Source: CLEMSON UNIVERSITY submitted to
DEVELOPMENT OF A FUNCTIONAL GENOMICS DATABASE FOR PEACH:A MODEL GENOME SPECIES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
0190366
Grant No.
2001-52100-11345
Project No.
SC-2002739
Proposal No.
2001-04424
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Sep 15, 2001
Project End Date
Mar 31, 2005
Grant Year
2001
Project Director
Abbott, A. G.
Recipient Organization
CLEMSON UNIVERSITY
(N/A)
CLEMSON,SC 29634
Performing Department
GENETICS, BIOCHEMISTRY & LIFE SCIENCE STUDIES
Non Technical Summary
The Rosaceae ranks third in economic importance. The most important crops include: apple (Malus), pear (Pyrus), raspberries/blackberries (Rubus), strawberries (Fragaria), and stone fruits (Prunus). Prunus has many important fruit crops [peach/nectarine, apricot, plum, sweet and sour cherry], nut [almond] and lumber trees [black cherry]. Despite the importance of these crops, identification of genes controlling important agronomic characters is limited. This is due to the lack of information and the underdevelopment of current genomic tools in these plants. In this project, we present a research strategy to address the deficiencies in Rosaceae genetics research. The objective of this work is the development of the requisite tools to speed the process of gene discovery and characterization in fruiting tree species. We have a partnership of five US institutions and one European laboratory focused on identification and characterization of genes important to fruit tree development and sustainability using peach as the genetics model. Development of educational programs using regional crops of importance is an ideal way to educate the public in the use and risks associated with application of the new technologies while demonstrating the importance of these tools for sustaining the production of safe local food products. For this reason, we are developing educational tools and workshops for high school educators and extension specialists highlighting peach genomics as an excellent example of crop improvement through genomics technology.
Animal Health Component
(N/A)
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011114108075%
2011212108025%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1212 - Almond; 1114 - Peach;

Field Of Science
1080 - Genetics;
Goals / Objectives
It is proposed that peach could serve as the model genome for Rosaceae genomics and provide a highly characterized "core" genome for subsequent comparative mapping and gene discovery. However, the rapid utilization of these genomics tools is not possible in the absence of a functional genomics database since this is required to directly provide candidate genes controlling important crop traits. Therefore, the centerpiece of this project is the development of a candidate gene database for the Rosaceae through the development a peach/almond EST unigene set and subsequent mapping of the important tissue specifically expressed ESTs on the developing physical map of peach and on the general Prunus genetic map database. The specific aims of this research are: 1.Develop an extensive peach EST database (target number, 4,000-6,000 unique ESTs) from a variety of vegetative and reproductive tissues of peach and almond. 2. Map up to 3,000 unique peach ESTs, as well as, the COS set of tomato EST loci on the developing peach physical map and initiate mapping a subset of these on the Prunus general map to provide a higher resolution genetic map and candidate gene resource. 3. Integrate the complete EST database into a developing International Prunus Genomics database. 4. Develop a science education outreach program for high school teachers, students, and SC extension personnel The integration of the structural and functional genomic tools will provide the resources to identify, characterize and manipulate through transgenic technologies, the genes important to sustainability of the Prunus and rosaceous crops. Of course, the use of genetically engineered materials is currently a subject of hot debate worldwide fueled by the lack of accurate knowledge in the public of the nature and risks of using such technologies. Development of educational outreach programs using regional crops of importance is an ideal way to educate the public: 1) in the use of these technologies; 2) in the risks associated with the application of the technologies; and 3) to the importance of these technologies for sustaining the production of safe local food products while minimizing the use of chemical control agents. For this reason, using this local crop model (peach), we propose development and implementation of a genomics educational outreach program for educators and students in the State of South Carolina with nationwide access through the World Wide Web.
Project Methods
In the case of specific aim one, we will develop a unigene set of peach ESTs 4,000-6,000. by sequencing 30,000 or more peach cDNA library inserts. We will focus our efforts sequencing efforts on three main libraries to begin with. These are the existing developing fruit library, almond seed library, and a newly, to be developed, shoot library using the Lambda Unizap II cDNA cloning vectors. We will make several other libraries of peach tissues (root, leaf, meristem) as proposed, which will be sources of ESTs for future continuing efforts. In specific aim two, we will map 3,000 peach ESTs and 1,000 tomato ESTs from the conserved ortholog set on the developing peach physical map by hybridizing labeled inserts of each EST on our library filters. Clones hybridizing to each EST will be fingerprinted and contigs containing the ESTs integrated into the developing physical map. To provide further anchor points for integration of genetic and physical maps, we will also map a subset of these ESTs on the Prunus general map using RFLP procedures . In specific aim 3, we are developing an object-oriented database. Specifically, the database manages a set of phenotypic and molecular markers with associated genotypes for peaches. In a particular data set, phenotype and genotype data for F2 family trees are recorded, along with the researcher that conducted the experiments, the testing technique used and the purpose of the study. A WWW interface allows researchers to query this data and add new data to the repository. Users can access these markers to create linkage maps by exporting data to MapMaker (Daley et al. 1993), a popular service available to genome scientists. This database is being expanded to accomodate BAC physical mapping data with integration of genetic marker and EST datasets . In addition the consortium intends to compile all the Prunus data in other laboratories into this database. Most members of the worldwide Prunus research community have been contacted and have agreed to the idea of a centralized database. A. Abbott has established a meeting in Narbonne France, June 2000 specifically to organize the integration of data from European laboratories into the growing peach physical database and to coordinate physical mapping research efforts. In specific aim 4, we will in house offer workshops to high school teachers and extension personnel on genomics in peach and develop in cooperation with South Carolina Educational Television a virtual genomics tour highlighting peach improvement as a local crop benefiting from this technology. This workshop manual will be compiled and made available to other institutions and general public through the Prunus Genomics web site and the virtual tour will be publicly available through the SCETV website and on CD rom.

