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Comparative sequence studies of the repeat elements in diverse insect species can provide useful information on how to make use of them for developing abundant markers that can be used in those species;
$ At the moment, a total of 8 species are in genome assembly stages and another 35 are in progress for genome sequencing;
$ Different molecular marker systems in the field of entomology are expected to provide new directions to study insect genomes in an unprecedented way in the years to come
Molecular Markers: MajorMolecular Markers: Majorapplications in insectsapplications in insectsSARAMITA DE (CHAKRAVARTI)SARAMITA DE (CHAKRAVARTI)M. Phil (Zoology), 2M. Phil (Zoology), 2ndndSemesterSemesterRoll: BUR MP ZOO No.: 2008 / 9Roll: BUR MP ZOO No.: 2008 / 9Registration No.: 2546 of 2008 – 2009Registration No.: 2546 of 2008 – 2009The University of BurdwanThe University of BurdwanBurdwan – 713 104Burdwan – 713 104West Bengal, IndiaWest Bengal, India
Concept of molecular markers inConcept of molecular markers ininsect ecology- prologueinsect ecology- prologueWhat makes insects so diverged and how the genes and geneticmake – up of insects contribute to their adaptable life – formsand most importantly how they may affect human life eitherdirectly or indirectly? (Speight et. al. 2005)Common visible markers including body colour, body spots,bands, hairs, spines were used as phenotypic markers – instudying of pattern of dispersal, mating behaviour andinheritance of genetic traits in insects.
Drawbacks of visible markers ---- visible phenotypes are relatively infrequent; hard to score; difficult and time – consuming to induce genetic mutations in laboratorypopulations; interfere with the overall fitness of the organism; they are rare;So use of them in a trait is problematic.
Protein Markers:Protein Markers:Electrophoretic pattern of allozymes were used to identify different alleles of a givengene; e.g. insecticide resistance, pathogen identification, chromosome mapping andeven in detection of prey in insect predators.DNA markers:DNA markers:Greater level of polymorphism could be obtained by using (Molecular markers)thereafter. Because mutations in introns or even in the codons of a gene canpotentially provide variation at the DNA level. DNA markers contributed- geneticrelatedness, phylogeny, population dynamics or gene and genome mapping ininsects.
Molecular marker is any kind of molecule indicating theexistence of a chemical or physical process.In biology and medicine, a molecular marker (biomarker)can be a substance native to the organism whosedetection indicates a particular disease state.In genetics, a molecular marker (genetic marker) is afragment of DNA sequence that is associated to a partof the genome.
DNA sequences are very highly specific;DNA sequences are very highly specific;they can be identified with the help of thethey can be identified with the help of theknown molecular markers which can find outknown molecular markers which can find outa particular sequence of DNA from a groupa particular sequence of DNA from a groupof unknown;of unknown;molecular biology;molecular biology;biotechnology ;biotechnology ;other scientific experiments to identify aother scientific experiments to identify aparticular sequence of DNA.particular sequence of DNA.
Elucidation of ecological interactions ;Elucidation of ecological interactions ; Possess taxonomic importance;Possess taxonomic importance; Discrimination of recently diverged taxa e.g. biotypes, races,Discrimination of recently diverged taxa e.g. biotypes, races,species,species,subspecies, cryptic species, sibling species and immature lifesubspecies, cryptic species, sibling species and immature lifestagesstagesthat show morphological differences.that show morphological differences.Importance of MolecularImportance of MolecularMarkersMarkers
Molecular Markers in InsectsInsects comprise the largest species composition;900,000 insect species approx. in the entire animal;nearly 75% of all the recorded animal species;possess a vast undiscovered genetic diversity and genepool that can be better explored using molecularmarker techniques.
Conventional MarkerSystem mt DNA (Mitochondrial DNA) Microsatellites RAPD - PCR (random amplified polymorphic DNA - PCR) ESTs (expressed sequence tags) AFLP (amplified fragment length polymorphism)They have contributed significantly for progresses towardsunderstanding genetic basis of insect diversity and formapping medically and agriculturally important genes andquantitative trait loci (QTL) in insect pests.
Novel Marker System Transposon display Sequence – specific amplification polymorphism (s – SAP) Repeat – associated PCR (RA – PCR)But use of some methodologies like whole – genomemicroarray and single nucleotide polymorphism (SNP)assays has not gained widespread popularity inentomological studies due to time consuming and cost –effective reasons.
