Location: Subtropical Horticulture Research2008 Annual Report
1a. Objectives (from AD-416)
The development and implementation of an international Marker Assisted Selection (MAS) program for cacao is the major objective of this project. This objective involves a combination of hypothesis-driven and non-hypothesis driven research and includes the training of scientists from cacao producing countries in plant breeding, genetics, and the use of molecular markers in a MAS program.
1b. Approach (from AD-416)
Single Nucleotide Polymorphism (SNP)s, both as single base pair substitutions and single base pair insertions/deletions (indels) are the most common sequence differences found between alleles. Methods have been developed for high-throughput detection of SNPs, but these methods require a priori knowledge of the SNP being assayed or sequence information surrounding the SNP. As more cacao EST sequence data become available, we can use it to screen for SNPs. In addition, SNP markers are completely portable and can be added into the growing international database (CocoaGenDB). Microsatellite allele calls are platform-dependent and there has been no way to efficiently share data among the research groups currently genotyping cacao. Once SNPs have been identified, they can be employed in a genetic assay that does not require electrophoresis or a dedicated molecular genetics facility staffed with highly trained technicians. Because breeding trials are established in cacao producing countries where such molecular genetics facilities do not exist, it is imperative to develop high throughput assays that can be performed and analyzed in the field to be able to use the molecular data for Marker Assisted Selection (MAS). Continue with existing field trials in Costa Rica, Ecuador, Brazil, and Ghana assisted by MAS to validate putative resistance to FP, BP, WB, Ceratocystis, CSSV and VSD. Establish new field trials in Cameroon, Ghana, Ivory Coast and Nigeria. Preventative breeding for Frosty Pod (FP) and Witches Broom (WB) in West Africa and South Asia. Using the markers flanking the Quantitative Trait Loci (QTL) for WB resistance on LG 1 and LG 9, selection of seedlings can be made from within families with ‘SCA6’ or ‘SCA12’ as a parent, that contain the genes conferring resistance to WB.
3. Progress Report
Progress on this project relates to National Program 301 Plant Genetic Resources, Genomics, and Genetics Improvement, Component 2: Crop Informatics, Genomics, and Genetic Analyses. An investigation into the use of our Single Nucleotide Polymorthism (SNP) markers to determine offtypes within a clonal population from Cameroon has begun. This population contains approximately 200 individuals comprised of 21 groups of clones. Microsatellites from 13 different loci had identified 53 offtypes. Using the 10 SNP markers from 6 different loci that we developed, we have already identified 87% of the offtypes. Plans are underway to continue to create more SNP markers through a variety of means including: conversion of microsatellites to SNPs, sequencing Conserved Orthologous Set genes across the diversity panel, and high throughput sequencing of the cacao genome. Efforts to develop a field SNP assay continue. The SNP assay was performed with a real-time polymerase chain reaction (PCR) machine and again with a standard thermocycler and platereader. Both methods worked equally well. This is important as a standard thermocycler and platereader are less cumbersome and more easily transported to a field site than a realtime PCR machine. A method to reliably isolate DNA from cacao seedlings in the field that requires minimal amounts of reagents, time and effort has been identified. This method from Epicentre extracts PCR amplifiable DNA from a leaf punch in a single 8 minute step. We have tested this extraction method with our SNP assay, and have successfully identified alleles. Additionally, discussions are continuing with GE Healthcare to convert the reagents needed for the SNP assay into a more stable form to allow better preservation in the field. A new database, on which we had been working for a few years, became ready for release to the public and was placed on a new server at the ARS Miami location with the address, http://184.108.40.206. The database will have a website as soon as it is assigned by the Information Technology Staff; we have requested http://shrsplantsciences.ars.usda.gov. Cocoa currently has the majority of the data in this database. The database is constantly growing, and has added a pedigree construction algorithm for crops for which data is available. The “CMap” function, a common piece of software in such databases, is currently being added for detailed comparison of pairs or sets of chromosomes of maps contained in the database. In November we moved into a new lab that was not, and is still not, fully functional. We have had serious limitations in our ability to perform molecular genetic analysis because of the non-functionality of many of the lab systems.
1. Sequence the genome of the clone Matina 1-6. Matina 1-6 clones were transferred to Clemson University Genomics Institute and three BAC libraries have been made. Significant progress has occurred toward the production of the physical map that is needed for the genome sequencing project. This accomplishment aligns with National Program 301 Plant Genetic Resources, Genomics and Genetic Improvement, Component 1 (Genetic Resource Management) and Component 2 (Crop Informatics, Genomics, and Genetic Analyses), Problem Statement 1B: Assess the Systematic Relationships and Genetic Diversity of Crop Genetic Resources.
2. Development of genetics database. A new genetics database was developed and is available on a server at the ARS-Miami facility at the address “http://220.127.116.11”. The website “http://shrsplantsciences.ars.usda.gov” has been requested to be assigned by the Information Technology Staff. Provides a unique resource for cacao researchers around the world. This accomplishment aligns with National Program 301 Plant Genetic Resources, Genomics and Genetic Improvement, Component 1 (Genetic Resource Management), Problem Statement 1B: Assess the Systematic Relationships and Genetic Diversity of Crop Genetic Resources.
3. Data Collection. Phenotypic and molecular data continued to be collected for all the breeding projects. New Specific Cooperative Agreements (SCAs) were developed and signed with several national and international cooperators in Central and South America, West Africa and South Asia. These new SCAs are funded from a grant from U.S. AID. A new position for a regional coordinator for West Africa was established and funded by NPS to coordinate all our projects with the four National Agriculture Research Services. Provides a unique resource for cacao researchers around the world. This accomplishment aligns with National Program 301 Plant Genetics Resources, Genomics and Genetic Improvement, Component 1 (Genetic Resource Management), Problem Statement 1B: Assess the Systematic Relationships and Genetic Diversity of Crop Genetic Resources.
4. Sequencing, Annotation, and Database Management of the Genome of Theobroma cacao. A new five-year trust agreement with Mars, Inc. is close to being signed. The primary goal of this project is to use the most advanced techniques available to sequence, assemble, annotate, and curate the cacao genome. The complete genome sequence would greatly enhance the efficiency of the Marker Assisted Selection by allowing the development of several thousand Single Nucleotide Polymorthism (SNP) markers. With proper development and support, these markers can be used in applied field breeding programs in developing countries. This accomplishment aligns to National Program 301 Genetic Resources, Genomics and Genetic Improvement, Component 1 (Genetic Resource Management), Problem Statement 1B: Assess the Systematic Relationships and Genetic Diversity of Crop Genetic Resources.
5. Significant Activities that Support Special Target Populations
Efombagan, I.B., Motamayor, J.C., Sounigo, O., Eskes, A.B., Nyasse, S., Cilas, C., Schnell Ii, R.J., Manzanares-Dauleux, M., Kolesnikova-Allen, M. 2008. Genetic diversity and structure of farm and genebank accessions of cacao (Theobroma cacao L.) in Cameroon revealed by microsatellite markers. Tree Genetics & Genomics. DOI 10.1007/s11295-008-0155-z