Progress 09/15/01 to 03/31/05

Outputs
We initiated a peach EST project with the goal of developing the unigene set for peach. Our efforts are centered on sequencing 30-40,000 cDNAs from libraries of developing fruit, shoot and seed. Our expectations were that these would resolve into 3-4,000 unigenes, however, we obtained this number from the first 15,000 sequences finished. The data summary for the completed analysis of 23,000 cDNAs from developing peach fruit , shoot and almond seed libraries is available at the CUGI website www.genome.clemson.edu. We are mapping peach ESTs on the developing physical/genetic map of peach and have determined that a significant portion of ESTs (13%), hybridized on our BAC libraries, were placed directly on genetically mapped anchored contigs in the physical map. From the current 15,000 sequences, a peach/almond unigene set has been initiated. Two hundred and seventy eight ESTs (13%) have been localized in 86 locations (involving 80 core markers) on the general Prunus genetic map by common hybridization with RFLP markers to BACs in the Nemared library. From our initial fruit unigene set, we have completed hybridizing in excess of 4000 peach ESTs onto the Nemared BAC library. We have finished the analysis of 2035 ESTs and are currently processing the data from the remainder. From this processed set, 278 ESTs have been directly located on the Prunus general genome map through common hybridization of mapped molecular markers and ESTs. BACs have been identified in the Nemared library for all but ~15% of these ESTs. Initial hybridizations of ~ 100 ESTs from these remaining orphan ESTs on the haploid Lovell BAC library have been 60% successful. Thus, we feel confident that upon completion of the physical map, virtually all EST locations will be identified. We are also mapping resistance gene analogues (RGAs) and resistance associated genes (RAGs). We have completed hybridizing over 80 different RGA/RAG genes and are currently positioning these on the physical map. We have already positioned one RAG in a contig that maps to a putative resistance gene location for Plum Pox Virus , demonstrating the potential for the transcript map of peach to provide candidate genes. For outreach, we have offered a two week summer workshop in 2002 and 2003 for highschool teachers through the SC Life Program. These workshops entitled Modern Genetics and Biotechnology for Teachers have provided up to date information on genomics and genetics utilizing the peach as a model local crop. In the laboratory of the workshop, teachers have extracted peach DNAs, run molecular marker analyses of their samples, sequenced peach ESTs and databased sequences. All lectures and associated genomics information is provided to the teachers both as handouts and powerpoint presentations for their future use in the classroom. We have also run an online web based course for Genetics and Genomics in the spring of 2003 as an initial effort to incorporate on-line educational approaches for outreach.

Impacts
The structural and functional genomics databases of peach serve as substrates for microsynteny analysis of regions of interest and for gene cloning investigations. With the integration of sequenced cDNA loci (EST loci), the physical map database immediately provides candidate genes located in the genetically marked intervals containing traits of interest. Having these associations has the potential to greatly speed the process of gene discovery. Rapid advances in molecular genetics and biotechnology of foods and other agricultural products require complementary public education that is accurate, easily understandable and non-biased. Students, teachers, and the general public cannot make informed decisions when their only education is through the popular press or misconceptions derived from casual observations of agricultural use of biotechnology at the rural/urban interface. Clemson University (CU) and the South Carolina DNA Learning Center (SCDNALC) are uniquely situated to provide this education, in part because CU is a land-grant institution with a long and robust history of molecular genetics, agricultural biotechnology, and K-12 Science education outreach. Utilization of the peach (a crop of local importance)as an educational tool underscores the importance of this research in the lives of the South Carolina public.

