Ranjana Bhattacharjee

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

The impact of SNP fingerprinting and parentage analysis on the effectiveness of variety recommendations in cacao

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.

Genomic Resources for Water Yam (Dioscorea alata L.): Analyses of EST-Sequences, De Novo Sequencing and GBS Libraries

Evidence for the impact of mislabeling and/or pollen contamination on consistency of field performance has been lacking to reinforce the need for strict adherence to quality control protocols in cacao seed garden and germplasm plot management. The present study used SNP fingerprinting at 64 loci to examine the diversity, labeling errors and parentage in 2551 trees obtained from six seed gardens, breeders clone collection and single-cross progenies and a sample of farmers’ trees in Ghana. Clone mislabeling was pervasive, both within the seed garden clones and among clones of the breeders’ active collection. Among the seed garden clones, mislabeled trees were assigned to other parental clones used in the seed garden, pointing to labeling errors prior to planting as the principal cause of mislabeling. Among the breeders’ clone collection, both homonymous and synonymous mislabeling were identified in addition to trees with unique genotypes. This implicates pre-planting labeling errors and rootstocks overtaking budded scions. Parentage analysis supported the Amelonado ancestry of farmers’ varieties but with significant contribution of Upper Amazon introductions. Parentage of recently developed clones and of progenies of controlled crosses showed evidence of both pollen contamination and effects of mislabeled parents. The observed patterns of unexpected parentage had direct effects on the consistency of the variety performance between trials and increased within-plot variability for families with mixed ancestry. The results provide a strong basis for mainstreaming SNP fingerprinting in cacao breeding programs to improve the efficiency of the variety development process.

Genome sequencing of the staple food crop white Guinea yam enables the development of a molecular marker for sex determination

The reducing cost and rapid progress in next-generation sequencing techniques coupled with high performance computational approaches have resulted in large-scale discovery of advanced genomic resources in several model and non-model plant species. Yam (Dioscorea spp.) is a major food and cash crop in many countries but research efforts have been limited to understand the genetics and generate genomic information for the crop. The availability of a large number of genomic resources including genome-wide molecular markers will accelerate the breeding efforts and application of genomic selection in yams. In the present study, several methods including expressed sequence tags (EST)-sequencing, de novo sequencing, and genotyping-by-sequencing (GBS) profiles on two yam (Dioscorea alata L.) genotypes (TDa 95/00328 and TDa 95-310) was performed to generate genomic resources for use in its improvement programs. This includes a comprehensive set of EST-SSRs, genomic SSRs, whole genome SNPs, and reduced representation SNPs. A total of 1,152 EST-SSRs were developed from >40,000 EST-sequences generated from the two genotypes. A set of 388 EST-SSRs were validated as polymorphic showing a polymorphism rate of 34% when tested on two diverse parents targeted for anthracnose disease. In addition, approximately 40X de novo whole genome sequence coverage was generated for each of the two genotypes, and a total of 18,584 and 15,952 genomic SSRs were identified for TDa 95/00328 and TDa 95-310, respectively. A custom made pipeline resulted in the selection of 573 genomic SSRs common across the two genotypes, of which only eight failed, 478 being polymorphic and 62 monomorphic indicating a polymorphic rate of 83.5%. Additionally, 288,505 high quality SNPs were also identified between these two genotypes. Genotyping by sequencing reads on these two genotypes also revealed 36,790 overlapping SNP positions that are distributed throughout the genome. Our efforts in using different approaches in generating genomic resources provides a non-biased glimpse into the publicly available EST-sequences, yam genome, and GBS profiles with affirmation that the genomic complexity can be methodically unraveled and constitute a critical foundation for future studies in linkage mapping, germplasm analysis, and predictive breeding.

Establishment of a cassava (Manihot esculenta Crantz) core collection based on agro-morphological descriptors

BackgroundRoot and tuber crops are a major food source in tropical Africa. Among these crops are several species in the monocotyledonous genus Dioscorea collectively known as yam, a staple tuber crop that contributes enormously to the subsistence and socio-cultural lives of millions of people, principally in West and Central Africa. Yam cultivation is constrained by several factors, and yam can be considered a neglected “orphan” crop that would benefit from crop improvement efforts. However, the lack of genetic and genomic tools has impeded the improvement of this staple crop.ResultsTo accelerate marker-assisted breeding of yam, we performed genome analysis of white Guinea yam (Dioscorea rotundata) and assembled a 594-Mb genome, 76.4% of which was distributed among 21 linkage groups. In total, we predicted 26,198 genes. Phylogenetic analyses with 2381 conserved genes revealed that Dioscorea is a unique lineage of monocotyledons distinct from the Poales (rice), Arecales (palm), and Zingiberales (banana). The entire Dioscorea genus is characterized by the occurrence of separate male and female plants (dioecy), a feature that has limited efficient yam breeding. To infer the genetics of sex determination, we performed whole-genome resequencing of bulked segregants (quantitative trait locus sequencing [QTL-seq]) in F1 progeny segregating for male and female plants and identified a genomic region associated with female heterogametic (male = ZZ, female = ZW) sex determination. We further delineated the W locus and used it to develop a molecular marker for sex identification of Guinea yam plants at the seedling stage.ConclusionsGuinea yam belongs to a unique and highly differentiated clade of monocotyledons. The genome analyses and sex-linked marker development performed in this study should greatly accelerate marker-assisted breeding of Guinea yam. In addition, our QTL-seq approach can be utilized in genetic studies of other outcrossing crops and organisms with highly heterozygous genomes. Genomic analysis of orphan crops such as yam promotes efforts to improve food security and the sustainability of tropical agriculture.

