Journal of Genetics (2019) 98:15 © Indian Academy of Sciences https://doi.org/10.1007/s12041-019-1072-7

RESEARCH ARTICLE

Genetic diversity and phylogeny analysis of Antheraea assamensis Helfer (: ) based on mitochondrial DNA sequences

MOUSUMI SAIKIA1∗ , RAMESH NATH2 and DIPALI DEVI1

1Seri-Biotech Unit, Life Sciences Division, Institute of Advanced Study in Science and Technology, Paschim Boragaon, Guwahati 781 035, India 2Department of Zoology, Dhing College, Dhing, Nagaon 782 123, India *For correspondence. E-mail: [email protected].

Received 20 June 2018; revised 1 October 2018; accepted 25 October 2018; published online 26 February 2019

Abstract. Antheraea assamensis Helfer, popularly known as Muga silkworm, the golden silk producer of northeast India is economically important and unique among the Saturniid silkworms. In this study, the genetic diversity and phylogeny of semi- domesticated and wild morphs of Muga silkworm collected from different geographical locations of northeast India were investigated based on the sequences of five mitochondrial loci, i.e. 12S rRNA, 16S rRNA, CoxI, Cytb and CR. All the five mitochondrial loci showed a strong bias towards higher ‘A’ and ‘T’ contents. Transitional substitutions were found to be more than the transversional substitutions. The rate of nucleotide substitution and average genetic divergence were found to be highest in CR sequences and lowest in 12S rRNA gene sequences among the morphs of Muga silkworm. The morphs collected from same geographical area had identical 12S rRNA, 16S rRNA, CoxI and Cytb gene sequences. Moreover, the 12S rRNA and 16S rRNA gene sequences of some semi-domesticated and wild morphs collected from different geographical locations were also found to be similar. In the phylogenetic trees generated based on the mitochondrial loci, mixing of semi-domesticated and wild morphs was observed as they shared the same group. The information generated in this study will help in formulating strategies to conserve the natural biodiversity present among these unique silkworms in northeast India. In addition, this will be useful in identifying diverse morphs of Muga silkworm, which will help in effective breeding programmes to improve its productivity.

Keywords. genetic diversity; morph; Muga; phylogeny; silkworm.

Introduction (Boore 1999). It encodes 37 genes and an A+T-rich region of variable length known as the control region in . Morphological similarities and differences have been used Mitochondrial DNA sequences have been widely used for to group and classify organisms. However, discriminating studies on population and molecular systematics of insects finer differences among morphs, strains, races, biotypes, as it has a number of specific biological properties, which breeds and populations is usually difficult due to the make mtDNA an appropriate marker for molecular bio- influence of environment on morphological characters. In diversity. Firstly, mtDNA is highly variable because of its recent years, DNA-based molecular markers are increas- high mutation rate, which can generate some signal about ingly, employed in diverse areas of biology, including population history over short time period. Secondly, it is , ecology, phylogenetic studies, population genet- maternally inherited which means that the whole genome ics and population dynamics in both plant and behaves as a single, nonrecombining locus. These unique systems because of their abundant polymorphism and the properties allow the development of universal primers and fact that they are independent of environmental conditions easy recovery from small or degraded biological samples (Behura 2006). A number of nuclear as well as mitochon- due to its high copy number in most cells with a different drial DNA markers have profound uses (Morin et al. 2004). evolution rate in different regions of mtDNA. These struc- The animal mitochondrial DNA (mtDNA) is a circu- tural and evolutional characteristics of mtDNA sequences lar and double-stranded molecule of 14–20 kb in length make it a marker of choice for various studies.

