Brief Summary of Minutes of Annual Meeting

1. Comments by SAES representative Dr. Bob Westerman (Oklahoma State University) began my commenting that he has been pleased with the projects accomplishments and progress to date. He reminded us that the current project expires on Sept. 30, 2005. In preparation for this expiration we need to determine the future path we wish to take. There are two main options: 1) continue as a Multi-State Project (MSP) or 2) become a State Coordinating Committee (SCC) (equivalent to the old SRIEGs). Either way we need to identify a writing committee to develop a proposal for establishing a Development Committee (DC). A DC is normally authorized for 2 years so that a more detailed MSP or SCC proposal can be written. During the time that a DC is in effect the group would continue much as it is. A proposal for forming a DC is relatively short (< 5 pages) but needs to be completed by the end of February so that the SAES Directors can consider at their spring meeting in late March/early April. There are disadvantages and advantages whichever way the group decides to go, with either a MSP or a SCC. The advantage of a MSP is that there is the option for money allocation by Experiment Stations and gives SAES Directors more flexibility in charging off salaries against CSREES allocations. MSP projects tend to be much more researcher driven. Both researchers and extension can be represented within a SCC. Participation (at least informally) by private industry in a MSP is allowed. Both MSPs and SCCs require annual reports and minutes but these are slightly more detailed in a MSP. While you dont have to have a Hatch project to be a member of a MSP, this is typically encouraged by administration for the reason noted above. Either way we choose to go, MSP or SCC, we need to form a DC and write a brief proposal so that it can be authorized. In writing the DC proposal (and ultimately an MSP or SCC proposal) the current projects objectives will need to be rewritten (or at least tweaked) and probably put in a broader societal context. It was decided by those present that an email vote on going forward as a MSP or as a SCC would be taken during early February so that the DC writing committee knows how to target the DC proposal. 2. Identify Development Committee to write project/timeline Given officer rotations, Peng Chee (2005 vice-chair) will chair the writing committee for the development of the DC proposal. Volunteers are sought to aid Peng. As mentioned earlier, this DC proposal needs to be completed by the end of February. Public researchers are encouraged to check with their administration on the benefits/disadvantages of being a MSP or a SCC. 3. Identify committee for S-304 final report John Yu (2005 chair) will take the lead in writing the S-304 Final Report. 4. Discuss a Vote of Support for the Cotton Incorporated Genetics Initiative While nobody claimed this agenda item, it was thought that this would be of benefit to Roy Cantrell (Cotton Incorporated), the cotton industry, and ultimately to many of us as he seeks continued support of the CI Genetics Initiative. This received a unanimous vote of support and Ted Wallace (outgoing chair) was charged with writing. It is important to show how monies from the initiative are being leveraged by what MSP funds do. 5. Overview of USDA Coordinated Agriculture Project Barbara Triplett informed the group about efforts underway to develop a proposal for the NRI funded CAP project specific for cotton. These are large projects covering 4-5 years, $4-5 million, and are multi-institutional. For the cotton CAP proposal to have any chance of success the cooperation/ contributions of traditional geneticists and breeders is essential. The overall goal of the CAP project is to translate genomics technology to plant breeders. A preliminary meeting in December 2004 was held and a follow up meeting is scheduled for later this spring (April/May). Two sites to check for more information on the project and to subscribe to future emailings regarding this project are: www.plantgenome.agtec.uga.edu/register and the Cotton Portal at http://gossypium.info. 6. State Reports Given limited time, state reports were not given at this time. An email soliciting reports and publications will be sent out in the next few weeks. These will be necessary to put together the 2004 Annual Report. 7. The next S-304 Meeting will take place in conjunction with the 2004 Breeder Tour tentatively scheduled for the 2nd full week in September in the Southeast. 