Plant Genome

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USDA NRICGP Abstracts of Funded Research, FY 1995

PLANT GENOME

This competitive grant program is part of the USDA Plant Genome Research Program. The Plant Genome Research Program was established in order to facilitate development of new or improved crop and forest species, thereby promoting sustainability and profitability of plant production and improvement of quality of food, fiber, and feed. To accomplish these goals, the program gives high priority to research for construction of genomic maps, and for detailed studies of specific regions of genomes, genes and genetic processes. The program also supports research on development of new methods or innovative approaches that have potential application to genome mapping, gene isolation or gene transfer in crop and forest species.

9500682 Targeted Gene Tagging by Generating Barley with Maize Ds at Defined Locations

University of California, Berkeley; Department of Plant Biology; Berkeley, CA 94720

This proposal centers on the development of a system for identification of genes important to understanding the basic biology and applications of barley. Through the isolation and characterization of these genes a better understanding of their function is gained. In addition the ability to use them to improve growth, disease resistance and feed and malting characteristics will be realized. The long-term prospects for this project involve the identification of genes important to manipulating not only barley but other small grain cereals, such as rice and wheat, and maize. Barley has an advantage for this purpose in being a self-pollinating crop and thereby useful in identifying certain types of genes that would be very difficult to identify in cross-pollinating crops, like maize. In addition, the complexity of the barley genome is lower than in wheat, making the identification of genes easier. The proposed research complements and extends the barley genome mapping project by affording the opportunity to isolate, characterize and further manipulate genes responsible for enhanced performance through molecular breeding. In addition, it also provides the opportunity to identify additional markers useful to breeders in facilitating the movement of characteristics from one barley variety to another. Both types of information will be useful in improving the growth, disease-resistance and feed and malting characteristics in barley and related cereals.

9500591 Molecular Tagging Genes for Nematode Resistance and Tree Growth in Peach

Virtually all peach trees grown in commercial orchards or home gardens are compound plants consisting of a scion variety selected for its fruit quality and a hardy, well-adapted rootstock. The availability of improved rootstocks that are both nematode resistant and provide control of the size and shape of the tree can eliminate the need for nematocides and reduce the cost associated with pruning or chemical size control. The size-controlling parent "Harrow Blood" and the nematode resistant parent "Okinawa" were hybridized and a segregating F2 progeny produced from a single F1 plant for the study of the inheritance of size control and nematode resistance. We intend to use the molecular markers that have been placed in the Prunus (peach x almond) linkage map that we have developed to locate the Mi gene for rootknot nematode resistance. Additional RFLP and RAPD markers will be added to the map to increase the marker density and uniformity of distribution among the linkage groups. The Mi gene will then be 'tagged' with closely-linked molecular markers to provide a rapid means of identifying resistant plants via markers rather than using screening methods that employ live nematodes. In a similar manner, we will study the inheritance of tree growth and size control in this population, then identify tightly-linked molecular markers for map placement and gene tagging. These methods will simplify selection and increase the efficiency of developing new rootstocks that have multiple desirable traits.

We will conduct simultaneous genetic, physical and transcriptional studies of the major cluster of resistance genes in lettuce, with particular focus on genes for resistance to downy mildew. We will study how resistance genes determine specificity and characterize the mechanisms generating variation. Lettuce downy mildew is an appropriate system for the molecular characterization of resistance gene clusters. Lettuce is genetically well-characterized for disease resistance; it is also one of the most important fresh vegetable crops and downy mildew is the most significant disease. In addition, several technologies will be developed that will aid in the cloning of more resistance genes from lettuce and other crops. Our genetic studies will characterize patterns and sites of genetic recombination as well as spontaneous mutants. We will generate a physical contig of overlapping clones through much of the region using large fragments of genomic DNA cloned in bacterial and yeast artificial chromosome vectors. Transcribed sequences will be identified using several techniques so that a variety of genes will be characterized. This may lead to the identification of other genes involved in disease resistance. To characterize mechanisms generating variation in resistance genes, we will analyze sequence divergence in genes distributed through the major cluster and the differences between clusters within a genotype and between genotypes. This will provide evidence for the evolutionary relationships between resistance genes and whether programmed genetic rearrangements generate new specificities. Determining mechanisms of resistance gene evolution will ultimately lead to engineering of new resistance specificities and to novel disease control strategies.