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The northeastern region has been recognized as the In the present study, the genetic diversity and phylogeny centre of seribiodiversity in India. The Indian golden among different morphs of Muga silkworm collected from silkworm, Antheraea assamensis Helfer (Lepidoptera: Sat- various geographical locations of northeast India were urniidae) popularly known as Muga silkworm, is endemic estimated using mitochondrial 12S ribosomal RNA (12S to northeast India. Being polyphagous, this feeds rRNA), 16S ribosomal RNA (16S rRNA), cytochrome oxi- on 15 different host plant . Silk produced by Muga dase subunit I (CoxI), cytochrome b (Cytb) and control silkworm is golden yellow in colour, which makes it region (CR) sequences. This information will provide effi- very attractive. This luminous golden Muga silk has now cient and effective measures for germplasm conservation secured geographical indications (GI) status, the recog- and utilization of these silkworms. nition under the intellectual property rights that it has its origin in the Assam region of the northeast India. Unlike the other Antheraea species, this species has the Materials and methods lowest chromosome number (n = 15) and ZZ/ZO sex chromosome system (Deodikar et al. 1962). Muga silk- Samples worm is known for its production of quality silk with natural golden colour, lustre, durability and resistant to After a brief survey on habitat of Muga silkworm, the UV radiation (Chowdhury 2001). It has better mechani- four morphs were collected from various geographical cal properties than other commercial silks, which impart locations of northeast India and were used in this study a wide range of use as a textile material (Devi et al. 2011). (table 1). The morphs were green (GM), blue (BM), orange The silk produced by Muga silkworm is touted as the sec- (OM) and wild (WM). They were coded as GM01, GM02, ond most expensive silk in the world after A. yamamai, GM03, GM04, GM05, GM06, BM01, BM02, OM01, and its cultivation contributes significantly to livelihoods WM01, WM02 and WM03 according to their geograph- of indigent peoples. Apart from the unique nature of ical origin (table 1). Twenty individuals per morph per the silk produced by the insect, its general morphology, locality were used for each experiment. As these silkworms behaviour and physiology are known to be different from are cultured for economic purposes throughout northeast those of other related species. Although Muga silkworm India, no specific permissions were required for the collec- is a monotypic species, it has four morphs, namely green, tions and experiments. In this study, national park or other blue and orange as semi-domesticated morphs and a wild protected area of land or sea, endangered and protected morph (Thangavellu et al. 1988). The first three morphs are species were not used. termed as semi-domesticated because during the life cycle of these morphs the larval period is completed in the trees Phenotypic characters under natural conditions and when the morphs are mature, they crawl down to the base of the tree. Further, they are The phenotypic characters like larval colour, cocoon picked up and are placed inside a room where they spin to colour, larval weight, cocoon weight, shell weight, shell form cocoons. They are kept there until the egg preparation ratio and voltinism of each sample were studied and val- stage. At the time of hatching, they are brought back to ues were expressed as mean ± standard deviation (SD), the tree again. In contrast, the whole life cycle is completed where n = 20. on the trees in case of the wild morph. Due to various reasons like overexploitation, deforestation, environmen- tal pollution, diseases etc., the population density of these Genomic DNA isolation morphs is reported to be showing a deteriorating trend. This depletion in population density results in low genetic Genomic DNA was extracted from the larval tissue of variation of this species (Goel and Krishna Rao 2004). each sample of Muga silkworm according to the stan- To develop a sustainable conservation programme, assess- dard procedure (Suzuki et al. 1972). Briefly, 1 g of larval ment of genetic variation and phylogenetic relationship tissue was ground in liquid nitrogen to a fine powder between and within species are a necessary prerequisite. and 10 mL of extraction buffer was added. The mix- Again, prior knowledge on genetic diversity among dif- ture was incubated at 37◦C for 2 h with occasional ferent populations of a single species can also be utilized swirling. The extraction buffer contained 0.1 M Tris- in breeding programmes for improving the productivity. HCl (pH 8), 0.25 M EDTA (pH 8), 0.01 M NaCl, Many studies have been carried out to assess the genetic 0.5% SDS and 100 µg/mL proteinase K. The DNA was diversity of silkworms (Kar et al. 2005; Velu et al. 2008; extracted twice with phenol:chloroform:isoamyl alcohol Chakraborty et al. 2015). Probably due to a narrow and (24:24:1) and once with chloroform. The supernatant endemic distribution of Muga silkworm, information on DNA was precipitated with 0.1 volume of 3-M sodium the genetic variation and phylogenetic relationships within acetate and two volumes of ice-cold absolute ethanol. Fol- this species is very scanty as compared with other insect lowing precipitation, the DNA was washed twice with species. 