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The decrease in agricultural productivity in successive cutting of sugarcane plants is associated with several extrinsic and intrinsic factors. However, no studies have focused on the physiological potential of sett roots in successive cuts in sugarcane culture. There have been no proteomic studies on sugarcane sett roots at different stages of cutting. In this study, the UPLC-ESI-TOF-MS system and bioinformatics tools were used to identify proteins of sett roots in the first and fifth cuts of sugarcane cultivar RB966928 in the sprouting stage. Differences in the proteome of sett roots of RB966928 in the first and fifth cuts detected in this study supports the hypothesis that the proteome of sett roots may change after successive cuts in sugarcane culture. A reduction in the number of proteins was observed in the roots of the fifth cut, whereas 34% of proteins, identified exclusively in the first cut, were absent in the fifth cut. Proteome analysis of sett roots in the first and fifth cuts showed that the changes after successive cuts were quantitative (number of proteins) and mainly qualitative. In this study, the detailed list of proteins identified in the first cut but absent in the fifth cut is relevant. The findings of this study may aid further research that employ biotic or abiotic elicitors to induce gene expression of essential proteins absent in sett roots of the fifth cut, and thus increasing the agricultural productivity and longevity of cane fields

The sugarcane variety RB92579 has excellent agricultural productivity, very low flowering, efficient water use, and a high content of sucrose. Despite its excellent agricultural productivity, the RB92579 has not been used as a direct parent in sugarcane improvement. The main goal of the present study was to investigate polymorphisms at the SSR and EST-SSR loci of the RB92579 sugarcane variety to evaluate its potential for breeding and generating new varieties and to guide better use by the industrial sector. A total of 92 samples of the RB92579 variety were collected from plants in the fourth cutting stage grown in two Brazilian states: Paraná (PR; South region) and Mato Grosso do Sul (MS; South-Central region). Four primers for DNA simple sequence repeats (SSRs) and eight primers for expressed sequence tags for simple sequence repeats (EST-SSR) were used for DNA amplification. The polymorphism occurrence in the 12 SSR loci was 28% in the PR and MS populations, with a total of 25 alleles and an average of 2.08 alleles/loci. High values for mean observed heterozygosity, a high value for genetic identity and a low level of population differentiation was found in samples from the PR and MS states. The number of polymorphisms in the EST-SSR and noncoding SSR loci as well as the genetic divergence was low. However, the high heterozygosity in both populations indicates that the RB92579 variety can be used as a parent to generate new cultivars. On the other hand, the low coefficient of genetic divergence and high identity coefficient indicate that there is genetic uniformity; therefore, there is no need for differential industrial adaptations for pretreatment or enzymatic hydrolysis of the sugarcane bagasse from RB92579 at the same cutting stage and planted in the two regions (PR and MS).

Public archives of next-generation sequencing data are growing exponentially, but the difficulty of marshaling this data has led to its underutilization by scientists. Here, we present ASCOT, a resource that uses annotation-free methods to rapidly analyze and visualize splice variants across tens of thousands of bulk and single-cell data sets in the public archive. To demonstrate the utility of ASCOT, we identify novel cell type-specific alternative exons across the nervous system and leverage ENCODE and GTEx data sets to study the unique splicing of photoreceptors. We find that PTBP1 knockdown and MSI1 and PCBP2 overexpression are sufficient to activate many photoreceptor-specific exons in HepG2 liver cancer cells. This work demonstrates how large-scale analysis of public RNA-Seq data sets can yield key insights into cell type-specific control of RNA splicing and underscores the importance of considering both annotated and unannotated splicing events., The increasing amount of raw RNA-seq data calls for new computational methods to mine information. Here, the authors present ASCOT, a computational resource to identify splice variants in RNA-seq data, and apply it to splicing patterns in neurons and unique splicing patterns in rod photoreceptors.

