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Genetic diversity in lac resin-secreting insects belonging to Kerria spp., as revealed through ISSR markers
Biochemical Systematics and Ecology 39 (2011) 112–120
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Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Genetic diversity in lac resin-secreting insects belonging to Kerria spp., as revealed through ISSR markers Dipnarayan Saha a,1, *, Sanjeev Kumar Ranjan a, Chandana Basu Mallick a, Ambarish Sharan Vidyarthi b, Ranganathan Ramani a a
Biotechnology Laboratory, Lac Production Division, Indian Institute of Natural Resins and Gums, Indian Council of Agricultural Research, Ranchi 834010, Jharkhand, India b Department of Biotechnology, Birla Institute of Technology, Mesra, Ranchi 835215, Jharkhand, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 September 2010 Accepted 29 January 2011 Available online 20 February 2011
The Indian lac insect Kerria lacca is harnessed in India for commercial production of lac, which has diversified industrial applications. Many of the geographical races of this species are under threat of extinction due to increasingly drastic local deviations in climate patterns. Thus, there is need for documentation and conservation of the lac insect biodiversity adapted especially for local climatic conditions and host species. The genetic diversity among twenty lines of commercially important Kerria spp. was analyzed using Inter-Simple Sequence Repeat (ISSR) technique. Seventeen ISSR primers produced 96.1% genetic polymorphism in the lac insect lines under study. The clustering dendrogram segregated the twenty lines into four major clusters with similarity coefficients between 0.25 and 0.81. The first three principal coordinates revealed 43.1% of the total genetic variation. The above results reveal significant genetic variability in the lines, which could be used for genetic improvement of lac insects. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Genetic diversity Inter-simple sequence repeats Kerria spp. Lac resin Scale insect
1. Introduction Members of the family Tachardiidae (¼Kerridae) are called lac insects and only Kerria spp. produces true lac (Sharma and Ramani, 1999; Ben-Dov, 2002). The Kerria lacca (Kerr.) is the commonly cultivated insect in India for lac production (Bahuguna and Shiva, 2002; Sharma et al., 2006) represented by infrasubspecific forms kusmi and rangeeni differing for their life cycle pattern, quality of lac resin, host preference and some morphological traits. The genetic variation has also been studied for productivity linked traits, such as sex ratio, resin cell weight, colour and size (Mishra et al., 1998, 2000; Sharma and Ramani, 2002). India is the leading producer of lac with annual production of about 16,495 tons (Pal et al., 2010). Lac yields three commercially important components: resin, dye and wax, which find applications in surface coatings, cosmetic, pharmaceutical, food and electrical industries (Ramani et al., 2007). The lac derived products are preferred due to their unique properties coupled with environmental safety. In India, the lac insects are distributed throughout the length and breadth of the country but in small isolated patches in different geographic locations. The intermixing of populations of lac insects is narrowed due to their restricted dispersal
Abbreviations: bp, base pairs; GI, Genotype index; h, hours; Ib, Band informativeness; kbp, kilo base pairs; ISSR-PCR, Inter-simple sequence repeatpolymerase chain reaction; MI, Marker index; min, minutes; PCOORD, Principal coordinates; Rp, Resolving power; s, seconds. * Corresponding author. Tel./fax: þ91 11 25842495. E-mail addresses: [email protected], [email protected] (D. Saha), [email protected] (S.K. Ranjan), [email protected] (C.B. Mallick), [email protected] (A.S. Vidyarthi), [email protected] (R. Ramani). 1 Present address: NRC on DNA Finger Printing, National Bureau of Plant Genetic Resources (ICAR), New Delhi 110012, India. 0305-1978/$ – see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2011.01.016
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ability. The rapidly changing ecosystem, depletion of host trees and discontinuation of lac cultivation by lac growers in some regions are major reasons for the erosion of lac insect biodiversity (Sharma et al., 2006). Thus, there is an urgent need to understand the structure of genetic diversity of lac insect and emphasize on its conservation. The DNA-based molecular markers have been widely used as a tool for assessing genetic diversity in number of insect species (Behura, 2006). Cook et al. (2002) and Schroer et al. (2008) have used DNA sequence variations of the nuclear small and large subunits of rDNA, 28S rDNA and mitochondrial gene cytochrome oxidase I (COI) to distinguish among different Paratachardina spp., the lobate lac scale producing pseudolac. Similar studies have been carried out in other coccids, like mealy bugs for analyzing phylogenetic relationships through DNA sequence variations of nuclear genes (Downie and Gullan, 2004; Hardy et al., 2008). Here we report the findings of ISSR-based genetic diversity analysis and genetic relationships within the twenty lines of commercially important lac insects. To the best of our knowledge, this is the first report of employing DNA markers (ISSR) in understanding the molecular diversity present in the populations of lac insects from India. 2. Materials and methods 2.1. Lac insect lines In the present study the twenty lac insect lines consisted of seventeen lines of K. lacca which included seven kusmi and rangeeni infrasubspecific forms from principal lac growing state Jharkhand, eight geographic races of rangeeni form and two inbred rangeeni lines. Apart from K. lacca two lines of K. chinensis and one K. sharda were also included (Table 1). Some of the above lines studied were those with naturally occurring yellow body colour variants found in K. lacca (Table 1). The original collection sites of the lac insect lines used in the study were depicted in Fig. 1A. The insect samples were derived from the cultures of respective collections maintained for several generations on Flemingia macrophylla, a lac host plant, under potted condition at the National Lac Insect Germplasm Centre at IINRG farm, Ranchi (23190 5100 N, 85 2201800 E, elevation w2080 ft above sea level). 2.2. Genomic DNA extraction The mature female insects were used in the study (Fig. 1B–D). This stage contains hundreds of developing embryos in the ovaries; besides, multiple-coitus (i.e. mating with many individuals) is normally observed in both sexes within the population (Chauhan and Mishra, 1970). Thus, the variation observed is assumed to represent the population-level differences. The gravid females in the resinous cover were washed with absolute alcohol for 48 h to dissolve resin and the insects were cleaned carefully under stereo microscope with the help of hairbrush for removing the waxy secretions. The cleaned insects were
Table 1 Lac insect lines used in the ISSR analysis. Sl No.
Insect line ID
Description of insect
Collection place/Source
Host plant from which collection was originally made
Rangeeni and kusmi subspecific forms of K. lacca from lac cultivated region 1 Klrsilli K. lacca, rangeeni Silli, Jharkhand 2 Klrranc K. lacca, rangeeni Ranchi, Jharkhand 3 Klrbokcr K. lacca, rangeeni (wild-type crimson) Bokaro, Jharkhand 4 Klrboky K. lacca colour variant (yellow) Bokaro, Jharkhand 5 Klkran K. lacca, kusmi Ranchi, Jharkhand 6 Klkselwb K. lacca, kusmi West Bengal
Schleichera oleosa Albizia saman Butea monosperma B. monosperma S. oleosa S. oleosa
Collections of wild populations of K. lacca from different geographic locations 7 Klgujsima K. lacca geographic race Simbalpani, Gujarat 8 Klrajpush K. lacca geographic race Pushkar, Rajasthan 9 Klapecho K. lacca geographic race Echoda, Andhra Pradesh 10 Klgujalci K. lacca, geographic race (yellow) Alcipur, Gujarat 11 Klgujsimz K. lacca, geographic race Simbalpani, Gujarat a K. lacca geographic race Kirnapur, Madhya Pradesh 12 Klmpkira 13 Klupbhat K. lacca geographic race Bhathat, Uttar Pradesh K. lacca geographic race Guna, Madhya Pradesh 14 Klmpguna
A. lebbek Z. mauritiana Peltophorum ferrugineum B. monosperma Ziziphus mauritiana B. monosperma Ficus infectoria B. monosperma
Colour form, inbred lines of K. lacca developed at IINRG 15 Klrcrm K. lacca colour variant (cream) 16 Klrinba K. lacca inbred line, rangeeni 17 Klrinbf K. lacca inbred line, rangeeni
Kundri, Jharkhand Kundri, Jharkhand Kundri, Jharkhand
B. monosperma B. monosperma B. monosperma
Other species of Kerria 18 Kcmegin 19 Ksorisar 20 Kcthaila
Nangpoh, Meghalaya, India Sarat, Orissa Thailand
F. religiosa A. saman Not known
a
Kerria chinensis (India) Kerria sharda (India) Kerria chinensis (Thailand)
Field collections directly used in the study.