8. Nominations/vote for new secretary There were no volunteer from those in attendance so nominees will be solicited over the next few weeks and voted on as appropriate Elections for the following posts were held and officers selected to serve for 2005 were: Chair John Yu Vice-chair Peng Chee Secretary To be determined at next annual meeting NEW BUSINESS Multistate Research Project (MRP) S-304 was scheduled to expire September 30, 2005. A request was e-mailed soliciting input regarding the continuation of participation in research efforts as a MRP or as a SSR (Southern Coordinating Committee), formerly known as SRIEG (Southern Research Information Exchange Group). All comments received were supportive of developing a new MRP. The development committee (P. Chee, G. Myers, W. Smith, and John Yu) drafted a pre-proposal and submitted a request for a new MSR. A request for extension was e-mailed to the Executive Director of the Southern Association of Agricultural Experiment Station (SAES) Directors who recommended that MRP S-304 be extended for one year, CSRESS concurred and approved the extension until September 30, 2006. A one year extension will allow for the development of a full proposal for the new MRP. Inputs for the new proposal will be solicited at the next meeting of S-304 participants.

OBJECTIVE 1 - To acquire, curate, characterize, and evaluate the species, races, and genetic types of Gossypium (cotton). The 196 Uzbekistan accessions were evaluated in the field for root-knot nematode resistance. The gall score average for the resistant check was 1.63 and for the susceptible check was 4.16 which is in line with expectations and previous ratings of these plants in other trials. As expected, a large majority of the Uzbekistan lines (157) were susceptible (gall score >=3) to highly susceptible (gall score = 5). Thirty one lines were moderately resistant (gall score >2 but <3). Eight lines could be classified as resistant with two of these (SA2654 and SA2791) being rated as more resistant than the resistant check (Stoneville LA887). Yield and fiber data was collected on 189 accessions from Uzbekistan. Lint yield for the accessions ranged from 129 to 739 lbs/a compared to 750 lbs/a for the check (PSC 355). Lint percent values for accessions ranged from 18.2% to 40.7% compared to 40.1% for the check. Most accessions performed poorly in terms of agronomic traits, however, a few accessions were identified with a competitive yield. Several accessions were used as parental material. Previously collected descriptor data will be submitted to the GRIN database The Cotton Cytogenetics Group (Stelly) is utilizing interspecific germplasm introgression (IGI) to expand the baseline genetic variability in cotton, and making it available to cultivar breeding programs. The Cotton Improvement Laboratory (Smith and Thaxton) strives to utilize such IGI lines as well as other sources to produce germplasm having improved quality, better yield potential, and resistance to biotic stresses such as seed/seedling diseases and abiotic stresses such as temperature and drought. G. barbadense, G. tomentosum and G. mustelinum carry genes that could improve agronomic traits in Upland cotton, even though they themselves perform poorly. In the G. tomentosum, individuals in segregating populations possessed superior fiber quality traits relative to the recurrent upland parent. The G. mustelinum populations also contained improved fiber quality phenotypes in an Upland background. Recent (2004) germplasm lines released because of their fiber quality package exhibited fiber length in the 35 and 36 staple range, fiber bundle strength as high as 36 g/tex, and 4.0 to 4.5 mic. Russell Kohel visited Uzbekistan and 800 Uzbek accessions were received for evaluation on an ARS-Uzbek project. These materials are being increased in Mexico, and they will be evaluated in Texas and Mississippi. Seed of 3,687 accessions were distributed from the Cotton Germplasm Collection to 85 domestic users and 44 accessions were distributed to 5 international users. These included governmental and commercial users. A. An 11-day survey (funded by USDA) was conducted in Mexico (Nov. 9-20, 2004) for two Gossypium species whose status was unknown. During this survey it was determined that Gossypium trilobum is a threatened species, whereas G. turneri, although it has a restricted distribution, is stable for the moment. New seed collections were made of both species. B. Several morphological traits were maintained and further characterized. Short fruiting branch. Twenty-eight selections of open-pollinated plants with the short fruiting branch phenotype made in 2003 were each planted to 50' progeny rows for evaluation and re-selection. New