Multi-State Project SDC-317:Genetic improvement approaches to sustained,

profitable cotton production in the United States

From October 1, 2007 to September 30, 2012.

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STATEMENT OF ISSUES AND JUSTIFICATIONSustained, profitable cotton production (Gossypium hirsutum L. and G. barbadense L.) in the U.S. is under increasing pressure from a number of sources. Foremost among these is the competition that natural fibers are facing from synthetic fibers for the manufacture of yarns and textiles. Nearly as significant is a fundamental shift that has taken place in the market for cotton lint, viz. a viz. from a primarily domestically consumed product to one in which nearly two-thirds of U.S. production must compete successfully on the world market with cotton lint produced by overseas countries. To remain competitive with synthetic fibers and with other cotton producing countries, further improvements in the genetic potential for yield and fiber quality are needed.

Failure to address the needs of our producers and their customers will lead to a marginalization of cotton production in the U.S. and the loss of one of our most important agricultural exports. This loss would be exacerbated since the infrastructure of the cotton industry cannot be reapportioned like the grain industry. Cotton is the leading textile fiber and second most important oilseed in the world. Ranked second in world cotton production, the U. S. grows about 14 million acres of cotton per year, with acreage in all southern states, from Virginia to California and from Kansas and Missouri to the lower Rio Grande Valley of Texas. The U.S. cotton industry is a $25 billion/yr industry and generates over 400,000 domestic jobs that are critically needed in farm-based communities. Clearly, the cotton crop is a significant contributor to the U.S. economy, but especially to that of rural America.

Analyses of historical cotton yield data indicate that genetic gain or progress in increasing yield has declined over the past several years (Meredith 2006). There is not a consensus as to the underlying cause of this decline in genetic gain potential. Previous responses to this reduction in progress included the 5 to 10 yr delay often seen as a common feature in conventional plant breeding efforts and the decline in publicly funded research for the development of improved cotton germplasm and cultivars. Such research in the past has had a significant, positive effect on cotton improvement in both the public and private sectors. Germplasm development efforts are often long-term and have little profit potential and thus not attractive to the private sector. Policy, funding, and regulatory changes over the last decade have also had an impact on the development of improved cotton cultivars/germplasm. Many of the efforts devoted by public institutions such as State Agricultural Experiment Stations and the USDA-ARS to develop cotton cultivars have been suspended and emphasis placed on germplasm improvement and characterization, redirected to other research initiatives, or the positions have been eliminated as the result of budgetary constraints. While germplasm development within the public sector will have future positive impacts on cotton cultivar development, it continues to limit the availability of elite improved genetic material for direct use in the short term. Regulatory changes and legal protection of intellectual property continue to restrict the free exchange of germplasm necessary for swift future improvement.

A third suggestion is that the focus of the cottonseed industry on the addition of highly beneficial transgenic traits, such as Bt and herbicide-resistance, have come at a cost in research time and effort on the overall improvement of yield and fiber quality of new cotton cultivars. Also, the increased reliance of the seed market on private breeding activities and the merging of those

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firms with other agricultural support companies may leave the cotton industry vulnerable to the financial stability of a small number of commercial breeding programs.

Whatever the underlying causes, genetic improvement is the best choice for increasing yield and improving fiber quality, and thus enhancing profitability for producers, manufacturers, marketers, etc. The challenges that face the cotton industry transcend state boundaries and can be most economically and efficiently addressed by linking the public research institutions (federal and state) with their multidisciplinary skills, and in partnership with private cotton researchers through a multi-state research project.

Continued genetic gain in all economic aspects of cotton production will require the utilization of both applied and molecular genetic approaches focused on identification of genes responsible for traits of interest, characterization of and use of exotic germplasm to expand the genetic base from which genetic gain is possible, and the incorporation of genes coding for traits of interest into phenotypic constructs where desirable epistatic interactions are maximized. These approaches follow current, progressive research techniques that are being used in cotton and other species.

RELATED, CURRENT, AND PREVIOUS WORKThe preceding MultiState Project (S-304) made progress on all of its objectives but additional work remains. Previous projects, S-258, S-77, and initially S-1 as well as S-304, have impacted every area of the genetic improvement of cotton. The Terminal Report for the S-304 MultiState Project can be found in Appendix A. No other cotton multistate project concerning the genetic improvement of cotton is currently found in the National Information Management and Support System (NIMSS) that is maintained by the University of Maryland.

A basic measure of success is the elite germplasm that was shared and exploited as part of the S-304 project. Contributions were made by and to both public and private cotton breeding programs. The new released elite germplasm lines and varieties include; from the USDA-ARS, Florence, SC: PD 94045 (Upland cotton germplasm lines); from the University of Georgia: GA96-211, GA98028, GA 98033, and GA98066 (Upland cotton germplasm lines) with GA 161 (an Upland cultivar); from the USDA-ARS in cooperation with the University of Arizona: 93252, 93260, 94217, 94218, and 94220 (G. barbadense germplasm lines); from Texas A&M University: TAM 94L-25, TAM 94J-3, TAM 94WE-37s, TAM 96WD-69s, TAM 98D-99ne, TAM 96WD-18, and TAM 98D-102 (Upland cotton germplasm lines) with Tamcot 22 (an Upland cultivar); from Texas Agricultural Experiment Station-MAR: CD3HG2CABS-1-91, CD3HGCBU8S-1-91, LBCBHGDPIS1-91, CUBQHGRPIS-1-92, PD23CD3HGS-1-93, CBD3HGDPIH-1-91, LBCHUD3HGH-1-91, CD3HGCULBH-1-91, CDRCIQCUBH-2-92, CDARCILBCH-1-92, and CUBQHGRPIH-1-92 (Upland cotton germplasm lines); from the University of Arkansas: Arkot 8712 (Upland cotton germplasm line); and from Mississippi State University: MISCOT 8806 and MISCOT 8839 (Upland cultivars). Additional germplasm lines and cultivars will be released in the near future that are directly related to S-304 activities. Publicly developed germplasm lines such as these are necessary more today than ever given the legal entanglements generated by private industry (and public Agricultural Experiment Stations in some cases) in efforts to gain intellectual property rights and thus reduce the free movement of germplasm among cultivar developers.

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Acquisitions of valuable and unique new cotton germplasm from plant explorations have been conducted in more than 20 nations. These and significant contributions from exchanges with Brazil, the Peoples Republic of China, India, and Zimbabwe as well as Russia and Uzbekistan have been made to the USDA Plant Germplasm Collection. These germplasms are available for exploitation by the cotton research community world wide without restrictions. Conservation of our genetic resources is an important aspect of previous Multistate Projects and must be continued to insure that valuable genes are not lost from the species.

Distribution of acquired accessions impacts germplasm and varietal improvement in all areas of cotton production worldwide. In total, 16,773 accessions were distributed to cotton researchers in the U. S. and other nations between 2001 and 2004 in response to 405 seed requests. Approximately 6,000 accessions maintained in the U.S. Cotton Germplasm Working Collection were increased by seed propagation, both to assure their preservation and to make sufficient quantities of seed available to others. More than 1,100 of the increased accessions were recent acquisitions from Uzbekistan and Russia. More than 1,000 G. hirsutum and G. barbadense accessions were utilized by participants in S-304 in identification of GRIN descriptors, yield potential, and insect, nematode, and disease resistance. Given that most of the cotton germplasm accessions have not been adequately evaluated, such activities within the extant accessions are a priority in this project.

Diversity is necessary as the foundation of genetic improvement. Geomorphological data with genetic fingerprinting is needed to understand the relationships between cotton lines and the population structure of the Gossypium species. The cotton around the Caribbean basin was evaluated for its genetic diversity and to determine the origin of the Florida wild cotton. The genetic diversity of the Gossypium species of the G genome, indicate that accessions from Western Australia are significantly different from eastern (Queensland) accessions. Genetic characterization of almost 300 different American and Russian cotton types reveal the genetic similarities and differences between these cottons of widely differing origin. Genetic diversity among almost 500 landraces was assessed to find that SSR marker data was more helpful than the morphological data in characterizing the diversity of the landraces according to their origin of collection. Diversity of these Mexican landraces was highest for accessions collected within the states of Guerrero, Yucatan, Oaxaca, Veracruz and Chiapas. Genetic diversity of 346 obsolete and current germplasm lines were clustered into three main groups which were typified by the Texas A&M University MAR program, the USDA-ARS Pee Dee program, and the Acala type. Information from diversity research will be useful to breeders as they identify parents for use in crosses to maximize genetic diversity in their crossing programs which will remain as the foundation of this collaboration of information.

Genetic maps of the cotton genome have been developed that provide a solid basis for future cotton improvement efforts. The molecular marker linkage maps that have been assembled for G. hirsutum and G. barbadense are not fully saturated even though there are some that have attained 26 linkage groups (Rong et al. 2004). These maps need to be more completely saturated and additional, new maps are needed to extend the utility of linkage maps since each segregating population is novel.

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During the period of the past project, cooperators have constructed components of an integrated genetic and physical map of the cotton genome. Cytogenetic tools provided a set of 17 chromosome substitution backcross lines (BCnF1) that are genetically identical except that each

differs by the replacement of a specific homologous pair of chromosomes from Pima 3-79 (G. barbadense) into Upland cotton (G. hirsutum). The interspecific backcrossed chromosome substitution lines provide unique opportunities to detect the effect of the group of genes that a specific chromosome carries and thus aid cotton genome mapping programs. It is also a novel resource to plant breeders to overcome the problems of genomic incompatibility at the whole genome level between the two species and create a unique set of chromosome comprehensive germplasm introgression products in Upland cotton. The set (26) needs to be completed to help finish the integration of the physical and molecular maps. Development of new chromosome substitution series for G. tomentosum and G. mustelinum will also be of great value.

More markers are needed in a system that provide polymorphisms within the populations to be studied, that are inexpensive, and have high throughput; all are necessary for effective breeding. A standardized 12-cotton DNA genotype panel, foundation for systematic screening of cotton DNA markers, was developed to assist in the development and mapping of additional cotton SSR markers (Blenda et al., 2006) including those recently derived from TM-1 BAC libraries. The standardized panel consists of 12 diverse genotypes including genetic standards, mapping parents, BAC donors, subgenome representatives, unique breeding lines, exotic introgression sources, and contemporary Upland cottons with significant acreage.

Even though over 1,000 SSR cotton primers have been developed from positive BAC clones and another 1,000 SSR cotton primers are being designed from small-insert genomic clones, more are needed. Also, the feasibility of using the Single Nucleotide Polymorphism (SNP) detection system (Koebner and Summers 2003) has been shown in other allopolyploids and is better suited for use in intraspecific marker-assisted breeding. Preliminary work has been completed in cotton and further work will be given a higher priority. Another method to develop markers uses techniques to screen differentially expressed genes. This has detected significant variation within the functional genes in cotton fiber quality which in turn will provide functional polymorphisms so we can select for the actual genes of these important characters.

Intraspecific and interspecific QTL mapping (Guo et al. 2003, Zhang et al. 2003, Mei et al. 2004, Chee et al. 2005a, Chee et al. 2005b, Draye et al. 2005) using the available markers for traits such as fiber quality have given a starting point for Marker Assisted Selection (MAS). These and new mapping populations are required to bring us realistically to the attractive benefits that MAS brings in stacking multiple genes needed to enhance the value of the new cultivars. Some of these interspecific populations from G. barbadense, G. tomentosum, G. mustelinum, and G. darwinii are also useful to develop near-isogenic introgression lines that can be used to extract useful traits. Many synthetic tetraploids, from hybrids made between the A genome (G. arboreum) accessions and D genome species (G. trilobum, G. raimondii and G. aridum) as well as crosses of G. herbaceum x G. aridum, G. davidsonii x G. anomalum and G. hirsutum x G. laxum, have been developed that are also available to obtain novel traits. A new synthetic hybrid, made from cross-pollinations of G. arboreum with a 2(ADD) genetic stock to make trispecies hybrids with 2(AD) genomic constituencies, was backcrossed to an elite line of upland cotton to begin development of an introgression population.