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Fig. 1. A) Locations of all the Kerria lines used in the present study. The abbreviations of collection from different states in the figure stand for GJ: Gujarat, AP: Andhra Pradesh, RJ: Rajasthan, OR: Orissa, WB: West Bengal, UP: Uttar Pradesh, CG: Chhattisgarh, MP: Madhya Pradesh, MEG: Meghalaya, JKD: Jharkhand. The places of original collections are indicated as per the serial numbers in Table 1. The collections from the Jharkhand state have been shown in the inset. B) Colony of mature females of K. lacca as lac encrustation. C) Individual lac cells with adult female K. lacca insect inside. D) Mature gravid female K. lacca insect devoid of resinous cover and having hundreds of embyos inside were used for DNA extraction.
frozen at 80 C in absolute ethanol for isolation of genomic DNA in fresh 1.5 ml eppendorf tubes using the protocol of De Barro et al. (1995). The insects were homogenized in TEN buffer [10 mM Tris–Cl (pH 8.0), 2 mM EDTA (pH 8.0) and 0.4 M NaCl] containing 0.2% SDS. The homogenate was purified by treating with RNase A (10 mg/ml) and Proteinase K (20 mg/ml) followed by sequential extraction with phenol:chloroform:isoamyl alcohol (25:24:1) and chloroform:isoamyl alcohol (24:1). The purified DNA was precipitated, air-dried and dissolved in 50 ml TE buffer (10 mM Tris–Cl, 1.0 mM EDTA, pH 8.5). The genomic DNA was analyzed and quantified on 1.0% agarose gel for subsequent use in PCR analysis. DNA extraction from each insect line was replicated twice. 2.3. ISSR amplification and agarose gel electrophoresis The genomic DNA extracted above was used as template in the PCR reactions for ISSR analysis. Fifty ISSR primers (11–21 bases) with di-, tri- and tetra-nucleotide repeats and 1–2 nucleotide anchored sequences at the 30 -end (Metabion Gmbh, Germany) were used for primer screening in four lac insect lines (Klgujsima, Klrranc, Klkselwb and Klmpkira, see Table 1 for codes used). Seventeen ISSR primers (Table 2) producing reproducible and clear bands across the four lac insect lines were selected for diversity analysis. The ISSR reactions were performed in 15.0 ml reaction mixtures containing 20 ng of template DNA, 1 Taq buffer [750 mM Tris–HCl (pH 8.8), 200 mM (NH4)2SO4, 0.1% (v/v) Tween-20 (Fermentas Inc., MD, USA)], 3.0 mM MgCl2 (Fermentas Inc., MD, USA), 0.2 mM of each dNTP mix (Fermentas Inc., MD, USA), 20 pmol (1.5 mM) of each primers and 1.5 units of Taq DNA polymerase (Fermentas Inc., MD, USA). All the PCR reactions were carried out in a thermal cycler (BioRad iCyclerÔ, USA) programmed with the following cycling conditions. Initial denaturation of template DNA was carried out at 95 C for 7 min followed by 38 cycles programmed for denaturation step at 95 C for 45 s, primer annealing step at specific Tm for the particular primer (Table 2) for 45 s, and DNA strand extension step at 72 C for 2 min. The final extension of the PCR products was carried out at 72 C for 7 min.
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Table 2 Details of amplified bands from ISSR analysis using 17 primers in 20 selected lac insect lines. Primer ID
Primer sequence (50 –30 )
Tm ( C)
No. of scored bands
Band size range (bp)
Band frequency
No. of polymorphic bands (%)
Issr3 Issr5 Issr6 Issr8 Issr11 Issr14 Issr15 Issr16 Issr17 Issr19 Issr21 Issr23 Issr32 Issr33 Issr35 Issr36 Issr41 Total
(AG)8-G (AG)8-TA (AG)8-C (CA)6-AC (CA)6-GG (CA)6-AG (CA)6-GT (GA)6-GG (GT)8-A (GAG)3-GC (GTG)3-GC (GA)6-TC (AC)8-C (CAG)5-A (ATG)6-C (CAA)5 (GACA)4
52 52 52 41 44 41 41 44 50 38 38 41 52 53 51 38 48
16 10 6 5 6 8 7 9 6 12 11 10 13 9 12 9 5 154
430–1300 350–1300 400–850 400–1150 350–1100 250–1200 300–1250 400–1000 500–1500 200–1400 280–1400 400–1500 350–2000 200–1800 300–2000 400–1450 400–1400
0.293 0.262 0.703 0.342 0.225 0.228 0.426 0.289 0.238 0.27 0.321 0.29 0.326 0.236 0.39 0.444 0.326
16 (100%) 10 (100%) 5 (83.3%) 5 (100%) 6 (100%) 8 (100%) 5 (71.4%) 9 (100%) 6 (100%) 12 (100%) 11 (100%) 10 (100%) 13 (100%) 9 (100%) 10 (83.3%) 8 (88.9%) 5 (100%) 148 (96.10%)
All the amplified products along with 100 bp DNA ladder (Fermentas Inc., MD, USA) were resolved on ethidium bromide (0.2 mg/ml) stained 1.5% agarose gel, prepared with 0.5 TBE buffer [45 mM Tris-borate, 1.0 mM EDTA (pH 8.0)] and electrophoresed in 0.5 TBE at 6 V cm1 for 2 h in BioRad submarine gel electrophoresis unit. Sufficiently resolved DNA bands were documented using Bioimaging system (Gene Genius, Syngene, UK) and processed for absence or presence of band scoring using the software program Genetool and GeneDirectory (Syngene, UK). The reproducibility of bands was assessed by repeating PCR reactions with replicated DNA from the lac insect lines and only the reproducible bands were scored. 2.4. Scoring of bands and data analysis Only the clear, unambiguous and reproducible bands present across the samples from two repeated ISSR reactions were used for scoring. At different band sizes the presence of bands were scored as ‘1’; while absence of bands or very faint bands were scored as ‘0’ (Fig. 2). The smeared and weak bands obtained with certain primers were excluded from the analysis. The information content of the ISSR primers was estimated through polymorphism information content (PIC), marker index (MI) and resolving power (Rp) using band informativeness (Ib). The MI was estimated according to Powell and Morgante (1996); MI ¼ Ibav EMR of a primer, where EMR is the product of number of polymorphic bands (i.e. absence of band atleast in one genotype at a particular locus) per primer and the fraction of polymorphic bands. The average band informativeness was calculated as Ibav ¼ 1/nS1 (2j0.5 pij) [where pi is the proportion of lines showing the ith amplicon, i ¼ 1 to n where ‘n’ is the total number of amplicons (Archak et al., 2003). The primer resolving power was calculated according to Prevost and Wilkinson (1999) as Rp ¼ SIb [where Ib (band informativeness) ¼ 1 (2j0.5 pij) for each amplicon]. The genotype index (GI) of a primer represents the proportion of the lines actually distinguished by the primer, i.e. the number of lines exhibiting unique fingerprints in pair wise comparisons divided by total number of lines analyzed (McGregor et al., 2000). The confidence of distinguishing the genotypes using the ISSR primers were estimated through analysis of probability of identical match by chance (Ramakrishana et al., 1994; Wetton et al., 1987). The analysis of genetic diversity was carried out using percent polymorphic bands (Ps); where Ps ¼ total polymorphic bands (p)/number of scored bands [p < 1.0 and p ¼ 1 q and q ¼ O(total no of null bands/total no bands)]. The mean number of band per loci (As) for each primer was calculated as total number of band frequencies [As ¼ S(1 q)]. The genetic relationship among the twenty lac insect lines was analyzed using the software program NTSYSpc version 2.02e (Exeter Software, New York) (Rohlf, 1998). The Jaccard’s similarity coefficient was employed to construct a clustering dendrogram using the unweighted pair group method with arithmetic average (UPGMA) (Sneath and Sokal, 1973). The data was further analyzed for principal coordinates (PCOORD) analysis (Sneath and Sokal, 1973) using the same software. The confidence of clustering in the dendrogram was evaluated through bootstrap analysis using the program WINBOOT (Yap and Nelson, 1996) with 1000 permutations. 