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Phase 3 (2007 Deadline)
Enhance Sugarcane Production in Pakistan by Modern Breeding Technology
Yong-Bao Pan, USDA-ARS, Sugarcane Research Unit, Houma, LA
Javed Iqbal, University of the Punjab, Lahore
Pakistani Funding (HEC): $ 71,000
US Funding (State): $ 85,000
Project Dates on US Side: September 1, 2008 - June 30, 2013
Project Overview
Sugarcane (Saccharum hybrids) is one of the most important cash crops in Pakistan. Before the initiation of this project, no research had been conducted in Pakistan on molecular marker identification for major diseases affecting sugarcane. The partners on this project have launched what they hope will be a long-term collaborative study to identify DNA markers that are closely linked to resistance genes for red rot, rust, and ratoon stunting disease and eventually for other diseases such as sugarcane mosaic virus and smut as well.
Major Results
- Hosted year-long training visits by three Pakistani PhD students to Dr. Pan’s laboratory, with another PhD student being advised by Dr. Pan via e-mail
- Organized month-long visits by Pakistani principal investigator Dr. Javed Iqbal and co-principal investigator Dr. Shahid Afghan to several US sugarcane research institutions
- Developed and published a paper on SSR-based DNA fingerprinting for screening of red rot-resistant and susceptible sugarcane cultivars from Pakistan
- Identified and published a paper on two SCAR markers associated with rust resistance in sugarcane using Bulk Segregant Analysis-Random Amplification Polymorphic DNA approach
- Identified and deposited to GenBank database 15 resistant gene analogues (RGA) and characterized nucleotide sequence variations between cultivars either resistant or susceptible to red rot or ratoon stunting diseases
Quarterly Update
During spring 2013, email communications continue between Dr. Pan and the PhD student Ms. Mehwish Khan regarding her dissertation research. A research paper from this project titled “Genetic variability among the brown rust resistant and susceptible genotypes of sugarcane by RAPD technique” was published on Pakistan Journal of Botany earlier this year. From June 12-14, 2013, Dr. Pan will present a poster paper entitled “Use of SSR Markers for DNA Fingerprinting and Diversity Analysis of Pakistani Sugarcane (Saccharum spp. hybrids) Cultivars” at the upcoming joint Annual Meeting of the American Society of Sugar Cane Technologists, to be held at Panama City Beach, FL. The paper is coauthored by Dr. Y.-B. Pan, U. Hameed, M. Khushi, S. Afghan and J. Iqbal. This project will soon be closed at the end of June 2013.
Progress Report Summaries
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2012 Show summary || Hide summary
Pakistani graduate student Mehwish Khan continues to make progress on her research experiments during her visit to Dr. Pan's lab, which began in May 2011 and has now been extended through May 2012. Her current work has advanced to sub-cloning, DNA sequencing, and sequence analysis using the DNAMAN software package. Ms. Khan attended the American Association of Sugar Cane Technology - LA Division Annual Meeting on February 7-8, 2012 in Lafayette, LA. On February 9 and February 23, she attended safety training classes and participated in seedling transplanting in greenhouse for two days in late February. Ms. Khan went back to Pakistan for a short visit on March 2; however, her visa process was delayed and was not able to get it until March 25. The other two students, Mr. Khushi Mohammad and Mr. Usman Hameed, are working on their dissertations. Although Dr. Iqbal has already completed his side of the project, a no-cost extension has been granted on the US side to allow Dr. Pan to continue his efforts through June 2012. Dr. Iqbal has extended an invitation for Dr. Pan to visit Lahore, Pakistan; however, Dr. Pan experienced difficulty in getting USDA-ARS approval for the travel.
Ms. Mehwish Khan returned to the USDA lab from Lahore, Pakistan and completed her one-year training on May 31, 2012. During her last month in USDA, she attended Louisana Sugarcane Extension and Research Meeting at LSU Sugar Station, ST. Gabriel, LA and visited the USDA-ARS Southern Regional Research Center. After this one-year training, she will continue her dissertation research at SBS under Professor Javed Iqbal. The Pakistani student Khushi Muhammad received his Ph.D. degree in May 2012 and now is a Assistant Professor at the Department of Genetics, Hazara University, Mansehra, Pakistan. The other student Mr. Usman Hameed has submitted his Ph.D. thesis to the University of Punjab on July 10. In addition, a peer-reviewed journal article "Use of simple sequence repeat markers for DNA fingerpriting and diversity analysis of sugarcane (Saccharum spp) cultivars resistant and susceptible to red rot" by U. Hameed, Y.B. Pan, K. Muhammad, S. Afghan and J. Iqbal was published in Genetics and Molecular Research 11(2): 1195-1204.