70% ethanol and dissolved in TE buffer. The RNAse Genetic diversity and phylogeny analysis of A. assamensis Page 3 of 12 15 = 20). n 1.88 Multivoltine 1.21 Multivoltine 0.97 Multivoltine 0.92 Multivoltine 3.70 Multivoltine 1.05 Multivoltine 1.10 Multivoltine 1.30 Multivoltine 1.76 Multivoltine 3.15 Univoltine/Bivoltine 2.12 Univoltine/Bivoltine 2.09 Univoltine/Bivoltine ± ± ± ± ± ± ± ± ± ± ± ± 0.12 9.30 0.06 11.70 0.16 12.15 0.09 11.78 0.09 9.51 0.10 10.37 0.08 9.09 0.15 8.04 0.09 9.66 0.060.19 14.50 8.98 0.29 8.55 ± ± ± ± ± ± ± ± ± ± ± ± SD, where number of samples are 20 ( ± 0.77 0.39 0.950.89 0.53 0.72 0.48 0.45 0.560.64 0.35 0.51 1.111.05 0.48 0.41 1.61 0.72 0.561.09 0.22 0.37 0.61 0.58 ± ± ± ± ± ± ± ± ± ± ± ± 4.19 4.62 3.95 3.82 3.68 4.92 5.36 5.10 7.12 1.59 6.82 6.78 golden brown golden brown golden brown golden brown golden brown golden brown golden brown golden brown golden yellow brown golden yellow golden yellow colour Cocoon weight (g) Shell weight (g) Shell ratio (%) Voltinism Cocoon 0.11 Light 0.19 Bright 0.13 Bright 0.15 Bright 0.21 Bright 0.11 Bright 0.25 Bright 0.13 Whitish 0.18 Whitish 0.120.23 Yellowish Light 0.22 Light ± ± ± ± ± ± ± ± ± ± ± ± 9.33 9.20 9.40 9.55 9.18 9.61 8.80 8.52 green green green green green green bluish bluish colour Larval weight (g) Larval GM04 Deep GM01 Deep GM02 Deep GM03 Deep GM05 Deep GM06 Deep BM01 Faint BM02 Faint OM01WM01 Orange GreenWM02 7.80 Green 9.90 WM03 Green 9.82 10.65 N N E N E N E E N N E N E N E E N E N E N E N E                         09 41 43 03 44 50 17 32 16 51 15 90 43 03 44 50 25 59 15 38 30 16 18 03 Co- ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ 26 92 92 91 92 90 93 94 92 90 91 90 93 26 26 26 26 26 26 27 26 26 25 25 ordinates Sample code Assam Assam Assam Assam Assam Assam Assam Lakhim- pur, Assam Assam Assam Megha- laya Assam Places of collection and phenotypic characters of the morphs of Muga silkworm. Table 1. Morph Location Green Khanapara, The values for larval weight (g), cocoon weight (g), shell weight (g) and shell ratio (%) are expressed as mean Green Mangaldoi, Green Udalguri, Green Goalpara, Blue Mangaldoi, Wild Hahim, Green Titabor, Green North Blue Udalguri, Orange Howley, Wild Tura, Wild Haflong, 15 Page 4 of 12 Mousumi Saikia et al. treatment was carried out by adding RNAse A (100 GenomeLab GeXp Genetic Analysis System (Beckman µg/mL) and incubated at 37◦C for 1 h. Further, DNA Coulter, USA). Sequence primers were the same as used was purified with phenol–chloroform–isoamyl alcohol, in PCR reaction. chloroform and ethanol precipitation as described earlier. Sequencing reactions were carried out in 20 µL vol- The extracted DNA was then checked on 0.8% agarose umes containing 2 µL of 3.2 pM primer, 8 µLofDye gel. The quantification of DNA was done by using Nan- Terminator Cycle Sequencing Quick Start master mix odropR (Thermoscientific). All the chemicals used in and 0.5–10 µL of 30–50 ng DNA template. The volume the study were of molecular grade specially procured was made up to 20 µL with distilled water. The cycling from Sigma Chemicals Company and Merck Millipore, conditions were as follows: 25 cycles of 96◦Cfor20s, India. 50◦C for 20 s, 60◦C for 4 min. It was done in a thermal cycler (Applied Biosystems, Germany). Freshly prepared stop solution/glycogen mixture was added to each reac- Amplification and sequencing of mitochondrial loci tion. The stop solution/glycogen mixture was prepared as follows (per sequencing reaction): 2 µL of 3 M sodium The genomic DNA isolated from each sample was used acetate (pH 5.2), 2 µL of 100 mM Na2-EDTA (pH 8) for amplification of mitochondrial loci. The mitochondrial and 1 µL of 20 mg/mL of glycogen (provided in the kit). loci were 12S rRNA, 16S rRNA, CoxI, Cytb and CR.The The sequencing reaction was mixed thoroughly and 60 primers for 12S rRNA (F: TACTATGTTACGACTTAT µL cold 95% (v/v) ethanol/dH2O from the freezer was and R: AAACTAGGATTAGATACCC) (Simon et al. added. The mixture was vortexed and centrifuged for 15 1994), 16S rRNA (F: GTGCAAAGGTAGCATAATCA min at 14,000 rpm at 4◦C. The flow through was discarded and R: TGTCCTGATCCAACATCGAG) (Hwang et al. and 200 µL of 70% (v/v) ethanol/dH2O from the freezer 1999a), CoxI (F: TGATCAAATTTATAATAC and R: was added to the precipitate. It was then centrifuged for GTAAAATTAAAATATAAC) (Hwang et al. 1999b), 2 min at 14,000 rpm at 4◦C. After centrifugation, the Cytb (F: TATGTACTACCATGAGGACAAATATC and supernatant was removed carefully with a micropipette. R: ATTACACCTCCTAATTTATTAGGAAT) (Sheppard Then the precipitate was vacuum dried for 10 min. The et al. 1994)andCR (F: GCAACTGCTGGCACAAAAT sample was resuspended in 40 µL sample loading solu- and R: TGAGGTATGAGCCCAAAAGC) (Arunkumar tion provided in the kit. The resuspended samples were et al. 2006) were synthesized by Genei (Bengaluru, India). transferred to the appropriate wells of the sample plate. Amplification was carried out in a thermal cycler (Applied Each of the resuspended samples was overlaid with one Biosystems, Germany) with 25 µL reaction mixture. The drop of light mineral oil. The samples were then loaded reaction mixture contained 2.5 µLbuffer(10× Taq DNA into the instrument. Sequences of both directions (for- polymerase buffer containing 15 mM MgCl2), 2.5 mM ward and reverse) were obtained from all samples of Muga dNTPs (from 10-mM stock), 10 mM each primer, 1 unit silkworm. of Taq DNA polymerase and 30 ng of genomic DNA. The PCR reaction conditions for 12S rRNA were as fol- ◦ ◦ lows: 1 cycle of 94 C for 5 min, 40 cycles of 94 C for 30 s Analysis of mitochondrial DNA sequences (denaturation), 50◦C for 1 min (annealing), 72◦C for 1 min (elongation), followed by 72◦C for 5 min. The PCR profiles The sequences of all mitochondrial loci were assem- for 16S rRNA, CoxI, Cytb and CR were identical to those bled using Contig Express software and aligned using for 12S rRNA except that the annealing temperatures were ClustalX 1.83 (Thompson et al. 1997). The sequences of 55◦Cfor16S rRNA,52◦CforCoxI and Cytb,and60◦C all samples were subjected to (basic local alignment search for CR. The amplified PCR products were resolved in a tool (BLAST) to perform sequence similarity searches. 