Multistate Research Project

S-304

from

Southern Regional Development Committee 98-01 (S-258)

A Proposal Entitled:

Development of Genetic Resources for Cotton

December 2000

TABLE OF CONTENTS

Cover Page	i

Table of Contents	1

Statement of Problem	2

Justification	3

Related Current and Previous Work	6

Critical Review of S-258	9

Objectives	24

Procedures	24

1.	To acquire, curate, characterize, and evaluate the species,

races, and genetic types of Gossypium (cotton).	24

2.	To develop, maintain, and distribute molecular genetic,

genetic, and cytogenetic tools for the evaluation and

enhancement of cotton germplasm	25

3.	To adapt and develop methodologies to evaluate, modify,

and utilize cotton germplasm.	27

4.	Germplasm enhancement for biotic and abiotic stress

resistance and agronomic traits.	28

5.	To refine and develop cotton breeding and variety

testing methodologies and techniques.	30

6.	To develop cotton bioinformatic systems.	34

Expected Outcomes	35

Organization	36

Signatures	38

References	39

Appendix 1. Participating Institutions by Objective	43

Appendix 2. Member and SY, PY, TY Commitment for Each SAES Cooperator	44

STATEMENT OF PROBLEM

Cotton (primarily Gossypium hirsutum L. and Gossypium barbadense L.) is the leading textile fiber in the world and the second most important oilseed. Widely grown in the U.S., it is a significant contributor to the U.S. economy. It is grown in 17 states from Virginia to California and from Missouri to the lower Rio Grande Valley of Texas, covering an estimated 15.7 million acres in 2000. In the U.S., cotton is the fourth most important crop (raw product value 5 to 6 U.S.$ billion/yr; economic impact U.S.$15 to 25 billion/yr), the most important industrial crop (the underpinning of the U.S. textile industry, i.e., several hundred thousand jobs of about U.S.$80 billion/yr), and the second most important oilseed crop. Clearly, productivity and profits from cotton fiber, cottonseed, and their products are important overall to the U.S. economy, but especially to that of rural America. Technological advances have enabled U.S. cotton producers to achieve unparalleled production efficiency. Approximately half of that efficiency is attributable to genetic improvement through breeding. The increases followed rapid producer adoption of new technologies that emerged from cotton research and development at public institutions and in private industry. Production in the U.S. has, however, been recently characterized as increasingly variable and unstable (Report of the American Cotton Producers, 1999). It is relevant, therefore, to determine the cause or causes of those problems and to propose appropriate solution(s). This proposal presents a coordinated plan of research to be undertaken during the 5 yr period 2000-2005 based upon a consensus of public and private cotton researchers.

Recent analyses of historical cotton yield data have indicated that increases in cotton have averaged 1.3% annually over the last 39 yr, or about 6 lb./acre/year. Unfortunately, this linear trend, upon further analysis, showed that much of the increase could be attributed to progress during the period 1970-1985. Since then, progress in increasing cotton yield has slowed and in fact was negative from 1992-1998. The question that arises from this is, "Have we reached a yield plateau?"

Several possible answers to this question have been suggested. A common response is that a 5 to 10 yr lag often occurs between a scientific discovery and its development into a technology usable by producers. Such a time lag (or longer) is a common feature in conventional plant breeding efforts. In recent times, cotton seed industry research has focused on the addition of traits to cotton using biotechnological tools. This has been successful in reducing the cost of production of cotton by providing the plant with increased resistance to some key insect pests [e.g. Bt genes(s)] and/or allowing more cost-effective weed control (e.g., glyphosate and buctril tolerance). These developments have come at a cost, however, in research time and effort on the improvement of conventional cotton varieties.

A third possible answer is in the decline of publicly funded research into the development of improved cotton germplasm and varieties. 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. The result over time has been a decline in the genetic diversity of commercial cotton varieties. This has a profound consequence on the stability of cotton yield and on the vulnerability of the crop to biotic fluctuations. An additional impact of this narrowing genetic base is that little variation is available for use in the development of new, higher yielding cotton varieties.

Policy, funding, and regulatory changes over the last decade have also had an impact on the development of improved cotton varieties/germplasm. The efforts devoted by the USDA-ARS to develop cotton varieties has been suspended and emphasis placed on germplasm improvement and characterization. While this will have future positive impacts on cotton variety development, it has in the short term limited the availability of elite improved genetic material for direct use in variety development. Funding changes at the public level have had one of the most pronounced impacts on the continuation of the historical trend toward improved cotton varieties and genotypes. There has also been a decline in the number of public scientists involved in basic cotton breeding (Report of the American Cotton Producers, 1999). Finally, several regulatory changes such as the Plant Variety Protection (PVP) Act, rulings by the Patent and Trademark Office, modifications of the PVP Act to include essentially derived language, and most recently the Convention of Biological Diversity have all served to divert and/or redirect both public and private research efforts into cotton improvement and to restrict the free exchange of germplasm necessary for future improvement efforts.

These possible answers point to a six point solution to address the problem of yield stagnation and loss of variability in cotton. These solutions address the constantly evolving biological pressures and industrial processing requirements that demand the genetic modification of U.S. cultivated cottons so that they can be advantageously produced and sold in an internationally competitive market. These solutions will require the concerted effort of public researchers at the Federal and State level as well as contributions from concerned private industry partners. In addition to proposing researchable objectives associated with applied cotton improvement, it is important that over the next 5 yr that a comprehensive set of molecular tools and resources for public cotton improvement is developed. This proposal also addresses the need for those resources to be immediately accessible and fully assimilated into the breadth of public cotton research and breeding infrastructure which generates U.S. varieties, germplasm, and professionals. Finally, the expected outcomes of the proposed research will help ensure the enhancement of cotton production and profitability for producers nationwide.

JUSTIFICATION

GENETIC GAIN AS A BASIS OF ECONOMIC GAIN: The importance of genetic improvement have long been undersold relative to other U.S. investments in cotton, particularly price support systems. Whereas price supports are a short-term fix, genetic improvements offer long-term base level improvements for U.S.-grown cottons. Breeding of economically important plants improves the economic return from those plants, e.g., more yield and/or quality for the same or reduced economic input. The biological prerequisites to breeding include genetic diversity, hybridization, genetic segregation, and phenotypic manifestation. Scientific requirements include a diversity of laboratory, greenhouse, field and specialized tests, and testing environments. Breeding programs necessarily integrate a very broad scope of supporting scientific and practical disciplines such as genetics, statistics, agronomy, pathology, entomology, physiology, reproductive biology, taxonomy, and so on. In the case of cotton, breeding results in improved productivity and quality of cotton and cottonseed. Because of its high value as a crop, foodstuff, and base for U.S. textile industries, economic gains from cotton breeding far exceed their cost.

As successful as cotton improvement efforts have been, however, the pace of innovation must accelerate to keep U.S. cotton production globally competitive and to meet the demands of increasing world population. The rate of genetic gain for cotton fiber yield from traditional breeding methods was estimated to be roughly 1.3%/yr (Lewis et al., 2000), i.e., an increase in the U.S. of about $50 million in raw product and $150 million to $200 million in economic impact per year. Unfortunately, investments in cotton breeding do not reflect the magnitude of returns. A recent publication (Report of the American Cotton Producers, 1999) highlights some disturbing trends in the improvement of cotton. Following a period of steady increases in cotton yield from 1970 through the mid 1980s, cotton yield increases leveled off and even become negative for a time. Primary causes of this decline most likely include reduced public staffing and funding for germplasm collection, introgression, genetics and breeding, and the diversion of commercial investments from conventional breeding to backcross breeding programs. Whereas the former elevate base performance, the latter merely add a trait or two by incorporating transgenes into existing elite genotypes.

Because genetic improvement is the most cost-efficient means of improving the profitability of economically important plants and because cotton is so valuable to the U.S., it is of paramount importance that the U.S. capitalizes on contemporary technologies for the improvement of U.S.- grown cottons. One of the rapidly developing, new technologies with potential to greatly accelerate cotton improvement is the broad area of genomics. Genes control all metabolic processes required to sustain life. Cotton can only be crossed with closely related plants. Wild relatives and more distant species are also sources of useful genes, but in many cases, the reproductive specificity of cotton prevent effective utilization of other plant genes. To do this, the U.S. must meld conventional and modern approaches to plant improvement by:

1.	Developing public structural and functional genomics resources for cotton that are made fully available to the public researcher in a timely (i.e., immediate) manner:

2.	Applying them to field-based and laboratory-based cotton breeding and genetic improvement efforts; and

3.	Strengthening those components of its cotton program that address germplasm, its analysis (e.g., markers, traits), usage (e.g., introgression), genetics and breeding.

GENETIC VARIATION IS TOO LIMITED: High-yielding cultivated upland cotton types have long been regarded as being rather uniform genetically and thus relatively vulnerable to potential pathogen or insect epidemics (Bowman et al., 1996). This has been demonstrated repeatedly with a number of technologies, most recently and perhaps most convincingly with isozymes and molecular DNA markers. Without genetic variation, genetic improvement is impossible. Empirically, we can deduce that the biggest constraint in cotton improvement has been the limited amount of genetic variation available for selection in the elite backgrounds present in breeder's nurseries. Increased genetic diversity must be made available to breeders in a usable form. To be incorporated into a varietal breeding program, the genetic diversity must be available in a form that allows expeditious incorporation of the trait, without inadvertent incorporation of undesirable genes. Otherwise, the elite genetic composition of the varietal breeding materials becomes "polluted" with agronomically unacceptable genes ("trash"). In essence, germplasm utilization thus requires extensive "pre-breeding" to generate genotypes that are "elite" or at least "advanced", i.e., free of large numbers of agronomically undesirable genes. The presence of such genes undermines the statistical probability of recovering genotypes of sufficient caliber to compete with existing varieties.

Opportunities to enlarge the scope of breeding activities continues to be extended by in vitro culture and molecular genetic technologies which allow genes to be captured from increasingly distant sources. In general in vitro culture allows for the transfer of larger numbers of genes, with relatively little precision; whereas, molecular genetic technologies allow for the transfer of a relatively small numbers of genes, with greater precision. These new technologies complement each other and more conventional methods of improvement.

NEED FOR COOPERATIVE WORK: Cotton is grown in the U.S. in a diversity of production environments and under a wide range of production systems. This is true for upland cotton which accounts for approximately 98.8% of the total production in the U.S., but also for American Pima cotton as well which accounts for the remainder (National Agricultural Statistics Service, 2000). Consequently, cotton researchers are strategically scattered across the U.S. Nevertheless, these researchers share commonalities that are best addressed through a cooperative research approach.

BENEFIT TO THE SOLUTION: The development of more detailed information regarding cotton genetics and cotton improvement procedures will allow present and future cotton researchers to meet demands for improved cotton yield and quality. New techniques, particularly those involving molecular genetics, offer additional approaches to genetic dissection and manipulation for genetic improvement and complement more traditional methods of germplasm usage. These methods take advantage of molecular techniques for analysis of genomes (structural genomics), genome expression (functional genomics), indirect selection for desired traits [marker assisted selection (MAS)], as well as gene cloning and genetic engineering The objectives of this proposal will allow for the development of these new techniques in addition to the refinement of existing techniques and the development of a uniform database of information for the extraction and input of research results.

Beyond the benefits afforded to cotton, success in this research will aid in the development of new genetic research tools to expedite and expand the scope of genetic improvement possible in other plants and animals. For cotton, as for these other organisms, there are new opportunities to enhance economic yield, lower production costs, increase plant productivity and reduce pesticide and other chemical inputs.

IMPACT ON SCIENCE: Once implemented for cotton, the methods will increase the rates of genetic improvement and will expand the scope of targets amenable to breeding and genetic engineering. Of particular importance for breeding are molecular markers and linkage maps for genetic dissection and MAS. Existing markers and maps are grossly inadequate for serious breeding applications in terms of their numbers, kinds, accessibility, and quality. This must be remedied by rapid expansion of effort by researchers who desire to share their products in an immediate and cost-effective manner. For longer term applications in breeding and genetics, comprehensive structural genomic and functional genomic resources are essential.