Dr. Pan has received his visa to Pakistan and his travel request has been approved by USDA. He will visit the School of Biological Science, University of the Punjab, Lahore, Pakistan in September, 2012.
2011 Show summary || Hide summary
Dr. Pan reports that one of his 2009-2010 visitors, Mr. Khushi Muhammad, submitted his Ph.D. dissertation on sugar cane rust to the University of the Punjab in July 2011. The other visitor during that period, Mr. Usman Hameed, is in the process of writing his dissertation on red rot and ratoon stunting disease of sugarcane and expects to submit it in December 2011. Since May 2011 Dr. Pan has been hosting another visitor, University of the Punjab doctoral candidate Ms. Mehwish Khan, at the USDA-ARS Sugarcane Research Laboratory in Houma. Ms. Khan is working to identify DNA markers associated with resistance to sugarcane smut disease. Dr. Pan also continues to advise another Pakistani student via e-mail. Ms. Ambreen Khadija Alvi, who is studying red rot, has completed her experimental work and expects to submit her Ph.D. dissertation by July 2012. Dr. Pan has received a no-cost extension through June 2012 to complete his project. In addition to the publications already cited in previous reports, he notes that he and his counterparts have six more papers currently in preparation. Recently, a peer-reviewed journal paper “Use of simple sequence repeat (SSR) markers for DNA fingerprinting and diversity analysis of sugarcane (Saccharum spp.) cultivars resistant and susceptible to red rot”, authored by U. Hameed, Y.-B. Pan, K. Muhammad, S. Afghan and J. Iqbal, has been accepted by Genetic and Molecular Research.
2010 Show summary || Hide summary
During the year 2010 the red rot work was continued by Ms. Ambreen Khadija Alvi in Dr. Iqbal’s lab in Pakistan. Of the 17 genotypes assessed, a total of 182 bands were generated by the 24 primers and the average number of bands produced by each primer was 7.6. Based on these data, similarity matrices were obtained by NTSYSpc and distance- homology matrices and a bootstrapped phylogenetic tree were obtained by DNAMAN. The 17 genotypes were grouped into five clusters. It was observed that all of the susceptible genotypes were grouped in the 2nd and 3rd clusters, whereas all of the resistant genotypes except HSF-240 and CSSG-676 were grouped separately from the susceptible ones. In addition, a scatter plot of the 17 genotypes was also made based on principle coordinate analysis (PCoA). The scatter plot showed that Groups I and IV were the least similar. Groups I and II contained only resistant genotypes. Group IV contained only susceptible genotypes. Group III contained both resistant and susceptible genotypes.
In August 2010, Dr. Pan hosted his counterpart Dr. Javed Iqbal from Pakistan for a two-week visit. During his visit, Dr. Iqbal gave two lectures, one at the USDA-ARS, Sugarcane Research laboratory and the other at the Sugar Research Station, Louisiana State University, and interacted with sugarcane researchers at both locations. Dr. Iqbal and Dr. Pan also visited the American Sugar Cane League Office and talked to Senior Agronomist Windell Jackson. Meanwhile, Dr. Pan is working with Dr. Iqbal and USDA staff on inviting another counterpart, Dr. Shahid Afghan, to visit the USDA-ARS, Sugarcane Research Laboratory for three months in 2011. Currently, Dr. Afghan is awaiting his visa. Another female graduate student, Ms. Mehwish Khan, has just initiated her doctoral work on a project entitled “Molecular Characterization of Resistant and Susceptible Sugarcane clones to Smut Disease”. Dr. Pan invited Ms. Khan to work in his laboratory for six months in 2011, and her background check was completed on February 28, which allows her to apply for a visa soon. So far, the collaborative research activities on this project have produced three publications, with two pending review, as well as six presentations at major symposia and conferences.