9500823 High Resolution Mapping of a Major Gene from a Durably Resistant Rice Cultivar

Breeding for durably disease resistant cultivars has been the priority in crop improvement for several decades. The long term goal of this project is to gain an understanding of the molecular mechanism of durable resistance to plant pathogens. We have shown in our previous studies that, although resistance in Moroberekan, a durably resistant rice cultivar to blast, is genetically complex, much of its qualitative resistance is attributable to the dominant Pi-5(t) locus. Pi-5(t) confers the broadest spectrum resistance of all blast loci studied, and is associated with complete resistance to at least sixteen isolates of the blast fungus. We propose to do high resolution mapping of this locus. Application of recently developed techniques such as amplified fragment length polymorphism (AFLP) analysis will greatly accelerate progress towards characterization of this locus. The specific objectives for this project are: 1) to identify AFLP markers tightly linked to Pi-5(t) locus; 2) to construct a high resolution map of the Pi-5(t) locus and; 3) to test the inheritance behavior of this locus in different genetic backgrounds. All plant resistance genes cloned to date confer resistance to only one race of the pathogen. The experiments outlined in this proposal will lead to isolation of the gene(s) at a broad spectrum locus from a durably resistant cultivar for the first time. Direct transfer of these genes to susceptible cultivars will provide an efficient method for utilizing Moroberekan genes. Finally, the elucidation of the molecular mechanism of durable blast resistance may reveal a general strategy of durable host resistance to fungal and other microbial pathogens.

USDA Agricultural Research Service; Plant Gene Expression Center; Albany, CA 94710

Plants and animals are constantly under pathogen attack. Plants, however, have evolved defense mechanisms distinct from the vertebrate immune system. Plant breeders first recognized that plant disease resistance was often controlled by simple dominant genes, termed R genes. Each plant R gene encodes resistance to a particular strain of pathogen. R genes are postulated to encode receptors that interact directly or indirectly with a pathogen produced ligand (also termed elicitor) and transmit signals that lead to induction of disease resistance. The recent molecular cloning of several plant R genes, that collectively confer disease resistance to a wide range of pathogens, revealed that many of the encoded R proteins have several features in common and indeed may encode pathogen receptors. The characterization of the molecular signals involved in pathogen recognition and the elucidation of the molecular events specifying the expression of resistance may lead to the development of extremely effective strategies for disease control. The results of these studies are predicted to positively impact agriculture. The goal of the proposal is to isolate the Tobacco Mosaic Virus (TMV) R gene, N', using established gene isolation procedures. The TMV coat protein is the N' elicitor molecule. The N'-TMV system of Nicotiana sylvestris offers an excellent model system for studying interactions between plant R proteins and pathogen ligands. Study of this system will greatly facilitate understanding of a specific plant-pathogen interaction, and ultimately provide information necessary to engineer stable resistance specificities leading to broad spectrum plant resistance.

Our goal is to determine the extent of similarity among pine genomes through the use of comparative genetic mapping. The genus Pinus contains over 100 species scattered throughout the Northern Hemisphere and its members are among the most economically important forest trees. Comparative mapping will contribute to the basic understanding of the evolution of pines, but there is also a considerable practical importance for establishing collinearity among pine genomes. If pine genomes are collinear, then mapping of genes in one species of pine will facilitate mapping and the eventual characterization of those genes in other pine species. This is perhaps more important for pines than agronomic plant groups because regional economic and ecological constraints preclude the widespread culture of a single forest tree species. Our specific objectives are: 1) construct low-density, genetic linkage maps for representative species from the genus Pinus using molecular genetic markers; 2) test for collinearity of linkage groups among pine species. The construction of genetic maps will be greatly facilitated by taking advantage of the unique biology of conifers. The relatively large megagametophyte of a conifer seed is genetically equivalent to the haploid egg cell. Therefore, open-pollinated seed from individual trees can be used for haploid segregation analysis, thereby eliminating the need for costly and time consuming hand-pollinations. The megagametophytes will be analyzed using homologous genes from each species, including isozyme and codominant PCR-based markers derived from cloned cDNAs. Our results will be electronically published in the Dendrome databases and thereby be of immediate use to the forest genetics research community.