Publications

  • Horn R., Lecouls A.C., Callahan A., Dandekar A., Garay L., McCord P., Howad W., Chan H., Verde I., Main D., Jung S., Georgi L., Forrest S., Mook J., Zhebentyayeva T., Yeisoo Y., Hye Ran K., Jesudurai C., Sosinski B., Arus P., Baird V., Parfitt D., Reighard G., Scorza R., Tomkins J., Wing R., Abbott A.G. (2005) Candidate gene database and transcript map for peach, a model species for fruit trees. Theoret. Appl. Genet. in press
  • Sook Jung, Christopher Jesudurai, Margaret Staton, Zhidian Du, Stephen Ficklin, Ilhyung Cho, Albert Abbott, Jeffrey Tomkins and Dorrie Main, Sept. 9, 2004. GDR Genome Database for Rosaceae): integrated web resources for Rosaceae genomics and genetics research BMC Bioinformatics 2004, 5:130


Progress 01/01/03 to 12/31/03

Outputs
We have initiated a peach EST project with the central goal of developing the unigene set for peach. Our current efforts are centered on sequencing 30-40,000 cDNAs from libraries of developing fruit, shoot and seed. Our original expectations were that these would resolve into 3-4,000 unigenes, however, we have obtained this number from the first 15,000 sequences finished. The data summary for the completed analysis of 15,000 cDNAs from developing peach fruit and almond seed libraries is available at the CUGI website www.genome.clemson.edu. We mapped peach ESTs on the developing physical/genetic map of peach and have determined that a significant portion of ESTs (11%) hybridized on our BAC libraries are placed directly on genetically mapped anchored contigs in the physical map. From the current 15,000 sequences, a peach/almond unigene set has been initiated. This currently annotated unigene set consists of 3,842 putative unique genes. We are currently sequencing characterizing an additional 8,000 ESTs from developing peach shoots and have available a root cDNA library for root EST sequencing. From our initial fruit unigene set, we have completed hybridizing in excess of 1,700 ESTs onto the Nemared BAC library. From this set, 184 ESTs have been directly located on the Prunus general genome map through common hybridization of mapped molecular markers and ESTs. BACs have been identified in the Nemared library for all but 15% of these ESTs. Initial hybridizations of 100 ESTs from these remaining orphan ESTs on the haploid Lovell BAC library have been 60% successful. Thus, we feel confident that upon completion of the physical map, virtually all EST locations will be identified. All the functional genomics resources are incorporated into the Prunus genomics website currently under construction at Clemson, www. genome.clemson.edu/gdr/. This website will will be the repository for worldwide Rosaceae genomics data and will house the Prunus genomics dataset as well as date of other important Rosaceae species. We have offered a two week summer workshop in 2002 and 2003 for highschool teachers through the SC Life Program. These workshops entitled Modern Genetics and Biotechnology for Teachers have provided up to date information on genomics and genetics utilizing the peach as a model local crop. In the laboratory of the workshop, teachers have extracted peach DNAs, run molecular marker analyses of their samples, sequenced peach ESTs and databased sequences. These hands on experiences are crucial for dispelling the mystique of this research and ground our educators with a real understanding of the technologies and their benefits in local economically important crops. All lectures and associated genomics information is provided to the teachers both as handouts and powerpoint presentations for their future use in the classroom.

Impacts
The structural and functional genomics databases of peach serve as substrates for microsynteny analysis of regions of interest and for gene cloning investigations. With the integration of sequenced cDNA loci (EST loci), the physical map database immediately provides candidate genes located in the genetically marked intervals containing traits of interest. Having these associations has the potential to greatly speed the process of gene discovery. Rapid advances in molecular genetics and biotechnology of foods and other agricultural products require complementary public education that is accurate, easily understandable and non-biased. Students, teachers, and the general public cannot make informed decisions when their only education is through the popular press or misconceptions derived from casual observations of agricultural use of biotechnology at the rural/urban interface. Clemson University (CU) and the South Carolina DNA Learning Center (SCDNALC) are uniquely situated to provide this education, in part because CU is a land-grant institution with a long and robust history of molecular genetics, agricultural biotechnology, and K-12 Science education outreach. Utilization of the peach (a crop of local importance)as an educational tool underscores the importance of this research in the lives of the South Carolina public.