The germination potential of the axillary buds is crucial for the installation of new sugarcane crop and is highly relevant for the maintenance of the crop during the regrowth cycles. In this way, 1-DE-UPLC-ESI-Q-TOF system of mass spectrometry in a shotgun approach evaluated total and differential proteome of germinating and dormant axillary buds of cultivar RB867515 in second ratoon. The current paper investigates whether differently expressed proteins can be detected in germinating axillary buds or not. The proteome of germinative and dormant axillary buds were obtained using TCA/acetone in triplicates with subsequent proteins and polypeptides pre-separation by SDS-PAGE and UPLC. Mass spectra were obtained via MS/MS and were confronted with Viridiplante mass spectra from NCBIprot, SwissProt and UniProtKB databases, using Mascot tool from Matrix Science. A total of 159 proteins were identified. In total, 46 proteins were exclusively detected in the germinating buds, while 41 proteins were exclusively reported in the dormant buds and 72 proteins were common in germinating and dormant buds. Different proteins, involved with biotic and abiotic stress, were detected at equivalent proportions in germinating (29.5%) and dormant (31.7%) buds, while contrasting proportions of proteins involved with energy metabolism were detected in germinating (22.7%) and dormant (12%) buds. Budding potential in the third cut seems to be determined by specific proteins related to energy metabolism and steps of the biosynthesis of primary and secondary metabolites for shoot growth and development. Seven enzymes (Aconitase, Succinate dehydrogenase, Pyruvate dehydrogenase, Fructose-6-phosphate 1-phosphotransgerase, Chacone synthase, Chalcone isomerase, and Monodehydroascorbate), one protein subunits (RuBisCo large subunit-binding protein) and one protein of Argonaute protein family (Tudor-Sn) supposedly involved with germinating potential of axillary buds and two enzymes (Hexokinase and Aldehyde dehydrogenase) supposedly involved with non-budding were highlighted.

Public archives of next-generation sequencing data are growing exponentially, but the difficulty of marshaling this data has led to its underutilization by scientists. Here we present ASCOT, a resource that allows researchers to summarize, visualize, and query alternative splicing patterns in public RNA-Seq data. ASCOT enables rapid identification of splice-variants across tens of thousands of bulk and single-cell RNA-Seq datasets in human and mouse. To demonstrate the utility of ASCOT, we first focused on the nervous system and identified many alternative exons used only by a single neuronal subtype. We then leveraged datasets from the ENCODE and GTEx consortiums to study the unique splicing patterns of rod photoreceptors and found that PTBP1 knockdown combined with overexpression of MSI1 and PCBP2 activates rod-specific exons in HepG2 liver cancer cells. Furthermore, we observed that MSI1 targets intronic UAG motifs proximal to the 5’ splice site and interacts synergistically with PTBP1 downregulation. Finally, we show that knockdown of MSI1 in the retina abolishes rod-specific splicing. This work demonstrates how large-scale analysis of public RNA-Seq datasets can yield key insights into cell type-specific control of RNA splicing and underscores the importance of considering both annotated and unannotated splicing events. ASCOT splicing and gene expression data tables, software, and interactive browser are available at http://ascot.cs.jhu.edu.