open-pollinated selections were made of individual plants with consistency of phenotype and good yield potential. This phenotype originated as a transgressive segregate from a A1xD2-1xAD1 trispecies hybrid. Glabrous thick leaf. Fifty-five rows (50'/row) were planted from seeds of plants grown the previous year and evaluated for chlorophyll content with a Spad meter. The two or three plants in each row with the highest chlorophyll content were self-pollinated and seed harvested at the end of the season. Phenotypic parameters including leaf thickness, specific chlorophyll content, photosynthesis, etc. will be measured next year. This phenotype originated as a segregate from a A1xD2-1xAD1 trispecies hybrid and was probably donated by the D2-1 species. Qualitative Traits in Cotton: Red-calyx, red anther, hairy anther, green fiber, brown fiber. Selected plants with good phenotypic expression of each of the qualitative traits listed were self-pollinated to maintain genetically stabilized homozygous lines. It was determined that the red calyx trait (introgressed from G. herbaceum) was pleiotropic with red petal spot but, as demonstrated previously, the red anther trait (D genome origin) is not associated petal spot. Selections based on yield potential were also made from these genetic lines. The hairy anther trait originated from a cross between G. hirsutum with the pilose trait and G. barbadense. For this trait to be expressed the pilose gene must be present, but all plants with pilose do not express the hairy anther trait. C. Species diversity. Three projects are concerned with evaluation of genetic diversity among taxa. Genetic diversity of Caribbean cotton. As a result of a survey and collection of wild cotton in Florida, 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. Seeds of more than 50 accessions of G. hirsutum collected near the coast of numerous island nations around the Caribbean were obtained and these, along with the collections from Florida were germinated and are growing in pots in the greenhouse. DNA for molecular analysis has been extracted from some of these. Diversity of the Gossypium species of the G genome. The objective of this project is to determine the diversity of G. australe, G. bickii and G. nelsonii and determine if hybridization between these species occurs in natural populations. In initial results the diversity of G. australe accessions from across Australia was measured with approximately 50 AFLP markers. This limited data set shows that accessions from Western Australia are significantly different from eastern (Queensland) accessions at the molecular level. Diversity among arborescent D-genome species. Diversity among the species with in section Erioxylum represented in germplasm collected in 2002, was measured by AFLP. DNA content of the Gossypium genus. Estimates of the nuclear DNA contents of the species in Gossypium are highly variable within a species, or have not been made. This information has significance in relation to molecular maps of the genomes, and in evolutionary understanding of the genus. Work was completed on the project to obtain an accurate estimation of the DNA content of a majority of the known Gossypium species based on flow cytometry. It was also determined that barley, when used as an internal standard, as conventionally recommended, gives an erroneously high estimation of the DNA content of cotton. Three external standards (rice, maize, & barley) were included in each run and a standard curve was constructed to assure accuracy of the results. Epigenetics of wide hybridization in cotton. Aspects of epigenetic responses in wide-hybrid introgression were examined. Reports for other plant species indicate that when dissimilar genomes are combined via hybridization, genome structure and normal gene expression patterns are disrupted. To investigate these issues in cotton, we assembled a panel of DNA composed of G. davidsonii, G. anomalum, and subsequent hybrids (non-doubled and doubled through F2) between these species. This panel was screened with SSR primers to assess the conservation of repetitive fractions of these genomes. Additionally, nuclear DNA contents for the lineage were measured. Complete genome conservation was observed for 20 SSR primer pairs. For the DNA content, a small reduction in genome size was observed in the G. davidsonii x G. anomalum F1 hybrid but no other epigenetic phenomena were observed through the F2 generation. Gossypium barbadense 3-79 chromosome substitution lines (CS-B lines) were evaluated directly and in the form of F2 progeny performance. A set of 434 Gossypium hirsutum