3. Results and discussion 3.1. Analysis of band-profiles and genetic diversity In the present investigation, out of fifty ISSR primers screened, seventeen produced satisfactory, clear and reproducible banding pattern in four lac insect lines viz. Klgujsima, Klrranc, Klkselwb and Klmpkira (Table 1) used for screening purpose. In the study lines, the seventeen ISSR primers amplified 154 scorable bands, of which 148 (96.1%) were polymorphic. The number
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Fig. 2. A representative DNA amplification profile generated through primers Issr3, 8 and 35, respectively in 20 lac insect lines.
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Table 3 Efficiency of analyzed ISSR primers in distinguishing lac insect lines. Primer ID
Genotype index (GI)
Average band informativeness (Ibav)
Marker index (MI)
Primer resolving power (Rp)
Issr3 Issr5 Issr6 Issr8 Issr11 Issr14 Issr15 Issr16 Issr17 Issr19 Issr21 Issr23 Issr32 Issr33 Issr35 Issr36 Issr41
0.90 0.15 0.20 0.05 0.15 0.55 0.00 0.20 0.25 0.80 0.35 0.75 0.45 0.40 0.55 0.00 0.20
0.53 0.44 0.22 0.40 0.50 0.44 0.40 0.38 0.47 0.45 0.45 0.63 0.53 0.61 0.33 0.29 0.26
8.50 4.40 0.90 2.00 3.00 3.50 1.43 3.40 2.80 5.40 4.90 6.30 6.90 5.50 2.78 2.05 1.30
8.5 4.4 1.3 2.0 3.0 3.5 2.8 3.4 2.8 5.4 4.9 6.3 6.9 5.5 4.0 2.6 1.3
of polymorphic bands produced by each primer was 5–16 (average 8.7). Except for four primers, viz. Issr6, 15, 35 and 36, all primers exhibited 100% polymorphic bands (Table 2). The band-sizes of amplified products were found between 200 bp and 2.0 kbp. The highest size difference (300–2000 bp) between the amplified products was obtained with the primer Issr35, while lowest size difference (400–850 bp) was obtained with Issr6. The minimum and maximum number of scorable band-sizes produced by the ISSR primers were five (by Issr8 and Issr41 primers) and sixteen (by Issr3 primer), respectively with an average band-size of 9.06 per primer. The range of average band frequency obtained per primer was 0.23 (Issr11)–0.70 (Issr6) (Table 2). 3.2. Resolving power of ISSR primers and discrimination of lac insect lines The usefulness of the primers used in the study is best analyzed through comparing the marker index (MI) and resolving power (Rp) values. The MI value was highest (8.5) and lowest (0.9) with Issr3 and Issr6 primers, respectively (Table 3). The band informativeness (Ib) calculated for Rp values ranged from 0.22 to 0.63, respectively for Issr6 and Issr23 primers. The highest Rp value calculated in terms of Ib was 8.5 observed for Issr3 primer, while it was lowest 1.3 for the primers Issr6 and Issr41. Five of the primers viz. Issr3, 19, 23, 32 and 33 produced Rp values more than five indicating the above primers were efficient in distinguishing the twenty lac insect lines through ISSR analysis (Table 3). The genotype index (GI), which was used to discriminate between the lac insect lines was recorded highest (0.9) for Issr3 and lowest (0.0) for Issr15 primer; the later could not exhibit even a single unique band in any lac insect lines under study. The value of the overall probability of identical match by chance was obtained 3.84 1023, which indicates about a good confidence level in the ISSR profiles of the twenty lac insect lines used in the study. A total of eleven unique bands were observed with ten different ISSR primers in seven lac insect lines (Table 4). The lac insect line Kcmegin, which is actually a K. chinensis from Meghalaya, India, exhibited maximum number of unique bands (4 out of 11) compared to other insect lines studied (Table 4). The above unique bands can be converted into specific markers, such as sequence characterized amplified Table 4 Lac insect lines identified through presence or absence of unique bands. ISSR primer ID
Lac insect lines
Number of band
By presence of unique band Issr3 Issr5 Issr8 Issr8 Issr14 Issr16 Issr17 Issr19 Issr32 Issr35 Issr41
Kcmegin Kcmegin Klapecho Klapecho Klmpgun Ksorisar Kcthaila Klmpkira, Klmpgun Kcmegin Klrranc Kcmegin
1 1 1 1 1 1 1 1 1 1 1
By absence of unique band Issr21 Issr21 Issr32
Klrajpush Klrinbf Klrinbf
1 1 1
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Fig. 3. UPGMA dendrogram of 20 lac insect lines analyzed through 17 ISSR primers. The similarity coefficient scale was provided below the diagram and the bootstrap values from 1000 replicates are given on the forks. The bootstrap values of more than 30% are indicated in the figure.
region (SCAR) or sequence tagged sites (STS) for more reliable identification of the lac insect lines. The lac insect line Klrajpush, which is a geographic race collected from Rajasthan, India, have exhibited unique absence of band with primer Issr41. Similarly, the insect line Klrinbf, which is inbred line of rangeeni form of K. lacca, exhibited unique absence of band with three different ISSR primers Issr21, 32 and 35. 3.3. Genetic relationships The data from the ISSR profiling were used to establish genetic relationships among the twenty lac insect lines through analysis of clusters and principal coordinates. The Jaccard’s similarity index between the pairs of lac insect lines ranged from 0.15 (between Klrinbf and Kcthaila) to 0.81 (between Klrajpush and Klgujalci) with a mean similarity index of 0.49. The clustering dendrogram differentiated the twenty lac insect lines into two distinct groups, I and II at 25% genetic similarity (Fig. 3). The high bootstrap values in the forks of major clusters revealed high confidence level in the UPGMA clustering. The similarity coefficient values of the dendrogram ranged from 0.25 to 0.81.