The sugar-energy sector plays an important role in the sustainability of energy and consumption resources, with sugarcane being the raw material for obtaining ethanol, sugar and energy. The current sugarcane cultivation system involves the initial planting called plant cane and successive regrowths (ratoons), where the cultivation is maintained in the average of six years and five cuts, in commercial crops. The productivity decrease is evident in the current system of cultivation and generally, it is not feasible to maintain the sugar cane after fifth cut. Extrinsic and intrinsic factors to sugarcane may be related to the productivity decrease, and the use of molecular tools such as proteomics, may contribute to the elucidation of the genetic and biochemical effects in plant response to external factors, such as cultural practices and edafoclimatic factors. The objective of this work was to know the effect of sugarcane cultivation on protein expression during the sugarcane cutting cycles, characterizing the total and differential proteome of axillary buds of the plant cane and fourth ratoon of the cultivar RB867515. The total proteins were extracted in triplicate by the modified TCA/acetone method for use in axillary sugarcane buds. Protein samples were submitted to denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and stained with comassie blue. Each lane of the polyacrylamide gel was fractionated and treated for purification and isolation of the polypeptides. Tryptic digestion was performed on each fragment with subsequent UPLC separation (Nano Acquity). The polypeptides were analyzed on Waters® Micromass® ESI-Q-Tof micro ™ mass spectrometer. The adaptation of the protein extraction protocol, performed in the present work, was effective in the extraction of total proteins in 200 mg of plant material. Protein samples from the axillary buds at different cut stages showed distinct bands patterns in the SDS-PAGE gel if it was promising for the proteomic study. Two hundred thirty five proteins were identified, of which 71 were differentially expressed in the plant cane, 52 in the fourth ratoon and 112 were expressed in both cuts. The proteins identified are part of the most diverse cellular structures and/or participate in biological processes as a response to stresses, metabolism of carbohydrates, lipids and nucleic acids, regulation of gene expression, etc. Stand out in each cut, proteins related mainly to biotic and abiotic stresses, regulation of gene expression, transcription factors and signal transduction. Many important proteins detected in the plant cane, mainly those that participate in defense responses of the plants, were not found in the fourth ratoon (five cut). In the fourth ratoon, were highlighted a set of proteins that indicate the influence of abiotic and biotic stresses and conditions of anoxia and/or hypoxia on the axillary buds of this cut, even in axillary buds not submitted to these conditions. The differential proteome of the plant cane and fourth ratoon represents changes at the molecular level with great potential for a considerable productivity and longevity decrease, found in the ages of more advanced cuts in the cane fields. O setor sucroenergético tem papel importante na sustentabilidade de recursos energéticos e de consumo, sendo a cana-de-açúcar, matéria prima para obtenção de etanol, açúcar e energia. O sistema atual de cultivo da cana-de-açúcar envolve o plantio inicial da cana-planta e sucessivas rebrotas (socas), onde o cultivo se mantem na média de seis anos e cinco cortes, em lavouras comerciais. A queda da produtividade é evidente no sistema atual de cultivo, sendo de forma geral, inviável a manutenção do canavial a partir do quinto corte. Fatores extrínsecos e intrínsecos à cana-de-açúcar podem estar relacionados com a queda da produtividade, sendo que, a utilização de ferramentas moleculares como a proteômica pode colaborar com a elucidação dos efeitos genéticos e bioquímicos relacionados à resposta da planta a fatores externos, como os tratos culturais e fatores edafoclimaticos. O objetivo neste trabalho foi conhecer o efeito do cultivo da cana-de-açúcar na expressão proteica ao longo dos ciclos de corte da cana-de-açúcar, caracterizando o proteoma total e diferencial de gemas axilares da cana-planta e da quarta soca da cultivar RB867515. As proteínas totais foram extraídas em triplicata pelo método TCA/acetona adaptado para utilização em gemas axilares de cana-de-açúcar. As amostras de proteínas foram submetidas à eletroforese em gel de poliacrilamida desnaturante (SDS-PAGE) e corado com azul de comassie. Cada lane do gel de poliacrilamida foi recortado, fracionado e tratado para purificação e isolamento dos polipeptídeos. Foi realizada a digestão tríptica em cada fragmento com posterior separação por UPLC (Nano Acquity). Os polipeptídeos foram analisados em espectrômetro de massas Waters® Micromass® ESI-Q-Tof micro™. A adaptação do protocolo de extração de proteínas realizada no presente trabalho se mostrou efetiva na extração de proteínas totais em 200 mg de material vegetal. As amostras de proteínas das gemas axilares nos diferentes estágios de cortes apresentaram distintos padrões de bandas no gel de SDS-PAGE indicando modificação do proteoma nos diferentes estágios de cortes. Foram identificadas um total de 235 proteínas, destas 71 foram diferencialmente expressas na cana-planta, 52 na quarta soca e 112 foram expressas em ambos os cortes. As proteínas identificadas fazem parte das mais diversas estruturas celulares e/ou participam de processos biológicos como resposta a estresses, metabolismo de carboidratos, lipídios, proteínas e ácidos nucleicos, regulação da expressão gênica, entre outros. Destacam-se entre as diferentemente expressas em cada corte, proteínas relacionadas principalmente a estresses bióticos e abióticos, regulação da expressão gênica, fatores de transcrição e transdução de sinais. Muitas proteínas importantes detectadas na cana-planta, principalmente relacionadas com respostas de defesa das plantas, não foram encontradas na quarta soca. Na quarta soca destacou-se um conjunto de proteínas que indicam a influência de estresses abióticos e bióticos e condições de anóxia e/ou hipóxia sobre as gemas axilares desse corte, mesmo em gemas axilares não submetidas a essas condições. O proteoma diferencial da cana-planta e quarta soca representam modificações ao nível molecular com grande potencial para queda considerável de produtividade e longevidade encontrada nas idades de cortes mais avançadas nos canaviais.

The reduction in sugarcane productivity in subsequent cutting stages may be related to a gradual decrease of the allele number and mean observed heterozygosity (HO) in the sugarcane ratoon. This hypothesis was tested assessing the number of alleles and HO values in 10 expressed sequence tag microsatellites (Est-SSR loci) of the sugarcane varieties RB72454 and RB867515 in different cutting stages. Changes of allele numbers in samples of different cutting stages were observed in seven and six EstSSR loci of the RB72454 and RB867515 varieties, respectively. Reduction of allele numbers was observed in the samples collected in the fourth and sixth cutting stages of the RB72454 variety. In contrast, an increase of the allele numbers was detected in the samples collected on fourth, sixth, and seventh cutting stages of the RB867515 variety. Unchanged allele numbers were observed only in EstB41, EstC84, and EstB130 loci of the RB72454 variety, and EstB41, EstC67, EstA68, and EstB130 loci of the RB867515 variety. The variety RB867515 has lower polymorphism and values of HO than the RB72454 variety in different stages of cutting. At molecular level, in Est-SSR loci, the RB72454 variety showed higher changes in subsequent stages of cutting than RB867515. The similarities and divergences at molecular level between varieties RB72454 and RB867515 observed in the 10 Est-SSR loci during subsequent cutting stages can not explain the reduced productivity frequently observed after subsequent cutting stages but showed that phenotypic and physiological changes after each cutting stage are also accompanied by changes at genomic level.