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Efforts to better utilize the available germplasm led to the conversion of race stocks of G. hirsutum to eliminate photoperiodism by a conversion program using backcrossing. This program needs to continue in order to make all of the photosensitive G. hirsutum Race Stocks photoinsensitive and thus more easily available to plant breeders world wide. Other segregating populations are being developed as a basic component of breeding as well as investigating academic questions within the breeding effort such as general- and specific-combining ability, and the relationship between genetic relatedness (as determined by molecular analyses) and yield, fiber, and agronomic data.

Even more distant introgression from the quarternary gene pool such as Bt cotton must be supported. A first step is to work toward developing a genotype-independent transformation technology to modify cotton cultivars. Not being able to transform any desired genotype is a bottleneck in providing elite trangenic cultivars to the growers and must be overcome. One of several approaches included an effort to increase the number of genotypes that can be genetically modified by increasing the alleles for regeneration potential in the cotton gene pool. Concurrently, elite breeding lines developed by the UGA cotton breeding program were found to have enhanced somatic embryogenesis, the basis of successful transformation (Sakhanokho et al. 2004). Additional improvements to callus induction medium increased the range of genotypes that can be transformed and regenerated as well as technical refinements that reduce the time needed to regenerate transformed plants to 6-8 months (Wilkins et al. 2004).

Genotypes with drought and heat resistance and resistance to pests must be identified and incorporated into lines with high yield and fiber quality potential. This must be accomplished to reduce risks and production costs in order to increase grower profit. We also have to react when new disease races emerge to find and introduce new resistance genes. Such is happening now as we look for highly resistant germplasm to FOV race 4 and develop genetic mapping populations to further study FOV resistance for this race and its heritability. It is also possible that new technologies and methodologies may unbalance the crop production scheme that is presently in place. With the success of Bt cotton, plant bug infestations is an example of an achievement creating new challenges. Obsolete cultivars, breeding lines, and nectariless isolines (F3 to F10) were grown under plant bug infested conditions. Even though the reduction in fiber quality due to plant bugs was minimal, the nectariless trait appeared to have conferred a yield advantage for a number of entries.

Nematode resistance is highly coveted because these pests are hidden underground and not directly visible. Researchers in Georgia, Mississippi, and Texas are looking for tightly linked markers to the resistance genes. Screening for nematode resistance is difficult and tedious so searching for molecular markers linked to resistance is a high priority. In Mississippi, several sequences homologous to fungal wilt resistance genes have also been cloned and are being further characterized since RKN resistant isolines are also known to show resistance to fungal wilt. Converted race stocks from within G. hirsutum are being used successfully to develop advanced strains and segregating populations with resistance to nematodes as well as screening for resistance to seed-seedling disease, cotton fleahopper, and silverleaf whitefly. Resistance sources for a second serious nematode pest, reniform nematodes, are coming from complex interspecific hybridization efforts using G. tomentosum, G. mustelinum, G. longicalyx and G. armourianum. Integrating resistance for both of these nematodes, plants from a cross of M315

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(root-knot resistant) and TX 110 (putatively reniform resistant) have been screened and resistant candidates identified for resistance to both nematodes.

While looking for desired traits such as increased yield, improved fiber quality, and enhanced resistance to abiotic and biotic stresses, improving breeding efficiency and effectiveness continues to be important. Evaluating the visual rating of yield showed a positive correlation between visual ratings and seed cotton yield (Bowman et al. 2004). Since some high-yielding genotypes are occasionally not visually chosen even though high-yielding genotypes are identified, placement of checks and use of the grid system should reduce the chances of eliminating these better lines. In cotton breeding, recent efforts to improve yield have focused increasingly on breeding for the improvement of specific yield components; notable among these is the number of fiber/seed and weight of fiber per seed. Further research on yield and fiber quality components will bring great advancement in selecting for increased yield and improved fiber quality. Characterization of traits that are not primary to yield and fiber quality such as the variability for bract trichomes will continue to be valuable to assess cultivars for improved performance in ginning. Methods to manipulate populations have been compared such as investigating the genetic gain from using the pedigree vs. the Single Seed Descent (SSD) plant breeding approach.

Numerous, essential yield tests to elucidate the cultivars of choice throughout the cotton belts are always taking place. Nothing can take the place of these tests; however it may be that the efficiency in selecting the best genetic material could be improved. A comparison of experimental design efficiency between Lattice and Randomized Complete Block is being analyzed. Nearest Neighbor Analysis (NNA), ANOVA, and TREND analyses were compared across five states on various trials, but each gave different results with no one design best overall. More work is required to determine the effectiveness of our statistical analyses. Improvement may also be accomplished by the use of early generation testing (EGT) methods. However, tests to predict advanced strain performance based on EGT of lint yield, lint percent, and fiber quality (micronaire, fiber length, fiber strength, fiber elongation, and fiber uniformity) lack consistent r values across generations and locations which suggest that a large environmental influence on both lint yield and fiber quality parameters exists (Jones and Smith 2006). Further refinements may be valuable.

Proper parental selection, a key step in breeding, is critical to continued progress for increased yield, enhanced fiber quality, and profitability in our new cultivars. Pedigree information has been updated and includes 283 new upland and 10 new Pima cultivars. Using this new information, genetic uniformity measured as Coefficient of Parentage (COP) has not increased since the introduction of transgenic varieties (Bowman et al. 2003). However, COP is not highly correlated to genetic uniformity based on molecular markers (Van Becelaere et al. 2005).

Practical interaction in cotton breeding between transgenic and conventional cotton leaves a lot of questions in keeping the two genetics separate. One third of the publicly developed strains in the 2002 Regional Breeders Testing Network were found to be an admixture with the RR transgene. Frequency of transgenes ranged from 0.230% to 1.892%. As this was only one of several possible transgenes (BG, BXN, LL, ?), observed frequencies are conservative as an estimate for overall GMO adulteration. The USA and European Union have proposed a 1%

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tolerance level for GMO contamination. Suggestions have been provided for screening publicly developed germplasm prior to release or exchange to limit the improper distribution of patented genes. Since cross-pollination earlier in breeding would be more problematic, GMO screening utilizing test strips in an early generation, followed by screening prior to the first sizable seed increase is suggested. GMO screening in combination with extra precautions against mechanical mixing and outcrossing should greatly reduce GMO contamination in publicly developed germplasm. Monitoring this problem must be maintained.

Another difficulty in working with cultivars that have transgenic technologies is the suggestion to separately test the cultivars of each transgenic system. Interaction of the cultivars with the transgenic systems was not detected indicating that the relative ranking of cultivars within systems should remain the same and may simplify field testing. However regarding main effects, cultivars need to be tested with the systems to reveal true yield potential for the growers.

Theoretical aspects of forward crossing were examined with such factors as relevance of transgenes, speed of market demand, capability of removing transgenes, genetic background, access to transgenes, and many other factors justifying whether one should forward cross. Backcrossing transgenes does have its advantages.

With all the breadth of data that this and the previous project develop, bioinformatic systems must be improved and maintained to drive our progress. A number of web-accessed databases have been developed to provide access to the data that has been gathered. This is a noteworthy aspect of the collaboration within the previous multi-state project. All of these sites have proven value and should be expanded and improved to be more user friendly.

Significant contributions to the CottonDB, http://www.cottondb.org, have been accomplished through addition of new information and reorganization. Data will contribute to the identification and exploitation of genetic variability at the molecular level for greatly improved efficacy in cotton germplasm improvement. Data from the Cotton Germplasm Collection is included in the GRIN database as well as in the CottonDB. The data classes were reorganized and updated with new information including cotton germplasm, cultivar trial, SSR clones and primers, BAC clones and fingerprints, and DNA sequences. Bioinformatic tools were incorporated into CottonDB for sequence blast and integration of genome maps. The USDA-ARS, College Station, TX also hosts the website of the International Cotton Genome Initiative, http://icgi.tamu.edu.

In collaboration with Cotton Incorporated, the standardized 12-cotton DNA genotype panel was established as the foundation of the Cotton Microsatellite Database (CMD), http://www.mainlab.clemson.edu/cmd/, for a systematic screening of cotton DNA markers developed by participants from the cotton community. At present it contains sequence, primer, mapping and homology data for nine cotton microsatellite projects (BNL, CIR, CM, JESPR, MGHES MUSB, MUSS/MUCS, NAU, and TMB). This provides a more efficient utilization of microsatellite resources and will help accelerate basic and applied research in molecular breeding and genetic mapping in Gossypium spp. The Cotton Functional Database, http://cfg.ucdavis.edu/microarrary.asp, at UC-Davis, Davis, CA allows access and manipulation of expression-related data that are now linked to clones, constructs, ESTs, genes, and SSRs. The Regional Breeders Testing Network (RBTN) provides a means for testing advanced breeding

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lines over a wide range of environments and also serves as vehicle for germplasm exchange among participants. The RBTN (http://cottonrbtn.com) has posted on its website the analyses for 2002, 2003, and 2004 with plans underway to include yield stability. The RBTN presently includes test sites in NC, GA, AL, MS, AR, LA, and TX. Other Cotton bioinformatics advances over the duration of the project include the development of “The Cotton Portal”, http://gossypium.info (which consist of various websites that provide query and display capabilities for performance trials, genetic maps, and comparative maps) and the Cotton Fiber Genomics Project, http://cottongenomics.tamu.edu).

FUTURE RESEARCH NEEDSThe results from S-304 established a solid basis for further research on the utilization of genetic resources to improve cotton. Current and additional germplasm accessions need to be evaluated and characterized for potential usefulness. Although considerable progress was made during the past five years, additional research is needed to develop new molecular tools to understand and manipulate our cotton germplasm. As additional sequencing of the cotton genome is completed and the sequencing data made available, research is needed to mine the data for useful genes. Enhancement of germplasm and cultivars through introgression and recombination for input and output traits must continue so that our industry remains competitive and profitable. This regional project needs to continue to provide both germplasm and methodology that will allow cotton breeders to address the needs for producers, industry, and the consumer by ensuring that this knowledge is available and disseminated on the internet web sites.

OBJECTIVESThe general goal of this Multi-State Project is to assist U.S. researchers in immediate and long-standing genetic needs of the U.S. cotton industry. To accomplish this, we will refocus the six areas of investigation established in S-304 into four by developing and exploiting new technologies relevant to germplasm, genetics and genomics, genetic improvement of cotton, and disseminating of information. The specific objectives of this proposal are:

1. Acquisition, curation, characterization, and evaluation of cotton germplasm for the improvement of cotton.

2. Understanding and manipulating the cotton genomes through traditional, cytogenetic, and molecular approaches.

3. Enhancing the profitability of cotton through germplasm enhancement, cultivar development, pest and disease resistance, and improved output traits.

4. Improvement of cotton bioinformatic systems and tools.

METHODSObjective 1: Acquisition, curation, characterization, and evaluation of cotton germplasm for the improvement of cotton (Chee, Percy, Yu, Weaver, etc.)Curating Gossypium accessions involves the coordinated efforts of researchers with a diverse set of skills. Acquisition may occur through plant exploration, the negotiated exchange of germplasm between international institutions, or the careful development of novel genetic combinations. Public and private sectors will acquire and contribute significant genetic material to the U.S. National Cotton Germplasm Collection which is comprised of the germplasm collected as seed during various expeditions, along with materials obtained as donations and

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exchanges with other germplasm banks around the world. The Collection, located in the USDA-ARS facility at College Station, TX, is a part of the National Plant Germplasm System (NPGS), and personnel in this ARS unit under the directive of Dr. Richard Percy are responsible for the acquisition, maintenance (including multiplication and preservation), and distribution of those germplasm resources. Data on the materials maintained is accessible through the Germplasm Resources Information Network (GRIN) [http://www.ars-grin.gov/npgs]. Multiple accessions of most of the 49 recognized Gossypium species are maintained as seed.