In the 1940's and 1950's, agriculture in the United States experienced unprecedented increases in productivity. The Green Revolution owes much of its success to the use of hybrid crop plants. When the female parts of one plant are fertilized by pollen from an unrelated plant, the resulting hybrid plants express "hybrid vigor" in terms of increased yield, growth, and resistance to plant pests and drought. Corn, like many other plants, will naturally self-pollinate, resulting in inbreeding. To prevent the production of inferior inbred plants in corn, plants are manually detasseled (tassels are structures that produce pollen). The ears of detasseled plants are then pollinated by an unrelated pollen donor to produce hybrid seed. This is an expensive, labor-intensive process. A genetically controlled trait that prevents pollen formation is called "male- sterility". In male-sterile plants no pollen is produced, but female flowers are perfectly normal, and are receptive to pollination by unrelated donors. Since these plants cannot self-pollinate, all resulting seed is hybrid. We are studying the genes that control male-sterility traits. Our hope is that the isolation of these genes will lead to novel ways to make hybrid plants, and thereby circumvent the detasseling process. In addition to optimizing the production of hybrid corn, these studies could provide the key to creating hybrid seed in other crops plants as well.

9500662 Physical Mapping of Genes and RFLP Markers on Tomato Pachytene Chromosomes

Colorado State University; Department of Biology and Department of Soil and Crop Sciences; Fort Collins, CO 80523-1878

In situ hybridization (ISH) is the most direct way to determine the location of DNA sequences and genes on chromosomes. Although ISH is usually performed on metaphase chromosomes and analyzed by light microscopy, spreads of synaptonemal complexes (SCs ~ pachytene chromosomes) offer an alternative substrate for ISH with a number of advantages. For example, SCs are 3-10 times longer than corresponding metaphase chromosomes so localization of hybridization sites should be 3-10 times more accurate on SCs. Also ISH on SC spreads can be analyzed by electron microscopy. We propose to use ISH to determine the location of single copy sequences on tomato SCs because: (1) Tomato's linkage maps are among the best available for plants, (2) each tomato SC can be identified based on its relative length and arm ratio, and (3) a physical recombination map has been constructed for each tomato SC. We propose to use ISH to localize sixteen restriction fragment length polymorphism loci (RFLP) on tomato SC 11. This study will permit alignment of the tomato genetic, RFLP, and physical recombination maps on SC 11 and subsequently provide information on the relationship between chromosome structure, gene location, and crossing over. In addition, this study will set the stage for similar studies on the chromosomes of other crop plants, provide useful information to plant breeders, and demonstrate the feasibility of using ISH on SC spreads to locate genes that have been inserted by genetic engineering.

9500756 Genetic Transformation of Mature Meristematic Tissues in Perennial Citrus Plants

University of Florida; Horticultural Sciences Department; Gainesville, FL 32611-0690

Genetic transformation (the use of cellular and molecular techniques to insert a single target gene into the DNA of an organism) of plants as a breeding technique may ultimately prove to be most valuable in woody perennial species because genetic improvement in these species is so difficult through conventional methods. However, transformation systems in perennial plants are generally quite inefficient. A major problem is that transgenic plants produced from woody perennial species are juvenile, and must be grown for many years before they reach maturity and their horticultural characteristics and fruit quality can be evaluated. We propose to develop a method for transforming mature meristematic (actively dividing) tissues of an important woody perennial genus, Citrus, that suffers from a prolonged juvenile period. The axillary buds found in leaf nodes of mature trees will be used as starting material in sterile culture experiments. Transgenic shoots that develop from these buds should also be mature since they will develop directly from already mature tissue that does not dedifferentiate. Agrobacterium-mediated transformation will be the method of choice; biolistic transformation methods will be evaluated if necessary. Factors that may influence the success or efficiency of the system, such as tissue source and preparation, sterilization procedures, and culture medium components, will be evaluated. An "on plant" method for Agrobacterium-mediated transformation will also be investigated. Putatively transformed plants from any of these experiments will be extensively characterized using standard techniques. The maturity of the transgenic plants must also be verified.