The research outlined in this proposal also affords additional benefits that positively impact the sustainability of U.S. agricultural research. Chief among these is that researchers involved in this project are primarily responsible for the training of the next generation of public and private breeders. Broad societal benefits relate to how the results of cotton breeding and genetic research into plant resistances to insects, pathogens, and abiotic stresses can reduce the need for pesticides and other chemicals which are expensive, pollute the environment, and require energy for synthesis and distribution.

RELATED CURRENT AND PREVIOUS WORK

This new project will expand and update the activities of its predecessor, S-258, entitled "Evaluate Cotton Germplasm to Identify Useful Characters for Genetic Mapping." That project descended from projects S-77, and before that S-1. Research conducted as part of these projects has touched virtually every aspect of cotton genetic improvement and has had major regional and U.S.-wide impact. A brief summary follows:

BREEDING: Research from the previous regional projects has contributed toward the success of cotton breeding programs throughout the U.S.. By affecting the goals, germplasm, methods, and staffing of U.S. public and private varietal breeding programs, it has resulted in major shifts and improvements in the characteristics of varieties grown in all parts of the U.S.. Many of these helped adapt cotton to changes in textile technologies and to genetically respond to abiotic and biotic pressures. Testing and selection methods for fiber yield and quality and a number of other traits, such as resistance systems to various insects and pathogens have been among the perennial targets. As noted below, the Regional Research Projects (RRPs) have facilitated organization, exchange, and usage of germplasm. Prevarietal breeding has provided breeders with germplasm and techniques suitable for development of genetically elite cultivars (Van Esbroeck et al.,1998). A number of specific methods for managing breeding materials have been developed, tested, or assessed, e.g., backcrossing, pedigree, single seed descent, doubled haploid, and modified recurrent selection methods.

GERMPLASM COLLECTIONS: Germplasm holdings of The National Cotton Germplasm Collection have been developed through collections in the field by U.S. scientists, sharing of collections with scientists in other countries, international goodwill, and specific agreements. The Collection now includes in excess of 8,000 accessions, including a broad array of wild 26- chromosome species from Australia, Asia, and Africa; cultivated 26-chromosome species; accessions from the New World including wild 26- and 52-chromosome species; wild types of U.S.-cultivated 52-chromosome species; and obsolete and current varietal types of the U.S.- cultivated 52-chromosome species. These are maintained and distributed from working collections maintained at College Station, TX and also maintained in specialized long-term storage facilities at Ft. Collins, CO.

TRAIT DISCOVERY, GENETIC ANALYSIS, AND BREEDING POTENTIAL: A wide range of traits and variants have been reported and characterized to varying degrees. Examples include developmental and morphological traits, proteins, fertility, isozymes, and resistances and tolerances to biotic and abiotic challenges. For many traits, studies established the inheritance (qualitative/quantitative, gene number, mode of action, interaction) and/or the heritability, genic or trait interactions, benefits, and deleterious effects. Where perceived to be of potential practical use, e.g., for "hairiness" (trichome density), gland density and distribution, or leaf shape (canopy), these studies have involved extensive experimental breeding and testing for not only the desired improvements, but also potential inadvertent negative effects.

MAPPING: Cotton lacks linkage maps that are significant from a breeding standpoint. Prior to the advent of molecular markers, mapping was restricted to conventional markers, e.g., morphological and isozyme traits. Isolation of cytogenetic stocks and their maintenance, development of markers [mostly Restriction Fragment Length Polymorphisms (RFLPs) and Randomly Amplified Polymorphic DNA (RAPDs)], development of a few mapping populations, and linkage mapping. The molecular marker linkage maps that have been assembled for the n=26 cottons, G. hirsutum and G. barbadense, are incomplete, involve many markers that are not readily available, and involve markers that are expensive to use. No published map has attained 26 linkage groups. Remarkable as it seems in this modern era, only cytogenetic markers allow identification of all of the 26 chromosomes of cotton. Cotton may have the dubious distinction of being the only crop for which that is true but, hopefully not for long. To date, all publically available maps for cotton are primitive and far from saturated (Reinisch et al., 1994; Yu et al., 1998; Shappley et al., 1998). Biological and other factors are involved. Cotton (G. hirsutum and G. barbadense) has a relatively large number of chromosomes, a large genome size, and (as predicted by cytogenetic data) a very high rate of recombination. The high rate of recombination imposes a need for very large numbers of markers to reach "closure". Moreover, constraints on access to and use of markers and data have limited the utility of many markers and map data, in some cases severely. Newer types of markers offer greater speed, portability, and lower cost than previous types. More recently, bacterial artificial chromosome (BAC) clones of cotton have been developed for map-based cloning efforts and an integrative genetic/physical mapping initiative (Yu et al. 2000). The BAC libraries consist of 115,584 clones, derived from a TM-1 isogenic line, covering 7.2X haploid AD genome equivalents. The average size of the DNA inserts is 143 kb, with a range of 90 to 210kb. In addition, genetic bridging of cotton with other model plants such as Arabidopsis has been investigated with Expressed Sequence Tag (EST) analysis. At least 10% of the conserved Arabidopsis EST singletons may be directly applicable to cotton functional genomics and gene discovery (Yu et al., unpubl.).

GERMPLASM INTROGRESSION: Introgression is the transfer of genes from one plant or species to another; the challenges are [1] to make the transfer and [2] to ferret out the agriculturally beneficial alleles from the disadvantageous ones. In the 20th century, wide crosses among Gossypium species were used extensively in cytogenetic studies to determine genomic relationships among species, but the task of introgressing wild species germplasm was been less adequately addressed. Introgression is a time- and resource-consuming task that requires sustained efforts. The benefits of such introgression are usually impressive and may be exemplified in cotton by the PeeDee cotton breeding program which spent decades introgressing, breeding, and selecting germplasm from the interploid interspecific tri-species hybrid (G. herbaceum, G. trilobum, and G. hirsutum) originally constructed by cytogeneticists. However, introgression projects have been an inadvertent victim of the heavy reliance on short-term competitive grants over the past 2 decades, because introgression requires a long time-frame (it cannot be completed during period of funding) and in many instances involves empirical presumptions about the existence and value of cryptic genes in the donor. Recent molecular marker work with interspecific introgression into tomato and other species increased the awareness of germplasm as a source for beneficial alleles. These studies have documented the fact that beneficial alleles exist cryptically in wild germplasm even in species that seem wholly worthless for the respective trait. In cotton, an example would be the development of the high strength PeeDee germplasm. It is thought that the genes for fiber strength came from G. trilobum yet typical members of this species have very short (often sparse) hairs on the seed. The new data underscore the reality and ramifications of "epistasis", a genetic term coined in 1902 by Punnett to describe the interaction of alleles at one gene locus with alleles at another.

In cotton, it has been possible to garner support for intraspecific and interspecific introgression among the 52-chromosome species. Intraspecific work has involved introgression of germplasm from wild accessions into the cultivated types, e.g., the G. hirsutum racestock conversion program (converts photoperiodic to photoperiod-insensitive materials). The interspecific work has primarily involved G. barbadense into G. hirsutum. Both species are cultivated and have number of complementary attributes, so such work is relatively easy for review panels to comprehend, rationalize, and justify. Examples have included development of interspecific F1 hybrids, F2 hybrids, recombinant inbreds, backcrosses in both directions (e.g., into upland and Pima types), backcross inbreds, and specific chromosome and chromosome arm substitutions. Among the more noteworthy examples of qualitative trait introgression are the methoxylation genes from G. raimondii, nectariless genes from G. tomentosum , hairiness alleles from G. tomentosum, G. raimondii, and G. armourianum, and bacterial blight resistance from G. arboreum, G. herbaceum, and G. anomalum (for a review, see Percy and Kohel, 1999; Endrizzi et al., 1984).

CRITICAL REVIEW OF S-258: There were several major accomplishments from the regional project S – 258. These major accomplishments listed under the two major objectives were as follows:

1.	Development of an integrated genomic linkage map of Gossypium

A cotton RFLP map of 705 loci at average marker spacing of 7 cM has been assembled. The cotton genome has a recombinational length of approximately 5000 cM. Consequently, a map with 26 linkage groups with markers spaced at 25 cM or less (e.g. ~a 25 cM map") will require more than 1000 markers. Nearly alI linkage groups have been assigned to "subgenomes" (A or D) and 14 linkage groups have been assigned to chromosomes. Three homoeologous chromosomes pairs in the A and D genomes were identified on the map. Chromosome pairs 1/15 and 6/25 were previously identified by classical evidence, whereas the homology of 5/20 is newly recognized. Genomic hybridization patterns of cDNAs and hypomethylated (pst1-digested) genomic sequences reflect the known polyploidy of tetraploid (n=26) cottons and suggest that "diploid (n=13) cottons may be paleopolyploid. Subject to a formal request, a subset of 89 markers that identify each chromosome arm in G. hirsutum has been made available to the scientific community. Two bacterial blight resistance genes in a Pima S-7 x Tamcot SP37 F2 population have been tentatively mapped to regions in the genome. (AES-TX).

Telosomic and disomic lines heterozygous for five genes linked on chromosome 12 were testcrossed, and progeny scored to test published hypothesized gene order and map distances of: centromere - 4 mu - ne, (nectarless) - 5 mu - Lf (Leaf fleck) - 2 mu - N, (Naked seed) - 28 mu - 9/2 (glandless) - 20 mu - bw, (withered bract). In pair-by-pair comparisons of the genes, the results from the telosomic and disomic progeny did not differ, suggesting that the telosome was not chromosome 12. The data were combined to estimate recombination frequencies, but the centromere:ne distance could not now be estimated. Also, Lf was not recovered in the expected 1:1 ratio. Calculated distances were: ne - 42 - Lf - 35 - N - 29 - 9/2 - 12 - bw,. The ne, - U, Lf - N, and ne, - N, map distances were significantly exaggerated, probably due to the inviability of Lf. The ne, -gl2, ne, - bw,, N, - 912, N, - bw,, and 9/2 - bw, distances were all close to the previously reported distances. (AES-AZ).

Putative segmental duplication-deficiencies (DPDFs) were derived from translocation heterozygotes involving chromosomes 8, 11, 13, 19, 21, 23, and/or 24. Hybrids have been or will be produced from crosses of the DPDFs to Pima 3-79 for identification of DPDF F' progeny and subsequent analysis of RFLPs. Results are expected to establish the chromosomal identity of RFLP linkage groups that presently remain anonymous. (AES-TX).

Three quantitative trait loci (QTL) associated with bacterial blight resistance genes were identified in interspecific cotton populations. Two QTLs map to A subgenome chromosomes. Applicability of fluorescent in situ hybridization (FISH) techniques to cotton meiotic and mitotic chromosomes was further improved. FISH, RFLP, and bacterial artificial chromosomes (BAC) technologies were integrated to form a set of tools for mapping and map-based cloning. Analysis of the distributions of repetitive sequences, sites of integration for alien genes in transformants, and many other applications are now feasible. Preliminary analyses have been made for retransposons, clustered and dispersed anonymous-function repeats, and rDNAs. BAC-FISH was shown to provide an invaluable approach to FlSHing with low-copy or unique DNA sequences. (AES-TX).

A combination of PCR and RFLP was utilized to identify genetic markers in intraspecific cotton populations. The PCR was used to amplify specific DNA sequences that code for important biological function and the amplified products were used as probes in a RFLP mapping program. This mapping strategy is being applied to a G. hirsutum F2 population from the cross of PD-3-14/'Zhongmian 12'. (AL-1890; ARS-SC).

A nested PCR technique was utilized to amplify 350 bp and 500 bp fragments of a specific fiber quality gene. This fiber protein gene is the only one yet characterized and it also plays a unique regulatory role during fiber development. Two strong bands in the homozygous parents putatively confirm two loci for this fiber quality gene in the cotton genome. (AL-1890).