The collection will be expanded and maintained as five subcollections representing different categories of germplasm by collaborative efforts of researchers across the cotton belt. Duplicate seed lots will be stored in the working collection at the Crop Germplasm Research Unit, College Station, TX, and in the base collection at the National Seed Storage Laboratory, Ft. Collins, CO. The collection consists of five subcategories:

1. OBSOLETE CULTIVARS (VARIETIES): Released cultivars, with rare exceptions, of G. hirsutum that are no longer commercially popular as well as recent releases that are not protected under PVP. Entries in this collection carry an "SA" number designation.

2. LANDRACE COLLECTION: Wild, feral, and "dooryard" (few or single plants, usually grown for home use) seed of G. hirsutum. Of these, 25% have been classified according to geographic-morphological races; palmeri, richmondi, punctatum, latifolium, marie-galante, morilli, and yucatanense. These are identified with a "TX-" designation.

3. G. barbadense COLLECTION: All germplasm is maintained with a "GB-" designation and includes cultivars (old and recent), dooryard, and wild accessions.

4. ASIATIC COLLECTION: Cultivated and wild A-genome cotton G. herbaceum and G. arboreum in this collection carry an "A1-" and "A2-"designation, respectively.

5. WILD SPECIES COLLECTION: currently, multiple accessions of three wild tetraploid species and 38 wild diploid species.

We will continue the exchange of collections with other domestic and international germplasm collections. The new and present cotton germplasm will be characterized and evaluated by public and private sectors and then documented as part of the bioinformatic systems that are also in development and part of this proposal. Given that a small percentage of cotton germplasm types have been adequately explored, evaluation of the extant germplasm is a priority. More accessions must be characterized and evaluated for information ranging from GRIN descriptors and yield, to disease and pest resistance, to tolerance of abiotic and biotic stresses. With nearly 10,000 accessions currently in the US cotton collection, the agronomic, physiological, pest, fiber, and molecular attributes of them cannot be determined by any one research program. Multi-state cooperation is needed to provide accurate, precise characterization and to coordinate activities on evaluation of a significant number of unique accessions with potentially useful genes. This project, through its annual meetings will provide a mechanism to approach the characterization of accessions in the germplasm bank systematically and ensure that duplicate efforts are mimimized. Additionally, recently proposed enhancements to the GRIN descriptor list (which includes cross references to data in other international cotton germplasm databases) will provide a more unified framework for incorporating data on the accessions, including molecular data. Institutions and individuals who have prior experience and commitment to these activities aare: Chee – GA and Yu – TX: genetic diversity based on DNA markers; Weaver – AL and Stewart -

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AR: reniform nematode resistance; Myers – LA, Gannaway and Smith – TX, Wallace and Thaxton – MS: fiber quality (HVI).

Seed increases to support proposed activities of Objective 1 will be made at three locations. Photoperiodic stocks will be increased at the Cotton Winter Nursery, Tecoman, Colima, Mexico. Day-neutral stocks will be increased at Rio Farms, Monte Alto, TX. Wild species will be increased in greenhouses at College Station, TX. Distribution of seed will be made from the working collection (USDA-ARS-TX)

Objective 2: Understanding and exploiting the cotton genome through traditional, cytogenetic, and molecular approaches (Chee, Stelly, Stewart, Turley, Magill, Zhang, etc.)The cotton genome is complex and requires the best efforts of research institutions across the U.S. to be fully utilized for the benefit of domestic producers. Traditional plant genetics research (e.g. mating designs, variance component estimation, heritability calculations, and inheritance studies) have made significant historical progress that has provided a scientific basis for germplasm and cultivar development efforts. Advances in genetic research methodology have made possible the dissection and analysis of plant genomes at the molecular level. Cytogenetic investigations to help understand the gross and fine structure of chromosomes provide a critical link in the development of a unified genetic map.

Additional efforts will be placed in developing new linkage maps for novel crosses and placing more markers on the present research populations. This project will help by providing a forum for research coordination and discussion which will foster increased collaborative efforts among laboratories in development of additional molecular markers and marker systems for a more complete saturation across the genome, increased refinement of the present genetic maps, and for a greater utility in breeding. Project participants will focus their efforts on using the SSR marker system because of their ease in use and greater polymorphic potential than previous systems which makes them more suitable for breeding elite lines. A common panel of publicly available SSRs will be compiled and shared among participating laboratories for use in various genetic and QTL mapping experiments.

The standardized 12-cotton DNA genotype panel from S-304, foundation for systematic screening of cotton DNA markers, was developed to assist in the development and mapping of additional cotton markers. The newly produced portable markers will be screened against this panel to provide a common starting point in genetic analysis and to identify a core marker set (highly polymorphic) so that a common point of interaction can be generated among multiple mapping populations. Continued development of portable markers such as RFLPs, RAPDs, AFLPs, SSRs, SNPs, and CAPs must continue, but not to exclude any additional subsequent systems. A web-distributed, group-approved, standardized nomenclature for cotton molecular markers, ESTs, cDNAs, etc. which includes embedded information as to lab source and/or developer will be established.

Development of cytogenetic stocks has not been completed to mark all of the G. hirsutum chromosomes by hypoaneuploidy. Those that have been developed and the other stocks in the Cotton Cytogenetic Collection will be maintained by Stelly lab (TX) which has committed itself to making these available to the community. Additional chromosome-

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doubled derivatives of interspecific F1 hybrids along with chromosome and chromatin substitution lines of the primary and secondary gene pool will be developed and placed in the Cotton Cytogenetic Collection. These can be used as points of targeted introgression so the evaluation of traits, markers, genes, expression, and linkage groups has great value.

Genetic diversity will be enhanced by these chromosome/chromatin substitution lines and other chromatin substitution lines developed using methodologies such as the Advanced Backcross QTL system (Tanksley & Nelson 1996). Both cytogenetic and molecular genetic systems will provide new combinations as well as introgressing both the tetraploid and diploid into the primary gene pool. Specifically, Chee (GA) will coordinate the effort to create new gene combinations by introgression using tetraploid cottons G. barbadense and G mustelinum, Stelly (TX) and Stewart (AR) will develop populations from diploid cottons G. arboreum and G. longicalyx. These populations and intraspecific populations (converted race stocks, race Yucatanense introgression, etc) will also be a central part of developing MAS using DNA markers linked to QTLs and qualitative genetic traits. Developing isoline series of these introgressions will maximize their utility. Additional research using molecular fingerprinting on the genetic population structure of Gossypium will provide further understanding on the relationships among the individual lines and accessions in the collection.

Cytogenetic/physical maps and molecular genetic/linkage maps have been integrated but we must refine and synthesize the existing and new linkage, physical, and cytogenetic maps into a more complete, valuable consensus map. To support these activities, the Yu (USDA-ARX-TX) and Zhang (NM) lab will each develop and distribute RIL mapping populations that are important components of this consensus map. Of further interest is to establish a comparative map with Arabidopsis, especially with regard to conserved sequences, regions of macro- and micro-synteny, and relative rearrangement.

Utilizing a plant system approach to discover potential genes within the germplasm collection by screening differentially expressed genes in cotton has proven to be successful. Further efforts will provide functional polymorphisms to select actual genes of important characters as well as a valuable resource to develop additional markers. Arabidopsis ESTs will be evaluated against ESTs from the cotton germplasm collection and the information from the cotton BAC library to look at cotton functional genomics. Exploring and expanding the utility of microarrays for comparative analyses of genotypes, physiological/ developmental stages, organs, tissues, and cell types to efficiently speed along the understanding of cotton genomics is a research area envisioned during this projects tenure

Tissue culture and transformation technologies are advancing but further improvements are yet possible in developing genotype-independent transformation technologies. Stacking alleles for regeneration potential in the cotton gene pool, finding enhanced somatic embryogenesis within current elite cultivars, and improving callus induction techniques are some of the methods that still have further potential (GA, TX?)

Objective 3: Enhancing the profitability of cotton through germplasm enhancement, cultivar development, pest and disease resistance, and improved output traits (Chee, Myers, Smith, Weaver, Zhang, etc)

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Germplasm enhancement efforts have become increasingly dependent upon the incorporation of new genetic diversity into usable backgrounds. In the case of exotic germplasm, we will use advanced crossing schemes and other introgression techniques to improve productivity potential and quality characteristics. Cultivar development research must increasingly use expanded testing and statistical techniques, e.g. spatial and genotype x environment analyses, to effectively identify stable, high yielding cultivars.. Improving output traits will require coordinated research efforts using a variety of techniques such as continued application of the Regional Breeders Testing Network (RBTN) (http://cottonrbtn.com). Thirteen breeding/genetics programs in North Carolina, South Carolina, Georgia, Alabama, Mississippi, Louisiana, Arkansas, Texas, and Arizona are now participating in the RBTN. The correlation of fiber properties to yarn performance is also being conducted by the members of the RBTN in collaboration with Cotton Incorporated (Cary, NC). Several public programs (USDA-ARS-AZ, CA and SC; LA, MS, GA) along with Cotton Incorporated are working to increase the heat tolerance of cotton with joint release of elite germplasm planned. This and similar coordinated efforts will remain important and serve as a measure of success.

Development of populations from both interspecific and intraspecific gene pools will continue to be essential to develop cotton germplasms. Members of this Multi-State Project will develop new populations and test methodologies to manipulate these populations with our present technologies, utilize recombination, and to decrease linkage drag in order to improve germplasm lines and cultivars. Methodologies useful in effectively selecting valuable traits introgressed from more distant gene pools such as the tetraploid and diploid Gossypium species will also be explored. Investigations to understand basic genetic questions that are directly useful in efficiently generating valuable parents will be initiated such as the relationship of the measures of general- and specific-combining ability with the genetic structure of the cotton germplasm pools.

Biotechnological innovations combined with germplasm evaluations will become important tools to surmount current and newly identified pests and to overcome abiotic and biotic stresses MAS will begin to be significantly employed using the QTL markers that have been previously found and those that will be located in further research from within this proposal. While traditional phenotypic selection will continue to be important, researchers in several of the states involvedin this project are looking into the development of selection indices that better correlate with end product (yarn and fabric) performance with important germplasm lines being developed for the wider cotton industry. This project will provide an additional mechanism for breeding programs and genetic research laboratories to work together to combine DNA markers and phenotypic screening approaches to provide greater efficiencies in selecting breeding lines with traits that are extremely tedious, time-consuming and expensive to manipulate. Initial focus will likely be on pathogenic soil nematodes (rootknot and reniform) with some additional efforts on fiber quality, heat tolerance, and drought resistance. For example, in the Chee (GA), Yu (USDA-ARS-TX), and Zhang (NM) labs, the focus will include a core of quality measures, specifically length, strength, fineness, elongation, length uniformity, and short fiber content, while in Stewart (AR), Stelly (TX) and Weaver (AL) lab, the focus will be on pests resistance such as reniform nematodes.

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Another approach is to re-examine how we achieve genetic gains, both in terms of methodology used to develop and evaluate new genotypes and in terms of the values of critical genetic parameters such as heritability. Genetic parameters are a function of the genetic populations they describe, so when genetic populations change, the genetic parameters of such traits as heritability of lint percentage and fiber quality of the population change. Many genetic parameters have been determined, but these studies were done some time ago and values may not apply to genetic populations currently in use. With the advent of new technology, such as greatly increased, readily available computer power, it may be feasible to utilize more sophisticated experimental design and data analysis than in the past. Design and analysis beyond the traditional ways may allow breeders to identify superior genotypes more accurately and dependably. Project participants will re-examine breeding methodologies and protocols for different post harvest analyses to compare overall effectiveness in the light of statistical advancements.

