Strains of Philosamia ricini Differ in their Mitochondrial 16S Ribosomal RNA and Cytochrome Oxidase Subunit I Gene Sequences

Abstract

Philosamia ricini, the Indian eri silkworm, contributes significantly to the production of commercial silk and is widely distributed in the Brahmaputra valley of the North-Eastern India. Strains of Philosamia ricini show wide variation in their phenotypic traits and are commercially exploited in these places, because of their high silk yield potential. In this study, the authors have analyzed the partial mitochondrial 16S ribosomal RNA (16S rRNA) and partial cytochrome oxidase subunit I (CoxI) gene sequences to assess the genetic diversity among five different strains of Philosamia ricini. Sequence analysis of mitochondrial 16S rRNA and mitochondrial CoxI genes showed variation among the strains. Except the Red cocoon variety, the sequence of the fragment of 16S rRNA gene was identical among White plain, White zebra, Blue plain and Blue zebra strains. Similarly, the sequence of the fragment of the CoxI gene grouped the strains into two distinct groups, (a) White plain and Red cocoon variety and (b) White zebra, Blue plain and Blue zebra strains.

Headnote

Abstract: Philosamia ricini, the Indian eri silkworm, contributes significantly to the production of commercial silk and is widely distributed in the Brahmaputra valley of the North-Eastern India. Strains of P. ricini show wide variation in their phenotypic traits and are commercially exploited in these places because of their high silk yield potential. In this study, we have analyzed the partial mitochondrial 16S ribosomal RNA (16S rRNA) and partial cytochrome oxidase subunit I (CoxI) gene sequences to assess the genetic diversity among five different strains of P. ricini. Sequence analysis of mitochondrial 16S rRNA and mitochondrial CoxI genes showed variation among the strains. Except the Red cocoon variety, the sequence of the fragment of 16S rRNA gene was identical among White plain, White zebra, Blue plain and Blue zebra strains. Similarly, the sequence of the fragment of the CoxI gene grouped the strains into two distinct groups, (a) White plain and Red cocoon variety and (b) White zebra, Blue plain and Blue zebra strains. For the first time, the mitochondrial gene sequences of different strains of P. ricini have been studied.

Keywords: Eri silkworm, genetic diversity, sequence analysis, strain.

1. Introduction

The Indian eri silkworm, Philosamia ricini (Lepidoptera: Saturniidae), a commercial silk producing insect, is reported to have originated in the Brahmaputra valley of Assam and Meghalaya15. In their natural habitats in North-Eastern India, the silkworms are geographically isolated and have morphological differences4. Different natural geographic strains of P. ricini are commercially exploited in these places because of their high silk yield potential. P. ricini is a polyphagous insect and feeds on a wide range of host plants. The primary food plant of this insect is castor (Ricinus communis L.), but it also feeds on a wide range of food plants such as Heteropanax fragrans Seem, Manihot utilissima Phol, Evodia flaxinifolia Hook, Ailenthus gradulosa Roxb etc. This silkworm is multivoltine and completes five to six generations per year depending on the climatic condition of the region6. Strains of P. ricini show wide variation in their phenotypic traits such as fecundity, voltinism, cocoon weight, cocoon shape, cocoon color, larval weight, larval color, silk ratio and silk quality. Due to over exploitation of the silkworms for commercial uses coupled with deforestation, most of these natural populations are dwindling rapidly and its preservation has become an important goal. In order to preserve the natural biodiversity present among these populations, attempts are being made to understand the genetic structure of each population. Assessment of the genetic diversity present within a species is a prerequisite for developing a sustainable conservation program.

The advent of molecular biological techniques clearly showed the advantages of molecular markers over morphobiochemical markers to analyze population diversity. As the molecular markers are stable and environmentally independent, they are increasingly being preferred to phenotypic traits to detect genetic variation not only among populations but also between individuals within a population. A number of DNA marker systems such as simple sequence repeats (SSR) 21, random amplified polymorphic DNA (RAPD) 5,7, inter-simple sequence repeats (ISSR) 17, 19and amplified fragment length polymorphism (AFLP)18 have been used to study the population genetics of different organisms including insects. The ISSR marker system was used to assess genetic diversity and differentiation among different commercially exploited populations of Samia cynthia ricini from North-Eastern states of India 2,12.

The metazoan mitochondrial genome is a circular, double stranded DNA molecule, which is known to have small molecular weight, rapid nucleotide mutation and rare recombination, and can be easily handled in a laboratory compared to nuclear DNA. Because of their structural and evolutional characteristics, mitochondrial DNA (mtDNA) sequences have been widely used as molecular markers in the study of molecular evolution in the past several decades 1,10,11,14. The sequences of 16S ribosomal RNA (16S rRNA) and cytochrome oxidase subunit I (CoxI) genes has been widely used for phylogenetic studies and sequence differences in these genes reflect strain variation 13, 20.