1.5% agarose gel and documented in Biodoc-lt imaging After alignment, invalid end sequences were trimmed. All system (UVP, UK). PCR products were purified with the sequences obtained in this study are deposited in Gen- Geneipure Quick PCR Purification kit (Merck Millipore, Bank. India). Electrophoretic analysis of PCR products based on Nucleotide frequencies, nucleotide pair frequencies, i.e. partially amplified 12S rRNA, 16S rRNA, CoxI, Cytb and the rate that one nucleotide was substituted by another CR resulted in a single band of 430, 383, 597, 530 and 680 nucleotide and the transition/transversion ratios, i.e. the bp, respectively, in length. The PCR products were purified number of transitions to the number of transversions for using the Geneipure Quick PCR Purification kit (Merck a pair of sequences were calculated by using MEGA6 Millipore, India). software (Tamura et al. 2013). The overall transition/ The purified PCR products of each loci of each sample transversion bias (R) was calculated for each dataset. were sequenced in both directions (forward and reverse). All positions containing gaps and missing data were The sequencing was done by using GenomeLab Dye eliminated from the dataset (complete-deletion option). Terminator Cycle Sequencing Quick Start kit (Beckman Insertion–deletion polymorphism (InDels) was estimated Coulter, USA). Sequencing was carried out on using DnaSP5 software (Librado and Rozas 2009). The Genetic diversity and phylogeny analysis of A. assamensis Page 5 of 12 15 same software was also used to detect conserved, variable, the sequences of GM05 and GM06 were identical. The parsimony informative and singleton sites. genetic distances among the morphs ranged from 0.003 to The genetic distances, i.e. the number of nucleotide 0.013 (table 2). The average number of pairwise nucleotide substitutions per site among sequences were calculated differences (k) was 1.77 and the nucleotide diversity i.e. the based on the maximum composite likelihood method average number of nucleotide differences per site between using MEGA6 software. To estimate the genetic diver- two sequences (Pi) was 0.003. sity among the morphs of Muga silkworms, the average The average size of the 16S rRNA gene fragment number of pairwise nucleotide differences among DNA analysed was 350 bp. The sequences were submitted to sequences (k) and nucleotide diversity (Pi), the average GenBank and the accession numbers are KU366545, numbers of nucleotide differences per site between two KU366546–KU366552 and KJ939435–KJ939438. Of the sequences were calculated. All these analyses were per- total 350 bp, six (1.71%) were polymorphic. All positions formed by using DnaSP software. containing gaps and missing data were eliminated from the dataset (complete-deletion option). From these six vari- able sites, there were four parsimony informative sites and Phylogenetic analysis two singleton variable sites. The nucleotide base compo- sitions varied among the samples, which showed higher The phylogenetic trees based on mitochondrial loci were ‘A’ and ‘T’ contents. The average A, T, C and G fre- constructed by using neighbour-joining (NJ) method with quencies were 41.8, 38.4, 6.5 and 13.4%, respectively. The Kimura 2 parameter model using MEGA6 software. Boot- overall transition/transversion ratio was R = 3.40. The strap analysis (1000 bootstrap replications) was employed 16S rRNA gene sequences of GM01, GM02, GM03, to test the reliability of the topologies of phylogenetic trees. BM01, BM02 and OM01 were found to be identical. In this study, the sequences of mori were retrieved No variation occurred among them. Further, GM04 and from GenBank (accession number: AB070264) and taken WM01 shared the same sequences. The distance matrix as an out group in the phylogenetic trees. obtained from the analysis of alignment of sequences revealed a range of genetic distance from 0.003 to 0.014 Results (table 3). The average number of pairwise nucleotide dif- ferences (k) was 2.02 and the nucleotide diversity, i.e. the Phenotypic variation average number of nucleotide differences per site between two sequences (Pi) was 0.006. The morphs of Muga silkworm showed variations in their The average size of CoxI gene analysed was 558 bp. phenotypic characters like larval colour, cocoon colour, The sequences were submitted to GenBank and the acces- larval weight, cocoon weight, shell weight, shell ratio and sion numbers are KU366553–KU366561 and KJ939439– voltinism (table 1;figure1). KJ939441. The sequences showed no InDels, the varia- tions among the sequences were due to nucleotide substi- tutions. Of the 558 bp, 539 sites were conserved and 19 Genetic diversity analysis (3.41%) were polymorphic sites. Of these 19 variable sites, there were 10 singleton sites and nine parsimony informa- The genetic diversity among the morphs of Muga silk- tive sites. The nucleotide base compositions showed higher worm were examined by using the sequences of the five ‘A’and ‘T’ contents. The average A, T, C and G frequencies mitochondrial loci, i.e. 12S rRNA, 16S rRNA, CoxI, Cytb were 28.8, 40.4, 17.0 and 13.8%, respectively. The overall and CR. A 383 bp of 12S rRNA gene sequences for all sam- transition/transversion ratio was R = 1.73. After the anal- ples of Muga silkworm was analysed. The sequences were ysis, it was found that the sequences of green and blue submitted to the GenBank and the accession numbers are morphs collected from Mangaldoi (GM02 and BM01) KU366533–KU366543 and KM205035. The 12S rRNA were identical. Similarly, the sequences of green and blue gene sequences showed no InDels, the variation among morphs collected from Udalguri (GM03 and BM02) were the sequences were due to nucleotide substitutions. Of the also found to be identical. The distance matrix obtained total 383 bp, 378 were conserved sites and five (1.31%) from the analysis of alignment of all sequences revealed a were variable. Of these five variable sites, four were parsi- range of genetic distance from 0.002 to 0.018 (table 4). The mony informative sites and one was singleton variable site. lowest genetic distance was found between green morph This mitochondrial gene showed a bias towards high ‘A’ collected from Mangaldoi, i.e. GM02 and green morph and ‘T’ contents. The average frequencies of A, T, C and collected from Udalguri, i.e. GM03 (0.002). Here, the high- G were 45.3, 39.5, 5.3 and 9.9%, respectively. The over- est genetic distance was observed between green morph all transition/transversion ratio was R = 0.103. The 12S collected from north Lakhimpur, i.e. GM06 and the wild rRNA gene sequences of GM01, GM02, GM03, BM01, morph collected from Haflong, i.e. WM03 (0.018). The BM02 and OM01 were found to be identical. Further, average number of pairwise nucleotide differences (k) was GM04 and WM01 shared the same sequences. Similarly, 5.23 and the nucleotide diversity, i.e. the average number 15 Page 6 of 12 Mousumi Saikia et al.

