Search

Global interest in sugarcane has increased significantly in recent years due to its economic impact on sustainable energy production. Sugarcane breeding and better agronomic practices have contributed to a huge increase in sugarcane yield in the last 30 years. Additional increases in sugarcane yield are expected to result from the use of biotechnology tools in the near future. Genetically modified (GM) sugarcane that incorporates genes to increase resistance to biotic and abiotic stresses could play a major role in achieving this goal. However, to bring GM sugarcane to the market, it is necessary to follow a regulatory process that will evaluate the environmental and health impacts of this crop. The regulatory review process is usually accomplished through a comparison of the biology and composition of the GM cultivar and a non-GM counterpart. This review intends to provide information on non-GM sugarcane biology, genetics, breeding, agronomic management, processing, products and byproducts, as well as the current technologies used to develop GM sugarcane, with the aim of assisting regulators in the decision-making process regarding the commercial release of GM sugarcane cultivars.

Expressed sequence tags derived markers have a great potential to be used in functional map construction and QTL tagging. In the present work, sugarcane genomic probes and expressed sequence tags having homology to genes, mostly involved in carbohydrate metabolism were used in RFLP assays to identify putative QTLs as well as their epistatic interactions for fiber content, cane yield, pol and tones of sugar per hectare, at two crop cycles in a progeny derived from a bi-parental cross of sugarcane elite materials. A hundred and twenty marker trait associations were found, of which 26 at both crop cycle and 32 only at first ratoon cane. A sucrose synthase derived marker was associated with a putative QTL having a high negative effect on cane yield and also with a QTL having a positive effect on Pol at both crop cycles. Fifty digenic epistatic marker interactions were identified for the four traits evaluated. Of these, only two were observed at both crop cycles.

Sucrose is the major product of photosynthesis in many higher plants. It is transported from the source tissue through the phloem to various sink tissues to support plant growth, development and reproduction. Knowledge on the signal transduction pathways involved in sucrose synthesis in mature leaves is limited. Using a microarray approach, we analyzed the expression profiles of 1920 sugarcane genes encoding signal transduction elements, transcription factors and stress-related proteins. We used individuals from a population segregating for sugar content and gene expression profiles were obtained from seven individuals with highest and seven with lowest sugar content. Surprisingly, from the 24 differentially expressed genes, 19 were more expressed in plants containing low-sugar content. Three of these genes encoded 14-3-3 like proteins, which have been found to reduce sucrose phosphate synthase (SPS) activity. Another encoded an SNF1-related protein similar to a protein kinase that phosphorylates SPS in vitro making it a target for the interaction with 14-3-3 proteins. The up-regulation of eight stress related genes in the lower sugar content plants supports a view that sugar levels modulate a complex signal transduction network that seems to involve responses that are related to stress. Evidence that hormone signaling is related to the sucrose content was also found. These data reinforced the usefulness of genomic approaches to uncover how sucrose metabolism can be regulated in sugarcane.

Microsatellites or simple sequence repeats (SSRs) are one of the most suitable markers for genome analysis as they have great potential to aid breeders to develop new improved sugarcane varieties. The development of SSR derived from expressed sequence tags (EST) opens new opportunities for genetic investigations at a functional level. In the present work, the polymorphism obtained with a subset of 51 EST-SSRs derived from SUCEST was compared with those generated by 50 genomic SSRs (gSSR) in terms of number of alleles, polymorphism information content, discrimination power and their ability to establish genetic relationships among 18 sugarcane clones including three Saccharum species (S. officinarum, S. barberi, S. sinense). The majority of EST-SSRs loci had four to six alleles in contrast to the seven to nine observed for the gSSRs loci. Approximately, 35% of the gSSRs had PIC values around 0.90 in contrast to 15% of the EST-SSRs. However, the mean discrimination power of the two types of SSR did not differ significantly as much as the average genetic similarity (GS) based on Dice coefficient. The correlation between GS of the two types of SSRs was high (r = 0.71/P = 0.99) and significant. Although differences were observed between dendrograms obtained with each SSR type, both were in good agreement with pedigree information. The S. officinarum clone IJ76-314 was grouped apart from the other clones evaluated. The results here demonstrate that EST-SSRs can be successfully used for genetic relationship analysis, extending the knowledge of genetic diversity of sugarcane to a functional level.

