Pollen DNA barcoding

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Microscopic image of Ligularia pollen Ligularia Pollen.jpg
Microscopic image of Ligularia pollen

Pollen DNA barcoding is the process of identifying pollen donor plant species through the amplification and sequencing of specific, conserved regions of plant DNA. Being able to accurately identify pollen has a wide range of applications though it has been difficult in the past due to the limitations of microscopic identification of pollen. [1]

Contents

Pollen identified using DNA barcoding involves the specific targeting of gene regions that are found in most to all plant species but have high variation between members of different species. The unique sequence of base pairs for each species within these target regions can be used as an identifying feature.

The applications of pollen DNA barcoding range from forensics, to food safety, to conservation. Each of these fields benefits from the creation of plant barcode reference libraries. [2] These libraries range largely in size and scope of their collections as well as what target region(s) they specialize in.

One of the main challenges of identifying pollen is that it is often collected as a mixture of pollen from several species. Metabarcoding is the process of identifying the individual species DNA from a mixed DNA sample and is commonly used to catalog pollen in mixed pollen loads found on pollinating animals and in environmental DNA (also called eDNA) which is DNA extracted straight from the environment such as in soil or water samples. [3]

Advantages

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Some of the principle constraints of microscopic identification are the expertise and time requirements. Identifying pollen via microscopy requires a high level of expertise in the pollen characteristics of the specific plants being studied. With expertise it can still be extremely difficult to identify pollen accurately with high taxonomic resolution. [1] The skills required to do DNA barcoding are much more common [4] making the approach easier to adopt. Pollen DNA barcoding is a technique that has grown in popularity due to the decreased costs associated with "next generation sequencing" (NGS) techniques [5] and is being continually improved in efficiency including through the use of a dual-indexing approach. [6] Some of the other major advantages include the savings in time and resources compared to microscopic identification. Identifying pollen is time-consuming, involving spreading pollen on a slide, staining the pollen to improve visibility, then focusing in on individual pollen grains and identifying them based on size, shape, as well as the shape and number of pores. [7] If a pollen reference library is not available, then pollen has to be collected from wild specimens or from herbarium specimens and is then added to a pollen reference library.

Rare plants visited by some pollinators can be difficult to determine, [8] by using pollen DNA barcoding researchers can uncover "invisible" interactions between plants and pollinators. [9]

Challenges

There are many challenges when it comes to genetic barcoding of pollen. The amplification process of DNA can mean that even small pieces of plant DNA can be detected included those from contaminants to a sample. Strict procedures to prevent contamination are important and can be facilitated by the hardiness of the pollen coat allowing the pollen to be washed of contaminants without damaging the internal pollen DNA.

DNA barcode reference libraries are still being built and standardized target regions are being gradually adopted. These challenges are likely due to the newness of DNA barcoding and will likely improve with the wider adoption of DNA barcoding as a tool used by taxonomists.

Determining the amount of each contributor to a mixed pollen load can be difficult to determine through the use of DNA barcoding. However, scientists have been able to compare pollen amounts via rank order. [10]

Alternatives

Innovations in automated microscopy and imagining software offer one potential alternative in the identification of pollen. Through the use of pattern-recognition software, researchers have developed software that can characterize microscopic pollen images based on texture analyzes. [11]

Target regions

There have been several different regions of plant DNA that have been used as targets for genetic barcoding including rbcL, [2] matK, [12] trnH-psbA, [13] ITS1 [14] and ITS2. A combination of rbcL and matK has been recommended for use in plant DNA barcoding. It has been found that trnL is better for degraded DNA and ITS1 is better for differentiating species within a genus. [15]

Applications

Butterfly foraging for nectar from a flower in the Chinese Himalayas Butterfly with Flower.jpg
Butterfly foraging for nectar from a flower in the Chinese Himalayas

Use in pollination networks

Being able to identify pollen is especially important in the study of pollination networks which are made up of all the interactions between plants and the animals that facilitate their pollination. [16] [17] Identifying the pollen carried on insects helps scientists understand what plants are being visited by which insects. Insects can also have homologous features making them difficult to identify and are themselves sometimes identified through genetic barcoding [18] (usually of the CO1 region [19] [20] ). Every insect that visits a flower is not necessarily a pollinator. [21] Many lack features such as hairs allowing them to carry pollen while others avoid the pollen-laden anthers to steal nectar. Pollination networks are made more accurate by including what pollen is being carried by which insects. Some scientists argue that pollination effectiveness (PE), which is measured by studying the germination rates of seeds produced from flowers visited only once by a single animal, is the best way to determine which animals are important pollinators [22] though other scientists have used DNA barcoding to determine the genetic origin of pollen found on insects and have argued that this in conjunction with other traits is a good indication of pollination effectiveness. [23] By studying the composition and structure of pollination networks, conservationists can understand the stability of a pollination network and identify which species are most important and which are at most risk to perturbation [24] leading to pollinator declines. [25]