The present study was focused on detecting DNA sequence variation among the five commercially exploited strains of P. ricini in the fragments of 16S ribosomal RNA (16S rRNA) and cytochrome oxidase subunit I (CoxI) genes.

2. Materials and Methods

P. Ricini Strains

Seeds of five morphologically distinct strains of P. ricini collected from different region of North-Eastern India were used for study. The strains were defined as: White plain (E1), White zebra (E2), Blue Plain (E3), Blue zebra (E4) and Red cocoon variety (E5) as shown in the figure 2.

Genomic DNA Isolation

Genomic DNA was isolated from fresh larvae of five different strains of P. ricini following the standard method 8. Briefly one gram of larval tissue was ground in liquid nitrogen to make it fine powder and 10 ml of extraction buffer was added. The mixture was incubated at 65o C for 2 h with occasional shaking. The extraction buffer contains 0.1 M Tris-HCL (pH 8), 0.25 M EDTA (pH 8), 0.01 M NaCl, 0.5% SDS and 100 µ g/ml Proteinase K. The mixture was then transferred to micro centrifuge tube and centrifuged at 10,000 rpm for 10 min. The supernatant was transferred to clean micro centrifuge tubes. To each tube same volume of Phenol: Chloroform: Isoamyl alcohol (25:24:1) was added and mixed by inverting the tubes. After mixing, the tubes were centrifuged at 10,000 rpm for 10 min. The upper aqueous phase was transferred to a clean micro centrifuge tube. To each tube 0.1 volume of 3M sodium acetate was added followed by two volumes of ice cold absolute ethanol. The tubes were inverted slowly several times to precipitate the DNA. The tubes were placed for 1 h at -20oC after the addition of ethanol to precipitate the DNA. Following precipitation, the DNA thread was washed twice with 70% ethanol and dissolved in TE buffer. The RNAse treatment was done by adding RNAse A (100 µ g/ml) and incubating at 37o for 1 h. DNA was purified with phenolchloroform-isoamyl alcohol and ethanol precipitation as described earlier.

The DNA was then visualized in a 0.8% agarose gel. Quantification of the DNA samples was done by using UV-Vis Spectrophotometer (Eppendorf, Germany).

PCR Amplification of 16S r RNA and CoxI Genes and Sequencing

A 383-bp region of 16S rRNA gene and a 597-bp region of CoxI gene were amplified by polymerase chain reaction (PCR). The primers used for amplification of the 383-bp fragment of 16S rRNA gene were forward 5'-GTGCAAAGGTAGCATAATCA-3' and reverse 5'TGTCCTGATCCAACATCGAG-3' 3. The primers for amplification of 597-bp fragment of CoxI were forward 5'-TGATCAAATTTATAATAC-3' and reverse 5'-GTAAAATTAAAATATAAC-3' 3. The PCR amplification was performed with 25 ml reaction mixture containing 2.5 ml buffer (10 × Taq DNA polymerase buffer), 2.5 mM dNTPs (from 10-mM stock), 25 pM of each primer, 1 unit of Taq DNA polymerase and 30 ng of genomic DNA. The PCR reaction conditions for 16S rRNA were as follows: 1 cycle of 94°C for 5 min, 40 cycles of 94°C for 30 sec (denaturation), 55°C for 1 min (annealing), 72°C for 1 min, followed by 72°C for 5 min. The PCR conditions for CoxI were identical to those for 16S rRNA except that the annealing temperature was 36oC. The amplified PCR products were visualized in a 1.5% agarose gel, in 1X TAE buffer (pH 8.0), stained with ethidium bromide (0.5µg/ml) and documented in a Biodoc-lt imaging system (UVP, UK). The PCR products were purified with the Geneipure Quick PCR Purification Kit.

Sequencing of the purified PCR products were performed at Merck Specialities Pvt. Ltd (Bangalore). Sequence primers were the same as used in PCR reaction. Sequence data were aligned using ClustalW - EBI online software.

3. Results and Discussion

Morphological Characteristics

The silkworm strains selected for the study varied considerably on the morphological traits (table 1). The color of the mature larvae was white in E1, E2 and E5, blue in E3 and E4 (figure. 2). Typical zebra markings were present on larvae of E2 and E4 whereas E1, E3 and E5 were plain without any markings (figure. 2).

The color of the cocoons recorded in this study was white in all the strains but red in E5 (figure. 2). Irrespective of sex the color of the moths was blackish grey (figure. 1a and 1b).