Figure 1. Larvae of different morphs of Muga silkworm (a) green, (b) blue, (c) orange and (d) wild.

Table 2. Pairwise genetic distances of the morphs of Muga silkworm based on 12S rRNA gene sequences.

GM01 GM02 GM03 GM04 GM05 GM06 BM01 BM02 OM01 WM01 WM02 WM03

GM01 0.000 GM02 0.000 0.000 GM03 0.000 0.000 0.000 GM04 0.003 0.003 0.003 0.000 GM05 0.005 0.005 0.005 0.008 0.000 GM06 0.005 0.005 0.005 0.008 0.000 0.000 BM01 0.000 0.000 0.000 0.003 0.005 0.005 0.000 BM02 0.000 0.000 0.000 0.003 0.005 0.005 0.000 0.000 OM01 0.000 0.000 0.000 0.003 0.005 0.005 0.000 0.000 0.000 WM01 0.003 0.003 0.003 0.000 0.008 0.008 0.003 0.003 0.003 0.000 WM02 0.010 0.010 0.010 0.008 0.005 0.005 0.010 0.010 0.010 0.008 0.000 WM03 0.008 0.008 0.008 0.005 0.013 0.013 0.008 0.008 0.008 0.005 0.008 0.000

of nucleotide differences per site between two sequences 21 variable sites, 10 were singleton and 11 were parsimony (Pi) was 0.010. informative sites. The average frequencies of A, T, C and A 475 bp of Cytb gene sequences for all samples of Muga G were 31.5, 42.9, 15.7 and 9.9%, respectively. The over- silkworm was analysed. The sequences were submitted all transition/transversion ratio was R = 5.91. Like CoxI to GenBank. The accession numbers are KU366570– gene sequences, the Cytb gene sequences of green and blue KU366578 and KM205036–KM205038. The obtained morphs collected from Mangaldoi (GM02 and BM01) partial Cytb gene sequences showed no InDels, the varia- shared same sequences. Similarly, the sequences of green tion among the sequences due to nucleotide substitutions. and blue morphs collected from Udalguri (GM03 and After the analysis of the gene sequences, 454 conserved BM02) were identical. The genetic distances ranged from sites and 21 polymorphic sites (4.42%) were found. Of the 0.002 to 0.030 (table 5). The genetic distance was lowest Genetic diversity and phylogeny analysis of A. assamensis Page 7 of 12 15

Table 3. Pairwise genetic distances of the morphs of Muga silkworm based on 16S rRNA gene sequences.

GM01 GM02 GM03 GM04 GM05 GM06 BM01 BM02 OM01 WM01 WM02 WM03

GM01 0.000 GM02 0.000 0.000 GM03 0.000 0.000 0.000 GM04 0.003 0.003 0.003 0.000 GM05 0.006 0.006 0.006 0.009 0.000 GM06 0.009 0.009 0.009 0.011 0.003 0.000 BM01 0.000 0.000 0.000 0.003 0.006 0.009 0.000 BM02 0.000 0.000 0.000 0.003 0.006 0.009 0.000 0.000 OM01 0.000 0.000 0.000 0.003 0.006 0.009 0.000 0.000 0.000 WM01 0.003 0.003 0.003 0.000 0.009 0.011 0.003 0.003 0.003 0.000 WM02 0.011 0.011 0.011 0.009 0.006 0.009 0.011 0.011 0.011 0.009 0.000 WM03 0.011 0.011 0.011 0.009 0.011 0.014 0.011 0.011 0.011 0.009 0.006 0.000

Table 4. Pairwise genetic distances of the morphs of Muga silkworm based on CoxI gene sequences.

