Traditionally, CIAT has focused on breeding for biotic stress resistance, especially for diseases and pests. New emphasis is being placed on breeding varieties for higher nutritional value under abiotic stresses. Several projects are currently under way to either develop and implement the needed genomic tools or incorporate genomic tools with breeding activities.

Among CIAT's priorities in genomic research are the use of existing marker resources in rice and beans, and the development of new PCR-based markers for beans, cassava, and Brachiaria grasses. Two main marker types have been developed at CIAT: sequence-characterized amplified region (SCAR) markers, and microsatellites or simple sequence repeats (SSRs). SSRs kits have been developed for beans, cassava, and Brachiaria grasses. Other marker systems such as AFLPs and RAPDs have been used for fine mapping; increasing the saturation of the common bean, cassava, and Brachiaria maps; and studying the diversity of bean and cassava gene pools.

Genetic Mapping

Common Bean

All new markers are mapped onto CIAT's principal mapping population, which now contains over 500 markers. A set of microsatellites is being put together to efficiently map other populations. Linked markers were identified for the bean golden mosaic virus, anthracnose, bacterial blight, and angular leaf spot, and are being used as part of a marker-assisted selection (MAS) program. Other mapping populations have been developed and are being used to tag quantitative trait loci (QTLs) for abiotic stress tolerance (e.g., low phosphorus, Al toxicity, and drought), micronutrient content (iron and zinc), and disease-and-pest resistance. Several of these CIAT populations are being analyzed in Belgium, Brazil, France, Germany, Mexico, and USA.

Cassava

A genetic linkage map of cassava was constructed, using an intraspecific F1 cross. To construct a male- and female-derived molecular genetic map, we used 150 RFLPs, 30 RAPDs, 110 SSRs, and 3 isoenzyme markers, segregating as single-dose restriction fragments (SDRFs). An additional 80 SSRs have since been added. Linked markers were identified for cassava mosaic disease (CMD), the most important disease of cassava in Africa and a potential threat to the crop in Latin America; cassava bacterial blight (CBB), caused by Xanthomonas axonopodis pv. manihotis and the most important disease of cassava (Manihot esculenta Crantz) worldwide; and early bulking. Cloning of the gene for resistance to CMD is ongoing.

Rice

Three mapping projects are under way: (1) fine mapping of rice blast and cloning of a Pi gene; (2) rice "hoja blanca" virus; and (3) advanced backcross mapping on several populations derived from crosses between elite rice genotypes and Oryza rufipogon, O. barthii, and O. glaberrima. The advanced backcrossing concentrates on QTL identification for agronomic traits. A MAS for rice blast is being implemented.

Brachiaria Grasses

SSR markers were developed and mapped along AFLP and rice RFLP for Brachiaria populations at CIAT for resistance to spittlebug, apomixis, and Al tolerance.

Genomic Libraries

To help develop additional microsatellite and SCAR markers, the Biotechnology Unit at CIAT has made several types of genomic libraries, including microsatellite-enriched libraries and unenriched total genomic libraries.

Common Bean

CIAT has made leaf cDNA libraries for common bean from a genotype that tolerates low phosphorus levels in soils and has resistance to multiple diseases, including anthracnose and angular leaf spot. More than 140,000 clones have been plated and picked into 384-well plates. Root cDNA libraries have been made from adventitious and basal roots grown under phosphorus deficiency. About 4000 clones from the libraries have been sequenced so far. Many of the expressed sequence tags (ESTs) have homologs in the soybean database.

Cassava

A project, carried out in collaboration with IRD and CNRS, both of France, is generating ESTs from four cDNA libraries constructed by using mRNA from a CBB-resistant genotype and a high starch genotype. A second project on SAGE for CMD is under way in collaboration with the Iwate Biotechnology Research Center in Japan. So far, more than 4000 ESTs have been developed for SAGE annotation.

Brachiaria Grasses

Root cDNA libraries are being developed from Al-resistant and susceptible parents to monitor gene expression patterns in root apices and to identify candidate genes for Al resistance.

Resistance Gene Analogs

CIAT has generated and analyzed resistance gene analogs, using degenerate primers for NBS-LRR, TIR, and P-loop regions for rice, common bean, cassava, and Brachiaria.

Insertion Mutagenesis Populations

CIAT is collaborating with Steve Dellaporta of Yale University on an Ac/Ds project on rice, funded by the USDA. We have recently, in conjunction with CIRAD and IRD, begun the evaluation of T-DNA rice population.

Bioinformatics and Database

CIAT is part of a consortium of CGIAR centers to develop bioinformatics tools linking mapping, QTL analysis, and germplasm evaluation. Emphasis is given to creating databases for managing genotype and genetic mapping information and establishing a sequence storage. Molecular marker data are being updated in the BeanGenes AceDB and CassavaDB Database and NCBI Genbank Database. A Laboratory Information Management System (LIMS) is in its final stage of development.

Microarray and Single Nucleotide Polymorphism Markers

CIAT established, in mid-2001, a DNA microarray facility that will develop new genetic marker systems based on the diversity array system, known as DarT and developed at CAMBIA, and generate gene expression DNA chips for biotic-abiotic stresses for beans, cassava, and Brachiaria spp. Single nucleotide polymorphism (SNP) markers are being developed for beans.

Outcomes of this research are expected to include the efficient development of varieties; better conservation of the germplasm bank, using the genomics approach; and development of genomic tools for beans and cassava, two crops that are receiving less attention than other commercial crops.

Future Plans

• To fully implement an automated, high throughput of markers for breeding and germplasm characterization
• Develop rice "knockout" populations, and clone-targeted genes
• Develop and use gene expression chips for biotic and abiotic stresses
• Develop a Euphorbiaceae chip
• Participate in a legume chip effort
• Strengthen the bioinformatics component
• Contribute to CIAT's soil microbial research
• Provide training for national programs.

Breeding-genomic Team

Steve Beebe, Daniel Debouck, César Martínez, Idupulapati Rao, Matthew Blair, Myriam Duque, Chike Mba, Silvia Restrepo,
Hernán Ceballos, Martin Fregene, John Miles, Joe Tohme