Fig. 4. A three-dimensional plot generated from the principle coordinates analysis of 20 lac insect lines. The axes of the planes were denoted in the figure. The variation from the first three coordinates account for 43.1% of the total variation. The numbers denoted in the figure pertains to the lac insect lines under study as per the serial numbers in Table 1.
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Table 5 Eigen value, percent of variability explained by each coordinate and the cumulative variability obtained from PCOORD analysis. Principal coordinates
Eigenvalue
Percent variability
Cumulative variability
1 2 3
1.93 1.33 1.20
18.20 12.54 11.39
18.20 30.74 42.13
The branch II of the dendrogram consisted two lines of K. chinensis; those are grouped distinct from the K. lacca and K. sharda in branch I. The branch IB consisted of a single line (Klrinbf), an inbred lac insect of rangeeni K. lacca, which might have diverged from the other insects in branch IA due to inbreeding. The branch IA consisted of a mixed group of seventeen lines comprising all the geographic races, two forms of K. lacca and one line of K. sharda. Grouping of K. sharda within the same cluster with K. lacca insects suggest its genetic closeness to K. lacca. A small sub-cluster of four insect lines in branch IAii, included two geographic races collected from Gujarat and one from Madhya Pradesh; both are adjoining states of India. An inbred line of rangeeni lac insect (Klrinba) was also grouped in this cluster. The branch IAi consisting of a mixed group of thirteen lac insect lines including five geographic races, five cultivated lines from the principal lac growing regions, two colour variants and one line of K. sharda. The K. sharda although is trivoltine in its life cycle pattern (Mishra and Sushil, 2000) it is clustered together with the bivoltine lac insect lines in branch IAi. Similarly the kusmi form of K. lacca did not clearly form a separate cluster from the rangeeni lines. The above results indicate that there is gene flow between these two forms, despite reduced scope of interbreeding due to life cycle asynchrony and differences in host preference. Interestingly, the yellow bodied and wild-type crimson colour variants are separated from each other in the dendrogram indicating a distinct genetic difference between them. The yellow insect differs from crimson wild type in their body colour due to a simple recessive gene mutation (Chauhan, 1967). The lac insects are sessile in nature and thus have limited dispersal capacity and restricted gene mixing. Thus, genetic similarity could be expected in relation with the geographic distances between the lines under study. Close grouping of lines from geographic proximity has been found in three instances: Klrajpush and Klgujalci (IAi); Klrboky and Klupbhat (IAi) and Klgujsimz and Klmpgun (IAii). Similar kind of study revealed a strong effect of geographic distance in genetic differentiation of Indian eri silkmoths (Samia cynthia ricini) of North-east India, which also have limited mobility in juvenile and adult stages (Vijayan et al., 2006). The PCOORD analysis demonstrated that the twenty lac insect lines can be separated distinctly from each other (Fig. 4). The first three principal coordinates explained 18.2%, 12.5% and 11.4% of the total variation of 43.1% (Table 5). The K. chinensis lines from Meghalaya and Thailand emerged as most divergent from the other lac insect lines in agreement with the UPGMA dendrogram. Among the K. lacca lines, two geographic races from Gujarat (Klgujsimz, Klgujsima) and two inbred lines of rangeeni insect (Klrinba, Klrinbf) grouped distinctly. A yellow colour variant lac insect line Klgujalci, which is collected from Gujarat, containing only yellow colour variant, remained distinct, while the remaining thirteen lac insect lines remained under a major cluster. The present study demonstrates the potential use of ISSR markers in characterization of genetic diversity in lac insects and has also yielded a few line-specific markers. However, other DNA markers like SSRs would be efficient in developing molecular tags for specific traits of lac insects; such as body colour variations, life cycle and host specificity for lac insect breeding programme. Nonetheless, the results revealed very high (96.1%) genetic polymorphism and also helped us to explain the wide genetic variability present within the twenty lines of lac insects used in the study. This natural variability of the lac insects can be harnessed in breeding of lac insects for high quality resin production. The ISSR markers identified would serve complementary in identification of lines for conservation as well as characterization of commercially important lac insect populations. Acknowledgements Authors express their gratitude to the reviewers for their critical reviewing of the article whose comments have been helpful in significant improvement in the manuscript. The work was supported by the Department of Biotechnology, Govt. of India, under DBT grant no. BT/012/30/NBDB/2002-II. We also acknowledge the technical help rendered by Sri AK Sinha and Sri Bhupal Kumar. References Archak, S., Gaikwad, A.B., Gautam, D., Rao, E.V.V.B., Swamy, K.R.M., Karihaloo, J.L., 2003. 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Cook, L.G., Gullan, P.J., Trueman, H.E., 2002. A preliminary phylogeny of the scale insects (Hemiptera: Sternorrhyncha: Coccoidea) based on nuclear smallsubunit ribosomal DNA. Mol. Phylogenet. Evol. 25, 43–52. De Barro, P.J., Sherratt, T.N., Brookes, C.P., David, O., Maclean, N., 1995. Spatial and temporal genetic variation in British field populations of the grain aphid Sitobion avenae (F.) (Hemiptera: aphididae) studied using RAPD-PCR. Proc. Roy. Soc. Lon. B. 262, 321–327. Downie, D.A., Gullan, P.J., 2004. Phylogenetic analysis of mealybugs (Hemiptera: Coccoidea: Pseudococcidae) based on DNA sequences from three nuclear genes, and a review of the higher classification. Syst. Entomol. 29, 238–259. Hardy, N.B., Gullan, P.J., Hodgson, C.J., 2008. A subfamily-level classification of mealybugs (Hemiptera: Pseudococcidae) based on integrated molecular and morphological data. Syst. Entomol. 33, 51–71. McGregor, C.E., Lambert, C.A., Greyling, M.M., Louw, J.H., Warnich, L., 2000. A comparative assessment of DNA fingerprinting techniques (RAPD, ISSR, AFLP and SSR) in tetraploid potato (Solanum tuberosum L.) germplasm. Euphytica 113, 135–144. Mishra, Y.D., Sushil, S.N., Bhattacharya, A., Sharma, K.K., 1998. Morphometric differences between strains of Indian lac insect, Kerria lacca (Kerr). J. Insect Sci. (India) 11, 171–172. Mishra, Y.D., Sushil, S.N., 2000. A new trivoltine species of Kerria Targioni-Tozzetti (Homoptera: Tachardiidae) on Schleichera oleosa (Lour.) Oken from eastern India. Oriental Insects 34, 215–220. Mishra, Y.D., Sushil, S.N., Bhattacharya, A., Kumar, S., Mallick, A., 2000. Genetic variations in some productivity linked traits of female lac insects, Kerria spp. (Homoptera: Tachardiidae). J. Ent. Res. 24, 121–124. Pal, G., Jaiswal, A.K., Bhattacharya, A., 2010. Lac Statistics at a Glance 2010. Indian Institute of Natural Resins and Gums, Ranchi, India. www.icar.org.in/ilri. Powell, W., Morgante, M., 1996. The comparison of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis. Mol. Breeding 2, 225–238. Prevost, A., Wilkinson, M.J., 1999. A new system of comparing PCR primers applied to ISSR fingerprinting of potato cultivars. Theor. Appl. Genet. 98, 107–112. Ramakishana, W., Lagu, M.D., Gupta, V.S., Ranjekar, P.K., 1994. DNA fingerprinting in rice using oligonucleotide probes specific for simple repetitive DNA sequences. Theor. Appl. Genet. 