Genomic Resources to Improve Fruit Size and Quality Traits in Cherry


PI: Amy Iezzoni, Co-PIs: Wayne Loescher, Dechun Wang, and Esther van der Knaap
Department of Horticulture and Department of Crop and Soil Science
Michigan State University, East Lansing, MI 48864
E-mail: iezzoni@msu.edu
Telephone: 517-355-5191 ext 391


Accomplishments:

The goal of this project is to develop the genomic resources necessary to implement marker-assisted selection for fruit size and quality traits in cherry breeding programs. We are using a linkage map based QTL strategy to be followed by QTL validation and allele mining utilizing software developed to analyze genotypic and phenotypic data from multigenerational pedigrees. An important resource is our sweet cherry pseudo-testcross mapping population that consists of 574 F1 progeny from reciprocal crosses between ‘Emperor Francis’ (EF) and ‘New York 54’ (NY).

  • The success rate for identifying Prunus SSR markers that could be placed on either the EF or NY maps was only 26%. To increase marker density, we developed 19 CAPS, four dCAPS and four InDel markers for cherry based on 101 Prunus ESTs that had been placed on the peach × almond bin map or peach physical map. In addition, four genederived markers representing orthologs of a tomato vacuolar invertase and fruit size gene, and two sour cherry sorbitol transporters were developed.
  • The EF and NY linkage maps constructed were 711.1 cM and 565.8 cM, respectively, with the average distance between markers of 4.94 cM and 6.22 cM. A total of 82 shared markers between the EF and NY maps and the Prunus reference map showed that the majority of the marker orders were the same with the Prunus reference map.
  • Because of our continuing need for more markers not only in cherry, but for comparative mapping within the Rosaceae, we initiated the development of Conserved Ortholgous Set (COS) markers. From a total of 91,000 Prunus ESTs, 4,852 putative COS were preselected after BLAST search against Arabidopsis single copy genes. The assembly of the Prunus COS candidates resulted in 1,758 Prunus COS of which 1,374 corresponded to Arabidopsis COS. These1,374 Prunus-Arabidopsis COS will form the basis of our future gene-based marker development.
  • A cherry pedigree set, consisting of 41 cultivars representing up to four familial generations was genotyped for 58 of the mapped SSR markers, and the marker data was entered into FlexQTL software. From two to eight alleles were identified for each marker locus.
  • The progeny from the mapping population and pedigree set were phenotyped for fruit weight, length, and width, mesocarp cell size and number, and fruit sorbitol, glucose and fructose contents.
  • Two fruit weight QTL on linkage groups (LG) two and six were identified from 2006 and 2007 data utilizing the NY × EF mapping population. The QTL on LG2 explained 30.3% of the phenotypic variance (LOD score = 9.9), while the QTL on LG6 explained 14.9% of the phenotypic variance (LOD score = 9).
  • FlexQTL analysis using the pedigree set also identified fruit size QTL on LG 2 and LG 6, plus additional fruit size QTL on LG 1 and LG 8.
Broad Impacts:
  • The newly developed and genetically mapped markers increase the choice of markers for cherry breeding programs and promote additional comparative mapping efforts in Prunus and other members of the Rosaceae family.
  • The apparent co-linearity of the cherry genome with that of the other diploid Prunus will facilitate the use of comparative mapping for QTL discovery and our ability to successfully utilize the forthcoming peach sequence.
  • Profitable sweet cherry production requires the utilization of varieties that produce large fruit. Our identification of fruit size QTL in the mapping population and breeding material (pedigree set) will lead to the ability to apply marker assisted selection for this critical trait, thereby increasing the efficiency of cherry breeding programs.
  • Our results suggest that pedigree genotyping will be a valuable approach for QTL discovery and validation within breeding material.
Deliverables:
  • Publications:
    • Olmstead, JW, Sebolt AM, Antonio Cabrera A, Sooriyapathirana SS, Hammar S, Iriarte Q, Wang D, Chen CY, van der Knaap E, Iezzoni AF (200x) Construction of an intraspecific sweet cherry (Prunus avium L.) genetic linkage map and synteny analysis with the Prunus reference map. Tree Genetics & Genome (in review).
    • Iezzoni, A, Olmstead J (2006) Agricultural Biology in the 21st Century: It’s about the genes. Compact Fruit Tree 39 (2): 12-15.
  • Posters:
    • Olmstead J, Iezzoni A, and Whiting M (2006) Fruit size QTL in sweet cherry: Cell number is under stronger genetic control than cell size. Third Intl. Rosaceae Genomics Conference, Napier, NZ, 19-22 Mar.
    • Olmstead J, and Iezzoni A (2006) Alignment of sweet cherry linkage groups with the Prunus reference map. Third Intl. Rosaceae Genomics Conference, Napier, NZ, 19-22 Mar.
    • Iezzoni A, Olmstead J, Sebolt A, Chen Y, Wang D (2006) Construction of a sweet cherry linkage map suitable for comparative mapping in Prunus. Plant & Animal Genomes XIV
    • Olmstead J, A Sebolt, Y Chen, S Hammar, G. Iriate, D Wang, E van der Knaap, and A Iezzoni (2007) Sweet cherry linkage maps constructed from a pseudo-testcross mapping population. Plant and Animal Genome XV. Abstract Guide, pg. 218.
    • Cabrera A, Kozik A, Sooriyapathirana S, Sebolt A, Hammar S, Olmstead JW, Iriarte G, Wang W, Zhang G, Iezzoni AF, van der Knaap E (2008) Development of gene-based markers for linkage map construction and QTL analysis in sweet cherry (Prunus avium L.) Plant and Animal Genome XVI.
  • Web site:
    • http://www.cherrygenetics.org/
  • Training:
    • Graduate Students [Antonio Cabrera & Gloria Iriarte, marker development and mapping of the EST-derived markers; James Olmstead, genotyping and linkage map construction; Suneth Sooriyapathirana, genotyping and generation of the FlexQTL data set],
    • Post-Doctoral associates [Yiwu Chen & Guorong Zhang, QTL and FlexQTL analyses],
    • Technical Staff [Audrey Sebolt & Sue Hammar, phenotyping and genotyping],
    • Under-Graduate Student [Elisabeth Loconto, phenotyping].
  • New Collaborations:
    • Dr. Alex Kozik (Genome and Biomedical Sciences Facility, University of California at Davis) selected and assembled the Prunus-Arabidopsis COS set.