In this study, we measured the genetic diversity within and among a set of 9 commercial sugarcane varieties used for alcohol and sugar production using 17 microsatellite DNA markers. The UGSM148 and UGSM59 primers were monomorphic for all 74 sugarcane samples. The estimated proportion of simple sequence repeated (SSR) polymorphic loci was 88.23%; 17 alleles were detected. The mean gene diversity of all SSR loci was 0.7279. The highest observed heterozygosity (HO) value was found in the RB72454 variety, whereas the lowest HO value was recorded in the SP813250 variety. The SP813250, RB845210, and RB835054 sugarcane varieties were the most genetically uniform varieties. An extremely high level of population differentiation was detected in the varieties exhibiting similar agronomic characteristics. Analysis of the genetic structure of the 9 sugarcane varieties using SSR markers was especially important to identify SSR loci with high levels of heterozygosity and to identify varieties showing the highest levels of heterozygosity. The monomorphic primers may be used to evaluate the genetic stability of sugarcane during cycles of vegetative multiplication, i.e., propagation via rhizomes.

Productivity of sugarcane (Saccharum spp.) crops varies at each cutting stage, reaching critical rates close to the fifth cut (fourth ratoon). Knowledge of proteins involved in the regrowth of sugarcane within the cutting process is important for the development of cultivars with greater longevity. The present study presents new information that the proteome of axillary buds is changed in successive cuts in sugarcane culture. Proteins were identified by UPLC-ESI-Q-TOF (ultra-high-performance liquid chromatography coupled with electrospray ionisation–quadrupole–time-of-flight) mass spectrometry and the Mascot tool. A reduction in the number of proteins was evident in the axillary buds of the fifth cut, as well as a reduction in the number of proteins exclusively detected in the axillary buds with the first cut, an indicator of reduction in the expression of genes that may be essential for the stability of culture development. The reduction in agricultural productivity, sprouting and tillering at advanced stages of the sugarcane crop is accompanied by alterations in axillary-bud gene expression, where 50% (52.35%) were expressed in either the axillary buds of the plant cane or the axillary buds of the fourth ratoon. All MS data are available via jPOST and ProteomeXchange with identifiers JPST000331 and PXD007957, respectively.

We analyzed 80 plants of the sugarcane (Saccharum spp) variety 'RB867515' in order to investigate its diversity and genetic structure at the molecular level. Four simple sequence repeat (SSR) loci (UGSM51, SMC1237, SEGMS1069, and UGSM38) and five expressed sequence tag (EST)-SSR loci (ESTA68, ESTB92, ESTB145, ESTC66, and ESTC84) were used as molecular markers. The polymorphic loci rate was 66.6%. A total of 17 alleles and an average of 1.88 alleles/locus were detected. The number of alleles in the EST-SSR loci was lower than the number of alleles in the SSRs of non-expressed loci. The mean observed heterozygosity among the nine SSR loci was 0.3291. Genetic structure analysis showed that 'RB867515' contains alleles from three ancestral groups (K = 3), but there is little admixing of alleles in the same plant (from 0.8 to 17.3%); only 1.88% of the plants shared alleles from two or three groups. ESTB92, ESTC84, and UGSM38 were monomorphic, but there was evidence of polymorphism in ESTA68, ESTB145, ESTC66, UGSM51, SMC1237, and SEGMS1069, indicating that 'RB867515' has variability at the molecular level and the potential to be used as a parent in breeding programs. The molecular variability observed in 'RB867515' indicates that the clone terminology that is used to identify this cultivar is inconsistent with the original meaning of "clone", which is defined as a sample of genetically identical plants.