Another advantage of pollen DNA barcoding is that it can be used to determine the source of pollen found on museum specimens of insects, [26] and these records of insect-plant interactions can then be compared to modern-day interactions to see how pollination networks have changed over time [27] due to global warming, land use change, and other factors.

Forensics

Being accurately able to identify pollen found on evidence helps forensic investigators identify which regions evidence originated from based on the plants that are endemic to those regions. [28] In addition to this, atmospheric pollen originating from illegal cannabis farms were successfully detected by scientists [29] which in the future could allow law enforcement officials to narrow down the search areas for illegal farms.

Ancient pollen

Due to the hardy structure of pollen which has evolved to survive being transported sometimes great distances while keeping the internal genetic information intact, the origin of pollen found mixed in ancient substrates can often be determined through DNA barcoding.

Food safety

Honeybees carry pollen as well as the nectar used in their production of honey. For food quality and safety concerns it is important to understand the plant providence of human-consumed bee products including honey, royal jelly, and pollen pellets. Investigators can test which plants honeybees foraged on and thus the origin of the nectar used in honey by collecting pollen packets from honeybees' corbicular loads and identify the pollen via DNA metabarcoding. [30]

See also

Related Research Articles

<span class="mw-page-title-main">Pollen</span> Grains containing the male gametophytes of seed plants

Pollen is a powdery substance produced by most types of flowers of seed plants for the purpose of sexual reproduction. It consists of pollen grains, which produce male gametes. Pollen grains have a hard coat made of sporopollenin that protects the gametophytes during the process of their movement from the stamens to the pistil of flowering plants, or from the male cone to the female cone of gymnosperms. If pollen lands on a compatible pistil or female cone, it germinates, producing a pollen tube that transfers the sperm to the ovule containing the female gametophyte. Individual pollen grains are small enough to require magnification to see detail. The study of pollen is called palynology and is highly useful in paleoecology, paleontology, archaeology, and forensics. Pollen in plants is used for transferring haploid male genetic material from the anther of a single flower to the stigma of another in cross-pollination. In a case of self-pollination, this process takes place from the anther of a flower to the stigma of the same flower.

<span class="mw-page-title-main">Honey bee</span> Colonial flying insect of genus Apis

A honey bee is a eusocial flying insect within the genus Apis of the bee clade, all native to mainland Afro-Eurasia. After bees spread naturally throughout Africa and Eurasia, humans became responsible for the current cosmopolitan distribution of honey bees, introducing multiple subspecies into South America, North America, and Australia.

<span class="mw-page-title-main">Forensic palynology</span> Forensic application of the study of particulate matter

Forensic palynology is a subdiscipline of palynology, that aims to prove or disprove a relationship among objects, people, and places that may pertain to both criminal and civil cases. Pollen can reveal where a person or object has been, because regions of the world, countries, and even different parts of a single garden will have a distinctive pollen assemblage. Pollen evidence can also reveal the season in which a particular object picked up the pollen. Recent research into forensic palynology has seen advancements in DNA barcoding from pollen, to the level of singular pollen molecules, allowing DNA profiles to be created from singular palynomorphs, streamlining the efficiency and accuracy of taxonomic identification.

<span class="mw-page-title-main">Pollinator</span> Animal that moves pollen from the male anther of a flower to the female stigma

A pollinator is an animal that moves pollen from the male anther of a flower to the female stigma of a flower. This helps to bring about fertilization of the ovules in the flower by the male gametes from the pollen grains.

<span class="mw-page-title-main">Pollination</span> Biological process occurring in plants

Pollination is the transfer of pollen from an anther of a plant to the stigma of a plant, later enabling fertilisation and the production of seeds. Pollinating agents can be animals such as insects, for example beetles or butterflies; birds, and bats; water; wind; and even plants themselves. Pollinating animals travel from plant to plant carrying pollen on their bodies in a vital interaction that allows the transfer of genetic material critical to the reproductive system of most flowering plants. When self-pollination occurs within a closed flower. Pollination often occurs within a species. When pollination occurs between species, it can produce hybrid offspring in nature and in plant breeding work.