Sugarcane (Saccharum spp.) is a clonally propagated outcrossing polyploid crop of great importance in tropical agriculture. Up to now, all sugarcane genetic maps had been developed using either full-sib progenies derived from interspecific crosses or from selfing, both approaches not directly adopted in conventional breeding. We have developed a single integrated genetic map using a population derived from a cross between two pre-commercial cultivars ('SP80-180' x 'SP80-4966') using a novel approach based on the simultaneous maximum-likelihood estimation of linkage and linkage phases method specially designed for outcrossing species. From a total of 1,118 single-dose markers (RFLP, SSR and AFLP) identified, 39% derived from a testcross configuration between the parents segregating in a 1:1 fashion, while 61% segregated 3:1, representing heterozygous markers in both parents with the same genotypes. The markers segregating 3:1 were used to establish linkage between the testcross markers. The final map comprised of 357 linked markers, including 57 RFLPs, 64 SSRs and 236 AFLPs that were assigned to 131 co-segregation groups, considering a LOD score of 5, and a recombination fraction of 37.5 cM with map distances estimated by Kosambi function. The co-segregation groups represented a total map length of 2,602.4 cM, with a marker density of 7.3 cM. When the same data were analyzed using JoinMap software, only 217 linked markers were assigned to 98 co-segregation groups, spanning 1,340 cM, with a marker density of 6.2 cM. The maximum-likelihood approach reduced the number of unlinked markers to 761 (68.0%), compared to 901 (80.5%) using JoinMap. All the co-segregation groups obtained using JoinMap were present in the map constructed based on the maximum-likelihood method. Differences on the marker order within the co-segregation groups were observed between the two maps. Based on RFLP and SSR markers, 42 of the 131 co-segregation groups were assembled into 12 putative homology groups. Overall, the simultaneous maximum-likelihood estimation of linkage and linkage phases was more efficient than the method used by JoinMap to generate an integrated genetic map of sugarcane.

Sugarcane microsatellites or simple sequence repeats (SSR) were developed in an economical and practical way by mining EST databases. A survey in the SUCEST (sugarcane EST) database revealed a total of 2005 clusters out of 43 141 containing SSRs. Of these, 8.2% were dinucleotide, 30.5% were trinucleotide, and 61.3% were tetranucleotide repeats. Except for dinucleotides, the CG-rich motif types were the most common. Differences in abundance of trinucleotide motif types were observed between EST-SSRs and those isolated from sugarcane genomic libraries. Among the different cDNA libraries used for EST sequencing, SSRs were more frequent in the ones derived from leaf roll (LR). Twenty-three out of 30 tested SSRs produced scorable polymorphisms in 18 sugarcane commercial clones. These EST-SSRs showed a moderate level of polymorphism with some SSRs producing unique fingerprints. The number of alleles observed among the 18 clones evaluated varied from 2 to 15, with an average of 6.04 alleles/locus. The polymorphism information content (PIC) values ranged from 0.28 to 0.90 with a mean of 0.66. The EST-SSRs screened over both parents (SP 80-180; SP 80-4966) and 6 F1individuals produced 52 segregating markers that could potentially be used for sugarcane mapping. The EST-SSRs were found in clusters that had significant homology to proteins involved in important metabolic pathways such as sugar biosynthesis, proving that EST-SSRs are a valuable tool for the construction of a functional sugarcane map.Key words: sugarcane, polyploid, expressed sequence tags (ESTs), microsatellites (SSRs), genetic mapping.