Maternal inheritance haploid status and highMaternal inheritance haploid status and highrate of evolutionrate of evolution Loci can be readily amplified by usingLoci can be readily amplified by usinguniversal primers designed from highlyuniversal primers designed from highlyconserved mt genesconserved mt genes (Lanave(Lanave et. al.et. al. 2002)2002);; Upon amplification these loci can be used forUpon amplification these loci can be used forgenotyping by RFLP by easy and simplegenotyping by RFLP by easy and simplerestriction digestions and gel electrophoresisrestriction digestions and gel electrophoresis(Behura(Behura et. al.et. al. 2001)2001);; mt DNA undergoes selective sweep, paternalmt DNA undergoes selective sweep, paternallinkage and even nuclear organizations, so inlinkage and even nuclear organizations, so ininsects these markers are difficult to use ininsects these markers are difficult to use inevolutionary and phylogenetic studies;evolutionary and phylogenetic studies;Fig. Structure of mt DNA
Easy – to – perform and easy – to –Easy – to – perform and easy – to –score procedurescore procedure (Black 1993)(Black 1993).. Reliability and reproducibilityReliability and reproducibilitypoor;poor; unsuitable for population studies.unsuitable for population studies.[E[Example inxample in Gypsy mothGypsy moth((Lymantria disparLymantria dispar Linnaeus),Linnaeus), it wasit wasfound that a fragment was present infound that a fragment was present inthe RAPD amplification products ofthe RAPD amplification products ofF1 progeny although it was notF1 progeny although it was notamplifiable from either parent].amplifiable from either parent].Fig. Standard PCRFig. Standard PCR
It combines the easiness of RAPD and the reliability of RFLP.It combines the easiness of RAPD and the reliability of RFLP. It is relatively time – consuming and often utilizes radiolabeled primers.It is relatively time – consuming and often utilizes radiolabeled primers. AFLP loci may contain repeats such as microsatellites in their sequences and mayAFLP loci may contain repeats such as microsatellites in their sequences and maypose difficulty in scoring the allelespose difficulty in scoring the alleles (Wong(Wong et. al.et. al. 2000)2000).. Advantage of AFLP system is that the selective PCR generates more numbers ofAdvantage of AFLP system is that the selective PCR generates more numbers ofmarker loci, on average 50 – 100 bands per primer pairs per sample.marker loci, on average 50 – 100 bands per primer pairs per sample. AFLP loci are highly reproducible and codominant in nature.AFLP loci are highly reproducible and codominant in nature. AFLP markers suitable for mapping of genes and QTL and to generate the linkageAFLP markers suitable for mapping of genes and QTL and to generate the linkagemaps of genesmaps of genes (Behura(Behura et. al.et. al. 2004; Ruppell2004; Ruppell et. al.et. al. 2004)2004) and can also be applied toand can also be applied tocDNA and the results can be used to detect differentially expresses genes incDNA and the results can be used to detect differentially expresses genes ininsectsinsects (Reineke(Reineke et. al.et. al. 2003)2003)..
Highly polymorphic in nature and are also abundant to generate large numbers of markersHighly polymorphic in nature and are also abundant to generate large numbers of markerscompared to AFLP system.compared to AFLP system. Large numbers of microsatellites can be isolated by generating a genomic library of smallLarge numbers of microsatellites can be isolated by generating a genomic library of smallfragments either amplified by PCR or generated by partial digestion of genomic DNA andfragments either amplified by PCR or generated by partial digestion of genomic DNA andenriched with simple sequences repeats.enriched with simple sequences repeats. Amenable for high throughput genotyping by non – radioactive labeling and scoring byAmenable for high throughput genotyping by non – radioactive labeling and scoring byautomated sequencing machines.automated sequencing machines. Initial set up a cost – effective than that of the AFLP, but this is often compromised inInitial set up a cost – effective than that of the AFLP, but this is often compromised inpopulation and ecological studies where more numbers of genotypes are required forpopulation and ecological studies where more numbers of genotypes are required formeaningful statistical analyses.meaningful statistical analyses. In some cases they are not neutral markers as they are involved in gene regulation, geneticIn some cases they are not neutral markers as they are involved in gene regulation, genetichitchhiking and sex – specific differential selection and hence, those loci may not be useful forhitchhiking and sex – specific differential selection and hence, those loci may not be useful forevolutionary and phylogenetic studies.evolutionary and phylogenetic studies.
