a universal library to which comparisons of unidentified taxa can be made. Here, we propose two DNA regions for barcoding plants and provide an initial test of their utility.
DNA barcoding follows the same principle as does the basic taxonomic practice of associating a name with a specific reference collection in conjunction with a functional understanding of species concepts (i.e., interpreting discontinuities in interspecific variation). Presently, some controversy exists over the value of DNA barcoding (7), largely because of the perception that this new identification method would diminish rather than enhance traditional morphology-based taxonomy, that species determinations based solely on the amount of genetic divergence could result in incorrect species recognition, and that DNA barcoding is a means to reconstruct phylogenies when it is actually a tool to be used largely for identification purposes (8-10). In support of barcoding as a species identification process, Besansky et al. (11), Janzen (12, 13), Hebert et al. (1-4), and Kress (14) have offered arguments for the utility of DNA barcoding as a powerful framework for identifying specimens. Our objective in this paper is not to debate the validity of using barcodes for plant identification, but rather to determine appropriate DNA regions for use in flowering plants.
A portion of the mitochondrial CO1 gene was deliberately chosen for use in animal identification when DNA barcoding was proposed (1), and its broad utility in animal systems has been demonstrated in subsequent pilot studies (1-5). The taxonomic limits of CO1 barcoding in animals are not fully known, but it has proven useful to discriminate among species in most groups tested (2). The choice of a DNA region usable for barcoding has been little investigated in other eukaryotes, whereas in prokaryotes, rRNA genes are favored for identifications (e.g., ref. 15). Among plants, especially angiosperms, DNA-based identifications, although not strictly through the use of DNA barcodes, have been creatively used to reconstruct extinct herbivore diets (16, 17), to identify species of wood (18), to correlate roots growing in Texas caves with the surface flora (19), and to determine species used in herbal supplements (20). However, some of these identifications have not been entirely successful at the species level, and DNA barcoding per se has not yet been applied to plants. The primary reason that barcoding has not been applied to plants by the emerging initiative is that plant mitochondrial genes, because of their low rate of sequence change, are poor candidates for species-level discrimination. The divergence of CO1 coding regions among families of flowering plants has been documented to be only a few base pairs across 1.4 kb of sequence (21, 22). Furthermore, plants rapidly change their mitochondrial genome structure (23), thereby precluding the existence of universal intergenic spacers that otherwise would be appropriately variable unique identifiers at the species level (e.g., ref. 24).
For plant molecular systematic investigations at the species level, the internal transcribed spacer (ITS) region of the nuclear ribosomal cistron (18S-5.8S-26S) is the most commonly sequenced locus (25). This region has shown broad utility across photosynthetic eukaryotes (with the exception of ferns) and fungi and has been suggested as a possible plant barcode locus (26). Species-level discrimination and technical ease have been validated in most phylogenetic studies that employ ITS, and a large body of sequence data already exists for this region (>36,000 angiosperm sequences were available in GenBank in December 2004, although these sequences have not been filtered for taxa, so it is not certain how many species are represented). However, the limitations of this nuclear region in some taxa are well established. ITS has reduced species-level variability in certain groups (especially recently diverged taxa on islands), divergent paralogues that require cloning of multiple copies, and secondary structure problems resulting in poor-quality sequence data (25, 27). In some cases, the preferential amplification of endophytic or contaminating fungi may occur, although this can be eliminated with plant-specific primer design (28, 29).
