Joe Tohme

Provitamin A Accumulation in Cassava (Manihot esculenta) Roots Driven by a Single Nucleotide Polymorphism in a Phytoene Synthase Gene

Cassava is a very important staple crop, especially in the arid tropics where it is a chief source of carbohydrates, but the provitamin A content in the storage root is insufficient to sustain a healthy life. The work presented shows that a single amino acid exchange in a conserved region of the enzyme phytoene synthase leads to substantive accumulation of β-carotene (provitamin A) in the roots. Cassava (Manihot esculenta) is an important staple crop, especially in the arid tropics. Because roots of commercial cassava cultivars contain a limited amount of provitamin A carotenoids, both conventional breeding and genetic modification are being applied to increase their production and accumulation to fight vitamin A deficiency disorders. We show here that an allelic polymorphism in one of the two expressed phytoene synthase (PSY) genes is capable of enhancing the flux of carbon through carotenogenesis, thus leading to the accumulation of colored provitamin A carotenoids in storage roots. A single nucleotide polymorphism present only in yellow-rooted cultivars cosegregates with colored roots in a breeding pedigree. The resulting amino acid exchange in a highly conserved region of PSY provides increased catalytic activity in vitro and is able to increase carotenoid production in recombinant yeast and Escherichia coli cells. Consequently, cassava plants overexpressing a PSY transgene produce yellow-fleshed, high-carotenoid roots. This newly characterized PSY allele provides means to improve cassava provitamin A content in cassava roots through both breeding and genetic modification.

Through the genetic bottleneck: O. rufipogon as a source of trait-enhancing alleles for O. sativa

This paper summarizes results from a decade of collaborative research using advanced backcross (AB) populations to a) identify quantitative trait loci (QTL) associated with improved performance in rice and to b) clone genes underlying key QTLs of interest. We demonstrate that AB-QTL analysis is capable of (1) successfully uncovering positive alleles in wild germplasm that were not obvious based on the phenotype of the parent (2) offering an estimation of the breeding value of exotic germplasm, (3) generating near isogenic lines that can be used as the basis for gene isolation and also as parents for further crossing in a variety development program and (4) providing gene-based markers for targeted introgression of alleles using marker-assisted-selection (MAS). Knowledge gained from studies examining the population structure and evolutionary history of rice is helping to illuminate a long-term strategy for exploiting and simultaneously preserving the well-partitioned gene pools in rice.

Cross-species amplification of cassava (Manihot esculenta) (Euphorbiaceae) microsatellites: allelic polymorphism and degree of relationship

Microsatellite amplification was performed on cassava (Manihot esculenta) and six other different species (all wild) of the Manihot genus. We used ten pairs of microsatellite primers previously developed from cassava, detecting 124 alleles in a sample of 121 accessions of the seven species. The number of alleles per locus ranged from four to 21 alleles, and allelic diversity was greater in the wild species than in cassava. Seventy-nine alleles, including unique ones, were detected in the wild species but were not found in the crop. The lower level of heterozygosity in some wild species probably resulted from a combination of fine-scale differentiation within the species and the presence of null alleles. Overall, microsatellite primers worked across the genus, but, with increasing genetic distance, success in amplifying loci tended to decrease. No accession of M. aesculifolia, M. carthaginensis, and M. brachyloba presented a banding pattern at locus Ga-140; neither did one appear for M. aesculifolia at locus Ga-13. Previous work with amplified fragment length polymorphism (AFLP) markers and this microsatellite analysis show that these three wild taxa are the most distant relatives of the crop, whereas the wild forms M. esculenta subsp. flabellifolia and M. esculenta subsp. peruviana appear to be the closest.