Tropical and subtropical plants are generally sensitive to cold and can show appreciable variation in their response to cold stress when exposed to low positive temperatures. Using nylon filter arrays, we analyzed the expression profile of 1,536 expressed sequence tags (ESTs) of sugarcane (Saccharum sp. cv SP80-3280) exposed to cold for 3 to 48 h. Thirty-four cold-inducible ESTs were identified, of which 20 were novel cold-responsive genes that had not previously been reported as being cold inducible, including cellulose synthase, ABI3-interacting protein 2, a negative transcription regulator, phosphate transporter, and others, as well as several unknown genes. In addition, 25 ESTs were identified as being down-regulated during cold exposure. Using a database of cold-regulated proteins reported for other plants, we searched for homologs in the sugarcane EST project database (SUCEST), which contains 263,000 ESTs. Thirty-three homologous putative cold-regulated proteins were identified in the SUCEST database. On the basis of the expression profiles of the cold-inducible genes and the data-mining results, we propose a molecular model for the sugarcane response to low temperature.

Expressed sequence tags (ESTs) offer the opportunity to exploit single, low-copy, conserved sequence motifs for the development of simple sequence repeats (SSRs). The authors have examined the Sugarcane Expressed Sequence Tag database for the presence of SSRs. To test the utility of EST-derived SSR markers, a total of 342 EST–SSRs, which represent a subset of over 2005 SSR-containing sequences that were located in the sugarcane EST database, could be designed from the nonredundant SSR-positive ESTs for possible use as potential genic markers. These EST–SSR markers were used to screen 18 sugarcane ( Saccharum spp.) varieties. A high proportion (65.5%) of the above EST–SSRs, which gave amplified fragments of foreseen size, detected polymorphism. The number of alleles ranged from 2 to 24 with an average of 7.55 alleles per locus, while polymorphism information content values ranged from 0.16 to 0.94, with an average of 0.73. The ability of each set of EST–SSR markers to discriminate between varieties was generally higher than the polymorphism information content analysis. When tested for functionality, 82.1% of these 224 EST–SSRs were found to be functional, showing homology to known genes. As the EST–SSRs are within the expressed portion of the genome, they are likely to be associated to a particular gene of interest, improving their utility for genetic mapping; identification of quantitative trait loci, and comparative genomics studies of sugarcane. The development of new EST–SSR markers will have important implications for the genetic analysis and exploitation of the genetic resources of sugarcane and related species and will provide a more direct estimate of functional diversity.

