Nagaraju's Laboratory at Centre for DNA Fingerprinting and Diagnostics, Hyderabad

Research

Molecular marker genetics of silkmoths (completed)



The domesticated silkmoth, Bombyx mori has been used as a model system for formal genetic studies since the discovery of Mendelian inheritance at the turn of the century because of its large body size, ease of rearing in the laboratory and economic importance. The well-developed genetics of this species includes more than phenotypically well characterized 400 mutations, mapped to 28 linkage groups or chromosomes, existence of hundreds of geographic races and genetically improved strains used for silk production which differ not only in Mendelian traits but also in quantitative traits such as body size, feeding duration, thermal tolerance and disease resistance. These genetic stocks had remained to be subjected to systematic analysis using modern genetic tools.

Molecular markers have several advantages over the traditional phenotypic markers that are available in silkworm genetic stocks. They are unaffected by environment, detectable in all stages of development and ubiquitous in number covering the entire genome. Development of molecular markers is important in silkworm for construction of linkage map, fingerprinting of strains for breeding, and marker-assisted selection.

Microsatellites (SSRs) which are short stretches of simple tandemly repeated mono, di-, tri-, tetra-, penta or hexanucleotide repeats such as (A)n, (CA)n, (GA)n, (GTA)n, (ATT)n, (GATA)n, (ATTTT)n, (ACGTCG)n distributed across genomes of most of the eukaryotes, fulfill these criteria. PCR products of different lengths can be amplified using primers flanking the variable microsatellite region. Allelic variations among individuals are based mostly on differences in the number of tandem repeats in a microsatellite array providing a ready source of polymorphism. By using the fluorescent-labelled primers and automated sequencers, the entire process of microsatellite analysis is automated. Due to these advantages, microsatellites have been used to construct linkage maps. In addition to their clear utility for practical applications, information about the distribution and variability of microsatellite sequences in the genome of a given species can help elucidate its genetic history from the standpoint of evolution and artificial selection. We undertook to survey, characterise, catalogue, analyse and map the microsatellite motifs in the silkworm genome.

Public release of silkworm genome sequences from Japanese and Chinese researchers has facilitated the anchoring of genetic markers onto the actual chromosomal locations as the next logical step. In our lab, efforts are on for identifying the contigs/scaffolds carrying the mapped loci as well as genes. This contributes to the identification of additional markers to generate a high-density genetic map as well as to position the known genes on the linkage groups.


Comparative genomics of silkmoths

The past couple of years have seen the accomplishment of genome sequences of species that are close relatives of many model organisms. When the order Lepidoptera is considered, which consists of many economically important insects such as, silkmoths, agriculture pests and beautiful butterflies, genomic resources are available only for the domesticated silkmoth, B mori.

Wild silkmoths are the least exploited at molecular level, mainly due to non-availability of genomic resources for any of these species. Any genomic resources generated for these species will be of immense use in comparative studies of insects in general and silkmoths in particular. One good example for usefulness of the wild silkmoth sequence data may be drawn from a study (Gandhe et al., 2006, BMC Genomics) from our lab, wherein construction of 1400 ESTs from a wild silkmoth, Antheraea mylitta led to the identification of several novel immune response genes.


A assama, one of the economically important wild silkmoths whose genome is among the least understood, is unique among saturniid moths. By virtue of the narrow ecological distribution of host food plant, A. assama is confined only to the Assam state of India. The silk proteins of this species have not been studied so far despite their unique properties of providing golden luster to the silk thread. In view of this, it is important to look at the transcriptome of this unique species in comparison with other species, which will throw light on the basic and applied aspects of domesticated and wild silkmoths. Comparative studies on expressed sequences of the B. mori and wild silkmoths with the other published genome sequences of insects and other model organisms could uncover lepidoptera-specific genes.

With the support of Department of Biotechnology, we sequenced and characterized 35,772 ESTs from A. assama. By using the EST sequences we have already characterized dsx, the double sex gene in the sex determination pathway. We have also identified and characterized silk proteins encoding genes whose comparative analysis is being carried.



Projects completed

Silkworm Genome Mapping

  • Survey and analysis microsatellites
  • Construction of genetic and physical maps
  • Mapping of QTLs on Z-chromosome

Silkmoth Genomics

  • Construction, characterization and analysis of expressed sequences from silkmoths.

Projects being pursued

  • Functional and comparative genomics of silkmoths.

Publications

  • Shimomura M, Minami H, Suetsugu Y, Ohyanagi H, Satoh C, Antonio B, Nagamura Y, Kadono-Okuda K, Kajiwara H, Sezutsu H, Nagaraju J, Goldsmith MR, Xia O, Yamamoto K, Mita K (2009) KAIKObase: An integrated silkworm genome database and data mining tools. BMC Genomics 10: 486.

  • Arunkumar KP, Mita K, Nagaraju J (2009) The Silkworm Z Chromosome is Enriched in Testis-Specific Genes. Genetics 182: 493-501.

  • Arunkumar KP, Kifayathullah L, Nagaraju J (2008) Microsatellite markers for the Indian golden silkmoth, Antheraea assama (Saturniidae: Lepidoptera). Molecular Ecology Resources 9: 268-270

  • Arunkumar KP, Tomar A, Daimon T, Shimada T, Nagaraju J (2008) WildSilkbase: An EST database of wild silkmoths. BMC Genomics 9: 338.

  • Archak S, Nagaraju J (2007) InSatDb: A Genomic Tool for Insect Geneticists. FLY 1:5, 279-281.

