Download RAPD marker system in insect study: A review

Indian Journal of Biotechnology
Vol 9, January 2010, pp 7-12
RAPD marker system in insect study: A review
Subodh Kumar Jain*, Bharat Neekhra, Divya Pandey and Kalpana Jain
Department of Biotechnology, Dr H S Gour University, Sagar 470 003, India
Received 30 September 2008; revised 2 June 2009; accepted 7 August 2009
Insects represent a major life form on earth. So far, nearly 0.9 million insect species are discovered, comprising 75% of
all the recorded animal species. Some of the insect species are easy to identify and categorize, while for others, it is difficult
because of their small size and morphological similarity. Moreover, it is further difficult to identify morphological variation
due to environmental factors by available traditional methods. To overcome these problems, the advanced molecular
techniques, viz., PCR (Polymerase Chain Reaction), RFLP (Restriction Fragment Length Polymorphism), RAPD (Random
Amplified Polymorphic DNA), and AFLP (Arbitrary Fragment Length Polymorphism) have been a great help. RAPD
markers have been used in gene mapping to characterize cultivars and species genetically, infer phylogeny and
biogeography of insect population and understand modes of evolution and evolutionary trajectories. Thus, RAPD markers
have become the most common yardsticks for measuring genetic differences between individuals, within and between
related species or population. The unprecedented advancements in modern molecular biology, particularly in those of DNA
marker technology, have created a wealth of technical know-how that finds useful application in molecular ecology research
in insects.
Keywords: Genetic variation, insects, molecular markers, PCR, RAPD
Introduction
Insects comprise the largest species composition in
the entire animal kingdom and possess a vast
undiscovered genetic diversity and gene pool that can
be better explored using molecular marker techniques.
Insect populations, even within a species, vary in their
behaviour and morphology that attributes to their
complex interaction with the environment1. Insects
are beneficial as they pollinate crops, act as natural
enemies of damaging pests, and produce useful
products for humans. Also, they are harmful as major
pests of food crops, vectors for transmitting deadly
diseases, and cause damage to our urban
infrastructure, environment, forest and natural
resources. The study of insect ecology is important to
understand their evolution and diversification, and
their influence on the functional and trophic links
between different components of associated habitats2.
In insects, DNA markers are used to provide raw
information, based on which an ecologist make
estimates of genetic diversity and gene flow between
species3. Molecular data provide the means to
differentiate sympatric species from allopatric4,5 and
___________
*Author for correspondence:
Tel: 91-7582-265450; Fax: 91-7582-264236
E-mail: [email protected]
parapatric species6,7, and modes of evolution and
evolutionary trajectories8. Also, diagnostic molecular
markers, based on linkage to certain traits9 or genes,
are used for diagnostic purposes of individual
insects10,11. The greater level of polymorphism could
be obtained by using DNA markers than by using
protein markers12. The DNA marker technology finds
useful applications of these markers especially in
molecular ecology research in insects13. Over the last
15 yr or so, DNA markers have made a significant
contribution to rapid rise of molecular studies of
genetic relatedness, phylogeny and population
dynamics14,15, and gene and genome mapping in
insects16,17. In molecular markers, RAPD-PCR is a
conceptually simple technique for estimation of
genetic diversity of organism18,19.
RAPD Markers
The RAPD markers method has been reported to be
an efficient tool to differentiate geographically and
genetically isolated population. It has been used to
verify the existence of population of species that have
arisen either through genetic selection under different
environmental conditions or as a result of genetic
drift20. However, there are several disadvantages that
must be taken into account when using the technique.
The most easily counteracted drawback is the
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INDIAN J BIOTECHNOL, JANUARY 2010
dominant mode of inheritance of RAPD bands, which
reduces the information provided by each locus.
Because each primer can amplify several loci and
there are many commercially available primers, the
loss of information per locus can be easily balanced
by using a high number of loci21.
