Candidate Genes for Fruit Softening in Prunus


NRI Award #2005-35300-15463
PI: Cameron P. Peace, Co-PIs: Ann M. Callahan, Thomas M. Gradziel, and Carlos H. Crisosto
Department of Horticulture and Landscape Architecture
Washington State University, Pullman, WA, 99164-6414
E-mail: cpeace@wsu.edu
Telephone: 509-335-6899


Accomplishments:

The first goal of the project is to elucidate the molecular genetic organization of the complex endopolygalacturonase (endoPG) locus in peach.

  • Re-screening of two Clemson peach BAC libraries specifically for this gene by collaborators (Tatyana Zhebentyayeva and Bert Abbott) at Clemson University identified numerous BACs with significant homology to the endoPG gene. Fingerprinting, subcloning, and sequencing of these BACs is revealing the basic organization of the Freestone-Melting flesh (F-M) locus for two of the four major alleles. The F allele appears to contain two copies of the gene, separated by less than 50-kb and perhaps less than 15-kb, and the f allele contains only one of the gene copies. EndoPG-like gene sequences also were detected upstream of the target endoPG genes.

The second goal is to understand the role of endoPG in Prunus fruit softening.

  • Complete co-segregation was verified on approximately 350 further progeny segregating for the freestone/clingstone and melting/non-melting flesh traits, greatly increasing our confidence that endoPG does indeed control F-M. Several other labs around the world have used the endoPG markers on peach mapping populations to confirm this gene-trait association across several hundred more progeny.
  • Allelic diversity was assessed in Prunus via PCR of a microsatellite in the endoPG gene. Over 200 different alleles and gene copies of endoPG were detected from a survey of approximately 650 accessions of Prunus (peach, almond, apricot, plum, sweet and tart cherry, and many closely related species). This included approximately 150 peach and nectarine varieties, for which the genotypic survey was capable of ascertaining the correct F-M fruit type for many varieties that previously had an unclear phenotype. Sweet cherry was the only species that has not shown abundant polymorphism for endoPG.
  • A subset of accessions was chosen for targeted sequencing. Phylogenetic analysis is underway, but preliminary results suggest that the endoPG gene tandem duplication controlling the F and M traits exists only within subgenus Amygdalus (peach, almond, and closely related species).
  • To better understand the qualitative and quantitative effects of the various alleles and gene copies in peach, fruit softening characteristics were examined in two years for about 70 varieties each year, followed by molecular physiological assays with tissue collected for each variety throughout ripening. These physiological experiments are confirming the role of endoPG in fruit texture determination, the role of each endoPG gene copy, and differences between alleles.
  • Major softening profile differences were observed between the four major endoPG locus alleles. Further distinct softening profiles were observed for many of the endoPG-SSR alleles and allelic combinations.
  • Gene expression assays of the examined varieties are revealing the allele-specific patterns of expression through ripening for the two gene copies at the locus, with differences detected between inner and outer fruit flesh.
  • Assays of endoPG enzyme activity of varieties appear to match PCR genotypes, gene expression levels, and fruit softening phenotypes.
  • Metabolite levels representing breakdown products of endoPG activity appear to be very low and difficult to detect in peach. Nevertheless, detailed oligo structure analysis is underway for representative of various endoPG alleles.

The third major goal of the project is to provide molecular tools to facilitate identification and/or development of new fruit quality phenotypes of important Prunus crops.

  • The endoPG PCR itself is already valuable in determining Freestone-Melting flesh fruit type of peach and nectarine varieties (i.e. predicting whether fruit will be freestone melting flesh, clingstone melting flesh, or clingstone non-melting flesh). Surveys of varieties of these crops have validated the gene-phenotype association. We are still examining the extent to which the gene controls similar traits in other Prunus crops.
  • Markers based on the endoPG locus can also distinguish between two types of non-melting flesh – “rubbery” canning peach types (typical California canning peaches) and firmer freshmarket types with a very long storage potential (typical in Spain and from breeding efforts in Florida).
  • EndoPG genotyping has helped in explaining the unusual texture phenotype of two canning peach breeding lines – one that exhibits an “air-free” pit in warm seasons (apparently associated with expression of the “Freestone” endoPG gene copy that is otherwise usually suppressed in non-melting canning cultivars), and another that exhibits very delayed ripening and softening (apparently not due to any novel endoPG allele).
  • EndoPG genotyping has helped in identifying possible effects of introgression of novel endoPG alleles from peach relatives. A certain peach-almond hybrid breeding line was the only melting flesh variety observed to retain significant firmness after six days of ripening. Another breeding line derived from a cross between peach and its wild relative P. mira, was the fastest-softening variety observed, and displayed a P. mira endoPG allele not observed in other varieties.
  • The project is not just concerned with the endoPG gene, and to discover further genes that may affect fruit softening in Prunus, 40 additional candidate genes for softening were screened via PCR across Prunus accessions. Most genes were polymorphic between species. More than half were polymorphic within at least one of the Prunus crops. About a quarter of the genes tested were also detected in apple, indicating high homology of gene sequence, and potentially function also.
  • The Stony hard trait (controlled by the Hd locus) was specifically investigated using the candidate gene approach based on size polymorphism of amplified gene sections. While we now have a much better understanding of the physiological interaction between Hd and F-M. Certain candidate genes for this trait are under investigation.
  • A “softening gene map” of Prunus was constructed, containing the genetic locations of more than 50 genes putatively involved in fruit softening. The locations of some of these genes coincide with reported QTLs for texture traits in Rosaceae tree fruit
  • Molecular genetic comparisons between peach and apple for endoPG and other softening candidate genes is broadening our understanding of the role of these genes in fruit softening.
Broad Impacts:

The massive genotypic diversity detected for endoPG in Prunus has important implications for fruit evolution and crop domestication in this genus, and represents much potential for genetic improvement of texture attributes in stone fruit. The bin-mapping strategy developed for Prunus at IRTA, Spain, which was used to develop the softening gene map, is now expanding in its use in Rosaceae, perhaps due to the reported success in this project. The project’s activities have allowed involvement in RosPOP, a consortium being established for the sharing of data and physical resources of mapping populations, and RosCAP, a coordinated strategy of the US Rosaceae genomics, genetics, and breeding community with international partners to enable the translation of genomics research into practical application in industry. As an early example of RosPOP, efforts are currently underway to comparatively map the distal end of Prunus linkage group 4 where the endoPG gene and F-M locus reside, involving more than a dozen Prunus mapping populations. An intergeneric partnership to comparatively map softening candidate genes between peach and apple, unexpectedly arising from this NRI project, is hoped to bridge the genomic gap between these important rosaceous crops. PCR tests for the endoPG gene are already being used in numerous peach breeding programs to better understand the fruit type of parents used.

Deliverables:
  • Publications
    • Peace CP, Callahan AM, Ogundiwin EA, Potter D, Gradziel TM, Bliss FA, Crisosto CH (2007). Endopolygalacturonase genotypic variation in Prunus. Acta Hort. 738:639-646
    • Zhebentyayeva TN, Jiwan D, Jun JH, Reighard GL, Lalli DA, Forrest S, Duncan J, Main D, Abbott AG, Callahan A, Scorza R (2007). Exploitation of structural and functional genomics databases for gene identification in peach. Acta Hort. 738:711-717
    • Peace C, Crisosto C (2006). Revealing the genetic control of internal breakdown in peach. Summerfruit Australia Quarterly 8: 20-21
    • Peace C (2006). Long-term approaches to increase peach fruit consumption. Compact Fruit Tree 39:15-17
  • Presentations
      • Oral Presentations
      • CSIRO Plant Industry Staff Seminar, Brisbane, Australia, November 2007. “Molecular genetics of flesh texture in stone fruit”
      • Istituto Agrario San Michele all'Adige, Trento, Italy, September 2007. “Functional genetic markers for characterising and breeding tree fruit”
      • Seminis Vegetable Seeds, Woodland, California, March 2007. “Endopolygalacturonase – a functional genetic marker for fruit softening and other quality traits in peach”
      • Horticulture Seminar Series, Pullman, Washington, February 2007. “Functional genetic markers for characterizing and breeding tree fruit”
      • North West Pear Research Review, Hood River, Oregon, February 2007 “The genomics, genetics, and breeding pipeline: channeling research into new cultivars”
      • Washington Tree Fruit Research Commission Apple Research Review, Wenatchee, Washington, January 2007: “The genomics, genetics, and breeding pipeline: channeling research into new cultivars”
      • Plant & Animal Genome XV Conference, San Diego, California, January 2007. “Candidate genes for fruit softening in Prunus”
      • 3rd International Rosaceae Genomics Conference, Napier, New Zealand, March 2006: “Fruit softening in Prunus: progress and prospects of the candidate gene approach”
      • International Fruit Tree Association 49th Annual Educational Conference, Hershey, Pennsylvania, March 2006. “Long-term solutions to increasing peach fruit consumption”
      • Plant & Animal Genome XIV Conference, San Diego, California, January 2006. “Prunus projects of the USDA CSREES National Research Initiative: synergies and progress”
      • American Society for Horticultural Science Annual International Conference 2005, Las Vegas, Nevada, June 2005. “Candidate genes for fruit softening in Prunus”
  • Posters
    • Plant & Animal Genome XV Conference, San Diego, CA, January 2007. “Deciphering the structure, function, and diversity of the endoPG locus in Prunus”
    • Plant & Animal Genome XV Conference, San Diego, CA, January 2007. “Physical structure of an endopolygalacturonase locus in peach”
    • Plant & Animal Genome XIV Conference, San Diego, California, January 2006. “Candidate gene analysis of Prunus fruit softening: research update”
    • International Rosaceae Genomics Conference, Napier, New Zealand, March 2006. “Endopolygalacturonase marker-assisted selection for novel fruit types in peach”
    • ISHS Symposium on Biotechnology of Temperate Fruit Crops and Tropical Species, Daytona Beach, Florida, October 2005. “Endopolygalacturonase genotypic variation in Prunus”