Identifying resistance gene analogs associated with resistances to different pathogens in common Bean

ABSTRACT A polymerase chain reaction approach using degenerate primers that targeted the conserved domains of cloned plant disease resistance genes (R genes) was used to isolate a set of 15 resistance gene analogs (RGAs) from common bean (Phaseolus vulgaris). Eight different classes of RGAs were obtained from nucleotide binding site (NBS)-based primers and seven from not previously described Toll/Interleukin-1 receptor-like (TIR)-based primers. Putative amino acid sequences of RGAs were significantly similar to R genes and contained additional conserved motifs. The NBS-type RGAs were classified in two subgroups according to the expected final residue in the kinase-2 motif. Eleven RGAs were mapped at 19 loci on eight linkage groups of the common bean genetic map constructed at Centro Internacional de Agricultura Tropical. Genetic linkage was shown for eight RGAs with partial resistance to anthracnose, angular leaf spot (ALS) and Bean golden yellow mosaic virus (BGYMV). RGA1 and RGA2 were associated with resistance loci to anthracnose and BGYMV and were part of two clusters of R genes previously described. A new major cluster was detected by RGA7 and explained up to 63.9% of resistance to ALS and has a putative contribution to anthracnose resistance. These results show the usefulness of RGAs as candidate genes to detect and eventually isolate numerous R genes in common bean.

The use of geographical information systems in biodiversity exploration and conservation

We describe a method for applying geographical information systems (GIS) to exploring biodiversity in the wild relatives of crop species and illustrate its application to the wild common bean (Phaseolus vulgaris L.). We use the latitude, longitude and altitude of the location of origin of each accession in a germplasm collection of wild P. vulgaris, along with long-term monthly mean values of rainfall, temperature and diurnal temperature range for about 10000 stations throughout Latin America to produce maps indicating areas with ‘bean-favouring’ climates. In a test case, these identify a new suitable area in Colombia where wild P. vulgaris has been reported in the literature, and two more areas which are strong candidates on other grounds. Dividing the ‘bean-favouring’ climates into clusters identifies areas that have similar climates but are geographically remote, where we can expect to find wild beans with similar ecological adaptation. We discuss the implications of these results for conserving and improving the common bean, and the application of these methods to other species.

Sequencing analysis of 20,000 full-length cDNA clones from cassava reveals lineage specific expansions in gene families related to stress response

BackgroundCassava, an allotetraploid known for its remarkable tolerance to abiotic stresses is an important source of energy for humans and animals and a raw material for many industrial processes. A full-length cDNA library of cassava plants under normal, heat, drought, aluminum and post harvest physiological deterioration conditions was built; 19968 clones were sequence-characterized using expressed sequence tags (ESTs).ResultsThe ESTs were assembled into 6355 contigs and 9026 singletons that were further grouped into 10577 scaffolds; we found 4621 new cassava sequences and 1521 sequences with no significant similarity to plant protein databases. Transcripts of 7796 distinct genes were captured and we were able to assign a functional classification to 78% of them while finding more than half of the enzymes annotated in metabolic pathways in Arabidopsis. The annotation of sequences that were not paired to transcripts of other species included many stress-related functional categories showing that our library is enriched with stress-induced genes. Finally, we detected 230 putative gene duplications that include key enzymes in reactive oxygen species signaling pathways and could play a role in cassava stress response features.ConclusionThe cassava full-length cDNA library here presented contains transcripts of genes involved in stress response as well as genes important for different areas of cassava research. This library will be an important resource for gene discovery, characterization and cloning; in the near future it will aid the annotation of the cassava genome.

Genetic diversity of traditional South American landraces of cassava (Manihot esculenta Crantz): An analysis using microsatellites