RAPD markers have been used in gene mapping to
genetically characterize cultivars and species, to
estimate genetic variability22, and to determine the
genetic structure of populations of various
organisms23. RAPDs are particularly useful to study
the genetic structure of populations because they
reveal polymorphisms in non-coding regions of
the genome24.
RAPD Marker Approach in Insects
Ecological research on insects provides invaluable
information on population structure, speciation, gene
flow and genetic diversity, and explanation on insect
diversity based on their interaction with
environmental factors, either biotic (including other
biological species) or abiotic. Many a time, molecular
marker data help to distinguish between different
species, where there is no other comprehensive way
available to do so.
DNA markers can unravel information to determine
parentage and kinship relations in insects. One of
innovative works done in this regard was to use
RAPD marker to determine paternity in two odonate
species of Anisopteran dragon flies, Anax parthenope
Sélys and Orthetrum coerulescens Fabricius (keeled
skimmer)25. RAPD banding patterns were used to
access paternity of ‘synthetic offsprings’ generated by
quantitative mixing of genomic DNA from putative
parents. This approach has been helpful to establish
the paternity of guarding males in species where no
information on mating histories of both males and
females are known. Using molecular markers, it has
been shown to how the females in some nonparthenogenetic insect species, such as, white pine
weevil—an important forest pest, carry sperms of
more than one male from one season to the
next26. The results of such studies provide explanation
on how the offsprings are produced in new habitats
where no males are available for mating and
as the basis of their colonization in new geographical
regions.
RAPD markers associated with the variations of
life cycle of aphids and breeding traits in these insects
have been converted into co-dominant sequences,
known as sequence characterized amplified region
(SCAR) markers27. Using these SCAR markers in
segregating and natural populations of known
breeding systems, a complete linkage was found in
segregating population. Apparently, the association in
field populations was on an average of 94%. Such
information has potential use for studying the role of
genetic mechanism of sexual and asexual mating
behaviour in dispersal and colonization of aphid
populations in geographical regions.
RAPD primers generated polymorphic bands for
the Caribbean fruit fly Anastrepha suspensa Loew,
indicating that polymorphic RAPD bands are useful
as genetic markers. The markers are potentially useful
for host and geographic population studies as they
relate to quarantine issues28. DNA profiling of thirteen
silkworm genotypes was studied by RAPD29.
Insect-Plant Interaction
One of the most appealing applications of
molecular markers in insect studies is that of insectplant interaction. Using RAPD-PCR with pooled
DNA from different strains (or biotypes) of Asian rice
gall midge, distinct loci specific to individual strains
were identified30. Confirming by Southern blotting
and sequencing, co-dominant SCAR markers were
developed; and using these diagnostic markers for
allele specific amplification, good correlation between
genotypes to the observed phenotypes of these
biotypes (ability/inability to attack host plants) was
established. An AFLP marker was further discovered
using bulk segregants method and the locus showed
linkage with γGm2 avirulence gene that interacted
with the corresponding resistance gene (Gm2) in rice.
Similarly, in Hessian fly, RAPD and AFLP
markers31,32 have been employed in combination with
bulk segregants analysis to identify major avirulence
genes, those condition the resistance mechanism in
wheat varieties cultivated in the United States33.
Using RAPD-PCR, it was found that major genetic
differences existed between winged and the wingless
phenotypes of the asexual adult aphids34. Similarly, in
natural populations of grain aphids, Sitobion avenae
Fabricius feeding on different hosts of grasses and
cereals, it has been clearly demonstrated that the
RAPD banding pattern could be correlated to host
adaptation35. These profiles could identify 'specialist'
genotypes found on specific grasses from the
'generalist' genotypes, colonizing on multiple host
types including cultivated cereals or native grasses.
JAIN et al.: RAPD MARKER SYSTEM IN INSECT STUDY
The degree of virulence of individual clones in pea
aphids in response to natural resistance in alfalfa has
been evaluated using RAPD markers36. These works
specifically establish that application of RAPD
markers are helpful in better understanding the
mechanistic and evolutionary basis for the genetic
interaction between insect pests and their host plants.