L. landraces of Mexico from the USDA-ARS Cotton Germplasm Collection was planted at Shafter, CA for phenotypic and genetic characterization of variability. Commonly used phenotypic characters were scored for each accession and tissue was bulked of each accession for DNA extraction. Preliminary results showed that significant variation still exists in the collection from the data collection. Diversity between and within accessions was the highest for accessions collected within the states of Guerrero, Yucatan, Oaxaca, Veracruz and Chiapas. The use of molecular markers is expected to reveal more variation not evident in the physical traits, and will become a valuable tool in the maintenance and utility of the diversity of the germplasm collection. Fusarium oxysporum f.sp. vasinfectum (FOV) Atk. Sny & Hans race 4 isolate has been identified in California soils. Disease expression of this race has been most severe in Pima cotton fields, but also has the capability to infect many Acala and Upland cotton varieties to a high degree depending upon inoculum levels. In order to assess the U.S. cotton gene pool and study the resistance inheritance of FOV race 4, in 2003 and 2004 more than 150 Pima and Acala/Upland commercial and experimental varieties, and improved germplasm were evaluated in known infested fields and in inoculated greenhouse assays. Preliminary results showed that most commercial Pima varieties grown in California were observed to be more susceptible to FOV race 4 (stand loss, stunting, etc) than any Upland cottons (G. hirsutum). However, tested Acala and non-Acala Upland cotton varieties were still infected by FOV race 4 at levels where plants would be expected to reproduce the fungus and expand inoculum. Highly resistant germplasm to FOV race 4 were identified on Pima for inoculum levels tested (field and greenhouse). Development of highly resistant germplasm and genetic mapping populations are ongoing in order to further study FOV resistance and its heritability. OBJECTIVE 2 - To develop, maintain, and distribute molecular genetic, genetic, and cytogenetic tools for the evaluation and enhancement of cotton germplasm. 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. D8-R lines that had previously been backcrossed to various elite cotton cultivars to improve their agronomic quality were self-pollinated to increase available seeds for evaluation. Reciprocal crosses were made between four elite cotton cultivars and the original D8 restorer containing the cytoplasm of G. trilobum and Rf2 and between the elite cultivars and B411R with the G. harknessii cytoplasm and RF1. The effect of cytoplasm was determined in replicated field trials at Las Cruces, NM. The D8 cytoplasm caused about a 20% reduction in yield whereas no detectable yield loss was associated with the D2 cytoplasm. Additional quantitative trait loci (QTL) were added to the intraspecific AFLP map of Gossypium hirsutum. Loci added were for yield and the within boll yield components: bolls per plant, number of fiber per seed, average weight per fiber, and number of seeds per boll. The number of QTL identified by composite interval mapping were 2, 1, 2, 2, and 0, respectively. An additional 2 markers were identified using interval mapping and 40 using single point analysis [AES-LA]. Using G. tomentosum chromosome substitution lines of G. hirsutum, AFLP linkage groups were able to be assigned to some of the monosomic and monotelodisomic genetic stocks. In the end, 53 AFLP markers were assigned to chromosomes and/or chromosome arms. At least one AFLP marker was assigned to each of 16 different chromosomes and 50 markers were localized to different chromosome arms. Nine of 53 common AFLP markers were found between the aneuploid stocks and the intraspecific cross used to develop the AFLP map. This allowed 8 linkage groups to be assigned to 6 different chromosomes In cooperation with Cotton Inc and cotton programs across the Cotton Belt, ARS in College Station, Texas, assembled, maintained, and distributed a standard germplasm panel of 12 diverse cotton genotypes for systematic evaluation of molecular genetic markers. We filled in the requests of the community for the permanent mapping population of 191 TM-1 x 3-79 RI lines. We also delivered 192 pairs of SSR primers that were developed from TM-1 BAC clones. Genetic mapping of SSR markers from this and other sources was conducted in the RI population. In cooperation with Texas A&M University, we assembled 5,000 physical contigs from 100,000 TM-1 BAC fingerprints. Interspecific germplasm developed in the Cotton Improvement Laboratory with G. barbadense, G. tomentosum