Resistance of bmr energy sorghum hybrids to sugarcane borer and fall armyworm / Resistência de híbridos de sorgo energia bmr à broca-da-cana-de-açúcar e lagarta-do-cartucho

Abstract The lower lignin content in plants species with energy potential results in easier cellulose breakdown, making glucose available for ethanol generation. However, higher lignin levels can increase resistance to insect attack. The objective of this work was to evaluate the susceptibility of a bmr-6 biomass sorghum (a mutant genotype with a lower concentration of lignin) to important pests of energy sorghum, Diatraea saccharalis and Spodoptera frugiperda. Experiments were performed in the laboratory and greenhouse to evaluate the development of these pests on the biomass sorghum bmr hybrids BR007, BR008, and TX635 and their respective conventional near-isogenic genotypes (without the bmr gene). The lignin content was higher in non-bmr hybrids, but the evaluated insect variables varied between treatments, not being consistent in just one hybrid or because it is bmr or not. The lowest survival of S. frugiperda was observed in the BR008 hybrid, both bmr and non-bmr. The S. frugiperda injury scores on plants in the greenhouse were high (>7) in all treatments. For D. saccharalis, there was no difference in larval survival in the laboratory, but in the greenhouse, the BR007 hybrid, both bmr and non-bmr, provided greater survival. Due the need to diversify the energy matrix and the fact that greater susceptibility of the bmr hybrids to either pests was not found in this study, these results hold promise for cultivation of these biomass sorghum hybrids for the production of biofuels.

Resumo O menor teor de lignina em espécies de plantas com potencial energético resulta na maior facilidade de quebra da celulose, disponibilizando glicose para geração de etanol. Porém, maiores teores de lignina representa um fator de resistência ao ataque de insetos. O objetivo deste trabalho foi avaliar como importantes pragas do sorgo energia, Diatraea saccharalis e Spodoptera frugiperda, se comportam quanto à alimentação e desempenho em sorgo bmr-6, um genótipo mutante com menor concentração de lignina. Foram realizados experimentos em laboratório e casa de vegetação, avaliando o desenvolvimento destas pragas nos híbridos de sorgo biomassa bmr 007, 008, TX635 e seus respectivos genótipos isogênicos convencionais (sem o gene bmr). O teor de lignina foi maior nos híbridos não bmr, mas nos parâmetros avaliados nos insetos, houve variação entre os tratamentos, não sendo consistente em apenas um híbrido e nem por ser ou não bmr. A menor sobrevivência de S. frugiperda foi verificada no híbrido BR008 tanto bmr quanto não bmr. As notas de injúria por S. frugiperda no sorgo em casa de vegetação foram altas (>7) em todos os tratamentos. Para D. saccharalis, não houve diferença significativa para a sobrevivência larval em laboratório, mas em casa de vegetação o híbrido BR007 tanto bmr quanto não bmr proporcionaram maior sobrevivência. Diante da necessidade de diversificar a matriz energética e o fato de que não foi comprovada neste estudo maior suscetibilidade dos híbridos bmr a ambas as pragas, estes resultados são promissores para o cultivo desses híbridos de sorgo biomassa para produção de biocombustíveis.

Expression analysis of transcription factors in sugarcane during cold stress / Análise de expressão de fatores de transcrição em cana-de-açúcar sob estresse por frio

