[Show abstract][Hide abstract] ABSTRACT: Cassava, originally from South America, is the fourth most important source of calories in the developing world after the cereal crops wheat,maize, and rice.Worldwide, it feeds an estimated 700 million people directly or indirectly. Cassava production has increased steadily for the last 50 years, with 242 MT harvested in 2012. The increase is likely to continue as farmers in more than 105 countries come to recognize the crop’s advantages. A semi-perennial root crop, cassava can stay in the ground for up to 3 years. This makes it an excellent food security crop: when all other crops have been exhausted, cassava roots can still be
harvested. It is naturally drought resistant and resilient to climatic changes, high temperatures, and poor soils, and in addition, cassava responds extremely well to high CO2 concentrations, making it a very important crop for the 21st century. Africa alone accounts for more than 55 % of the world’s production, and cassava is the first food crop in fresh tonnage before maize and plantain in sub-Saharan Africa. Cassava is also an important source of income, especially for women in sub-Saharan Africa (SSA). Furthermore, cassava is the second most important source of starch in the world. Cassava is thus a highly valuable crop
for the world today and in the future. It is critical that it should not be compromised by viral diseases.
[Show abstract][Hide abstract] ABSTRACT: Since their initial discovery, transposons have been widely used as mutagens for forward and reverse genetic screens in a range of organisms. The problems of high copy number and sequence divergence among related transposons have often limited the efficiency at which tagged genes can be identified. A method was developed to identity the locations of Mutator (Mu) transposons in the Zea mays genome using a simple enrichment method combined with genome resequencing to identify transposon junction fragments. The sequencing library was prepared from genomic DNA by digesting with a restriction enzyme that cuts within a perfectly conserved motif of the Mu terminal inverted repeats (TIR). Paired-end reads containing Mu TIR sequences were computationally identified and chromosomal sequences flanking the transposon were mapped to the maize reference genome. This method has been used to identify Mu insertions in a number of alleles and to isolate the previously unidentified lazy plant1 (la1) gene. The la1 gene is required for the negatively gravitropic response of shoots and mutant plants lack the ability to sense gravity. Using bioinformatic and fluorescence microscopy approaches, we show that the la1 gene encodes a cell membrane and nuclear localized protein. Our Mu-Taq method is readily adaptable to identify the genomic locations of any insertion of a known sequence in any organism using any sequencing platform.
PLoS ONE 01/2014; 9(1):e87053. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent advances in high-throughput sequencing (HTS) technologies and computing capacity have produced unprecedented amounts of genomic data that have unraveled the genetics of phenotypic variability in several species. However, operating and integrating current software tools for data analysis still require important investments in highly skilled personnel. Developing accurate, efficient and user-friendly software packages for HTS data analysis will lead to a more rapid discovery of genomic elements relevant to medical, agricultural and industrial applications. We therefore developed Next-Generation Sequencing Eclipse Plug-in (NGSEP), a new software tool for integrated, efficient and user-friendly detection of single nucleotide variants (SNVs), indels and copy number variants (CNVs). NGSEP includes modules for read alignment, sorting, merging, functional annotation of variants, filtering and quality statistics. Analysis of sequencing experiments in yeast, rice and human samples shows that NGSEP has superior accuracy and efficiency, compared with currently available packages for variants detection. We also show that only a comprehensive and accurate identification of repeat regions and CNVs allows researchers to properly separate SNVs from differences between copies of repeat elements. We expect that NGSEP will become a strong support tool to empower the analysis of sequencing data in a wide range of research projects on different species.
Nucleic Acids Research 01/2014; · 8.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Many areas critical to agricultural production and research, such as the breeding and trait mapping in plants and livestock, require robust and scalable genotyping platforms. Genotyping-by-sequencing (GBS) is a one such method highly suited to non-human organisms. In the GBS protocol, genomic DNA is fractionated via restriction digest, then reduced representation is achieved through size selection. Since many restriction sites are conserved across a species, the sequenced portion of the genome is highly consistent within a population. This makes the GBS protocol highly suited for experiments that require surveying large numbers of markers within a population, such as those involving genetic mapping, breeding, and population genomics. We have modified the GBS technology in a number of ways. Custom, enzyme specific adaptors have been replaced with standard Illumina adaptors compatible with blunt-end restriction enzymes. Multiplexing is achieved through a dual barcoding system, and bead-based library preparation protocols allows for in-solution size selection and eliminates the need for columns and gels.