The extent and structure of the genetic variability of traditional varieties of cassava (Manihot esculenta Crantz) have been little documented, despite considerable evidence for this cropś great varietal diversity in traditional agroecosystems. We used microsatellite markers to assess the genetic structure of traditional landraces of sweet and bitter cassava collected from five South American sites. As reference, we used a sample of 38 accessions from a world collection of cultivated cassava. For a total of 10 loci examined, we found 15 alleles that were not represented in this sample. Ten of these had been previously detected in wild Manihot species. The geographical structure of genetic variability was weak, but the genetic differentiation between bitter and sweet landraces was significant, suggesting that each form had evolved separately after domestication. Our results showed that traditional landraces form an important source of genetic diversity and merit more attention from managers of crop genetic resources.RésuméA extensão e a estruturação da variabilidade genética de variedades tradicionais de mandioca (Manihot esculenta Crantz) têm sido pouco documentadas, apesar de existirem evidências sugerindo uma grande diversidade varietal desta cultura em agroecosistemas tradicionais. No presente trabalho, foram usados marcadores de DNA, do tipo microssatélite, para avaliar a estrutura genética de variedades tradicionais de mandioca brava e mandioca de mesa coletadas em cinco localidades da América do Sul. Como referência, usou-se um conjunto de 38 acessos de uma coleção mundial de germoplasma de mandioca. Entre as variedades tradicionais, foram encontrados 15 alelos que não estavam presentes nesta amostragem da coleção mundial. Dez destes alelos já haviam sido detectados em espécies silvestres de Manihot. Apenas uma leve estruturação geográfica da variabilidade foi observada. No entanto, foi evidenciada uma diferenciação genética entre variedades bravas e de mesa, sugerindo que cada forma tenha evoluído separadamente após a domesticaç~ao. Nossos resultados mostram que variedades tradicionais constituem uma importante fonte de diversidade genética e deveriam receber maior atenção no manejo de recursos genéticos de plantas cultivadas.

Application of genomics-assisted breeding for generation of climate resilient crops: progress and prospects

Climate change affects agricultural productivity worldwide. Increased prices of food commodities are the initial indication of drastic edible yield loss, which is expected to increase further due to global warming. This situation has compelled plant scientists to develop climate change-resilient crops, which can withstand broad-spectrum stresses such as drought, heat, cold, salinity, flood, submergence and pests, thus helping to deliver increased productivity. Genomics appears to be a promising tool for deciphering the stress responsiveness of crop species with adaptation traits or in wild relatives toward identifying underlying genes, alleles or quantitative trait loci. Molecular breeding approaches have proven helpful in enhancing the stress adaptation of crop plants, and recent advances in high-throughput sequencing and phenotyping platforms have transformed molecular breeding to genomics-assisted breeding (GAB). In view of this, the present review elaborates the progress and prospects of GAB for improving climate change resilience in crops, which is likely to play an ever increasing role in the effort to ensure global food security.

BAC-end Sequence Analysis and a Draft Physical Map of the Common Bean ( Phaseolus vulgaris L.) Genome

Common bean (Phaseolus vulgaris L.) is a legume that is an important source of dietary protein in developing countries throughout the world. Utilizing the G19833 BAC library for P. vulgaris from Clemson University, 89,017 BAC-end sequences were generated giving 62,588,675 base pairs of genomic sequence covering approximately 9.54% of the genome. Analysis of these sequences in combination with 1,404 shotgun sequences from the cultivar Bat7 revealed that approximately 49.2% of the genome contains repetitive sequence and 29.3% is genic. Compared to other legume BAC-end sequencing projects, it appears that P. vulgaris has higher predicted levels of repetitive sequence, but this may be due to a more intense identification strategy combining both similarity-based matches as well as de novo identification of repeats. In addition, fingerprints for 41,717 BACs were obtained and assembled into a draft physical map consisting of 1,183 clone contigs and 6,385 singletons with ~9x coverage of the genome.

A unigene catalogue of 5700 expressed genes in cassava

Two economically important characters, starch content and cassava bacterial blight resistance, were targeted to generate a large collection of cassava ESTs. Two libraries were constructed from cassava root tissues of varieties with high and low starch contents. Other libraries were constructed from plant tissues challenged by the pathogen Xanthomonas axonopodis pv.manihotis. We report here the single pass sequencing of 11 954 cDNA clones from the 5’ ends, including 111 from the 3’ ends. Cluster analysis permitted the identification of a unigene set of 5700 sequences. Sequence analyses permitted the assignment of a putative functional category for 37% of sequences whereas ~ 16% sequences did not show any significant similarity with other proteins present in the database and therefore can be considered as cassava specific genes. A group of genes belonging to a large multigene family was identified. We characterize a set of genes detected only in infected libraries putatively involved in the defense response to pathogen infection. By comparing two libraries obtained from cultivars contrasting in their starch content a group of genes associated to starch biosynthesis and differentially expressed was identified. This is the first large cassava EST resource developed today and publicly available thus making a significant contribution to genomic knowledge of cassava.