the lac insects ( coccoidea : tachardiidae (= kerriidae ) ) have been commercially harnessed to yield three useful products , viz . , resin , wax and dye , which have found a remarkably wide range of applications in food , pharmaceuticals , cosmetics , perfumes , varnishes , paints , polishes , adhesives , jewellery and textile dyes , since ancient times ( dave , 1950 ; sarkar , 2002 ; ramani et al . , 2007 ) .
the importance of this commodity lies in its safety for human use , and as a renewable and eco - friendly resource .
lac production is confined to a few south , southeast and east asian countries in the tropical forest region ( ramani et al . , 2007 ) with india
is the leading lac - producer , with an annual production of about twenty thousand tons ( pal et al . , 2011 ) .
they are phytosuccivorous and sessile ; only the crawlers and adult males are free moving .
they thrive well only on certain plant species known as lac hosts ( kapur , 1962 ; varshney , 1985 ) .
information on the taxonomy of lac insects is based on a monograph and its supplement ( chamberlin 1923 , 1925 ) , as well as subsequent works by kapur ( 1958 ) , varshney ( 1977 ) and kondo and gullan ( 2007 ) .
ninety species , under nine genera , have so far been reported worldwide ( varshney , 2009 ) .
distribution is mainly restricted to tropical and subtropical regions between the latitudes 40 n and 40 s ( kapur , 1962 ) .
nearly all the production comes from the indian lac insect kerria lacca , represented by two infrasubspecific forms , viz . , kusmi and rangeeni , which differ by host preference , life - cycle pattern , the quality and amount of lac produced , etc .
other minor species are k. sharda ( mishra and sushil , 2000 ) and k. chinensis
. palas ( butea monosperma ) , ber ( ziziphus mauritiana ) and kusum ( schleichera oleosa ) are the most common hosts used for lac production in india ( roonwal 1962 ) , which is mainly restricted to the states of jharkhand , chhattisgarh , madhya pradesh , west bengal , maharashtra , besides a few others ( pal et al . , 2011 ) .
notwithstanding , wild populations of kerria are distributed throughout the length and breadth of the country , except in the colder regions ( varshney 1977 ) .
the taxonomy of coccoids is based on adult - female morphology ( varshney , 1977 ; kondo and gullan , 2007 ) .
even so , they are highly degenerate , and undergo tremendous changes in size and shape during the post - metamorphic stage . differentiated populations , due to geographic separation and host - choice , and which have not diverged morphologically , pose an additional challenge to identification .
molecular approaches would therefore serve as useful complementary tools for characterizing such lac - insect taxa with greater reliability .
a wide range of markers are employed for understanding insect - population genetics ( behura , 2006 ) .
rapd - pcr is widely used for identifying cultivars , clones , natural populations , etc . despite the limitation posed by reproducibility , unless reaction conditions are stringent ( baruffi et al . , 1995 and bertin et al . , 2007 )
, this technique offers the advantages of simplicity , independence from prior dna sequence information , and the evaluation of a large number of loci across the genome ( hadrys et al . , 1992 ; lynch and milligan , 1994 ; weising et al . , 2005 ) , besides providing the basis for developing more reliable scar ( sequence characterized amplified regions ) markers ( kethidi et al . , 2003 ) .
the technique has already been widely employed for assessing the genetic diversity of other insect populations ( reyes and ochando , 1998 ; castiglioni and bicudo , 2005 ; dvorak et al . , 2006 ; lopes - da - silva and vieira , 2007 ; martins et al .
, 2007 ; magaa et al . , 2007 ; karam et al . , 2007 ; sosa - gomez et al . , 2008 ;
this constitutes a report on genetic diversity in lac - insect populations belonging to the genus kerria , the true lac - producing insects , from different parts of india , using the rapd technique ( williams et al . , 1990 ; welsh and mcclelland , 1990 ) .
the study material included lac - insect populations collected from different parts of the country , both natural and cultured populations , besides inbred lines derived from k. lacca .
the insects used in the study were obtained from lac - insect cultures maintained at the research farm , iinrg campus , ranchi ( 231951  n 852218  e ; elevation 2080 ft ) .
a few were also drawn from collections of natural field populations ( table 1 ; figure 1 ) .
the lac - insect cultures were maintained on a common lac host flemingia macrophylla , under potted conditions .