9500785 Genetic and Physical Analysis of the Mla Resistance-Gene Cluster in Barley

To offset yield loss caused by fungal pathogens, cereal-breeding programs devote significant effort to the incorporation of genes for resistance. Powdery mildew of barley is an ideal system for investigating resistance to fungal pathogens in small grains. In barley, there are many genes conferring resistance to powdery mildew. Each of these genes provides specific resistance to various isolates of the pathogen. Detailed, high-resolution, genetic maps are necessary to carry out these investigations. Previously, we generated a detailed genetic map based on a large population of recombinants in the region surrounding the Mla (powdery mildew) resistance-gene cluster. To add to resolution of this map, we propose to identify additional molecular markers tightly linked to Mla. The specific objectives of this project are: 1) to use newly-obtained powdery mildew isolates to position additional Mla alleles that are present in the recombinant population, 2) to use these recombinants to analyze physical to genetic distance in specific regions near the Mla locus, 3) to generate additional tightly-linked molecular markers, and 4) to use these markers in the design of a feasible cloning strategy for Mla alleles. The availability of a cloned Mla allele will facilitate experiments designed to understand the molecular mechanisms of resistance to obligate fungal pathogens. This will increase our understanding of how genes for resistance evolve and how long they may be effective in breeding programs.

9500891 Use of Arabidopsis DNA Markers to Isolate a Disease Resistance Gene from Soybean

We are investigating the molecular basis of disease resistance gene specificity in plants. The effectiveness of naturally occurring disease resistance genes is often limited by there extreme specificity; a single resistance gene is typically effective against only specific pathogen strains. However, greater use of naturally occurring disease resistance genes is dictated by a lack of environmentally sound pesticides. The knowledge gained from our studies could lead to engineering of disease resistance genes with broader specificity. As a first step towards understanding specificity, we have been comparing disease resistance genes in soybean and Arabidopsis that confer resistance to bacterial blight disease, which is caused by Pseudomonas syringae. In Arabidopsis, resistance to specific strains of P. syringae is controlled by a single gene designated RPM1. In soybean, resistance to specific P. syringae strains is controlled by the gene RPG1. There are at least 4 different versions of RPG1, each of which is specific to a different strain of P. syringae. We wish to determine whether RPM1 and RPG1 are similar genes. If they prove to be similar, this would demonstrate that Arabidopsis disease resistance genes can be used as a tool to isolate disease resistance genes from commercially valuable crop plants, such as soybean. By comparing the amino acid sequences of RPM1 and the four different versions of RPG1 we will gain insight into which specific amino acid sequences are important for conferring specificity. This in turn should help us design plant disease resistance genes with new and broader specificities.

The Hessian fly is a major pest of wheat and causes yearly losses in wheat production in the United States. To date, 26 Hessian fly resistance genes have been identified in wheat. For the past 40 years genetic resistance has been used to reduce Hessian fly damage to wheat. The biological interaction between wheat and the fly is highly specific, demonstrating a gene-for-gene relationship for host resistance and virulence in the insect. Because wheat is the primary host for this insect, the widespread use of resistant cultivars has resulted in a strong selection pressure and the evolution of new virulent biotypes of the fly. Wise management of these resistant genes is important to maximize their longevity and effectiveness. One strategy is to pyramid two or more resistance genes into elite cultivars as a means to significantly extend the durability of resistance. This has not been feasible in the case of the wheat-Hessian fly interaction because of the difficulties in verifying the presence of more than one gene in a particular line. To do this, we are applying DNA marker technologies to identify markers closely linked with a number of highly effective Hessian fly resistance genes. To date, we have identified more than 20 DNA markers associated with ten Hessian fly resistance genes. The incorporation of DNA-based technologies into wheat breeding strategies should greatly accelerate the selection process in development of new cultivars with more durable resistance to insect pests.

9500814 Drought Tolerance in Sorghum: Mapping of QTLs and Analysis of Near-Isogenic Lines

Purdue University, West Lafayette; Department of Horticulture; West Lafayette, IN 47907-1165