A silver staining method for the AFLP technique in cotton has been developed which has the following advantages over radioactive dNTP techniques: 1) it is less expensive, simpler and faster with direct detection of DNA fragments; 2) radiolabeled compounds were not used; 3) it does not require Southern blotting and 4) it can resolve DNA fragments over a larger size range than RFLP or RAPD techniques. Continuing efforts to develop molecular markers in cotton using chromosome substitution lines have produced four AFLP markers ranging from 400 bp to 560 bp on chromosome 16 (primer pair E-AGC and M-CTC), a 450 bp AFLP marker on chromosome 17 (E-AGC and M-CTT primer pair) and a 320 bp AFLP marker on chromosome 25 (E-AGC and M-CTA primer pair). A 1140 RFLP (HindIII) marker from a microsatellite probe also was found to be located on the short arm of chromosome 6 and a 1460bp RFLP (HindIII) marker of a cDNA probe was located on chromosome 20. Studies of microgametophyte development using chromosomal deficient stocks indicate that some genes responsible for pollen development are located on chromosome 12, 17 and 25. (AL-1890).

An F2 population from a trispecies hybrid, Gossypium arboreum x G. trilobum x G. hirsutum is being investigated for segregation of morphological and molecular markers. A total of 216 poly morphic markers (194 AFLP, 19 RAPD, 3 morphological) were scored, of which 85 had normal Mendelian inheritance. Using two point linkage analysis, 11 linkage groups were identified that spanned 523 cM with an average distance of approximately 17 cM between markers. Pilose (H2), known to be on chromosome 6, was linked to one of the groups. (AES-AR; AES-NM).

A genetic map is under construction in the population TM1 X NM24016 using SSR, AFLP, SAMPL and RAPD markers. Dr. Ben Burr of Brookhaven National Lab has identified and mapped 113 SSR polymorphisms in the population. The level of polymorphism in this interspecific G. hirsutum x G. barbadense population is intermediate between that previously observed for TM 1 x 3-79 (Kohel) and MD51 x HQ95 (Meredith). (AES-NM).

Research continues to map a G. hirsutum x G. barbadense F2 population and to conduct QTL analysis of fiber strength. Twenty-seven linkage groups were identified and one significant QTL was found. Analyses are continuing and markers are being added from collaborators' projects. (ARS-TX).

The expanded probe set (192 clones detecting about 250 loci) is available from Dr. Paterson, as stabs in microtiter format. All prior requesters of earlier probe sets, as well as new reguesters, have been forwarded the expanded set. Under the renewed grant, we anticipate that a significant number of additional DNA markers will be added to the existing map, and also that a formal revision of the map will be published in a peer-reviewed journal. (AES-TX).

An RFLP linkage map with 120 loci was developed from an F2 population of 96 plants from the cross of MARCABUCAG8US-1-88 by HS 46. These 120 loci were grouped into 31 linkage groups covering 865cM. In addition 18 non-linked loci were observed. (USDA-MS).

An F2 population of a trispecies hybrid, Gossypium arboreum x G. trilobum x G. hirsutum, was used to develop a genome map. A total of 426 polymorphic markers (332 AFLP's, 91 RAPDs and 3 morphological) were identified. Segregation patterns among 90 F2 progeny established 43 linkage groups spanning 6,450 cM. Markers were identified that were linked to three morphological traits: R1, T1, N1. Approximately 50% of the markers exhibited non-Mendelian segregation. Divergence among the genomes probably results in gamete selection. (AES-AR; AES-NM).

A cotton molecular framework map, consisting of 213 RFLP and 142 RAPD markers, has been constructed, by use of 171 F2 progeny from an interspecific cross between two improved cottons, G. hirsutum L. acc. TM-1, and G. barbadense L. acc. 3-79. These 355 loci are assembled into 41 linkage groups and cover 3,955 cM of the cotton genome. Two thirds of the linkage groups have been assigned to their respective genomic origin (A vs. D) or chromosomal identity (1 through 26) by use of diploid and aneuploid cottons. The mapping information reveals chromosomal rearrangements including possible transposition in cotton. Currently, TM-1 X 3-79 mapping population has generated over 800 DNA markers, including 198 SSRs (collaborating with Ben Burr's group at BNL) and 279 AFLPs (collaborating with Sam Reddy's group at TAMU). An integrated molecular genetic map of cotton may be expected in 1998 to consist of 1,000 DNA markers (RFLPs, RAPDs, AFLPs, SSRs, and ESTs) from this mapping population. A new version of CottonDB, the cotton genome database, is assembling cotton genomics information from various sources for cotton community. (USDA-TX;AES-TX; Brookhaven National Lab).

2.	Identify germplasm possessing desired traits and develop appropriate test populations for mapping

The first criteria for germplasm evaluation is maintenance and distribution. Plants grown for seed increase included 425 accessions at Tecoman, Mexico; 220 accessions at Rio Farms, Inc., Monte Alto, TX; 200 Asiatic accessions at College Station, TX; 60 Asiatic accessions at Fayette\/ille, AR, and 175 accessions with low fecundity in greenhouses at College Station or screen cage at Weslaco, TX. All accessions were evaluated for agronomic characteristics. Seeds of 1246 accessions were distributed nationally and internationally, and germplasm was provided to participants of this regional project. (ARS-TX, AES-AR).

Development of techniques and molecular tools are also essential for mapping. Specific primers (20 to 25-mer) were synthesized for about 70 genes. Results from these indicated that many of the low copy genes are very conserved across species (AD1, AD2). (1890-AL). Procedures were developed for preparation of extremely large numbers of perfectly spread mitotic metaphases (and other nuclear stages) from root tips of all Gossypium species. Procedures were established for genomic in situ hybridization (GISH), fluorescent in situ hybridization (FISH) using single or dual labeling, and FISH analysis of diploids and tetraploids for repetitive and single-copy sequences, including sequences cloned into bacterical artificial chromosomes (BACs). (AES-TX).

Several populations were constructed to support mapping of fiber quality parameters. Two populations were derived from hybrids of Chinese cultivars, Simian 2 and Zhongmian 12, with relatively unimproved fiber properties and PD-3-14, a germplasm line with excellent fiber properties. (ARS-SC). The parents and F2 populations are currently being screened for polymorphic markers. (1890-AL). Other populations involved hybrids between G. hirsutum and G. barbadense. Genetic diversity of 16 near-homozygous genotypes derived from interspecific hybridization was investigated using RAPDs and morphological characters. PCR on genomic DNA primed with 80 random decamer primers generated 135 RAPDs. Twenty-one stable morphological traits, chosen to distinguish typical G. hirsutum from G. barbadense, were measured on the genotypes. Dendrograms for genetic distance (RAPDs) and taxonomic distance (morphological data) both produced two clusters, one resembling G. hirsutum and one G. barbadense. Classification of all genotypes based on the two methods gave similar results with a correlation of 0.63 between methods. Several genotypes were identified that were genetically and phenotypically distant from typical G. hirsutum and G. barbadense. The germplasm developed as part of this project will be made available to all cotton researchers in 1995. (AES-NM). Screenings of RAPD-PCR DNA fragments for association with fiber strength were continued. Aneuploid DNA fragment screens were made to provide chromosome anchors for QTL analysis. (ARS-TX). Pima and Sea Island cottons were crossed with two Pee Dee germplasm lines, PD 5363 and PD-3-14. The F2 will be screened for G. hirsutum growth habit and G. barbadense fiber properties. (ARS-SC). F2 populations of seven MAR strains, representing the MAR hybrid pools, crossed with Pima S7 were produced. These populations will be used to map genes controlling fiber quality traits. Parents have also been selected and crosses made among MAR strains and Pima S7 for mapping genes controlling drought tolerance and photosynthetic capacity. (AES-TX).

F2 populations of Pima S-7 by Tamcot CD3H and SP37 were screened for bacterial blight resistance to four race of Xanthomonas campestris pv mahacoarum. RFLP analysis of resistant and susceptible progeny is currently in progress. Crosses were made between isogenic lines, each containing a different bacterial blight resistant gene(s) (B2, B3, B2B3, B2B6, B2B3B6, B4, B7) and Pima S-7. An additional cross was made between the Pima parent and S295, an African line with resistance to the virulent races of bacterial blight found in Africa. The F2 populations will be grown under field conditions in 1995, and RFLP analysis will be conducted. (AES-TX).

Several screens for unique traits were conducted. A preliminary screen of 18 Gossypium species was conducted for constitutive and induced resistance to spider mites. Resistance was not related to genome, and level of induced and constitutive resistance within a species was not correlated. G. australe and G. /axam exhibited high levels of both forms of resistance, while others were high in one type but low in the other. (AES-CA). Asiatic germplasm (G. arboreum) with excellent resistance to reniform nematode was identified among 200 accessions. Forty accessions of Gossypium (30 species) were evaluated for quality and quantity of terpenoid aldehydes. Species high in total or specific terpenoid aldehydes were identified. (AES-AR).

F2 populations were developed from trispecies crosses involving (G. arboreum x G. trilobum) and G. hirsutum TM-1 and T-586. The latter population will be evaluated for morphological traits and polymorphisms with the objective of relating A2 and D8 physical maps to the diploid AD map. Asiatic cottons with identified biotic resistances were hybridized with hexaploid (G. turneri x G. barbadense) to give trispecies tetraploids compatible with cultivated cotton. Some of these have been crossed with G. hirsutum. (AES-AR).

Two kinds of semigamous lines are in development that allow mass extraction of doubled haploids. Tests are in progress for homozygosity and seed increase. (AES-TX). Additional semigamous lines are being selected for homozygosity that contain the cytoplasms of 14 different Gossypium species. (AES-AR).

Thirty accessions of G. arboreum and G. herbaceum were grown for seed increase and evaluated for agronomic characteristics. Seed of 85 accessions of Gossypium species were distributed nationally and internationally, and leaf tissue of six species was supplied to molecular labs in Mississippi and California. An F2 involving [(G. arboreum x G. trilobum) x G. hirsutum T-586] was evaluated for selected morphological traits to determine if segregation would be normal in the trispecies hybrid. Molecular mapping of this population has begun. A backcross population (G. arboreum 3*/G. Iongicalyx) was screened for reniform nematode resistance (RN). A hybrid of G. Iongicalyx/G. armourianum was made and the chromosome number doubled. Because the F1 genome is close to the A subgenome of upland cotton, a triple hybrid cross with cotton should force recombination to transfer the immunity of the F1 to upland cotton. Morphological markers are being introgressed from diploid species hybrids with upland cotton. Progeny from crosses of upland cotton (4X) on to triploid hybrids (3X) involving Kimberly species of Gossypium were shown to be pentaploid. D8 cms in cotton has two levels of dysfunction, one sporophytic and one gametophytic, the latter being evident only in plants heterozygous for the restorer allele. Test crosses established that the nonrestorer allele is not transmitted through the male gamete when in the D8 cytoplasmic background, even though the pollen looks normal. It is transmitted through the female. When in the AD1 cytoplasmic background both restorer and non-restorer alleles are transmitted through the pollen. Segregation of the restorer allele was inconsistent among test crosses. (AES-AR).

Twenty-one species of Gossypium were screened for constitutive and induced resistance to Tetranychid spider mites. Constitutive resistance appeared to be an evolutionarily labile trait, found throughout the phylogenetic tree and occurring in all geographic regions where native cottons are found. Resistance appeared to be the ground plan with repeated losses. In contrast, induced resistance to mites was less commonly observed among the 21 species. It appeared to have arisen 4 times and never been lost, based on the most parsimonious solution. This most parsimonious solution posits that induced resistance is derived, rather than ancestral. Induced resistance is more restricted geographically than constitutive resistance, with 2 originations in the New World and 2 originations in Australia. It is of interest that G. hirsutum is in the cluster of species that showed the strongest induced resistance. These data showed no suggestion of a negative correlation between levels of constitutive and induced resistance. (AES-CA).