the cultures , enclosed in synthetic mesh sleeves to exclude parasite and predator infestation , were regularly sprayed with fungicide ( carbendazim , 0.01% ) to maintain cleanness .
field - collected insects were carefully screened , in order to select only healthy ones .
the lines studied mainly included the most commonly used species for lac production , viz . , k. lacca ( kusmi and rangeeni infrasubspecific forms ) , a collection of k. sharda , and two collections of k. chinensis ( india and thailand ) , as well as inbred and crossbred lines , and collections from wild populations collected from all over of india .
these lines were divided into four groups , depending on the nature ( table 1 ) .
mature female insects were kept in 100% ethanol for 48 h at room temperature , so as to dissolve the resinous covering , whence they were individually cleaned with sable - hair brushes under a stereo - zoom microscope , and serially washed with alcohol to eliminate waxy secretions .
the cleaned insects were kept in 200 l absolute ethanol in 1.5 ml microcentrifuge tubes and stored in 80 c freezer .
dna was extracted from mature females , adopting a phenol - chloroform procedure described by de barro et al .
the extracted dna was individually quantified with a shimadzu uv - vis 1700 spectrophotometer using a dna program pack , and checked by electrophoresis on 1% agrose gel together with 100 bp dna ladder - plus ( fermentas , germany ) .
we screened 120 decamer primers ( operon bio - technologies gmbh , germany ) for satisfactory amplification of products , using three selected lines .
rapd - pcr was carried out with 48 samples of pooled genomic dna from three female insects , to shortlist primers exhibiting polymorphism and reproducibility .
for each of these primers , annealing temperatures and other parameters were standardized by repeated experiments ( table 2 ) .
all the rapd reactions were done in 25 l of reaction mixtures containing 20 ng of template dna , 1x taq buffer [ 750 mm tris - hcl ( ph 8.8 ) , 200 mm ( nh4)2so4 , 0.1% ( v / v ) tween 20 ; fermentas gmbh , germany ] , 2.5 mm of mgcl2 ( fermentas gmbh , germany ) , 0.2 mm of each dntp mix ( fermentas gmbh , germany ) , 20 pmol of each primer , and 1.5 units of taq dna polymerase ( fermentas gmbh , germany ) .
all the pcr reactions were carried out in a thermal cycler ( biorad icycler , usa ) programmed with the following cycling conditions : initial denaturation of template dna was carried out at 95 c for 5 min followed by 35 cycles programmed for denaturation step at 95 c for 1 min , primer annealing step at specific tm for the particular primer ( table 2 ) for 45 s , and extension step at 72 c for 2 min .
the final extension of the pcr products was carried out at 72 c for 7 min .
all pcr amplified products were resolved on 2% agarose gel containing 0.5 g / ml , ethidium bromide , prepared with 0.5 x tbe buffer [ 45 mm tris - borate , 1.0 mm edta ( ph 8.0 ) ] and electrophoresed in 0.5 x tbe at 4 v cm   for 2 h in an amersham submarine electrophoresis unit .
either fermentas 100 bp ladder ( 1001000 ) or 100 bp plus ladder ( 1003000 ) were used as reference , depending on the band - size range .
sufficiently resolved dna bands were documented using the bioimaging system ( gene genius , syngene , u k ) , through genesnap . clear and unambiguous bands present across the dna samples from 48 lac - insect lines at a particular locus ( based on size ) were scored as 1 , whereas their absence or only a very faint outline were scored as 0 , to so generate a binary matrix , to be used for analysis . for line diagnosis and the analysis of marker discrimination power ,
the average band frequency obtained for each primer , marker index ( mi ) and resolving power ( rp ) , using band informativeness ( ib ) , were calculated ( prevost and wilkinson 1999 ) . 