An advantage of the ITS region is that it can be amplified in two smaller fragments (ITS1 and ITS2) adjoining the 5.8S locus, which has proven especially useful for degraded samples. The quite conserved 5.8S region in fact contains enough phylogenetic signal for discrimination at the level of orders and phyla (29), although identification at this taxonomic level is not the concern of barcoding. Alignments are trivial to optimize for 5.8S due to the few indels found in plants and fungi (30). In contrast for phylogenetic reconstruction, ITS or any rapidly evolving noncoding region can require complex sequence alignment for homology assessments. Thus, the 5.8S locus can serve as a critical alignment-free anchor point for search algorithms that make sequence comparisons for both phylogenetic and barcoding purposes. The utility of conserved regions such as 5.8S to generate a pool of nearest neighbors for refined comparisons will be critical for effective database searches, especially when comparing a sequence that has no identical match in a sequence library. GenBank blast searches with our ITS data (see below) returned correct matches for the sequences in GenBank. This success suggests that despite alignment concerns, current search algorithms will be fast and effective at using ITS for species-level identifications, given an adequate database for comparison. For all of these reasons, ITS, even with its recognized limitations, is a prime candidate as an effective locus for DNA barcoding in plants.
However, the recognition that ITS has certain functional limitations for DNA barcoding of plants is a compelling argument that a search for additional loci is warranted. For phylogenetic investigations, the plastid genome has been more readily exploited than the nuclear genome and may offer for plant barcoding what the mitochondrial genome does for animals. It is a uniparentally inherited, nonrecombining, and, in general, structurally stable genome. Universal primers are available for a number of loci and intergenic spacers that are evolving at a variety of rates. The plastid locus most commonly sequenced by plant systematists for phylogenetic purposes is rbcL, followed by the trnL-F intergenic spacer, matK, ndhF, and atpB (e.g., refs. 31-33). rbcL has been suggested as a candidate for plant barcoding (34), even though it has generally been used to determine evolutionary relationships at the generic level and above. Besides rbcL and atpB, all of the latter plastid loci have been used at the species level with various degrees of success. Most of them (except the trnL-F spacer) require full-length sequences of >1 kb to yield enough sequence length to discriminate species. Most relevant to plant barcoding, no region of the plastid genome has been found to have the high level of variation seen in most animal CO1 barcodes, although a few intergenic spacers have shown more promise than any plastid locus now in general use (33).
When evaluating other genetic loci appropriate for plant DNA barcoding, three criteria must be satisfied: (i) significant species-level genetic variability and divergence, (ii) an appropriately short sequence length so as to facilitate DNA extraction and amplification, and (iii) the presence of conserved flanking sites for developing universal primers. With regard to sequence length, we note that in CO1 barcoding systems, the 600- to 700-bp length fortuitously matches high-quality sequence data from average capillary sequencer reads, although it is expected that routine read length will improve with new technology. An important rationale for using short sequences also resides in the need to obtain useful data from potentially degraded samples found in museum specimens. Amplicon size and gene copy number have been shown to account for much of the variability of amplification success: smaller sizes and increased copy number promote greater success with PCR, presumably by increasing the likelihood that a desired sequence has been preserved (18).
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Determining Suitable Regions of the Genome. To screen for appropriate levels of sequence divergence in the plastid genome, we chose two closely related flowering plant species for comparison, Atropa belladonna and Nicotiana tabacum (Solanaceae). Both species have complete sequence data available for their plastid genomes (35-37). Twenty-nine additional complete plastid genomes spread across a wide range of plant groups are also available for comparison: algae (five genera in various families), mosses and liverworts (three genera in different families), ferns and relatives (three genera in different families), gymnosperms (two species in the genus Pinus), and angiosperms (eight genera in eight different families, two genera in the Fabaceae, and four genera and several cultivars in the Poaceae). We selected Nicotiana and Atropa, even though they belong to different subfamilies of Solanaceae (38), because they represent the most closely related taxa among the genomes available in the angiosperms. The complete plastid genomes of the taxa in the Fabaceae and the Poaceae include cultivars, hybrids, and more distantly related genera. We aligned the Nicotiana and Atropa genomes, and raw divergence levels (i.e., number of base-pair discordances divided by length of sequence under consideration) were individually estimated across all genes, introns, and intergenic spacers (Fig. 1). Plastid regions with raw sequence differences ≥2% (Table 1) were categorized as the most variable segments, and therefore the most promising of the plastid genome for DNA barcoding when normalized for length. The nuclear ITS region and plastid rbcL gene were used as baseline comparisons for these chloroplast test regions (Table 1). To further narrow down the number of remaining regions usable for barcoding purposes, we applied a sequence criterion of 300-800 bp and a stable presence across multiple plastid genomes of both monocots and dicots.
