In planta gene expression analysis of Xanthomonas oryzae pathovar oryzae, African strain MAI1

<p>Abstract</p> <p>Background</p> <p>Bacterial leaf blight causes significant yield losses in rice crops throughout Asia and Africa. Although both the Asian and African strains of the pathogen, <it>Xanthomonas oryzae </it>pv. <it>oryzae </it>(<it>Xoo</it>), induce similar symptoms, they are nevertheless genetically different, with the African strains being more closely related to the Asian <it>X. oryzae </it>pv. <it>oryzicola </it>(<it>Xoc</it>).</p> <p>Results</p> <p>Changes in gene expression of the African <it>Xoo </it>strain MAI1 in the susceptible rice cultivar Nipponbare were profiled, using an SSH <it>Xoo </it>DNA microarray. Microarray hybridization was performed comparing bacteria recovered from plant tissues at 1, 3, and 6 days after inoculation (dai) with bacteria grown <it>in vitro</it>. A total of 710 bacterial genes were found to be differentially expressed, with 407 up-regulated and 303 down-regulated. Expression profiling indicated that less than 20% of the 710 bacterial transcripts were induced in the first 24 h after inoculation, whereas 63% were differentially expressed at 6 dai. The 710 differentially expressed genes were one-end sequenced. 535 sequences were obtained from which 147 non-redundant sequences were identified. Differentially expressed genes were related to metabolism, secretion and transport, pathogen adherence to plant tissues, plant cell-wall degradation, IS elements, and virulence. In addition, various other genes encoding proteins with unknown function or showing no similarity to other proteins were also induced. The <it>Xoo </it>MAI1 non-redundant set of sequences was compared against several <it>X. oryzae </it>genomes, revealing a specific group of genes that was present only in MAI1. Numerous IS elements were also found to be differentially expressed. Quantitative real-time PCR confirmed 86% of the identified profile on a set of 14 genes selected according to the microarray analysis.</p> <p>Conclusions</p> <p>This is the first report to compare the expression of <it>Xoo </it>genes <it>in planta </it>across different time points during infection. This work shows that as-yet-unidentified and potentially new virulence factors are appearing in an emerging African pathogen. It also confirms that African <it>Xoo </it>strains do differ from their Asian counterparts, even at the transcriptional level.</p

Análisis de la diversidad genética de accesiones de Theobroma cacao L. del banco de conservación a cargo de Corpoica

The genetic diversity present in the germplasm bank of Theobroma cacao L. conserved in the experimental station “La Suiza” of the Colombian Corporation for Agricultural Research –Corpoica– in Santander Province was studied. To that end, 100 cacao genotypes were characterized using isozymes, RFLPs, RAPDs and SSRs, using 25 microsatellites previously published in GenBank. The percent of amplification obtained was 100% which permitted identifying 168 alleles. Levels of polymorphism ranged from 2 to 14 alleles per locus, with an average of 6.72. Nei- Li’s similarity values and genetic distances among the 100 genotypes were obtained using the Ntsys® software version 2.1, and a dendrogram was constructed using the Upgma algorithm. The values obtained were greater than 0.45, and the dendrogram identified two main genetic groups and several internal subgroups. These results are an important advance in the state of knowledge of the genetic diversity of Theobroma cacao accessions preserved on germplasm banks and are very useful for developing and implementing genetic improvement and conservation programs based on genetic information and phenotypic traits favoring better quality, higher crop production and profitability in Colombia.  Se estudió la diversidad genética presente en el banco de germoplasma de Theobroma cacao L. que se conserva en la Estación Experimental ‘La Suiza’ de la Corporación Colombiana de Investigación Agropecuaria –Corpoica– en el departamento de Santander. A tal fin se caracterizaron 100 genotipos de cacao mediante isoenzimas, RFLPs, RAPDs y SSR, utilizando 25 microsatélites publicados previamente en el GenBank. El porcentaje de amplificación obtenido fue del 100% lo que permitió identificar 168 alelos. Los niveles de polimorfismo variaron entre 2 y 14 alelos por locus con un promedio de 6,72. Las tasas de similaridad y distancia genética de Nei y Li entre los 100 genotipos se obtuvieron mediante el programa Ntsys v2.1® y se construyó un dendrograma con el algoritmo Upgma. Los valores obtenidos fueron superiores a 0,45 y en el dendrograma se identificaron dos grupos genéticos principales y varios subgrupos internos. Los resultados obtenidos se consideran un avance importante en el conocimiento de la diversidad genética de accesiones de Theobroma cacao L. conservadas en bancos de germoplasma y son de gran utilidad para desarrollar e implementar programas de mejoramiento y conservación del cacao basados en información genética relativa a características fenotípicas que favorezcan una mejor calidad, mayor producción y rentabilidad del cultivo en Colombia.  