Screening of a core set of cotton lines containing various amounts of G. hirsutum and G. barbadense introgression continued. Of the RAPDs analyzed, 12 were classified as "species specific". These types of markers are being mapped and will be used to quantify introgression in wide crosses. Two F2 populations are being studied extensively in the program. TM1/NM24016 F2 was grown in 1995 in the field at Las Cruces,NM and data was recorded on and DNA extracted from 120 plants. NM24016 is a line derived from the introgression program, exhibiting many traits of both G. hirsutum and G. barbadense. Segregation within this population appears normal and all plants were selfed for further study. This population provides a unique opportunity to map stomatal conductance as it ranged from 180 to 680 mol m-2s-1, while typical values for TM1 and NM24016 were 500 and 300 mol m-2s-1, respectively. Upland cotton generally has significantly higher stomatal conductance than Pima. The F2 from the cross 'Acala 1517-95'/NM24052 was used for genetic analysis of fiber strength. NM 24052 is derived from Del Cerro and Triple Hybrid stock and has stelometer fiber strength of 33 g/tex which exceeds 'Pima S-6'. Segregating progeny in 1995 were identified in this population that approached 30 g/tex fiber strength while maintaining a desirable upland phenotype. (AES-NM).

Interspecific F1 and F2 populations from crosses of 'Pima S-7' with various Upland cottons were produced to be utilized in mapping genes for drought tolerance, photosynthetic capacity, insect resistance, and other agriculturally important genes. The cotton cytogenetics collection was maintained and distributed on request. Testing and application was partially completed for two new chromosome manipulation procedures, the "Monosome Walk" and "DNA Shuffle". New aneuploids, prospectively deficient for all or part of the respective chromosomes, were derived in a strategic manner for the 7 chromosomes for which monosomes and telosomes are lacking in the Collection. The HEHP project, on development of systems for mass-extraction of doubled-haploids was mothballed in early 1995 due to lack of industry support. Statistical evidence was obtained for inadequacy of the prevailling method of CPFA analysis. Development of a new analytical procedure, more amenable to automation and smaller samples was partially developed. New stain-clearing procedures were developed for video-based observation of megagametophytes. Other procedures were developed for isolation of intact megagametophytes free of sporophytic tissue. (AES-TX).

QTL anaiysis of an intraspecific G. hirsutum F2 population created between germplasm line PD-3-14 and Chinese cultivar Simian 2 was continued. Traits measured in the field include iint yield components, fiber, and arealometer properties. Screening interspecific germplasm with desirable fiber properties based on the rate of in vitro fiber cell elongation was tested on PD germplasm. (AL-1890; ARS-SC) Ovule culture and confocal microscopy were utilized to characterize a mutant line where fiber ceases elongation at a juvenile stage of development. (AL-1890; ARS-TX).

Recombinant inbred lines were initiated in PD/Sea Island interspecific populations and NILS were initiated in PD/Chinese intraspecific populations to support mapping efforts. (ARS-SC).

Screening of a G. hirsutum/G. barbadense (TM1/3-79) population with RFLP and RAPDs was conducted. DNA from this population was provided to cooperators to expand the molecular marker database. F3 and F4 progeny were produced for verification of fiber properties and marker phenotypes. The cotton genome database, CottonDB, was loaded onto the NAL Plant Genome Gopher and Web sites. Seed of 1369 accessions has been distributed to geneticists, breeders, and other scientists and includes both national (1242 accessions) and international (127 accessions) requests. Seed of 1176 accessions of G. arboreum were obtained from India as part of a seed exchange agreement made in 1992. A trip to Russia, Uzbekistan, and India established preliminary contact with personnel in charge of the Russian and Uzbekistan cotton seed collections for possible seed exchanges in the future. Materials obtained from India are being grown in Mexico this season. (ARS-TX).

A replicated experiment containing 118 F2 3 progeny from the mapping population TM1 X NM24016 was grown and evaluated at Las Cruces, NM and Maricopa, AZ. The segregation for 2 fiber quality traits and stomatal conductance in the F3 progeny was extensive. The progeny mean fiber length was 29.7mm with a range from 26.9mm to 33.5mm. The mean fiber strength was 21.3 g/tex with a range from 16.5 to 27.6 g/tex. A preliminary QTL analysis in this population has identified at least one QTL region (LOD = 2.36) which explains 26% of the variation in fiber length. (AES-NM).

A replicated experiment containing 120 F23 progeny from the cross of Acala 1517-95 and the putative high-strength line NM24052 was grown and evaluated at Las Cruces and Artesia, NM. Progeny fiber strength averaged 25.4 g/tex over the-two locations, and ranged from 19.4 to 29.3 g/tex. The population exhibited G. hirsutum plant phenotypes and should have great potential for future fiber strength genetic gains. Unfortunately, it behaves as a typical G. hirsutum intraspecific cross and DNA polymorphism is limited. However, DNA pools are being constructed of the high and low fiber strength progeny for a Bulk Segregant Analysis. (AES- NM).

QTL analysis of an intraspecific G. hirsutum population created between gerrnplasm line PD-3-14 and Chinese cultivar Simian 2 was continued on a second set of 45 F2 plants. Traits measured in the field include lint yield components, fiber and arealometer properties. Little polymorphism has been found with RFLP. Genetic analysis indicated that the polymorphic bands of 1240 bp and 1180 bp RFLP markers using an AT-ACC (HindIII) probe were allelic and segregated in a 1:2:1 ratio following a normal Mendelian ratio for a single locus. However, the unique band of a 2720 bp RFLP marker using microsatellite probe (HindIII) segregated in a 3:1 ratio. Linkage analysis indicated that these two RFLP markers segregated independently. None of the RFLP loci identified are linked. The population is now being screened for AFLP markers. A recombinant inbred line (RIL) population is in development from the same cross. F5 seed from each member of the RIL population was sent to the Mexico winter nursery for selfing. Fourteen germplasm lines obtained by S. Saha from South Africa were evaluated in a replicated field test for yield, fiber, arealometer, Soxhlet extract, and wax content properties. (AL- 1890; ARS-SC).

Test crosses confirmed that the inheritance of D8 cytoplasmic male sterility (CMS) is restored by a single gene supposedly from the original D8 parent. We also confirmed that the CMS condition involves interaction between the D8 c,ytoplasm and the AD1 nucleus at both the sporophytic and microgametophytic levels. Plants heterozygous for the restorer gene produce two genotypic pollen types, but only pollen carrying the restorer gene is functional. Failure of the non-restorer pollen to accomplish fertilization was not related to competition between the two pollen genotypes. The D8 restorer segregates normally in the female. Both D2-2 restorers were found to restore the D8 cytoplasm (but not vise versa) and segregate normally in both male and female gametes. A RAPD marker was identified that segregated at a maximum of 12 cM from the D8 restorer locus. (AES-AR).

Seventy genotypes of G. arboreum, selected for their high micronaire values (>7.0), were grown in SE Arkansas to evaluate regional adaptation, agronomic traits, yield potential, and fiber characteristics suitable for non-woven applications. Several lines were identified that possessed excellent yield potentials (between 2 and 3 bales/acre) and agronomic characteristics suitable for standard cotton production methods. Data on fiber evaluations have not yet been received. (AES-AR).

A project was begun to detect and isolate genes involved in osmotic adjustment in response to water stress. The differential display technique for detection of unique gene expression between two physiologically different tissues is being used to compare gene expression among susceptible and tolerant genotypes with and without water-stress. Several PCR products unique to a stress resistant genotype or associated with water-stress have been identified. Two fragments have been isolated for characterization beginning with DNA sequencing. (AES-AR).

Populations have been created and analyses begun in two projects to detect and isolate genes involved in root knot nematode resistance and photoperiodic response. RFLPs and RAPDs are being analyzed using Bulk Segregant Analysis.(ARS-MS).

An interspecific G. hirsutum x G. barbadense F2 mapping population is being maintained as a live collection in the greenhouse for cooperative use. (ARS-TX).

DNA marker genotyping of an intraspecific G. hirsutum population created between germplasm line PD-3-14 and Chinese cultivar Simian 2 was continued. AFLP markers from several pairs of primers differentiated PD-3-14 and Simian 2. Genotyping of the F2 individuals is in progress. A recombinant inbred line (RIL) population of 58 entries is in development from the same cross. F6 seed from each member of the RIL population was sent to the Mexico winter nursery for selfing, while F, seed was generated in the field in 1997. Fourteen germplasm lines obtained by S. Saha from South Africa were evaluated for the second year in a replicated field test for yield and fiber properties. Several of the South African germplasms have high fiber strength among other desirable fiber properties. (AL-1890; ARS-MS; ARS-SC).

G. hirsutum germplasm developed by AES-TX with one chromosome of a homologous pair putatively substituted with its G. barbadense homolog were evaluated for fiber properties at Florence, SC and Mississippi State, MS.

Gerrnplasm with high micronaire reading (about 6.0) and short 2.5% span length (about 26mm) were identified. This germplasm may be useful in elucidating DNA marker-phenotype associations. (ARS-MS; TX-AES; ARS-SC). High fiber strength (>25 g/tex) germplasm with reduced leaf trichomes derived from G. hirsutum x G. barbadense crosses has been identified. This germplasm is the result of attempts to stably introgress G. barbadense level fiber strength into G. hirsutum. The germplasm has two or more doses of the upland parent but maintains the G. barbadense cytoplasm. Agronomic evaluations and continued selection for smooth leaf and upland phenotype are in progress. (ARS-SC; AES-NM; ARS-AZ).

In collaboration with John Gannaway, the Str-l gene from G. barbadense that confers improved fiber strength to G. hirsutum is being introgressed into several G. hirsutum cultivars. In collaboration with Wayne Smith, we are now completing the data analysis for 24 advanced-backcross populations that are each segregating for a small number of G. barbadense (Pima S6) chromosome segments, in a background that is otherwise G. hirsutum (TAM 2111). In collaboration with John Gannaway and Jonathan Wendel, several additional advanced-backcross populations are under development.Several prior QTL mapping projects are now completed. An experiment based on mapping of genes controlling fiber-related traits has been accepted for publication . A second project involving mapping of genes controlling bacterial blight reaction is submitted .Two additional experiments, involving about 20 other quantitative traits, are in progress. (AES-TX).

Preliminary genetic tests indicate that the cytoplasm male sterile restorers D2-2 (R~ ) and D8 (R~ ) are not allelic but are closely linked. For each restorer, a RAPD marker was identified that was linked to it. (AES-AR).

Four genotypes with different sensitivity to water-stress were used to detect differentially expressed genes in response to stress. More than 60 gene products unique to a stress-resistant genotype or associated with water-stress were identified and sequenced. Several of these relate to metabolic events associated with adjustment to water deficit by the tolerant genotypes. The differential display technique was utilized to identify gene expression related to the semigamy trait. Thus far, 31 transcripts are recovered that may be related to semigamy. Among 6 cloned and sequenced, one is a kinetochore protein gene. (AES-AR,USDA-MS),

A detailed RFLP map was used to map genes affecting presence and density of leaf trichomes. A QTL on an A-subgenome chromosome that imparts dense leaf pubescence is inferred to be the Hl locus. A second QTL at the homologous location of a D-subgenome chromosome fits the description of the Sml locus. Two additional QTLs explained significant phenotypic variation in leaf pubescence. Some QTLs appeared to be specific to particular cotton development stages; for example, the A-2 locus reduced hairiness only in young leaves while A-3 increased hairiness in mature leaves. A single locus associated with variation in trichome density on the stem did not correspond to the genes/QTLs affecting leaf trichomes, suggesting that these traits may largely be controlled by different genes. (AES-TX).

The limited number of genotypes amenable to regeneration via somatic embryogenesis is a significant obstacle to genetic engineering in cotton. We have developed segregating test populations to undertake both classical and molecular genetic analyses of the inheritance of somatic embryogenesis in cotton. Test populations involving the highly regenerable line Coker 312 in combination with genetic marker stocks (TM-1, TM-582 and TM-586) have been developed. Tissue culture protocols to reliably regenerate plants from Coker 312 have been tested. Currently, RAPD primer combinations that can reliably detect DNA polymorphisms between the genotypes involved in the test populations are being screened. Other work on the genomic organization of ribosomal DNA (rDNA) in cotton has found that the rDNA structure of cotton is similar to that in most other plants and that a low level of rDNA variability is detected in cotton. Restriction site variation in cotton rDNA is useful in distinguishing between Old World and New World cottons. (AES-LA).

