Peter O. Aikpokpodion

An improved semiautomated rapid method of extracting genomic DNA for molecular marker analysis in cocoa,Theobroma cacao L

DNA extraction is a time-consuming and expensive component of molecular marker analysis, constituting about 30–60% of the total time required for sample processing. Furthermore, the procedure for extracting high-quality DNA from tree species such as cocoa differs from extraction protocols suitable for other crop plants. This is accompanied by problems in collecting leaf tissues from field-grown cocoa trees, where storage facilities are not available and where transporting samples to laboratory for immediate refrigeration is usually impossible. We preserved cocoa leaf tissues in the field in an NaCl-CTAB-azide solution (as described in Rogstad, 1992), which did not require immediate refrigeration. This method also allowed preservation of leaf tissues for a few days during transportation and protected leaf tissues from bacterial and fungal attacks. Once transported to the laboratory, the samples were stored at 4°C for almost 1 y. To isolate good-quality DNA from stored leaf tissues, a rapid semiautomated and relatively high-throughput protocol was established. The procedure followed a modified CTAB/β-mercaptoethanol method of DNA extraction in a 96-well plate, and an automated system (i.e., GenoGrinder 2000) was used to grind the leaf tissues. The quality of DNA was not affected by long storage, and the quantity obtained per sample was adequate for about 1000 PCR reactions. Thus, this method allowed isolation of about 200 samples per day at a cost of

Population structure and molecular characterization of nigerian field genebank collections of cacao, Theobroma cacao L.

0.60 per sample and is a relatively high-throughput, low-cost extraction compared with conventional methods that use manual grinding and/or expensive kits.

Genetic identity and diversity of Nigerian cacao genebank collections verified by single nucleotide polymorphisms (SNPs): a guide to field genebank management and utilization

Abstract Inadequate knowledge of the population structure and diversity present often hamper the efficient use of germplasm collections. Using a high through-put system, twelve microsatellite loci were used to analyze genetic diversity and population structure in a national field genebank repository of 243 cacao accessions grouped into 11 populations based on their known sources. Based on multi-locus profiles, the Bayesian method was used for individual assignment to verify membership in each population, determine mislabeling and ancestry of some important accessions used in breeding program. A total of 218 alleles was revealed with a mean number of 18.2 alleles per locus. Gene diversity (He = 0.70) and allelic richness (4.34 alleles per locus) were highest in the F1 hybrid population. Differential mating system was suggested as responsible for the observed deficit and excess of heterozygotes observed among the populations. Analysis of molecular variance showed that within-population variance accounted for 63.0% of the total variance while the rest 37% was accounted for by the among-population variance. Cluster dendrogram based on UPGMA revealed two main subsets. The first group was made up of the Amelonado/Trinitario ancestry and the other of Nanay/Parinari ancestry. We found that Nanay and Parinari populations were the major source of Upper Amazon genes utilized while a large proportion of genetic diversity in the field genebank remained under-utilized in development of improved cultivars released to farmers in Nigeria. This study showed that the presence of alleles of the Upper Amazon Forasteros (Nanay, Parinari and Iquitos Mixed Calabacillo) genetic materials in the locally available accessions predated the formal large scale introduction of Upper Amazon materials in 1944. This is the first report of population structure of field genebank collections of cacao in Nigeria since more than seven decades of formal cacao breeding research.

Genetic diversity in African nutmeg (Monodora myristica) accessions from South Eastern Nigeria

Nigeria is the sixth largest cacao producer in the world. Field performance and quality of cacao hybrid families is largely dependent on the genetic integrity of parental clones obtained in field genebank collections. However, information on the impact of mislabeling on seed garden output in Nigeria is lacking. Using 63 single nucleotide polymorphism (SNP) markers, we analyzed 1457 cacao trees sampled from seven major field genebank plots in Nigeria to assess the genetic integrity in Nigerian cacao germplasm. The procedure of multilocus matching with known reference clones revealed up to 78% mislabeling in recently introduced international germplasm. A high rate of mislabeling was also revealed in the West African local selections and breeding lines, using Bayesian assignment test. The problem of mislabeling has been attributed to errors from the sources of introduction, pre-planting labeling errors, and rootstocks overtaking budded scions due to poor field management. The analysis of genetic diversity revealed a good representation of the available cacao germplasm groups in Nigerian field genebanks, indicating that the genetic base of Nigeria cacao germplasm has been significantly widened through germplasm introductions. However, only a small proportion of the available germplasm in the genebank have been utilized for variety development. This study proved the utility of SNP markers for cleaning up the genebanks and reducing offtypes; thereby providing a strong basis for improving the accuracy and efficiency in cacao genebank management and breeding, as well as for mobilizing improved varieties to cacao farmers in Nigeria.

Genetic diversity assessment of sub-samples of cacao, Theobroma cacao L. collections in West Africa using simple sequence repeats marker

Twenty-one accessions of African nutmeg ( Monodora myristica Gaertn. Dunal), an endangered spice plant, were collected from the South-East and South-South regions of Nigeria and analyzed for genetic diversity using random amplified polymorphic DNA (RAPD) markers. Twenty-one (21) decamer primers were tested out of which 10 that gave reproducible band patterns were selected for the study. A total of 77 bands were generated, ranging from 3 for OPB17 to 13 for OPT07, and were all polymorphic. The mean polymorphic information content ( PIC ) and genetic diversity ( H e ) were 0.673 and 0.697, respectively, indicating high genetic variation among the accessions. Cluster analysis delineated the accessions into four major groups. The maximum similarity index (0.88) based on Dice coefficient was recorded between AGL-01 and CRS-01 while the least (0.13) was between UGA-02 and EKW 01. The derived data was thus able to determine the extent of molecular variation underlying RAPD size polymorphism. Results obtained from this study proved that RAPD could be successfully used as a molecular tool for diversity study in M. myristica . The distributive pattern of genetic variation of M. myristica accessions provides important baseline data for conservation and improvement strategies for this species. Keywords: African nutmeg, random amplified polymorphic DNA (RAPD), genetic variation, polymorphic information content (PIC), similarity index, Monodora myristica African Journal of Biotechnology , Vol 13(42) 4105-4111