New evidence on the nutritional and functional benefits of biofortified crops

Unravelling the genetic mechanism of high betacarotene acumulation in cassava

Luis Augusto Becerra Lopez-Lavalle1,2, Tatiana Ovalle1, Claudia Perea1, Meike Anderson2, Hernan Ceballos1

1 Centro Internacional de Agricultura Tropical (CIAT), Cali, Colombia. 2 CGIAR Research Program on Roots Tubers and Bananas

Introduction: Nearly two billion poor people suffer micronutrient deficiencies. Africa is the continent with the highest percentage of people affected. A strategy to efficiently and cost effectively mitigate micronutrient deficiencies is to genetically improve staple crops to increase their nutritional content. Cassava (Manihot esculenta Crantz) is a staple food for more than 500 million people in the world. In the past 10 years, CIAT in collaboration with HarvestPlus has made continuous efforts to increase the content of beta-carotene [provitamin A (pVA)] in cassava. In cassava, pVA content is naturally very low and likely to be controlled by several genes. To accelerate the selection of high pVA cassava genotypes, we are developing a SNP-based genetic map to unravel the genetic location of high pVA and characterize all other genes involved in its accumulation, as well as, unravel the mechanism of its control. The use of next generation sequencing for SNP discovery and whole genome characterization, offers to expand the crop’s potential for genomic exploration of genetic features that will not allow elucidating the chromosomal location of high pVA. Two pseudo F2 high pVA segregating populations (GM372 and GM3736) were established at CIAT and over three years evaluated for pVA segregation using colorimetry, NIRS and HPLC methodology. The association of the biochemical data with the RAD sequencing based genetic map and variability of the genetic information of the gene(s) responsible of the b-carotenoid pathway through Whole Genome Sequencing will allow us to develop molecular markers linked with high pV A in cassava. keywords: Next Generation Secuencing, Beta-carotene, Cassava. Next generation Sequencing (RAD-seq): Development of a high pVA genetic map Currently two pseudo-F2 mapping populations, with the highest pVA accumulation potential, have been selected to develop SNP based molecular markers that will enable identifying the position of high pVA in this study. Nearly 11,000 SNPs markers have been mined from a reduced representation RAD sequencing libraries to construct a pVA molecular map aiming at determining the location of high pVA loci. The rapid discovery of thousands of SNPs is a fundamental step toward the reconstructions of chromosomes including ordering and distance calculation of the loci defined by these SNP markers. Using JoinMap® v4, we constructed a 17 linkage groups SNP-based ultra-dense genetic map. This genetic map will be fully annotated with against the cassava reference genome (Van Ooijen 2006, Prochnik, et al. 2012).’ Quantitative genetic analysis of high pVA content in cassava. CIAT’s breeding program has advanced with the identification of cassava genotypes with high β-carotene using the classical mass recurrent selection approach with a minor modification (Ceballos, et al. 2013). This method is not only used for pVA, but for breeding any other agronomical important traits in this crop. Genotypes with higher levels of pVA, developed using conventional cassava breeding methods have now reached its maximum at around 15μg of β-carotene. Molecular markers might offer to accelerate the process for selecting parents with high pVA potential. The broad sense heritability of the pVA trait; which is an important input to estimate QTL regions have been estimated at nearly 70%. Also, we measured the difference in levels of several carotenoids and compare it against those obtained from botanical seed (un-replicated) and the first clonally propagated material (replicated)..These results confirm the stability of the carotenoid accumulation in cassava. Data analysis showed that the expression of carotenoid is robust over time on one location is stable both for total carotenoid, as well as, β-carotene. Unravel the genetic mechanism of Carotenoid accumulation - Molecular Marker discovery Our result from three years of phenotypic characterization showed that the variability of β-carotene in cassava is quantitative in nature and very stable. Similarly, the accumulation of dry matter does not influence the way in which β-carotene is accumulated in the cassava roots. The next step in this process is to undertake a quantitative trait loci (QTL) analysis using the GM3732 and GM3736 segregating progeny of CR87 and BRA1A. This will identify specific genomic regions in the cassava genome were import genes for high pVA accumulation are located and mark their location with molecular markers. With an understanding of the number of genes involved in high pVA accumulation, how they interact and where there are located in the genome, cassava breeders can maximize the genetic potential of their breeding lines, insuring that the resistance gains made to date will continue.