88, 402–406. Ramani, R., Baboo, B., Goswami, D.N., 2007. Lac – an introduction. Indian Lac Research Institute, Ranchi. Rohlf, F.J., 1998. NTSYS-PC: Numerical Taxonomy and Multivariate Analysis System Version 2.0. Department of Ecology and Evolution, State University of New York. Schroer, S., Pemberton, R.W., Cook, L.G., Kondo, T., Gullan, P.J., 2008. The genetic diversity, relationships, and potential for biological control of the lobate lac scale, Paratachardina pseudolobata Kondo & Gullan (Hemiptera: Coccoidea: Kerriidae). Biol. Cont. 46, 256–266. Sharma, K.K., Ramani, R., 1999. An update on synoptic catalogue of lac insects (Homoptera: Tacchardidae). J. Bom. Nat. Hist. Soc. 96, 438–443. Sharma, K.K., Ramani, R., 2002. Genetic variability in lac insects. In: Kumar, K.K., Ramani, R., Sharma, K.K. (Eds.), Recent Advances in Lac Culture. Indian Lac Research Institute, Ranchi, India, pp. 43–47. Sharma, K.K., Jaiswal, A.K., Kumar, K.K., 2006. Role of lac culture in biodiversity conservation: issues at stake and conservation strategy. Curr. Sci. 91, 894–898. Sneath, P.H.A., Sokal, R.R., 1973. Numerical taxonomy. W.H. Freeman and Company, San Francisco. 147–157. Vijayan, K., Anuradha, H.J., Nair, C.V., Pradeep, A.R., Awasthi, A.K., Saratchandra, B., Rahman, S.A.S., Singh, K.C., Chakraborti, R., Urs, S.R., 2006. Genetic diversity and differentiation among populations of the Indian eri silkworm, Samia cynthia ricini, revealed by ISSR markers. J. Insect Sci. 6, 30. Available online: www.insectscience.org/6.30. Wetton, J.H., Carter, R.E., Parkin, D.T., Walters, D., 1987. Demographic study of a wild house sparrow population by DNA fingerprinting. Nature 327, 147–149. Yap, I.P., Nelson, R.J., 1996. IRRI Discuss Paper Ser. 14. IRRI, Manila, Philippines.
Contents lists available at ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Genetic diversity in lac resin-secreting insects belonging to Kerria spp., as revealed through ISSR markers Dipnarayan Saha a,1, *, Sanjeev Kumar Ranjan a, Chandana Basu Mallick a, Ambarish Sharan Vidyarthi b, Ranganathan Ramani a a
Biotechnology Laboratory, Lac Production Division, Indian Institute of Natural Resins and Gums, Indian Council of Agricultural Research, Ranchi 834010, Jharkhand, India b Department of Biotechnology, Birla Institute of Technology, Mesra, Ranchi 835215, Jharkhand, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 September 2010 Accepted 29 January 2011 Available online 20 February 2011
The Indian lac insect Kerria lacca is harnessed in India for commercial production of lac, which has diversified industrial applications. Many of the geographical races of this species are under threat of extinction due to increasingly drastic local deviations in climate patterns. Thus, there is need for documentation and conservation of the lac insect biodiversity adapted especially for local climatic conditions and host species. The genetic diversity among twenty lines of commercially important Kerria spp. was analyzed using Inter-Simple Sequence Repeat (ISSR) technique. Seventeen ISSR primers produced 96.1% genetic polymorphism in the lac insect lines under study. The clustering dendrogram segregated the twenty lines into four major clusters with similarity coefficients between 0.25 and 0.81. The first three principal coordinates revealed 43.1% of the total genetic variation. The above results reveal significant genetic variability in the lines, which could be used for genetic improvement of lac insects. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Genetic diversity Inter-simple sequence repeats Kerria spp. Lac resin Scale insect
1. Introduction Members of the family Tachardiidae (¼Kerridae) are called lac insects and only Kerria spp. produces true lac (Sharma and Ramani, 1999; Ben-Dov, 2002). The Kerria lacca (Kerr.) is the commonly cultivated insect in India for lac production (Bahuguna and Shiva, 2002; Sharma et al., 2006) represented by infrasubspecific forms kusmi and rangeeni differing for their life cycle pattern, quality of lac resin, host preference and some morphological traits. The genetic variation has also been studied for productivity linked traits, such as sex ratio, resin cell weight, colour and size (Mishra et al., 1998, 2000; Sharma and Ramani, 2002). India is the leading producer of lac with annual production of about 16,495 tons (Pal et al., 2010). Lac yields three commercially important components: resin, dye and wax, which find applications in surface coatings, cosmetic, pharmaceutical, food and electrical industries (Ramani et al., 2007). The lac derived products are preferred due to their unique properties coupled with environmental safety. In India, the lac insects are distributed throughout the length and breadth of the country but in small isolated patches in different geographic locations. The intermixing of populations of lac insects is narrowed due to their restricted dispersal
Abbreviations: bp, base pairs; GI, Genotype index; h, hours; Ib, Band informativeness; kbp, kilo base pairs; ISSR-PCR, Inter-simple sequence repeatpolymerase chain reaction; MI, Marker index; min, minutes; PCOORD, Principal coordinates; Rp, Resolving power; s, seconds. * Corresponding author. Tel./fax: þ91 11 25842495. E-mail addresses: [email protected], [email protected] (D. Saha), [email protected] (S.K. Ranjan), [email protected] (C.B. Mallick), [email protected] (A.S. Vidyarthi), [email protected] (R. Ramani). 1 Present address: NRC on DNA Finger Printing, National Bureau of Plant Genetic Resources (ICAR), New Delhi 110012, India. 0305-1978/$ – see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2011.01.016
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ability. The rapidly changing ecosystem, depletion of host trees and discontinuation of lac cultivation by lac growers in some regions are major reasons for the erosion of lac insect biodiversity (Sharma et al., 2006). Thus, there is an urgent need to understand the structure of genetic diversity of lac insect and emphasize on its conservation. The DNA-based molecular markers have been widely used as a tool for assessing genetic diversity in number of insect species (Behura, 2006). Cook et al. (2002) and Schroer et al. (2008) have used DNA sequence variations of the nuclear small and large subunits of rDNA, 28S rDNA and mitochondrial gene cytochrome oxidase I (COI) to distinguish among different Paratachardina spp., the lobate lac scale producing pseudolac. Similar studies have been carried out in other coccids, like mealy bugs for analyzing phylogenetic relationships through DNA sequence variations of nuclear genes (Downie and Gullan, 2004; Hardy et al., 2008). Here we report the findings of ISSR-based genetic diversity analysis and genetic relationships within the twenty lines of commercially important lac insects. To the best of our knowledge, this is the first report of employing DNA markers (ISSR) in understanding the molecular diversity present in the populations of lac insects from India. 2. Materials and methods 2.1. Lac insect lines In the present study the twenty lac insect lines consisted of seventeen lines of K. lacca which included seven kusmi and rangeeni infrasubspecific forms from principal lac growing state Jharkhand, eight geographic races of rangeeni form and two inbred rangeeni lines. Apart from K. lacca two lines of K. chinensis and one K. sharda were also included (Table 1). Some of the above lines studied were those with naturally occurring yellow body colour variants found in K. lacca (Table 1). The original collection sites of the lac insect lines used in the study were depicted in Fig. 1A. The insect samples were derived from the cultures of respective collections maintained for several generations on Flemingia macrophylla, a lac host plant, under potted condition at the National Lac Insect Germplasm Centre at IINRG farm, Ranchi (23190 5100 N, 85 2201800 E, elevation w2080 ft above sea level). 2.2. Genomic DNA extraction The mature female insects were used in the study (Fig. 1B–D). This stage contains hundreds of developing embryos in the ovaries; besides, multiple-coitus (i.e. mating with many individuals) is normally observed in both sexes within the population (Chauhan and Mishra, 1970). Thus, the variation observed is assumed to represent the population-level differences. The gravid females in the resinous cover were washed with absolute alcohol for 48 h to dissolve resin and the insects were cleaned carefully under stereo microscope with the help of hairbrush for removing the waxy secretions. The cleaned insects were
Table 1 Lac insect lines used in the ISSR analysis. Sl No.
