Application of the latest molecular marker technology to cassava improvement greatly enhances the ongoing breeding effort aimed at increasing productivity for small holder farmers of SSA.
As a prelude to molecular marker assisted breeding, IITA has embarked on the development of a vast range of genetic, genomic, and breeding resources.
Development of markers for major diseases and quality traits that are expensive and cumbersome to measure
Summary of completed work
Mapping a different source of R gene for the various variants cassava mosaic virus, namely, ACMV, EACMV and UGV. Phenotype data has been collected for four populations segregating for CMD resistance. Mapping population derived from TMS 30555 (S-improved) x TME 4 (R-land race) will be utilised for mapping an additional source of resistance. Mapping populations will be developed for the east-African variants.
The first endeavor in developing and utilizing molecular markers in cassava improvement was targeted to improve CMD resistance. A number of RF funded projects were carried out to halt the threat posed by the cassava mosaic disease (CMD), the most important disease of cassava in Africa, which is caused by any one or a combination of the whitefly transmitted cassava mosaic begomoviruses. In the latest project, the main objective of the project was to tag all sources of natural resistance to CMD and utilize it to pyramid CMD resistant genes in cassava germplasm. Molecular markers developed will be used to combine different sources of resistance genes into elite parental lines for use in developing durable disease and pest resistant germplasm for several African countries. The end result will be establishment of a protocol for molecular marker-assisted pyramiding of resistance genes.
Cuttings derived from crosses and selfed progenies were planted in early July, 2007 at two locations,and Onne, in Nigeria to establish mapping populations and experiments for allelism tests. Field evaluation of host plant resistance to CMD and other traits will be performed as from August, 2007.
Mapping populations developed using different source of CMD resistance genes have been developed. Genotyping of these mapping populations will be carried out while the plants await field evaluation for phenotyping of disease and other agronomic traits. The result from the resistance profiles, test of allelism and host-plant resistance evaluation will be used for selection and hybridization of parental materials via marker-assisted selection (MAS).
Resistance profiling of the parental clones for their response to various cassava begomoviruses/strains from Africa and India, and cassava brown streak viruses/strains from east and central Africa, is still in progress. This task is being undertaken by the German Collection of Microorganisms and Cell Cultures (DSMZ), Plant Virus Division, Braunschweig, Germany (Dr Stephan Winter’s Lab) since we cannot introduce different strains and variants of these viruses into Nigeria. In the last report, resistance profiles of several genotypes were reported. Additional information has indicated that a good number of improved IITA varieties and resistant African landraces respond with recovery from infections with ACMV or ICMV as a non symptomatic phase of infection. Most of the genotypes were susceptible to EACMV species. TME 4, 96/1089A and 96/0529A are excellent sources of resistance with differential resistance characters - resistance is directed against all cassava viruses and their strain(s) including ICMV and /or SLCMV infections in India>
To pyramid all the above CMD resistance genes via marker-assisted selection (MAS) and three cycles of genetic crosses without resort to field evaluations and to select recombinants before flowering.
Sequence alignment of cloned plant R genes and analysis of structure of the R genes revealed that most R genes encode homologous proteins with similar domains. The most common domains are the nucleotide-binding site (NBS) and leucine-rich repeats (LRR). The NBS-LRR containing proteins that contain an amino-terminal TIR (Toll/interleukin receptor) are termed TIR-NBS-LRR, whereas those with an coiled-coil (CC) amino-terminal are called CC-NBS-LRR. These conserved regions have been utilized to design degenerate primer for amplification of putative resistance gene analogs (RGAs) (Gedil, 2001, Radwan, 2005). Such homology-based identification of RGAs have been successfully utilized as short-cut method of resistance gene tagging. Therefore, we are planning to use the RGA approach to develop putative markers for BSA analysis. Degenerate primers were designed in the regions of TIR-NBS-LRR and CC-NBS-LRR based on published sequences of plant R genes. Oligos have been ordered recently. In cassava, cloning of RGA has been done using degenerate primers derived from three motifs (NBS, LRR, Protein kinase) with the aim of identifying marker for CBB resistance (citation).
