INTERNATIONAL CENTRE FOR GENETIC ENGINEERING AND BIOTECHNOLOGY ARUNA ASAF ALI MARG, NEW DELHI - 110067 Consortium Leader

1. GENOMICS OF COTTON BOLL AND FIBRE DEVELOPMENT National Agricultural Innovation Project (Indian Council of Agricultural Research) INTERNATIONAL CENTRE FOR GENETIC ENGINEERING AND BIOTECHNOLOGY ARUNA ASAF ALI MARG, NEW DELHI - 110067 Consortium Leader NATIONAL RESEARCH CENTRE ON PLANT BIOTECHNOLOGY PUSA CAMPUS, NEW DELH I- 110012 UNIVERSITY OF AGRICULTURAL SCIENCES DHARWAD - 580005

2. National Agricultural Innovation Project (NAIP) of Indian Council of Agricultural Research (ICAR) accords high priority to generation and transfer of innovative agricultural technologies. The overall objective of the NAIP is to facilitate an accelerated and sustainable transformation of the Indian agriculture, so that it can support poverty alleviation and income generation through collaborative development and application of agricultural innovations by the public organizations in partnership with farmers, the private sector and other stakeholders. The specific objective of the NAIP under the component 4 is to build capacity to undertake basic and strategic research in frontier areas of agricultural sciences. Present project is funded by the NAIP to develop cotton genotypes with good quality fiber and insect resistance. Cultivated Species Cotton is the most important fibre crop in India occupying 9 mha area which represents world highest acreage of 34 mha under cotton. In four of the cultivated cotton species, two are A-genome diploid species (Gossypium arboretum L. and G. herbaceum L.) and two are allotetraploid AD-genome species (G. barbadense L. and G. hirsutum L.). G. hirsutum L. is the most widely cultivated species and accounts for more than 90% of the world cotton lint production. Fibre morphogenesis can be divided into four overlapping development stages: Fibre initiation, Primary cell wall synthesis (fibre expansion and elongation), Secondary cell wall synthesis and Maturation. The growth and development of cotton plant is highly influenced by both biotic and abiotic factors and directly influence the fibre yield and quality. Biotic factors Abiotic factors Insects Drought In the recent years, rapid development of genomic tools and resources such as expression profiling of elongating fiber cells has provided clues to facilitate the understanding of cotton production. This opens up opportunities to isolate genes encoding fiber quality and strength. Efforts to isolate these genes will facilitate our ability to manipulate fibre characters that the textile industry is looking forward. Promoters that are highly active during boll development will sustain the expression of foreign proteins such as Bt toxins in the bolls providing effective protection against Cotton bollworm and Pink bollworm. Similarly, boll-specific promoters will facilitate hyper-expression of osmotolerant genes in developing bolls thus imparting protection to the physiological and biochemical processes involved in fibre development. • OBJECTIVES • Identification of genes in the developing boll that are highly influenced under Bollworm attack and drought and nutritional stresses. • Construction of EST database of Gossypium hirsutum from tissues derived from various stages of boll development. • Assignment of functions to key genes involved in cotton fibre length and quality. • Isolation and characterization of promoters for the genes expressed specifically in bolls and are strongly up regulated.

3. Different boll developmental stages Isolate proteins from various stages of fiber development Gel spots Trypsin fragments Proteome and EST data bases, genome analysis Masspec data 2DE MALDI-TOF: Peptide mapping Studies on the influence of various factors during the boll development can provide vital information on the expression profile of various genes and their role at molecular level that is necessary for understanding the cotton fibre formation and its development under various adverse conditions. The project envisages identification and isolation of genes and promoters involved in fibre development and characterization of their responses to biotic and abiotic stress factors. Towards this objective novel approaches will be followed which include: • Global expression profiling of boll-specific ESTs under biotic and abiotic stress conditions. • Identification of the corresponding gene sequences by bioinformatics tool • PCR-based genome walking to isolate boll-specific and high expressing promoter sequences and • validation using reporter gene expression in cotton. • Development of cotton boll proteome database and identify proteins whose accumulation is • influenced by abiotic and biotic factors using mass spectrometric analysis. • Functional validation of candidate genes involved in the fibre development using expression and • gene silencing approaches in cotton. • The deliverables: • Genes and promoters that are specific to boll development. • Genes associated with fiber development. • Genes and promoters that are expressed in response to biotic and abiotic stress factors during boll development. • Validated boll-specific promoters useful for the development of transgenic cotton • Novel methods for the genomic analysis. Expected output and impact of the project: • The most important outcome of the project is a fundamental understanding of gene expression during flower, square, boll and fiber development in cotton. The knowledge thus gained will have profound implications in applied cotton breeding, transgenic development and biotechnology of cotton leading towards trait introduction and improvement. • Expression of insecticidal proteins such as Bt toxins under the control of boll-specific promoter will sustain their expression thus providing effective control of cotton Bollworms. • The project will generate fundamental knowledge about the gene function and the influence of biotic and abiotic factors on fiber development.

4. ICGEB, New Delhi Proteome analysis: * Global analysis of protein expression during boll development under normal and stress conditions * Validation of genes/promoters NRCPB, New Delhi Transcriptome analysis: * Global analysis of gene expression during boll development under normal & stress conditions * Validation of genes/promoters GENOMICS OF COTTON BOLL AND FIBER DEVELOPMENT • UAS, Dharwad • Production of cotton plants under biotic and abiotic stress conditions for proteomic and transcriptomic studies • Isolation of transcripts & cDNA for transcripome studies • Validation of genes/promoters • Execution of the programme will help identify and isolate genes and respective promoters that will lead to better fiber quality, improved marketability and value to the farmer. • Well-trained human resource in the frontier technologies such as functional genomics through DNA microarrays, proteomics and transcriptomics. • The novel genes and promoters identified will be protected in the form of patents by the NAIP. CONSORTIUM IMPLEMENTATION COMMITTEE CONSORTIUM ADVISORY COMMITTEE Prof A. R. Reddy, Chairman-CAC,Vice-Chancellor, Yogi Vemana University, Kadapa, A.P. (Expert on functional genomics of crop plants) Prof Amit Ghosh, Director, Indian Institute of Advanced Research, Gandhinagar (Expert on Microbial genetics and molecular biology of Vibrio cholerae) Dr B. M. Khadi, Director, Central Institute of Cotton Research, Nagpur (Expert on cotton breeding and biotechnology) Estimated total cost of the project : Rs. 755.50 lakhs