Genetics of reproductive self-compatibility in Theobroma cacao L.

Authors: Royaert, Stefan; Martinez, Kathleen; PHILLIPS, WILBERTH - Catie Tropical Agricultural Research; LOPES, UILSON - M & M Mars Company - Brazil; Kuhn, David; Schnell Ii, Raymond; MOTAMAYOR, JUAN - M & M Mars Company - United States

Submitted to: Proceedings of the International Cocoa Producer's Conference
Publication Type: Abstract Only
Publication Acceptance Date: 11/16/2009
Publication Date: 2/28/2011
Citation: Royaert, S.E., Cariaga, K.A., Phillips, W., Lopes, U., Kuhn, D.N., Schnell Ii, R.J., Motamayor, J.C. 2011. Genetics of reproductive self-compatibility in Theobroma cacao L.. Proceedings of the International Cocoa Producer's Conference. 1.

Interpretive Summary: Self-incompatibility (SI) in plants prevents self-fertilization. Highly specific recognition events lead to the rejection of self-pollen. These specific events occur at different stages of the fertilization process, causing interruption in pollen germination or pollen tube growth, fusion of sperm cell and egg cell, or even arrest in embryo development. The main effect in an agricultural setting is yield reduction and it also affects progress in plant breeding because of the impossibility of performing certain crosses. SI in the chocolate tree, Theobroma cacao L., is sporo-gametophetic, and so far, no genes regulating this phenomenon have been identified. The aim of this project is to identify genes involved in SI using different approaches such as QTL mapping, candidate gene mapping, differential gene expression and microscopy. To understand the genetics of SI we are studying two different mapping populations. A preliminary analysis on available data from 79 individuals out of 256 trees from the cross Pound-7 x UF-273, identified a QTL on chromosome 4, but further data collection is necessary to confirm its statistical significance. The position of the markers overlaps with the position of a QTL for Black Pod resistance. A second approach used is the candidate gene approach. A cDNA database was searched for homology with SI genes known in other plant species and 14 candidate genes were selected for sequencing. Sequence polymorphisms, mainly single nucleotide polymorphisms (SNP), were identified and analyzed in a subset of the CATIE ‘Pound 7 x UF 273’ mapping population. Preliminary results, with less than half of the sequences analyzed, showed no correlation between identified SNPs and SI. The third approach is to identify differentially expressed genes or sequences using RNA of unpollinated and pollinated flowers, harvested at different time points. The RNA is being sequenced on an Illumina Genome Analyzer in search of sequences to study and to determine their expression levels. Up- or down-regulated gene sequences will be analyzed for SNPs related to SI and subsequently mapped. Lastly, confocal laser scanning microscopy is used in a preliminary experiment to study morphological differences in intact ovules, unpollinated or pollinated with compatible or incompatible pollen. SNPs and other sequence polymorphisms correlated with self-compatibility or self-incompatibility will be used for marker assisted selection and allele mining of the available germplasm for breeding purposes.

Technical Abstract: Self-incompatibility (SI) in plants prevents self-fertilization. Various events are involved at different stages of this process, from interruption in pollen germination, pollen tube growth, syngamy or even to an arrest in embryo development. The main effect is yield reduction. SI also affects progress in hybrid development because of the impossibility of performing certain crosses. SI in Theobroma cacao L. is sporo-gametophetic, and so far, no genes regulating this phenomenon have been identified. To attempt identification of genes involved in SI a candidate gene approach was undertaken. The ESTtik cDNA database was searched for homology with SI genes known in other plant species and 55 ESTs were identified (E-values between e-111 and e-20). These 55 ESTs presented 27 different candidate genes of which 14 were selected for sequencing. Five genes, identified in Papaver rhoeas, are involved in the S-glycoprotein mechanism. Three of the 5 genes are MAP kinases, the other 2 are pyrophosphatases. Another 6 genes, found in Petunia integrifolia ssp. inflata, are involved in the S-RNase mechanism. All these genes (RBX1, SBP1, SK1, SK2, SK3 and CUL1-C) are part of the SCF-complex, which plays an important role in the protein degradation process. Two other genes, POP2 and CLP, involved in pollen tube growth and proteolysis, respectively were identified in Arabidopsis thaliana. The last candidate gene, MOD, is an aquaporin-related gene identified in Brassica rapa,. First, candidate genes in cacao were amplified by PCR and sequenced in two self-compatible and two self-incompatible clones. Sequence polymorphisms, mainly single nucleotide polymorphisms (SNP), were identified. A total of 27 SNPs were found, a few others are still to confirm. Secondly, identified SNPs were analyzed in a subset of the CATIE ‘Pound 7 x UF 273’ mapping population, and were mapped using the same population. Preliminary results, with less than half of the sequences analyzed, showed no correlation between certain SNPs and SI so far. Furthermore, RNA of unpollinated and pollinated (using compatible or incompatible pollen) flowers, harvested at different time points will be sequenced on an Illumina Genome Analyzer in search of more sequences to study. The sequences will be analyzed for SNPs in expressed genes related to SI. Lastly, confocal laser scanning microscopy will be used to study morphological differences in intact ovules, unpollinated or pollinated with compatible or incompatible pollen. SNPs and other sequence polymorphisms, which will hopefully turn out to be correlated with self-compatibility or self-incompatibility will be transformed into useful markers for marker assisted selection and allele mining of the available germplasm for breeding purposes.