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Download as PowerPoint SlideFig. 1. Plastid genome variation between deadly nightshade (A. belladonna; shown) and tobacco (N. tabacum), adapted from Shinozaki et al. (35). Shown are a complete genome (A), loci with ≥1% sequence difference between species (B), and loci with ≥2% sequence difference between species (C). The letters in C correspond to spacer regions listed in Table 1.
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In this window In a new windowTable 1. Sampled loci in plastid genomes of Atropa belladonna and Nicotiana tabacum that were found to have base-pair sequence divergences ≥2%
Selecting Taxa for Testing. To empirically test the regions identified as most appropriate for barcoding in our comparison of the plastid genomes of Atropa and Nicotiana (Table 1), we selected two sets of flowering plant taxa. The first taxon set consisted of 2 or 3 species in each of eight genera spread across seven families of plants for a total of 19 species (Table 2 and Table 3, which is published as supporting information on the PNAS web site). The second taxon set included a geographically circumscribed flora comprised of taxa that are not closely related but represent a broad range of angiosperms in 50 plant families, including 83 species in 72 genera (Table 3). The selection of the two taxon sets was made so as to test each locus for appropriate sequence length and divergence, primer success across a wide taxonomic spectrum, and the viability of routinely extracting DNA from dried herbarium specimens, compared with fresh or silica-dried tissue. The species in the first taxon set were selected because they represent a diverse set of species pairs across the angiosperms (including monocots and dicots) with various levels of phylogenetic distance as previously shown in research by the authors using other genetic markers (W.J.K. and K.J.W., unpublished data). In addition, high-quality DNA extractions from living plants, silica-dried tissue, and/or herbarium specimens were readily available for these taxa. The genera were not selected randomly and were not biased a priori toward low or high levels of interspecific divergence. The second taxon set was selected to represent a floristic sample that would be used in a typical plant DNA barcoding project. The samples were taken from Plummers Island, MD, a National Park Service habitat reserve in the Potomac River that has been studied and inventoried by biologists in the Washington, DC, area for >100 years, making it an appropriate test site for barcoding trials. For the Plummers Island taxa, tissue samples were taken from dried leaves only on herbarium specimens located in the U.S. National Herbarium (Smithsonian Institution) collected between 1960 and 2000 (Table 3). These samples were used to compare ITS and rbcL as standards to the best plastid regions identified in the tests of taxon set one. A smaller set of older herbarium collections of Erysimum cheiranthoides (Brassicaceae) prepared as early as 1897 were compared with more recent collections made as recently as 1997 from the same populations to empirically test the relationship between specimen preservation status, age, and DNA quality (see Fig. 2, which is published as supporting information on the PNAS web site).
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In this window In a new windowTable 2. Sequence length and percent sequence divergence for nine plastid regions sampled for eight genera from taxon set one
DNA Analysis. New DNA extractions were performed with the DNeasy Plant Mini kit (Qiagen, Valencia, CA) after tissue disruption of 0.5-1 cm2 of leaf tissue in a FastPrep FP-120 bead mill (Qbiogene, Carlsbad, CA). DNA extractions followed manufacturer's protocols with the modification of buffer AP1 lysis conditions by the addition of 0.4 mg of proteinase, 15 mg of DTT, and incubation at 42°C for 12 h on a rocking platform. This method can easily be scaled up to a 96-well format for large-scale (high-throughput) barcoding purposes. Amplification by PCR used puReTaq Ready-To-Go PCR beads (Amersham Pharmacia Biosciences) and direct sequencing of purified PCR products used bigdye 3.1 software on a 3100 sequencer, both from Applied Biosystems. Universal primers for selected genes and intergenic spacers were taken from investigations described in refs. 39-41 and Table 4, which is published as supporting information on the PNAS web site. Comparative rbcL data were generated for the Plummers Island flora by splitting the gene into two overlapping fragments (1f-724r and 636f-1368r), because test amplifications on a portion of the samples netted only 31% success as a full-length fragment vs. 94% as two pieces.