High and low polymorphisms in simple sequence repeats of expressed sequence tag (EST-SSR) for specific proteins and enzymes, such as β-amylase, cellulose synthase, xyloglucan endotransglucosylase, fructose 1,6-bisphosphate aldolase, and fructose 1,6-bisphosphatase, were used to illustrate the genetic divergence within and between varieties of sugarcane (Saccharum spp.) and to guide the technological paths to optimize ethanol production from lignocellulose biomass. The varieties RB72454, RB867515, RB92579, and SP813250 on the second stage of cutting, all grown in the state of Parana (PR), and the varieties RB92579 and SP813250 cultured in the PR state and in Northeastern Brazil, state of Pernambuco (PE), were analyzed using five EST-SSR primers for EstC66, EstC67, EstC68, EstC69, and EstC91 loci. Genetic divergence was evident in the EstC67 and EstC69 loci for β-amylase and cellulose synthase, respectively, among the four sugarcane varieties. An extremely high level of genetic differentiation was also detected in the EstC67 locus from the RB82579 and SP813250 varieties cultured in the PR and PE states. High polymorphism in SSR of the cellulose synthase locus may explain the high variability of substrates used in pretreatment and enzymatic hydrolysis processes, which has been an obstacle to effective industrial adaptations.

The purpose of this study was the molecular characterization of nine varieties of sugarcane grown in the northern region of Paraná, and used by one of the units producing sugar and ethanol, analyzing microsatellite loci. The analysis of polymorphism in microsatellite loci of these varieties should provide information on the variability present within each cultivar and genetic similarity and divergence between them. Forty primers were tested of which 17 were used for analysis of nine varieties of sugarcane, because they presented better definition. Among the 17 primers used only primers were monomorphic UGSM59 UGSM148 and being useful for the study of genetic uniformity of varieties. With the primers UGSM35, SEGMS240, SEGMS1069,SMC226CG, ESTB130, SMC1237FL, SMC165A, UGSM117, ESTB41, ESTB145, SMC1047HA, SMC2017FL, ESTC66, mSSCIR56 and ESTB50, were found a total of 70 alleles ranging from 2 to 6 alleles, a average of 4.12 alleles per locus. The analysis of polymorphism at 15 SSR loci of Saccharum spp showed that the loci Smc226CG, Smc1237FL, Smc1047HA, mSscir56 and EstB60 showed higher values for the estimates of observed heterozygosity, and in situ Smc1047HA found the largest number of alleles. The varieties RB72454, RB92579 and RB867515 showed the highest values of heterozygosity was observed. The calculated genetic diversity showed greater genetic identity among the varieties RB92579 and RB72454 with 86.31% similarity and the lowest similarity was found between the varieties and RB845210 RB835054. The SSR loci in varieties of sugarcane are promising markers for assessing the actual genetic diversity at the molecular level of varieties that have uniform agronomic characteristics, contradicting the assumption that there is genetic uniformity in commercial varieties of sugarcane. O objetivo do presente trabalho foi a caracterização molecular de nove variedades de cana-de-açúcar cultivadas na região norte do Paraná, e utilizadas por uma das unidades produtoras de etanol e açúcar, analisando locos microssatélites. A análise do polimorfismo em locos microssatélites dessas variedades deve informar sobre a variabilidade presente dentro de cada cultivar e a divergência e similaridade genética entre as mesmas. Foram testados 40 primers dos quais 17 foram utilizados para análise das 9 variedades de cana-de-açúcar, por apresentarem melhor definição. Dos 17 primers utilizados apenas os primers UGSM148 e UGSM59 foram monomórficos sendo úteis para o estudo de uniformidade genética das variedades. Com os primers UGSM35, SEGMS240, SEGMS1069, SMC226CG, ESTB130, SMC1237FL, SMC165A, UGSM117, ESTB41, ESTB145, SMC1047HA, SMC2017FL, ESTC66, mSSCIR56, e ESTB50, foi encontrado um total de 70 alelos variando de 2 a 6 alelos, sendo uma média de 4,12 alelos por locos. A análise do polimorfismo nos 15 locos SSR de Saccharum spp mostrou que os locos Smc226CG, Smc1237FL, Smc1047HA, mSscir56, e EstB60 apresentaram os valores maiores para as estimativas de heterozigosidade observada, e no loco Smc1047HA foi encontrado o maior número de alelos. As variedades RB72454, RB92579 e RB867515 apresentaram os valores mais altos de heterozigosidade média observado. A diversidade genética calculada mostrou maior identidade genética entre as variedades RB92579 e RB72454 com 86,31% de similaridade e a menor similaridade encontrada foi entre as variedades RB845210 e RB835054. Os locos SSR em variedades de cana-de-açúcar são marcadores promissores para avaliar a real diversidade genética em nível molecular de variedades que apresentam características morfoagronômicas uniformes, contrariando o postulado de que existe uniformidade genética em variedades comerciais de cana-de-açúcar. 44 f