Drought is a major environmental factor that limits crop yield. A long term solution to this problem is the development and utilization of plants with increased tolerance to drought. Drought tolerance is controlled by many genes and its expression is dependent on the timing and severity of the stress. These factors make it difficult to identify plants with good drought tolerance. Our approach to this problem is to identify and characterize individual genetic components that influence drought tolerance in sorghum, one of the most drought tolerant grain crops. To this end, we developed a recombinant inbred (RI) population from two sorghum lines (TX7078 and B35) with contrasting patterns of drought tolerance. This population expresses a range of reactions to drought stress at different stages of development. We will measure the relative performance of the RI lines in fully irrigated conditions and under drought stress, and use molecular markers to assess the genetic contribution of each parent. This will allow identification of genetic loci that influence drought tolerance in sorghum. To further understand the role of specific loci in drought tolerance, near-isogenic lines will be produced that differ only in the region of the genome that contains the gene of interest. These lines will then be compared for drought tolerance in a number of environments. This research will identify genetic components that contribute to drought tolerance in sorghum and should lead to an understanding of the physiological and biochemical mechanisms that sorghum uses to adapt to drought.

This project seeks to introduce any cloned gene into any line of alfalfa by transferring the gene into alfalfa pollen grains. The transfer into the pollen is to be accomplished using a "gene gun"; relatively new technology that shoots microscopic "bullets" carrying genes, into tissue. The procedure to be followed is to collect pollen from alfalfa plants, spread it over the surface of a nylon membrane, bombard it with microscopic tungsten particles coated with a gene of interest, transfer the pollen to flowers of male-sterile alfalfa plants, collect the seeds that result, grow the seeds into plants, and assay the plants for the presence of the gene. Initial studies will use a gene that produces an easily-detected color change in the plant leaves, but virtually no other effect. This will allow for rapid optimization of the system. This project has been designed to use relatively inexpensive and simply-operated equipment to facilitate the transfer of the technology, once developed, to the private sector. It is envisioned that this technology will bring genetic engineering into the mainstream of plant breeding and plant improvement, even in smaller, private crop breeding companies. Also, because the technology requires nothing unique to alfalfa, it is likely that the techniques developed in this research will have broad applicability to most, if not all, plant species.

9500575 High Resolution Mapping and Positional Cloning Tools to Study Soybean Cyst Nematode Resistance

University of Minnesota; Department of Plant Pathology and Department of Agronomy and Plant Genetics; St. Paul, MN 55108-6030

Soybean cyst nematode causes one of the country most destructive plant diseases. Genetic resistance to this parasite provides the best control strategy. Research on soybean cyst nematode can potentially be revolutionized through the use of genome mapping and gene cloning, but these techniques are poorly developed in soybean. In this project, essential foundations for cloning the soybean cyst resistance gene will be developed through high resolution genome mapping experiments. Additionally, a system for testing candidate resistance genes in culture will also be developed. The primary goals of the project include: 1) Precisely locating the most important soybean cyst nematode resistance gene based on tightly linked DNA markers, 2) Physically mapping the region surrounding the soybean cyst nematode resistance gene by examining very large fragments of DNA; in the process, the chromosomal organization of two soybean relatives (common bean and mungbean) will also be investigated, and 3) Developing a root transformation system for soybean that can be used to test gene sequences in culture for their effectiveness against the nematode. In addition to establishing a foundation for cloning soybean cyst nematode resistance genes, this work will also: 1) Create valuable genome mapping, cloning, and transformation tools for the soybean research community, 2) Provide insights into the use of genome mapping and gene cloning techniques with complex genetic traits, 3) Examine parallel genome structure between soybean and related legume species, and 4) Complement research now underway in the soybean cyst nematode parasite.

USDA Agricultural Research Service; University of Missouri; Plant Genetics Research Unit; Columbia, MO 65211-0001

The corn earworm is a major insect pest on many crops in the southern United States. A flavonoid compound called maysin provides resistance to earworm in corn silks. This project is designed to identify the genes that determine maysin concentration and therefore earworm resistance in corn silks. The approach is quantitative trait loci analysis using restriction fragment length polymorphisms (RFLP) to determine the genotype of plants in segregating populations. This projects differs from similar studies in the use of RFLP probes for specific genes from the pathway determining the trait. The knowledge gained from this project will allow more efficient selection of corn earworm resistant varieties. This project is of broader scientific interest in that the pathway to synthesis of the chemicals directly related to this agronomic trait are largely known, allowing the identification of the types of genes and their interactions that determine the trait. There are two major types of genes that control biochemical pathways, structural genes that code for the enzymes that perform the chemistry, and regulatory genes that control the levels of expression of the structural gene products. This project is designed to determine the relative importance of these two classes for determining this trait, and therefore serve as a model for traits where the biochemistry is unknown.