An improved method of DNA extraction from plants for pathogen detection and genotyping by polymerase chain reaction

International Institute of Tropical Agriculture maintains 2544 cassava accessions (Manihot esculenta Crantz) from 28 countries in its field bank. Being vegetatively propagated, this poses challenges in maintenance in terms of cost as well as in labour requirements. A core collection representing the range of phenotypic diversity present in the entire collection would enhance the conservation aspects and increase the potential for its exploitation in crop improvement programmes. The present study aimed to establish a core collection using 40 agro-morphological traits evaluated at two locations using a different number of accessions in each location. To meet the challenges generated by the types of variables and include maximum diversity in the core collection, a sequential strategy based on five major concepts was used: hierarchical multiple factor analysis allowing the mixture of variables of different kinds; three-way analysis that included the effect of genotype £ environment interaction in the clustering process; linear discriminant function to assign all those individuals who were included in one location but not in the other to the groups that were generated from the common number of accessions evaluated in both locations; and D-allocation method to select samples from each cluster. The representativeness of the core subset to the entire collection was further estimated by comparing means and variances, range, and distances between accessions. The established cassava core collection consisted of 428 accessions that conserved 15% higher phenotypic diversity with no redundancies. The phenotypic diversity represented in this core collection will be a guide to users of cassava germplasm in their crop improvement programmes.

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

Polymerase chain reaction (PCR)-based applications in plant molecular biology and molecular diagnostics for plant pathogens require good quality DNA for reliable and reproducible results. Leaf tissue is often the choice for DNA extraction, but the use of other sources such as tubers, stems, or seeds, is not uncommon. The extraction of DNA from different tissue sources often requires different protocols. In this study, a simple protocol was established for the extraction of DNA from leaves, tubers, stems, seeds and even fungal mycelia. The protocol is simple and suitable for high-throughput DNA extraction using automated tissue grinders. It yielded large quantities of DNA (0.4 μg to 2 mg DNA from 100 mg tissue) of high quality from seeds of maize, soybean, and cowpea, tubers of yam, tuberous roots of cassava, and leaf tissues of banana and plantain, yam, cassava, maize, okra, mango, and other species. DNA was successfully used for the detection of fungal and viral pathogens and the genotyping of yam and cassava by PCR. Keywords : DNA isolation, plant tissues, PCR amplification, pathogen detection, high throughput DNA extraction African Journal of Biotechnology Vol. 12(15), pp. 1894-1901

Carotenoid profiling of yams: Clarity, comparisons and diversity

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.

Understanding the genetic diversity and population structure of yam (Dioscorea alata L.) using microsatellite markers

Screening carotenoids of elite accessions of yam (Dioscorea spp.) used in the global yam breeding program has been conducted to quantitatively determine the carotenoid composition of the crop. Comparisons to previous data reporting cerotenoid levels in yam has been made, in order to deduce greater perspectives across multiple studies. Characterisation of complex species and accession -specific profiles have shown a rich base of diversity that can inform breeding strategies. Key findings include; (i) the identification of accessions rich in β-carotene which can aid provitamin A biofortification, (ii) Data disputing the commonly held belief that yellow Guinea yam (D. cayennensis) has higher β-carotene content than that of white Guinea yam (D. rotundata), and (iii) the tentative identification of C25-epoxy-apocarotenoid persicaxanthin with potential implications for tuber dormancy.

Re-defining the yam (Dioscorea spp.) core collection using morphological traits

Yams (Dioscorea sp.) are staple food crops for millions of people in tropical and subtropical regions. Dioscorea alata, also known as greater yam, is one of the major cultivated species and most widely distributed throughout the tropics. Despite its economic and cultural importance, very little is known about its origin, diversity and genetics. As a consequence, breeding efforts for resistance to its main disease, anthracnose, have been fairly limited. The objective of this study was to contribute to the understanding of D. alata genetic diversity by genotyping 384 accessions from different geographical regions (South Pacific, Asia, Africa and the Caribbean), using 24 microsatellite markers. Diversity structuration was assessed via Principal Coordinate Analysis, UPGMA analysis and the Bayesian approach implemented in STRUCTURE. Our results revealed the existence of a wide genetic diversity and a significant structuring associated with geographic origin, ploidy levels and morpho-agronomic characteristics. Seventeen major groups of genetically close cultivars have been identified, including eleven groups of diploid cultivars, four groups of triploids and two groups of tetraploids. STRUCTURE revealed the existence of six populations in the diploid genetic pool and a few admixed cultivars. These results will be very useful for rationalizing D. alata genetic resources in breeding programs across different regions and for improving germplasm conservation methods.