Publications

  • Horn R., Lecouls A.C., Callahan A., Dandekar A., Garay L., McCord P., Howad W., Chan H., Verde I., Main D.,Jung S., Georgi L., Forrest S., Mook J., Zhebentyayeva T., Yeisoo Y., Hye Ran K., Jesudurai C., Sosinski B.,Arus P., Baird V., Parfitt D., Reighard G., Scorza R., Tomkins J., Wing R., Abbott A.G. (2004) Candidate gene database and transcript map for peach, a model species for fruit trees. Genome Res. (submitted)
  • Horn R., Zhebentyayeva T., Mook J., Swire-Clark G., Garay L., McCord P., Howad W., Chan H., Jung S.,Abbott A. (2004) Development of a physical and transcript map for peach: a model tree species for Rosaceae. Plant, Animal and Microbe Genome Conference XII, 10.-14. January 2004, San Diego, USA,(http://www.intl-pag.org/12/abstracts/W23_PAG12_104.html
  • Horn R., Lecouls A.C., Main D., Callahan A., Dandekar A., Wing R., Sosinski B., Arus P., Parfitt D., Abbott A. (2004) Candidate gene database and transcript map for peach: a model genome species for Rosaceae. Plant, Animal and Microbe Genome Conference XII, 10.-14. January 2004, San Diego, USA.(http://www.intl-pag.org/12/abstracts/P5h_PAG12_590.html )
  • Sook Jung, Albert Abbott, Christopher Jesudurai, Jeff Tomkins and Dorrie Main. 2003. Fequency, Type, Localization and Annotation of SSRs in Rosaceae ESTs. Genome Resources (Submitted).


Progress 01/01/02 to 12/31/02

Outputs
We have initiated a peach EST project with the goal of developing an extensive peach EST database for identification and cloning of genes important to tree development. Our current efforts are centered on sequencing 30-40,000 cDNAs from libraries of developing fruit, shoot and seed. The data summary for the completed analysis of the first 16,000 cDNAs from developing fruit peach and almond seed libraries are available at the CUGI website (http://www.genome.clemson.edu/projects/peach) and the Arizona genome website (http://www.genome.arizona.edu). So far, we sequenced and analyzed more than 16,000 cDNAs (13,331 for peach and 3451 for almond). The ESTs were assembled into 1309 contigs and 3500 singletons for peach and 145 contigs and 788 singletons for almond. From the current 16,000 sequences, a peach/almond unigene set has been initiated. This annotated unigene set consists of 4,576 putative unique genes. For a random subset of 175 sequences the distribution of the EST sequences according to their function was analyzed. Using BLAST, 62% of the sequences showed a significant match to known sequences, 12% matched to unknown sequences and 22% had no homology in the Genbank including the EST libraries. These sequences are of special interest as they are unique to peach as a model for fruit tree species. The sequences with matches represented genes involved in biosynthesis/degradation (26%), ripening/stress (9%), allergens (3%), regulatory proteins (12%), structural RNAs (2%), ribosomal proteins (3%) and miscellaneous (9%). This distribution according to the putative function fits very very well to the expectations of functions needed in developing peach fruit. We have successfully mapped 381 peach and almond ESTs on the developing physical/genetic map of peach and have determined that 14% of the ESTs (53) hybridized are placed directly on the genetically anchored contigs in the physical map. On average 3.5 BACs hybridized to an EST probe, resulting in a total number of 1368 BACs that we detected by the EST probes. In addition, 224 RFLP, AFLP and SSR markers from peach as well as RFLP markers from almond, Prunus ferganensis and cherry and ESTs from tomato were mapped 1136 BACs. So far, a total of 605 probes detected 2504 BACs (average 4.0 BACs/ probe) At the moment, we are beginning analysis of peach shoot and root EST libraries, as well as developing libraries of specialized additional tissues such as flower buds, graft zones and almond seed pellicle. We have also offered our first outreach summer education course for high school teachers and extension agents. We had a complete program of two weeks of classroom and laboratory experience for 12 teachers in the state. The course was well received and we have utilized its structure to design an implement a distance education course through the web which is currently being offered this spring. We will offer another course this summer and intend to continue this as part of the developing DNA learning center at Clemson . We have initiated construction of a virtual field trip through the genomics sciences in cooperation with the South Carolina ETV network. This will be showcased on the SCETV website.