Arabidopsis LEC1 and LEC2 Orthologous Genes Are Key Regulators of Somatic Embryogenesis in Cassava

High genotype-dependent variation in friable embryogenic callus (FEC) induction and subsequent somaclonal variation constitute bottlenecks for the application and scaling of genetic transformation (GT) technology to more farmer- and industry-preferred cassava varieties. The understanding and identification of molecular factors underlying embryogenic development in cassava may help to overcome these constraints. Here, we described the Arabidopsis thaliana LEAFY COTYLEDON (LEC) LEC1 and LEC2 orthologous genes in cassava, designated as MeLEC1 and MeLEC2, respectively. Expression analyses showed that both, MeLEC1 and MeLEC2, are expressed at higher levels in somatic embryogenic (SE) tissues in contrast with differentiated mature tissues. The rapid expression increase of MeLEC genes at early SE induction times strongly suggests that they are involved in the transition from a somatic to an embryonic state, and probably, in the competence acquisition for SE development in cassava. The independent overexpression of the MeLEC genes resulted in different regenerated events with embryogenic characteristics such as MeLEC1OE plants with cotyledon-like leaves and MeLEC2OE plants with somatic-like embryos that emerged over the surface of mature leaves. Transcript increases of other embryo-specific regulating factors were also detected in MeLECOE plants, supporting their mutual interaction in the embryo development coordination. The single overexpression of MeLEC2 was enough to reprogram the vegetative cells and induce direct somatic embryogenesis, which converts this gene into a tool that could improve the recovery of transformed plants of recalcitrant genotypes. The identification of MeLEC genes contributes not only to improve our understanding of SE process in cassava, but also provides viable alternatives to optimize GT and advance in gene editing in this crop, through the development of genotype-independent protocols

Cultivos trotamundo: Una contribución del ICA y la Alianza Bioversity-CIAT para la investigación agrícola

La investigación científica y el desarrollo tecnológico es el trabajo creativo como resultado de la colaboración de múltiples instituciones, que ha existido por décadas en una gran sinergia entre el Instituto Colombiano Agropecuario (ICA) y la Alianza Bioversity-CIAT. Esta colaboración ha contribuido significativamente con la distribución y promoción alrededor del mundo de materiales genéticos de arroz, forrajes, fríjol y yuca con mejores características agronómicas, y al mismo tiempo en el mantenimiento y conservación de la diversidad de estos cultivos. Los registros de los programas de mejoramiento genético y el banco de germoplasma de la Alianza muestran que en colaboración ambas instituciones han distribuido cerca de 400.000 materiales genéticos que contienen germoplasma base, líneas avanzadas, o variedades mejoradas a diversos países en todo el mundo, siguiendo la normatividad internacional y certificación fitosanitaria emitidas por el ICA. Los resultados muestran que los materiales distribuidos han sido solicitados por una gran diversidad de instituciones en diferentes países y con múltiples propositos en la investigación y la Ingenería Agronómica. Finalmente, en este documento resaltamos los mayores logros alcanzados por la Alianza y el ICA, lo cuales han contribuido significativamente con la investigación científica y la seguridad alimentaria a nivel mundial

Análisis de expresión génica durante la respuesta de defensa de la yuca a la bacteriosis vascular (añublo bacteriano)

La bacteriosis vascular de la yuca es una destructiva enfermedad en Sur AmØrica y África, causada por la bacteria Xanthomonas axonopodis pv. manihotis (Xam). Produce pérdidas entre el 12% y 100% de los cultivos. Algunos estudios se han realizado a nivel bioquímico y citoquímico para conocer las respuestas de defensa de la yuca a Xam; sin embargo, las bases moleculares de los mecanismos de defensa no han sido aún caracterizadas. Con el propósito de identificar genes diferencialmente expresados durante la respuesta de la planta al patógeno, se ha construido una librería sustractiva, usando el método de Sustracción Diferencial en Cadena (DSC), con 1536 clones de dos variedades resis­tentes (MBRA 685 y SG 107-35). De esta librería fueron seleccionados al azar 110 clones para ser secuenciados y realizar búsquedas de similitud en bases de datos públicas. El análisis de secuencia mostró 14 clones con similitud a genes previamente reportados como involucrados en procesos de defensa en plantas, 70 clones con similitud a genes de plantas sin función conocida o que no presentaron similitud, representando nuevos genes potencialmente involucrados en las respuestas de defensa de la yuca. Finalmente fueron construidos microarreglos de ADNc, usando los clones seleccionados de las librerías sustractivas para confirmar su expresión diferencial durante el desarrollo de la infección. Palabras clave: yuca, añublo bacteriano, identificación de genes, librerías sustractivas, microarreglos.Cassava bacterial blight (CBB) caused by Xanthomonas axonopodis pv. manihotis (Xam) is a destructive disease in the South América and África and yield losses range between 12 and 100%. Cytochemistry and biochemistry of defense response to CBB have been well studied. However, the response of the plant to pathogen attack at the molecular and cellular level remains uncharacterized. Identification of genes associated with defense responses is one of most critical steps leading to the elucidation of disease resistance mechanisms in cassava. In this study, we identified differentially expressed genes during pathogen attack by subtractive hybridization, using the Differential Subtraction Chain method (DSC). A population of cDNA obtained from infected plants was used as "treatment" and a population of cDNA obtained from healthy plants was used as "control". 1536 clones were isolated from the resistant varieties (MBRA 685 and SG 107-35). Of these, 110 randomly selected clones were sequenced and a homology search was conducted. The sequence analysis showed that 14 cDNA clones shared homology with plant genes involved in defense responses, 70 clones were either homologous to plant genes of unknown function or showed no homology, representing new genes potentially involved in cassava defense responses. A cDNA microarray was constructed by spotting the clones identified from our subtractive libraries. Other clones potentially involved in cassava defense responses were also included. The cassava defense cDNA microarray was used to confirm the differential expression of the clones. Keywords: cassava, bacterial blight, gene expression, subtractive library, microarrays