Transcription factors (TF) are a wide class of genes in plants, and these can regulate the expression of other genes in response to various environmental stresses (biotic and abiotic). In the current study, transcription factor activity in sugarcane was examined during cold stress. Initially, RNA transcript reads of two sugarcane cultivars (ROC22 and GT08-1108) under cold stress were downloaded from SRA NCBI database. The reads were aligned into a reference genome and the differential expression analyses were performed with the R/Bioconductor edgeR package. Based on our analyses in the ROC22 cultivar, 963 TF genes were significantly upregulated under cold stress among a total of 5649 upregulated genes, while 293 TF genes were downregulated among a total of 3,289 downregulated genes. In the GT08-1108 cultivar, 974 TF genes were identified among 5,649 upregulated genes and 283 TF genes were found among 3,289 downregulated genes. Most transcription factors were annotated with GO categories related to protein binding, transcription factor binding, DNA-sequence-specific binding, transcription factor complex, transcription factor activity in RNA polymerase II, the activity of nucleic acid binding transcription factor, transcription corepressor activity, sequence-specific regulatory region, the activity of transcription factor of RNA polymerase II, transcription factor cofactor activity, transcription factor activity from plastid promoter, transcription factor activity from RNA polymerase I promoter, polymerase II and RNA polymerase III. The findings of above results will help to identify differentially expressed transcription factors during cold stress. It also provides a comprehensive analysis of the regulation of the transcription activity of many genes. Therefore, this study provides the molecular basis for improving cold tolerance in sugarcane and other economically important grasses.

Fatores de transcrição (FT) são uma ampla classe de genes em plantas e podem regular a expressão de outros genes em resposta a vários estresses ambientais (estresses bióticos e abióticos). No presente estudo, a atividade do fator de transcrição na cana-de-açúcar foi examinada durante o estresse pelo frio. Inicialmente, as leituras de transcrição de RNA de duas cultivares de cana-de-açúcar (ROC22 e GT08-1108) sob estresse frio foram baixadas do banco de dados SRA NCBI. As leituras foram alinhadas em um genoma de referência e as análises de expressão diferencial foram realizadas com o pacote R / Bioconductor edgeR. Com base em nossas análises no cultivar ROC22, 963 genes TF foram significativamente regulados positivamente sob estresse pelo frio entre um total de 5.649 genes regulados positivamente, enquanto 293 genes TF foram regulados negativamente entre um total de 3.289 genes regulados negativamente. No cultivar GT08-1108, 974 genes TF foram identificados entre 5.649 genes regulados positivamente e 283 genes TF foram encontrados entre 3.289 genes regulados negativamente. Os fatores de transcrição, em sua maioria, foram anotados com categorias GO relacionadas à ligação de proteína, ligação de fator de transcrição, ligação específica de sequência de DNA, complexo de fator de transcrição, atividade de fator de transcrição em RNA polimerase II, atividade de fator de transcrição de ligação de ácido nucleico, atividade de corepressor de transcrição, sequência específica da região reguladora, atividade do fator de transcrição da RNA polimerase II, atividade do cofator do fator de transcrição, atividade do fator de transcrição do promotor do plastídio, atividade do fator de transcrição do promotor da RNA polimerase I, polimerase II e RNA polimerase III. As descobertas dos resultados acima ajudarão a identificar fatores de transcrição expressos diferencialmente durante o estresse pelo frio. Ele também fornece uma análise abrangente da regulação da atividade [...].

Expression analysis of transcription factors in sugarcane during cold stress / Análise de expressão de fatores de transcrição em cana-de-açúcar sob estresse por frio

Abstract Transcription factors (TF) are a wide class of genes in plants, and these can regulate the expression of other genes in response to various environmental stresses (biotic and abiotic). In the current study, transcription factor activity in sugarcane was examined during cold stress. Initially, RNA transcript reads of two sugarcane cultivars (ROC22 and GT08-1108) under cold stress were downloaded from SRA NCBI database. The reads were aligned into a reference genome and the differential expression analyses were performed with the R/Bioconductor edgeR package. Based on our analyses in the ROC22 cultivar, 963 TF genes were significantly upregulated under cold stress among a total of 5649 upregulated genes, while 293 TF genes were downregulated among a total of 3,289 downregulated genes. In the GT08-1108 cultivar, 974 TF genes were identified among 5,649 upregulated genes and 283 TF genes were found among 3,289 downregulated genes. Most transcription factors were annotated with GO categories related to protein binding, transcription factor binding, DNA-sequence-specific binding, transcription factor complex, transcription factor activity in RNA polymerase II, the activity of nucleic acid binding transcription factor, transcription corepressor activity, sequence-specific regulatory region, the activity of transcription factor of RNA polymerase II, transcription factor cofactor activity, transcription factor activity from plastid promoter, transcription factor activity from RNA polymerase I promoter, polymerase II and RNA polymerase III. The findings of above results will help to identify differentially expressed transcription factors during cold stress. It also provides a comprehensive analysis of the regulation of the transcription activity of many genes. Therefore, this study provides the molecular basis for improving cold tolerance in sugarcane and other economically important grasses.