<span class="mw-page-title-main">Entomophily</span> Form of pollination by insects

Entomophily or insect pollination is a form of pollination whereby pollen of plants, especially but not only of flowering plants, is distributed by insects. Flowers pollinated by insects typically advertise themselves with bright colours, sometimes with conspicuous patterns leading to rewards of pollen and nectar; they may also have an attractive scent which in some cases mimics insect pheromones. Insect pollinators such as bees have adaptations for their role, such as lapping or sucking mouthparts to take in nectar, and in some species also pollen baskets on their hind legs. This required the coevolution of insects and flowering plants in the development of pollination behaviour by the insects and pollination mechanisms by the flowers, benefiting both groups. Both the size and the density of a population are known to affect pollination and subsequent reproductive performance.

<span class="mw-page-title-main">Palynivore</span> Group of herbivorous animals

In zoology, a palynivore /pəˈlɪnəvɔːɹ/, meaning "pollen eater" is an herbivorous animal which selectively eats the nutrient-rich pollen produced by angiosperms and gymnosperms. Most true palynivores are insects or mites. The category in its strictest application includes most bees, and a few kinds of wasps, as pollen is often the only solid food consumed by all life stages in these insects. However, the category can be extended to include more diverse species. For example, palynivorous mites and thrips typically feed on the liquid content of the pollen grains without actually consuming the exine, or the solid portion of the grain. Additionally, the list is expanded greatly if one takes into consideration species where either the larval or adult stage feeds on pollen, but not both. There are other wasps which are in this category, as well as many beetles, flies, butterflies, and moths. One such example of a bee species that only consumes pollen in its larval stage is the Apis mellifera carnica. There is a vast array of insects that will feed opportunistically on pollen, as will various birds, orb-weaving spiders and other nectarivores.

<span class="mw-page-title-main">Pollination syndrome</span> Flower traits that attract pollinators

Pollination syndromes are suites of flower traits that have evolved in response to natural selection imposed by different pollen vectors, which can be abiotic or biotic, such as birds, bees, flies, and so forth through a process called pollinator-mediated selection. These traits include flower shape, size, colour, odour, reward type and amount, nectar composition, timing of flowering, etc. For example, tubular red flowers with copious nectar often attract birds; foul smelling flowers attract carrion flies or beetles, etc.

<span class="mw-page-title-main">Western honey bee</span> European honey bee

The western honey bee or European honey bee is the most common of the 7–12 species of honey bees worldwide. The genus name Apis is Latin for 'bee', and mellifera is the Latin for 'honey-bearing' or 'honey-carrying', referring to the species' production of honey.

<span class="mw-page-title-main">DNA barcoding</span> Method of species identification using a short section of DNA

DNA barcoding is a method of species identification using a short section of DNA from a specific gene or genes. The premise of DNA barcoding is that by comparison with a reference library of such DNA sections, an individual sequence can be used to uniquely identify an organism to species, just as a supermarket scanner uses the familiar black stripes of the UPC barcode to identify an item in its stock against its reference database. These "barcodes" are sometimes used in an effort to identify unknown species or parts of an organism, simply to catalog as many taxa as possible, or to compare with traditional taxonomy in an effort to determine species boundaries.

<span class="mw-page-title-main">Environmental DNA</span> DNA sampled from the environment rather than directly from an individual organism

Environmental DNA or eDNA is DNA that is collected from a variety of environmental samples such as soil, seawater, snow or air, rather than directly sampled from an individual organism. As various organisms interact with the environment, DNA is expelled and accumulates in their surroundings from various sources. Such eDNA can be sequenced by environmental omics to reveal facts about the species that are present in an ecosystem — even microscopic ones not otherwise apparent or detectable.

<span class="mw-page-title-main">Floral scent</span>

Floral scent, or flower scent, is composed of all the volatile organic compounds (VOCs), or aroma compounds, emitted by floral tissue. Other names for floral scent include, aroma, fragrance, floral odour or perfume. Flower scent of most flowering plant species encompasses a diversity of VOCs, sometimes up to several hundred different compounds. The primary functions of floral scent are to deter herbivores and especially folivorous insects, and to attract pollinators. Floral scent is one of the most important communication channels mediating plant-pollinator interactions, along with visual cues.

