J. Steven Brown

Analysis of clonal germplasm from five Saccharum species: S. barberi, S. robustum, S. officinarum, S. sinense and S. spontaneum. A study of inter- and intra species relationships using microsatellite markers

In order to study relationships within and among species of the highly complex polyploid genus Saccharum, 30 clones from 5 species (S. barberi, S. officinarum, S. robustum, S. sinense, and S. spontaneum) and 6 cultivated clones were analyzed using 15 sugarcane simple sequence repeat (SSR) markers and two multivariate statistical methods. The origins of two species, S. barberi and S. sinense, are unclear, and it has been hypothesized that they arose from crosses of S. officinarum × S. spontaneum. S. robustum is suggested to be ancestral to S. officinarum, which, when crossed with S. spontaneum, principally comprise the genomes of commercial cultivars. Although our analyses supported S. robustum as being an ancestor of S. officinarum, and show the composition of commercial clones as expected, our results concerning the species S. barberi and S. sinense indicated them to be much less related to the main Saccharum germplasm pool than previous molecular marker investigations, suggesting that introgression from other genera may exist in their ancestry. The differing results of our analysis of S. barberi and S. sinense are likely attributable to more equal and larger numbers of clones studied in each species. Given the extremely high ploidy, the use of only 15 SSR markers enabled clear distinction of Saccharum species and clones, and also identified likely mislabeled accessions. In addition, the replication of DNA extraction, polymerase chain reactions, and fragment electrophoresis increased the robustness of our analysis.

Development of single nucleotide polymorphism markers in Theobroma cacao and comparison to simple sequence repeat markers for genotyping of Cameroon clones

Single nucleotide polymorphism (SNP) markers are increasingly being used in crop breeding programs, slowly replacing simple sequence repeats (SSR) and other markers. SNPs provide many benefits over SSRs, including ease of analysis and unambiguous results across various platforms. We have identified and mapped SNP markers in the tropical tree crop Theobroma cacao, and here we compare SNPs to SSRs for the purpose of determining off-types in clonal collections. Clones are used as parents in breeding programs and the presence of mislabeled clones (off-types) can lead to the propagation of undesired traits and limit genetic gain from selection. Screening was performed on 186 trees representing 19 Theobroma cacao clones from the Institute of Agricultural Research for Development (IRAD) breeding program in Cameroon. Our objectives were to determine the correct clone genotypes and off-types using both SSR and SNP markers. SSR markers that amplify 11 highly polymorphic loci from six linkage groups and 13 SNP markers that amplify eight loci from seven linkage groups were used to genotype the 186 trees and the results from the two different marker types were compared. The SNP assay identified 98% of the off-types found via SSR screening. SNP markers spread across multiple linkage groups may serve as a more cost-effective and reliable method for off-type identification, especially in cacao-producing countries where the equipment necessary for SSR analysis may not be available.

A Composite Linkage Map from Three Crosses Between Commercial Clones of Cacao, Theobroma cacao L.

In this paper, we report the construction of the first composite map of cacao from linkage data of one F2 and two F1 mapping populations with a high number of codominant markers in common. The combination of linkage information from all three maps results in the currently most precise estimates of marker locations and distances between markers, especially in densely marked areas. JoinMap®V4 software was used for all marker quality assessment and mapping. Individual (sub-composite) maps and the composite map contained 10 major linkage groups, corresponding to the number of cacao chromosomes. Homogeneity of marker placement was very high among sub-composite maps, the composite map, and the designated “reference” map. Care was exercised in the re-creation of sub-composite maps and the composite map to include only markers with acceptable mapping quality parameters. The composite map places more markers with higher precision than any individual map. This research clearly demonstrates for the first time a very high level of marker homogeneity among commercial cacao clones compared to other species. The observed homogeneity between different maps, including the composite one, is probably due to a narrow genetic base of commercial cacao clones. Markers linked to identified quantitative trait loci (QTLs) are more likely to retain linkage in other commercial clones, rendering the QTLs in cacao potentially more stable than in other species.