QTLs for fiber quality properties including bundle fiber strength, fiber length, and fiber fineness have been identified in 3-79, an extra long staple cotton. We have detected three QTLs for bundle fiber strength (Sf), three QTLs for fiber length (Lf), and five QTLs for fiber fineness (Ff) in different linkage groups. These QTLs explain about 35% to 50% of the total genetic variance for fiber characteristics in the F2 population. Characterization of these QTLs is underway in the intraspecific G. hirsutum crosses involving Pee Dee and Acala cottons. Molecular tags for monogenic traits have been identified for glandless cotton and photoperiod sensitivity. High-resolution mapping of the glandless locus on the long arm of chromosome 12 is underway by use of a larger segregating population resulting from a cross between TM-I and ESP, a pair of NlLs.Two independent insertions of the gene for 2, 4-D resistance were found to be at the same or very close locations. Both inserts were linked to N1 on chromosome 12. The F2 of the cross between the mutants did not reveal any recombination with 900+ plants. Crosses were made with telosomic stocks to determine arm location. (USDA-TX).

Seed of 128 accessions were added to the Collection in 1997. Seed of 399 accessions were successfully increased at the Cotton Winter Nursery (CWN), Tecoman, Mexico for the 1996-97 growing season. Seed of 195 accessions were increased at Rio Farms, Monte Alto, Texas, and included 192 G. hirsutum and 3 G. herbaceum. Seed of 367 accessions were sent for seed increase to the CWN for the 1997- 98 growing season, included were (163 G. hirsutum race stocks, 28 G. hirsutum varieties, 11 G. barbadense, and 14 G. herbaceums). Also sent were 148 miscellaneous stocks from NSSL for increase to renew stocks with deteriorating viability. Seed of 1575 accessions were distributed in 102 seed orders. Dr. Albert Kassianenko, Head, Oil Seeds Crops, VIR Institute, St. Petersburg, Russia, was able to visit our laboratory in November of 1997, with funds provided by OICD. We have agreed to exchange germplasm from our respective Collections. Initially the Russians want to send their entire collection to insure that it is preserved. This will add approximately 6000 to 7000 accessions to our Collection if most of the Russian seeds are viable. (USDA-TX).

QTL analysis for stomatal conductance (Gs) was conducted on the F2:3 progeny derived from the mapping population TMI x NM24016 utilizing DNA markers (RAPDs and SSRs). Two QTLs ( LOD = 2.0 and 3.8) were detected for Gs at Maricopa. These QTLs explained about 12% ofthe variation in the trait. Both intervals were from NM24016 and negatively affected Gs. It is suspected, but not yet verified, that these regions are derived from G. barbadense. This is one of the first physiological traits that have been subjected to QTL analysis. Gs is a heritable trait and seems to be significantly associated with lint yield in heat stress environments.(AES-NM, USDA-AZ).

3. MAJOR PUBLICATIONS:

Saha, S. and D.M. Stelly. 1993. Chromosomal location of Phosphoglucomutase 7 locus in Gossypium hirsutum. J. Hered. 85:35-39.

Samora, P.J., D.M. Stelly and R.J. Kohel. 1993. Localization and mapping of the Le1 and Gl2 loci of cotton (Gossypium hirsutum L.). J. Hered. 85:152-157.

El-Zik, K.M. and P.M. Thaxton. 1994. Breeding for resistance to bacterial blight of cotton in relation to races of the pathogen. In G. Constable and N. Forrester (eds.), Proc.First World Cotton Research Conference, February 13-17, Brisbane, Australia.

Park, Y.H. and R.J. Kohel. 1994. Effect of concentration of MgCI2 on random-ampimed DNA polymorphism. Biotechniques 16:652-655.

Reinisch, A.J., J. Dong, C. Brubaker, D.M. Stelly, J.F. Wendel and A.H. Paterson. 1994. A detailed RFLP map of cotton (Gossypium hirsutum x G. barbadense): Chromosome organization and evolution in a disomic polyploid genome. Genetics 138:829847.

Stanton, M.A., C.S. Rothrock and J.McD. Stewart. 1994. Response of A-genome cotton germplasm to the seedling disease pathogens, Rhizoctonia solani and Pythium ultimum. GRACE 41:9-12.

Stanton, M.A., J.M. Stewart, A.E. Percival and J.F. Wendel. 1994. Morphological diversity and relationships in the A-Genome cottons, Gossypium arboreum and G. herbaceum. Crop Sci. 34:519-527.

Calhoun, D.S., D.T. Bowman and O.L. May. 1994. Pedigrees of upland and Pima cotton cultivars released between 1970 and 1990. Tech. Bulletin 1017, MAFES.

Hanson, R.E., M.S. Zwick, S. Choi, M.N. Islam-Faridi, T.D. McKnight, R.A. Wing, H.J. Price, and D.M. Stelly.1995. Fluorescent in situ hybridization of a bacterial artificial chromosome. Genome 38:646-651.

Reyes-Valdes, M.H., and D.M. Stelly. 1995. A maximum likelihood algorithm for genome mapping of cytogenetic loci from configuration data. Proc. Nat. Acad. Sci. 92:9824

Saha, S., A. Kakani, V.T. Sapra, A. Zipf, and G.C. Sharma.1995. Confocal laser scanning light microscopy with optical sectioning: Application in plant science research. pp. 171-183. In: P.M. Grasshoft (ed.) Technology transfer of plant biotechnology: Current topics in plant molecular biology. Vol. 4 CRC Press.

Stelly, D.M., H.J. Price, and T.D. McKnight. 1995. Molecular-meiotic cytogenetic analysis of cotton. pp.148-156.1n B. Gill and J. Raupp (ed.) Classical and Molecular Cytogenetic Analysis of Cereal Genomes.

Faver, K.L., T.J. Gerik, K.M. El-Zik, and P.M. Thaxton.1996. Response of two cotton cultivars to late season water stress. ll. Leaf gas exchange and assimilation capacity. Crop Sci 36:922-928.

Gerik, T.J., K.L. Faver, K.M. El-Zik, and P.M. Thaxton.1996. Response of two cotton cultivars to late season water stress. l. Plant growth, water use, and yield. Crop Sci. 36: 914-921.

May, O.L., C.C. Green, T.W. Culp, and D.S. Howle. 1996. Registration of PD-3-14 germplasm line of upland cotton. Crop Sci. 36:1718.

Tatineni, V., R.G. Cantrell and D.D. Davis. 1996. Genetic diversity in elite cotton germplasm determined by morphological characteristics and RAPDs. Crop Sci. 36:186-192.

Callahan, F. E., Jenkins, J. N., Creech, R. G., and Lawrence, G. W. 1997. Changes in cotton root proteins correlated with resistance to root knot nematode development. J. Cotton Sci. I :39-47.

Feng, X., S. Saha, and K.M. Soliman. 1997. DNA fingerprinting using AFLP. Focus 19: (1)

Jenkins, Johnie, N., McCarty, Jack C., Jr., Buehler R. E., Kiser, J., Williams, C. and Wofford, T. 1997. Resistance of cotton with delta-endotoxin genes from Bacillus thuringiensis var. kurstaki on selected Lepidopteran insects. Agron. J. 89:768-780.

Ma, Din-Pow, Lui, His-Chou, Tan, Hingpoh, Creech, R. G., Jenkins, J. N. and Chang, Yung-Fu. 1997. Cloning and characterization of a cotton lipid transfer protein gene specifically expressed in fiber cells. Biochimica et Biophysica Acta 1344: 111-114.

Mann, J.E., S.G. Turnipseed, M.J. Sullivan, P.H. Adler, J.A. DuRant and O.L. May. 1997. Effects of early season loss of flower buds on yield, quality, and maturity of cotton in South Carolina. J. Econ. Ent. 90:1324-1331.

May, O.L., and D.S. Howle. 1997. Registration of PD 93009, PD 93019, PD 93021, PD 93030, PD 93034, and PD 93057 germplasm lines of upland cotton. Crop Sci. 37: 1031-1031.

May, O.L., and D.S. Howle. 1997. Registration of PD 93007, PD 93043, and PD 93046 germplasm lines of upland cotton. Crop Sci. 37: 1030.

Robinson, Michael, Jenkins, Johnie N., and McCarty, Jack C., Jr., 1997. Root knot nematode resistance of F2 cotton hybrids from crosses of resistant germplasm and commercial cultivars. Crop Sci. 37: 1041-1046.

Robinson, A. F., and A. E. Percival. 1997. Resistance to Meloidogyne Incognita Race 3 and Rotylenchulus reniformis in Wild Accessions of Gossypium hirsutum and G. barbadense from Mexico. Sup. to the J. Nematol. 29(4S): 746-755.

Saha, S. and A. Zipf. 1997. Genetic diversity and phylogenetic relationship in cotton based on isozyme markers. J. Crop Production. 1: 79-93.

Saha, S., F.E. Callahan, D.A. Dollars and J.B. Creech. 1997. Effect of Lyophilization of cotton tissue on The quality of extractable DNA, RNA and Protein. J. Cotton Sci. 1:10-14.

Vant-Hoff, J., and S. Saha.1997. Cotton fiber cells can undergo cell division. Am. J. Bot. 84:1231-1235.

Bauer,P.J., O.L.May, and J.C.Camberato. Seeding date and K fertility effects on cotton yield and fiber properties. J. of Prod. Ag. (In press).

May, O.L. and J.A. DuRant. 1998. Development of cotton germplasm for reduced insecticide use production systens. J. Sust. Agric. 11:39 49.

May, O.L. and R.A. Taylor. 1998. Breeding cotton with higher yarn tenacity. Textile Res. J. 68: 302-307.

Nepomuceno, A.L., D.M. Oosterhuis and J. McD. Stewart. 1998. Physiological responses of cotton to water deficit induced by polyethylene glycol. Environ. and Exper. Bot. 40:29-41.

Van Esbroeck, G., D.T. Bowman, D.S. Calhoun, and O.L. May. 1998. Changes in genetic diversity of cotton in the U.S. from 1970 to 1995. Crop Sci. 38:33-37.

Van Hoft, J. and S. Saha. 1998. Growth and mitotic potential of multi-celled fibers of cotton. Am. J. Bot. 85(1):25-29.

Altaf, M.K., J.McD. Stewart, and J.B. Murphy. 1999. Evaluation of the Gossypium gene pool for foliar terpenoid aldehydes. Crop Sci. 39:253-258.

Kakani, A., S. Saha, V. T. Sapra, A. Zipf and D. M. Stelly. 1999. Genetic mechanism and chromosomal location of polen-specific gene(s) in Gossypium. Crop Sci. 39:668-673.

May, O.L., and G.M. Jividen. 1999. Genetic modification of cotton fiber properties with single- and high-volume instruments. Crop Sci. 39:328-333.

Van Esbroeck, G.A., D.T. Bowman, O.L. May, and D.S. Calhoun. 1999. Genetic similarity indices for ancestoral cotton cultivars and their impact on genetic diversity estimates of modern cultivars. Crop Sci. 39: 323-328.

APPLICATION OF RESULTS:

The 700+ loci map based on AD1xAD2 provides a base from which to work with other populations. The level of polymorphism exhibited by genotypes intermediate between G. hirsutum and G. barbadense could be exploited in genetic mapping populations to tag economically important traits, such as fiber quality. These genotypes also could serve as a germplasm source of unique alleles in G. hirsutum that are derived from G. barbadense. The full spectrum of molecular cytogenetic analytical techniques are now available for applicable to the cotton genus. These techniques complement conventional (organism-based) and other (gel-based) contemporary methods of genetic analysis and manipulation. The semigamy lines can increase the rate of genetic improvement, facilitate genotype dependent research, and facilitate QTL mapping.

