Final Report: Discovery and Evaluation of SNP Variation in Resistance-Gene Analogs and Other Candidate Genes in Cotton

EPA Grant Number: R829479C009
Subproject: this is subproject number 009 , established and managed by the Center Director under grant R829479
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program
Center Director: Schumacher, Dorin
Title: Discovery and Evaluation of SNP Variation in Resistance-Gene Analogs and Other Candidate Genes in Cotton
Investigators: Paterson, Andrew
Institution: University of Georgia
EPA Project Officer: Lasat, Mitch
Project Period: May 1, 2003 through August 31, 2003
RFA: The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program (2001) RFA Text |  Recipients Lists
Research Category: Targeted Research , Hazardous Waste/Remediation

Objective:

The objective of this research project was to explore approaches for single nucleotide polymorphism (SNP) discovery in cotton, a complex polyploid genome that has many economic and environmental impacts. SNP explorations will shed new light on fundamental research questions important to planning future studies of variation at multitudes of genetic loci, in larger numbers of accessions (such as germplasm banks).

Summary/Accomplishments (Outputs/Outcomes):

DNA loci to be screened for SNPs were drawn from the most detailed cotton genetic map available (described in Rong, et al., 2003). We gave first priority to sequence-tagged sites derived from genomic DNA, which contained simple-sequence repeats, to increase the likelihood of polymorphism. Polymerase chain reaction (PCR) primers were designed using standard techniques, optimized on a single A-genome accession, A1-97, then used to amplify the diploid panel, including Gossypium arboreum (A2), G. herbaceum (A1), G. trilobum (D8), G. raimondii (D5), G. sturtianum (C1), and outgroup G. kirkii. A 5-µL aliquot of each primer product was run on a 1 percent agarose gel to determine if the products were clean and concentrated enough for sequencing. Optimization of PCR amplifications were performed through the manipulation of variables such as annealing temperature and salt concentration. The MJ Scientific PTC-200 Gradient Thermocycler was instrumental in the optimization of PCR because of its ability to simultaneously test the reaction efficiency of multiple annealing temperatures for a single template. PCR products were only deemed suitable for sequencing if single, high-quality bands were clearly observed in the agarose electrophoresis gels.

It is noteworthy that amplification success/failure has in some cases shown phylogenetic congruity; for example, A-genome works and D-genome fails, or vice versa; or A and D work, but C genome fails, etc. The key to obtaining adequate sequence quality has been PCR product cleanup. Three different procedures for the cleanup of PCR products prior to sequencing were attempted. Currently, the Exo/Sap procedure is being used because it is considerably less expensive than the Millipore plate alternative. Finished-cycle, sequencing reaction products are cleaned for sequencing using Sephadex filter plates prepared in our laboratory. The products first are treated with a dilute (2.2 percent) sodium dodecyl sulfate solution and then transferred to the Sephadex plates. The cleaned products are filtered directly into Perkin-Elmer MicroAmp Optical 96-well reaction plates. The filtered sequence reaction products then are placed directly on an ABI Prism 3700 automated DNA sequencer (Applied Biosystems, Foster City, CA). Information pertaining to each read, such as primer used and genotype, is associated with each chromatogram file by importing plate records containing all of the desired information. As of August 27, 2003, a total of 2,542 sequence reads have been attempted for almost 400 different primer-pair combinations (almost twice the 207 primer pairs that had been evaluated as of the last quarterly report).

Completed sequence runs are moved to a desktop computer for further analysis. Backups of raw sequence data are placed on the laboratory's Novell server, "Bigboy," as well as on CD-ROM. Chromatogram files are imported to a desktop computer with the Red Hat Linux operating system, and loaded into the Phred software package (Ewing, et al., 1998). This program is used to call bases and generate quality values for each base of each read. These data then are processed using a small program written in our laboratory using the programming language, Python. This program calculates quality statistics for each read and removes all reads that do not meet the minimum threshold of 100 base pairs, with a Phred-assigned quality score (Q-score) greater than 16, and a minimum of 60 percent of the bases greater than Q-score 16. A multifaceted SNP discovery pipeline has been developed, integrating Phred/Phrap, Consed, and PolyBayes.

A total of 1,164 candidate polymorphisms were phylogenetically distributed as follows. As expected, the majority of polymorphisms segregated the A, C, and D genomes from the outgroup, G. kirkii. A total of 365 polymorphic loci effectively segregated the A genome from the D genome. These data are from all sequencing done prior to August 29, 2003.

Conclusions:

The level of polymorphism between the subgenomes of tetraploid cotton appears to be sufficient to provide for the design of nested primer sets that can be used to screen tetraploid cotton at single loci for SNPs.