Genetic and Molecular Characterization of Self-Incompatibility and Self-Compatibility in Tetraploid Sour Cherry

POSTER #: 29

Accomplishments:

The ultimate goal of this project was to develop molecular markers for selfcompatibility (SC) and self-incompatibility (SI) that could be used for marker assisted selection for SC sour cherry progeny in breeding programs. To accomplish this goal we had to first determine the genetic and molecular basis for the genotype-dependent control of SI and SC in sour cherry.

  • The genetic control of SI and SC in tetraploid sour cherry was determined.
    • Genetic analyses of natural sour cherry selections identified seven independent, nonfunctional S-haplotypes with disrupted pistil component (stylar-S) and/or pollen component (pollen-S) function.
    • We developed and validated a genetic model that demonstrated that the transition from SI to SC in sour cherry is caused by the accumulation of non-functional S-haplotypes. In this model, a match between a functional pollen-S in the 2x pollen and its cognate functional S-RNase in the style, results in an incompatible reaction. A similar reaction would occur regardless of whether the pollen contained a single functional pollen-S gene or two different pollen-S genes. Thus for successful self-fertilization, the 2x pollen must contain two non-functional S-haplotypes.
  • The molecular changes resulting in the non-functional S-haplotypes in sour cherry were identified.
    • Molecular characterizations of the non-functional S-haplotype variants identified in the genetic studies determined that the losses of function were due to structural alterations of the S-RNase, SFB or S-RNase upstream sequences.
    • These structural alternations included the presence of transposable element insertions, deletions and nucleotide changes resulting in premature stop codons.
  • Molecular markers for the identification of SC progeny individuals at the seedlings stage were developed.
    • We designed dCAPS markers and S-haplotype specific primer pairs based on either the length polymorphisms between S-haplotypes, differential S-haplotype sequences, or differential restriction enzyme cut sites. These primer pairs can discriminate among the non-functional and wild-type S-haplotypes thereby enabling the identification of the S-haplotypes present in a sour cherry individual.
Broad Impacts:
  • The S-haplotype markers developed in this project are currently being used to determine whether sour cherry individuals are SC or SI. In a sour cherry breeding program, the ability to discriminate between SI and SC individuals at the seedling stage so that SI individuals can be discarded prior to field planting, dramatically increases the breeding program’s efficiency and cost-effectiveness.
  • The transition from SI to SC is regarded as one of the most prevalent transitions in Angiosperm evolution, having profound impacts on the genetic structure of populations. Yet, the identity and function of mutations that resulted in the breakdown of SI in nature were not well understood. This work provided the first detailed genetic description of the breakdown of S-RNase mediated gametophytic SI in a polyploid species that exhibits genotype-dependent loss of SI. Our finding that sour cherry is SI when only one nonfunctional S-haplotype is present has significant evolutionary implications since nonfunctional S-haplotypes would be maintained in the population without causing an abrupt shift to SC.
  • Our demonstration that heteroallelic sour cherry pollen is SI is counter to the well documented phenomena in the Solanaceae where SC accompanying polyploidization is frequently due to the SC of heteroallelic pollen. This finding was one of the first pieces of evidence leading to the current view that the pollen-S differs between Prunus (Rosaceae) and the Solanaceae.
Deliverables:
  • Publications:
    • 1. Hauck NR, Yamane H, Tao R, Iezzoni AF (2006) Accumulation of non-functional S-haplotypes results in the breakdown of gametophytic self-incompatibility in tetraploid Prunus. Genetics 172:1191-1198
    • 2. Hauck NR, Ikeda K, Tao R, Iezzoni AF (2006) The mutated S1-haplotype in sour cherry has an altered S-haplotype specific F-box protein gene. J Hered 97:514-520
    • 3. Tsukamoto T, Hauck NR, Tao R, Jiang N, Iezzoni AF (2006) Molecular characterization of three non-functional S-haplotypes in sour cherry (Prunus cerasus). Plant Mol Biol 62:371-383
    • 4. Tsukamoto T, Tao R, Iezzoni AF (2007) PCR markers for mutated S-haplotypes enable discrimination between self-incompatible and self-compatible sour cherry selections. Mol Breed [Published online: 9 June 2007].
  • Presentations:
    • 1. Iezzoni AF, Hauck NR, Tsukamoto T, and Tao R (2006) Self-compatibility in tetraploid sour cherry (Prunus cerasus) results from the accumulation of non-functional Shaplotypes. Third Intl. Rosaceae Genomics Conference, Napier, NZ, 19-22 Mar.
    • 2. Potter D, Tsukamoto T, Chin SW, Iezzoni A (2007) Phylogeny of self-incompatibility genes in Prunus (Rosaceae). Botanical Society of America Annual Meeting, Chicago, IL.
  • Meeting Abstracts for Poster Presentations:
    • 1. Hauck NR, Ikeda K, Tao Ryutaro, Iezzoni AF (2005) Molecular characterization of nonfunctional S-haplotypes in sour cherry. Plant and Animal Genome XIII. Abstract Guide, pg.
    • 2. Tsukamoto T, Tao R, Iezzoni AF (2008) Genetic and molecular characterization of self-(in)compatibility in tetraploid sour cherry. Plant and Animal Genome XVI.
  • Training:
  • PhD student [Nathanael Hauck], Post-doctoral Associate [Tatsuya Tsukamoto], Technical [Audrey Sebolt]. Drs. Hauck and Tsukamoto were lead scientists on this project as evidenced by their first author publications.
Collaborations:

As our findings had significant evolutionary implications, we have developed collaborations with the following evolutionary biologists: Dr. Daniel Potter, Univ. of California- Davis; Drs. Cristina and Jorge Vieira, University of Porto, Portugal