Seed of cottons from India may contain valuable genes for agriculturally important traits. Fine mapping of bacterial blight resistance genes will provide tools for breeders to use marker assisted selection in the deployment of gene combinations that will result in higher and more durable resistance. The nested PCR technology shall aide the identification and cloning of genes of biological importance that will be beneficial for germplasm screening and transformation. A map saturated with RFLP markers will ultimately provide the resolution necessary to break down quantitative traits into individual loci and accelerate their introgression into commercially acceptable cultivars. The identification of genes associated with stomatal conductance could benefit efforts to improve heat tolerance of upland and pima cotton. The cotton genome database containing DNA marker and other pertinent information should provide ready access to information for breeders, geneticists, and molecular biologists. The discovery of immunity to reniform nematode in G. Iongicalyx and its relatively simple inheritance ultimately will allow the introgression of resistance into G. hirsutum. The amelioration of significant yield-loss due to reniform nematode would positively impact cotton production. The monosome walk and DNA shuffle chromosome manipulation procedures will enhance map-based cloning of cotton genes. New aneuploids for chromosomes for which they previously did not exist will significantly impact efforts to map genes to specific chromosomes.

The mapping of genes associated with fiber length and strength will permit cotton breeders to tag these in marker-assisted -selection programs. This is especially useful in interspecific populations where introgression of new genetic variability for fiber quality traits is being attempted. This will enhance the genetic variability in G. hirsutum and allow "pyramiding" of fiber quality genes to further improve fiber quality traits. (AES-NM).

A map of Gossypium involving the A2 and D8 species will relate the genomic arrangement of these species to that of upland cotton, and facilitate the introgression of traits from these species into cotton. (AES-AR)

The development of a molecular framework map, and eventually integrated molecular map, of the cotton genome serves as a foundation that is essential to individual gene mapping projects and exotic germplasm evaluations. This publicly accessible genetic map is also useful in developing consensus linkage maps based on "anchor" markers for each chromosome. This genetic map provides a backbone to an ultimate physical mapping of the cotton genome. Molecular markers linked to fiber quality QTLs and major agronomic/physiological genes would be useful in marker-assisted selection (MAS) of cotton breeding programs. A map of Gossypium involving the A2 and D8 species will relate the genomic arrangement of these species to that of upland cotton, and facilitate the introgression of traits from these species into cotton. In addition to understanding the physiological basis of water-stress tolerance, identification of associated genes will sirnplify enhancement and maintenance of this trait in breeding programs through MAS.

FUTURE RESEARCH NEEDS: The results from S-258 established a solid basis for further research on the utilization of genetic resources to improve cotton. Additional germplasm collections of cotton need to be acquired, characterized and evaluated for potential usefulness. Although considerable progress was made during the past five years, additional research is needed to develop new molecular tools for the evaluation and improvement of cotton germplasm. As additional sequencing of the cotton genome is completed and the sequencing date made available research is needed to mine the data for useful genes.

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.

OBJECTIVES

The overall goal of this new project is to enable U.S. researchers to address the short- and long- term genetic needs of the U.S. cotton industry. To do so, we will augment the five main areas of investigation established in S-258 by developing and assimilating new technologies relevant to germplasm, genetics, genomics, and genetic improvement of cotton. The specific objectives of this proposal are:

1.	To acquire, curate, characterize, and evaluate the species, races, and genetic types of Gossypium (cotton).

2.	To develop, maintain, and distribute molecular genetic, genetic, and cytogenetic tools for the evaluation and enhancement of cotton germplasm.

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

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

5.	To refine and develop cotton breeding and variety testing methodologies and techniques.

6.	To develop cotton bioinformatic systems.

PROCEDURES

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

The U.S. National Cotton Germplasm Collection is comprised of the germplasm collected as seed during various expeditions, along with materials obtained as donations and exchanges with other germplasm banks around the world. It is maintained by the USDA-ARS at College Station, TX. The Collection is a part of the National Plant Germplasm System (NPGS), and personnel in this ARS unit 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 under five subcollections representing different categories of germplasm. 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). These are, with rare exceptions, released cultivars 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. Accessions in this collection originated from 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 of this tetraploid species is maintained with a "GB-" designation and includes cultivars (old and recent), dooryard, and wild accessions.

4.	ASIATIC COLLECTION. The cultivated and wild A-genome cotton G. herbaceum and G. arboreum are maintained in this collection. The former species carries an "A1-" designation while the latter carries an "A2-"

5.	WILD SPECIES COLLECTION. Currently, multiple accessions of three wild tetraploid species and 38 wild diploid species are maintained in the collection.

Seed increases 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.

Objective 2: To develop, maintain, and distribute molecular genetic, genetic, and cytogenetic tools for the evaluation and enhancement of cotton germplasm.

Advances in genetic research methodology have made possible the dissection and analysis of plant genomes at the molecular level. An area of research that promises to be important to applied efforts in plant breeding in the near future is in the area of genetic/molecular markers. Molecular markers, like sign posts and road maps, aid plant breeders in incorporating new genes into improved cotton cultivars. Development of additional markers will accelerate this process. Efforts to develop such markers are underway in several research settings (public and private) in the U.S. and worldwide. The main goals of this objective are to develop a universally "agreed upon" and "adhered to" system of nomenclature for these markers and a mechanism to share them among researchers. To do this, the following detailed objectives are proposed:

1.	Create and distribute on the web recommendations for a group-approved, standardized nomenclature for cotton molecular markers, ESTs, cDNAs. This nomenclature should include embedded information as to lab source and/or developer.

2.	Create a "running list" (user-undatable) of publicly available markers, donor; summary list of donors and respective contributions; interface this with maps to facilitate their usage.

3.	Develop a large number of portable markers. Marker types would include RFLPs, RAPDs, AFLPs, SSRs, SNPs, CAPs and any others developed during the period of this proposal.

4.	Develop a large number of TM-1 BAC clones to build an integrative genetic and physical mapping infrastructure during the period of this proposal.

5.	The identification of "Core" marker sets (highly polymorphic) so that a common interface can be generated among multiple mapping populations.

6.	The development and maintenance of mapping populations. This would include:

a.	A highly polymorphic public reference population for mapping low-polymorphism markers.

b.	Other ad hoc public mapping/breeding populations.

7.	The refinement and synthesis of existing and new linkage, physical, and cytogenetic maps into a consensus map. Such a map would make use of available marker technology as well as information on QTLs and ESTs. Of further interest is the establishment of a comparative map with Arabidopsis, especially with regard to conserved sequences, regions of macro- and micro-synteny, and relative rearrangement. This subobjective includes the maintenance of a web site containing this information.

8.	Mutants can provide powerful tools for the investigation of the cotton genome. Research covered in this proposal request would undertake their characterization, maintenance, and distribution and to maximize their utility through isoline development.

9.	Utilize wide crosses to access valuable traits in the secondary and tertiary gene pools.

10.	Maintain, develop, and utilize cytogenetic stocks. Specific objectives include:

a.	Interspecific F1 hybrids and chromosome-doubled derivatives.

b.	Maintain Cotton Cytogenetic Collection.

Maintain and distribute the Cotton Cytogenetic Collection: Working plant collections will be grown and seed collections will be maintained at College Station in two storage sites, and for long-term storage at Ft. Collins, CO. Seed and plants will be distributed upon request.

1.	Mark all G. hirsutum chromosomes by hypoaneuploidy.

2.	The primary goal will be to find, create, or obtain some sort of deficiency for each chromosome (whole or part), so that all linkage groups can be identified as to chromosome of origin. The secondary goal will be to obtain complete coverage for each chromosome, e.g., through a primary monosome, both telosomes, or the complementing segmental duplication- deficiencies from a single translocation. Presently, some coverage on just 2 to 3 more chromosomes is needed to meet the first objective; coverage remains incomplete on 10 or fewer of the 26 chromosomes. Cytogenetic types providing the desired coverage are being induced by irradiation and generated by two methods of directed chromosome manipulation that yield translocation-derived segmental or whole-chromosome aneuploids), all of which have been effective for other chromosomes or chromosome segments.

3.	Produce, maintain, and distribute chromosome and chromatin substitution lines (intact germplasm introgression), their tissue, and/or DNA: The monosomes, telosomes, and critical segmental aneuploids (those uniquely marking a chromosome) will be crossed to produce cytogenetically identified aneuploid F1 hybrids useful for mapping as well as subsequent introgression. After repeated backcrossing and aneuploid recovery, an advanced generation will be inbred for seed increase such that the germplasm can be tested.

4.	Evaluate (traits, markers, genes, expression and linkage groups): Backcross derived seed of chromosome-substitution lines (see above) will be used for trait detection and small-scale testing, e.g., for chromosome-specific fiber traits.

5.	Use chromatin substitution lines as point of targeted introgression: Lines with advantageous traits will be used as parents in breeding efforts to further localize and identify the genes, markers allowing indirect selection, and introgression of the gene(s) into elite breeding germplasm.

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

The evaluation and utilization of methodologies for analyses of cotton germplasm has been limited. Previous regional projects (such as S-258) have only scratched the surface of the available developing technologies to characterize and implement the vast stores of genetic variability within the cotton germplasm collection. The evaluation and utilization of the cotton germplasm collection has had only limited progress to date due to the complexity of the genome and the limited amount of funding available. To date, molecular tools such as RFLPs (Reinisch et al., 1994; Shappley et al., 1998), RFLPs and RAPDs (Yu et al., 1999), SSRs (Xiao, unpublished) and AFLPs (Reddy et al., 1997 Xiao, unpublished) have been utilized to evaluate selected cotton germplasm. QTL identification has been demonstrated by Jiang et al. (1998), Yu et al. (1998), and Shappley et al.(1998) thereby establishing the potential to discover new genes to enhance cotton production.

Within this proposal a concerted effort will be presented to create a process to hone down the large collection into a workable set which will provide detailed characterization, evaluation, and the utilization of valuable diverse genotypes in cotton improvement. The processes will combine molecular tools, model system genotyping, functional genomics, phenotypic screens, and physiological analyses to identify specific genotypes for integration into breeding programs and thus the improvement of cotton varieties in the marketplace. In addition, investigations into the genetics and physiological basis of tissue culture and somatic embryogenesis will be undertaken. The major objectives are as follows:

1.	Determine genetic relationships among selected cotton germplasm. Within the large assortment of genotypes within the cotton germplasm collection, it is necessary to pare down the number to a subset of genetic outliers that will provide the maximum potential for success within the program. Selections will be based on molecular techniques using a combination of AFLPs, RFLPs, SSRs, RAPDs, SNPs, and ESTs. These molecular analyses will determine the relationships within the collection and provide information on their relationships with historical and current cultivars. A subset of genotypes will be selected based on these data for further characterization.

2.	Utilize a plant system approach to discover potential genes within the germplasm collection. This will involve the construction, ordering, and distribution of BAC and EST libraries in cotton. To this end the Arabidopsis genome will be utilized as a model system to discover complementary conserved DNA sequences within the selected subset to use as potential candidates for cotton improvement. 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.

3.	Develop microarrays for comparative analyses of genotypes, physiological/ developmental stages, organs, tissues, and cell types.

4.	Tissue culture and transformation technologies.

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

Quantitative trait loci have been identified using populations of both the interspecific and intraspecific hybrids of the allotetraploids G. hirsutum and G. barbadense. Greater variability exists between G. hirsutum and G. barbadense than within each species. Reinisch et al. (1994) made use of the interspecific hybrid's variability to prepare an RFLP map. This map has been employed by several groups of experimenters. Jiang et al. (1998) used RFLP markers from the interspecific map to find QTL for various traits of fiber quality (elongation, length, strength and thickness), yield components and earliness. Wright et al. (1998) identified one A genome and five D genome chromosomes which had genes that conferred resistance to Xanthomonas campestris pv. malvacearum. A complementary F2 population was derived from a cross between two homozygous lines G. hirsutum acc. TM-1 and extra-long-staple G. barbadense acc. 3-79 at the USDA-ARS, Crop Germplasm Research Unit (TX). Because neither of these two genetic standards is photoperiodic, all filial generations are amenable to QTL analysis of quantitative traits that are maturity-dependent, i.e., many economically important fiber and seed traits (Yu et al., 1998). Segregation data of 171 F2 individuals of the TM-1 x 3-79 cross were obtained for a collection of over 1,000 genetic markers (Yu et al., 1997; Reddy et al., 1997; Burr et al., unpubl.; Pepper et al., unpubl.). Thirteen QTLs for fiber quality properties (strength, length, and fineness) have been identified in 3-79 (Yu et al., 1998; Kohel et al., 2000). These QTLs explained the phenotypic variance ranging 30% to 60% for each fiber property in the F2 population. Ulloa and coworkers (2000) obtained variation by crossing a G. hirsutum cultivar with a line that contained a significant introgression of G. hirsutum and G. barbadense (Cantrell et al., 1998). From this population, QTL for stomatal conductance were associated with markers (Ulloa et al., 2000). Though marker discovery has been more arduous with intraspecific crosses, populations derived from intraspecific hybrids of G. hirsutum have revealed QTL for lint yield and fiber strength (Meredith, 1994). Kloth (1995) used a morphological marker, pilose (T1), to identify loci influencing fiber length, micronaire, perimeter, and wall thickness.