GM01 GM02 GM03 GM04 GM05 GM06 BM01 BM02 OM01 WM01 WM02 WM03

GM01 0.000 GM02 0.007 0.000 GM03 0.009 0.002 0.000 GM04 0.014 0.007 0.009 0.000 GM05 0.011 0.004 0.005 0.011 0.000 GM06 0.013 0.005 0.007 0.013 0.009 0.000 BM01 0.007 0.000 0.002 0.007 0.004 0.005 0.000 BM02 0.009 0.002 0.000 0.009 0.005 0.007 0.002 0.000 OM01 0.014 0.011 0.013 0.004 0.014 0.016 0.011 0.013 0.000 WM01 0.014 0.007 0.009 0.004 0.011 0.013 0.007 0.009 0.007 0.000 WM02 0.016 0.009 0.011 0.005 0.013 0.014 0.009 0.011 0.009 0.005 0.000 WM03 0.013 0.013 0.014 0.014 0.016 0.018 0.013 0.014 0.014 0.014 0.013 0.000

between green morph collected from Mangaldoi (GM02) revealed a range of genetic distance from 0.003 to 0.042 and green morph collected from Udalguri (GM03). The among the morphs (table 6). Minimum divergence was highest genetic distance value was observed between green found between green morph (GM02) and blue morph morph collected from north Lakhimpur (GM06) and wild (BM01) collected from Mangaldoi (0.003) and maximum morph collected from Haflong (WM03). The average num- was between GM06 and WM03 (0.042). The average num- ber of pairwise nucleotide differences (k), i.e. the number ber of pairwise nucleotide differences (k) i.e. the number of base differences per sequence from averaging over all of base differences per sequence from averaging over all sequence pairs was 5.44 and the nucleotide diversity (Pi), sequence pairs was 6.45 and the nucleotide diversity (Pi), i.e. the average number of nucleotide differences per site i.e. the average number of nucleotide differences per site between two sequences was 0.012. between two sequences was 0.019. The average size of CR fragment analysed was 334 bp. The sequences were submitted to GenBank and the acces- sion numbers are KU366562–KU366569 and KM205040– Phylogenetic analysis KM205043. The obtained partial gene sequences showed no InDels, the variation among the sequences was due to The phylogenetic relationships among the morphs of nucleotide substitutions. Of the total 334 bp, 22 (6.59%) Muga silkworm collected from different locations were were polymorphic sites. From these polymorphic sites, studied based on the sequences of five mitochondrial loci. there were 12 singleton and 10 parsimony informative The sequences of B. mori were taken as out group. The sites. The nucleotide base compositions varied among the topology of the tree was tested by bootstrapping. The boot- morphs, which showed a strong bias towards higher ‘A’and strap values are shown at the nodes of the trees. The trees ‘T’ contents. The average frequencies of A, T,C and G were were supported by high bootstrap values. 40.3, 50.1, 7.1 and 2.5%, respectively. The overall transi- The phylogenetic relationships among the morphs of tion/transversion ratio was R = 0.39. The distance matrix Muga silkworm collected from different locations of obtained from the analysis of alignment of all sequences northeast India were studied based on the mitochondrial 15 Page 8 of 12 Mousumi Saikia et al.

Table 5. Pairwise genetic distances of the morphs of Muga silkworm based on Cytb gene sequences.

GM01 GM02 GM03 GM04 GM05 GM06 BM01 BM02 OM01 WM01 WM02 WM03

GM01 0.000 GM02 0.004 0.000 GM03 0.006 0.002 0.000 GM04 0.008 0.004 0.006 0.000 GM05 0.008 0.004 0.006 0.008 0.000 GM06 0.011 0.006 0.008 0.011 0.006 0.000 BM01 0.004 0.000 0.002 0.004 0.004 0.006 0.000 BM02 0.006 0.002 0.000 0.006 0.006 0.008 0.002 0.000 OM01 0.004 0.008 0.006 0.008 0.013 0.015 0.008 0.006 0.000 WM01 0.008 0.004 0.006 0.004 0.008 0.011 0.004 0.006 0.008 0.000 WM02 0.023 0.019 0.021 0.019 0.023 0.025 0.019 0.021 0.023 0.015 0.000 WM03 0.027 0.023 0.025 0.023 0.027 0.030 0.023 0.025 0.027 0.019 0.013 0.000

Table 6. Pairwise genetic distances of the morphs of Muga silkworm based on CR sequences.

GM01 GM02 GM03 GM04 GM05 GM06 BM01 BM02 OM01 WM01 WM02 WM03

GM01 0.000 GM02 0.006 0.000 GM03 0.009 0.009 0.000 GM04 0.012 0.006 0.015 0.000 GM05 0.009 0.009 0.006 0.015 0.000 GM06 0.030 0.024 0.027 0.030 0.021 0.000 BM01 0.009 0.003 0.006 0.009 0.006 0.021 0.000 BM02 0.009 0.009 0.006 0.009 0.006 0.027 0.006 0.000 OM01 0.009 0.009 0.012 0.009 0.006 0.027 0.012 0.012 0.000 WM01 0.027 0.021 0.030 0.015 0.024 0.033 0.024 0.024 0.018 0.000 WM02 0.033 0.027 0.036 0.021 0.030 0.039 0.030 0.030 0.024 0.018 0.000 WM03 0.030 0.030 0.039 0.024 0.033 0.042 0.033 0.033 0.027 0.021 0.015 0.000