2009 Show summary || Hide summary
Dr. Pan worked hard in cooperation with USDA staff to process visa invitations for two Pakistani graduate students, Mr. Usman Hameed and Mr. Khushi Mohammad, to come to Louisiana for training. Although the Pakistani students encountered frustrating delays in the agency clearance and visa process, Usman Hameed finally received his visa on June 22, 2009, and arrived in Louisiana on July 30 to begin what was initially planned to be a 6-month visit. Due to his strong performance and with the consent of his supervisor back in Lahore, his visit has now been extended to a full year, through June 2010. As the focus of his research project Mr. Hameed has selected two subgroups of five clones each from the LCP 85-384 mapping population based on both ratoon stunting disease (RSD) response and QTL data such that the two subgroups differ only in responses to RSD infection. Two groups of Pakistani cultivars, either resistant or susceptible to RSD infection, are also involved in the study. Resistant gene analogue (RGA) primers were chosen based upon literature review from sugarcane and other related crops. As of January 2010, he had finished screening 21 RGA primer pairs, six of which have produced PCR products. Direct sequencing of these PCR products was not satisfactory, so Mr. Hameed is in the process of cloning them into a TA cloning vector.
The second Pakistani student, Mr. Khushi Muhammad, received his visa in early September 2009 and traveled to Louisiana on September 9 to begin his one-year visit. He had identified two subgroups of six clones each from the LCP 85-384 mapping population based on both rust disease response and QTL data such that the two subgroups differed only in responses to rust infection. Four rust resistant and four rust susceptible Pakistani varieties were also included in the study. Genomic DNA samples were extracted from the leaf tissue of LCP 85-384 and the self progeny and then quantified and calibrated before bulking. Four DNA bulks were prepared, two from the rust resistant (R) subgroups and two from the rust susceptible (S) subgroups, and those four bulked DNA samples along with LCP 85-384 DNA (positive control) and water (negative control) were screened with RAPD primers. Similarly, two Bulked DNA samples were generated from the Pakistan cultivars. One from four rust resistant cultivars and the other from four rust susceptible cultivars. A total of 319 RAPD primers were screened by polymerase chain reactions by bulk segregant analysis (BSA). One RAPD marker OPN-6 was identified against rust resistant from the LCP 85-384mapping population. Another RAPD marker BE-03 was identified from the Pakistan cultivars. These unique RAPD bands were excised from the gels, sub-cloned into a TA cloning vector, and sequenced. Based on the DNA sequences, Mr. Muhammad was able to design a sequence-characterized amplified region (SCAR) marker, which was validated on rust resistant and susceptible progeny of LCP 85-384 mapping population as well as commercially grown Pakistani cultivars.
Besides working hard on their primary research projects described above and getting acclimated to life in southern Louisiana, Usman Hameed and Khushi Mohammad also were involved in three other activities. They spent a day at the USDA-ARS Sugarcane Research Unit's farm to participate in sugarcane planting activity with other USDA staff. They also visited the Louisiana State University main campus, a sugarcane genetics laboratory, and the crossing facility at the Sugar Research Station. Finally, they learned from Dr. Pan how to use the genotyping software GeneMapper and used it to analyze the genotyping files of 21 SSR markers produced by the USDA-ARS Mid-South Area Genomics Laboratory on 20 Pakistan varieties. A joint publication entitled “Use of SSR markers for DNA fingerprinting and diversity analysis of Pakistan sugarcane (Saccharum spp.) cultivars was prepared on the SSR-based molecular genotyping work. Before returning to Pakistan, the two students successfully gave oral presentations of their research progress at the 40th Annual Joint Meeting of the American Society of Sugar Cane Technologists held during June 17-18, 2010 in Panama City, Florida. The title of Usman’s presentation was “Isolation and Characterization of Resistant Gene Analogs from RSD Resistant and Susceptible Sugarcane Clones: A Progress Report” (Journal of American Society of Sugar Cane Technologists, Vol. 30: 153) and Khushi’s presentation was entitled “Identification of RAPD Marker Associated with Brown Rust Resistance in Sugarcane” (Journal of American Society of Sugar Cane Technologists, Vol. 30: 154).
2008 Show summary || Hide summary
In July and September 2008, Dr. Pan submitted requests to his agency (USDA) for clearance to travel to Pakistan on the project, but both requests were denied due to security concerns, and this remained the case in 2009.
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