1a.Objectives (from AD-416):
There are three primary objectives of this research. The first objective is to obtain the transcriptome of the fastidious pathogen of cacao, Oncobasidium theobromae, presumptive causal agent of vascular streak dieback (VSD). The second objective is to obtain the transcriptome of the cacao pathogen, referred to as Colletotrichum gloeosporioides, causal agent of anthracnose. The third objective is to map QTLs for resistance/tolerance to VSD and develop markers for marker-assisted breeding. Additional objective may be developed based on results gathered, including obtaining the genome sequences of the identified pathogens, and characterization of the resistance mechanisms involved in cacao’s responses to infection by these pathogens.
1b.Approach (from AD-416):
Pyrosequencing sequences small DNA fragments but is capable of sequencing very large numbers of small sequences in a very short time. Bioinformatic techniques allow for the assembling of these short sequences into larger sequences and ultimately into linkage groups. This can be done at progressively lower cost as techniques are improved. The transcriptome Oncobasidium theobromae will be determined using this technique. Since Oncobasidium theobromae cannot be cultured, our approach will be to isolate total RNA from infected cacao stems and sequence using pyrosequencing and other techniques. Colletotrichum gloeosporioides can be cultured. For this objective we will obtain both pure cultures and infected stem tissues. Total RNA will be isolated from these samples and sequenced. Finally, for the third objective, linkage mapping based on SNP markers will be applied to identify QTLs responsible for resistance/tolerance. Two mapping populations from different parental clones will be used.
A series of cacao stem sections, some showing symptoms of vascular streak dieback and some healthy stems, were collected in Indonesia and sent to the ARS laboratory in Beltsville, MD. Before the samples were sent, they were freeze dried and vacuum packed. RNA will be extracted from the samples and the RNA sequenced to identify the expressed genes of the pathogen(s) causing the symptoms. The cacao germplasm collection maintained in ICCRI was genotyped using 54 SNP markers. These SNP markers are DNA markers that are capable of identifying cacao trees at the molecular level. Based on the SNP profiles, verification of international clones was carried out and parentages in advanced breeding lines were analyzed. Crosses were made between parental clones that are resistant or susceptible to VSD to create a F1 mapping population.