12S rRNA gene sequences. As the sequences of GM01, The phylogenetic relationships among the morphs of GM02, GM03, BM01, BM02 and OM01 were identical, Muga silkworm was also investigated by using CoxI gene only the sequence of GM01 was taken in the phylogenetic sequences. The CoxI gene sequences of GM02 and BM01 analysis. Similarly, the sequence of GM04 was considered were the same. Therefore, only GM02 was taken for phy- as WM01 shared the same sequence with GM04. Further, logenetic analysis. Likewise, the sequences of GM03 and the sequences of GM05 and GM06 were identical. Thus, BM02 were found to be identical for which GM03 was only the sequence of GM05 was used in the construction used for the construction of the phylogenetic tree. The of the phylogenetic tree. The phylogenetic tree consisted of phylogenetic tree generated based on CoxI gene sequences three groups (figure 2a). The first group included GM01, formed of two major groups, – I and II (figure 2c). Group GM04 and GM05. The wild morphs collected from Tura I consisted of GM01, GM02, GM03, GM05, GM06 and (WM02) and Haflong (WM03) came out to form the sec- OM01 and group II included GM04, WM01, WM02 and ond and third groups, respectively. WM03. The phylogenetic relationships among the morphs of A phylogenetic tree was constructed based on Cytb Muga silkworm were studied based on 16S rRNA gene gene sequences to study the phylogenetic relationships sequences. The sequences of GM01, GM02, GM03, BM01, among the morphs of Muga silkworm collected from var- BM02 and OM01 were identical. Therefore, only GM01 ious geographical locations of northeast India (figure 2d). was taken in the phylogenetic analysis. Similarly, GM04 The Cytb gene sequences of GM02 and BM01 were the was considered for the analysis as the sequences of GM04 same. Therefore, only GM02 was considered for phyloge- and WM01 were the same. The phylogenetic tree consisted netic analysis. Likewise, the sequences GM03 and BM02 of three groups: groups – I, II and III (figure 2b). Group I were found to be identical for which GM03 was used comprised of GM01, GM04, GM05 and GM06. The wild for the study. The tree consisted of two groups. The first morphs WM02 and WM03 branched off independently to group comprised of the semi-domesticated morphs GM01, form groups II and III, respectively. GM02, GM03, GM04, GM05, GM06, OM01 and WM01. Genetic diversity and phylogeny analysis of A. assamensis Page 9 of 12 15

Figure 2. Phylogenetic trees constructed based on (a) 12S rRNA,(b)16S rRNA,(c)CoxI,(d)Cytb and (e) CR sequences. The sequences of B. mori were taken as out group.

