Location: Sustainable Perennial Crops Laboratory
2019 Annual Report
Objectives
Objective 1: Develop genomic tools for assessing pathogen genetic diversity and determining the basis for virulence in support of improved cacao breeding. [NP303, C2, PS2A]
Sub-objective 1.A. Identify the genome structure of established and emerging pathogens of cacao to determine the genetic basis for their virulence in cacao.
Sub-objective 1.B. Develop and use genomic tools to characterize the regional distribution of Ceratobasidium theobromae genetic diversity.
Sub-objective 1.C. Combining genotyping by sequencing and whole genome sequencing data to assess the genetic diversity of P. megakarya and evaluate the evolution/selection of virulence-associated genes.
Objective 2: Identify cacao defense genes through transcriptome analysis, and develop tools to exploit these genes for the improvement of cacao breeding. [NP303, C2, PS2A]
Sub-objective 2.A. Identify the cacao defense gene complement by assessing changes in gene expression of elite cacao clones responding to infections by diverse pathogens.
Sub-objective 2.B. Identify components of the cacao defense transcriptome with potential for providing durable tolerance against infections by diverse pathogens.
Approach
Diseases caused by Moniliophthora roreri, Phytophthora megakarya, Ceratobasidium theobromae, and Cocoa Swollen Shoot Virus reduce cacao yields, impacting farmers’ profits, and increase the costs of chocolate products. Emerging pathogens such as Marasmiellus scandens and Lasiodiplodia theobromae further threaten cacao production. While progress is being made in breeding tolerance to some cacao diseases, these efforts are hindered by an undefined pathogen genetic diversity and a limited understanding of cacao genes that confer disease tolerance. Therefore, this project will develop tools including pathogen genome and transcriptome sequences and associated single-nucleotide polymorphism panels characterizing pathogens and their variants pertinent to breeding programs and an annotated cacao transcriptome database identifying cacao genes linked to disease tolerance. The genome and transcriptome sequences of established and emerging cacao pathogens will be acquired and their genetic diversity will be determined. We will also characterize the expression of the cacao transcriptome during interactions between five cacao pathogens and eight genetically diverse clones. Using advanced mathematic approaches, we will identify differences and commonalities in the cacao responses associated with tolerance to pathogens. The most common traits associated with tolerance to cacao diseases relate to polyphenol biosynthesis and cell wall development. Gene expression and other measures associated with these specific traits will be evaluated during cacao developmental processes in leaves and pods and during the disease interactions previously mentioned. Once incorporated into an annotated cacao transcriptome database, these data along with gene sequence variations will provide new markers to help accelerate cacao breeding and increase the likelihood of obtaining sustainable disease tolerance.
Progress Report
Progress has been made over the last year on Objective 1: Develop genomic tools for assessing pathogen genetic diversity and determining the basis for virulence in support of improved cacao breeding. [NP303, C2, PS2A], Sub-objective 1.A. Identify the genome structure of established and emerging pathogens of cacao to determine the genetic basis for their virulence in cacao. We have continued our efforts studying vascular streak dieback of cacao in Indonesia, which is one of the main diseases limiting cacao production in Southeast Asia. Vascular streak dieback is caused by the fungus Ceratobasidium theobromae. The fungus C. theobromae cannot be maintained in pure culture which has made its study difficult. This work was initiated through collaborations with the Indonesian Cocoa and Coffee Research Institute and the University of Hasanuddin and includes collaborators from all major cacao producing countries in Southeast Asia. We completed assembly of the C. theobromae draft genome sequence and have submitted the genome to Genbank. We have prepared a manuscript describing the work which is now under review. Once accepted, this will the first available genome sequence for this pathogen. Through the same collaborations, we have obtained the genome sequence for Lasiodiplodia theobromae, the pathogen causing tip die back and have submitted its genome sequence to Genbank. Recent efforts have also focused on charactering the genome and genetic diversity associated with the thread blight pathogen Marasmiellus scandens. This species has turned out to be more complex than expected and we have now began sequencing 8 genetically diverse isolates of the pathogen. We also have cultures and DNA from more than 60 isolates of the genus Marasmius (contains Marasmiellus scandens) in the laboratory and have assessed their biological and genetic diversity.
Substantial progress has also been made toward accomplishing Sub-objective 1.C. Combining genotyping by sequencing and whole genome sequencing data to assess the genetic diversity of P. megakarya and evaluate the evolution/selection of virulence-associated genes. Much of this past year’s efforts have been spent resequencing some of the major pathogens of cacao. This has included use of long sequence PacBio techniques. Working with our collaborators at the University of California, Davis and MARS Incorporated, we have obtained 21 Phytophthora palmivora genomes using short read Illumina technology and 2 genomes using long read technology. We have obtained 19 P. megakarya genomes using short read technology and 3 genomes using long read technology. We are currently preparing this work for publication. We have also obtained 19 genome sequences for Moniliophthora roreri and 4 genome sequences for M. perniciosa using short read technologies.
Over the past year we have set the stage for accomplishing Objective 2: Identify cacao defense genes through transcriptome analysis, and develop tools to exploit these genes for the improvement of cacao breeding. [NP303, C2, PS2A]. Sub-objective 2.A. Identify the cacao defense gene complement by assessing changes in gene expression of elite cacao clones responding to infections by diverse pathogens. Agreements have been put in place with the International Cocoa Quarantine Centre (ICQC), Reading, U.K. and the Cocoa Research Centre (CRC), the University of the West Indies, Trinidad and Tobago. These institutions will provide RNA for sequencing isolated from diverse cacao clones after infection with the Cocoa Swollen Shoot Virus or Moniliophthora perniciosa, causal agent of witches’ broom disease. In addition, we have acquired all the cacao clones required for the proposed research to be carried out in Beltsville, Maryland. All three sites are at the stage of propagating material before beginning inoculations with the various pathogens.
Accomplishments