ib=1(2|0.5pi|)where pi is the proportion of lines showing the i band , and i = 1 to n where
primer resolving power ( rp ) was also calculated using the following formula 
 rp=ibwhere ib is band informativeness , as calculated above .
marker index ( mi ) is the parameter for determining the utility of the marker in distinguishing different genotypes .
the estimation of mi was by applying the following formula provided by archak et al .
mi=1/n ib x emrwhere emr ( effective multiplex ratio ) is the product of the number of polymorphic bands ( i.e. a band absent in at least one genotype at a particular locus ) per primer and the fraction of polymorphic bands . in order to study the genetic relationships among kerria lines ,
the scored binary data matrix was analyzed using the ntsyspc version 2.02e software program ( exeter software , new york , usa ) ( rohlf , 1998 ) .
the dendrogram was generated by applying the neighbor - joining method ( saitou and nei , 1987 ) , using midpoint rooting .
only wild - type insects were considered for assessing the variation of similarity indices , in the case of mixed populations .
the same software was used for principal coordinate ( pcoord ) analysis ( sneath and sokal , 1973 ) of the data .
the confidence level for distinguishing genotypes , using the selected primers , was estimated through the analysis of probability of identical match by chance ( pimc ) , as proposed by wetton et al .
the insects used in the study were obtained from lac - insect cultures maintained at the research farm , iinrg campus , ranchi ( 231951  n 852218  e ; elevation 2080 ft ) .
a few were also drawn from collections of natural field populations ( table 1 ; figure 1 ) .
the lac - insect cultures were maintained on a common lac host flemingia macrophylla , under potted conditions .
the cultures , enclosed in synthetic mesh sleeves to exclude parasite and predator infestation , were regularly sprayed with fungicide ( carbendazim , 0.01% ) to maintain cleanness .
field - collected insects were carefully screened , in order to select only healthy ones .
the lines studied mainly included the most commonly used species for lac production , viz . , k. lacca ( kusmi and rangeeni infrasubspecific forms ) , a collection of k. sharda , and two collections of k. chinensis ( india and thailand ) , as well as inbred and crossbred lines , and collections from wild populations collected from all over of india .
these lines were divided into four groups , depending on the nature ( table 1 ) .
mature female insects were kept in 100% ethanol for 48 h at room temperature , so as to dissolve the resinous covering , whence they were individually cleaned with sable - hair brushes under a stereo - zoom microscope , and serially washed with alcohol to eliminate waxy secretions .
the cleaned insects were kept in 200 l absolute ethanol in 1.5 ml microcentrifuge tubes and stored in 80 c freezer .
dna was extracted from mature females , adopting a phenol - chloroform procedure described by de barro et al .
the extracted dna was individually quantified with a shimadzu uv - vis 1700 spectrophotometer using a dna program pack , and checked by electrophoresis on 1% agrose gel together with 100 bp dna ladder - plus ( fermentas , germany ) .
we screened 120 decamer primers ( operon bio - technologies gmbh , germany ) for satisfactory amplification of products , using three selected lines .
rapd - pcr was carried out with 48 samples of pooled genomic dna from three female insects , to shortlist primers exhibiting polymorphism and reproducibility .
for each of these primers , annealing temperatures and other parameters were standardized by repeated experiments ( table 2 ) .
all the rapd reactions were done in 25 l of reaction mixtures containing 20 ng of template dna , 1x taq buffer [ 750 mm tris - hcl ( ph 8.8 ) , 200 mm ( nh4)2so4 , 0.1% ( v / v ) tween 20 ; fermentas gmbh , germany ] , 2.5 mm of mgcl2 ( fermentas gmbh , germany ) , 0.2 mm of each dntp mix ( fermentas gmbh , germany ) , 20 pmol of each primer , and 1.5 units of taq dna polymerase ( fermentas gmbh , germany ) .
all the pcr reactions were carried out in a thermal cycler ( biorad icycler , usa ) programmed with the following cycling conditions : initial denaturation of template dna was carried out at 95 c for 5 min followed by 35 cycles programmed for denaturation step at 95 c for 1 min , primer annealing step at specific tm for the particular primer ( table 2 ) for 45 s , and extension step at 72 c for 2 min .
the final extension of the pcr products was carried out at 72 c for 7 min .
all pcr amplified products were resolved on 2% agarose gel containing 0.5 g / ml , ethidium bromide , prepared with 0.5 x tbe buffer [ 45 mm tris - borate , 1.0 mm edta ( ph 8.0 ) ] and electrophoresed in 0.5 x tbe at 4 v cm   for 2 h in an amersham submarine electrophoresis unit .
either fermentas 100 bp ladder ( 1001000 ) or 100 bp plus ladder ( 1003000 ) were used as reference , depending on the band - size range .
sufficiently resolved dna bands were documented using the bioimaging system ( gene genius , syngene , u k ) , through genesnap .
clear and unambiguous bands present across the dna samples from 48 lac - insect lines at a particular locus ( based on size ) were scored as 1 , whereas their absence or only a very faint outline were scored as 0 , to so generate a binary matrix , to be used for analysis . for line diagnosis and the analysis of marker discrimination power ,
the average band frequency obtained for each primer , marker index ( mi ) and resolving power ( rp ) , using band informativeness ( ib ) , were calculated ( prevost and wilkinson 1999 ) . 

