Project	Molecular Ecology and Phylogenetics
Investigators PhD Scholars Technical Staff Research Associates	K Sriramana, KP Arunkumar and Sunil Archak, M Muthulakshmi S Johny
Collaborator	R Gadagkar, IISc, Bangalore
Funding Agency	Department of Biotechnology, Govt of India, New Delhi

Evolution can be defined in terms of genetics as a change in the frequencies and types of genes in a population. Molecular data can be used to make a phylogenetic tree that illustrates phenetic relationships. Phenetics also known as numerical taxonomy has been used to understand the phylogeny of individuals, groups or species. Molecular phylogenetics attempts to determine the rates and patterns of change occurring in DNA and proteins and to reconstruct the evolutionary history of genes and organisms. In our laboratory, we have used patterns of DNA variation to study the evolutionary relationships among individuals, groups or species.

Silk moth

The utility of multilocus RFLPs and three PCR-based techniques, Random Amplified Polymorphic DNA (RAPD), Inter-Simple Sequence Repeat-PCR (ISSR-PCR) and simple sequence repeats (SSRs) for genetic characterization was examined using 13 diverse silkworm strains. All four approaches successfully discriminated the 13 silkworm varieties but differed in the amount of polymorphism detected. The usefulness of each system was examined in terms of number of loci revealed (effective multiplex ratio, EMR) and the amount of polymorphism detected (diversity index, DI). For example, the six multilocus RFLP probes produced 180 products of which 97% were polymorphic; 15 SSR loci gave rise to an average of 8 alleles each, of which 86% were polymorphic. The ISSR-PCR produced 39 fragments of which 76.98% were polymorphic. The highest diversity index was observed for ISSR-PCR (0.957) and the lowest for RAPDs (0.744). The RAPD, ISSR-PCR and RFLP assays clearly separated the diapausing and non-diapausing silkworm varieties. These results are discussed in terms of choice of appropriate marker technology for different aspects of silkworm genome analysis.

Thirteen diverse strains of the silkworm Bombyx mori were analysed using the simple sequence repeat anchored polymerase chain reaction (SSR-anchored PCR) or Inter-SSR-PCR (ISSR-PCR). A set of four 5'-anchored and two 3B"-anchored repeat primers amplified a total of 239 bands out of which 184 (77%) were polymorphic. The 5'-anchored primers revealed more distinct polymorphic markers than the 3'-anchored primers and the ISSR-PCR method showed greater variability than RAPDs. The strain-specific pattern was shown to be inherited and segregated in a Mendelian fashion. A dendrogram constructed using the UPGMA method revealed two distinct groups, one comprising non-diapausing and one comprising diapausing strains. These results suggest that the ISSR-PCR method is potentially useful for genetic fingerprinting of silkworm genotypes and as a mapping tool in the silkworm.

The silkmoths belong to two families, Bombycidae and Saturniidae. In the present study, molecular phylogeny of these silkmoths was derived using three mitochondrial genes, 12S rRNA, 16S rRNA and COI and, Control Region (CR). The NJ and MP analyses showed two distinct clades, one clade consisting species of the Bombycidae and another with species of Saturniidae. Among saturniid moths, A. pernyi was closely related to A. roylei and A. assama was more close to A. mylitta and A. yamamai whereas, S. C. ricini was found to be more distant from other saturniid moths. The mitochondrial CR showed length polymorphism with indels. The NJ and MP analyses for complete mitochondrial genome sequences of B. mori (Strains Aojuku, C₁₀₈, Backokjam and Xiafang), Japanese mandarina, Chinese mandarina and A. pernyi showed two distinct clades of B. mori strains and B. mandarina with A. pernyi forming an outgroup. Pairwise distances revealed that all the strains of B. mori studied were more close to Chinese mandarina than to Japanese mandarina. The molecular clock analysis suggested the divergence of B. mori from Chinese mandarina about 15.7 MYA. Japanese mandarina and Chinese mandarina shared a common ancestor about 24.8 MYA. The divergence time between Saturniidae and Bombycidae was found to be about 160.9 MYA. The analyses based on chromosome number, genetic distance derived from complete mitochondrial genome sequence analysis and presence of a 250 bp insertion only in Japanese mandarina, suggested that B. mori must have originated from Chinese mandarina.

Nosema

The pathogenicity, mode of transmission, tissue specificity of infection and the small subunit rRNA (SSU-rRNA) gene sequences of the three new microsporidian isolates from the silkworm Bombyx mori were studied. Out of the three, NIK-2r revealed life cycle features and SSU-rRNA gene sequence similar to Nosema bombycis, suggesting that it is N. bombycis. The other two, NIK-4m and NIK-3h, deferred from each other as well as from N. bombycis. NIK-4m was highly pathogenic and did not show any vertical transmission, in accordance with the apparent lack of gonadal infection, whereas NIK-3h was less pathogenic and vertical transmission was not detected but could not be excluded. Phylogenetic analysis based on SSU-rRNA gene sequence placed NIK-3h and NIK-4m in a distinct clade that included almost all the Vairimorpha species and Nosema species that infect lepidopteran and nonlepidopteran hosts, while NIK-2r was included in a clade containing almost all the Nosema isolates that infect only lepidopteran hosts. Thus, we have presented molecular evidence that one of the three isolates is in fact the type species N. bombycis, while the other two isolates are Vairimorpha spp. There was distinct separation of microsporidian isolates infecting only lepidopteran hosts and those infecting lepidopteran and non-lepidopteran hosts, reflecting possible co-evolution of hosts and microsporidian isolates.