Agrobacterium tumefaciens is established as a vector for gene transfer in many dicotyledonous plants but is not accepted as a vector in monocotyledonous plants, especially in the important Gramineae. The use of Agrobacterium to transfer genes into monocot species could simplify the transformation and improvement of important crop plants. In this report we describe the use of Agrobacterium to transfer a gene into corn, the regeneration of plants, and detection of the transferred genes in the F(1) progeny. Shoot apices of Zea mays L. variety Funk's G90 were cocultivated with A. tumefaciens EHA 1, which harbored the plasmid pGUS3 containing genes for kanamycin resistance (NPT II) and beta-glucuronidase (GUS). Plants developed from these explants within 4 to 6 weeks. Fluorometric GUS assays of leaves and immature seeds from the plants exhibited low GUS activity. Both NOS and GUS gene fragments were amplified by polymerase chain reaction in the DNA isolated from the F(1) generations of one of the original transformed plants. Southern analysis showed both GUS and NPT probes hybridized to DNA in several of the F(1) progeny, demonstrating the incorporation of GUS and NPT II genes into high molecular weight DNA. These data establish successful gene transfer and sexual inheritance of the genes.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2014-02-26T17:29:02Z No. of bitstreams: 1 WOS000245799300001.pdf: 721026 bytes, checksum: a95bd81c2e9b89892963ba8568987d0e (MD5) Made available in DSpace on 2014-02-26T17:29:02Z (GMT). No. of bitstreams: 1 WOS000245799300001.pdf: 721026 bytes, checksum: a95bd81c2e9b89892963ba8568987d0e (MD5) Previous issue date: 2007-03-13 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:17:30Z No. of bitstreams: 1 WOS000245799300001.pdf: 721026 bytes, checksum: a95bd81c2e9b89892963ba8568987d0e (MD5) Made available in DSpace on 2014-05-20T13:17:30Z (GMT). No. of bitstreams: 1 WOS000245799300001.pdf: 721026 bytes, checksum: a95bd81c2e9b89892963ba8568987d0e (MD5) Previous issue date: 2007-03-13 Background: Sugarcane is an increasingly economically and environmentally important C4 grass, used for the production of sugar and bioethanol, a low-carbon emission fuel. Sugarcane originated from crosses of Saccharum species and is noted for its unique capacity to accumulate high amounts of sucrose in its stems. Environmental stresses limit enormously sugarcane productivity worldwide. To investigate transcriptome changes in response to environmental inputs that alter yield we used cDNA microarrays to profile expression of 1,545 genes in plants submitted to drought, phosphate starvation, herbivory and N-2-fixing endophytic bacteria. We also investigated the response to phytohormones (abscisic acid and methyl jasmonate). The arrayed elements correspond mostly to genes involved in signal transduction, hormone biosynthesis, transcription factors, novel genes and genes corresponding to unknown proteins.Results: Adopting an outliers searching method 179 genes with strikingly different expression levels were identified as differentially expressed in at least one of the treatments analysed. Self Organizing Maps were used to cluster the expression profiles of 695 genes that showed a highly correlated expression pattern among replicates. The expression data for 22 genes was evaluated for 36 experimental data points by quantitative RT-PCR indicating a validation rate of 80.5% using three biological experimental replicates. The SUCAST Database was created that provides public access to the data described in this work, linked to tissue expression profiling and the SUCAST gene category and sequence analysis. The SUCAST database also includes a categorization of the sugarcane kinome based on a phylogenetic grouping that included 182 undefined kinases.Conclusion: An extensive study on the sugarcane transcriptome was performed. Sugarcane genes responsive to phytohormones and to challenges sugarcane commonly deals with in the field were identified. Additionally, the protein kinases were annotated based on a phylogenetic approach. The experimental design and statistical analysis applied proved robust to unravel genes associated with a diverse array of conditions attributing novel functions to previously unknown or undefined genes. The data consolidated in the SUCAST database resource can guide further studies and be useful for the development of improved sugarcane varieties. Univ São Paulo, Inst Quim, Dept Bioquim, BR-01498 São Paulo, Brazil Univ São Paulo, BIOINFO Nucl Pesquisas & Bioinformat, BR-05508 São Paulo, Brazil Univ Estadual Campinas, Ctr Biol Mol & Engn Genet, BR-13081970 Campinas, SP, Brazil Univ Fed Rio de Janeiro, Inst Bioquim Med, BR-21941 Rio de Janeiro, Brazil Univ São Paulo, Escola Super Agr Luiz Queirzo, Dept Genet, BR-05508 Piracicaba, SP, Brazil Ctr Tecnol Canavieira, Piracicaba, SP, Brazil Univ Estadual Paulista, Fac Ciências Agrarias & Vet Jaboticabal, Dept Tecnol, Jaboticabal, SP, Brazil Univ São Paulo, CENA, BR-05508 São Paulo, Brazil Univ Estadual Paulista, Fac Ciências Agrarias & Vet Jaboticabal, Dept Tecnol, Jaboticabal, SP, Brazil