Insect line ID
Description of insect
Collection place/Source
Host plant from which collection was originally made
Rangeeni and kusmi subspecific forms of K. lacca from lac cultivated region 1 Klrsilli K. lacca, rangeeni Silli, Jharkhand 2 Klrranc K. lacca, rangeeni Ranchi, Jharkhand 3 Klrbokcr K. lacca, rangeeni (wild-type crimson) Bokaro, Jharkhand 4 Klrboky K. lacca colour variant (yellow) Bokaro, Jharkhand 5 Klkran K. lacca, kusmi Ranchi, Jharkhand 6 Klkselwb K. lacca, kusmi West Bengal
Schleichera oleosa Albizia saman Butea monosperma B. monosperma S. oleosa S. oleosa
Collections of wild populations of K. lacca from different geographic locations 7 Klgujsima K. lacca geographic race Simbalpani, Gujarat 8 Klrajpush K. lacca geographic race Pushkar, Rajasthan 9 Klapecho K. lacca geographic race Echoda, Andhra Pradesh 10 Klgujalci K. lacca, geographic race (yellow) Alcipur, Gujarat 11 Klgujsimz K. lacca, geographic race Simbalpani, Gujarat a K. lacca geographic race Kirnapur, Madhya Pradesh 12 Klmpkira 13 Klupbhat K. lacca geographic race Bhathat, Uttar Pradesh K. lacca geographic race Guna, Madhya Pradesh 14 Klmpguna
A. lebbek Z. mauritiana Peltophorum ferrugineum B. monosperma Ziziphus mauritiana B. monosperma Ficus infectoria B. monosperma
Colour form, inbred lines of K. lacca developed at IINRG 15 Klrcrm K. lacca colour variant (cream) 16 Klrinba K. lacca inbred line, rangeeni 17 Klrinbf K. lacca inbred line, rangeeni
Kundri, Jharkhand Kundri, Jharkhand Kundri, Jharkhand
B. monosperma B. monosperma B. monosperma
Other species of Kerria 18 Kcmegin 19 Ksorisar 20 Kcthaila
Nangpoh, Meghalaya, India Sarat, Orissa Thailand
F. religiosa A. saman Not known
a
Kerria chinensis (India) Kerria sharda (India) Kerria chinensis (Thailand)
Field collections directly used in the study.
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Fig. 1. A) Locations of all the Kerria lines used in the present study. The abbreviations of collection from different states in the figure stand for GJ: Gujarat, AP: Andhra Pradesh, RJ: Rajasthan, OR: Orissa, WB: West Bengal, UP: Uttar Pradesh, CG: Chhattisgarh, MP: Madhya Pradesh, MEG: Meghalaya, JKD: Jharkhand. The places of original collections are indicated as per the serial numbers in Table 1. The collections from the Jharkhand state have been shown in the inset. B) Colony of mature females of K. lacca as lac encrustation. C) Individual lac cells with adult female K. lacca insect inside. D) Mature gravid female K. lacca insect devoid of resinous cover and having hundreds of embyos inside were used for DNA extraction.
frozen at 80 C in absolute ethanol for isolation of genomic DNA in fresh 1.5 ml eppendorf tubes using the protocol of De Barro et al. (1995). The insects were homogenized in TEN buffer [10 mM Tris–Cl (pH 8.0), 2 mM EDTA (pH 8.0) and 0.4 M NaCl] containing 0.2% SDS. The homogenate was purified by treating with RNase A (10 mg/ml) and Proteinase K (20 mg/ml) followed by sequential extraction with phenol:chloroform:isoamyl alcohol (25:24:1) and chloroform:isoamyl alcohol (24:1). The purified DNA was precipitated, air-dried and dissolved in 50 ml TE buffer (10 mM Tris–Cl, 1.0 mM EDTA, pH 8.5). The genomic DNA was analyzed and quantified on 1.0% agarose gel for subsequent use in PCR analysis. DNA extraction from each insect line was replicated twice. 2.3. ISSR amplification and agarose gel electrophoresis The genomic DNA extracted above was used as template in the PCR reactions for ISSR analysis. Fifty ISSR primers (11–21 bases) with di-, tri- and tetra-nucleotide repeats and 1–2 nucleotide anchored sequences at the 30 -end (Metabion Gmbh, Germany) were used for primer screening in four lac insect lines (Klgujsima, Klrranc, Klkselwb and Klmpkira, see Table 1 for codes used). Seventeen ISSR primers (Table 2) producing reproducible and clear bands across the four lac insect lines were selected for diversity analysis. The ISSR reactions were performed in 15.0 ml reaction mixtures containing 20 ng of template DNA, 1 Taq buffer [750 mM Tris–HCl (pH 8.8), 200 mM (NH4)2SO4, 0.1% (v/v) Tween-20 (Fermentas Inc., MD, USA)], 3.0 mM MgCl2 (Fermentas Inc., MD, USA), 0.2 mM of each dNTP mix (Fermentas Inc., MD, USA), 20 pmol (1.5 mM) of each primers and 1.5 units of Taq DNA polymerase (Fermentas Inc., MD, USA). All the PCR reactions were carried out in a thermal cycler (BioRad iCyclerÔ, USA) programmed with the following cycling conditions. Initial denaturation of template DNA was carried out at 95 C for 7 min followed by 38 cycles programmed for denaturation step at 95 C for 45 s, primer annealing step at specific Tm for the particular primer (Table 2) for 45 s, and DNA strand extension step at 72 C for 2 min. The final extension of the PCR products was carried out at 72 C for 7 min.