Impacts
The generation and mapping of EST sequences provides an invaluable resource for gene identification and characterization in all of the economically important Rosaceas species. If the genomic positions of EST sequences and qualitative or quantitative trait loci were available in a single core map, it would be possible to readily identify candidate genes of interest. A genetically mapped EST database would provide framework, mapping probes for use in other Rosaceae species mapping efforts currently underway, as well as, provide the EST sequences for functional genomics studies. The second objective is the development and implementation of an educational outreach program for educators and students in the State of South Carolina. Public understanding of biotechnology is a necessary factor in the public making wise decisions about the use of this new technology. Because this area is so new, most high school agricultural ecucation and biology teachers, as well, as, county extension agents, have received little formal education about biotechnology techniques and issues. Training for highschool teachers in Genomics and its modern applications is the first and key step in developing public understanding. The use of peach as a regional example will enhance our educational program by grounding its principles in a crop whose importance has significant impact on the lives of people in the southeastern US.

Publications

  • Abbott, A., L. Georgi, D. Yvergniaux, M. Inigo, B. Sosinski, Y. Wang, A. Blenda, and G. Reighard. 2002. Peach: The model genome for Rosaceae. Acta Horticulturae 575: 145-155.
  • Georgi, L. L., Y. Wang, D. Yvergniaux, T. Ormsbee, M. Inigo, G. L. Reighard, and A. G. Abbott. 2002. Construction of a BAC library and its application to the identification of simple sequence repeats in peach (Prunus persica [L.] Batsch). Theoretical and Applied Genetics 105: 1151-1158.
  • Hurtado, M.A., C. Romero, S. Vilanova, A.G. Abbott, G. Llacer, ML. Badenes. 2002. Genetic linkage maps of two apricot cultivars (Prunus armeniaca L.) and mapping of PPV (sharka) resistance. Theoretical and Applied Genetics 105: 182-191
  • Salava, J, Y. Wang, B. Krska, J. Polak, P. Kominek, W. Miller, W. Dowler, GL. Reighard, A. G. Abbott. 2002. Identification of molecular markers linked to resistance of apricot (Prunus armeniaca L.) to plum pox virus. Zeitschrift fur Pflanzenkrankheiten und Pflanzenschutz-Journal of Plant Diseases and Protection, 109 (1): 64-67
  • Wang, Y., L. Georgi, G. L. Reighard, R. Scorza, and A. G. Abbott. Genetic mapping of the evergrowing gene in peach [Prunus persica (L.) Batsch]. 2002.Journal of Heredity 93: 352-358.
  • Wang,Y., L. L. Georgi, T. N. Zhebentyayeva, G. L. Reighard, R. Scorza and A. G. Abbott. 2002. High throughput targeted SSR marker development in peach [Prunus persica (L.) Batsch]. Genome 45: 319-328.


Progress 01/01/01 to 12/31/01

Outputs
We have initiated this project as proposed and have completed the initial development and arraying of two of the projected three cDNA libraries. These are the peach developing fruit library and the almond seed library. The third library(peach shoot) is currently being constructed. These libraries have been submitted to the Clemson University Genomics Institute for sequencing which is currently underway. In addition we have obtained 500 COS tomato EST clones from S. Tanksley's group and these are being hybridized on the peach BAC library for assignment onto the developing peach physical map. We have also started hybridizing peach ESTs on the BAC library. Once the EST sequencing is complete, a unigene set will be obtained and this unigene set will be distributed to the physical mapping partners for mapping. Development of a Prunus genomics website has begun. In the outreach and extension program, we have developed a summer post graduate workshop in Genetics and Genomics for high school teachers. This has been advertised and 10 teachers selected. This two week workshop is scheduled for July 15-25. In addition, we have begun the development of a virtual tour field trip in Genomics that will be showcased with the SCETV website in association with the SC Life program.

Impacts
The generation and mapping of EST sequences provides an invaluable resource for gene identification and characterization in all of the economically important Rosaceae species. If the genomic positions of EST sequences and qualitative or quantitative trait loci (QTLs) were available in a single core map, it would be possible to readily identify candidate genes that could be immediately tested to determine whether that gene (EST) controls/ contributes to the phenotypic trait of interest. A genetically mapped EST database would provide framework, mapping probes for use in other Rosaceae species mapping efforts currently underway as well as provide the EST sequences for functional genomics studies (i.e., microarray technology). The second objective is the development and implementation of an educational outreach program for educators and students in the State of South Carolina. Public understanding of biotechnology is a necessary factor in the public making wise decisions about the use of this new technology. Because this area is so new, most high school agricultural education and biology teachers, as well as, county extension agents, have received little formal education while in college about biotechnology techniques and issues. Training for highschool teachers in Genomics and its modern applications is the first and key step in developing public understanding. The use of peach as a regional example will enhance our educational program, by grounding its principles in a crop whose importance has significant impact on the lives of people in the southeastern US.

Publications

  • No publications reported this period