Insect Pathogen Interaction
Triatoma infestans Klug (Redvuiidae: Triatominae)
is a major insect vector of Chagas disease in many
South American countries and transmits the
Trypanosoma cruzi Chagas, the causal agent. Mixed
and pure clones of T. cruzi in the gut of T. infestans
have been studied by using RAPD profiles to provide
information on the vectorial ability of the insects37.
Similarly, molecular markers were applied to
determine the vectorial ability of mosquitoes16 by
means of mapping quantitative trait loci (QTL) that
determined if a species could transmit the malaria
parasite39,40.
Insecticide Research
Insecticidal resistance is another important focus in
entomological research and bears medical and
agricultural importance. Molecular markers are used
for identification and mapping of resistance genes in
insects against insecticides. Using random amplified
DNA markers, genetic loci have been mapped in
lesser grain borer, Rhyzopertha dominica Fabricius
that determines high level resistance to phosphine40.
Diagnostic PCR based markers, such as, SCAR,
derived from specific RAPD loci were used to
identify Trialeurodes vaporariorum West. and
Helicoverpa armigera Hubner prey in the gut of
Dicyphus tamaninii Wagner41,42, which provide
information to understand the prey-predator parasite
trophic interactions in insects.
Genetic Diversity
Genetic differentiation among six Florida
populations of Diaprepes abbreviatus L. was
determined using protein and random amplified
polymorphic DNA-polymerase chain reaction
(RAPD-PCR) markers. RAPD-PCR data showed
significant
differentiation
among
population,
consistent with the hypothesis of three independent
introductions of D. abbreviatus into Florida. The data
indicate that D. abbreviatus populations, once
introduced, have generally remained in one locality
with limited dispersal to new areas43.
9
The genetic structure of cotton bollworm,
H. armigera Hubner (Lepidoptera: Noctidae) was
studied in the eastern Mediterranean. Moths were
sampled in six locations (five in Israel, and one in
Turkey) and their genetic relationship was analysed
using RAPD-PCR. Three 10-oligonucleotide primers
revealed 84 presumptive polymorphic loci that were
used to estimate population structure. Results revealed
low level of genetic distance among Israeli and
Turkish populations. Although no isolation by
geographical distance was detected44.
To examine gene flow using other genetic methods,
RAPD-PCR polymorphism in 28 presumptive loci of
Aedes albifasciatus Mac. from populations in central
Argentina was analysed. Allele frequencies were
estimated assuming that RAPD products segregate as
dominants and that genotype frequencies at those loci
were in Hardy-Weinberg equilibrium45.
RAPD-PCR technique was useful for revealing
genetic variation in screw worm fly, Cochliomyia
hominivorax Coquerel populations (one of the most
important agents of traumatic myiasis throughout
neotropical regions), otherwise not detected by others
techniques and could represent an efficient method for
understanding the genetic structure and population
genetic phenomena of this important pest46.
Callosobruchus maculatus Fabricius shows
polymorphism in male as well as females on the basis
of colour pattern of elytron and pygidium. Genomic
DNA of all these polymorphic forms has been
analysed using RAPD-PCR technique with two
random decanucleotide primers P12 and P17. This
study revealed that the normal and abnormal forms of
males of C. maculatus in the population were quite
distinct at genetic level47.
A genetic map of the red flour beetle (Tribolium
castaneum Herbst) integrating molecular with
morphological markers was constructed using a
backcross population of 147 siblings. The process of
converting RAPD markers to sequence-tagged site
markers was initiated; 18 RAPD markers were cloned
and sequenced, and single strand conformational
polymorphisms were identified for 4 of the 18. The
map positions of all 4 coincided with those of the
parent RAPD markers48.
RAPD markers for genetic characterization were
examined using 13 diverse silkworm strains. The
RAPD assay clearly separated the diapausing and
non-diapausing silkworm varieties49.