A preliminary study at the Delta Research and Extension Center indicated a moderate positive correlation between early generation performance and performance of descendent pure lines (Barut, 1998). However, tests to predict advanced strain performance based on EGT of lint yield, lint percent, and fiber quality (micronaire, fiber length, fiber strength, fiber elongation, and fiber uniformity) lacked consistent r values across generations and locations which suggest that a large environmental influence on both lint yield and fiber quality parameters exists (Jones & Smith, 2006). Smith (Texas A&M, TX) will further study this issue in cooperation with other breeders.

Even though visual rating of yield showed a positive correlation to seed cotton yield, additional fine-tuning is possible. Since some high-yielding genotypes are occasionally chosen that do not have a high visual rating, placement of checks and use of the grid system will be researched to reduce the chances of eliminating these better lines. Given that selection for yield per se has resulted in shifts among yield components (Bridge et al., 1971), integration of a visual rating with specific yield components not associated with the strong visual whiteness of the lint of open bolls may provide a superior index and save time in selecting elite breeding lines. Further selection will be prioritized for yield components with a relatively low environmental main effect/high genotypic main effect. The main effects and genotype by environment interaction of these traits will be reassessed and then corroborated across regional breeding programs (LA, MS)

Objective 4: Improvement of cotton bioinformatic systems and tools (Percy, Yu, Gingle etc.)Bioinformatics is playing an increasingly critical role in crop improvement. Keeping track of all the data that is increasingly been gathered requires a dedicated effort. Updates and improvements of bioinformatics systems will serve the cotton research community with greater efficiency. In addition to maintaining data from various types of cotton research, long-term management and coordination beyond the life of an individual grant is needed to sustain the utilization of the information and resources from the cotton germplasm and genome project.

The CottonDB, http://www.cottondb.org, which is housed in and maintained by the USDA-ARS in College Station, TX, will continue to be the keystone for documenting cotton genetic information. New data will continually be added along with improvements in the presentation and organization of the site. Data in the CottonDB from the Cotton Germplasm Collection will be included in the GRIN database. In addition, participants engaged in objective 2 in this project will submit data to contribute to the identification and exploitation of genetic variability at the

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molecular level for greatly improved efficacy in cotton germplasm improvement. Additional bioinformatic tools will be incorporated into CottonDB. The available annual reports of the previous cotton multi-state projects will be organized and archived as historical documentation for posterity in Adobe Acrobat PDF files.

Utilizing the standardized 12-cotton DNA genotype panel will be a foundation of the Cotton Microsatellite Database (CMD), http://www.mainlab.clemson.edu/cmd/, for a systematic screening of all cotton DNA markers developed by participants from the cotton community. The collection of all publicly available cotton SSR markers will be placed into a readily accessible web-enabled database to provide a more efficient utilization of microsatellite resources. These resources are available to the participants of this project as well as the general cotton research community.

Other cotton bioinformatic websites such as “The Cotton Portal”, http://gossypium.info (which consist of various websites that provide query and display capabilities for performance trials, genetic maps, and comparative maps) will be improved as the bioinformatics field advances. The Cotton Functional Database, http://cfg.ucdavis.edu/microarrary.asp, will be expanded as additional items such as clones, constructs, ESTs, genes, and SSRs are input. It will be improved to be more user friendly, allowing access and manipulation of expression-related data. The Regional Breeders Testing Network (RBTN) will offer a means for testing advanced breeding lines over a wide range of environments and also serve as vehicle for germplasm exchange among participants. The RBTN (http://cottonrbtn.com) will post on its website the analyses for all the years in existence with additional analyses such as yield stability.

MEASUREMENT OF PROGRESS AND RESULTS; OUTPUTS : Objective 1: Systematic and expanded characterization of the germplasm accessions for standard agronomic GRIN descriptors and opportunistic screenings for resistance to abiotic and biotic stresses; genetic materials which include seed and DNA stocks that are developed during the course of this project will be delivered to the appropriate clients: growers, researchers, or other interested parties.

Objective 2: Development and refinement of the molecular and cytogenetic inter- and intra-specific maps; development of more portable markers in systems with high throughput potential; developing genotype-independent transformation technologies; utilizing a plant system approach to discover potential genes within the germplasm collection; genetic materials, which include genomic clones and primer sequences that are developed during the course of this project will be delivered to the appropriate client.

Objective 3: Greatly enhanced genetic diversity of domesticated cotton derived from both the primary and secondary gene pools; reassessed traditional breeding designs and analytical methodologies while reviewing breeding techniques used with other species; continuing to develop breeding populations while developing protocols to utilize Marker-Assisted Breeding genetic materials which include elite breeding lines and cultivars that are developed during the course of this project will be delivered to the appropriate client.

Objective 4: Development and improvement of the bioinformatic systems to disseminate the information gathered in this project.

MEASUREMENT OF PROGRESS AND RESULTS; OUTCOMES/PROJECT IMPACTS :

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Objective 1: Maintenance of the cotton germplasm collection by continued acquisition, increase, and distribution of cotton accessions. Goal of increasing the characterization of the number of accessions in the collection to 20 percent.

Objective 2: Completed cytogenetic physical map, more completed saturation of molecular linkage maps, increased numbers of portable, high throughput, polymorphic molecular markers, an integrated physical and linkage map, enhanced transformation technologies.

Objective 3: Large numbers of released breeding lines with improved genetic diversity for yield, fiber quality, and tolerance to abiotic and biotic stresses, improved cotton breeding methodologies including utilizing MAS.

Objective 4: Maintained, up-to-date, user-friendly bioinformatic resources that provide timely access to the data developed by this project.

General Outcomes: Development of distributed elite breeding lines and cultivars; disseminated sources of resistance for abiotic and biotic stresses which include resistance to drought, heat, nematodes, plant bugs, and Fusarium; and any other improvements, including information that will decrease production costs, are direct project impacts and will be measured ultimately by the utilization of the breeding lines/cultivars purchased by the cotton grower. Quantification of this is practically impossible since the commercial seed companies now consider pedigrees and genetic resources as company secrets. The development of knowledge, tools, and basic genetic resources will have strong impacts in research steps that are initially somewhat removed from directly impacting the cotton industry but ultimately will have even greater impacts on the industry. Success in achieving these objectives will ultimately have a positive impact on cotton production and fiber quality which in turn will affect the profitability of U.S. cotton by making it more competitive against synthetic fibers and within the world market.

MILESTONES: Year Milestone2008 Completion of the physical cytogenetic map prior to integration of

the physical and linkage map.

Most of what we learn in one objective can be applied to other objectives in serial or in parallel with the harvest of the basic facts, a common trait in investigating such in a rich research area.

PROJECTED PARTICIPATION: see Appendix E (see guidelines)

OUTREACH PLAN:The release of the elite germplasm lines and cultivars will be published in the Journal of Plant Registrations (CSSA). Characterization and evaluation of germplasm accessions will be placed on-line using the new GRIN descriptor list via CottonDB. All cotton DNA markers will be placed on-line in the CMD. The RBTN web site hosts a U.S. meta-environmental trial analysis for public breeder to help address issues of yield and stability. The information will be reported within appropriate media including the annual MultiState project reviews, web-sites, refereed journals; popular articles; presentations and posters in state, national, and international meetings and conferences; breeder tours; producer workshops; and field days but not excluding other suitable means of communication. The collaborative nature of the research will be emphasized

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via such output as web sites, regional bulletins, multi-authored journal articles, joint germplasm releases, etc.

ORGANIZATION AND GOVERNANCEThe regional technical committee plans and coordinates research and performs other functions as specified in the "Manual for Cooperative Regional Research." The membership of the regional technical committee will include representatives from each participating Land Grant Institution USDA-ARS, and private industry. Appointments will be made by the appropriate administrator. The committee will also have an administrative advisor as appointed by the SAES and a CSREES representative; both of these positions will be nonvoting. Each participating public or private research group will have one vote. All voting members of the technical committee will be eligible to hold office. Regional technical committee offices will consist of a chair, a vice-chair, and a secretary. An executive committee, composed of current office holders, will conduct necessary business between annual meetings in consultation with the administrative advisor.

The regional technical committee will meet annually to summarize and critically evaluate progress, discuss results, and to plan data analyses, activities, reports, and publications. Notices for all meetings will be sent to participants. Meeting notices will also be sent by the administrative advisor to appropriate administrators. The chair will be responsible for compiling an annual report. The secretary will be responsible for preparing and distributing the minutes of meetings.

The scientific cooperators involved in this effort have developed an RRP that addresses key issues through a plan that orchestrates diverse genetic and breeding research efforts at multiple sites. Success of any major crop genetic improvement program, whether public or private, requires such multidisciplinary teamwork, extensive resources (sustained financial support, personnel, and facilities) and time. While many of the participating AES and ARS programs serve state and regional needs in an idiosyncratic manner, they also comprise a large part of the national public effort. Each group brings to the table unique research capabilities, technologies, and/or germplasm resources. This RRP builds on past interagency collaborations (S-304, S-258, S-77, S-1, and others) for the common good of the nation and offers greater success for each of the RRP’s respective state components. So as to avoid redundancy, extend the ramifications, and reduce cost, the RRP will take advantage, where possible, of external research components in the U.S. and elsewhere that are not official participants in the group effort.

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Wilkins TA. 2004. Functional genomics of cell elongation in developing cotton fibers. Plant Mol. Biol. 54:911-929.

Barut A. 1998. Early generation bulk testing for predicting F4:5 line performance in cotton. M.S. thesis. Miss. State Univ., Starkville.

Blenda A., J. Scheffler, B. Scheffler, M. Palmer, J.-M. Lacape, J. Z. Yu, C. Jesudurai, S. Jung, S. Muthukumar, P. Yellambalase, S. Ficklin, M. Staton, R. Eshelman, M. Ulloa, S. Saha, B. Burr, S. Liu, T. Zhang, D. Fang, A. Pepper, S. Kumpatla, J. Jacobs, J. Tomkins, R. Cantrell, and D. Main. 2006. CMD: A Cotton Microsatellite Database resource for Gossypium genomics. BMC Genomics 7:132 doi:10.1186/1471-2164-7-132.

Bourland FM, Johnson JT, Jones DC. 2005. Registration of Arkot 8712 germplasm line of cotton. Crop Sci. 45: 1173-1174.

Bowman DT, Bourland FM, Myers GO, Wallace TP, Caldwell WD. 2004. Visual selection for yield in cotton breeding programs. J. Cotton Sci. 8(2):62-68.

Bowman DT, May OL, Creech JB. 2003. Genetic uniformity of the U.S. Upland cotton crop since the introduction of transgenic cottons. Crop Sci. 43: 515-518.

Bridge RR, Meredith Jr WR, Chism JF. 1971. Comparative performance of obsolete and current varieties of upland cotton. Crop Sci. 11:29-32.

Chee P, Draye X, Jiang C-X, Decanini L, Delmonte TA, Bredhauer R, Smith CW, Paterson AH. 2005a. Molecular dissection of phenotypic variation between Gossypium hirsutum and G. barbadense (cotton) by a backcross-self approach: I. Fiber Elongation. Theor. Appl. Genet. 111:757-763

Chee P, Draye X, Jiang C-X, Decanini L, Delmonte TA, Bredhauer R, Smith CW, Paterson AH. 2005b. Molecular dissection of phenotypic variation between Gossypium hirsutum and G. barbadense (cotton) by a backcross-self approach: III. Fiber Length. Theor. Appl. Genet. 111:772-781.

Draye X, Chee P, Jiang C-X, Decanini L, Delmonte TA, Bredhauer R, Smith CW, Paterson AH. 2005. Molecular dissection of phenotypic variation between Gossypium hirsutum and G. barbadense (cotton) by a backcross-self approach: II. Fiber Fineness. Theor. Appl. Genet. 111:764-771.

Guo WZ, Zhang TZ, Shen XL, Yu JZ, Kohel RJ. 2003. Development of SCAR marker linked to a major QTL for high fiber strength and its molecular marker assisted selection in Upland cotton. Crop Sci. 43:2252-2256.