and G. mustelinum are being distributed to the molecular geneticist for evaluation. In 2004, 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. Molecular marker technology has progressed rapidly in the 1990s with the construction of the first genetic map of cotton published in 1994. Our lab has collaborated with Dr. Andrew Paterson to expand the cotton map to include over 2500 loci. This high-density genetic map, which covers all 26 chromosomes of the tetraploid cotton genome, provides new insights into the structure, function, and evolution of the cotton genome. A majority of the loci was from RFLPs, although a few came from PCR based technology such as SSRs or STS. Data was also presented that shows the feasibility of converting the mapped loci into a Single Nucleotide Polymorphism (SNP) detection system that is better suited for use in marker-assisted breeding. To add to knowledge of gene expression during cotton fiber development, a cDNA library (prepared from Gossypium hirsutum cv Delta Pine 90) biased toward genes expressed at 20 DPA compared to 6 DPA was constructed and sequenced in collaboration with Curt Wilkerson, Michigan State University. In this case where plants were growing under fairly cool conditions, 20 DPA represented a very early stage of secondary wall deposition. From 9,121 high quality EST sequences (GenBank Accession numbers CO490611  CO499850; called G.h.fbr-sw ESTs), 3,420 unigenes were assembled within this one library, and the sequences were contributed to a comprehensive assembly of cotton ESTs, (J. Udall and J. Wendel, Iowa State University, and coworkers, http://agcol.arizona.edu/pave/cotton/. in which the library was called GH_SCW). About 15% of the translated G.h.fbr-sw 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. Based on the comprehensive assembly including primary and secondary wall fiber genes, cotton fiber expresses genes with significant similarity (25% of the proteome of this model plant. Of these, 600 were sequenced only from the secondary wall stage of fiber development. The G.h.fbr-sw library was not sequenced to completion, indicating that this is a minimal estimate of the uniqueness of secondary-wall-specific expression in cotton fiber. Therefore, secondary wall deposition in cotton fiber is accomplished through a substantial number of genes that are apparently uniquely expressed at that stage, as well as a large complement of expressed genes that are shared with the primary wall stage. These novel secondary wall sequences add to the growing platform for functional genomics of cotton fiber. The support of the NSF Plant Genome Program and Cotton Incorporated, Cary, NC is gratefully acknowledged. 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 3-79. 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. DNAs of G. barbadense chromosome substitution BCnF1 stocks were created and distributed to several genome-mapping labs. Backcross-mediated chromosome substitution with 3-79 into TM-1 was advanced for more recently discovered G. hirsutum hypoaneuploids. Prospectively new G. hirsutum hypoaneuploids continue to be identified and tested. The identification of a new monosomic G. hirsutum was reported, H21. An expressed sequence tag (EST) database, developed from a diploid cotton species (Gossypium arboreum L.) (http://cfgc.ucdavis.edu), contains ~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 (MUSS and MUCS) primer pairs were designed and tested for PCR amplification. Based on PCR amplification, the transferability of the microsatellite markers was high in that 83% of the primer pairs successfully amplified products from eight Gossypium species, including both diploid (A and D) and tetraploid accessions and cultivars (total 23 entries). High interspecific transferability can be due to the sequence conservation of the gene-coding region of the genome. Polymorphic PCR-amplified DNA fragments among species of cotton were observed for 311 (62.7 %) MUSS and 251 (48.0%) MUCS, while 457 (44.6 %) were monomorphic. 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 (Table 1). 