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Pakistan-US Science and Technology Cooperation Program                                                            
Phase 4 (2009 Deadline)

Characterization and Utilization of Gossypium arboreum as a Source of Resistance Against Begomoviruses that are a Threat to the Cotton Crop in Pakistan and the United States

Peng W. Chee, University of Georgia
Shahid Mansoor, National Institute for Biotechnology and Genetic Engineering
Pakistani Funding (HEC): $106,000
US Funding (Department of State): $115,522
Project Dates: July 18, 2011 - July 18, 2014 (Extended through February 28, 2015)
Project Overview
Cotton leaf curl disease (CLCuD) complex is currently endemic throughout Pakistan and epidemic in Western India. Yield losses due to cotton leaf curl virus are as high as 100 percent in some cotton fields in Pakistan. Over the last ten years, the introduction of CLCuD resistant or tolerant cotton varieties in Pakistan produced by conventional breeding and selection methods has shown promise in helping manage the disease. However, in 2001 resistance was broken, with previously resistant varieties showing symptoms of CLCuD in the Vehari district of Pakistan, and in 2009-10 severe symptoms of cotton leaf curl disease appeared in several other districts. In this project, two cotton species resistant to cotton leaf curl disease complex will be crossed by the Cotton Research Institute in Faisalabad, a collaborating institution on the project, and the resulting plants will be tested for resistance to the disease. Scientists at NIBGE and the University of Georgia will use bioinformatics and molecular tools to identify polymorphism in genes that are involved in virus-host interactions such as those that interact with geminiviruses. This project should lay the foundation for addressing one of the most devastating diseases of cotton, which represents a shared concern of Pakistan (where it is already crippling cotton production in some areas) and the United States (where all germplasm appears to be vulnerable). 
Quarterly Update
In 2013,  the Pakistani PI Dr Shahid Mansoor, traveled to Co-PI Paterson Laboratory in Athens, Ga with funding from the Fulbright Program and established a RNA extraction procedure that works well for cotton. The RNA collected were sent to LC Sciences, which is based in Houston, TX and to the Boyce Thompson Institute at Cornell University for short RNA sequencing to determine the levels and diversity of short RNA (siRNA and miRNA) in G. arboreum and G. herbaceum. Preliminary analysis of miRNA isoforms miR482 (the most common miRNA that regulate R gene expressions) showed that this miRNA has many isoforms in G. arboreum and G. herbaceum, the two diploid species resistant to cotton leaf curl disease. In addition, the expression levels of miR482 were higher in different tissues of G. arboreum as compared to G. hirsutum. A comprehensive analysis of the two dataset is currently in progress.

Ms. Mychele Da Silva, a new PhD student, started working on this project. Since the cotton leaf curl virus is not found in the U.S., the team's approach to identify genes involved in plant disease-host interaction would require challenging the cotton plants with other disease causing pathogens such as root-knot nematodes, which is prevalent in all cotton production regions in the U.S. Ms. Da Silva has established a greenhouse inoculation procedure for the root-knot nematodes pathogen and will conduct an experiment to determine if resistance to nematode can be used as a model system to analyze the cotton genome for differences in R gene expression between the infected and non-infected (control) plants via sequencing.

The team have received seeds from different accessions of G. arboreum, G. herbaceum and G. gossypioides from the USDA cotton germplasm collection in College Station, TX. Plants from the three species above are currently growing in the greenhouse and will be cross-hybridized to develop genetic populations to dissect the genetics of resistance to cotton leaf curl virus.

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