Casuarina

Inter simple sequence repeat polymerase chain reaction (ISSR-PCR) was used for the genetic analysis of the six species of Allocasuarina, five species of Casuarina and twelve superior performing selections of C. equisetifolia L. We also fingerprinted C. equisetifolia L. selections using Fluorescent-ISSR-PCR (FISSR-PCR), an improvised ISSR-PCR assay.

The ISSR analysis provided information on the frequency of various simple sequence repeats in the casuarina genome. The di-nucleotide repeats were more common, among which (CA)_n and its complementary nucleotide (GT)_n repeat motifs amplified relatively higher number of bands with an average of 6.0B13.5 and 6.3B11.8 respectively. Eleven species of casuarinas were amplified with 10 primers anchored either at 5' or 3' end. A total of 253 PCR products were obtained and all were polymorphic, out of which 48 were specific to Allocasuarina and 36 were specific to Casuarina genus. Genetic similarity among the species was 0.251. A UPGMA dendrogram grouped all the Casuarina species together. The 12 superior performing selections of C. equisetifolia L. produced 57 polymorphic ISSR markers while the FISSR assay revealed 105 polymorphic markers. The primer CRR(ATT)₄ distinguished all the selections. DNA profiles obtained with ISSR and FISSR assays would serve as a reference library for the establishment of clonal identity in casuarinas.

Morus

The genus Morus, known as mulberry, is a dioecious and cross-pollinating plant that is the sole food for the domesticated silkworm, Bombyx mori. Traditional methods using morphological traits for classification are largely unsuccessful in establishing the diversity and relationships among different mulberry species because of environmental influence on traits of interest. As a more robust alternative, PCR based marker assays including RAPD and ISSR were employed to study the genetic diversity and interrelationships among twelve domesticated and three wild mulberry species.

RAPD analysis using 19 random primers generated 128 discrete markers ranging from 500-3000 bp in size. One-hundred-nineteen of these were polymorphic (92%), with an average of 6.26 markers per primer. Among these were a few putative species-specific amplification products which could be useful for germplasm classification and introgression studies. The ISSR analysis employed six anchored primers, 4 of which generated 93 polymorphic markers with an average of 23.25 markers per primer. Cluster analysis of RAPD and ISSR data using the WINBOOT package to calculate the Dice coefficient resulted into two clusters, one comprising polyploid wild species and the other with domesticated (mostly diploid) species. These results suggest that RAPD and ISSR markers are useful for mulberry genetic diversity analysis and germplasm characterization, and that putative species-specific markers may be obtained which can be converted to SCARs after further studies.

Cattle

Molecular characterization of cattle breeds is important for the prevention of germplasm erosion by cross breeding. The Indian zebu cattle have their significant role in evolution of present day cattle breeds and development of some of the exotic breeds. Microsatellites are the best available molecular tools for characterization of cattle breeds. The present study was carried out to characterize two Indian cattle breeds, Ongole and Deoni, using microsatellite markers.

Using five di- and five tri-nucleotide repeat loci, 17 Ongole and 13 Deoni unrelated individuals were studied. Of the ten loci, eight revealed polymorphism in both the breeds. The di-nucleotide repeat loci were found to be more polymorphic (100%) than tri-nucleotide repeat loci (60%). A total of 39 polymorphic alleles were obtained at 4.5 alleles per locus in Ongole and 4.1 in Deoni. The average expected heterozygosity was 0.46 (B10.1) and 0.50 (B10.1) in Ongole and Deoni breeds, respectively. The PIC values of the polymorphic loci ranged from 0.15 to 0.79 in Ongole and 0.13 to 0.80 in Deoni breeds. Six Ongole specific and three Deoni specific alleles were identified. The two breeds showed a moderate genetic relationship between themselves with a FST value of 0.117 (P = 0.01). This preliminary study shows that microsatellite markers are useful in distinguishing the two zebu breeds namely, Ongole and Deoni. Further studies of other zebu breeds using many microsatellite loci with larger sample sizes can reveal the genetic relationships of Indian breeds.

Rice

The objective of the present study was to make use of efficient molecular marker systems to reveal genetic relationships in traditional and evolved Basmati (EB) and semidwarf non-Basmati (NB) rice varieties. A subset of three rice groups was analyzed by using 19 simple sequence repeat (SSR) loci and 12 inter-SSR-PCR primers. A total of 70 SSR alleles and 481 inter-SSR-PCR markers were revealed in 24 varieties from the three groups. The lowest genetic diversity was observed among the traditional Basmati varieties, whereas the EB varieties showed the highest genetic diversity by both the marker assays. The results indicated that the subset of aromatic rice varieties analyzed in the present study is probably derived from a single land race. The traditional Basmati (TB) and semidwarf NB rice varieties used in the present study were clearly delineated by both marker assays. A number of markers, which could unambiguously distinguish the TB varieties used in the present study from the evolved and NB rice varieties, were identified. The potential use of these markers in Basmati rice-breeding programs and authentication of TB varieties used in the present study are envisaged.

We are also implementing a collaborative project on Molecular Ecology and Systematics under the programme National Bioresource Development Board. We have oninitiated 'Genetic analysis of ecotypes of tropical tasar silkmoth Antheraea mylitta'. Under this project we are also providing training and infrastructure facilities to carry out molecular analyses, to selected ecology projects. At present two projects viz. "Molecular ecology of the primarily eusocial wasp Ropalidia marginata". (isolation and application of SSR markers for kinship studies) and "Identification and functional analysis of genes related to gall midge resistance in rice" (isolation and application of microsatellite based markers, SSR and ISSR for biotype identification; tagging avirulence genes) are being provided research support.