Objective
Cassava breeding is constrained by limited genetic variation for value-added (speciality) traits, resistance to diseases, and important post-harvest traits. The present breeding work largely depends on African germplasm collection and Latin-American introductions. With the aim of broadening the genetic base of the germplasm pool and expanding the industrial uses of cassava, the program embarked on radiation mutation of selected elite lines as well as wide crosses involving wild Manihot species and castor bean. In collaboration with IAEA, three different methods of irradiation have been used for inducing mutations to effect subtle changes to the genetic make-up of half-sib seeds obtained in a polycross scheme of elite, well-adapted cassava varieties from IITA and NARS. While evaluation of the radiation mutant is underway, as an integral part of IITA’s cassava breeding program, we have initiated a pilot study on EMS-induced mutation. Detailed phenotypic and genotypic characterization of mutants entails development and application of cost-effective high throughput screening technologies such as TILLING (McCallum et al. 2000). In this project, we are proposing to perform traditional chemical mutagenesis that can directly be applied to cassava improvement via mutation breeding followed by TILLING, a practical approach for rapid discovery of induced as well as natural polymorphisms in plants.
The number of mutants to be screened is limited by the extent of funding in this project. Large number of seeds has to be mutagenized to discover a mutation in a particular gene, for instance, in beta carotene gene. The focus of this project will be a single trait. Once the protocol is established at the end of the project, it can be easily adapted to discover mutations for other important traits and to other IITA mandate crops. TILLING has been successfully applied in different crops as demonstrated by several recent publications and ongoing activities in various programs. Scientists and donors alike are very well aware of the potential of this robust technology of reverse genetics.
Chemically induced point mutations provide genetic variation of otherwise intractable traits such as beta-carotene, protein content, improved starch quality and quantity, antinutritional factors, and resistance to diseases (e.g. CBSD, CBB) thereby broadening the genetic base.
Access to gene technologies for the improvement of cassava through genetic modification or for molecular marker based cassava improvement is a constraint, since most genes or technologies have been developed and are owned by other institutions. In some cases, the technologies are protected by intellectual property rights with inherent limitations on access and use.
DNA microarrays are an established tool for gene expression profiling in plants and animals (Rensink and Buell, 2005; Zhu T, Salmeron J, 2007). The basic principle of DNA microarray is the hybridization of samples to immobilized DNA molecules with tremendous speed (Schena, 2000). For example, comparisons can be made between control and diseased or stressed tissues and subjected to microarray analysis to detect differentially expressed genes. These genes can then be isolated from the cDNA library and utilized to develop molecular markers or further characterized for genetic transformation.
IITA, in collaboration with USDA-Fargo, has developed and characterized a cassava EST collection of 18,166 sequences. Analysis of a total of 18,166 ESTs resulted in the identification of 8,577 unique gene clusters comprising 5,383 singletons and 3,194 clusters. The unigene clusters were screened for the presence of short tandem repeats for further development as microsatellite markers. A total of 592 clusters contained 645 repeats, representing 3.3% of the ESTs queried. Stretches of di-, tri-, and tetrameric nucleotide repeats were identified using parameters that would detect dimeric motifs with seven or more repeats, trimeric motifs with six or more repeats, and tetra- or pentameric motifs with four or more repeats. Perfect and near perfect repeats with slight repeat pattern deviations were scored. This allows for the possibility that there may be a perfect repeat pattern at a locus within different cassava varieties and cultivars. This figure is similar to the rate of microsatellite discovery in other species such as grape, sugarcane and switchgrass where the frequency of EST-derived SSRs was between 2.5 and 3.8%. We detected 186 perfect di-nucleotides, 131 perfect tri-nucleotides, 1 perfect tetra-nucleotide, and 2 perfect penta-nucleotides. In addition, there were 264 imperfect di-nucleotides, 57 imperfect tri-nucleotides, 4 imperfect tetra-nucleotides and 1 imperfect penta-nucleotide. The different classes of EST-SSRs are summarized in Table 4. Dinucleotide repeats represented 70% of the total number of microsatellites. Of these (TC)n and (AT)n were the most common. Trinucleotide repeats were also detected with (GAA)n repeats being the most common class. Only one (imperfect) (GC)n repeat was returned and tetra- and pentanucleotide repeats were also uncommon (Lokko et al., 2007).
A total of 346 primer pairs could be designed from this non-redundant EST-SSR set, after eliminating microsatellite loci with insufficient flanking sequences, duplicates of previously developed cassava SSR (provided by M. Fregene, CIAT) and removal of identical ESTs (in some cases more than one microsatellite locus was present in the same EST). Primers were designed using the Primer3 software with the following parameters: annealing temperature 57-63oC; product size 100-400bp; primer size 18-28 bases; GC content 20-80%.
In 2007, an oligo DNA chip was designed and a DNA chip comprising approximately 14,000 unigenes will become available in 2007.
Genomic resources available in Public databases (insert NCBI output here)
EST clustering/unigene
SNP development
Alternative markers - COS
Genome sequence
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