Moth color Blackish grey Blackish grey Blackish grey Blackish grey Blackish grey

Voltinism Multivoltine Multivoltine Multivoltine Multivoltine Multivoltine

Analysis of 16S rRNA Gene Sequences

The band of 383-bp of 16S rRNA was amplified under standardized PCR conditions (figure.3). PCR product of 383-bp fragment of 16S rRNA of all five strains was sequenced and compared with the sequence obtained from Genebank databases with accession number AY601280.1 (Samia cynthia ricini, mitochondrial 16S ribosomal RNA gene). This sequence was considered as master sequence. It showed very low genetic diversity among all the strains of P. ricini. The sequences of E1, E2, E3 and E4 strains were identical with the master sequence. There was only one variable site found in E5 strain. This variation was a deletion of 'T' occurred at position 48. The other parts of the sequences were similar. No transition or transversion occurred (figure. 5).

Analysis of CoxI Gene Sequences

The band of 595-bp fragment of CoxI was amplified under standardized PCR conditions (figure. 4). These sequences were compared with the sequence of Genebank databases with accession number KC195912.1 (Samia cynthia ricini, mitochondrial cytochrome c oxidase subunit I gene) which was taken as master sequence. It showed variation among the strains of P. ricini. The sequences of E1, E5 and the master sequence were identical but differ from that of E2, E3 and E4. Variation occurred at position 379 which was a transition (A/G). Sequences of the three strains i.e. E2, E3 and E4 were identical. No deletion or insertion occurred in the CoxI gene sequences of all the strains (figure. 6).

The lanes are M= DNA ladder of 100-bp, N= Negative control, E1= White plain, E2= White zebra, E3= Blue plain, E4= Blue zebra, E5= Red cocoon variety.

The lanes are M= DNA ladder of 100-bp, N= Negative control, E1= White plain, E2= White zebra, E3= Blue plain, E4= Blue zebra, E5= Red cocoon variety.

The sequences of five strains were aligned with master sequence. Only those nucleotides that differ from the master sequence at these positions are shown. Dotes (.) denote the identity with the master sequence.' -' indicates absence of any nucleotide.

The sequences of five strains were aligned with master sequence. Only those nucleotides that differ from the master sequence at these positions are shown. Dotes (.) denote the identity with the master sequence.

The geographically isolated strains of P. ricini have shown significant phenotypic variation however differences of 16S rRNA and CoxI gene sequences have been observed only in few cases. Multiple sequence alignment of 16S rRNA gene revealed low genetic divergence among five different strains of P. ricini (figure. 5). It showed only one base difference in E5 strain, whose cocoon color was red. The sequences of the four different strains i.e. E1, E2, E3, E4 were identical. The high genetic similarity among these strains is due to the continuous multiplication by inbreeding and/or genetic mixing though human intervention . The increased rate of inbreeding and genetic drift can be considered as p9 ossible causes of erosion in genetic diversity in these strains of P. ricini . The sequence analysis of the CoxI gene among the strains also revealed only one base difference (figure. 6). There was no variation found between the sequences of E1 and E5. The sequences of other three strains i.e. E2, E3 and E4 showed great homology. The high genetic similarity was observed between E1 and E5 and among E2, E3 and E4 strains of P. ricini as they were similar in phenotypic traits, such as the larval color was white in E1 and E5 strains and the cocoon color was white in E2, E3 and E4. It suggests their common origin and later succession into different strains by adapting to the varying climatic c12 onditions .

The genetic variation revealed among the strains points to the fact that the populations have already differentiated into separate genetic pools. Hence, these different gene pools should be conserved separately and maintained without any inter-mixing. The use of forest areas for farming and human dwellings has been the cause of forest fragmentation in North-Eastern India. This, besides contributing to genetic erosion, has negatively affected the survival of many flora and fauna, 16 including the P. ricini . Transportation of populations for multiplication and unscientific breeding between P. ricini strains leads to genetic mixing of the populations. Such human interventions should be avoided at the core germplasm level to maintain purity of the germplasm.

The phenotypic and genetic diversity revealed in this study is of much use in formulating strategies to conserve the diversity present in these unique silkworms. This study revealed that the mitochondrial genes can be used as potential markers to evaluate genetic diversity among populations. Since P. ricini is believed to have originated in the Brahmaputra valley of North-Eastern India and this is the region where maximum genetic diversity is expected, the populations identified as genetically divergent have to be conserved in situ. The high economic value of the silk industry emphasizes the need for urgent measures in this direction. Increased gene flow and habitat-area expansion are required to maintain higher genetic variability and conservation of the original P. ricini gene pool.

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