Use of commercial kits for preparation of cDNA libraries andUse of commercial kits for preparation of cDNA libraries andautomated sequencing methods has made it possible toautomated sequencing methods has made it possible togenerate large sets of ESTs in a relatively fast and efficientgenerate large sets of ESTs in a relatively fast and efficientmanner.manner. Mainly used for transcriptosomes analysesMainly used for transcriptosomes analyses (Nakabachi(Nakabachi et. al.et. al.2005)2005), integrated linkage mapping of insect genomes, integrated linkage mapping of insect genomes(Graham(Graham et. al.et. al. 2004)2004)..
Major Applications of ConventionalMajor Applications of ConventionalMarker Systems in studying insectMarker Systems in studying insectecologyecologyDNA markers are used ---- to provide raw information based on which an ecologist makesestimates or predicts migration and colonization history (Bosio et. al.2005); to infer phylogeny and biogeography of insect populations; to provide the means to differentiate sympatric species from allopatricspecies and parapatric species (Margonari et. al. 2004); to understand modes of evolution and evolutionary trajectories (Prasadet. al. 2005); for diagnostic purposes of the individual traits (Ullman et. al. 2003).
Major ApplicationsMajor Applications Mating, parentage and kinship;Mating, parentage and kinship; Insect – plant interaction;Insect – plant interaction; Insect – pathogen interaction;Insect – pathogen interaction; Insecticide resistance;Insecticide resistance; Prey, predator and parasites;Prey, predator and parasites; Gene, genome and QTL mapping;Gene, genome and QTL mapping; Behavioural studies;Behavioural studies; Comparative genomics and cytogeneticsComparative genomics and cytogenetics..
Mating, parentage and kinship -Mating, parentage and kinship - DNA markers can unravel information to determine parentage and kinshipDNA markers can unravel information to determine parentage and kinshiprelations in insects;relations in insects; RAPD markers:RAPD markers: determined paternity in two odonate species of anisopterandetermined paternity in two odonate species of anisopterandragonflies (dragonflies (Anax parthenopeAnax parthenope Julius Braner) and keeled skimmersJulius Braner) and keeled skimmers((Orthetrum coerulescensOrthetrum coerulescens)) [Hadreys[Hadreys et. al.et. al. 1993]1993];; RAPD markers:RAPD markers: showed how the females in some non – parthenogeneticshowed how the females in some non – parthenogeneticinsect species such as white – pin weevil (an important forest prey), carryinsect species such as white – pin weevil (an important forest prey), carrysperms of more than one male from one season to the nextsperms of more than one male from one season to the next (Lewis(Lewis et. al.et. al.2002)2002);;
Microsatellite markers:Microsatellite markers: used in bush cricketused in bush cricket Requena verticalisRequena verticalis Walker toWalker tofind out that the ejaculated sperm quantity between males is the determiningfind out that the ejaculated sperm quantity between males is the determiningfactor in establishing paternity identity of the offsprings;factor in establishing paternity identity of the offsprings; Microsatellite markers:Microsatellite markers: in identification of egg paternity to validatein identification of egg paternity to validatetheoretical models that predict the influence on parental care ontheoretical models that predict the influence on parental care onreproductive success in some insects like golden bugs (reproductive success in some insects like golden bugs (PhyllomorphaPhyllomorphalaciniatalaciniata Villers)Villers) [Garcia – Gonzalenz[Garcia – Gonzalenz et. al.et. al. 2003]2003];; Microsatellite markers:Microsatellite markers: used to test the polyandry as a mating strategy inused to test the polyandry as a mating strategy incricket (cricket (Gryllus bimaculatususeGryllus bimaculatususe de Geer) – to minimize genetic inbreeding ofde Geer) – to minimize genetic inbreeding oftheir brood;their brood;
Microsatellite markers:Microsatellite markers: measured genetic diversitymeasured genetic diversitybetween sexual and asexual aphid populations;between sexual and asexual aphid populations; RAPD markers:RAPD markers: associated with life cycle variation andassociated with life cycle variation andbreeding traits in some insects.breeding traits in some insects.
Insect – plantInsect – plantinteraction -interaction -• DNA markers provide utility in tagging and mapping genes inimportant crop plants that provide resistance to damaging insectpests;• These are also useful in characterizing avirulence genes in theinsects interacting with the host plants (Harris et. al. 2003);• RAPD – PCR: identified distinct loci specific to individual strains(or biotypes) of Asian Rice gall midges (Orseolia oryzae Wood –Mason), were identified (Behura et. al. 2000);
• RAPD and AFLP markers: employed to identify majoravirulence genes in Hessian fly (Behura et. al. 2004);• RAPD – PCR: established a major difference existed betweenwinged phenotypes and the wingless phenotypes of theasexual adult aphids;
• RAPD banding pattern: showed the differences between thefeeding habits on different hosts of grasses and cereals of thegrain aphids (Sitobion avenae Fabricus);• Microsatellite markers: studied genetic basis of host plantassociation in lettuce root aphid (Pemphigus bursariusLinnaeus) [Miller et. al. 2005];• RAPD markers: evaluated degree of virulence(aggressiveness) of individual clones in pea aphids in responseto natural resistance in alfa alfa (Bournoville et. al. 2000).