Previous SectionNext SectionResults
In our comparison of the Atropa and Nicotiana plastid genomes, the most variable regions that tentatively met the barcode criteria were nine intergenic spacers: trnK-rps16, trnH-psbA, rp136-rps8, atpB-rbcL, ycf6-psbM, trnV-atpE, trnC-ycf6, psbM-trnD, and trnL-F (listed in order of decreasing variability; Table 1 and Fig. 1). By comparison, ITS had a much higher divergence value (13.6%) than any of the plastid regions, and rbcL was by far the lowest in divergence (0.83%). Although three spacers (atpB-rbcL, ycf6-psbM, and psbM-trnD) were slightly to moderately longer than our 800-bp cutoff, we included them in our further analysis because of their high interspecific variability.
The results of our intrageneric tests across eight genera in the first taxon set demonstrated conspicuous differences between the nine plastid regions with respect to our three barcoding criteria: amplification success, sequence length, and sequence divergence. Only three regions (trnH-psbA, rp136-rpf8, and trnL-F) were successfully amplified for all eight genera and 19 species; the other regions, including ITS, could not be amplified in one or more taxa (Table 2). Sequence length in the nine plastid regions ranged from 204 to 1,240 bp, with mean length in all but two (ycf6-psbM and psbM-trnD) falling within our 300- to 800-bp optimum length criterion (Table 2). ITS had the highest between-species sequence divergence values in four of the five genera successfully amplified (Table 2), with a mean sequence divergence of 2.81% across the five genera. trnH-psbA ranked first in divergence value in six of the eight genera and in 11 of the 14 species pairs, compared with the other eight plastid regions; trnV-atpE and trnC-ycf6 ranked highest for the remaining two genera and three species pairs (Table 2). trnH-psbA ranked highest (1.24%) in mean percent sequence divergence across all genera, whereas trnV-atpE (0.29%) and ycf6-psbM (0.30%) ranked lowest (Table 2).
In our broader taxonomic sampling of the Plummers Island flora in which only herbarium material was used, none of the loci could be successfully amplified for all of the 83 species tested, which we suggest may be related to primer design or to more fundamental changes in gene structure during herbarium specimen preparation and storage (see ref. 33). Amplification success was highest for trnH-psbA (100%), followed by rbcL (5′ half; 95%), and ITS (88%, although high-quality sequence data were not obtained from all ITS amplifications). We could not detect any general correlation between specimen age and amplification success, indicating that herbarium specimens in apparently good condition and as old as 20 years can be successfully used to establish DNA-sequence reference libraries. Moreover, amplification of full-length ITS was possible (results not shown) for the five specimens of Erysimum cheiranthoides collected between 1897 and 1997 (Fig. 2), indicating that significantly older specimens also may be used.
Because of the high sequence divergence value in the majority of genera in our taxon set one and the high amplification success of the trnH-psbA spacer in all of our test samples, this region became the focus of our examination of the plastid genome for further analyses of barcoding potential. The trnH-psbA amplicon ranged from 247 to 1,221 bp, whereas the intergenic spacer alone (excluding primer-binding regions and small regions of flanking exon) ranged from 119 to 1,094 bp across 53 families of flowering plants, including both the Plummers Island species and the taxonomic groups (extremes were Thalictrum and Trillium, respectively; see Table 2 and Table 5, which is published as supporting information on the PNAS web site). Most taxa (92%) had amplicons falling between 340 and 660 bp, which is within our suggested length criterion for successful barcoding. All species in our sampling had unique trnH-psbA spacer sequences, which is very relevant to the question of using this gene for barcoding plants.