<span class="mw-page-title-main">Pedro Jordano</span>

Pedro Diego Jordano Barbudo is an ecologist, conservationist, researcher, focused on evolutionary ecology and ecological interactions. He is an honorary professor and associate professor at University of Sevilla, Spain. Most of his fieldwork is done in Parque Natural de las Sierras de Cazorla, Segura y Las Villas, in the eastern side of Andalucia, and in Doñana National Park, where he holds the title of Research Professor for the Estación Biológica Doñana, Spanish Council for Scientific Research (CSIC). Since 2000 he has been actively doing research in Brazil, with fieldwork in the SE Atlantic rainforest.

<span class="mw-page-title-main">Aquatic macroinvertebrate DNA barcoding</span>

DNA barcoding is an alternative method to the traditional morphological taxonomic classification, and has frequently been used to identify species of aquatic macroinvertebrates. Many are crucial indicator organisms in the bioassessment of freshwater and marine ecosystems.

<span class="mw-page-title-main">Algae DNA barcoding</span> Technique used for species identification and phylogenetic studies

DNA barcoding of algae is commonly used for species identification and phylogenetic studies. Algae form a phylogenetically heterogeneous group, meaning that the application of a single universal barcode/marker for species delimitation is unfeasible, thus different markers/barcodes are applied for this aim in different algal groups.

Microbial DNA barcoding is the use of DNA metabarcoding to characterize a mixture of microorganisms. DNA metabarcoding is a method of DNA barcoding that uses universal genetic markers to identify DNA of a mixture of organisms.

<span class="mw-page-title-main">Fish DNA barcoding</span>

DNA barcoding methods for fish are used to identify groups of fish based on DNA sequences within selected regions of a genome. These methods can be used to study fish, as genetic material, in the form of environmental DNA (eDNA) or cells, is freely diffused in the water. This allows researchers to identify which species are present in a body of water by collecting a water sample, extracting DNA from the sample and isolating DNA sequences that are specific for the species of interest. Barcoding methods can also be used for biomonitoring and food safety validation, animal diet assessment, assessment of food webs and species distribution, and for detection of invasive species.

<span class="mw-page-title-main">DNA barcoding in diet assessment</span>

DNA barcoding in diet assessment is the use of DNA barcoding to analyse the diet of organisms. and further detect and describe their trophic interactions. This approach is based on the identification of consumed species by characterization of DNA present in dietary samples, e.g. individual food remains, regurgitates, gut and fecal samples, homogenized body of the host organism, target of the diet study.

<span class="mw-page-title-main">Metabarcoding</span> Genetic technique for identifying organisms in mixed samples

Metabarcoding is the barcoding of DNA/RNA in a manner that allows for the simultaneous identification of many taxa within the same sample. The main difference between barcoding and metabarcoding is that metabarcoding does not focus on one specific organism, but instead aims to determine species composition within a sample.

<span class="mw-page-title-main">Plant–animal interaction</span> Relationships between plants and animals

Plant-animal interactions are important pathways for the transfer of energy within ecosystems, where both advantageous and unfavorable interactions support ecosystem health. Plant-animal interactions can take on important ecological functions and manifest in a variety of combinations of favorable and unfavorable associations, for example predation, frugivory and herbivory, parasitism, and mutualism. Without mutualistic relationships, some plants may not be able to complete their life cycles, and the animals may starve due to resource deficiency.

References

  1. 1 2 Bell, Karen L.; de Vere, Natasha; Keller, Alexander; Richardson, Rodney T.; Gous, Annemarie; Burgess, Kevin S.; Brosi, Berry J. (2016-04-13). "Pollen DNA barcoding: current applications and future prospects". Genome. 59 (9): 629–640. doi: 10.1139/gen-2015-0200 . hdl: 1807/72815 . ISSN   0831-2796. PMID   27322652.
  2. 1 2 Bell, Karen L.; Loeffler, Virginia M.; Brosi, Berry J. (2017-03-01). "An rbcL Reference Library to Aid in the Identification of Plant Species Mixtures by DNA Metabarcoding". Applications in Plant Sciences. 5 (3): 1600110. doi:10.3732/apps.1600110. PMC   5357121 . PMID   28337390.
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  30. ↑ Galimberti, Andrea; Mattia, Fabrizio De; Bruni, Ilaria; Scaccabarozzi, Daniela; Sandionigi, Anna; Barbuto, Michela; Casiraghi, Maurizio; Labra, Massimo (2014-10-08). "A DNA Barcoding Approach to Characterize Pollen Collected by Honeybees". PLOS ONE. 9 (10): e109363. Bibcode:2014PLoSO...9j9363G. doi: 10.1371/journal.pone.0109363 . ISSN   1932-6203. PMC   4190116 . PMID   25296114.
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