Location: Genomics and Bioinformatics Research2013 Annual Report
1a. Objectives (from AD-416):
Mitigate the threat of Cotton Leaf Curl Virus (CLCV) to the Pakistani cotton crop. Develop resistant germplasm as well as detection and screening methods needed to increase Pakistan’s and the U.S.’s level of preparedness for CLCV.
1b. Approach (from AD-416):
ARS will work with ICARDA and other selected agencies as well as National Cotton Council and U.S. universities to implement the collaborative project. Objectives will include: Identify new sources of resistance; transfer resistance genes; develop new tools to identify, characterize, and monitor different strains of Cotton Leaf Curl Virus (CLCV); transfer new techniques, genetic resources, and expertise to Pakistani scientists.
3. Progress Report:
This project represents additional funds added to the project 6402-21310-003-09R, where this report can also be found. The funding for this project goes towards the following projects: 6402-21310-003-11S, Stable Introgression of Cotton Leaf Curl Virus Resistance into Cultivated Cotton and Germplasm Enhancement; 6402-21310-003-10S, Development of Cotton Leaf Curl Virus (CLCuV) Diagnostic Tools and Monitoring of Clcuv; 6402-21310-003-12S, Increase of Cotton Germplasm with Emphasis on Non-Cultivated Species for Examination of Important Disease Resistance Traits; 6402-21310-003-13S, Identification and Introgression of Cotton Leaf Curl Virus Resistance into Cultivated Cotton; 6402-21310-003-14S, Enhancing Cotton Germplasm, Improving Resistance to Cotton Leaf Curl Virus and Supporting Cotton Best Management Practices for Small Farmers, where additional details can be found. Added to this overall project are the sub-projects: 6402-21310-003-24s, using bioinformatic tools to investigate the basis of resistance/tolerance in cotton species to cotton leaf curl virus (CLCuV); 6402-21310-003-25s, development of diagnostic screening tools and evaluation of transgenic tomato plants for cotton leaf curl virus (CLCuV) resistance. During the past year, a comprehensive international research program continued to combat Cotton Leaf Curl Virus (CLCuV) by developing resistant cotton lines and studying the virus itself. The CLCuV strain found in Pakistan developed within the country and is the most virulent form known in the world. Germplasm from the USDA ARS Cotton Germplasm Collection is continuously being propagated and sent to Pakistan for screening to see if it carries any resistance. In 2011 over 500 accessions of wild relatives of cultivated cotton were sent to Pakistan and screened. All were resistant to the virus. The result is surprising and may indicate that the diploid wild relatives to be a non-host, for the virus versus carrying resistance. The overall goal here is to identify novel forms of resistance, genetically map the resistance to identify a DNA marker associated with resistance, and then integrate the resistant trait into cultivated cotton. If the diploid species are non-host, it may mean a new approach for solving the problem could be attempted. In 2012, approximately 1,400 cotton accessions were sent to Pakistan for screening. This included upland cotton, additional wild species and introgression lines between upland cotton and a diploid. Sentential plots and random selections were established in Pakistan to trap new isolates of the virus for characterization and to establish protocols for to see if new forms of the virus are evolving. DNA samples from the viral samples were sent to the USA for DNA analysis. Project partners continue to make progress and the research objectives set for the program are being met. For the second year, seed was increased at the Cotton Winter Nursery, Tecoman, Mexico, and sent to Pakistan to screen for CLCuV. Early results indicate that all the Gossypium (G.) arboreum accessions were resistant including several accessions that have already been crossed with special lines designed to transfer traits from the diploid G. arboreum to tetraploid upland cotton. In late August, a U.S. project review team visited several locations and observed tetraploid lines that were not exhibiting CLCuV symptoms. Accessions symptom free at 120 days will be further evaluated to confirm their resistance. In parallel, crosses are being made between the putative resistant accessions and elite U.S. germplasm as the first step in developing resistant elite lines for U.S. growers. Pakistani scientists will use the material for crossing with their own adapted lines. As an alternative strategy, transgenic plants have also been created using a technology designed to induce resistance in the plant. Sentinel plots were established to track the spread of CLCuV in Pakistan. Tissue samples were collected from the plots and are being evaluated for the presence and concentration of the virus in the samples. Development continues on new diagnostic techniques, to detect and quantify the virus, with further testing occuring in the 2013 field season, which runs from Bevruary through November. During their visit, the U.S. review team also met with Pakistani partners and evaluated research progress and facilities. The visit culminated in a two day workshop organized jointly by ICARDA, ARS and FAS with the theme “Enhancing Cotton Germplasm, Improving Resistance to Cotton Leaf Curl Virus (CLCuV) Disease, Supporting Cotton Best Management Practices (BMPs) for Small Farmers and Capacity Building of Pakistani Cotton Researchers”. The workshop was attended by researchers, extension agents, government officials and farmers. Technical Status Report: 1,488 accessions from the Cotton Winter Nursery and Stoneville, Mississippi were packaged and sent to Pakistan. The material reached Pakistan in time for the Pakistani partners to quickly divide and repackage the seed so that it reached all the locations in time to be planted for CLCuV resistance screening. It was a major logistics and team effort and everyone did their part to make it succeed. The accessions were planted in late June to maximize the chance of infection with CLCuV. 1,386 accessions were screened at four locations (Faisalabad, Verhari, Multan, and Sakrand). Seed was also increased at Sakarand where the incidence of CLCuV is low. Due to the low number of seed available, the 102 introgression lines from Stoneville were only grown at one location. The accessions were evaluated for CLCuV symptoms at 30, 60, 90 and 120 days after planting to fully access each line’s level of susceptibility/resistance. Data on morphological traits and agronomic characters were also collected. The end of August, U.S. project team members [ARS (3 scientists), University of Arizona and Texas A&M] traveled to Pakistan to review the screening plots at three locations. The plots were at the ~70 day mark and there were several G. hirsutum accessions that did not exhibit any symptoms while the susceptible spreader rows and the other G. hirsutum accessions all exhibited severe symptoms. This is the first step in developing CLCuV resistant elite lines for U.S. growers. Pakistani scientists will use the material for crossing with their own adapted lines. The two virus portions of the project are collaborating with the Genomics and Bioinformatic Research Unit for the DNA sequencing portion of the project. As each virus is over 2,000 bp it requires several reactions to sequence one virus clone on both DNA strands. The virus clones from the University of Punjab, Lahore are cloned by the University of Arizona and the purified DNA is sent for sequencing. Over 13,648 DNA sequencing reactions were conducted. If the sequencing had been done by Pakistani cooperators or commercially, the estimated cost would have been ~$80,000 compared to a much lower in-house costs of ARS ~$19,000. Research activities during this reporting period were monitored by the ADODR through email communications with the cooperator and through in-person discussions with the cooperator in Washington D.C. and U.S.A. Embassy in Pakistan, phone conversations, and weekly conference calls.