Source: CLEMSON UNIVERSITY submitted to NRP
CHROMOSOME MAPPING IN PEACH AND ITS APPLICATION TO FRUIT QUALITY MAINTENANCE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
NRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0189842
Grant No.
2001-35301-11033
Cumulative Award Amt.
(N/A)
Proposal No.
2001-01884
Multistate No.
(N/A)
Project Start Date
Sep 1, 2001
Project End Date
Aug 31, 2004
Grant Year
2001
Program Code
[(N/A)]- (N/A)
Recipient Organization
CLEMSON UNIVERSITY
(N/A)
CLEMSON,SC 29634
Performing Department
GENETICS, BIOCHEMISTRY & LIFE SCIENCE STUDIES
Non Technical Summary
The goal of our research is to develop peach as a model genetic resource for the identification, characterization and cloning of important genes of Rosaceae species. Toward this goal, we developed saturated, molecular marker linkage maps in several peach crosses segregating for both simple and complex traits. Fruit quality is the single most important character to the industry, therefore, our past research efforts have emphasized development of a map database for important fruit quality traits and flower bud cold hardiness. The mapped molecular markers will be used to tag and eventually isolate genes associated with important traits controlling fruit development and bud hardiness. For the purpose of gene discovery in Rosaceae, it is imperative to have a physical database for a model Rosaceae species. A physical map serves as an ideal tool to identify cloned genomic regions containing important genes, facilitating the process of gene marking and gene discovery. To develop such a genomics resource database in Rosaceae, we propose: 1. To construct a BAC physical map of peach to serve as a genome database and source of cloned material for genome comparison and gene discovery studies in Rosaceae. 2. To integrate the marker database of the general Prunus genetic maps onto the peach physical map, utilizing markers tagging important gene-containing regions in our peach maps and maps of other Prunus species. Integration of the Rosaceae genetic map resources and the peach physical map will speed the process of gene discovery in this important plant family.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20111141080100%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1114 - Peach;

Field Of Science
1080 - Genetics;
Goals / Objectives
The goal of our research is to develop peach as a model genetic resource for the identification, characterization and cloning of important genes of Rosaceae species. Toward this goal, we developed saturated, molecular marker linkage maps in several peach crosses segregating for both simple and complex traits. As fruit quality is the single most important character to the industry, we have focused a great deal of effort on developing a map database for important fruit quality traits and flower bud cold hardiness. The mapped molecular markers will be used to tag and eventually isolate genes associated with important QTL controlling fruit development and bud hardiness. This map resource will facilitate the maintenance of fruit quality traits in breeding lines where other important characters, such as disease and pest resistance or tree architecture, are being introgressed. In cooperation with colleagues around the world, we are utilizing our peach genetic resources to identify and characterize conserved, agronomically important genomic regions in other species of the Rosaceae. Currently we are addressing questions of whether there is structural and functional conservation of gene order among the genomes of peach and other family members, for loci homologous to our mapped fruit quality traits. For the purpose of addressing these questions and facilitating the process of comparative genomics and gene discovery in Rosaceae, it is imperative to have a physical database for a model Rosaceae species. A physical map serves as an ideal tool to compare maps of different species and to identify cloned genomic regions containing important gene loci thus facilitating the process of gene marking and gene discovery in related species. For this reason, we propose to build a physical map database for peach and to integrate into this physical map important gene-containing regions from other Rosaceae species. Specific Aims: 1. Construction of a BAC physical map of peach to serve as a genome database and source of cloned material for genome comparison and gene discovery studies in Rosaceae. 2. Integrate the marker database of other Prunus species onto the peach consensus map resource via our peach physical map, utilizing markers tagging important gene-containing regions in our peach maps and maps of other Prunus species.
Project Methods
Specific aim 1: We have produced a BAC library in the vector pBeloBAC11 utilizing DNA from the root stock cultivar `Nemared'. This library has served as a clone resource to begin building a BAC physical contig map of peach using mapped molecular markers from maps worldwide. For BAC fingerprinting physical mapping approaches, we are also constructing at least one new library utilizing a haploid peach variety and two enzymes and Sau 3A1. BAC library construction: Our original BAC library was produced by partial digestion of high molecular weight DNA between 150kb-500kb using the six base cutters Hind III and subsequent ligation into pBeloBAC11. High molecular peach DNA was isolated by methods outlined in ten Lohuis et al. 1993. Partially digested DNA is gel purified by standard agarose gel electroelution and fragments ligated into pBeloBAC11. These are introduced into E. coli cells by electroporation and clones are robotically arrayed into 384 well plates and stored at -80. Clone libraries are robotically macroarrayed on filters (Hybond N+) for hybridization screening. For the additional BAC library of the haploid peach variety, we are using standard protocols currently employed by the CUGI (for details, see http://www.genome.clemson.edu). b. BAC fingerprinting of peach BAC clones: BAC DNA will be prepared and fingerprinted using BAC fingerprinting technologies. Digested DNA samples are electrophoresed visualized by either fluorescence or labeling. Gel images are recorded and uploaded to, and archived in, a Sun Ultra 30 workstation. Gel images are imported into the IMAGE program (Sanger Center) for sizing of each band with respect to a molecular weight standard (1 to 32 kilobases) run every 5th lane. The resulting band sizes associated with each clone name are imported into the fingerprint analysis program FPC (C. Soderlund, Sanger Center) for physical map assembly. Specific Aim 2: We have available approximately 1,000 markers from various Prunus maps. We intend to map most of these markers on the physical map. Initial hybridizations of a small sample (20 RFLPs) of these markers on our first BAC library have revealed the expected average number of overlapping clones. Methods: We will employ standard probe labeling and hybridization conditions as we have used for all our RFLP studies (Rajapakse et al. 1995). BAC filters will be prepared of peach BAC tiling paths by standard methods and procedures utilized in the Clemson University Genomics Institute available at (http://www.genome.clemson.edu). AFLP marker probes are obtained by: excision of the AFLP band from the gel, reamplification of the fragment, and labeling of the amplified fragments by primer extension methods. These AFLP probes are hybridized on our BAC filters to detect the appropriate corresponding BACs. Microsatellite markers are physically mapped, by using BAC clone pools as DNA samples for PCR utilizing the SSR primers. Alternatively, since these markers are sequence generated, overgo probes that exclude the repeat region can be developed from the regions adjacent to the microsatellite locus.