May OL. 2001. Registration of PD94045 germplasm line of Upland cotton. Crop Sci. 41:279-280.

May OL. 2004. Registration of GA98028 Upland cotton germplasm line. Crop Sci. 44:1882-1883.

May OL, Cantrell RG, Jones DC. 2005. Registration of GA98066 Upland cotton germplasm line. Crop Sci. 45:1175-1176.

May OL, Chee PW, Sakhanokho H. 2004. Registration of GA98033 Upland cotton germplasm line. Crop Sci. 44:2278-2279.

May OL, Davis RF, Baker SH. 2001. Registration of GA 161 cotton. Crop Sci. 41:1995-1996.May OL, Davis RF, Baker SH. 2004. Registration of GA96-211 Upland cotton germplasm line.

Crop Sci. 44:700-701.

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Mei M, Syed NH, Gao W, Thaxton PM, Smith CW, Stelly DM, Chen ZJ. 2004. Genetic mapping and QTL analysis of fiber-related traits in cotton (Gossypium). Theor. Appl. Genet. 108:280–291.

Haigler CH, Zhang D, Wilkerson CG. 2005. Biotechnological improvement of cotton fiber maturity. Physiologia Plantarum 124:285-294.

Jones DG and Smith CW. 2006. Early Generation Testing in Upland Cotton. Crop Sci. 46:1-5.Koebner RMD and Summers RW. 2003. 21st century wheat breeding: plot selection or plate

detection? Trends in Biotechnology 21:59-63.Kohel RJ, Stelly DM, Yu J. 2002. Tests of six cotton (Gossypium hirsutum L.) mutants for

association with aneuploids. J. Heredity 93:130-132.Liu S, Saha S, Stelly D, Burr B, Cantrell RG. 2000. The use of cotton aneuploid for the

chromosomal assignment of microsatellite loci. J. Heredity 91:326-332.Meredith WR. 2006. Obsolete conventional vs. modern transgenic cultivar performance

evaluations. 2006 Beltwide Cotton Conferences, San Antonio, Texas - January 3 - 6, 2006.Park YH, Alabady MS, Ulloa M, Sickler B, Wilkins TA, Yu J, Stelly DM, Kohel RJ, El-Shihy

OM, Cantrell RG. 2005. Genetic mapping of new cotton fiber loci using EST-derived microsatellites in an interspecific recombinant inbred line cotton population, Molecular Genetics and Genomics. 274(4): 428 - 441.

Rong J, Abbey C, Bowers JE, Brubaker CL, Chang C, Chee PW, Delmonte TA, Ding X, Garza JJ, Marler BS, Park C, Pierce GJ, Rainey KM, Rastogi VK, Schulze SR, Trolinder NL, Wendel JF, Wilkins TA, Williams-Coplin D, Wing RA, Wright RJ, Zhao X, Zhu L, Paterson AH. 2004. A 3347-locus genetic recombination map of sequence-tagged sites reveals features of genome organization, transmission, and evolution of cotton (Gossypium). Genetics 166:389-417.

Rong J, Pierce GJ, Waghmare VN, Rogers CJ, Desai A, Chee PW, May OL, Gannaway JR, Wendel JF, Wilkins TA, Paterson AH. 2005. Genetic mapping and comparative analysis of seven mutants related to seed fiber development in cotton. Theor. Appl. Genet. 111(6):1137-1146.

Saha S, Wu J, Jenkins JN, McCarty Jr JC., Gutierrez OA, Stelly DM, Percy RG, Raska DA. 2004. Effect of Chromosome Substitutions from Gossypium barbadense L. 3-79 into G. hirsutum L. TM-1 on Agronomic and Fiber Traits. J. Cotton Sci. 8:162–169.

Sakhanokho,HF, Ozias-Akins P, May OL, Chee PW. 2004. Induction of somatic embryogenesis and plant regeneration in select Georgia and Pee Dee cotton (Gossypium hirsutum L.) lines. Crop Sci. 44:2199-2205.

Smith CW. 2003a. Registration of TAM 94L-25 and TAM 94J-3 germplasm lines of upland cotton with improved fiber length. Crop Sci. 43: 742-743.

Smith CW. 2003b. Registration of TAM 94WE-37s smooth-leaf germplasm line of upland cotton with improved fiber length. Crop Sci. 43: 743-744.

Stelly D. 2004. Aneuploid mapping in polyploids. Encyclopedia of Plant and Crop Science. Marcell Dekker, Inc.

Tanksley SD, Nelson JC. 1996. Advanced backcross QTL analysis: A method for the simultaneous discovery and transfer of valuable QTLs from unadapted germplasm into elite breeding lines. Theor. Appl. Genet. 92: 191-203

Thaxton PM, El-Zik KM. 2003. Registration of eleven multi-adversity resistant (MAR-7A) germplasm lines of upland cotton. Crop Sci. 43: 741-742.

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Thaxton PM, Smith CW, Cantrell R. 2005a. Registration of ‘Tamcot 22’ high-yielding upland cotton cultivar. Crop Sci. 45: 1165-1166.

Thaxton PM, Smith CW, Cantrell R. 2005b. Registration of TAM 98D-102 and TAM 98D-99ne Upland Cotton Germplasm Lines with High Fiber Strength. Crop Sci. 45: 1668.

Thaxton PM, Smith CW, Cantrell R. 2005c. Registration of TAM 96WD-18 Upland Cotton Germplasm Line with Improved Fiber Length and Strength. Crop Sci.45: 1172.

Thaxton PM, Smith CW, Cantrell R. 2005d. Registration of TAM 96WD-69s Glabrous Upland Cotton Germplasm Line. Crop Sci. 45: 1172-1173.

Van Becelaere G, Lubbers EL, Paterson AH, Chee PW. 2005. Pedigree- vs. DNA Marker-Based Genetic Similarity Estimates in Cotton. Crop Sci. 45:2281–2287

Wallace TP, White BW, Hollowell JE. 2002. Registration of ‘MISCOT 8806’ Cotton. Crop Sci. 42: 2216-2217.

Wallace TP, White BW, Hollowell JE. 2005. Registration of ‘MISCOT 8839’ Cotton. Crop Sci. 45:1167-1168.

Wilkins TA, Arpat AB. 2005. The cotton fiber transcriptome. Physiol Plantarum 124(3):295-300.Wilkins TA, Mishra R, Trolinder NL. 2004. Agrobacterium-mediated transformation and

regeneration of cotton. J Food Agric Environ 2:179-187Zhang TZ, Yuan YL, Yu JZ, Guo WZ, Kohel RJ. 2003. Molecular tagging of a major QTL for

fiber strength in Upland cotton and its marker-assisted selection. Theor. Appl. Genet. 106:262-268.

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APPENDIX A

Project No: S-304

Project Title: Development of Genetic Resources for Cotton

Period Covered: December 2000 – January 2006

Date of This Report: June 2, 2006

Annual Meeting Dates: 08/29/2001 01/09/2002 09/14/2003 01/05/2005 09/11/2005

Participants:http://www.lgu.umd.edu/lgu_v2/homepages/saes.cfm?trackID=1254#3

Brief summary of minutes of annual meetings:http://www.lgu.umd.edu/lgu_v2/homepages/saes.cfm?trackID=1254#3

ACCOMPLISHMENTS AND IMPACTS

Objective 1: To acquire, curate, characterize, and evaluate the species, races, and genetic types of cotton.

IMPACTAcquisition and distribution of germplasm impact population development and varietal improvement in every cotton growing region of the world. Significant contributions have been made to the collection of germplasm available for exploitation by the cotton research community. Characterization of germplasm resulted in the identification of important sources of fiber quality traits as well as resistance to diseases and insects. A number of the accessions characterized have been used as parental material in several different breeding programs and no doubt will eventually be introgressed with commercial material destined to the U.S. cotton producer. Information added to the GRIN database will facilitate worldwide exploitation of newly acquired germplasm.

ACCOMPLISHMENTS Acquisition, Maintenance, Distribution: Increased concerns that genetic vulnerability in cotton was a major contributor to the apparent recent decline of cotton yields in the U.S., highlighted the need to significantly expand and develop the cotton germplasm base available in this country. This is especially true given that less than 1% of cotton germplasm types have been adequately explored. Scientists in the Crop Germplasm Research Unit at the Southern Plains Agricultural Research Center, College Station, TX, in cooperation with Texas A&M University and the National Cotton Council of America, undertook work to increase important cotton genetic types and distribute them appropriately. Approximately 6,000 accessions maintained in the U.S. Cotton Germplasm Collection were increased by seed propagation, both to assure their preservation and to make sufficient quantities of seed available to others. More than 1,100 of the increased accessions were recent acquisitions from Uzbekistan and Russia. In total, 16,773

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accessions were distributed to cotton researchers in the U. S. and many other nations between the years 2001 and 2004 in response to 405 seed requests. Plant explorations have been conducted in more than 20 nations, and valuable and unique new cotton germplasm has been secured, added to the U.S. Cotton Germplasm Collection, and in some cases distributed to others. Cotton seed exchanges have been accomplished with Brazil, the Peoples Republic of China, India, Russia, Uzbekistan, and Zimbabwe.

Characterization: Project participants utilized a large numbers of accessions in projects aimed at characterizing unexplored germplasm to facilitate usefulness of the GRIN database and aid breeders in exploitation of new sources of genetic variability. More than 1,000 G. hirsutum and G. barbadense accessions were utilized in evaluations that collected information ranging from GRIN descriptors and yield, to insect, nematode, and disease resistance.

In 2001, 79 converted race stocks of G. hirsutum were backcrossed to an elite breeding line (TAM 94L-25) to produce the BC3F1 generation in a photoperiod conversion program. Fifty accessions in the working collection from India, Pakistan and Uzbekistan were evaluated for bacterial blight resistance and insect resistance, as well as being characterized as to species, leaf type, and level of pubescence. Fifty-nine G. barbadense accessions obtained from a Russian collection were evaluated for GRIN descriptors, fiber traits, and yield performance in Arizona. Efforts to develop an improved, non-genotype specific regeneration system in cotton led to the identification of an improved Upland cotton cultivar (G. hirsutum), a Gossypium arboreum line and a G. barbadense line with regeneration capacities. In 2002, sixty-nine G. barbadense accessions obtained from Uzbekistan were evaluated for GRIN descriptors, fiber traits, and yield performance in Arizona. In 2002 and 2003, 197 accessions recently acquired form Uzbekistan were characterized and evaluated for GRIN descriptors, fiber traits, and yield performance in Louisiana and Mississippi. These accessions also were evaluated for Root-Knot Nematode resistance at the Louisiana location. Thirty one lines were found to be moderately resistant and eight lines were classified as resistant.

In 2003, 155 accessions rescued from a closed-out breeding program in Shafter, CA were characterized and evaluated for GRIN descriptors, fiber traits, and yield performance in Louisiana and Mississippi. Also in 2003, 70 converted race stock lines were evaluated for robustness of seedling root development.

In 2004, a project was initiated to determine the genetic diversity of the cotton around the Caribbean basin and, if possible, determine the origin of the Florida wild cotton. Also in 2004, the genetic diversity of the Gossypium species of the G genome was measured with approximately 50 AFLP markers and initial results indicate that accessions from Western Australia are significantly different from eastern (Queensland) accessions at the molecular level. A population of Gossypium barbadense chromosome substitution lines (CS-B lines) were evaluated and phenotyped for agronomic and fiber trait expression. A set of 434 Gossypium hirsutum L. accessions from the USDA-ARS Cotton Germplasm Collection of Mexican origin were evaluated for phenotypic and genetic variability. Diversity between and within accessions was the highest for accessions collected within the states of Guerrero, Yucatan, Oaxaca,

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Veracruz and Chiapas. In 2003 and 2004 more than 150 Pima and Acala/Upland commercial and experimental varieties, and improved germplasm were evaluated for resistance.