Determination of DNA sequences of expressed genes is a powerful way to learn about the genetic basis of growth and development in plants, animals and lower organisms. Many of the genes thus identified are completely novel, with no similarity to any previously described gene, while others can tentatively be assigned functions based on sequence similarity to genes described in other organisms. Placement of both novel and identified genes on genetic linkage maps would provide additional information valuable for later studies to assign functions to new genes. The objective of this project is to test a hierarchical approach to mapping genes expressed during wood formation in loblolly pine. Wood, the secondary xylem of trees, is important both in the global carbon cycle and as the raw material for a multi-billion dollar industry, yet little is known of the genes that control wood formation. This project will provide to public databases the partial sequences of 1000 genes expressed during wood formation in pine, along with formation on the tissue-specificity and abundance of expression of these genes, and genetic map locations for about 100 of the genes. This is part of a long-term research effort to understand the genetic control of wood formation, in order to provide both answers to basic questions and potential opportunities to manipulate wood properties for fiber or structural uses.

9500625 Fine Structure Mapping of Disease Resistance Related Genes in Common Bean

Bean rust, caused by the pathogen Uromyces appendiculatus, is a severe pathogen of common bean (Phaseolus vulgaris L.) in both temperate and tropical climates. The degree to which the pathogen is a field problem is actually increasing in the bean growing regions of the United States. One method of controlling the pathogen is through genetic resistance. A number of regions of the genome contain genes which provide such resistance. One such region is called Ur- 3. This genetic region contains one or several genes which provide resistance to 14 different races of the bean rust pathogen. Resistance can be expressed as immunity or hypersensitive flecking on the leaf tissue, and the control of these two resistance types also appears to be under genetic control. The goal of this research project is to analyze the genetic and molecular organization of the Ur-3 region, and the genes that control resistance type. A series of experiments will be performed to identify a number of molecular markers tightly associated with the Ur-3 region and the gene controlling resistance type. These experiments will incorporate the most recent marker techniques such as RFLP and RAPDs. The Ur-3 region will also be mapped with regards to a series of bean rust races to which it provides resistance. This combined phenotypic and molecular map will give insight into the organization of this complex locus. The scientific outcome of this research will be a better understanding of the genetic organization of genes that provide multiple race resistance and control of resistance type.

This research is part of an overall effort to genetically and physically map sequences in the maize genome, clone these sequences based on their map position, and ultimately transfer sequences into other maize genotypes or other species. The goal of this project is to produce DNA-clone libraries for specific maize chromosomes, chromosome arms, and parts of chromosome arms. Chromosome specific libraries can be used in a number of applications including chromosome painting, saturation of specific genetic regions with molecular markers, physical genome mapping, and efficiently identifying a sequence from a known genomic location. These applications will enhance understanding of the maize genome and the ability to manipulate it, thereby increasing the efficiency by which improved cultivars can be produced. In addition, maize can serve as a model system to increase understanding of monocot genomes in general. Maize has chromosomes which vary over two-fold in length, and is a species rich in cytogenetic stocks. Both of these factors will facilitate the isolation of chromosome specific sequences. Expected results from this funding period are: 1) flow sorting and development of chromosome specific libraries for two of the ten maize chromosomes, 2) refinement of flow-sorting methodology to enhance separation of the eight remaining maize chromosome pairs, 3) preliminary development of procedures to use chromosome-specific libraries for chromosome painting, molecular marker localization, physical map construction, and specific sequence isolation.

9500652 Structural and Functional Analysis of a Region of the Arabidopsis Genome

Cold Spring Harbor Laboratory ; Cold Spring Harbor, NY 10024; Grant 95-37300-1578; $230,000; 3 Years

The genomes of most important crops are extremely large and complex. Much of this DNA is repetitive and does not encode essential proteins. In contrast, the Arabidopsis thaliana genome is 1/30th to 1/50th the size of crop genomes. It contains genes similar to those found in higher plants, including major crops, but fewer repetitive sequences. Once a gene is identified in Arabidopsis , it's possible to isolate it from other plants too. We will sequence a small region of the Arabidopsis genome, and use this sequence information to identify plants with genes that have been interrupted by a transposable element. As a starting point, we have chosen the PROLIFERA gene region on chromosome 4 that we have recently cloned from Arabidopsis. Analysis of these plants should provide valuable knowledge about tissue and developmental stages in which this region functions. This should allow us to rapidly identify and characterize similar genes in crops too. Using our strategy and Arabidopsis may allow us to obtain this information cheaper and faster than possible with other plant systems. Our strategy might be a more cost effective way to systematically study the genes and genomes of higher plants and provide the knowledge infrastructure for molecular plant breeding in the 21st century.