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Table 2 Details of amplified bands from ISSR analysis using 17 primers in 20 selected lac insect lines. Primer ID
Primer sequence (50 –30 )
Tm ( C)
No. of scored bands
Band size range (bp)
Band frequency
No. of polymorphic bands (%)
Issr3 Issr5 Issr6 Issr8 Issr11 Issr14 Issr15 Issr16 Issr17 Issr19 Issr21 Issr23 Issr32 Issr33 Issr35 Issr36 Issr41 Total
(AG)8-G (AG)8-TA (AG)8-C (CA)6-AC (CA)6-GG (CA)6-AG (CA)6-GT (GA)6-GG (GT)8-A (GAG)3-GC (GTG)3-GC (GA)6-TC (AC)8-C (CAG)5-A (ATG)6-C (CAA)5 (GACA)4
52 52 52 41 44 41 41 44 50 38 38 41 52 53 51 38 48
16 10 6 5 6 8 7 9 6 12 11 10 13 9 12 9 5 154
430–1300 350–1300 400–850 400–1150 350–1100 250–1200 300–1250 400–1000 500–1500 200–1400 280–1400 400–1500 350–2000 200–1800 300–2000 400–1450 400–1400
0.293 0.262 0.703 0.342 0.225 0.228 0.426 0.289 0.238 0.27 0.321 0.29 0.326 0.236 0.39 0.444 0.326
16 (100%) 10 (100%) 5 (83.3%) 5 (100%) 6 (100%) 8 (100%) 5 (71.4%) 9 (100%) 6 (100%) 12 (100%) 11 (100%) 10 (100%) 13 (100%) 9 (100%) 10 (83.3%) 8 (88.9%) 5 (100%) 148 (96.10%)
All the amplified products along with 100 bp DNA ladder (Fermentas Inc., MD, USA) were resolved on ethidium bromide (0.2 mg/ml) stained 1.5% agarose gel, prepared with 0.5 TBE buffer [45 mM Tris-borate, 1.0 mM EDTA (pH 8.0)] and electrophoresed in 0.5 TBE at 6 V cm1 for 2 h in BioRad submarine gel electrophoresis unit. Sufficiently resolved DNA bands were documented using Bioimaging system (Gene Genius, Syngene, UK) and processed for absence or presence of band scoring using the software program Genetool and GeneDirectory (Syngene, UK). The reproducibility of bands was assessed by repeating PCR reactions with replicated DNA from the lac insect lines and only the reproducible bands were scored. 2.4. Scoring of bands and data analysis Only the clear, unambiguous and reproducible bands present across the samples from two repeated ISSR reactions were used for scoring. At different band sizes the presence of bands were scored as ‘1’; while absence of bands or very faint bands were scored as ‘0’ (Fig. 2). The smeared and weak bands obtained with certain primers were excluded from the analysis. The information content of the ISSR primers was estimated through polymorphism information content (PIC), marker index (MI) and resolving power (Rp) using band informativeness (Ib). The MI was estimated according to Powell and Morgante (1996); MI ¼ Ibav EMR of a primer, where EMR is the product of number of polymorphic bands (i.e. absence of band atleast in one genotype at a particular locus) per primer and the fraction of polymorphic bands. The average band informativeness was calculated as Ibav ¼ 1/nS1 (2j0.5 pij) [where pi is the proportion of lines showing the ith amplicon, i ¼ 1 to n where ‘n’ is the total number of amplicons (Archak et al., 2003). The primer resolving power was calculated according to Prevost and Wilkinson (1999) as Rp ¼ SIb [where Ib (band informativeness) ¼ 1 (2j0.5 pij) for each amplicon]. The genotype index (GI) of a primer represents the proportion of the lines actually distinguished by the primer, i.e. the number of lines exhibiting unique fingerprints in pair wise comparisons divided by total number of lines analyzed (McGregor et al., 2000). The confidence of distinguishing the genotypes using the ISSR primers were estimated through analysis of probability of identical match by chance (Ramakrishana et al., 1994; Wetton et al., 1987). The analysis of genetic diversity was carried out using percent polymorphic bands (Ps); where Ps ¼ total polymorphic bands (p)/number of scored bands [p < 1.0 and p ¼ 1 q and q ¼ O(total no of null bands/total no bands)]. The mean number of band per loci (As) for each primer was calculated as total number of band frequencies [As ¼ S(1 q)]. The genetic relationship among the twenty lac insect lines was analyzed using the software program NTSYSpc version 2.02e (Exeter Software, New York) (Rohlf, 1998). The Jaccard’s similarity coefficient was employed to construct a clustering dendrogram using the unweighted pair group method with arithmetic average (UPGMA) (Sneath and Sokal, 1973). The data was further analyzed for principal coordinates (PCOORD) analysis (Sneath and Sokal, 1973) using the same software. The confidence of clustering in the dendrogram was evaluated through bootstrap analysis using the program WINBOOT (Yap and Nelson, 1996) with 1000 permutations. 3. Results and discussion 3.1. Analysis of band-profiles and genetic diversity In the present investigation, out of fifty ISSR primers screened, seventeen produced satisfactory, clear and reproducible banding pattern in four lac insect lines viz. Klgujsima, Klrranc, Klkselwb and Klmpkira (Table 1) used for screening purpose. In the study lines, the seventeen ISSR primers amplified 154 scorable bands, of which 148 (96.1%) were polymorphic. The number
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Fig. 2. A representative DNA amplification profile generated through primers Issr3, 8 and 35, respectively in 20 lac insect lines.
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Table 3 Efficiency of analyzed ISSR primers in distinguishing lac insect lines. Primer ID
Genotype index (GI)
Average band informativeness (Ibav)
Marker index (MI)
Primer resolving power (Rp)
Issr3 Issr5 Issr6 Issr8 Issr11 Issr14 Issr15 Issr16 Issr17 Issr19 Issr21 Issr23 Issr32 Issr33 Issr35 Issr36 Issr41
0.90 0.15 0.20 0.05 0.15 0.55 0.00 0.20 0.25 0.80 0.35 0.75 0.45 0.40 0.55 0.00 0.20
0.53 0.44 0.22 0.40 0.50 0.44 0.40 0.38 0.47 0.45 0.45 0.63 0.53 0.61 0.33 0.29 0.26
8.50 4.40 0.90 2.00 3.00 3.50 1.43 3.40 2.80 5.40 4.90 6.30 6.90 5.50 2.78 2.05 1.30
8.5 4.4 1.3 2.0 3.0 3.5 2.8 3.4 2.8 5.4 4.9 6.3 6.9 5.5 4.0 2.6 1.3
of polymorphic bands produced by each primer was 5–16 (average 8.7). Except for four primers, viz. Issr6, 15, 35 and 36, all primers exhibited 100% polymorphic bands (Table 2). The band-sizes of amplified products were found between 200 bp and 2.0 kbp. The highest size difference (300–2000 bp) between the amplified products was obtained with the primer Issr35, while lowest size difference (400–850 bp) was obtained with Issr6. The minimum and maximum number of scorable band-sizes produced by the ISSR primers were five (by Issr8 and Issr41 primers) and sixteen (by Issr3 primer), respectively with an average band-size of 9.06 per primer. The range of average band frequency obtained per primer was 0.23 (Issr11)–0.70 (Issr6) (Table 2). 3.2. Resolving power of ISSR primers and discrimination of lac insect lines The usefulness of the primers used in the study is best analyzed through comparing the marker index (MI) and resolving power (Rp) values. The MI value was highest (8.5) and lowest (0.9) with Issr3 and Issr6 primers, respectively (Table 3). The band informativeness (Ib) calculated for Rp values ranged from 0.22 to 0.63, respectively for Issr6 and Issr23 primers. The highest Rp value calculated in terms of Ib was 8.5 observed for Issr3 primer, while it was lowest 1.3 for the primers Issr6 and Issr41. Five of the primers viz. Issr3, 19, 23, 32 and 33 produced Rp values more than five indicating the above primers were efficient in distinguishing the twenty lac insect lines through ISSR analysis (Table 3). The genotype index (GI), which was used to discriminate between the lac insect lines was recorded highest (0.9) for Issr3 and lowest (0.0) for Issr15 primer; the later could not exhibit even a single unique band in any lac insect lines under study. The value of the overall probability of identical match by chance was obtained 3.84 1023, which indicates about a good confidence level in the ISSR profiles of the twenty lac insect lines used in the study. A total of eleven unique bands were observed with ten different ISSR primers in seven lac insect lines (Table 4). The lac insect line Kcmegin, which is actually a K. chinensis from Meghalaya, India, exhibited maximum number of unique bands (4 out of 11) compared to other insect lines studied (Table 4). The above unique bands can be converted into specific markers, such as sequence characterized amplified Table 4 Lac insect lines identified through presence or absence of unique bands. ISSR primer ID
Lac insect lines
Number of band
By presence of unique band Issr3 Issr5 Issr8 Issr8 Issr14 Issr16 Issr17 Issr19 Issr32 Issr35 Issr41
Kcmegin Kcmegin Klapecho Klapecho Klmpgun Ksorisar Kcthaila Klmpkira, Klmpgun Kcmegin Klrranc Kcmegin
1 1 1 1 1 1 1 1 1 1 1
By absence of unique band Issr21 Issr21 Issr32
Klrajpush Klrinbf Klrinbf
1 1 1
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Fig. 3. UPGMA dendrogram of 20 lac insect lines analyzed through 17 ISSR primers. The similarity coefficient scale was provided below the diagram and the bootstrap values from 1000 replicates are given on the forks. The bootstrap values of more than 30% are indicated in the figure.