Objective 2: To develop, maintain, and distribute molecular genetic, genetic, and cytogenetic tools for the evaluation and enhancement of cotton germplasm. IMPACTAn integrated genetic and physical map of the cotton genome has been developed that provides a solid basis for future cotton improvement efforts. Furthermore, a standardized 12-cotton DNA genotype panel, the foundation for systematic screening of cotton DNA markers, was developed. A set of backcrossed chromosome substitution lines (BCnF1) was developed that provide a

unique opportunity to detect the effect of the group of genes that a specific chromosome carries and thus will also aid cotton genome mapping programs. Genetic materials (seed and DNA stocks, genomic clones, and primer sequences) developed during the course of this project are being delivered to cotton researchers worldwide for the identification, mapping, and cloning of cotton genes that will lead to the improvement of fiber properties, agronomic performance and stress tolerance. An innovative molecular method was developed to screen differentially expressed genes in cotton and it has detected significant variation within the functional genes in cotton. This variation will be useful in the development of cotton fabrics with enhanced and/or unique functional, performance, aesthetic and comfort characteristics.

ACCOMPLISHMENTSIntegrated genomic map: During the period of this project, cooperators have constructed components of an integrated genetic and physical map of the cotton genome.  Three complementary libraries of high-quality, large insert bacterial artificial chromosome (BAC) clones were developed. The feasibility of AD cotton BAC contig assembly and sorting by use of A or D subgenomic specific DNA markers and other techniques was demonstrated. Approximately 100,000 cotton BAC TM-1 fingerprints were generated and 5,000 BAC physical contigs were assembled. Information on BAC clones, DNA markers, SSR primers, and related information has been entered into CottonDB.

Development was completed of a Recombinant Inbred (RI) population between the G. hirsutum and G. barbadense genetic standards, TM-1 and 3-79, respectively. This permanent mapping population consists of 191 TM-1 x 3-79 RI lines.

Molecular markers: Molecular marker technology has progressed rapidly in the 1990s with the construction of the first genetic map of cotton published in 1994. Currently, the interspecific cotton map includes over 2500 loci, many of which were added during the course of this project. This high-density genetic map covers all 26 chromosomes of the tetraploid cotton genome. A majority of the loci was from RFLPs, although a few came from PCR based technology such as SSRs or STS. Research on the feasibility of converting mapped loci into a Single Nucleotide Polymorphism (SNP) detection system, better suited for use in marker-assisted breeding, is underway.

The development and mapping of cotton SSR markers derived from TM-1 BAC libraries continues. Currently, over 1,000 cotton SSR primers have been developed from positive BAC

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clones and another 1,000 cotton SSR primers are being designed from small-insert genomic clones.

Quantitative Trait Loci (QTL) mapping: Efforts to develop a intraspecific QTL map in cotton for yield components and fiber quality traits continued. Twenty AFLP combinations yielded 200 polymorphic markers used to construct a 1773 cM linkage map containing 28 linkage groups. QTL loci for fiber quality, yield and the within boll yield components have been identified. Using G. tomentosum chromosome substitution lines of G. hirsutum, AFLP linkage groups were assigned to some of the monosomic and monotelodisomic genetic stocks. Common AFLP markers were found between the aneuploid stocks and the intraspecific cross used allowing 8 linkage groups to be assigned to 6 different chromosomes. An intraspecific Gossypium barbadense RIL population of 144 F6.8 lines (PS-6/89590) was characterized for yield, fiber, and agronomic properties in replicated tests in California, Arizona, and New Mexico. An introgressed G. hirsutum RIL population of 98 lines (NM24016/TM-1) was increased for public seed distribution. And several plant populations have been developed between Pima S-7 and Hartz 1220, Stoneville 132, Lankart 57, Stoneville 213 and nine MAR germplasm lines for use in molecular mapping.

Genetics Tools for the evaluation and enhancement of cotton germplasm

Tools for seedling (a)biotic stress resistance: The genetic material from a Pima S-7/Acala 44 cross is being used to evaluate seed-seedling resistance and fiber quality. Some 617 polymorphic fragments amplified from 53 primer pairs have been selected for mapping.

120 F3 progeny derived from single seed decent from a Pima S-7/Acala 44 cross have been used to evaluate cold tolerance and fiber quality (using AFIS). Data from this experiment is also being used in the construction of cotton linkage maps.

Molecular tool for functional genome analysis: Among many other molecular methods in cotton the analysis of differential gene expression is very difficult due to many challenges. Focus has been on what cotton genes are required to make fiber, how each gene contributes to fiber shape and its physiochemical structure and how these relate to the plant’s agronomic properties. We have found that about 14,000 unique gene sequences define the cotton fiber transcriptome in 7-10 days post anthesis (DPA) developing cotton fibers and that the transcriptome is high conserved. Using microarray expression analysis, we have begun to map the developmental switches and regulatory circuits important for fiber development.

Complementary research on gene expression during cotton fiber development, with a biased toward genes expressed at 20 DPA compared to 6 DPA, was constructed using a cDNA library (prepared from G. hirsutum cv Delta Pine 90) and sequenced. This represents a very early stage of secondary wall deposition. From 9,121 high quality EST sequences, 3,420 unigenes were assembled within this one library. About 15% of the translated ESTs had no significant match to the Arabidopsis proteome, and 150 had no similarity to proteins in the non-redundant database. Such genes are indicative of processes that make the cotton fiber distinctive. Conversely, cotton fiber expresses genes with significant similarity (>25%) to the proteome of Arabidopsis. Some 600 unique secondary-wall-specific genes were expression in cotton fiber.

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An expressed sequence tag (EST) database, developed from a diploid cotton species Gossypium arboreum L., containing ~14,000 non-redundant sequences from 7-10 dpa (days post anthesis) fiber transcripts and their homologous gene functions, was used to develop microsatellite markers. A total of 1232 EST-derived microsatellite primer pairs were designed and 83% successfully amplified products from eight Gossypium species, including both diploid and tetraploid accessions and cultivars. Such high interspecific transferability could be due to sequence conservation of the gene-coding region of the genome. Between G. hirsutum L. and G. barbadense L., 202 markers were polymorphic, while the polymorphism within these two tetraploid species was low - 1.4 % and 1.5 %, respectively. Locations of 40 microsatellite markers were delimited to 19 cotton chromosomes and/or 17 chromosome arms by hypo-aneuploid deficiency analysis. Polymorphic markers between G. hirsutum and G. barbadense showed significant sequence similarity to genes or putative genes with known function including endo-β-1,4-glucanses, cytochrome P450-like protein, expansin, RING zinc finger protein, and ABC transporter, which are related to fiber.

Cytogenetic Tools for the evaluation and enhancement of cotton germplasm

Chromosome substitution lines: These lines are genetically identical except that each differs by the replacement of a specific homologous pair of chromosomes from Pima 3-79 (Gossypium barbadense) into Upland cotton (G. hirsutum). The interspecific backcrossed chromosome substitution lines provide a novel resource to plant breeders to overcome the problems of genomic incompatibility at the whole genome level between the two species and create a unique set of chromosome comprehensive germplasm introgression products in Upland cotton. To this end, a germplasm release was made of 17 chromosome substitution backcross lines, each being quasi-isogenic to TM-1, but substituted for one chromosome or one chromosome arm of the non-photoperiodic G. barbadense line 3-7. Prospectively new G. hirsutum hypoaneuploids continue to be identified and tested. The identification of a new monosomic G. hirsutum was reported, H21. Development was advanced for new chromosome substitution series for G. tomentosum and G. mustelinum Various BCnF1 chromosome substitution stocks from these programs and DNAs were distributed.

Radiation Hybrids: During the course of this project a new approach to avail cotton of the advantages of hybrid mapping was developed. This was dubbed the Wide-cross Whole-genome Radiation Hybrid mapping (WW-RH) method and was used to create two radiation hybrid panels for physical mapping. Experiments were completed that prove the feasibility and utility of whole-genome radiation hybrid mapping of 52-chromosome species genomes of Gossypium and also for integrative physical mapping of linkage groups onto cotton chromosomes by molecular cytogenetic means (FISH) with marker-selected BACs. These prove the feasibility of unequivocally identifying linkage group-to-chromosome relationships, hooking up the extra linkage groups, and determining arm relationships.

Alloplasmic lines (AD3, AD4, AD5, A2, C1, D3-d, D8, E1) in the G. barbadense semigamy (Se) nuclear background were maintained. A partial set of molecular markers were developed to distinguish among the various alloplasmic lines based on chloroplast DNA markers. Markers to distinguish among A- type cytoplasms have not been identified.

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Objective 3: To adapt and develop methodologies to evaluate, modify, and utilize cotton germplasm.

IMPACTUse of new DNA and RNA technolgies will eventually provide more useful information to evaluate, modify and untilize cotton germpalm, and to better understand this complex genome. However, one of the biggest chanllenges is to transalate and/or adapt and develop suitable methodolgies to be used in cotton germplam research. One of the most popular new approaches in cotton germplasm research, in 2001, was the use of molecular markers. However, the limited number of molecular markers in cotton limited the amount of reseach in this area a that time. In 2005 the number of molecular markers increased, and approximately 3,500 microsatellite can now be used for diverse genome analyses and genetic diversity studies for the improvement of the cotton crop.

ACCOMPLISHMENTSThe molecular marker linkage maps that have been assembled for the n=26 cottons, G. hirsutum and G.barbadense are incomplete and involve a large number of RFLP markers which have limited use in breeding, in 2001. No published map has attained 26 linkage groups. Several groups from the private and public sector avocated to increase the number of molecular markers in cotton. The ARS MS locations completed screenning of over 500 polymorphic DNA markers covering more than 50% of the chromosomes (primarily RFLP and SSR markers, and a few AFLP) against RI lines and intraspecific populations. In addition, in conjunction with Dr. Roy Cantrell at New Mexico State University, a mapping population of plants was developed in order to associate useful traits within the Converted Race Stocks with SSR markers. Plants are being grown currently to produce the BC3 or final backcross. (AES-TX A&M, AES-NM)

In 2002, with new genetic research tools such as molecular markers, we are able to better understand the genetic parameters associated with desirable production and quality traits in cotton. Genetic characterization of almost 300 different American and Russian cotton types with appropriate molecular markers clearly revealed the genetic similarities and differences between these cottons of widely differing origin.

An innovative approach to increasing the number of genotypes that can be genetically modified, getting a step closer towards a genotype-independent transformation technology, was developed. The strategy incorporates the increase in the alleles for regeneration potential in the cotton gene pool. A new callus induction medium was developed that increases the range of genotypes that can be transformed and regenerated. Technical refinements have been made that reduce the time needed to regenerate transformed plants to 6-8 months. These techniques are in use to increase yield and fiber quality of cotton (UC-Davis-CA).

Expression profiling using cotton fiber long oligonucleotide microarrays revealed major and minor developmental switches that control fiber growth and development. Using this developmental framework as the foundation, comparative genomics studies identified genes deemed of pivotal importance to fiber development, and as molecular determinants of fiber traits (UC-Davis). A list of candidate genes and Cotton ESTs are being generated and assabled for

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functional analysis and to develop into molecular markers for genetic mapping in the cotton community. Cotton chips are under development in order to be distributed to the cotton community.

In 2004, an analysis of 346 obsolete and current germplasm lines was undertaken (UGA) using a variety of molecular markers. Based upon the results, a dendogram was presented outlining the relationships amongst them. Genotypes were clustered into three main groups: Eastern, Delta and Pee Dee. Information from this will be useful to breeders as they identify parents for use in crosses to maximize genetic diversity in their crossing programs. In addition, UGA lab has initiated a project to evaluate elite breeding lines developed by the cotton breeding program for somatic embryogenesis. The results showed that somatic embryogenic ability is present in several elite lines. The line with the greatest embryo production, GA98033, was released in 2004 as public germplasm line. GA98033 combines high yield potential, acceptable fiber quality, resistance to fusarium wilt and conducive to plant regeneration through tissue culture somatic embryogenesis. Therefore, in additional to being useful to breeders as a parental source with high yield potential and acceptable fiber quality, GA98033 can potentially be useful to molecular biologists as a recipient of transgenic traits.