9500764 Evaluation and Application of a New BAC Library Vector Designed for Transfer of Large DNA Inserts to Plants

This project evaluates new technology for transferring high molecular weight DNA to plants. A new vector has been constructed that is based on the binary (BIN) plant transformation vector and the bacterial artificial chromosome (BAC) vector, and is referred to as BIBAC. Agriculturally important traits such as disease resistance, growth habit, and yield are encoded by one or more genes. In order to establish that a gene responsible for a specific characteristic has been isolated from a plant genomic library, it is necessary to introduce the candidate DNA into a plant in which that trait can be observed. This is accomplished by plant transformation. Currently the candidate DNA must be moved from the library vector to a plant transformation vector. This is the most tedious and risky step in plant gene identification. The BIBAC vector circumvents this problem because candidate DNA clones can be introduced directly into the bacteria that are used to transform plants. The development of a BIBAC vector that can reliably transform high molecular weight constructs into plants will have a number of important applications in plant science and agriculture. It will accelerate the identification of agriculturally important genes and make it possible to introduce valuable traits into plants without dragging along deleterious traits (a common problem for classical plant breeders). The BIBAC will make it possible to introduce desirable traits into plants in such a way that they will be inherited all together, and to identify the genes that contribute to quantitative traits such as yield, fruit size and flavor.

9500763 Isolation of Megabase-sized DNA and Construction of Physical Maps in Arabidopsis and Rice

Cornell University; Section of Biochemistry, Molecular and Cell Biology; Ithaca, NY 14853

Rice is an important crop world wide, including the United States. It is estimated that the loss of rice crops due to diseases and insect pest amounts to at least 10 billion dollars annually. Much of the loss can be avoided if rice plants can be disease resistant and insect resistant. The genes coding for these traits are present in many wild rice species. It is important to locate and then characterize these genes first by physical mapping and then by cloning them. Arabidopsis has been a model plant for genome-related analysis because it has a very small genome (100 mb), a short generation time, and an extensive RFLP map. It is easy to produce mutants in Arabidopsis and methods are available for transformation/regeneration. Despite its small size, Arabidopsis probably contains all the genes present in other plants. Thus, almost any information obtained with Arabidopsis can be applied to other plants and facilitate a better understanding of plants with larger genome sizes. The long term goal of the proposed project is to develop a physical map of rice and Arabidopsis. After the completion of this work, it would be easier to clone the different resistance-conferring genes from domestic as well as wild rice species. Finally, these genes could be transferred to important rice cultivars to produce agronomically valuable transgenic rice plants.

Oregon State University; Department of Crop and Soil Science; Corvallis, OR 97331-3002

The goal of this project is to develop a genetic map for domesticated sunflower (Helianthus annuus L.). Sunflower is a source of seed and oil with food and non-food uses, and is one of the most important oilseed crops in the world. The worldwide sunflower hybrid seed industry is second only to maize. We are using a variety of DNA markers to construct the genetic map of sunflower. Two of our major aims are (i) to develop a bank of hypervariable sequence-based DNA markers and (ii) to disseminate mapping resources and the genetic map to laboratories around the world. These resources can be used by plant breeders, geneticists, and the seed industry to (i) DNA fingerprint genetic stocks and varieties, (ii) study the ancestries and pedigrees of genetic stocks, (iii) map and characterize economically important genes, e.g., oil quality and disease resistance genes, and (iv) develop superior varieties through marker-aided breeding. Genetic maps anchor DNA markers to coordinates which can be used to find and track important genes and develop superior genotypes (varieties). We are presently using the map to pinpoint genes underlying oil biosynthetic pathways. These genes are important determinants of oil quality. The genetic base for domesticated sunflower is narrow. One of our goals is to create resources for broadening the genetic base (decreasing the genetic vulnerability) of sunflower. DNA markers can be used to efficiently transfer superior genes for specific traits from exotic to elite germplasm while minimizing the transfer of inferior genes for other traits.