region (SCAR) or sequence tagged sites (STS) for more reliable identification of the lac insect lines. The lac insect line Klrajpush, which is a geographic race collected from Rajasthan, India, have exhibited unique absence of band with primer Issr41. Similarly, the insect line Klrinbf, which is inbred line of rangeeni form of K. lacca, exhibited unique absence of band with three different ISSR primers Issr21, 32 and 35. 3.3. Genetic relationships The data from the ISSR profiling were used to establish genetic relationships among the twenty lac insect lines through analysis of clusters and principal coordinates. The Jaccard’s similarity index between the pairs of lac insect lines ranged from 0.15 (between Klrinbf and Kcthaila) to 0.81 (between Klrajpush and Klgujalci) with a mean similarity index of 0.49. The clustering dendrogram differentiated the twenty lac insect lines into two distinct groups, I and II at 25% genetic similarity (Fig. 3). The high bootstrap values in the forks of major clusters revealed high confidence level in the UPGMA clustering. The similarity coefficient values of the dendrogram ranged from 0.25 to 0.81.
Fig. 4. A three-dimensional plot generated from the principle coordinates analysis of 20 lac insect lines. The axes of the planes were denoted in the figure. The variation from the first three coordinates account for 43.1% of the total variation. The numbers denoted in the figure pertains to the lac insect lines under study as per the serial numbers in Table 1.
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Table 5 Eigen value, percent of variability explained by each coordinate and the cumulative variability obtained from PCOORD analysis. Principal coordinates
Eigenvalue
Percent variability
Cumulative variability
1 2 3
1.93 1.33 1.20
18.20 12.54 11.39
18.20 30.74 42.13
The branch II of the dendrogram consisted two lines of K. chinensis; those are grouped distinct from the K. lacca and K. sharda in branch I. The branch IB consisted of a single line (Klrinbf), an inbred lac insect of rangeeni K. lacca, which might have diverged from the other insects in branch IA due to inbreeding. The branch IA consisted of a mixed group of seventeen lines comprising all the geographic races, two forms of K. lacca and one line of K. sharda. Grouping of K. sharda within the same cluster with K. lacca insects suggest its genetic closeness to K. lacca. A small sub-cluster of four insect lines in branch IAii, included two geographic races collected from Gujarat and one from Madhya Pradesh; both are adjoining states of India. An inbred line of rangeeni lac insect (Klrinba) was also grouped in this cluster. The branch IAi consisting of a mixed group of thirteen lac insect lines including five geographic races, five cultivated lines from the principal lac growing regions, two colour variants and one line of K. sharda. The K. sharda although is trivoltine in its life cycle pattern (Mishra and Sushil, 2000) it is clustered together with the bivoltine lac insect lines in branch IAi. Similarly the kusmi form of K. lacca did not clearly form a separate cluster from the rangeeni lines. The above results indicate that there is gene flow between these two forms, despite reduced scope of interbreeding due to life cycle asynchrony and differences in host preference. Interestingly, the yellow bodied and wild-type crimson colour variants are separated from each other in the dendrogram indicating a distinct genetic difference between them. The yellow insect differs from crimson wild type in their body colour due to a simple recessive gene mutation (Chauhan, 1967). The lac insects are sessile in nature and thus have limited dispersal capacity and restricted gene mixing. Thus, genetic similarity could be expected in relation with the geographic distances between the lines under study. Close grouping of lines from geographic proximity has been found in three instances: Klrajpush and Klgujalci (IAi); Klrboky and Klupbhat (IAi) and Klgujsimz and Klmpgun (IAii). Similar kind of study revealed a strong effect of geographic distance in genetic differentiation of Indian eri silkmoths (Samia cynthia ricini) of North-east India, which also have limited mobility in juvenile and adult stages (Vijayan et al., 2006). The PCOORD analysis demonstrated that the twenty lac insect lines can be separated distinctly from each other (Fig. 4). The first three principal coordinates explained 18.2%, 12.5% and 11.4% of the total variation of 43.1% (Table 5). The K. chinensis lines from Meghalaya and Thailand emerged as most divergent from the other lac insect lines in agreement with the UPGMA dendrogram. Among the K. lacca lines, two geographic races from Gujarat (Klgujsimz, Klgujsima) and two inbred lines of rangeeni insect (Klrinba, Klrinbf) grouped distinctly. A yellow colour variant lac insect line Klgujalci, which is collected from Gujarat, containing only yellow colour variant, remained distinct, while the remaining thirteen lac insect lines remained under a major cluster. The present study demonstrates the potential use of ISSR markers in characterization of genetic diversity in lac insects and has also yielded a few line-specific markers. However, other DNA markers like SSRs would be efficient in developing molecular tags for specific traits of lac insects; such as body colour variations, life cycle and host specificity for lac insect breeding programme. Nonetheless, the results revealed very high (96.1%) genetic polymorphism and also helped us to explain the wide genetic variability present within the twenty lines of lac insects used in the study. This natural variability of the lac insects can be harnessed in breeding of lac insects for high quality resin production. The ISSR markers identified would serve complementary in identification of lines for conservation as well as characterization of commercially important lac insect populations. Acknowledgements Authors express their gratitude to the reviewers for their critical reviewing of the article whose comments have been helpful in significant improvement in the manuscript. The work was supported by the Department of Biotechnology, Govt. of India, under DBT grant no. BT/012/30/NBDB/2002-II. We also acknowledge the technical help rendered by Sri AK Sinha and Sri Bhupal Kumar. References Archak, S., Gaikwad, A.B., Gautam, D., Rao, E.V.V.B., Swamy, K.R.M., Karihaloo, J.L., 2003. 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