Twenty-one F2 populations were developed from diallel crosses involving seven Upland cottons, TM-1, 7235, SG125, Fibermax 832, CAMD-E, MD51, and DPL50. The populations were grown in the ARS field plot in College Station, Texas, for measurements of fiber properties and isolation of genomic DNA from individual F2 plants. This project continues to develop molecular descriptors with portable PCR-based DNA markers that will be at the foundation of germplasm characterization.

Morphological trait and SSR marker data were collected from a field planting of over 400 landrace accessions of Gossypium hirsutum, and all known D diploid genome species collected from recent exploration trips (Drs. Ulloa and Stewart). Thirty morphological traits were evaluated and scored on a selection of landraces in Shafter, CA. DNA was extracted from tissue of over 400 of these selected landraces (previously planted in 2004), and 85 newly evaluated landraces. Selected SSR markers (75) for which chromosomal locations are validated were assayed on DNA of the landrace accessions. Genetic diversity among the landraces was assessed with preliminary data on 6 morphological traits (pooling data from 3 environments, two in 2004) and 67 SSR markers. The variation in the morphological data and SSR data were separately used to cluster the accessions by relatedness. SSR marker data was more helpful than the morphological data in characterizing the diversity of the landraces according to their origin of collection. This research will help update the morphological characterization of the collection, integrate portable mapped SSRs to understand the genetic diversity of G. hirsutum on a genome basis, and reveal unique sources of genetic variation in the collection (USDA-ARS-CA).

In collaboration with Cotton Incorporated, a standardized 12-cotton DNA genotype panel was established that is at the foundation of the Cotton Microsatellite Database (CMD) for a systematic screening of cotton DNA markers developed by participants from the cotton community. The CMD is a curated and integrated web-based relational database providing centralized access to all publicly available cotton microsatellites (http://www.cottonssr.org). At present it contains sequence, primer, mapping and homology data for nine cotton microsatellite

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projects (BNL, CIR, CM, JESPR, MGHES MUSB, MUSS/MUCS, NAU, and TMB). The standardized panel consists of 12 diverse genotypes including genetic standards, mapping parents, BAC donors, subgenome representatives, unique breeding lines, exotic introgression sources, and contemporary Upland cottons with significant acreage. The collection of all publicly available cotton SSR markers into a centralized, readily accessible web-enabled database provides a more efficient utilization of microsatellite resources and will help accelerate basic and applied research in molecular breeding and genetic mapping in Gossypium spp. The CMD is a true cotton community effort and definitely a tremendous resource of microsatellite markers with approximately 3,500 markers to date for the community that can be used for diverse genome analyses and genetic diversity studies for the improvement of the cotton crop.

Objective 4: Germplasm enhancement for biotic and abiotic stress resistance and agronomic traits.

IMPACTGenotypes with drought resistance and resistance to pests such as seed/seedling diseases, bronze wilt, whiteflies, plant bugs, and nematodes need to be identified and incorporated into lines with high yield and fiber quality potential either through genetic resistance or with morphological traits such as smooth leaf and nectariless. Significant accomplishments have been made in developing and releasing germplasm with some levels of pest resistance, fiber quality with especially with nematodes and plant bugs using the nectariless trait. This research will provide the cotton growers and industry with the new tools to combat pests, thus reducing production cost and risk, and increase grower profit.

ACCOMPLISHMENTSGermplasm releases: Research from this regional project has contributed to the success of release of new germplasm and varieties. In 2001, PD 94045 cotton was released by university of Georgia for its combination of desirable fiber properties, yield potential, and wide adaptation. Five germplasm lines of cotton (G. barbadense), designated as 93252, 93260, 94217, 94218, and 94220 were d released by the USDA-ARS in cooperation with the Univ. of Arizona in 2001. All five lines produce significantly longer and stronger fiber than is currently available in commercial American Pima cultivars. The lines possess agronomically acceptable yield potentials, maturity intervals, and plant heights, and exhibit good levels of heat tolerance. The cotton improvement laboratory at Texas A&M Univ. released of four germplasm lines and one new cultivar in 2004. These were TAM 96WD-69s (glabrous), TAM 98D-99ne (nectariless), TAM 96WD-18 and TAM 98D-102 and Tamcot 22, a conventional cultivar. TAM 96WD-18 and TAM 98D-102 have excellent fiber packages and competitive agronomic performance.

Interspecific hybridization: Genome-wide introgression efforts from G. tomentosum, G. mustelinum, G. longicalyx and G. armourianum were advanced 1-2 generations. Generation means analysis field evaluations were conducted for G. tomentosum and G. mustelinum introgression products in 2003-2005.

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In 2004, interspecific diploid hybrids were made between the A genome (G. arboreum) accessions and D genome species (G. trilobum, G. raimondii and G. aridum). As a part of the project to enhance the secondary germplasm pool, trispecies hybrids were developed with a D3-compatible AD1: (B1 x D3-d x AD1). The resulting hybrids will be evaluated for trait segregation in 2005. New synthetic hybrids were made from cross-pollinations of G. arboreum with a 2(ADD) genetic stock to make trispecies hybrids with 2(AD) genomic constituencies. One of these was backcrossed to an elite line of upland cotton to begin development of an introgression population.

Screening converted race stocks: Converted race stocks have been used to develop advanced strains and segregating populations and for screening for resistance to seed-seedling disease, cotton fleahopper, silverleaf whitefly, nematode resistance. Of the 150 accessions evaluated, twenty-four accessions had galling index scores of less than 3 and ten TX accessions (TX-1028, TX-1483, TX-1437, TX-1355, TX-2311, TX-2324, TX-695, TX-2362, TX-1585, and TX-1240) had gall scores not significantly different than the resistant check. Two of these, TX-1028 and TX-1483 had average gall scores lower than the resistant check. Several of the resistant accessions were from countries outside the center of diversity for Upland cotton, indicating that useful diversity can arise outside of germplasm centers.

Screening for nematodes: Researchers have been very interested in developing germplasm that is resistant to nematodes. F3 plants from a cross of M315 (root-knot resistant) and TX 110 (putatively reniform resistant) were screened for resistance to both nematodes. The goal was to obtain a single plant that has resistance to both. The development of markers that can be assigned to resistance to these pests is continuing. Additional efforts by several projects were underway to introgress the high level of resistance for reniform nematode found in G. longicalyx. Using the G. longicalyx as the immunity source, reniform nematode-immune BC3F1, BC4F1, and BC5F1 G. hirsutum backcross segregates were identified. Their tissue was sampled for nuclear genomic DNA preparation, and some were backcrossed and inbred. Preliminary co-segregation analysis was initiated for markers.

DNA markers for root-knot nematode resistance genes: Researchers in Mississippi detected about 23 polymorphic SSR markers between RKN and susceptible lines. Pest/disease resistance gene homologs in cotton are being identified and mapped to aid in location of these and other potential nematode resistance genes. Several sequences homologous to fungal wilt resistance genes have been cloned and are being further characterized due to possibility that they are tightly linked to RKN resistance genes (i.e. RKN resistant isolines are known to show resistance to fungal wilt). Screening for Bronze Wilt: A project was begun in 1998 in Arizona with the objectives of determining the heritability of tolerance to bronzing, bronze wilt, or early foliar decline (EFD), documenting the detrimental effects of EFD upon yield and fiber quality, demonstrating the relationship between EFD severity and plant maturity, and creating earlier, tolerant germplasm. Results showed that selection may be feasible in unreplicated early generation progeny rows, planted at multiple locations. From the results of this investigation it appears that the impact of early foliar decline upon yield and fiber quality is dependent upon the time of initial onset and speed of progression of the disorder.

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Screening for nectariless: Over 400 nectariless progeny rows (F3 to F10) where grown in 2003 and the top performing entries were selected for evaluation under plant bug infested conditions. A wide range of germplasm was represented in this material including obsolete varieties, breeding lines, and nectariless isolines. A field evaluation of TPB resistance in lines of nectariless cotton was established in 2004-05 on Delta Branch Research Station, Stoneville, MS. Reduction in fiber quality due to plant bugs was minimal. The nectariless trait, however, appears to have conferred a yield advantage for a number of entries.

Fiber Improvement and heat tolerance: In 2003 and 2004, three upland genotypes (TAM 94L-25, FM 832, and TTU 202) were crossed in a half diallel with Tamcot CAMD-E, and Acala 1517-99 to determine if the genes for near long staple in TAM 94L-25, Fibermax 832, and TTU 202 are allelic and determine transgressive segregation in the F2 generation. In 2005, G. hirsutum germplasm was evaluated for fiber quality and heat tolerance in Arizona. Three of the original 90 germplasm lines were released in 2005 on the basis of their yield and fiber. The Az project continues incorporating fiber quality traits from G. barbadense introgression sources, as well as from Acala sources. In 2004, selection was made within F3 populations of two way or double crosses, thus incorporating two sources of fiber improvement and two sources of heat adaptation within each segregating population.

OBJECTIVE 5: To refine and develop cotton breeding and variety testing methodologies and techniques. IMPACTCotton breeding efforts have been enhanced with the revelation that 1) separate trials are not needed for transgenic and conventional cotton cultivars 2) visual selection for yield is possible and lower yielding lines can be eliminated prior to harvest 3) there have been three primary sources of improved fiber strength genes in upland cotton and 4) backcrossing is an acceptable method of inserting transgenes into improved germplasm. Genetic uniformity has not been compromised with the advent of transgenic cotton cultivars.

ACCOMPLISHMENTSUpdate information on genetic relationships: Pedigree information has been updated and includes 283 new upland and 10 new pima varieties. Genetic uniformity has not increased since the introduction of transgenic varieties. Fiber strength has increased since 1980 due to introduced genetic diversity by New Mexico State University and the USDA Pee Dee Program and from recombination. Quantify advantages and disadvantages of divergent variety development strategies: Populations have been developed (Auburn) from six families with 300 pedigree lines and 195 SSD lines for yield testing in a study to compare genetic gain between SSD and pedigree selection.

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Develop guidelines for efficient and discriminating genotype evaluation: In a study among public cotton breeders, a positive correlation between visual ratings and seed cotton yield was indicated, suggesting visual selection is a viable option for breeders.GGE Biplot was evaluated for its ability to identify best environments for selection and was found to corroborate historical information. NNA, ANOVA, and TREND analyses were compared across five states on various trials and each gave different results with no one best. Further studies are needed.

Evaluate the need to incorporate systems testing in variety trials for transgenic technologies: Interaction by varieties with technologies were not detected indicating that the relative ranking of cultivars within systems should remain the same. Investigate the consequences of forward crossing of transgenic parents: Theoretical aspects of forward crossing were examined with such factors as relevance of transgenes, speed of market demand, capability of removing transgenes, genetic background, access to transgenes, and many other factors justifying whether one should forward cross. Backcrossing transgenes does have its advantages.

Objective 6: To develop cotton bioinformatic systems.

IMPACTSignificant contributions to the CottonDB have been accomplished through addition of new information and reorganization. Data will contribute to the identification and exploitation of genetic variability at the molecular level for greatly improved efficacy in cotton germplasm improvement.

ACCOMPLISHMENTSMaintenance of CottonDB was continued from its origin in 1995 as part of the USDA, ARS National Plant Genome Database System (NPGDS). It was maintained as mirrored sites as part of the central plant genome databases and a cotton database at http://cottondb.tamu.edu until the NPGDS was terminated in 2001. ARS at College Station, TX continued the maintenance and development of CottonDB. CottonDB data classes were reorganized and updated with new information including cotton germplasm, variety trial, SSR clones and primers, BAC clones and fingerprints, and DNA sequences. Some bioinformatic tools were incorporated into CottonDB for sequence blast and integration of genome maps.

