Molecular Markers Assisted Cocoa Genebank Management

Cacao (Theobroma cacao L.)(2n=2x=20), is an important Sterculiaceae tropical evergreen plant origin from Central and South America, as early as3,000years ago was domesticated and cultivated by the Mayans. Now it is widely planted in Africa, Southeast Asia and Latin America. Cacao is one of the world’s major crops, cocoa beans, are the main resource of chocolate industry, with the global annual production about4million tons. The world’s major cocoa growing areas cultivated mainly in Latin America, South America, West Africa, Southern Asia and other tropical along the equator, but this region is also important for biodiversity conservation areas.The conservation of cacao germplasm requires maintaining living trees in genebanks within tropical regions. There are two international public cacao collection, one is the International Cacao Genebank, Trinidad (ICG, T) and the other is International CATIE Cacao Collection (IC3). ICG, T is managed by the Cocoa Research Unit (CRU) of the University of West Indies and IC3by the Centro Agronomico Tropical de Investigacion y Ensenanza (CATIE) of Costa Rica (Beakele and Beakele,1996). Germplasm held in these two international collections is available for breeders via the international quarantine center located at the University of Reading in UK. These genebanks are supported by both public and industrial funding. Information of cacao germplasm held in these international collections is currently keeping in the International Cacao Germplasm Database (ICGD) which is hosted by the University of Reading in UK, as well as Centre de Cooperation Internationale en Recherche Agronomique pour le Developpement (CIRAD) in France. Since cocoa seed can not be stay alive for a long time, either to be stored under low temperature conditions. Therefore, only in the field of cocoa germplasm vivo preservation. This kind of offsite mode management has brought many problems. One major problem is mislabeling or label lost will cause to the confusion in evaluation, identification and breeding process of cocoa. The major rate of mislabeling within current international cocoa offsite repository is around30%to40%by average. The most urgent task of cocoa Repository Management is to correct the mislabeling in cacao collection fileds. That will be great help to assist documented pedigree and evaluation of genetic variaties.It is commonly accepted that there are three groups of cocoa germplasm. The first group is Criollo, which was originally distributed in Central America and the Caribbean, and is well known for its "fine-flavor" beans. The second group is Forastero, which includes diverse wild and more recently cultivated populations originally from South America. Today Forastero is widely grown in West Africa and Brazil, and accounts for approximately90%of the cocoa beans produced in the world. Finally, there is the Trinitario group, which is believed to be the result of hybridizations between Criollo and Forastero types in the18th century. The original hybrid zone is the island of Trinidad.The SSR (Simple Sequence Repeat) polymorphic markers and SNP (Single Nucleotide Polymorphism) markers are used to explore diverse populations of the best molecular identification means, and provide accurate and reliable molecular basis for the correction of mislabeling germplasm for theoretical guidance and support.Experiment I. Assessing genetic identity and population structure of Trinitario cocoa germplasm using microsatellite markersIn this experiment, SSR markers were used to characterize a Trinitario cacao collection in the International Genebank in Trinidad. Trinitario cocoa is indigenous to the island of Trinidad and is believed to have had a very small number of founding parents in the18th century. However, rarely known about the individual identity and population structure in this group of cocoa germplasm.This study was aimed at assessing the population structure and the extent of mislabeling in an indigenous Trinitario collection, and tested the hypothesis that the Trinitario were founded by a few progenitors. Multilocus microsatellite data were generated using15pairs of SSR primers. We used a capillary electrophoresis as previously described (Saunders et al,2004) using a CEQTM8000genetic analysis system (Beckman Coulter Inc.). Data analysis was performed using the CEQTM8000Fragment Analysis software version7.0.55according to manufacturers" recommendations (Beckman Coulter Inc). SSR fragment sizes were automatically calculated to two decimal places by the CEQTM8000Genetic Analysis System. Allele calling was performed using the CEQTM8000bining wizard software (CEQTM8000software version7.0.55, Beckman Coulter Inc.).Synonymous mislabeling was identified by matching multilocus genotypes among individuals. Population structure was assessed by a Bayes method of sample partition. Heterozygosity deficiency was estimated as indication of recent population bottleneck. All microsatellite loci were polymorphic, with the number of alleles per locus ranging from2to10(mean=5.8). The mean expected heterozygosity ranged from0.307to0.656, with an average of0.552. The multi-locus fingerprints unambiguously identified all59individuals and three of them were found to be synonymous. Probability of identity predicted that the top7loci were necessary, yet sufficient, to distinguish with greater than99%certainty between the Trinitario individuals. Wilcoxon and Sign tests showed no heterozygosity excess. The distance-based cluster analysis, multi-dimensional scaling based on genetic relatedness, and Bayesian cluster method all showed that there is population admixture in the ICS Trinirario germplasm.Analysis showed, ICS2, ICS83and ICS95three clones in15alleles are exactly match, by checking the relative position and compared to trees, it is determined that this three should be correct as the same lable. Moreover, only by the seven loci mTcCIRll, mTcCIR15, mTcCIR40, mTcCIR37, mTcCIR24, mTcCIR33and mTcCIR12can tell whether the same basic lines repetition. Population structure analysis was results showed by PowerMarker that the samples ICS1, ICS10, ICS35, ICS46, ICS57, ICS77, ICS80and ICS100other eight clones may be due to off-site preservation mislabeled and mixed ICS groups of individuals.It shows that SSR fingerprinting is highly efficient to identify cacao accessions. We demonstrated that the Trinitario cocoa is comprised of more than one source population thus should not be treated as a single uniform genetic group in conservation and breeding. Experiment II. Genetic diversity and parentage in farmer varieties of cacao from Honduras and Nicaragua as revealed by single nucleotide polymorphism (SNP) markersAlthough SSR markers are efficient tool for cacao germplasm characterization, this system has several drawbacks for large scale DNA fingerprinting. Compared to SSR markers, SNP assays can be done without separating