ib=1(2|0.5pi|)where pi is the proportion of lines showing the i band , and i = 1 to n where  n is the total number of bands . primer resolving power ( rp ) was also calculated using the following formula 
 rp=ibwhere ib is band informativeness , as calculated above .
marker index ( mi ) is the parameter for determining the utility of the marker in distinguishing different genotypes .
the estimation of mi was by applying the following formula provided by archak et al .
mi=1/n ib x emrwhere emr ( effective multiplex ratio ) is the product of the number of polymorphic bands ( i.e. a band absent in at least one genotype at a particular locus ) per primer and the fraction of polymorphic bands . in order to study the genetic relationships among kerria lines ,
the scored binary data matrix was analyzed using the ntsyspc version 2.02e software program ( exeter software , new york , usa ) ( rohlf , 1998 ) .
the dendrogram was generated by applying the neighbor - joining method ( saitou and nei , 1987 ) , using midpoint rooting .
only wild - type insects were considered for assessing the variation of similarity indices , in the case of mixed populations .
the same software was used for principal coordinate ( pcoord ) analysis ( sneath and sokal , 1973 ) of the data . the confidence level for distinguishing genotypes , using the selected primers , was estimated through the analysis of probability of identical match by chance ( pimc ) , as proposed by wetton et al .
out of the 120 rapd primers screened , 26 produced the satisfactory , clear and reproducible banding patterns used herein .
these produced 173 loci in the 48 lines studied , of which 169 ( 97.7% ) were found to be polymorphic .
the maximum size - range of amplified products for a single primer was obtained with oph5 ( 4502.0 kbp ) , whereas the minimum , i.e. , one single band ( 600 bp ) , was obtained with ops12 . the number of bands produced by the primers ranged from 1 ( ops 12 and ops 16 ) to 12 ( ops 19 ) , with an average of 6.7 per primer .
polymorphism in the bands produced by all the rapd primers , except for ops 9 , ops 10 , ops 19 and opt 16 , was 100% .
the primers used , their sequences , tm , number of scored bands , band - size range and the number of polymorphic bands , are given in table 2 .
the total number of amplified bands in all the forty - eight lac - insect lines generated , when using all the twenty - six rapd primers , was 3,380 , with an average of 70.4 bands per line .
the number of bands amplified through all the primers in all the lines ranged from 33 ( li036 ) to 95 ( li025 ) .
the resolving power ( rp ) of the primers screened ranged from 0.08 ( ops9 ) to 4.08 ( opa13 ) , with a mean of 1.97 .
a total 33 unique bands were generated , with 15 rapd primers ( ops9 , ops10 , ops13 , ops15 , ops17 , ops19 , opt7 , opt16 , oph5 , oph12 , oph19 , opb15 , opb18 , opa2 and opa9 ) in eight lac - insect lines ( li002 , li010rr , li09b , li068 , li05 , li044 , li087 and li069 ) .
the highest number of unique bands ( 21 out of 33 ) , compared to the remainder , was in line li002 , kerria chinensis from india . only three ( li002 , li003 and li015 ) presented unique null bands with four rapd primers , opt16 , ops19 , ops10 and ops9 . in line
, ops19 , ops10 and ops9 . out of the 48 lac - insect lines analyzed , nine , viz . ,
li009a , li002 , li068 , li043 , li085 , li026 , li027 , li003 and li028 , could be identified by the presence of two bands by eight primers . with most of the primers , lines
li002 and li068 , k. chinensis lines , respectively from india and thailand , were found to share a common pattern , by the presence or absence of two bands .