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INDIAN J BIOTECHNOL, JANUARY 2010
Genetic Map
In insects, RAPD and RFLP markers have been
extensively used to generate genetic maps. RAPDbased linkage maps have been constructed for
genomes of honey bee50, Silkworm51, beetle48,52 and
sawfly53. Analysis of RAPD markers to generate
linkage maps in a haplodiploid parasitic wasp Bracon
(Habrobracon) hebetor Say and a diploid mosquito,
A. aegypti Linn. revealed segregation of co-dominant
alleles at markers that appeared to segregate as
dominant (band presence/band absence) marker
appeared invariant on agarose gel54. To permit quick
identification of arthropods, random amplified
polymorphic DNA typing (RAPD) was used to
support classical morphological and medico-legal
analysis of maggots on a human corpse55.
Insect Behaviour Study
The social behaviours of honey bee are polygenic
traits and are influenced by more than one gene
referred to as ATL (allele trait loci). The two major
QTLs (quantitative trait loci) that determine the
foraging behaviour in honey bee have been identified
by employing RAPD markers in backcross population
between bees collecting nectar and those collecting
pollen50. Exploiting similar procedures with molecular
markers in honey bee, colony level behaviours, such
as, stinging behaviour, body size, pheromone alarm
level, traits for reversal learning and hygienic
behaviour, have also been dissected at the level of
specific genomic regions56.
One of the most common neotropical sting less
bees is Tetragonisea angustula Latreille, popularly
known in Portuguese as jatai. To determine the
genetic distance between T. angustula populations
from 25 localities in three different Latin American
countries, 18 primers were used to generate 218
RAPD markers57.
RAPD-PCR has been applied to reveal genetic
variation in four aphid species, the green bug
(Schizaphis graminum Rondani), the pea aphid
(Acyrthosiphon pisum Harris), the Russian wheat
aphid (Diuraphis noxia Mordvilko) and the brown
ambrosia aphid (Uroleucon ambrosiae Thomas), and
large amounts of genetic variation were detected
among individuals in each of these species58.
The RAPD technique has been successfully used to
detect the effects on DNA induced by benzo(a)
pyrene59, mitomycin C60, ultraviolet radiation61
and
17-β-estradiol
(estrogen)/4-n-nonylphenol
(Xenoestrogen)62 in aquatic species under in vitro
and in vivo condition.
Concluding Remarks
RAPD markers are well suited for genetic mapping,
plant and animal breeding applications, and DNA
fingerprinting, with particular utility for studies on
population genetics. RAPD markers can also provide
an efficient assay for polymorphism, which should
allow rapid identification and isolation of
chromosome-specific DNA fragments. Hybrid cell
lines or genetic stocks carrying deletions or additions
of large chromosomal segments could be screened
relative to appropriate controls to identify the region
of the genome carrying the deletions or additions.
Like most molecular markers, the information content
of an individual RAPD marker is very low. It is only
when many of the anonymous markers are used to
define a genome that they begin to have utility. High
density genetic maps comprised of molecular markers
have lead to the identification of several previously
unidentified loci of biological importance.
RAPD markers hold promise for the automation of
the genome mapping, extending the power of genetic
analysis to organisms which lack an ample number of
phenotypic markers to completely describe their
genome. Genetic mapping using RAPD markers has
several advantages over other methods, such as:
(i) A universal set of primers can be used for
genomic analysis in a wide variety of species.
(ii) No preliminary work, such as, isolation of cloned
DNA probes, preparation of filters for
hybridization or nucleotide sequencing is
required.
(iii) Each RAPD marker is the equivalent of an
information transfer in collaborative research
programmes. Perhaps the most significant
advantage of this method is that the determination
of genotype can be automated. Genetic maps
consisting of RAPD markers can be obtained
more efficiently, and with greater marker density,
than by RFLP or targeted PCR-based methods.
Acknowledgement
Authors are grateful to Professor R C Sobti, Vice
Chancellor, Punjab University, Chandigarh for
guidance and encouragement, and University Grant
Commission, New Delhi for financial assistance
(F.No. 33-236/2007 SR).
JAIN et al.: RAPD MARKER SYSTEM IN INSECT STUDY
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