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 barbadense showed segnificant sequence similarity to genes or putative genes with known function including endo-²-1,4-glucanses, Cytochrome P450-like protein, expansin, RING zinc finger protein, and ABC transporter, which are related to fiber. PIC values ranged from 0.12 to 0.74. Analysis of relationship among 23 accessions generated three major clusters which grouped separately the different species. Besides being of value for genome analysis per se, the origin of these SSR markers from fiber-derived ESTs and their demonstrated portability, suggest that they may also be useful for marker-assisted selection in breeding of fiber quality-related genes. This information will be useful to other geneticist working with cottons and will facilitate future efforts to map the cotton genome. Funding for this project was provided by Cotton Inc. These microsatellite markers are made public through the Cotton Microsatellite Database (CMD), http://www.mainlab.clemson.edu/cmd/projects/muss/index.shtml To exploit the cotton sequence in the public domain, simple and complex SSRs derived from fiber ESTs were identified as potential DNA markers for genetic mapping and screening germplasm collections. The SSR candidate list is posted on the labs web page (http://cfgc.ucdavis.edu). Several groups are working from this list, and new fiber loci have been mapped, increasing the number of fiber genes on the molecular map. The discovery of cotton SSRs was recently expanded to screen the new global assembly of cotton ESTs (Udall et al., 2005). Mapping of fiber mutants identified new fiber QTLs and characterized new fiber loci as candidate genes for functional analysis. OBJECTIVE 3 - To adapt and develop methodologies to evaluate, modify, and utilize cotton germplasm. The CIL (Texas) evaluated the use of early generation testing (EGT) methods to predict advanced strain performance at College Station (CS) and Weslaco (WS), TX based on lint yield, lint percent, micronaire, fiber length, fiber strength, fiber elongation, and fiber uniformity. Associations (P=.05) were present for fiber length and fiber strength. At WS, F2 EGT lint yield data were associated with the number of plants and strains selected through the F6 generation, yet no associations were found for fiber quality characteristics. Lack of consistent r values across generations and locations suggest a large environmental influence on both lint yield and fiber quality parameters. 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. We continued to develop molecular descriptors with portable PCR-based DNA markers that will be at foundation of germplasm characterization. In 2004, an investigation was initiated to determine the relationship between genetic relatedness (as determined by molecular analyses) and general combining and specific combining ability in U.S. and foreign Gossypium barbadense cultivars. Yield, fiber, and agronomic data were obtained from replicated tests of diallel F1 lines at Maricopa, AZ and Las Cruces, NM. The limited number of regenerable cotton varieties has not only exacerbated the narrow genetic base of commercial cotton varieties, but also remains a major obstacle for the routine use of gene transformation for cotton improvement. Our lab has initiated a project to evaluate elite breeding lines developed by the UGA 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. 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. A list of candidate genes has been generated for functional analysis and to develop into molecular markers for genetic mapping. Cotton fiber chips are distributed to the cotton community on a cost recovery basis OBJECTIVE 4 - Germplasm enhancement for biotic and abiotic stress resistance and agronomic traits. Diploid hybrids were made between the A genome (G. arboreum) accessions and D genome species (G. trilobum, G. raimondii and G. aridum). These need to be doubled to convert these to allotetraploids and restore fertility for crossing with cotton. One hybrid (A2 x D8) had doubled sectors after colchicine treatment and a flower was obtained with some fertile pollen. This was crossed as male to AD1 cotton and two plants were obtained. Prior to boll maturity the two F1 plants died with symptoms similar to that shown by lethal gene complementation, but four BC1 embryos were obtained by rescue culture. One of the plants died with symptoms similar to the trispecies hybrid, whereas the other three matured, developed bolls and yielded BC2F1 and BC1F2 seeds. The death of the tri-species hybrids and one of the BC1 plants suggests that a genetic lethal complementation was present. As a part of the project to enhance the secondary germplasm pool we developed a trispecies hybrids with a D3-compatible AD1: (B1 x D3-d x AD1). These hybrids were self-pollinated and also backcrossed to the same AD1 to produce F2 and BC1 seeds. These 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. Cotton improvement laboratory is an aggressive plant breeding program headquartered at the flagship campus of Texas A&M University centers around improving the genetic variability, yield potential, agronomic