  • Archak S, Meduri E, Sravana KP and Nagaraju J (2007) InSatDb: A microsatellite database of fully sequenced insect genomes. Nucleic Acids Research 35: D36 - D39.

  • Meglecz E, Anderson SJ, Bourguet D, Butcher R, Caldas A, Cassel-Lundhagen A, d'Acier AC, Dawson DA, Faure N, Fauvelot C, Franck P, Harper G, Keyghobadi N, Kluetsch C, Muthulakshmi M, Nagaraju J, Patt A, Petenian F, Silvain JF, Wilcock HR (2007) Microsatellite flanking region similarities among different loci within insect species. Insect Molecular Biology 16:175-85.

  • Miao XX, Xub SJ, Li MH, Li MW, Huang JH, Dai FY, Marino SW, Mills DR, Zeng P, Mita K, Jia SH, Zhang Y, Liu WB, Xiang H, Guo QH, Xu AY, Kong XY, Lin HX, Shi YZ, Lu G, Zhang X, Huang W, Yasukochi Y, Sugasaki T, Shimada T, Nagaraju J, Xiang ZH, Wang SY, Goldsmith MR, Lu C, Zhao GP, Huang YP (2005) Simple sequence repeat-based consensus linkage map of Bombyx mori. Proceedings of the National Academy of Sciences USA 102: 16303-16308.

  • Nagaraja GM, Mahesh G, Satish V, Madhu M, Muthulakshmi M, Nagaraju J (2005) Genetic mapping of Z chromosome and identification of W chromosome-specific markers in the silkworm, Bombyx mori. Heredity 95: 148-157.

  • Prasad MD, Muthulakshmi M, Madhu M, Archak S, Mita K, Nagaraju J (2005) Survey and analysis of microsatellites in the silkworm, Bombyx mori: frequency, distribution, mutations, marker potential and their conservation in heterologous species. Genetics 169: 197-214.

  • Prasad MD, Muthulakshmi M, Arunkumar KP, Madhu M, Sreenu VB, Pavithra V, Bose B, Nagarajaram HA, Mita K, Shimada T, Nagaraju J (2005) SilkSatDb: a microsatellite database of the silkworm, Bombyx mori. Nucleic Acids Research 33: D403-406.

  • Nagaraju J (2002) Recent advances in silkworm biology. Current Science (Editor, Special Issue) 83: 409-471.

  • Nagaraju J (2002) Application of genetic principles for improving silk production. Current Science 83: 409-414.

  • Nagaraju J, Goldsmith MR (2002) Silkworm genomics - progress and prospects. Current Science 83: 415-425.

  • Nagaraju J, Reddy KD, Nagaraja GM, Sethuraman BN (2001) Comparison of multilocus RFLPs and PCR-based marker systems for genetic analysis of the silkworm, Bombyx mori. Heredity 86: 588-597.

  • Nagaraju J (2001) Identification of a gene associated with Bt resistance in the lepidopteran pest, Heliothis virescens and its implications in Bt transgenic-based pest control. Current Science 81: 746-747.

  • Nagaraju J (2000) Recent advances in molecular genetics of the silk moth, Bombyx mori. Current Science 78: 746-747.

  • Nagaraju J, Klimenko V, Couble P (2000) The silkworm Bombyx mori, a model genetic system, In: Encyclopedia of Genetics; Reeves E, editor. London, UK: Fitzroy Dearborn.

  • Reddy KD, Nagaraju J, Abraham EG (1999) Genetic characterization of the silkworm Bombyx mori by simple sequence repeat (SSR)-anchored PCR. Heredity 83: 681-687.

  • Reddy KD, Abraham EG, Nagaraju J (1999) Microsatellites in the silkworm, Bombyx mori: abundance, polymorphism, and strain characterization. Genome 42: 1057-1065.

  • Sharma A, Niphadkar MP, Kathirvelu P, Nagaraju J, Singh L (1999) DNA fingerprinting variability within and among the silkworm Bombyx mori genotypes and estimation of their genetic relatedness using Bkm-derived probe. Journal of Heredity 90: 315-319.

  • Nagaraju J, Singh L (1997) Assessment of genetic diversity by DNA profiling and its significance in silkworm, Bombyx mori. Electrophoresis 18: 1676-1681.

  • Nagaraju J, Sharma A, Sethuraman BN, Rao GV, Singh L (1995) DNA fingerprinting in silkworm Bombyx mori using banded krait minor satellite DNA-derived probe. Electrophoresis 16: 1639-1642.

  • Nagaraja GM, Nagaraju J (1995) Genome fingerprinting of the silkworm, Bombyx mori, using random arbitrary primers. Electrophoresis 16: 1633-1638.

  • Nagaraju J, Abraham EG (1995) Purification and characterisation of amylase in tasar silkworm, Antheraea mylitta. Comparative Biochemistry And Physiology - Part B 110B: 201-209.

  • Nagaraju J, Kumar PT (1995) Effect of selection of cocoon filament length in divergently selected lines of the silkworm, Bombyx mori. Journal of Sericulture Science Japan 64: 103-109.

  • Abraham EG, Nagaraju J, Datta RK (1992) Biochemical studies of amylase in silkworm, Bombyx mori (I) Comparative analysis in diapausing and non-diapausing strain. Insect Biochemistry and Molecular Biology 22: 867-873.

  • Nagaraju J, Jolly MS (1986) Interspecific hybrids of Antheraea pernyi and A. roylei - A cytogenetic reassessment. Theoretical and Applied Genetics 72: 269-273.


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