Investing wisely to end hunger and strengthen agriculture, with no region left behind: Latin America

Latin America and the Caribbean economies began to slow down and stagnate during the 2008 economic crisis. This situation has been aggravated by the current health crisis, which is expected to set back regional development by almost ten years. Projected gross domestic product (GDP) was expected to shrink by 9.1% by the end of 2020, falling to values similar to those observed in 2010. In addition, poverty has increased like never before in only one year, reaching an estimated total of 231 million people, about 38% of all the population, with almost two out of every five poor people living in extreme poverty, and who are more exposed to food insecurity. However, the extent of the current economic crisis is quite heterogeneous among countries. For some, it is coupled with pre-pandemic conditions of political instability, social unrest, high poverty rates, and extreme food insecurity. Countries from the Dry Corridor (El Salvador, Nicaragua, Honduras, and Guatemala) were already facing a continuing exodus of people trying to escape from their precarious economic situations aggravated by environmental disasters such as the recent hurricanes. Countries facing political instability and social unrest are likely to suffer more from the uncertainty in their economic systems and their populations are prone to leave, while areas with erratic climate conditions have had to deal with high-risk agriculture and face similar consequences. If no immediate actions are taken, this problem could be exacerbated, creating an even greater flow of outward migration, risking the region’s stability and its potential to become a major food producer for the world. Agriculture is one of the most relevant sectors for its potential to continue contributing to food security, poverty reduction, and resilience to counteract external shocks such as climate uncertainty or the current health crisis

Identification of a Rice stripe necrosis virus resistance locus and yield component QTLs using Oryza sativa × O. glaberrima introgression lines

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>We built a set of 64 chromosome segment substitution lines carrying contiguous chromosomal segments of African rice <it>Oryza glaberrima </it>MG12 (acc. IRGC103544) in the genetic background of <it>Oryza sativa ssp. </it>tropical <it>japonica </it>(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 <it>Rice stripe necrosis virus </it>was located between SSR markers RM202-RM26406 (44.5-44.8 cM) on chromosome 11.</p> <p>Conclusions</p> <p>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.</p

Flexible and Scalable Genotyping-by-Sequencing Strategies for Population Studies

Background: 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. Results: 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. Conclusions: 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

Expression of the Aeluropus littoralis AlSAP gene enhances rice yield under field drought at the reproductive stage

We evaluated the yields of Oryza sativa L. 'Nipponbare' rice lines expressing a gene encoding an A20/AN1 domain stress-associated protein, AlSAP, from the halophyte grass Aeluropus littoralis under the control of different promoters. Three independent field trials were conducted, with drought imposed at the reproductive stage. In all trials, the two transgenic lines, RN5 and RN6, consistently out-performed non-transgenic (NT) and wild-type (WT) controls, providing 50–90% increases in grain yield (GY). Enhancement of tillering and panicle fertility contributed to this improved GY under drought. In contrast with physiological records collected during previous greenhouse dry-down experiments, where drought was imposed at the early tillering stage, we did not observe significant differences in photosynthetic parameters, leaf water potential, or accumulation of antioxidants in flag leaves of AlSAP-lines subjected to drought at flowering. However, AlSAP expression alleviated leaf rolling and leaf drying induced by drought, resulting in increased accumulation of green biomass. Therefore, the observed enhanced performance of the AlSAP-lines subjected to drought at the reproductive stage can be tentatively ascribed to a primed status of the transgenic plants, resulting from a higher accumulation of biomass during vegetative growth, allowing reserve remobilization and maintenance of productive tillering and grain filling. Under irrigated conditions, the overall performance of AlSAP-lines was comparable with, or even significantly better than, the NT and WT controls. Thus, AlSAP expression inflicted no penalty on rice yields under optimal growth conditions. Our results support the use of AlSAP transgenics to reduce rice GY losses under drought conditions. (Résumé d'auteur