The use of markers to introgress genes into elite lines is pending. The major barrier is availability of worthwhile genes. However, the USDA, ARS group at Mississippi State University seems on the verge of finding alleles for root-knot nematode resistance. Germplasm accessions and commercial lines with resistance to root-knot nematode have been identified (Robinson et al., 1977), proteins have been correlated with resistance to root-knot nematodes (Callahan et al., 1997) and prepared an RFLP map (Shappley et al. 1996, 1998) is under development. Work is also underway on identifying genes and gene products that would lead to enhanced fiber properties and resistance to diseases such as aflatoxin produced by Aspergillus flavus.

Traditional applied plant breeding approaches have made great progress in improving cotton yield, quality, and resistance to an array of abiotic and biotic stresses. The foundation for this progress has been the identification of desirable genetic variation. Efforts over the next 5 year period will be made to characterize accessions within the cotton germplasm collection not previously investigated for traits of interest. In certain instances, desirable genetic variation is not readily accessible and research into methods to make it more readily usable will be undertaken. Both efforts are seen as crucial to maintaining a broad genetic base in cotton.

1.	Detection and mapping of qualitative and quantitative trait loci.

a.	Marker-assisted selection is a prized goal. For this to be practical, QTL must be assigned to intervals bracketed by markers. Quantitative trait loci are assigned to markers by the use of a segregating population which is produced from parental plants with ample variation in both markers and quantitative traits. Parental plants are selected on the assumption that divergence of phenotype reflects divergence of genotype for QTL. This assumption is extended to the anonymous phenotypes of the DNA markers and marker variation between candidate parental lines is not generally assessed beforehand. However, if the candidate lines are carefully chosen, prior analysis of marker genotypes may not be strictly necessary: U.S. cotton varieties show marker variation based on region of origin (Meredith 1994). Abundant variation can always be found by creating interspecific crosses, but segregation distortion in these combinations complicate analyses. Once two cotton varieties or lines are crossed, the hybrid is self-pollinated; and an F2 generation is produced. Frequently, but not necessarily, the F2 plants are used to screen markers. Most quantitative traits are not reliably evaluated on a single plant basis and, thus, require a replicated experimental design employing the progeny of F2 plants to estimate the phenotypic value of the quantitative trait. To produce sufficient seed for replicated designs several schemes are used, all based on individual harvest of F2 plants. Choices include the use of F3 progeny rows, inbreeding to near-homozygosity to develop single or multiple inbred lines (recombinant inbreds) from each plant in the F2, and populations created by intermating the plants in an F3 progeny row.
b.	Data will be analyzed with a variety of publicly available computer programs. New markers will be checked for linkage and, if prior information is available, oriented with respect to previously utilized and discovered markers. For each quantitative trait, the data is tested to insure that significant variation exists between F2-derived progeny. Lastly, the marker and quantitative trait data are combined and tested to determine if the QTL lies between linked markers or are adjacent to markers that are not yet associated with a linkage group.

2.	Characterize the germplasm subsets by phenotypic and physiological analyses to identify specific traits of interest.

a.	A series of experiments will be conducted to determine the potential of selected genotypes against biotic and abiotic stresses. Biotic stresses will include resistance to diseases and insect pests, and measurements will be taken to quantify the specific responses. Abiotic stresses will include extremes of temperature, salinity, and nutrients. Measurements for evaluation will include growth and development and physiological processes such as photosynthetic rates and nutrient accumulation in specific plant parts.

b.	Additional characterization to discover genetic variants in fiber traits and cottonseed components will be undertaken. Fiber characteristics such as length, strength, elongation, micronaire, and uniformity will be investigated from the germplasm pool. In addition, seed quality traits such as amino acid and fatty acid content and their respective specific chemical components will be measured.

3.	Development and utilization of techniques to broaden the genetic base of cotton.

a.	Day-neutral conversion program.

b.	Interspecific hybridization.

c.	Methods of gene introgression.

The analysis of quantitative trait loci and their association with molecular markers is expected to produce substantial benefits to cotton breeding. The association of markers with quantitative traits provides an opportunity to understand the organization of the cotton genome and the interaction of the two genomes in the allotetraploid. Such understanding will allow improved results with transformation and traditional breeding. In the near term, knowing which QTLs are tethered to markers will bring a new level of efficiency to such projects as breeding cotton for improved fiber traits, disease resistance, and other traits of agronomic importance. Efficiencies are expected to come in the form of improved means of selecting parental plants, early generation testing for desirable alleles, or the transfer of genes to agronomically elite lines from variation-rich, but agronomically poor, germplasm lines.

Objective 5: To refine and develop cotton breeding and variety testing methodologies and techniques.

Many questions addressed by the research proposed in this section may seem to have been answered by previous research. However, plant breeding and genetics are dynamic activities that must respond to the challenges of cotton production. Two major factors make it prudent to revisit some of these questions. First, a "yield plateau" or even decline over the last decade has been noted by several leading scientists and commodity groups (Report of the American Cotton Producers, 1999). This suggests some fundamental change in the germplasm pool in active use by plant breeders since genetic gains in yield by traditional means do not seem to be occurring. One approach to solving this yield plateau problem is to broaden the germplasm base as addressed in other sections of this proposal. Another approach is to re-examine how we go about achieving 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. When genetic populations change, as seems to be the case recently, the genetic parameters of the population change and the approach to further manipulate the population may also require change.

The second factor that justifies re-visiting these questions is the advent of new technology. Utilizing new molecular genetic technology is addressed in another section of the proposal. Advances in information technology offer potential for progress in traditional field evaluation of germplasm. In addition to facilitating data acquisition, the vast computing power now widely available makes it feasible to utilize more sophisticated experimental design and data analysis than in the past. Design and analysis beyond the traditional randomized complete block (RCBD) may allow breeders to identify superior genotypes more accurately and dependably.

The first objective, updating pedigree information, builds on previous work and keeps breeders up to date. This document also provides historic information to future generations of cotton breeders.

Cotton breeders currently use a variety of breeding strategies, but the choice of method is seldom based on solid comparison with other methods. The second objective of the proposed research is to provide breeders with quantitative information on contrasting breeding strategies and on methods that are not in wide use in cotton. Comparisons among methods in breeding cotton for disease resistance have been made (Baker and Sappenfield, 1985), but few such comparisons are available in breeding for yield.

Early-generation, bulk testing has proven useful in some crops and situations (Cregan and Busch, 1977; Singh et al., 1990), but not in others (e.g., Fowler and Heyne, 1955). This approach or modifications of it have been used by some commercial and public breeders, but its utility has not been empirically measured to any great extent in cotton (Green and Culp, 1989). A preliminary study at the Delta Research and Extension Center indicated a moderate positive correlation between early generation performance and performance descendent pure lines (Barut, 1998), but this finding should be corroborated.

The heritability of lint percentage and fiber quality has been determined in numerous genetic populations. However, these studies were done some time ago, and values may not apply to genetic populations currently in use.

At early stages of variety development, when seed are too scarce for replicated testing, breeders traditionally evaluate a large number of genotypes by visual examination. Although this practice is often hallowed as the "art" of breeding, greater progress might be achieved by the application of science (i.e., objective yield determination) at this point. Field variability often masks genetic differences in the absence of replication. Various techniques have been proposed to account and/or adjust for field variability. Calhoun (1997) demonstrated that modified augmented designs (MAD) could be used to reduce the influence of field variability when such variability was fairly large. This finding needs to be corroborated and other methods need to be evaluated.

Among cotton breeders, the RCBD and simple analysis of variance (ANOVA) are used almost exclusively in replicated yield trials. Bowman (1989) demonstrated that more sophisticated designs and analyses can reduce and/or account for field variability that can mask genetic differences, even in replicated trials. These findings need to be corroborated in other regions by calculating the relative efficiency (Cochran and Cox, 1957) of the various designs. Furthermore, the techniques and computer code for field layout and data analysis of these more sophisticated designs needs to be made more accessible to the general community of cotton breeders.

The concept of increasing yield by selecting for increases in specific yield components was examined in some depth in the 1970s (e.g., Worley et al. 1976). The general consensus was that compensatory shifts among yield components precluded progress in final yield. Nevertheless, selection for yield per se has resulted in shifts among yield components (Bridge et al., 1971). Lewis et al. (2000) has argued fairly convincingly that (indirectly) measuring fiber number (or fiber weight) per seed, rather than yield per se, may be more effective in identifying genetically high yielding varieties because management and environment have a large impact on seed number per hectare, but a relatively small impact on fiber per seed. This finding needs to be corroborated in other regions and methods to economically determine fiber per seed must be developed.

The advent of genetically engineered cotton varieties has raised several questions about how to measure or use them for both producers and plant breeders. One difficulty posed is how to evaluate the new technology in official variety trials. To date, genetically engineered cotton varieties have been tested in trials that may mask the benefits of the value added traits. Alternative variety testing methods may need to be developed that can provide producers with information they can use in their variety selection efforts. Additionally, cotton breeders are faced with a new challenge in their variety development efforts due to the wide spread adoption of transgenic cotton in the marketplace and in the improved germplasm base: the merits and demerits of forward crossing of transgenic genotypes. The objectives of this research are:

1.	Update information on the genetic relationships among potential parental germplasm to aid in identifying divergent parents for use in artificial hybridization: Information on the pedigrees of commercial and non-commercial germplasm released since 1995 will be collected from the Plant Variety Protection Office, official release notices, and other sources as necessary. This information will be used to update the MAFES bulletin, "Pedigrees of Upland and Pima Cotton Cultivars Released between 1975 and 1995" (Calhoun et al., 1997), and the USDA technical bulletin, "Coefficients of parentage for 260 cotton cultivars released between 1970 and 1990" (Bowman et al. 1997).

2.	Quantify advantages and disadvantages of divergent variety development strategies.

3.	Compare genetic gain from selection in pedigree breeding schemes (plant-to-row selection each generation) with that of a bulk breeding scheme (plant selection delayed until F4 or F5):

a.	A cooperative project will be initiated whereby at least two cooperators in each of three regions (Southeast, Mid-south, and Southwest) each begin with four hybrid populations of their choosing. Strains will be developed from the four populations by 1) the pedigree method (plant-to-row selections in the F2 to F4) and 2) the single seed descent (SSD) method (SSD from the F2 to F4). In the F4, plants will be selected in both groups and seed planted as F5 progeny rows. Selected progeny rows will be harvested, and seed will be used to plant F4:6 replicated yield trials. The specific size of the breeding populations will be determined by the resources of each cooperator, but resource allocation for the pedigree vs. SSD treatments will be constant. The number of F5 progeny rows from the SSD treatment will equal the total number of progeny rows used during pedigree selection. For example, if 100 progeny rows were planted in each generation from F2 to F5 in the pedigree treatment, 400 F5 progeny rows will be planted in the SSD treatment. An equal number (20 to 30) of F5 progeny rows will be harvested from each treatment and included in F6 yield trials. These procedures are designed to keep resource allocation approximately equal between the two treatments. Analysis of the F6 yield trial will compare between treatments (i.e., breeding method) as well as among F6 strains.

b.	Determine the value of measuring performance of unselected early generation (F2 to F4) bulk populations in identifying those with a high probability of producing pure lines with superior yield and/or fiber quality: Each cooperator in this project will begin with a minimum of 20 hybrid populations. All populations will be advanced by SSD (or a variant, single boll descent) from the F2 to F5. In each generation, a duplicate sample unselected bulk (from SSD harvest) of all populations, along with regional checks, will be planted in replicated yield trials at one or more locations. In the F4, a minimum of 30 plants per population will be harvested (in addition to the F4 SSD). Seed from individual F4 plants will be planted in an unreplicated yield trial of F5 progeny rows using site-
appropriate experimental design (e.g. modified augment design). Performance (lint yield, lint percentage, fiber length, fiber strength, and micronaire) of F5 progeny rows will be compared in various ways to performance of the SSD populations from which they were derived. Comparisons will include but not be limited to correlation of individual F5 row performance with performance of SSD bulks in each generation (N = 30 x P, where P = number of hybrid populations) and performance of F5 rows, averaged across populations (families), with performance of SSD bulks (N = P).

c.	Determine relationship between values for lint yield, lint percentage, and fiber quality parameters measured in single plants to those values measured in descendant progeny rows: Cooperators will harvest a minimum of 30 plants from each of 10 populations of their choosing in the F2, F3, and F4 generations. From each plant, cooperators will determine lint yield, lint percentage, fiber length, fiber strength, and micronaire. Seed from all plants will be planted the subsequent year and samples from each progeny row will be collected to determine lint yield, lint percentage, and fiber properties. Parent- offspring regression will be used to determine heritability for each trait in each generation. Regression analysis will be used to determine if there is a closer relationship between parents and offspring in various stages of generation advance.