Progress 09/01/01 to 08/31/04

Outputs
We are constructing a peach physical map utilizing these current BAC library resources. We are employing essentially the strategies utilized to develop the Drosophila physical map and others. The approach utilizes a combination of hybridization of mapped markers and BAC fingerprinting and in our case hybridization of EST sequences as well. With the current Prunus molecular marker map resources, we have completed hybridizing 210 low-copy mapped RFLP markers, 4,000 peach fruit ESTs, resistance gene analogs, and other cDNAs and specific AFLP markers. We completed BAC fingerprinting approximately 25,000 BACs (20,000 from the ?Nemared? library and 5,000 from the haploid ?Lovell? library from which approximately 15,000 have been used to construct an initial physical map. Although the average insert size of the physical mapping clones from both libraries is perceived to be somewhat small, ~60 kb for one library, ~85 kb for the other, in fact physical mapping with these libraries has proceeded satisfactorily. We have utilized FPC (V4.7) to construct and initial physical map of the peach genome. Initially, we construct the map at a cutoff of e -10to e-12 and tolerance 5 to obtain all high confidence contigs. These are merged by testing end clones at cutoff values ranging from e-8- e-11. As we have a significant amount of hybridization data, we can in many cases establish merges based on common hybridization of BACs in different contigs. In other cases where we have no other data but the fingerprint data, we make note of the merge points for further testing. At this juncture, the framework map developed from the smaller insert 'Nemared' library is composed of 1135 contigs containing approximately 9000 clones. Estimations from FPC evaluation of the data suggest that the coverage of the peach genome is 64%. We are currently analyzing an additional 5000 fingerprints from this library that were detected by 4000 EST and 600 genetic marker hybridizations and commencing random fingerprinting of the second library (constructed with the 4 base cutter Sau 3A1) which has inserts of ~85kb. Interestingly, clones detected by EST hybridizations seem on inspection to comprise many of the larger insert clones as judged by the number and range of fragment sizes in the fingerprints and thus, contigs covering the gene space of peach are the most highly supported and the largest. We currently have 780 contigs that contain at least one EST. Of course, we are fortunate that the peach genome is small in comparison to many other plant genomes currently being physically mapped and this certainly works in our favor. As we have included marker hybridization data from the general Prunus genetic map, the developing physical map is anchored to the genetic map. From initial analysis of the integrated genetic/physical map, we already have evidence for duplication of some regions of the peach genome. The developing physical map is housed in the Prunus genome website within the Genome Database for Rosaceae (GDR) www.genome.clemson.edu/gdr/.

Impacts
This physical map is already being utilized by researchers to identify and characterize genes important to fruit tree growth, development and sustainability. Genes involved with dormancy, disease resistance, and fruit quality have already been identified and are current targets for study. This map has also great utility for comparative genomics not just in fruit trees but with other important deciduous trees such as poplar and we are currently incorporating research groups on these species into our program.