pair - wise jaccard s similarity indices varied from 0.15 ( li068 , li003 ) to 0.81 ( li012 , li015 ; li029 , li032 ; li048 , li032 ) , with a mean of 0.58 for all the cultivated lines , geographic races of k. lacca and two other species ( groups i , ii and iv in table 1 ) .
the index varied between 0.27 ( li036 , li085 ) and 0.81 among the 33 cultivated lines and geographic races of k. lacca ( groups i and ii in table 1 ) , with a mean of 0.61 , indicative of significant intra - specific genetic diversity .
the six inbred lines of k. lacca ( rangeeni ) , derived from a single mother population , presented divergence with a mean similarity index of 0.77 ( range : 0.620.92 ) .
analysis of the above data showed that the probability of identical chance matching was 4.74 x 10 , thus indicative of a high level of reliability .
the dendrogram generated from the jaccard s similarity matrix , by using the neighbor - joining method , appears in figure 2 .
basically , it resolves the lines into two major clusters ( nodes a and b ) , as well as outgroups comprising eight lines under six branches ( 1 to 6 ) .
the cluster originating from node a , the major one comprising 32 lines , basically includes cultivated lines of the rangeeni form , geographic races and inbred lines of k. lacca .
subcluster a1 is a heterogeneous group of seven lines , comprised of two ( li003 and li031 ) from jharkhand state , two ( li42 and li36 ) from chhattisgarh state , two from andhra pradesh state , and a cream recombinant mutant line ( li001a ) .
the subcluster from node a2 is comprised of 15 lines , mainly from the eastern region , and consists of six lines derived from cultivated populations of four adjoining lac - growing states ( jharkhand , west bengal , chhattisgarh and madhya pradesh ) , six inbred lines ( also developed from a cultivated population from jharkhand ) , two geographic races from northern and eastern india , and the wild - type color form of a recombinant line ( li001b ) .
the subcluster of ten lines originating from a3 , is mainly composed of those from western india ( five from gujarat and rajasthan ) , one from the northern state of uttar pradesh ( li72b ) , two crossbred lines ( li058 and li061 ) , and the yellow mutant of a kusmi line from orissa ( li078 ) , whereas subclusters a2 and a3 mainly consist of lines from eastern and western regions of india , respectively .
, seven of the kusmi form and one of the rangeeni of k. lacca from the southern state of karnataka .
these forms of k. lacca appear to have originated through a common ancestor at o. kusmi insects are naturally distributed mainly in the eastern part of peninsular india , whereas the rangeeni are distributed countrywide .
these two forms differ as to host preference -rangeeni insects are characterized by their ability to thrive well on butea monosperma , and kusmi on schleichera oleosa ( varshney , 1977 ) .
based on crosses between the two , it has been shown that the kusmi form is genetically endowed for survival on s. oleosa ( chauhan and mishra , 1970 ) .
the remaining eight lines form a series of outgroups ( branches 16 ) , which include one of the kusmi form ( li005 ) , one collection from the central state of madhya pradesh ( li085 ) , and a group of three yellow mutant lines ( 9b , 19a and 13 ) of k. lacca , besides two lines of k. chinensis ( li068 and li002 ) and one of k. sharda .
the dendrogram differentiated the species of kerria studied , as well as li085 , represented as an outgroup , and which requires re - examination as to taxonomic status .
three color mutants in k. lacca ( white , yellow and cream ) , which affect body and resin color , have been shown to be recessive ( chauhan , 1977 ; chauhan and mishra , 1977 ; ramani , 2002 ) . in the present study ,
the dendrogram generated always separated colour mutants ( yellow and cream ) from their wild - type counterparts in the same population , due to their distinctive rapd banding profiles , indicative of their distinct genetic makeup .
principal component analysis revealed that 27.2% of the variation could be attributed to the first three components .
figure 3 presents the 3-d plot of the principal coordinate analysis of the similarity matrix data of the lines studied .
the k. lacca populations are well - spread over the dimensions , thereby indicating their diversity .
two lines of k. chinensis derived from different geographic locations ( li002 and li068 ) , as well as k. sharda ( li066 ) , are well separated from those of k. lacca , thus consistent with the nj dendrogram . according to pcoord analysis
, the seven kusmi lines of k. lacca , which formed a group at node b in this dendrogram , appeared to spread out .
the lines li075 ( kalamati , west bengal ) and li077 ( hesadih , jharkhand ) appeared distinct .