Journal Articles on this Report : 1 Displayed | Download in RIS Format

Other subproject views: All 1 publications 1 publications in selected types All 1 journal articles
Other center views: All 211 publications 48 publications in selected types All 44 journal articles
Type Citation Sub Project Document Sources
Journal Article Rong JK, Abbey C, Bowers JE, Brubaker CL, Chang C, Chee PW, Delmonte TA, Ding XL, Garza JJ, Marler BS, Park C-H, Pierce GJ, Rainey KM, Rastogi VK, Schulze SR, Trolinder NL, Wendel JF, Wilkins TA, Williams-Coplin TD, Wing RA, Wright RJ, Zhao XP, Zhu LH, Paterson AH. A 3347-locus genetic recombination map of sequence-tagged sites reveals features of genome organization, transmission and evolution of cotton (Gossypium). Genetics 2004;166(1):389-417. R829479 (2006)
R829479C009 (Final)
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  • Supplemental Keywords:

    single nucleotide polymorphism, SNP, resistance-gene, cotton, polymerase chain reaction, PCR, PTC-200 Gradient Thermocycler, amplification, phylogenetic, phyton, polymorphism, A genome, D genome, polymorphic loci., Scientific Discipline, TREATMENT/CONTROL, Sustainable Industry/Business, Geochemistry, Genetics, Technology, New/Innovative technologies, Agricultural Engineering, bioengineering, resistant-gene analogs, plant genes, cotton, environmental engineering, biotechnology, plant biotechnology, SNP variation, single nucleotide polymorphism

    Relevant Websites:

    http://www.plantgenome.uga.edu Exit
    http://www.cpbr.org Exit

    Main Center Abstract and Reports:

    R829479    The Consortium for Plant Biotechnology Research, Inc., Environmental Research and Technology Transfer Program

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R829479C001 Plant Genes and Agrobacterium T-DNA Integration
    R829479C002 Designing Promoters for Precision Targeting of Gene Expression
    R829479C003 aka R829479C011 Biological Effects of Epoxy Fatty Acids
    R829479C004 Negative Sense Viral Vectors for Improved Expression of Foreign Genes in Insects and Plants
    R829479C005 Development of Novel Plastics From Agricultural Oils
    R829479C006 Conversion of Paper Sludge to Ethanol
    R829479C007 Enhanced Production of Biodegradable Plastics in Plants
    R829479C008 Engineering Design of Stable Immobilized Enzymes for the Hydrolysis and Transesterification of Triglycerides
    R829479C009 Discovery and Evaluation of SNP Variation in Resistance-Gene Analogs and Other Candidate Genes in Cotton
    R829479C010 Woody Biomass Crops for Bioremediating Hydrocarbons and Metals
    R829479C011 Biological Effects of Epoxy Fatty Acids
    R829479C012 High Strength Degradable Plastics From Starch and Poly(lactic acid)
    R829479C013 Development of Herbicide-Tolerant Energy and Biomass Crops
    R829479C014 Identification of Receptors of Bacillus Thuringiensis Toxins in Midguts of the European Corn Borer
    R829479C015 Coordinated Expression of Multiple Anti-Pest Proteins
    R829479C016 A Novel Fermentation Process for Butyric Acid and Butanol Production from Plant Biomass
    R829479C017 Molecular Improvement of an Environmentally Friendly Turfgrass
    R829479C018 Woody Biomass Crops for Bioremediating Hydrocarbons and Metals. II.
    R829479C019 Transgenic Plants for Bioremediation of Atrazine and Related Herbicides
    R829479C020 Root Exudate Biostimulation for Polyaromatic Hydrocarbon Phytoremediation
    R829479C021 Phytoremediation of Heavy Metal Contamination by Metallohistins, a New Class of Plant Metal-Binding Proteins
    R829479C022 Development of Herbicide-Tolerant Energy and Biomass Crops
    R829479C023 A Novel Fermentation Process for Butyric Acid and Butanol Production from Plant Biomass
    R829479C024 Development of Vectors for the Stoichiometric Accumulation of Multiple Proteins in Transgenic Crops
    R829479C025 Chemical Induction of Disease Resistance in Trees
    R829479C026 Development of Herbicide-Tolerant Hardwoods
    R829479C027 Environmentally Superior Soybean Genome Development
    R829479C028 Development of Efficient Methods for the Genetic Transformation of Willow and Cottonwood for Increased Remediation of Pollutants
    R829479C029 Development of Tightly Regulated Ecdysone Receptor-Based Gene Switches for Use in Agriculture
    R829479C030 Engineered Plant Virus Proteins for Biotechnology