Publications

  • T. Zhebentyayeva1 , R. Horn1 , J. Mook1 , A. Lecouls1 , L. Georgi1 , G. Swire-Clark2 , W.V. Baird2, G. Reighard2 and A. Abbott1. 2005. A physical framework of the peach genome. Acta Horticulturae, 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
  • Bielenberg DG, Wang Y, Fan S, Reighard GL, Scorza R, and Abbott AG. 2004. A deletion affecting several gene candidates is present in the peach Evergrowing mutant. Journal of Heredity, :95(5): 436-444
  • Horn R., Lecouls A.-C., Callahan A., Dandekar A., Garay L., McCord P., Howad W., Chan H.,Verde I., Ramaswamy K., Main D., Jung S., Georgi L., Forrest S., Mook J., Zhebentyayeva T.N., Yu Y., Kim H.R., Jesudurai C., Sosinski B.A., 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. Theor and Appl. Genet in press


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

Outputs
Specific aim 1: Construct BAC physical map of peach. Specific Aim 2: Integrate the marker database of the general Prunus genetic maps onto the peach physical map, utilizing markers tagging important gene-containing regions in our peach maps and maps of other Prunus species. We are constructing a peach physical map utilizing these current BAC library resources. We are employing essentially the strategies utilized to develop the Drosophila physical map and others. The approach utilizes a combination of hybridization of mapped markers and BAC fingerprinting and in our case hybridization of EST sequences as well. With the current Prunus molecular marker map resources, we have completed hybridizing 210 low-copy mapped RFLP markers, 1,700 peach fruit ESTs, Resistance gene analogs, and other cDNAs and specific AFLP markers. We completed BAC fingerprinting approximately 20,000 BACs (15,000 from the Nemared library and 5,000 from the haploid Lovell library from which approximately 15,000 have been used to construct an initial physical map. We have utilized FPC (V4.7) to construct and initial physical map of the peach genome. We are utilizing essentially the same strategy of others to construct the map. Initially, we construct the map at a cutoff of e -10to e-12 and tolerance 5 to obtain all high confidence contigs. These are then merged by testing end clones at a cutoff values ranging e-8- e-11. As we have a significant amount of hybridization data we can in many cases establish merges based on common hybridization of BACs in different contigs. In other cases where we have no other data but the fingerprint data, we make note of the merge points for further testing. At this juncture the framework map is composed of 1000 contigs containing approximately 8000 clones. Our estimates based on an average insert size of 60 kb, and average 60% degree of overlap in contigs, we estimate that we have covered 80% or better of the peach genome in high confidence contigs. We are currently seeding in singletons (for which we have approximately 7,000 remaining) and merging contigs at lower cutoff scores to fill in as much of the map as we can from our initial efforts. Preliminary estimates from trial merges of contigs suggests that we will end up with approximately 800-900 contigs with an average of 12 clones/contig upon completion of this initial map. As we have included marker hybridization data from the general Prunus genetic map, the developing physical map is anchored to the genetic map. From initial analysis of the integrated genetic/physical map, we already have evidence for duplication of some regions of the peach genome. The developing physical map is housed in the Prunus genome website within the Genome Database for Rosaceae (GDR) www.genome.clemson.edu/gdr/.

Impacts
This physical map is already being utilized by researchers to identify and characterize genes important to fruit tree growth, development and sustainability. Genes involved with dormancy, disease resistance, and fruit quality have already been identified and are current targets for study. This map has also great utility for comparative genomics not just in fruit trees but with other important deciduous trees such as poplar and we are currently incorporating research groups on these species into our program.