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The ITS and rbcL loci provide a baseline against which to compare other genes and intergenic spacers in our directed search for sequences to use in plant DNA barcoding. Besides ITS, those single-copy nuclear genes or their introns that are gaining prominence in species-level molecular systematics studies (e.g., leafy, waxy, pistillata, and RPB2), also were considered. However, because of the lack of universal primers (either published or with potential development by using current information) and poor success by using existing primers, these loci have been eliminated as potential barcode loci. The poor success by using existing primers is probably due to the difficulty of amplifying genes with low numbers of copies in degraded samples and the frequent need to clone PCR products before sequencing. We, therefore, turned our attention to the plastid genome in search of the most variable sequences that would also meet the criteria needed for maximum utility (i.e., variability, universal primers, and short length) and that could be used in place of or in addition to the ITS region. The significantly greater length of rbcL (usually 1,428 bp; Tables 1 and 2) causes problems because it is necessary to use four primers for double-stranded sequencing of the entire gene. Although this number of primers is equivalent to that needed if a two-loci system is used for barcoding purposes, the level of interspecific variation we observed in rbcL is less than the variation detected in either ITS or trnH-psbA alone (Table 2). Furthermore, this gene has been previously discounted for discrimination at the species level (e.g., refs. 31, 42, and 43).
We suggest that the trnH-psbA intergenic spacer is the best plastid option for a DNA barcode sequence that has good priming sites, length, and interspecific variation. In our trials across a diverse set of genera in seven plant families, three plastid regions (trnH-psbA, rp136-rpf8, and trnL-F) ranked highest with respect to amplification success and appropriate sequence length, but trnH-psbA demonstrated nearly 3 times the percentage sequence divergence of these other two regions (1.24% in trnH-psbA vs. 0.44% in both rp136-rpf8 and trnL-F; Table 2). The two spacers with the next highest mean sequence divergence after trnH-psbA (atpB-rbcL at 0.63% and trnC-ycf6 at 0.55%) could not be amplified in one or more of the test genera. In only one genus (Solidago; Asteraceae), exceptionally low sequence divergence in trnH-psbA prevented discrimination among the three species tested, although insertion/deletion differences still allowed us to distinguish among the species. This lack of sequence divergence between taxa was true for one or more species pairs in ITS and all other plastid spacers, except atpB-rbcL, in our test sample. In only 2% of our samples did homopolymer regions adversely affect sequence quality in trnH-psbA.
For a number of reasons, we refrained from a statistical test of differences among mean sequence divergences of the nine spacer regions. First, the sample size in our survey was too restricted to provide a meaningful statistical test (although the standard error of the mean of trnH-psbA does not directly overlap with the means of any of the other spacers). More importantly, as pointed out by Shaw et al. (33), genera within and between families of plants are phylogenetically nonequivalent, i.e., lineages recognized as genera may have quite different divergence rates depending on the various life history traits of the included species. Therefore, statistical comparisons between genera with respect to genetic distance are not valid or warranted at this time. Our intent in calculating these mean percent divergences across loci is to provide a qualitative evaluation of each spacer region for barcoding purposes. In this respect, we consider the high divergence value of trnH-psbA, which permits species discrimination in the largest number of taxa we tested (six of the eight genera and 11 of the 14 species pairs), as strong support for its use as a plant barcode.
The universality of trnH-psbA for differentiating among all flowering plant species clearly needs further investigation (see below), especially in taxa with extremely short spacers that may not contain enough sequence variation for species-level discrimination (e.g., Thalictrum and Solidago in our study and Minuartia in ref. 33). This spacer region also is present in other nonflowering land plants. In a search of GenBank, we found that the trnH-psbA spacer has been successfully amplified in angiosperms, gymnosperms, ferns, mosses, and liverworts, although we do not know at this time the degree of between-species divergence. Further study is needed to determine whether this plastid region is as variable in the nonflowering plants as we have shown for our test angiosperms, and therefore whether it is of broad utility as a barcode across the total spectrum of land plants.