The Cotton Functional Database at UC-Davis was expanded and improved to be more user friendly, allowing access and manipulation of expression-related data that is now linked to clones, constructs, ESTs, genes, and SSRs.

Members of S-304 participated in a Cotton Incorporated lead initiative to develop new publicly available microsatellite markers. As part of this initiative, a cotton microsatellite database (CMD) was developed for the program. Microsatellite marker data is available at this site and CottonDB.

Data from the Cotton Germplasm Collection was included in the GRIN database and CottonDB.

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MAJOR PUBLICATIONS

Arpat AB, Waugh M, Sullivan JP, Gonzales M, Frisch D, Main D, Wood T, Leslie A, Wing RA, Wilkins TA. 2004. Functional genomics of cell elongation in developing cotton fibers. Plant Mol. Biol. 54: 911-929.

Blenda, A., J. Scheffler, B. Scheffler, M. Palmer, J.-M. Lacape, J. Z. Yu, C. Jesudurai, S. Jung, S. Muthukumar, P. Yellambalase, S. Ficklin, M. Staton, R. Eshelman, M. Ulloa, S. Saha, B. Burr, S. Liu, T. Zhang, D. Fang, A. Pepper, S. Kumpatla, J. Jacobs, J. Tomkins, R. Cantrell, and D. Main. 2006. CMD: A Cotton Microsatellite Database resource forGossypium genomics. BMC Genomics 7:132 doi:10.1186/1471-2164-7-132.

Bourland FM., Johnson JT, Jones DC. 2005. Registration of Arkot 8712 germplasm line of cotton. Crop Sci. 45: 1173-1174.

Bowman DT, Bourland FM, Myers GO, Wallace TP, Caldwell WD. 2004. Visual selection for yield in cotton breeding programs. J. Cotton Sci. 8(2):62-68.

Bowman DT, Gutierrez OA. 2003. Sources of fiber strength in the U.S. Upland cotton crop from 1980-2000. J. Cotton Sci. 7:86-94.

Bowman DT, May OL, Creech JB. 2003. Genetic uniformity of the U.S. Upland cotton crop since the introduction of transgenic cottons. Crop Sci. 43: 515-518.

Cedroni ML, Cronn RC, Adams KL, Wilkins TA, Wendel JF. 2003. Evolution and expression of MYB genes in diploid and polyploidy cotton. Pl. Molec. Biol. 51: 313-325.

Chee PW, Rong J, Williams-Coplin D, Schulze SR, Paterson AH. 2004. EST derived PCR-based markers for functional gene homologues in cotton. Genome 47:449-462.Feng CD, Stewart J.McD, Zhang JF. 2004. STS markers linked to the Rf1 fertility restorer gene of cotton. Th. Appl. Genet. 110(2): 237-243.

Frelichowski JE, Palmer MB, Main D, Tomkins JP, Cantrell RG, Stelly DM, Yu J, Kohel RJ, Ulloa M (2006) Cotton genome mapping with new microsatellites from Acala ‘Maxxa’ BAC-ends. Mol. Genet. Genom. 275 (5) 479-491.

Gao W, Chen ZJ, Yu JZ, Raska D, Kohel RJ, Womack JE, Stelly DM. 2004. Wide-cross whole-genome radiation hybrid (WWRH) mapping of cotton (Gossypium hirsutum L.). Genetics. 167 (3): 1317-1329.

Guo WZ, Zhang TZ, Shen XL, Yu JZ, Kohel RJ. 2003. Development of SCAR marker linked to a major QTL for high fiber strength and its molecular marker assisted selection in Upland cotton. Crop Sci. 43: 2252-2256.

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Haigler CH, Zhang D, Wilkerson CG. 2005. Biotechnological improvement of cotton fiber maturity. Physiologia Plantarum 124: 285-294. He L, Du C, Covaleda L, Xu Z, Robinson AF, Yu JZ, Kohel RJ, Zhang HB. 2004. Cloning, characterization, and evolution of the NBS-LRR-encoding resistance gene analogue family in polyploidy cotton (Gossypium hirsutum L.) Mol. Pl. Microbe Interactions. 17(11) :1234-1241.

Hendrix B and Stewart J.McD. 2005. Estimation of the nuclear DNA content of Gossypium species. Annals of Botany. 95: 789-797.

Kakani VG, Reddy KR, Koti S, Wallace TP, Vara Prasad PV, Reddy VR, Zhao D. 2005. Differences in in vitro pollen germination and pollen tube growth of cotton cultivars in response to high temperatures. Annals of Botany 96(1):59-67.

Karaca M, Saha S, Callahan F, Jenkins JN, Read JR, Percy RG. 2004. Molecular and cytological characterization of a cytoplasmic-specific mutant in pima cotton (Gossypium barbadense L.). Euphytica 139:187-197.

Karaca M, Saha S, Jenkins JN, Zipf A, Kohel R, Stelly DM. 2002. Simple sequence repeat (SSR) markers linked to the Ligon Lintless (Li1) mutant in cotton. J. Hered. 93:221-224.

Khan MA, Myers GO, Stewart J. McD. 2002. Molecular markers, genomics, and cotton improvement. In: M.S. Kang (ed.) Crop Improvement: Challenges in the Twenty-First Century. (pp. 253-284). Food Products Press, Binghampton, NY.

Kohel RJ, Bird LS. 2002. Inheritance and linkage analysis of the yellow pulvinus mutant of cotton. J. Cotton Science. 6: 115-118.

Kohel RJ, Stelly DM, Yu J. 2002. Tests of six cotton (Gossypium hirsutum L.) mutants for association with aneuploids. J. Heredity 93:130-132.

Liu S, Saha S, Stelly D, Burr B, Cantrell RG. 2000. The use of cotton aneuploid for the chromosomal assignment of microsatellite loci. J. Heredity 91:326-332.

Lu HJ, Myers GO. 2002. Genetic relationships and discrimination of ten influential Upland cotton varieties using RAPD markers. Theor. Appl. Genet. 105:325-331.

May OL, Bourland FM, Nichols RL. 2003. Challenges in testing transgenic and nontransgenic cotton cultivars. Crop Sci. 43: 1594-1601.

May OL, Chee PW, Sakhanokho H. 2004. Registration of GA98033 upland cotton germplasm line. Crop Sci. 44:2278-2279.

Mei M, Syed NH, Gao W, Thaxton PM, Smith CW, Stelly DM, Chen ZJ. 2004. Genetic mapping and QTL analysis of fiber-related traits in cotton (Gossypium). Theor. Appl. Genet. 108:280–291.

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Park YH, Alabady MS, Ulloa M, Sickler B, Wilkins TA, Yu J, Stelly DM, Kohel RJ, El-Shihy OM, Cantrell RG. 2005. Genetic mapping of new cotton fiber loci using EST-derived microsatellites in an interspecific recombinant inbred line cotton population, Molecular Genetics and Genomics. 274(4): 428 - 441.

Paterson AH, Boman RK, Brown SM, Chee PW, Gannaway JR, Gingle AR, May OL, Smith CW. 2004. Reducing the genetic vulnerability of cotton. Crop Sci. 44:1900-1901.

Percy RG. 2003. Comparison of bulk F2 performance testing and pedigree selection in thirty Pima cotton populations. J.Cotton Sci. 7:170-178.

Reddy OU, Pepper AE, Abdurakmonov I, Saha S, Jenkins J, Brook T, El-Zik KM. 2001. New dinucleotide and trinucleotide microsatellite resources for cotton genome research. J. Cotton Science. 5(2):103-113.

Rong J, Abbey C, Bowers JE, Brubaker CL, Chang C, Chee PW, Delmonte TA, Ding X, Garza JJ, Marler BS, Park C, Pierce GJ, Rainey KM, Rastogi VK, Schulze SR, Trolinder NL, Wendel JF, Wilkins TA, Williams-Coplin D, Wing RA, Wright RJ, Zhao X, Zhu L, Paterson AH. 2004. A 3347-locus genetic recombination map of sequence-tagged sites reveals features of genome organization, transmission and evolution of cotton (Gossypium). Genetics 166:389-417.

Rong J, Pierce GJ, Waghmare VN, Rogers CJ, Desai A, Chee PW, May OL, Gannaway JR, Wendel JF, Wilkins TA, Paterson AH. 2005. Genetic mapping and comparative analysis of seven mutants related to seed fiber development in cotton. Theor. Appl. Genet. 111(6): 1137 - 1146.

Saha S, Karaca M, Jenkins JN, Lang D. 2000. Improved methods for screening cotton cDNAs using fluorochrome-labeled AFLP- and SSR-specific primers. In: Emerging Technologies in Cotton Breeding, J.N. Jenkins and S. Saha (eds). Science Publishers Inc., Enfield, New Hampshire, USA, pp 239-255.

Saha S, Karaca M, Jenkins J, Zipf AE, Reddy OU, Kantety R. 2003. Simple sequence repeats as useful resources to study transcribed genes of cotton. Euphytica. 130(3): 355-364.

Saha S, Wu J, Jenkins JN, McCarty Jr JC., Gutierrez OA, Stelly DM, Percy RG, Raska DA. 2004. Effect of Chromosome Substitutions from Gossypium barbadense L. 3-79 into G. hirsutum L. TM-1 on Agronomic and Fiber Traits. J. Cotton Sci. 8:162–169.

Sakhanokho,HF, Ozias-Akins P, May OL, Chee PW. 2004. Induction of somatic embryogenesis and plant regeneration in select Georgia and Pee Dee cotton (Gossypium hirsutum L.) lines. Crop Sci. 44:2199-2205.

Senchina DS, Alvarez I, Cronn RC, Liu B, Rong J, Noyes RD, Paterson AH, Wing RA, Wilkins TA, Wendel JF. 2003. Rate variation among nuclear genes and the age of polyploidy in Gossypium. Molec. Biol. Evol. 20: 633-643.

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Smith CW. 2003. Registration of TAM 94L-25 and TAM 94J-3 germplasm lines of upland cotton with improved fiber length. Crop Sci. 43: 742-743.

Smith CW. 2003. Registration of TAM 94WE-37s smooth-leaf germplasm line of upland cotton with improved fiber length. Crop Sci. 43: 743-744.

Stelly D. 2004. Aneuploid mapping in polyploids. Encyclopedia of Plant and Crop Science. Marcell Dekker, Inc.

Thaxton PM, El-Zik KM. 2003. Registration of eleven multi-adversity resistant (MAR-7A) germplasm lines of upland cotton. Crop Sci. 43: 741-742.

Thaxton PM, Smith CW, Cantrell R. 2005. Registration of ‘Tamcot 22’ high-yielding upland cotton cultivar. Crop Sci. 45: 1165-1166.

Thaxton PM, Smith CW, Cantrell R. 2005. Registration of TAM 98D-102 and TAM 98D-99ne Upland Cotton Germplasm Lines with High Fiber Strength. Crop Sci. 45: 1668.

Thaxton PM, Smith CW, Cantrell R. 2005. Registration of TAM 96WD-18 Upland Cotton Germplasm Line With Improved Fiber Length and Strength. Crop Sci.45: 1172.

Thaxton PM, Smith CW, Cantrell R. 2005. Registration of TAM 96WD-69s Glabrous Upland Cotton Germplasm Line. Crop Sci. 45: 1172-1173.

Udall JA, Swanson JM, Haller K, Rapp RA, Sparks ME, Hatfield J, Yu Y, Wu Y, Dowd C, Arpat AB, Sickler BA, Wilkins TA, Guo JY, Chen XY, Scheffler J, Taliercio E, Turley R, McFadden H, Payton P, Klueva N, Allen R, Zhang D, Haigler C, Wilkerson C, Suo J, Schulze SR, Pierce ML, Essenberg M, Kim H, Llewellyn DJ, Dennis ES, Kudrna D, Wing R, Paterson, AH, Soderlund C, Wendel JF. A global assembly of cotton ESTs. Genome Res. 2006 16: 441-450.

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