Publications

  • Zhebentyayeva T., Horn R., Mook J., Lecouls A.C., Georgi L., Swire-Clark G., Baird V., Reighard G., Abbott A. (2004) Towards an integrated physical/ genetic map of peach genome: a model Rosaceae species. Plant, Animal and Microbe Genome Conference XII, 10.-14. January 2004, San Diego, USA, (http://www.intl-pag.org/12/abstracts/P5h_PAG12_591.html)
  • Zhebentyayeva T.N., Horn R., Mook J., Lecouls A.-C., Georgi L., Swire-Clark G., Baird V., Reighard G., Abbott A. Development of an integrated physical/genetic map for peach. Southeastern Professional Fruit Worker?s Conference. October 14-16, 2003. Clemson University, Clemson, SC, USA
  • Abbott A.G., Ballard R.E., Callahan A., Sosinski B.R., Arus P., Wing R., Reighard G.L., Baird W.V., Parfitt D.E., Dandekar A. (2003) Structural and functional genomics in peach: a plant genome model. Plant, Animal and Microbe Genome Conference XI, 11.-15. January 2003, San Diego, USA, (http://www.intl-pag.org/11/abstracts/W23_W166_XI.html)
  • Zhebentyayeva T., Lecouls A.C., Jung S., Mook J., Georgi L., Horn R., Swire-Clark G., Baird V., Reighard G., Abbott A. (2003) Development of an integrated physical/genetic map for peach: a model genome species. Plant, Animal and Microbe Genome Conference XI, 11.-15. January 2003, San Diego, USA, (http://www.intl-pag.org/11/abstracts/P5h_P544_XI.html )
  • 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
For the development of the genetically anchored physical map, we used a combination of hybridization of mapped markers and BAC (Bacterial Artificial Chromosome) fingerprinting. Two BAC libraries are available. One is based on a HindIII partial digest of the diploid peach variety Nemared (2n=16) and has 44,160 BACs with an average insert size of 50-70kb giving a 8.8 fold genome coverage. The second one was developed from a Sau3AI partial digest of a haploid peach variety of Lovell (Plov 2-1N) with 34,560 BACs with an average insert size of 80kb and a 9.2 fold genome coverage. We have completed hybridizing low-copy RFLP (Restriction Fragment Length Polymorphism) markers and specific AFLP (Amplified Fragment Length Polymorphism) markers to the first BAC library. The development of an EST (Expressed Sequence Tag) candidate gene database will provide additional resources to increase the marker density of the general Prunus map. In addition to the AFLP and RFLP markers used for anchoring the physical map, EST sequences will be mapped on the physical map. The EST sequences used as probes represent 3,842 distinct peach genes. More than 16,000 ESTs from peach fruit mesocarp tissue have been sequenced and annotated. BAC fingerprinting was performed using two restriction enzymes (HindIII and HaeIII) to digest the BAC DNA. Scored fragments ranged in size from 153 to 2235bp based on a Lambda-Sau3AI digested marker. Scanned images of the gel autoradiographs were submitted to the Image program to produce normalized band files for the FPC (fingerprinted contigs) program. FPC analyzed these files and developed contigs under highly stringent conditions (Tolerance = 7; Cutoff = 1e-14) based on clones with high similarity. The preliminary framework map is composed of 58 contigs developed from successful fingerprints of 660 BAC clones from the Nemared library that hybridized to 143 anchored markers of the genetic map. Many of these contigs contained clones with identical markers or markers within a few centiMorgans of each other on the physical map. In addition, these contigs can be used as a foundation to construct further contigs from random BAC clones or as further verification of contig integrity. More than 11,500 random BAC clones, and an additional 820 BAC clones that hybridized to 381 EST sequences that were not included in the random set of BAC clones, have been fingerprinted from the Nemared BAC library. About 6,900 BAC clones from the Lovell BAC library have also been fingerprinted to assist with final contig assembly. Clones from the second library are being used to obtain a better coverage of the genome and will provide potentially larger overlapping regions due to differences in library construction. Processing of the fingerprints is progressing rapidly at this time, with about 25 percent of all fingerprints being completely submitted to the FPC software for contig analysis. Once the final contigs have been developed, we will provide public access to this information on the developing Prunus genomics website (http://www.genome.clemson.edu/projects/peach).

Impacts
The centerpiece of this work is the development of a high-density physical map in peach. It is proposed that peach would serve as the "rice of the Rosaceae" and provide a highly characterized "core" genome for the subsequent comparative mapping and gene discovery. Additionally, a physical map database would be invaluable to the Rosaceae research community to accelerate ongoing research efforts in linkage mapping, candidate gene/QTL associations, and functional genomics. The long range goal of this effort would be development of genetically improved Rosaceous fruit, ornamental and forest varieties to enhance the sustainability of U.S. argiculture.

Publications

  • 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.
  • 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.


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

Outputs
We have started the construction of the peach physical map using the BAC library developed in our laboratory of DNA from the variety Nemared. Currently, more than 200 RFLP markers located on several Prunus species genetic maps, including peach, almond, cherry, Prunus ferganensis and the general Prunus core map 'Texas' x 'Earlygold', have been hybridized on this library. A total of 1100 BAC clones (an average of 5 clones per marker) have been identified. This initial genetically anchored partial physical map of the peach genome will be completed by BAC fingerprinting of this library and another constructed from DNA of a haploid accession of the variety Lovell. We have begun the construction of the anchored marker contigs by BAC fingerprinting. This should cover approximately 4% of the peach genome. In addition, two unigene sets of tomato and peach ESTs provided by S. Tanksley and A. Callahan have also been used in order to increase the marker density of the map and to assist the final assembly of the physical contigs.

Impacts
The centerpiece of this work is the development of a high-density physical map in peach [Prunus persica (L.) Batsch It is proposed that peach would serve as the "rice of the Rosaceae" and provide a highly characterized "core" genome for subsequent comparative mapping and gene discovery. Additionally, a physical map database would be invaluable to the Rosaceae research community to accelerate ongoing research efforts in linkage mapping, candidate gene/QTL associations, and functional genomics. The long range goal of this effort would be development of genetically improved Rosaceous fruit, ornamental and forest varieties to enhance the sustainability of U.S. agriculture.

Publications

  • No publications reported this period