Detection of Invasive Species 16.5 PCR-Based Methods Utilized for Routine Identification and/or Surveillance 445
Part B | 16.5
to undergo relatively rapid rates of evolution [16.64,
65]. Mitochondria are generally inherited maternally,
making them particularly useful as a species-specific
marker for the delineation of closely related species
e.g., [16.35, 39]. The identification of different strains
(i. e., population variants) has important implications
for understanding the geographical origin and patterns
of spread of marine pest species, potentially providing
a better understanding of patterns and means of translocation. Mitochondrial DNA has proven particularly
useful in understanding the genetic structure of populations due to their maternal inheritance which provides
tracers to patterns of colonization and its smaller effective population size (when compared to the nuclear
genome [16.66–68]).
Like mitochondria, chloroplasts have a circular
genome that is smaller than the nuclear genome. Typically, chloroplast genomes display lower levels of
heterogeneity than mitochondrial genomes; this is in
part due to mitochondria having larger genomes which
encode for less genes than chloroplasts [16.69, 70]. The
ribulose-bisphosphate carboxylase (rbcL) gene is of interest for use as a diagnostic marker of algae. Hanyuda
et al. [16.71] reported that the intron in the rbcL
gene was highly variable amongst different species and
strains of Caluerpa, and [16.32] used a rbcL PCR-based
assay to identify invasive strains of Caulerpa taxifolia.
Freshwater et al. [16.33] also used a rbcL gene PCRbased assay to discriminate invasive Gracilaria from
nearly morphologically identical native species.
16.5 PCR-Based Methods Utilized for Routine Identification
and/or Surveillance
A wide range of molecular-based techniques have
been employed for the identification of species at
adult and/or larval stages of marine invasive species
or related species (Table 16.1). The advantages, limitations, and uses of these techniques for marine
pest research and detection are summarized in Table 16.2, and the limitations of these approaches are
further discussed in the following section. PCR has
revolutionized many areas of biological research, including species and strain delineation. PCR can amplify minute amounts of template DNA, and its high
specificity makes it highly effective for species and
strain identification for a wide range of organisms.
PCR with direct sequencing is a gold standard technique; however, it takes several days to obtain results, and a relevant question is whether other PCRbased procedures can be used as reliably in the same
amount of time. The relatively low cost of equipment and reagents makes PCR accessible to small
laboratories.
16.5.1 PCR-Based Methods
Species Detection Through Design of Specific
Primers
PCR utilizing genus or species specific primers for marine pests has been utilized using a wide range of techniques: one-step reactions e.g. [16.23, 24, 30, 35, 38],
nested PCR [16.37], and multiplex [16.44] and nested
multiplex PCR [16.43, 75].
Random Amplified Polymorphic DNA (RAPD)
RAPD (RAPD) is based on the amplification of random fragments of genomic DNA using single primers
of arbitrary sequence. RAPD offers rapid molecular assessment in that it does not require the design of specific primers that bind exclusively to
one sequence type. It essentially scans the entire
genome without prior knowledge of sequence information. RAPD will produce a specific banding pattern
when amplicons are subjected to electrophoresis. It
can be particularly useful in discriminating between
closely related species or strains and understanding
sequence variation between individuals [16.52]. Zhou
and Gao [16.22] utilized RAPD for the identification
of mitten crab (Eriocheir sinensis) populations, but it
has not otherwise been widely utilized for marine pest
identification.
Restriction Fragment Length Polymorphism
(RFLP)
Restriction (RFLP) endonucleases are used to cut
DNA at precise sequences producing specific bands
which are visualized via electrophoresis. When RFLP
is combined with PCR, a fragment will be amplified via PCR before being digested, using the restriction endonucleases to give a characteristic pattern based on the amplified fragment. RFLP has
not been widely used for the identification and/or
surveillance of marine pests. Scholin and Anderson [16.49] used PCR-RFLP to distinguish between
Part B | 16.5
to undergo relatively rapid rates of evolution [16.64,
65]. Mitochondria are generally inherited maternally,
making them particularly useful as a species-specific
marker for the delineation of closely related species
e.g., [16.35, 39]. The identification of different strains
(i. e., population variants) has important implications
for understanding the geographical origin and patterns
of spread of marine pest species, potentially providing
a better understanding of patterns and means of translocation. Mitochondrial DNA has proven particularly
useful in understanding the genetic structure of populations due to their maternal inheritance which provides
tracers to patterns of colonization and its smaller effective population size (when compared to the nuclear
genome [16.66–68]).
Like mitochondria, chloroplasts have a circular
genome that is smaller than the nuclear genome. Typically, chloroplast genomes display lower levels of
heterogeneity than mitochondrial genomes; this is in
part due to mitochondria having larger genomes which
encode for less genes than chloroplasts [16.69, 70]. The
ribulose-bisphosphate carboxylase (rbcL) gene is of interest for use as a diagnostic marker of algae. Hanyuda
et al. [16.71] reported that the intron in the rbcL
gene was highly variable amongst different species and
strains of Caluerpa, and [16.32] used a rbcL PCR-based
assay to identify invasive strains of Caulerpa taxifolia.
Freshwater et al. [16.33] also used a rbcL gene PCRbased assay to discriminate invasive Gracilaria from
nearly morphologically identical native species.
16.5 PCR-Based Methods Utilized for Routine Identification
and/or Surveillance
A wide range of molecular-based techniques have
been employed for the identification of species at
adult and/or larval stages of marine invasive species
or related species (Table 16.1). The advantages, limitations, and uses of these techniques for marine
pest research and detection are summarized in Table 16.2, and the limitations of these approaches are
further discussed in the following section. PCR has
revolutionized many areas of biological research, including species and strain delineation. PCR can amplify minute amounts of template DNA, and its high
specificity makes it highly effective for species and
strain identification for a wide range of organisms.
PCR with direct sequencing is a gold standard technique; however, it takes several days to obtain results, and a relevant question is whether other PCRbased procedures can be used as reliably in the same
amount of time. The relatively low cost of equipment and reagents makes PCR accessible to small
laboratories.
16.5.1 PCR-Based Methods
Species Detection Through Design of Specific
Primers
PCR utilizing genus or species specific primers for marine pests has been utilized using a wide range of techniques: one-step reactions e.g. [16.23, 24, 30, 35, 38],
nested PCR [16.37], and multiplex [16.44] and nested
multiplex PCR [16.43, 75].
Random Amplified Polymorphic DNA (RAPD)
RAPD (RAPD) is based on the amplification of random fragments of genomic DNA using single primers
of arbitrary sequence. RAPD offers rapid molecular assessment in that it does not require the design of specific primers that bind exclusively to
one sequence type. It essentially scans the entire
genome without prior knowledge of sequence information. RAPD will produce a specific banding pattern
when amplicons are subjected to electrophoresis. It
can be particularly useful in discriminating between
closely related species or strains and understanding
sequence variation between individuals [16.52]. Zhou
and Gao [16.22] utilized RAPD for the identification
of mitten crab (Eriocheir sinensis) populations, but it
has not otherwise been widely utilized for marine pest
identification.
Restriction Fragment Length Polymorphism
(RFLP)
Restriction (RFLP) endonucleases are used to cut
DNA at precise sequences producing specific bands
which are visualized via electrophoresis. When RFLP
is combined with PCR, a fragment will be amplified via PCR before being digested, using the restriction endonucleases to give a characteristic pattern based on the amplified fragment. RFLP has
not been widely used for the identification and/or
surveillance of marine pests. Scholin and Anderson [16.49] used PCR-RFLP to distinguish between