Our findings on the properties of trnH-psbA agree with Shaw et al. (33) in their extensive survey of noncoding plastid DNA for phylogenetic purposes. By applying our barcode criteria (i.e., length considerations and universality) to the framework of their study, we conclude that trnH-psbA has greater potential for species-level discrimination than any other locus they analyzed. Similar to our results, they demonstrated that trnH-psbA amplified and sequenced easily with an average length of 465 bp across the 30 taxa they surveyed. Although this region was the second most variable of the 21 spacers they tested in terms of potentially informative characters, they ranked its utility for phylogenetic purposes as low (tier 3) because of its short length. Our analysis of the number of nucleotide substitutions within genera across all taxa in the 21 plastid regions presented by Shaw et al. (33) indicates that the trnH-psbA spacer has the highest percentage nucleotide difference (0.0135 difference per base pair), even though at least 8 of the 21 other regions showed a greater total number of nucleotide substitutions because of their longer length. The interspecific nucleotide differences in trnH-psbA ranged from 18% to 105% higher than that of the other eight most variable plastid regions. Because short sequence length is an important criterion for barcoding, the high frequency of nucleotide differences of trnH-psbA, in combination with its relatively short length, is a significant advantage. Other studies also have shown a high percentage of interspecific divergence for trnH-psbA, and in most cases, the highest in all plastid regions tested (e.g., refs. 44-48).
Despite this high level of interspecific variation, trnH-psbA has found only limited use in species-level phylogenetic reconstruction because of the short length as well as the difficulty of alignments resulting from a high number of indels (e.g., refs. 49-51). In contrast with the problems of indels for phylogenetic construction, we suspect that indels will ultimately enhance the information needed for species identifications, once the appropriate informatics tools for barcoding are developed. In the set of species we sampled, sequences were alignable within genera, but problematic above that rank. In the one case (Solidago) where sequence divergence was not sufficient to separate species, the presence of unique indels allowed easy discrimination among the taxa. Blaxter (34) advocates ease of alignment as a criterion when evaluating the utility of barcode loci. We do not consider difficulty of alignment to be a major obstacle to the applicability of either ITS or trnH-psbA for the primary purpose of DNA barcoding, i.e., identification. Although ease of alignment is desirable, it is not necessary for barcoding. Searches in GenBank by using our data from both loci with a blast search returned correct identities at both the gene and species level. blast searches are anchored and canalized by conserved regions in both loci, 5.8S in ITS and the small region of flanking exon for trnH-psbA. Intraspecific variation in both ITS and trnH-psbA is known to be relatively low, compared with interspecific variation (27, 52), although in the present study, our intraspecific sampling was insufficient to address this issue.
The extraction of DNA from specimens in herbarium collections was highly successful. This success may be due to the specimens having been air-dried and in a good state of preservation as evidenced by the generally green appearance of the leaves selected for extraction (Fig. 2). Plant voucher specimens vary in how and when they are dried after being pressed. If specimen-drying facilities are not immediately available, especially in humid tropical climates, botanists often treat pressed specimens with ethanol to temporarily preserve them against fungal attack and degradation. Alcohol has been shown to be detrimental to recovering high-quality DNA (53), although how it will affect the short sequences needed for barcoding is unknown. We are encouraged by the fact that museum specimens of insects dried from ethanol storage readily yield CO1 sequences. A more thorough investigation and optimization of methods to extract high-quality barcode DNA from herbarium collections in a high-throughput format will be critical to efficiently build a sequence-database library for plant DNA barcodes. Our positive results by using well preserved specimens indicate that the a priori selection of apparently undegraded plant samples will be an important determinant of success. Fortunately, herbaria often have more than one specimen per species among which to select for successful DNA barcoding.
We have shown here that there are gene sequences suitable for DNA barcoding of flowering plants. It may be necessary to employ more than one locus to attain species-level discrimination across all flowering plant species. Algorithms for combining barcoding sequences from two or more DNA regions to yield species-level unique identifiers are now needed. We believe that ITS and trnH-psbA serve as good starting points for large-scale testing of DNA barcoding across a large sample of angiosperms. A good test would be to expand taxon sampling through the application of both ITS and trnH-psbA to barcode the estimated 8,000 species of flowering plants of Costa Rica (54).
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