4.	Develop guidelines for efficient and discriminating genotype evaluation.

5.	Evaluate various methods of nonreplicated evaluation:

a.	Visual evaluation vs. yield measurement: Plots in replicated trials of new, fairly unfamiliar strains will be evaluated visually and assigned a numeric value for yield potential by one or more persons. Plots will then be harvested for yield determination. Visual evaluations and objective yield measurements will then be correlated on an individual plot basis and on an entry-mean basis. In addition, comparisons will be made of entries that would have been discarded on visual vs. measured evaluation.

b.	Compare various methods to remove or adjust for spatial field variability in small-plot yield trials in cotton (comparison of experimental designs): Several yield trials will be set up in rectangular lattice designs. Results will be analyzed using the lattice model and traditional analysis of variance as if the design had been an RCBD. Control of experimental error will be compared between the two methods.

c.	Comparison of post-mortem data analyses: Existing variety test data will be analyzed using traditional analysis of variance, nearest neighbor analysis, and trend analysis. Differences among analyses in terms of relative efficiency, experimental error, and measured entry performance will be examined.

6.	Determine the value of yield component analysis in place of, or in addition to, traditional yield estimation: A replicated yield trial consisting of at least 20 diverse genotypes will be conducted across the Cotton Belt. Prior to harvest, three 25-boll samples will be collected from each plot. Samples will include first position bolls from nodes 10 and less, from nodes 11 to 15 and from nodes 16 and above. Samples will be ginned taking care to avoid loss of seed or lint during the process. Seed number and weight, lint weight, fiber length, and micronaire (linear density) will be determined for each sample and used to determine fiber number and fiber weight per seed. These parameters will be compared to traditional yield measurement in terms of entry rank, experimental error, and genotype x environment interaction.

7.	Evaluate the need to incorporate systems testing in variety trials for transgenic technologies.

8.	Investigate the genetic consequences of forward crossing of transgenic parents.

Updated information on pedigrees and genetic relationships among potential parental material will allow breeders to make more genetically divergent crosses thereby increasing the probability of generating new, unique genetic combinations. Quantifying costs and benefits of different breeding strategies will allow breeders to make informed decisions regarding the choice of strategies, rather than relying solely on habit or training. This information should result in more efficient utilization of resources and therefore greater benefit (to breeding entities, producers, and ultimately to consumers) at a given level of input. A document will be developed that outlines advantages, disadvantages, and appropriate applications of alternative experimental designs and data analyses relative to RCBD/traditional ANOVA with specific reference to cotton and to issues encountered in various regions. The document will include software available (including SAS and required SAS code) to facilitate these designs and analyses. Language and explanations in the document will be geared toward non-statisticians. Developing a user friendly guide to experimental design and analysis would allow non-statistically oriented breeders to get the most (in terms of identifying superior genotypes) out of the data they generate. Yield component analysis offers the promise of a more discriminating and stable method of evaluating genotypes. In combination, these outcomes could help overcome the current stagnation in yield improvement.

Objective 6: To develop cotton bioinformatic systems.

Advancements in information technology over the last decade have been impressive and now allow for the development of sophisticated, yet user friendly, programs for data collection, synthesis, and querying. Such bioinformatics tools are already being used to some degree, but are anticipated to become increasingly more important in future cotton improvement efforts. Over the next 5 yr period, existing information systems for cotton, namely CottonDB, should be enhanced to handle the large volume of classical and molecular genetic data being generated through the cotton research produced by this project and the cotton research community at large. The goals for this objective are:

1.	Continued curation of CottonDB.

2.	The development of user friendly data input and research collaboration linkage tools.

3.	The establishment of linkages among plant genomic and cotton web sites.

4.	The coordination and standardization of data and nomenclature for cotton.

5.	Development of genomics analysis techniques (including graphical genotypes).

6.	Methodologies for presenting and preserving variety testing data.

7.	Development of internet information exchange sites devoted to cotton.

8.	Information exchange (annual meetings, biennial Cotton Breeder's Tour; symposia, etc.)

EXPECTED OUTCOMES

The primary goal of this research is to improve the yield, fiber quality, and profitability of cotton production. The objectives of the research conducted under this Regional Research Project will address numerous important and/or unresolved issues relevant to increasing the agronomic potential of cotton. Success in achieving these objectives is expected to have a positive impact on cotton production and the work should be considered incomplete until it has done so. Several of the objectives will focus on germplasm enhancement and characterization, topics critical to improving cotton productivity. By the end of the proposed 5 yr period of coordinated work, it is anticipated that a significantly greater percentage of the cotton germplasm currently maintained in the U.S. will be characterized for standard Gossypium agronomic descriptors and additional traits such as insect and disease resistance. A more detailed and integrated genetic map of cotton will also be produced using both molecular genetic and cytogenetic techniques. Such information will help revolutionize the trait-specific improvement of cotton.

Marker-assisted selection has been a tantalizing promise of molecular biology for many years, but has not seen widespread utilization. With the development of a highly saturated cotton genetic map and the development of transportable, tightly linked markers (both of which are expected outcomes of the research outlined in this proposal), it is anticipated that such selection will be more commonly practiced in cotton within the next 5 yr period. This will allow cotton breeders and geneticists to tackle more effectively and efficiently those significant problems facing U.S. cotton producers and processors. Notable examples will likely include increased resistance to pests (e.g., root-knot and reniform nematodes), reduction of gossypol in seed, improved fiber strength and length, and higher yield.

Increasing the efficiency with which new cotton varieties are developed will allow plant breeders to more readily address the problems faced by cotton producers and consumers. A recent study (Bowman, 2000) found a great similarity in the procedures used by both public and private cotton breeders. This may represent the synthesis of over a century's worth of experience into a streamlined procedure, but it is deserving of critical evaluation. The availability and merits of increasingly sophisticated statistical analysis techniques should be examined. These two issues are a core component of this proposal.

New tools for the collection and dissemination of both historical and current data have been developed. This proposal contains several elements for data synthesis, curation, and for making the results of this research available for use by cotton producers primarily through the use of the internet. Future progress in cotton improvement is dependent on such activities.

Not explicitly addressed an objective in this research proposal, but critical to the future of U.S. cotton production and profitability, are the opportunities that regional project activities afford for the formal and informal interaction amongst cotton researchers. At one level, this includes professional information exchanges and collaborative research initiatives that are enhanced by personal contact. Just as important, but more nebulous, are the casual contacts established.

Finally, an expected outcome of the contacts and collaborations that arise from this regional research project is that greater mutual respect and appreciation will result between public and private cotton breeders and geneticists. In a break with the past, cotton researchers from the public and private sectors developed this proposal. An invitation to all U.S. cotton researchers to participate in the research objectives outlined above has been extended. Through our combined efforts we hope to keep U.S. cotton competitive, environmentally benevolent, and of the highest quality.

ORGANIZATION

The 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 SAYS, 1890 institution, USDA-ARS laboratory, and private company. Appointments will be made by the appropriate administrator. The committee will also have an administrative advisor as appointed by the ASES 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-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 takes advantage, where possible, of external research components in the U.S. and elsewhere that are not official participants in the group effort. Together, we can achieve results that individually we could not.

TITLE:	Development of Genetic Resources for Cotton

S/Johnny C. Wynne	1/23/01

Administrative Advisor	Date

S/T. J. Helms for W. H. Brown	1/26/01

Chairperson, Southern Regional	Date

Association of Directors

______________________________	__________

Administrator, Cooperative State	Date

Resource, Education, and Extension

Service

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Reinisch, A.R., J.M. Dong, C. Brubaker, D. Stelly, J. Wendel, and A.H. Patterson, A.H. 1994. A detailed RFLP map of cotton, Gossypium hirsutum X Gossypium barbadense: Chromosome organization and evolution in a disomic polyploid genome. Genetics 138:829- 847.

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Robinson, M., J.N. Jenkins, and J.C. McCarty, Jr. 1997. Root knot nematode resistance of F2 cotton hybrids from crosses of resistant germplasm and commercial cultivars. Crop Sci. 37:1041-1046.

Shappley, Z.W., J.N. Jenkins, W.R. Meredith, and J.C. McCarty, Jr. 1998. An RFLP linkage map of upland cotton, Gossypium hirsutum L. Theor. Appl. Genet. 97:756-761.

Shappley, Z.W., J.N. Jenkins, C.E. Watson, Jr., A.L. Kahler, and W.R.Meredith. 1996. Establishment of molecular markers and linkage groups in two F2 populations of upland cotton. Theor. Appl. Genet. 92:915-919.

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Appendix 1. Participating Institutions by Objective

Institution	Obj. 1	Obj. 2	Obj. 3	Obj. 4	Obj. 5	Obj. 6
SAES Cooperators/Institution
Auburn University
Louisiana State University	X	X	X
Mississippi State University	X	X	X	X	X
New Mexico State University	X	X
North Carolina State University	X
Oklahoma State University
Texas A&M University	X	X	X	X	X	X
University of Arkansas	X	X	X
University of Arizona	X	X	X
University of California, Davis	X	X	X	X
University of Florida
University of Georgia	X	X
Alabama A&M	X	X	X
Non-SAES Cooperators (Public)
Texas Tech University	X	X	X	X
USDA/ARS - Mississippi State	X	X	X
USDA/ARS - Texas A&M	X	X	X	X	X
USDA/ARS - Jamie Whitten	X	X	X
USDA/ARS - SRRC	X
USDA/ARS - Arizona	X	X	X
Non-SAES Cooperators (Private)
Cotton Incorportated	X
CPCSD
Delta and Pine Land	X	X
Monsanto	X	X
Novartis	X	X
National Cotton Council	X
Olvey & Associates
Phytogen	X	X
Stoneville Pedigreed Seed	X	X
Appendix 2. Member and SY, PY, TY Commitment for Each SAES Cooperator

SAES Cooperators	Member	SY	PY	TY
Auburn University	David Weaver	0.1	0	0
Louisiana State University	Gerald Myers	0.1	0.15	0
Mississippi State University	John Creech

Ted Wallace	.10

.10	0.2

0	0.2

0
New Mexico State University	Roy Cantrell	0.1	0	0.1
North Carolina State University	Daryl Bowman	0.1	0	0
Oklahoma State University	J. C. Banks	0.1	0	0
Texas A&M University	David Stelly

Peggy Thaxton

C. Wayne Smith	0.5

0.2

0.1	0

0

0	1.0

0.1

0
University of Arkansas	M. Stewart	0.75	2.0	0
University of Arizona	Hal Mozer
University of California, Davis
University of Georgia	O. Lloyd May	0.1	0	0.1
Alabama A&M University	Khairy Soliman

Allan Zipf	0.1

0.1	0

0	0

0