performance, and fiber quality of germplasm or cultivars adapted to Texas while enhancing resistance to biotic pests, such as insects, nematodes, and diseases, and abiotic stresses such as drought. The CIL developed and released of four germplasm lines and one new cultivar in 2004. These were TAM 96WD-18, TAM 96WD-69s, TAM 98D-99ne, and TAM 98D-102. TAM 96WD-18 and TAM 98D-102 have excellent fiber packages and competitive agronomic performance, while TAM 96WD-69s is a glabrous genotype expressing a measurable level of fleahopper resistance and TAM 98D-99ne carries the nectariless trait in a good agronomic background for central and south Texas. Tamcot 22 was released as a conventional cultivar. The CIL (Smith and Thaxton), collaboratively with Dr. Jim Starr, Plant Pathologist, has developed an interspecific population that is segregating for both root-knot and reniform (a problem in the LRGV) nematode resistance. The donor parent for reniform resistance is a G. barbadense. Additional efforts (Stelly) are underway to introgress the high level of resistance found in G. longicalyx. Converted race stocks have been in our basic breeding program with a number of advanced strains and segregating populations developed. Breeding programs have been evaluating the BC2F2 populations for yield and fiber quality. The race stocks are being screened for resistance to cotton fleahopper, silverleaf whitefly, nematode resistance. A project is underway to simultaneously improve G. hirsutum germplasm for fiber quality and heat tolerance. In 2003, 90 lines selected for heat adaptation and fiber quality at Maricopa, AZ were evaluated in non-replicated tests at Tifton, GA, Maricopa, AZ, and Shafter, CA. In 2004, 16 of these lines - along with check cultivars - were evaluated in replicated tests at the above locations. On the basis of their yield and fiber performance, three of the 16 lines have been selected for public release in 2005. The project continues, incorporating fiber quality traits from Gossypium 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. The nectariless trait in cotton has been well documented as a host plant resistance trait through a reduction in tarnished plant bug (TPB), Lygus lineolaris (Palisot de Beauvois). Changes in insect management technology (boll weevil eradication and transgenic Bt cotton) have allowed TPB to become a key pest. Improved methods of managing TPB in cotton are needed to fully exploit the potential benefits of the new insect management environment in cotton production. Over 400 nectariless progeny rows (F3 to F10) where grown in 2003 and the top performing entries 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 on Delta Branch Experiment Station, Stoneville, Mississippi. Approximately 90 nectariless progeny rows were selected for evaluation in 2004 for yield and fiber quality when grown under plant bug infested and controlled (split-plot design) conditions and compared to a conventional nectaried commercial check variety. TPB infestation and damage data (insect counts, square shed, and bloom injury) and crop production data (square and bloom production, and yield) were evaluated by calculating ratios of data for infested/controlled plots and ranking the ratios from those inferring greatest resistance to those inferring least resistance to TPB. The top performing entries (upper 50 percent) have been selected for repeating the evaluation in 2005. 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. A second year of testing will help determine if testing under plant bug infested vs. controlled conditions is useful in discriminating among susceptible and more tolerant genotypes. Using the African species G. longicalyx as the immunity source, euploid 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. Genome-wide introgression efforts from G. tomentosum, G. mustelinum, G. longicalyx and G. armourianum were advanced 1-2 generations. Generation means analysis field evaluation experiments conducted for G. tomentosum and G. mustelinum introgression products. Improved methods in cotton transformation were published, and a pipeline for high-throughput cotton transformation to test gene function, and determine the impact of ectopic expression on fiber traits has been developed. OBJECTIVE 5  To refine and develop cotton breeding and variety testing methodologies and techniques. The ability of the program GGEbiplot to identify the best environments for selection (discriminating environments) was evaluated. Results corroborated historical anecdotal information on the best environments for selection. During 2004, we grew approximately 2000 F3:4 lines (pedigree method) and approximately 200 F3:4 lines from single-seed descent populations. Based on fiber data from individual F3 plants collected the previous year, a selection index was applied to the pedigree lines based on a combination of upper quartile length of fibers (inches, by fiber weight) (UQL), short fiber content (count) (SFC) and lint weight seed-1 (LWS). From each population the best 50 F4 rows from F3 plants with the highest UQL, lowest SFC, and highest LWS were selected. One hundred ninety-five F4 lines derived from the single-seed descent populations were also harvested without selection. This leaves us with a minimum of 300 lines derived by pedigree and 195 lines derived by SSD for future evaluation. We will begin yield-testing of these lines to compare effects of inbreeding method on estimates of genetic variance, heritability, and mean performance for a variety of fiber quality, yield, and yield-related traits. Update information on genetic relationships. Pedigree information has not been updated since the 1997 publication. Genetic uniformity has been examined since the introduction of transgenic cultivars and it was found that the level of diversity in the field was not compromised. Fiber strength has increased since 1980 and the sources of fiber strength genes were determined to come from two public breeding programs Quantify advantages and disadvantages of divergent variety development strategies. SSD v. Pedigree . David Weaver ( Auburn) has completed growing F3-derived F4 populations ( 6 different ones) this past summer. F5 progeny rows will be grown next summer and selected rows will be yield tested the following summer. Develop guidelines for efficient and discriminating genotype evaluation. Visual selection versus yield measurement. This work was performed by David Caldwell, Gerald Myers, Ted Wallace, Fred Bourland, and Daryl Bowman. There is a positive correlation between visual ratings and seed cotton yield suggesting a viable option for breeders. Compare spatial designs for efficiency; compare lattice designs with randomized, complete block designs. Work has been done by Wayne Smith and others and needs to be compiled. Compare post-mortem analyses- NNA, Trend, Lattice. Data are available and needs to be compiled. Evaluate the need to incorporate systems testing in variety trials for transgenic technologies. Interactions by varieties with technologies were not detected indicating that the relative ranking of cultivars within system (transgenic) should remain the same. However, the systems may need to be tested with cultivars to reveal true yield potential for the growers. Investigate the consequences of forward crossing of transgenic parents. This may best be evaluated over the long term by the private sector. OBJECTIVE 6 - To develop cotton bioinformatic systems. PEG-induced gene expression in cotton roots (Hendrix) The objective of this study was to develop a gene expression model of drought tolerance in cotton based on responses of the drought tolerant cultivar Siokra L23 to osmotic shock induced by polyethylene glycol (PEG). A PEG-induced phenotype was described for the plants based on time-course measurements of photosynthesis and leaf temperature. A gene expression profile was then developed from root RNA extractions taken at 0, 1, 4, 24, and 96 h after stress initiation utilizing cDNA-AFLP and cotton-fiber-based microarray chips. Siokra L23 progressed through two phases of PEG-induced stress that we designated response (0 to 48 h) and recovery (48 to 96 h). Forty-eight and 363 PEG-responsive genes were identified in the cDNA-AFLP and microarray analysis, respectively. Global gene expression profiles revealed an adaptation to the stress after 96 h. Functional categories played distinct roles in the response and recovery phases. The expression profiles of aquaporins suggested that osmotic adjustment occurred between 4 and 24 h. Sucrose synthase and several genes in the glycolytic pathway supported the hypothesis that sucrose was a solute involved in this osmotic adjustment. The expression patterns observed in this study provide insight into the mechanisms by which Siokra L23 responds to osmotic shock. ARS in College Station, Texas, re-organized CottonDB data classes and updated them 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 has been 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. These databases will be linked to the genetic map via the Cotton Portal. Expression data in the public domain can be downloaded into a MIAMI-compliant database for view by the community.

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