The second group contained the wild morphs WM02 and (WM03) combined together to form the second WM03. group. The phylogenetic relationships among the morphs of Muga silkworm collected from different locations were studied based on the mitochondrial CR sequences. The Discussion phylogenetic tree consisted of two groups (figure 2e). The first group comprised of all the semi-domesticated In this study, the genetic diversity was analysed among morphs, i.e. GM01, GM02, GM03, GM04, GM05, GM06, the morphs of Muga silkworm by using the sequences of BM01, BM02, OM01 and the wild morph WM01. The five mitochondrial loci, two ribosomal RNA genes, 12S wild morphs collected from Tura (WM02) and Haflong rRNA and 16S rRNA, two protein coding genes, CoxI 15 Page 10 of 12 Mousumi Saikia et al. and Cytb, and the noncoding region, CR. Additionally, analysis, 13 samples of C. japonica showed low genetic these sequences were used to determine the phylogenetic diversity, although a higher genetic diversity of these sam- relationships among them. These mitochondrial loci were ples had been revealed by RAPD and ISSR markers (Cao selected as they were proved to be good enough for genetic et al. 2007; Yang et al. 2008). These results were consistent variation and phylogenetic study (Arif and Khan 2009). with the findings in A. pernyi (Liu et al. 2006; Zhu et al. All the five mitochondrial loci showed a strong bias 2008). This discrepancy between studies might be due to towards a higher AT content, as it was in the case of the difference in the mode of inheritance. A pair of diploid other insects (Li et al. 2005; Arunkumar et al. 2006). can inherit maximum of four different types of However, results in this study showed that AT bias was nuclear gene, but they can only inherit one mitochon- relatively low in CoxI and Cytb genes as compared to the drial DNA type to the subsequent generation. Therefore, other loci. Such results were also reported in the case of nuclear DNA would show less effect in the general back- Drosophila (Nagaraja et al. 2004). It was reported that the ground of the subsequent genome during the course of mtDNA of insects exhibited a significantly larger propor- breeding compared to mitochondrial DNA. Although, tion of A and T nucleotides as compared with C and G further study may provide accurate reason for the low (Simon et al. 1994; Whitfield and Cameron 1998). Our genetic diversity in terms of mitochondrial DNA, another findings confirmed the earlier reports. Detailed analyses possible explanation appears to be related to the processes of the distribution patterns of A/T nucleotides in coding of breeding for better morphs. During the course of breed- and noncoding regions of the function of codon positions ing, cross between the morphs over generations may result and of secondary structures had led several authors to sug- in a few individual with desired characters but most with gest the existence of directional mutation pressure towards unwanted. These unwanted silkworms will be discarded. high A and T contents in insect mtDNA (Clary and Wol- Although the nuclear DNA loci, which encode the wanted stenholme 1985; Crozier and Crozier 1993). A hypothesis phenotypes, are selected, mitochondrial DNA genotypes for this AT richness is that strand-specific compositional would show the equivalent results of population bottle- bias (a predominance of G↔A transitions, perhaps the neck. Therefore, theoretically, the selected morphs may result of asymmetries in stem base pairing capabilities) has possess and will be fixed with the mitochondrial DNA led to an increase in ‘A’ content followed by an increase in genotype that was most frequent originally over the gener- T content (Dowton and Austin 1997). The base compo- ations. An extensive cross for breeding among the different sition bias is obviously reflected in the substitution bias morphs and the resultant discard of unwanted individu- toward A’s and T’s. The high AT bias could explain the als may further have eliminated the least mitochondrial strikingly low transition/transversion ratio (Whitfield and DNA diversity possessed by the morphs. The observation Cameron 1998). The transition bias was accompanied by that the process of breeding may have caused mitochon- AT richness in the mitochondrial genes of these silkworms, drial DNA homogeneity was also found in domesticated which causes an apparently lower transition bias ratio as strains of B. mori (Kim et al. 2000). Li et al. (2005) reported contended in Drosophila (Tamura 1992). Strong A/G and identical Cytb sequences of seven strains of B. mori from T/C transition biases in all mitochondrial loci suggested China. that the transitional substitutions occur more readily than After the analysis of mitochondrial loci, it was observed transversional substitutions (Brown et al. 1979). A much that the morphs collected from same geographical area higher rate of transitions was reported in closely related had identical 12S rRNA, 16S rRNA, CoxI and Cytb gene Hawaiian Drosophila (DeSalle et al. 1987). sequences. Moreover, the 12S rRNA and 16S rRNA gene The number of polymorphic sites varied among the sequences of some semi-domesticated and wild morphs mitochondrial loci, which suggested different mutation collected from different geographical locations were found rates in various loci. The 12S rRNA gene showed lowest to be identical indicating that the ribosomal genes were polymorphism, while CR showed highest polymorphism more conserved that the other mitochondrial loci. In among the other mitochondrial loci studied. This study the phylogenetic trees generated based on the mitochon- revealed a low genetic diversity among the morphs. On the drial loci, mixing of semi-domesticated and wild morphs contrary, several studies on nuclear markers like RAPD, was observed. The reason of the mixing between the SSR and ISSR showed comparatively high genetic diver- semi-domesticated and the wild morphs was unclear. The sity. Neog et al. (2010) found 94.82% polymorphism observed mixing may be due to migration or anthro- among 11 populations of Muga silkworm by using 36 pogenic introduction of wild parents to invigorate semi- RAPD primers. Similarly, Singh et al. (2012)andArunku- domesticated morphs. Since Muga silkworm is a mono- mar et al. (2012) used SSR and ISSR markers to study typic species and it is, confined to the narrow geographical genetic diversity among different populations of Muga location, most often this silkworm is forced to inbreeding silkworm and found significantly high polymorphism. In depression. The genetic divergence and phylogenetic rela- case of other silkworm species, Li et al. (2009) studied the tionship among wild and domestic silkworms have been genetic diversity of japonica (Lepidoptera: Sat- studied by many researchers (Kim et al. 2000; Arunkumar urniidae) by using mitochondrial DNA sequences. In the et al. 2006). Generally, there was consensus on the aspect Genetic diversity and phylogeny analysis of A. assamensis Page 11 of 12 15 that domestic silkworms were evolved from wild silk- vigour (Nagaraju and Goldsmith 2002). Information worms (Yoshitake 1968). However, opinions differ on the on genetic diversity can reduce the risk by magnitude of genetic divergence according to the molecu- helping to develop appropriate population management lar markers used. The phylogenetic relationships between programmes that can minimize the risks implied through B. mori and B. mandarina species were investigated based inbreeding. on 16S rRNA and 12S rRNA genes (Hwang et al. 1999a). This study suggested that the mitochondrial DNA The results showed that B. mori mtDNA large and small sequences are suitable enough for resolving genetic vari- rRNA genes were highly homologous to those of B. man- ation and phylogenetic relationships among the morphs darina by about 98 and 99%, respectively. Kim et al. (2000) of Muga silkworm. The phenotypic and genetic informa- found genetic homogeneity in the domestic silkworm tion revealed in this study will help to conserve the natural B. mori and wild silkworm B. mandarina using mitochon- biodiversity present among the morphs of this unique silk- drial CoxI gene sequences. The sequence divergence and worm in northeast India. In addition, this may be useful in phylogenetic relationships based Cytb gene sequences of identifying diverse morphs of Muga silkworm, which will 14 strains of B. mori and two strains of B. mandarina help in effective breeding programmes. were analysed and found that the sequences of B. mori and B. mandarina clustered into the same group (Li et al. Acknowledgement 2005). 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Corresponding editor: H. A. Ranganath