Resumo Fatores de transcrição (FT) são uma ampla classe de genes em plantas e podem regular a expressão de outros genes em resposta a vários estresses ambientais (estresses bióticos e abióticos). No presente estudo, a atividade do fator de transcrição na cana-de-açúcar foi examinada durante o estresse pelo frio. Inicialmente, as leituras de transcrição de RNA de duas cultivares de cana-de-açúcar (ROC22 e GT08-1108) sob estresse frio foram baixadas do banco de dados SRA NCBI. As leituras foram alinhadas em um genoma de referência e as análises de expressão diferencial foram realizadas com o pacote R / Bioconductor edgeR. Com base em nossas análises no cultivar ROC22, 963 genes TF foram significativamente regulados positivamente sob estresse pelo frio entre um total de 5.649 genes regulados positivamente, enquanto 293 genes TF foram regulados negativamente entre um total de 3.289 genes regulados negativamente. No cultivar GT08-1108, 974 genes TF foram identificados entre 5.649 genes regulados positivamente e 283 genes TF foram encontrados entre 3.289 genes regulados negativamente. Os fatores de transcrição, em sua maioria, foram anotados com categorias GO relacionadas à ligação de proteína, ligação de fator de transcrição, ligação específica de sequência de DNA, complexo de fator de transcrição, atividade de fator de transcrição em RNA polimerase II, atividade de fator de transcrição de ligação de ácido nucleico, atividade de corepressor de transcrição, sequência específica da região reguladora, atividade do fator de transcrição da RNA polimerase II, atividade do cofator do fator de transcrição, atividade do fator de transcrição do promotor do plastídio, atividade do fator de transcrição do promotor da RNA polimerase I, polimerase II e RNA polimerase III. As descobertas dos resultados acima ajudarão a identificar fatores de transcrição expressos diferencialmente durante o estresse pelo frio. Ele também fornece uma análise abrangente da regulação da atividade de transcrição de muitos genes. Portanto, este estudo fornece base molecular para melhorar a tolerância ao frio em cana-de-açúcar e outras gramíneas economicamente importantes.

Phenotypic Characterization and Genetic Diversity of Sugarcane Varieties Cultivated in Northern Karnataka of India based on Principal Component and Cluster Analyses

Abstract: Sugarcane is a major commercial crop grown in India and across the world. Hence, several elite varieties have been developed now-a-days to overcome many obstacles including abiotic stresses and diseases. The present study was undertaken to screen genetic variation among twenty four sugarcane varieties that are commonly cultivated across Northern Karnataka, India with reference to physicochemical characters. Experiment was conducted in triplicate following randomized complete block design (RCBD) at S. Nijalingappa Sugar Institute, Belagavi, Karnataka, India during February 2016-17. Physiological parameters such as internode length, stalk height, plant height, stalk girth, number of internodes, single cane weight, single cane volume of juice, cane yield and recovery were investigated. Further, statistical techniques such as principal component analysis and agglomerative hierarchical clustering were performed to characterize the twenty four varieties. Among twenty four sugarcane varieties studied, Co 86032 and CoC 671 were found to be elite varieties with respect to sugar recovery and cane yield, whereas varieties such as Co 86032 and Com 0265 were found to be best with respect to cane yield only. Based on the results obtained, eight varieties, viz., Co SNK 09232, Com 0265, Co 86032, Co SNK 09293, Co SNK 07680, CoC 671, Co 13006 and Co 2001-15 were found to be good with respect to overall qualities. Further studies need to be involved with molecular marker that would help in identification of elite varieties which could substantially contribute to construction of genetic resources library that may in turn find maximum use in molecular breeding.