Insecticide Resistance -Insecticide Resistance - Molecular markers are used for identification andmapping of resistance genes in insects againstinsecticides; Microsatellite markers: in mapping experimentsidentifies QTL in Anopheles gambiae (a major malariavector in Africa) that determines the DDT resistancephenotypes (Ranson et. al. 2000);
RFLP markers: discovery of the Kdr trait (associatedwith DDT resistance in houseflies Musca domestica); PCR markers: to examine expression pattern of thecytochrome P450 genes near a DDT resistance gene inDrosophila (Brandt et. al. 2002);
RAPD markers: mapping of genetic loci in lesser grainborer (Rhyzopertha dominica Fabricus) – thatdetermines high level resistance to phosphine(Schlipalius et. al. 2002); AFLP markers: in the identification of resistance loci inColorado potato beetle to pyrethroid (Hawthorne 2001)and in diamond back moth to Bt toxins.
Gene, genome and QTLGene, genome and QTLmapping -mapping -Genetic linkage mapping based on recombinationfrequency uses molecular markers for tagging andmapping of specific genes and QTL in insects;QTLs – stretches of DNA – closely linked to the genesthat underlie the trait in question.When more than one gene govern a trait (polygenictrait), markers identify those links to the chromosomalregion containing the genes.
QTLs are molecularly identified (by PCR or AFLP) tohelp map regions of the genome that contain genesinvolved in specifying a quantitative trait;AFLP markers: used to generate genetic maps ofsilkworms, Colorado potato beetles, red flour beetles,European corn borers, Hessian flies, butterflies;RAPD markers: for genomes of honeybees, silkworms,beetles, sawflies;
Microsatellite markers: for mapping of genomes of Drosophila mojavensis and in honeybeesand also in bumblebees and ants to identify genes responsible for diversity in foraging rangeand mating behaviour (Knight et. al. 2005), host – parasitization (Solignac et. al. 2005),colonization (Jensen et. al. 2005) and kinship relation (Troutti et. al. 2005);AFLP markers: used to map QTL governing aging in Drosophila melanogaster (Luckinbill andGoldenberg 2002), foraging behaviour in honeybees (Ruppell et. al. 2004), susceptibility inbeetles to tapeworm parasites (Zhong et. al. 2005), variation in pheromone composition inHeliothis sp (Groot et. al. 2004) and Bt resistance in diamond back moths (Heckel et. al. 1999);AFLP markers: employed in pea aphids (Acyrthosiphon pisum) [Braendle et. al. 2005] to tracethe phenotypic traits of wing size , in moth species (Thaumetopoea pityocampa and T.wilkinsoni) to study genetics of male dispersal (Salvato et. al. 2002) and in Venturia canesceuswasps to investigate the genetic basis of haplodiploidy (Reineke and Lobmann 2005);
Behavioural studies -ESTs: used as expression markers in microarrayformats to predict the nursing and foraging behaviour inthe individual honeybees (Apis mellifera) [Whitfield et.al. 2003];RAPD markers: determined two QTLs responsible forthe foraging behaviour in honeybees in backcrosspopulations between bees collecting nectar and thosecollecting pollen;
mt DNA: traced the movement of females between coloniesin Australian meat ants (Iridomyrmex purpureus Smith);AFLP markers: employed in bumble bees to study geneticbasis of ecological implications of foraging range and nestdensity behaviours (Knight et. al. 2005);Microsatellite markers: used in dissecting the guarding andstinging behaviours in honeybees (Arechavaleta – Velascoet. al. 2003).
Concluding RemarksConcluding Remarks$ Comparative sequence studies of the repeat elements in diverseinsect species can provide useful information on how to make useof them for developing abundant markers that can be used inthose species;$ At the moment, a total of 8 species are in genome assembly stagesand another 35 are in progress for genome sequencing;$ Different molecular marker systems in the field of entomology areexpected to provide new directions to study insect genomes in anunprecedented way in the years to come.