    • UC Davis Plant Sciences Symposium, Davis, California, September 2005. “Molecular genetics of fruit quality at Kearney Agricultural Center”
  • Meeting Abstracts
    • Callahan AM, Zhebentyayeva TN, Peace CP (2007). Physical structure of an endopolygalacturonase locus in peach. Plant & Animal Genome XV Abstracts P-475
    • Peace CP, Callahan AM, Ogundiwin EA, Potter D, Zhebentyayeva T, Abbott A, Aradhya M, Badenes ML, Ieezoni AF, Gradziel TM, Crisosto CH, Bliss FA (2007). Deciphering the structure, function, and diversity of the endoPG locus in Prunus. Plant & Animal Genome XIV Abstracts P-785
    • Gradziel T, Peace CP, Crisosto C (2006). Endopolygalacturonase marker-assisted selection for novel fruit types in peach. 3rd International Rosaceae Genomics Conference Abstracts J3, p117
    • Peace CP, Ogundiwin EA, Gradziel TM, Potter D, Weeks C, Badenes ML, Iezzoni AF, Bliss FA, and Crisosto CH (2006). Fruit softening in Prunus: progress and prospects of the candidate gene approach. 3rd International Rosaceae Genomics Conference Abstracts OP24, p44
    • Peace CP, Callahan AM, Ogundiwin EA, Potter D, Gradziel TM, Bliss FA, Crisosto CH (2006). Candidate gene analysis of Prunus fruit softening: research update. Plant & Animal Genome XIV Abstracts P-493, p225
    • Peace CP, Abbott AG, Dai W, Iezzoni AF, Arus P, Baird WV, Callahan AM, Crisosto CH, Gradziel TM, Loescher W, Main D, Reighard G, Sosinski B, Tomkins J, van der Knaap E, Walla JA, Wang D (2006). Prunus projects of the USDA CSREES National Research Initiative: synergies and progress. Plant & Animal Genome XIV Abstracts W-131, p37
    • Peace CP, Callahan AM, Ogundiwin EA, Potter D, Gradziel TM, Bliss FA, Crisosto CH (2005). Endopolygalacturonase genotypic variation in Prunus. International Symposium on Biotechnology of Temperate Fruit Crops and Tropical Species Abstracts p114
  • Web sites
    • http://www.bioinfo.wsu.edu/gdr/community/funding/peace.php
    • http://www.ars.usda.gov/research/projects/projects.htm?ACCN_NO=409851
  • Community resources generated (sequences, populations, plant materials, etc)
    • Candidate gene sequences, primers, and assay conditions, and DNA of population parent cultivars have been supplied to researchers requesting them
    • Numerous DNA sequences representing genes and alleles of the endoPG locus will soon be available after quality testing
    • EndoPG genotypes of peach and nectarine cultivars, which describe their fruit texture type, are being prepared for industry distribution. This information will inform breeders, growers, packers, processors, transporters, marketers, and consumers of the genetic predisposition of each cultivar.
  • Training:
    • UC Davis, Kearney Agricultural Center
    • Daniel Edge-Garza (technical, UC Davis) – contributed to the understanding of the functional role of endoPG alleles by locating and obtaining fruit samples, performing softening measurements, supervising summer work crew for the postharvest lab, and tissue preparation and dissemination to collaborators in 2006; contributed to the understanding of endoPG genotypic diversity by locating and obtaining leaf samples for DNA extraction, performing and training others in DNA extraction, PCR, and gel running. Jose Soto (technical, UC Davis) – contributed to the understanding of the diversity and function of PCR alleles by performing softening measurements, obtaining leaf samples for DNA extraction, and performing DNA extraction, PCR, and gel running. Robbie James (technical, UC Davis) – contributed to the understanding of the functional role of endoPG alleles by performing softening measurements, phenotypic data entry and preliminary analysis. Michael Pitchford (technical, UC Davis) – contributed to the understanding of the functional role of endoPG alleles by performing softening measurements. Rebecca Cooper (technical) – contributed to the understanding of the functional role of endoPG alleles by locating and obtaining fruit samples and performing softening measurements. Andrea Gamberini (graduate, visiting PhD student from University of Bologna, Italy) – contributed to the understanding of the endoPG genotypic diversity through candidate gene nomination and primer design, PCR, and gel running. George Manganaris (postdoctoral, UC Davis) – contributed to the understanding of the functional role of endoPG alleles by performing endoPG enzyme activity measurements. Ebenezer Ogundiwin (postdoctoral, UC Davis) – contributed to the understanding of allelic diversity and function of endoPG and other candidate genes, by performing bin-mapping PCR and gels, supervising the molecular lab, and helping with manuscript and poster preparation.