DNA by size, and therefore can be automated in high throughput assay format. The di-allelic nature of SNPs offers a much lower error rate in allele calling. Moreover, there is a higher consistency in allele calling across laboratories. While SNP markers have been widely used in plant varietal identification in many other crops, the efficacy of using SNP markers for cacao genotype identification and diversity assessment remain to be investigated. Large scale genotyping using a small set of SNP markers is still in great demand by the cacao community for a broad range of research and field applications.Experiment Ⅲ. Verification of genetic identity of introduced cacao germplasm in Ghana using single nucleotide polymorphism (SNP) markersCocoa is an important global commodity with an annual production that exceeded4million tons in2010, of which75%was produced in West Africa. Ghana alone produced850,000tons of cacao, accounting for21%of the world’s total output in2010(FAOSTAT, http://faostat3.fao.org/home/index.html).In the present study, high-throughput genotyping with SNP markers was used to fingerprint160cacao trees in the CRIG germplasm collection. These accessions had been originally introduced from international germplasm collections. The study also included100international clonesas references. DNA concentration was determined with a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE). Based on the level of polymorphism and on their distribution across the ten chromosomes in cacao,54SNP markers were selected from1560candidate SNPs that had been developed using cDNA sequences from a wide range of cacao organs (Argout et al.2008). SNP genotyping was performed at the Human Genetics Division Genotyping Core facility, Washington University, St. Louis, using MALDI-TOF mass spectrometry (Sequenom, Inc., San Diego, CA). Key descriptive statistics for measuring the infonnativeness of the SNP markers were calculated, including observed heterozygosity, expected heterozygosity, minor allele frequency, Inbreeding coefficient and probability of identity (Evett and Weir1998; Waits et al.2001). The program GenAlEx6.2(Peakall and Smouse2006;2012) was used for computation.For the identification of mislabeling (off-type), SNP profile of100reference trees maintained in the International Cacao Genebank, Trinidad (ICG,T) were used in the analysis. The genetic identity of the100reference trees has been characterized by both SNP (Zhang, personal communication and SSR fingerprinting (Zhang et al.2009b; Motilal et al.2010; Johnson et al.2009). Pairwise multilocus matching was applied among each pair of individual trees, including the reference trees from the International germplasm collections, using the same program. Accessions with same names as the reference trees, but not matching them, were declared off-types. For the multilocus matching, the option to ignore missing data was used. Discriminating power of the SNP loci was computed using the probability of identity (PID, Waits et al.2001) option implemented in the same computer program.The multilocus SNP profiles, generated by the Sequenom Mass Spectrometry platform, were compared with the SNP profiles of reference trees maintained in the international cacao collections. Comparison of the multilocus SNP profiles with the reference accessions identified seven intraclonal mislabelings in accessions NA79, PA150and IMC76. The multilocus matching also found that AMAZ-2and PA303were mislabeled. With all53loci considered, the combined probability of identity was in the order of10-9. Overall, the procedure of multilocus matching with known reference trees led to the identification of149true-to-type trees out of160tested samples. Based on the value of delta K, the model-based approach of STRUCTURE indicated K=5as the most probable number of genetic clusters. The tested cacao accessions from the Ghana cacao collection, as well as the100reference accessions were stratified as germplasm groups of Amelonado, IMC, SCA/Ucayali, Morona, Nanay and Parinari respectively. The assigned memberships for all the tested trees from Ghana were compatible with their known parentage germplasm groups. The assignment test of the T clones confirmed their recorded parental germplasm groups. The parental group of PA, IMC (which includes parental clones NA32and NA34) were clearly reflected in the admixed ancestry profiles of T60, T63, T65and T79. A full genetic background of IMC was revealed for accession T85/799, supporting its recorded parentage of IMC60and NA34(a member of the IMC germplasm group; Motamayor et al.2008). In addition, admixed ancestry of IMC and Amelonado was revealed for T16/613family, which not only supported the recorded parentage of IMC24, but also detected that the other parent came from the Amelonado group.Of the eight candidate parent-offspring relationships, the results of parentage inference confirmed six pairs at the95%confidence level and one pair (NA34-T80/799) at the80%confidence level (Table4). For offspring T16/613, only one parent (Amelonado22) was identified at the>80%confidence level because the reference genotype of maternal parent IMC24was not available. The result of parent-offspring assignment supported the outcome of model-based clustering analysis by the STRUCTURE program.The comparison unambiguously identified mislabeled trees. For materials introduced as hybrid seeds without an available reference genotype, parentage analysis and model-based assignment were applied to verify their recorded parentage and genetic background. Our study shows that a small set of polymorphic SNP markers can provide a robust and accurate result for cacao genotype identification. This protocol can be applied for large-scale genotyping of cacao as well as for many other crops.In summary, it shows that SSR fingerprinting is highly efficient to identify cacao accessions Trinatario from International Cocoa Cocoa ICS collection through this experiment. We demonstrated that the Trinitario cocoa is comprised of more than one source population thus should not be treated as a single uniform genetic group in conservation and breeding duplicate identification. The first study using single nucleotide polymorphism SNP markers for cocoa germplasm into polymorphism identified in single nucleotide polymorphisms prove that the level of technology will be the International Cocoa Genebank resource management and maintenance an important tool that can be achieved in a short time for a large number of high-throughput screening of single nucleotide loci cultivars, as well as wild cocoa farm genetic resources identified. For the first time using SNP markers, as a reliable basis for identification of mislabling and other historical legacy of offsite resources issues. Identification of single nucleotide polymorphisms to distinguish parental genetic relationship with hybrids and provide reliable and efficient identification for cacao world collections.