A panel of eight restriction enzymes was selected for testing on B73 maize and Nipponbare rice genomic DNA. Quality of the data was demonstrated by identifying that the vast majority of reads from each enzyme aligned to restriction sites predicted in silico. The link between enzyme parameters and experimental outcome was demonstrated by showing that the sequenced portion of the genome was adaptable by selecting enzymes based on motif length, complexity, and methylation sensitivity. The utility of the new GBS protocol was demonstrated by correctly mapping several in a maize F2 population resulting from a B73 × Country Gentleman test cross.
This technology is readily adaptable to different genomes, highly amenable to multiplexing and compatible with over forty commercially available restriction enzymes. These advancements represent a major improvement in genotyping technology by providing a highly flexible and scalable GBS that is readily implemented for studies on genome-wide variation.
[Show abstract][Hide abstract] ABSTRACT: Seed sterility and grain discoloration limit rice production in Colombia and several Central American countries. In samples of discolored rice seed grown in Colombian fields, the species Burkholderia glumae and B. gladioli were isolated, and field isolates were compared phenotypically. An artificial inoculation assay was used to determine that while both bacterial species cause symptoms on rice grains, B. glumae is a more aggressive pathogen, causing yield reduction and higher levels of grain sterility. To identify putative virulence genes differing between B. glumae and B. gladioli, four previously sequenced genomes of Asian and US strains of the two pathogens were compared with each other and with two draft genomes of Colombian B. glumae and B. gladioli isolates generated for this study. While previously characterized Burkholderia virulence factors are highly conserved between the two species, B. glumae and B. gladioli strains are predicted to encode distinct groups of genes encoding type VI secretion systems, transcriptional regulators, and membrane sensing proteins. This study shows that both B. glumae and B. gladioli can threaten grain quality although only one species affects yield. Furthermore, genotypic differences between the two strains are identified that could contribute to disease phenotypic differences.
[Show abstract][Hide abstract] ABSTRACT: The study of microRNAs (miRNAs) in plants has gained significant attention in recent years due to their regulatory role during development and in response to biotic and abiotic stresses. Although cassava (Manihot esculenta Crantz) is tolerant to drought and other adverse conditions, most cassava miRNAs have been predicted using bioinformatics alone or through sequencing of plants challenged by biotic stress. Here, we use high-throughput sequencing and different bioinformatics methods to identify potential cassava miRNAs expressed in different tissues subject to heat and drought conditions. We identified 60 miRNAs conserved in other plant species and 821 potential cassava-specific miRNAs. We also predicted 134 and 1002 potential target genes for these two sets of sequences. Using real time PCR, we verified the condition-specific expression of 5 cassava small RNAs relative to a non-stress control. We also found, using publicly available expression data, a significantly lower expression of the predicted target genes of conserved and nonconserved miRNAs under drought stress compared to other cassava genes. Gene Ontology enrichment analysis along with condition specific expression of predicted miRNA targets, allowed us to identify several interesting miRNAs which may play a role in stress-induced posttranscriptional regulation in cassava and other plants.
International journal of genomics. 01/2013; 2013:857986.
[Show abstract][Hide abstract] ABSTRACT: The genetic factors responsible for diversity in storage root of cassava are being studied by several groups. The few available studies is disperse and are not sufficient to accurate predictions of genetic determinant or regulation on the large diversity recently observed in our laboratory. With a microarray-based transcript we accessed the diversity of sucrose/starch conversion in sugary and pigmented cassava. A cDNA chip developed for Euphorbiacea family, based on two species (leaf spurge and cassava), was used. Out of 161 transcripts, differentially expressed among 9 representative accessions, we identified unique sub set of transcripts related to pigmented, sugary, commercial and Colombian landraces. Network of regulatory genes identified putative biological pathways that differentiate pigmented and sugary cassava from commercial cassava. Mining genes related to metabolic pathways for starch and carotenoid revealed that genes coding for enzymes in starch degradation was upregulated in sugary cassava in relation to normal cassava. At the same time it was observed that genes coding for enzymes on ABA synthesis is also highly regulated in cassava.