spurious larval emergence , a phenomenon whereby some crawler emergence takes place during a non - typical period ( nov  dec ) in kusmi populations , is indicative of interbreeding in kusmi and rangeeni forms .
oviposition peaks in the segregating progenies of crosses of these two forms correspond to the parental types .
four lines of the rangeeni form of k. lacca , li19a & b , li020 , li085 and li087 , tended to form a cluster at variance with the dendrogram .
lac insects , by depending on perennial trees for survival , are specialists in the preference for host - plants with limited dispersal .
they may thus become locally adapted , thereby forming genetically distinct geographic and host races without morphological differentiation .
human intervention , with systematic lac - cultivation and the transport of insect populations across various regions , also exerts an influence on dispersal and interpopulation gene flow .
thus , divergence in k. lacca , can be expected , through being both the most commonly used species for lac - production and widely distributed in india .
intermixing of cultivated populations is also expected in lac - producing areas , due to the transportation of insects within the cultivated areas , discernible in line - clustering in node a2 from the principal lac growing region .
the six inbred lines derived from the same mother population also diverged from each other , with only li024 and li020 remaining similar .
the two forms of kerria lacca , kusmi and rangeeni , commonly referred to as ` strains in lac - insect literature , are distinct as regards certain commercial and biological traits .
based upon morphological characteristics , they were allocated as infrasubspecific forms by varshney ( 1977 ) .
even so , they hybridize freely under laboratory conditions , thereby producing viable progeny . premating barriers , such as differences in host preference and asynchrony in sexual - maturity periods , due to differences in life - cycle patterns , prevent interbreeding under natural conditions .
nevertheless , through occasional period - overlaps during the rainy - season generation of both forms , interbreeding is possible .
a probable illustration is a subgroup of the a1 node cluster , comprising two collections from geographically close locations , i.e. , li042 , a line of the rangeeni form , collected from mainpur , chhattisgarh , and li036 a line of the kusmi form from kurubhatta , also chhattisgarh .
spurious crawler emergence in li077 , indicative of interbreeding in these two forms , has already been discussed .
the usefulness of rapd - pcr for assessing genetic diversity in indian lac insects has been demonstrated .
considering the general recommendation of fifty polymorphic markers to establish precise genetic distances ( nei , 1978 ) , 26 rapd primers having produced 169 polymorphic bands is sufficient for distinguishing species to complement pertinent taxonomic studies .
the dendrogram generated from the similarity matrix also throws light on the interrelationships of the otus investigated .
the above molecular evidence supports the current status of kusmi and rangeeni , as infraspecific forms of k. lacca .
color mutants also need to be examined in greater detail , in order to understand the basis for their differentiation . based on rapd profiles , the lac - insect populations of k. lacca collected from different locales presented considerable variation .
further studies using other markers and dna sequence variation , are likely to throw fresh light on these populations and their respective relationships .
 the lac insects ( homoptera : tachardiidae ) , belonging to the genus kerria , are commercially exploited for the production of lac . 
 kerria lacca is the most commonly used species in india . 
 rapd markers were used for assessing genetic variation in forty - eight lines of kerria , especially among geographic races , infrasubspecific forms , cultivated lines , inbred lines , etc . 
 , of k. lacca . 
 in the 48 lines studied , the 26 rapd primers generated 173 loci , showing 97.7% polymorphism . by using neighbor - joining , 
 the dendrogram generated from the similarity matrix resolved the lines into basically two clusters and outgroups . 
 the major cluster , comprising 32 lines , included mainly cultivated lines of the rangeeni form , geographic races and inbred lines of k. lacca . 
 the second cluster consisted of eight lines of k. lacca , seven of the kusmi form and one of the rangeeni from the southern state of karnataka . 
 the remaining eight lines formed a series of outgroups , this including a group of three yellow mutant lines of k. lacca and other species of the kerria studied , among others . 
 color mutants always showed distinctive banding patterns compared to their wild - type counterparts from the same population . 
 this study also adds support to the current status of kusmi and rangeeni , as infraspecific forms of k. lacca . 
Introduction
Material and Methods
Lac-insect collection and culture
Insect processing and DNA isolation
RAPD amplification and gel electrophoresis
Data analysis
Results and Discussion