The aim of the Gordon Conference on Quantitative Genetics and Biotechnology is to foster the interaction of scientists with an interest in applications of quantitative genetics and biotechnology from different specialty areas. The goal is that the conference may provide insights into uses and limitations of various tools that are not obvious in the individual disciplines. Particular attention will be paid to the location, characterization, and exploitation of genes affecting quantitative characters. This is one of the few meetings that brings together the specific mix of disciplines of people with a primary interest in plant or animal breeding, and in evolutionary biology and human genetics. The conference will include eight prominent workers in the field of plant quantitative genetics. They will present their latest findings in the studies of pine, maize and other crop species.

9500895 High Density Mapping and Isolation of Genes Regulating Tomato Fruit Ripening

Texas A&M University; Department of Horticultural Sciences; College Station, TX 77843-2133

Fruit ripening represents a developmental process both unique to plants and of great agricultural importance. Although fruit ripening is usually associated with desirable changes in color, flavor, and texture, it also imparts negative characteristics including susceptibility to post-harvest pathogenesis (rotting), and decreased resistance to mechanical stresses encountered during harvest and shipping. United States crop losses resulting from post-harvest diseases alone amount to millions of dollars annually, excluding the economic and environmental cost associated with fumigation, controlled atmosphere storage, and intensive harvest and handling practices. A greater understanding of the molecular, genetic, and physiological basis of ripening will promote our understanding of plant development and provide the specific genetic tools required to modify fruit and vegetable crops for enhanced quality, yield, nutritional value, and resistance to mechanical stress and post-harvest diseases. The specific goal of this project is to isolate two genes designated rin and nor via mutant analysis to regulate the ripening process during tomato fruit development. Information gained from analysis of these genes will broaden our understanding of how genes regulate changes during plant development in general and fruit ripening in particular. In addition, the rin and nor genes will likely prove useful as tools with which to genetically modify ripening characteristics of tomatoes and additional fruit species with similar ripening processes (including apple, pear, melon, squash, peach, avocado, and many others). The ability to enhance desirable ripening characteristics while minimizing negative attributes will positively impact the competitiveness, productivity, and economic and environmental sustainability of United States agriculture.

Forest trees are the dominant organisms in many ecosystems and have tremendous economic value for lumber, paper, fuel, and recreation. While the demand for forest products is increasing, there is also a growing desire to protect remaining native forests from overharvesting. One way to reduce harvesting pressure on natural forests is to use intensive management of fast-growing trees to produce large volumes of wood on a relatively small land base, primarily marginal farmland. An integral part of intensive forest management is tree breeding and genetics. Although most forest tree species are undomesticated and still contain large reserves of genetic variation, the long interval between generations slows the progress of tree breeding. Molecular genetic methods, including genome mapping, promise to greatly increase the speed and precision of tree improvement. This project proposes to study the genetics of rapid tree growth in hybrid poplar. These hybrids grow more than 10 feet in height per year, and are harvested within 7 years of planting. Using chromosome maps of hybrid poplars and their offspring, we will locate the regions of the genome responsible for rapid growth and for adaptation to the various growing environments in North America. This information will be used to test the hypothesis that tree growth can be predicted at the seedling stage, making it possible to identify superior trees without extensive field testing.

Crops such as cabbage, turnip, beets, and winter wheat are biennials and have genes that promote vegetative growth by setting up a cold requirement for flowering (vernalization). These genes could confer very useful growth habits if introduced into other crops. For example, they may improve the feed quality and winter survival of some forage crops, or permit fall planting of some crops thereby reducing herbicide use and expanding crop rotation options. We previously located genes for the vernalization requirement on the chromosomes of three Brassica species (cabbage, turnip rape, and oilseed rape) using DNA markers. We now propose to further define these genes by placing them in a common genetic background and testing their specific effects on plant growth. We also will screen over 1000 DNA fragments to find markers that are tightly linked to the vernalization genes. These markers can be used to compare vernalization genes from different species to determine if they are the same or different. We will use this information in future projects for cloning vernalization genes and transferring them to crop species that do not contain these types of genes.