Second Scientific Conference of the Global Cassava Partnership for the 21st Century - GCP21-II, Kampala, Uganda; 06/2012
[Show abstract][Hide abstract] ABSTRACT: The genetic factors responsible for diversity in storage root of cassava are being studied by several groups. The few available studies is disperse and are not sufficient to accurate predictions of genetic determinant or regulation on the large diversity recently observed in our laboratory. With a microarray-based transcript we accessed the diversity of sucrose/starch conversion in sugary and pigmented cassava. A cDNA chip developed for Euphorbiacea family, based on two species (leaf
spurge and cassava), was used. Out of 161 transcripts, differentially expressed among 9 representative accessions, we
identified unique sub set of transcripts related to pigmented, sugary, commercial and Colombian landraces. Network of regulatory genes identified putative biological pathways that differentiate pigmented and sugary cassava from commercial cassava. Mining genes related to metabolic pathways for starch and carotenoid revealed that genes coding for enzymes in starch degradation was upregulated in sugary cassava in relation to normal cassava. At the same time it was observed that genes coding for enzymes on ABA synthesis is also highly regulated in cassava
Second Scientific Conference of the Global Cassava Partnership for the 21st Century, Uganda 18-22 June 2012; 06/2012
[Show abstract][Hide abstract] ABSTRACT: We report here the molecular and phenotypic features of a library of 31,562 insertion lines generated in the model japonica cultivar Nipponbare of rice (Oryza sativa L.), called Oryza Tag Line (OTL). Sixteen thousand eight hundred and fourteen T-DNA and 12,410 Tos17 discrete insertion sites have been characterized in these lines. We estimate that 8686 predicted gene intervals--i.e. one-fourth to one-fifth of the estimated rice nontransposable element gene complement--are interrupted by sequence-indexed T-DNA (6563 genes) and/or Tos17 (2755 genes) inserts. Six hundred and forty-three genes are interrupted by both T-DNA and Tos17 inserts. High quality of the sequence indexation of the T2 seed samples was ascertained by several approaches. Field evaluation under agronomic conditions of 27,832 OTL has revealed that 18.2% exhibit at least one morphophysiological alteration in the T1 progeny plants. Screening 10,000 lines for altered response to inoculation by the fungal pathogen Magnaporthe oryzae allowed to observe 71 lines (0.7%) developing spontaneous lesions simulating disease mutants and 43 lines (0.4%) exhibiting an enhanced disease resistance or susceptibility. We show here that at least 3.5% (four of 114) of these alterations are tagged by the mutagens. The presence of allelic series of sequence-indexed mutations in a gene among OTL that exhibit a convergent phenotype clearly increases the chance of establishing a linkage between alterations and inserts. This convergence approach is illustrated by the identification of the rice ortholog of AtPHO2, the disruption of which causes a lesion-mimic phenotype owing to an over-accumulation of phosphate, in nine lines bearing allelic insertions.
[Show abstract][Hide abstract] ABSTRACT: This study evaluated P. edulis f. edulis intraspecific variation at DNA level using AFLPs and SSRs. About 60 specimens were collected from Colombian commercial plantations
in the departments of Antioquia, Boyacá, Cundinamarca, Huila, Quindío and Tolima. Ten specimens were also included from Corpoica’s
La Selva research center Passiflora germplasm bank. A non-commercial purple passion fruit plant and three species from the Passiflora genus (Passiflora maliformis, Passiflora edulis f. flavicarpa and Passiflora ligularis) were included as controls. The six most informative ones were selected from the initial screening of 49 different AFLP primer
combinations; 52–92 DNA fragments for each combination in each genotype (total 419 fragments) were obtained by using these
six combinations. 17 SSR primers reported in P. edulis f. flavicarpa and Passiflora alata were tested on purple passion fruit. Eight of them were successfully amplified but none showed polymorphism. Relationships
among individuals were assessed on AFLP data using cluster analysis by Dice coefficient, UPGMA algorithm and the multiple
correspondence analysis ordination method based on Greenacre measurement. The similarity coefficients ranged from 0.75 to
1.00 giving an average of 0.96 within the different purple passion fruit materials, showing low genetic variability for the
purple passion fruit material used in this study.