Optimization of RT-PCR reactions in studies with genes of lignin biosynthetic route in Saccharum spontaneum

ABSTRACT Saccharum spontaneum has been used for the development of energy cane a crop aimed to be used for the production of second-generation ethanol, or lignocellulosic ethanol. Lignin is a main challenge in the conversion of cell wall sugars into ethanol. In our studies to isolate the genes the lignin biosynthesis in S. spontaneum we have had great difficulty in RT-PCR reactions. Thus, we evaluated the effectiveness of different additives in the amplification of these genes. While COMT and CCoAOMT genes did not need any additives for other genes there was no amplification (HCT, F5H, 4CL and CCR) or the yield was very low (CAD and C4H). The application of supplementary cDNA was enough to overcome the non-specificity and low yield for C4H and C3H, while the addition of 0.04% BSA + 2% formamide was effective to amplify 4CL, CCR, F5H and CCR. HCT was amplified only by addition of 0.04% BSA + 2% formamide + 0.1 M trehalose and amplification of PAL was possible with addition of 2% of DMSO. Besides optimization of expression assays, the results show that additives can act independently or synergistically.

Sugars levels of four sugarcane genotypes in different stem portions during the maturation phase

ABSTRACT Maturation is a characteristic of sugarcane plant (Saccharum spp.) and even when grown under the same soil and climate conditions the varieties differ on the maturation curve. Thus, studies that allow establishing maturation curves of different sugarcane genotypes in the local soil and climate may indicate the proper harvesting period to ensure better quality of the raw material. This study aimed to analyze the levels of soluble sugars during the maturation phase and assess the technological and productivity indexes of four irrigated sugarcane genotypes in the region of Rio Largo, Alagoas. The experiment was conducted in randomized blocks in a 4 x 2 x 5 factorial: four genotypes (RB92579, RB98710, RB99395 and RB961003), two stem portions (internodes 1-4 and internodes 5-8) and five seasons (82, 49, 25, 13 and 3 days before harvesting), each treatment with three replications. Internodes 1-4 showed the highest levels of reducing sugars, while the largest accumulation of sucrose and total soluble solids occurred in internodes 5-8. RB99395 genotype showed more stability in the sugar levels during sugarcane maturation, which can indicate early maturation and high agricultural yield.

Abundance and diversity of resistance genes in the sugarcane transcriptome revealed by in silico analysis

Resistance genes (R-genes) are responsible for the first interaction of the plant with pathogens being responsible for the activation (or not) of the defense response. Despite their importance and abundance, no tools for their automatic annotation are available yet. The present study analyzed R-genes in the sugarcane expressed sequence tags database which includes 26 libraries of different tissues and development stages comprising 237,954 expressed sequence tags. A new annotation routine was used in order to avoid redundancies and overestimation of R-gene number, common mistakes in previous evaluations. After in silico screening, 280 R-genes were identified, with 196 bearing the complete domains expected. Regarding the alignments, most of the sugarcane’s clusters yielded best matches with proteins from Oryza sativa, probably due to the prevalence of sequences of this monocot in data banks. All R-gene classes were found except the subclass LRR-NBS-TIR (leucine-rich repeats, nucleotide-binding site, including Toll interleukin-1 receptors), with prevalence of the kinase (Pto-like) class. R-genes were expressed in all libraries, but flowers, transition root to shoot, and roots were the most representative, suggesting that in sugarcane the expression of R-genes in non-induced conditions prevails in these tissues. In leaves, only low level of expression was found for some gene classes, while others were completely absent. A high allelic diversity was found in all classes of R-genes, sometimes showing best alignments with dicotyledons, despite the great number of genes from rice, maize and other grasses deposited in data banks. The results and future possibilities regarding R-genes in sugarcane research and breeding are further discussed.