    • UC Davis, Davis campus
    • Eric Wada (graduate, UC Davis) – contributed to the understanding of the endoPG genotypic diversity and phylogeny by training and supervising three undergraduate students. Hoang Yen Nguyen, Paulina Walichiewicz, and Fei Yian Yoong (undergraduate, UC Davis) – contributed to the understanding of the endoPG genotypic diversity and phylogeny by cloning and sequencing of endoPG PCR products, and editing and analysis of sequences. John Sung (undergraduate) and Yuan Kejun (postdoc) - contributed to the understanding of the functional role of endoPG alleles by conducting endoPG enzyme activity measurements and sample purification for oligo analysis.

    • USDA-ARS, Appalachian Fruit Research Station
    • Anthony Rugh (technical, AFRS) – contributed to the understanding of the physical organization of the endoPG locus by BAC mapping, BAC subcloning, and sequencing of peach. Jessica Lauffer (undergraduate, University of Washington) – contributed to the physical organization of the endoPG locus through PCR of several cultivars of peach to determine primers that could distinguish functional alleles. Kenneth Smith (undergraduate, West Virginia University) – contributed to the physical organization of the endoPG locus by BAC mapping of plum.

Collaborations:
  • Interaction between all four PDs: Peace, Crisosto, and Gradziel were already collaborating on other projects, but formal collaboration with Callahan was enabled with this project
  • Project support enabled the originally planned collaborations with Marisa Badenes (IVIA, Spain), Amy Iezzoni (Michigan State University, USA), Dan Potter, David Slaughter, and John Labavitch (UC Davis, USA)
  • Tatyana Zhebentyayeva and Bert Abbott (Clemson University, USA) for peach endoPG locus physical isolation and sequencing
  • Laura Georgi (Clemson University, USA) for endoPG locus physical isolation from plum
  • Mallikarjuna Aradhya, Clay Weeks, and Ed Stover (National Clonal Germplasm Repository at Davis, USA) for endoPG diversity assessment in the Davis Prunus germplasm collection
  • Closer interaction with the California stone fruit industry regarding molecular genetics research
  • Dick Okie (USDA-ARS, Georgia) for endoPG and Hd analysis in additional peach germplasm
  • John Clark (University of Arkansas, USA) for endoPG and other softening candidate gene analysis in additional peach germplasm
  • Jim Olmstead and Matt Whiting (Washington State University, USA) for candidate gene testing of sweet cherry germplasm
  • Werner Howad and Pere Arus (IRTA, Spain) for details and DNA for the bin-mapping of candidate genes
  • Fabrizio Costa (University of Bologna, Italy), Rozemarijn Dreesen (KU Leuven, Belgium), Eric van de Weg (Plant Research International, Netherlands), and Pere Arus (IRTA, Spain) for apple-peach comparative mapping of softening candidate genes
  • Maria Teresa Dettori, Ignazio Verde, and Elisa Vendramin (ISF, Italy) for verification of endoPG co-segregation with F-M in other Prunus populations, elucidation of F-M interaction with Hd, and candidate gene analysis and mapping of Hd
  • Elisabeth Dirlewanger (INRA, France) for verification of endoPG co-segregation with F-M in other Prunus populations and mapping of further candidate genes
  • More than a dozen institutions in the US and Europe for a comparative mapping consortium for the endoPG region in Prunus and apple
  • Takashi Haji (NIFTS, Japan) for elucidation of F-M interaction with Hd, and candidate gene analysis and mapping of Hd
  • Involvement in RosPOP – PD Cameron Peace is the current director of RosPOP, and co- Liaison with Werner Howad (IRTA, Spain) for RosPOP-Prunus
  • Hosted Andrea Gamberini, a PhD student from the University of Bologna, Italy, for screening endoPG and other candidate genes across additional germplasm