Genetic Resources and Crop Evolution 01/2012; · 1.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A strong postzygotic reproductive barrier separates the recently diverged Asian and African cultivated rice species, Oryza sativa and O. glaberrima. Recently a model of genetic incompatibilities between three adjacent loci: S(1)A, S(1) and S(1)B (called together the S(1) regions) interacting epistatically, was postulated to cause the allelic elimination of female gametes in interspecific hybrids. Two candidate factors for the S(1) locus (including a putative F-box gene) were proposed, but candidates for S(1)A and S(1)B remained undetermined. Here, to better understand the basis of the evolution of regions involved in reproductive isolation, we studied the genic and structural changes accumulated in the S(1) regions between orthologous sequences. First, we established an 813 kb genomic sequence in O. glaberrima, covering completely the S(1)A, S(1) and the majority of the S(1)B regions, and compared it with the orthologous regions of O. sativa. An overall strong structural conservation was observed, with the exception of three isolated regions of disturbed collinearity: (1) a local invasion of transposable elements around a putative F-box gene within S(1), (2) the multiple duplication and subsequent divergence of the same F-box gene within S(1)A, (3) an interspecific chromosomal inversion in S(1)B, which restricts recombination in our O. sativa×O. glaberrima crosses. Beside these few structural variations, a uniform conservative pattern of coding sequence divergence was found all along the S(1) regions. Hence, the S(1) regions have undergone no drastic variation in their recent divergence and evolution between O. sativa and O. glaberrima, suggesting that a small accumulation of genic changes, following a Bateson-Dobzhansky-Muller (BDM) model, might be involved in the establishment of the sterility barrier. In this context, genetic incompatibilities involving the duplicated F-box genes as putative candidates, and a possible strengthening step involving the chromosomal inversion might participate to the reproductive barrier between Asian and African rice species.
PLoS ONE 03/2011; 6(3):e17726. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The angiosperm circadian clock has been well established from molecular-genetic studies in a temperate plant model. Conservation
of clock function is less explored in plants from the tropics. Cassava (Manihot esculenta) is a staple crop grown in the tropics that has been of limited research interest, and more generally, research on photoperiod
and clock genes has been sparse. EARLY FLOWERING 4 (AtELF4) of the temperate plant Arabidopsis thaliana (Arabidopsis) has been reported to be required for photoperiod perception and circadian function. Here, we describe our start
to identify circadian and photoperiod genes in cassava with an account on the characterization of its ELF4 gene (MeELF4). After isolating MeELF4, a phylogenetic study was conducted and it was found to cluster within the ELF4 subclade of the ELF4/EFL super-family. Similar to studies in temperate plants, MeELF4 was shown to be an evening-expressed gene in cassava. This collectively suggested to us that MeELF4 could be a functional ortholog of AtELF4. To test this, complementation studies of MeELF4 were performed in the Arabidopsis elf4 mutant. Hypocotyl-length measurements and flowering-time analysis were performed. MeELF4-complementation transgenics in the elf4 background were restored to the wild-type growth habit, suggesting a total rescue of photoperiodic perception. To expand
on the molecular role of MeELF4 in the resulting transgenic-complementation lines, the CCA1 and CCR2 promoter-luciferase markers where respectively introduced and bioluminescence-imaging experiments revealed a restoration
of circadian-regulated gene expression. The collective results showed that the cassava gene MeELF4 is a functional clock ortholog of AtELF4.
[Show abstract][Hide abstract] ABSTRACT: 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.
The Plant Cell 10/2010; 22(10):3348-56. · 9.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: MOTIVATION: We developed a technique and a tool for degenerate primer design based on multiple local alignments employing the MEME algorithm supported with electronic PCR. The objective is to find adequate primers starting from sequences with poor global similarity. We show an example of its application in our laboratory to find sequences in Brachiaria with similarity to ESTs related to apomixis.