Analysis of slipped sequences in EST projects

Slippage is an important sequencing problem that can occur in EST projects. However, very few studies have addressed this. We propose three new methods to detect slippage artifacts: arithmetic mean method, geometric mean method, and echo coverage method. Each method is simple and has two different strategies for processing sequences: suffix and subsequence. Using the 291,689 EST sequences produced in the SUCEST project, we performed comparative tests between our proposed methods and the SUCEST method. The subsequence strategy is better than the suffix strategy, because it is not anchored at the end of the sequence, so it is more flexible to find slippage at the beginning of the EST. In a comparison with the SUCEST method, the advantage of our methods is that they do not discard the majority of the sequences marked as slippage, but instead only remove the slipped artifact from the sequence. Based on our tests the echo coverage method with subsequence strategy shows the best compromise between slippage detection and ease of calibration.

Sucrose synthase molecular marker associated with sugar content in elite sugarcane progeny

We describe the development and application of an expressed sequence tag (EST)-derived restriction fragment length polymorphism (RFLP) marker for sugarcane elite genotypes which can be used for quantitative trait loci (QTL) tagging for sugar content. EST-derived RFLP markers for proteins involved in sucrose metabolism have been used in Southern analysis for mapping and gene tagging in elite sugarcane clones. A single dose marker, obtained from a sucrose synthase EST associated with sugar content at the alpha = 0.01 probability level, is presented for sugarcane breeding. Utilization of EST homologues to known genes for generation of molecular markers accelerated the identification of a QTL controlling an important trait-sugar content. Sugarcane bacterial artificial chromosome (BAC) clones hybridizing to the sucrose synthase EST were identified.

Putative pyrophosphate phosphofructose 1-kinase genes identified in sugar cane may be getting energy from pyrophosphate

Pyrophosphate-dependent phosphofructokinase (PPi-PFK) has been detected in several types of plant cells, but the gene has not been reported in sugar cane. Using Citrus paradisi PPi-PFK gene (AF095520 and AF095521) sequences to search the sugar cane EST database, we have identified both the alpha and beta subunits of this enzyme. The deduced amino acid sequences showed 76 and 80 similarity with the corresponding alpha and beta subunits of C. paradisi. A high degree of similarity was also observed among the PFK b subunits when the alignment of the sugar cane sequences was compared to those of Ricinus communis and Solanum tuberosum. It appears that alpha and beta are two distinct subunits; they were found at different concentrations in several sugar cane tissues. It remains to be determined if the different gene expression levels have some physiological importance and how they affect sucrose synthesis, export, and storage in vacuoles. A comparison between the amino acid sequences of b PFKs from a variety of organisms allowed us to identify the two critical Asp residues typical of this enzyme's activity site and the other binding sites; these residues are tightly conserved in all members of this protein family. Apparently, there are catalytic residues on the b subunit of the pyrophosphate-dependent enzyme

Optimizacion del proceso de embriogenesis somatica en variedades venezolanas de caña de azucar / Improvement of somatic embryogenesis in sugarcane venezuelan cultivars

En muchas variedades de caña de azúcar, se induce un alto porcentaje de callo embriogénico (70 %), al cultivar ôin vitroö explantes de hojas jóvenes en un medio constituido por las sales de Murashige y Skoog ,y 13 M de ácido 2,4 diclorofenoxiacético (2,4-D). Sin embargo, en las variedades venezolanas V78-1 y V75-6 el porcentaje de callo embriogénico producido en estas condiciones,es menor (30 %). Con el fin de optimizar el proceso de embriogénesis somática en estas dos variedades, se indujo la formación de callo embriogénico utilizando diferentes medios: Medio C3 (13 M de 2,4-D); Medio C7 (31.5 M de 2,4-D); Medio Cd (30 M de Dicamba) y medio C5BA (22.5 M de 2,4-D y 22.2 M de Benciladenina). Después de 45 días, en el medio Cd se observó el mayor porcentaje de calloembriogénico para ambas variedades (90 %). La capacidad morfogenética de estos callos, quedó evidenciada al ser transferidos al medio de regeneración (sin hormonas), donde a los cuatro días ya se observaba la formación de plantas a partir de los embriones somáticos obtenidos en el medio Cd, mientras que los embriones provenientes de los medios C3, C7 y C5BA, comenzaban a desarrollar plantas a los ocho días.