[Show abstract][Hide abstract] ABSTRACT: Xanthomonas oryzae pathovar oryzae (Xoo) and X. oryzae pv. oryzicola (Xoc) cause bacterial diseases in rice: leaf blight and leaf streak, respectively. Although both the Asian and the African strains of Xoo induce similar symptoms, they are genetically different, with the African Xoo strains being more closely related to the Asian Xoc. To identify the sequences responsible for differences between African and Asian Xoo strains and their relatedness to Xoc strains, a suppression-subtractive hybridization (SSH) procedure was performed, using the African Xoo MAI1 strain as a tester and the Philippine Xoo PXO86 strain and Xoc BLS256 strain as drivers. A nonredundant set of 134 sequences from MAI1 was generated. Several DNA fragments isolated by SSH were similar to genes of unknown function, hypothetical proteins, genes related to the type III secretion system, and other pathogenicity-related genes. The specificity of various fragments was validated by Southern blot analysis. SSH sequences were compared with several xanthomonad genomes. In silico analysis revealed SSH sequences as specific to strain MAI1, revealing their potential as specific markers for further epidemiological and diagnostic studies. SSH proved to be a useful method for rapidly identifying specific genes among closely related X. oryzae strains.
[Show abstract][Hide abstract] ABSTRACT: S(1) is the most important locus acting as a reproductive barrier between Oryza sativa and O. glaberrima. It is a complex locus, with factors that may affect male and female fertility separately. Recently, the component causing the allelic elimination of pollen was fine mapped. However, the position and nature of the component causing female sterility remains unknown. To fine map the factor of the S(1) locus affecting female fertility, we developed a mapping approach based on the evaluation of the degree of female transmission ratio distortion (fTRD) of markers. Through implementing this methodology in four O. sativa x O. glaberrima crosses, the female component of the S(1) locus was mapped into a 27.8-kb (O. sativa) and 50.3-kb (O. glaberrima) region included within the interval bearing the male component of the locus. Moreover, evidence of additional factors interacting with S(1) was also found. In light of the available data, a model where incompatibilities in epistatic interactions between S(1) and the additional factors are the cause of the female sterility barrier between O. sativa and O. glaberrima was developed to explain the female sterility and the TRD mediated by S(1). According to our model, the recombination ratio and allelic combinations between these factors would determine the final allelic frequencies observed for a given cross.
[Show abstract][Hide abstract] ABSTRACT: Developing new population types based on interspecific introgressions has been suggested by several authors to facilitate the discovery of novel allelic sources for traits of agronomic importance. Chromosome segment substitution lines from interspecific crosses represent a powerful and useful genetic resource for QTL detection and breeding programs.
We built a set of 64 chromosome segment substitution lines carrying contiguous chromosomal segments of African rice Oryza glaberrima MG12 (acc. IRGC103544) in the genetic background of Oryza sativa ssp. tropical japonica (cv. Caiapó). Well-distributed simple-sequence repeats markers were used to characterize the introgression events. Average size of the substituted chromosomal segments in the substitution lines was about 10 cM and covered the whole donor genome, except for small regions on chromosome 2 and 4. Proportions of recurrent and donor genome in the substitution lines were 87.59% and 7.64%, respectively. The remaining 4.78% corresponded to heterozygotes and missing data. Strong segregation distortion was found on chromosomes 3 and 6, indicating the presence of interspecific sterility genes. To illustrate the advantages and the power of quantitative trait loci (QTL) detection using substitution lines, a QTL detection was performed for scored traits. Transgressive segregation was observed for several traits measured in the population. Fourteen QTLs for plant height, tiller number per plant, panicle length, sterility percentage, 1000-grain weight and grain yield were located on chromosomes 1, 3, 4, 6 and 9. Furthermore, a highly significant QTL controlling resistance to the Rice stripe necrosis virus was located between SSR markers RM202-RM26406 (44.5-44.8 cM) on chromosome 11.
Development and phenotyping of CSSL libraries with entire genome coverage represents a useful strategy for QTL discovery. Mapping of the RSNV locus represents the first identification of a genetic factor underlying resistance to this virus. This population is a powerful breeding tool. It also helps in overcoming hybrid sterility barriers between species of rice.