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2014-02-26T17:22:38Z No. of bitstreams: 1 WOS000230919700003.pdf: 708129 bytes, checksum: 81dfe301960a8a0aef14a8a21d2abd09 (MD5) Made available in DSpace on 2014-02-26T17:22:38Z (GMT). No. of bitstreams: 1 WOS000230919700003.pdf: 708129 bytes, checksum: 81dfe301960a8a0aef14a8a21d2abd09 (MD5) Previous issue date: 2005-02-28 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:17:32Z No. of bitstreams: 1 WOS000230919700003.pdf: 708129 bytes, checksum: 81dfe301960a8a0aef14a8a21d2abd09 (MD5) Made available in DSpace on 2014-05-20T13:17:32Z (GMT). No. of bitstreams: 1 WOS000230919700003.pdf: 708129 bytes, checksum: 81dfe301960a8a0aef14a8a21d2abd09 (MD5) Previous issue date: 2005-02-28 A collection of 237,954 sugarcane ESTs was examined in search of signal transduction genes. Over 3,500 components involved in several aspects of signal transduction, transcription, development, cell cycle, stress responses and pathogen interaction were compiled into the Sugarcane Signal Transduction (SUCAST) Catalogue. Sequence comparisons and protein domain analysis revealed 477 receptors, 510 protein kinases, 107 protein phosphatases, 75 small GTPases, 17 G-proteins, 114 calcium and inositol metabolism proteins, and over 600 transcription factors. The elements were distributed into 29 main categories subdivided into 409 sub-categories. Genes with no matches in the public databases and of unknown function were also catalogued. A cDNA microarray was constructed to profile individual variation of plants cultivated in the field and transcript abundance in six plant organs (flowers, roots, leaves, lateral buds, and 1(st) and 4(th) internodes). From 1280 distinct elements analyzed, 217 (17%) presented differential expression in two biological samples of at least one of the tissues tested. A total of 153 genes (12%) presented highly similar expression levels in all tissues. A virtual profile matrix was constructed and the expression profiles were validated by real-time PCR. The expression data presented can aid in assigning function for the sugarcane genes and be useful for promoter characterization of this and other economically important grasses. Univ São Paulo, Inst Quim, Dept Bioquim, BR-05508900 São Paulo, Brazil Univ São Paulo, Inst Matemat & Estatist, Dept Estatist, BR-05508090 São Paulo, Brazil Univ Estadual Campinas, Inst Biol, Dept Genet & Evolucao, BR-13083970 Campinas, SP, Brazil Univ Estadual Campinas, Ctr Biol Mol & Engn Genet, BR-13083970 Campinas, SP, Brazil Ctr Tecnol Copersucar, BR-13400970 São Paulo, Brazil Univ Estadual Paulista, Fac Ciências Agr & Vet Jaboticabal, Dept Tecnol, Ctr Estocagem Clones,BCCCtr, BR-14870000 São Paulo, Brazil Univ Estadual Paulista, Fac Ciências Agr & Vet Jaboticabal, Dept Tecnol, Ctr Estocagem Clones,BCCCtr, BR-14870000 São Paulo, Brazil

Background - Sucrose content is a highly desirable trait in sugarcane as the worldwide demand for cost-effective biofuels surges. Sugarcane cultivars differ in their capacity to accumulate sucrose and breeding programs routinely perform crosses to identify genotypes able to produce more sucrose. Sucrose content in the mature internodes reach around 20% of the culms dry weight. Genotypes in the populations reflect their genetic program and may display contrasting growth, development, and physiology, all of which affect carbohydrate metabolism. Few studies have profiled gene expression related to sugarcane's sugar content. The identification of signal transduction components and transcription factors that might regulate sugar accumulation is highly desirable if we are to improve this characteristic of sugarcane plants. Results - We have evaluated thirty genotypes that have different Brix (sugar) levels and identified genes differentially expressed in internodes using cDNA microarrays. These genes were compared to existing gene expression data for sugarcane plants subjected to diverse stress and hormone treatments. The comparisons revealed a strong overlap between the drought and sucrose-content datasets and a limited overlap with ABA signaling. Genes associated with sucrose content were extensively validated by qRT-PCR, which highlighted several protein kinases and transcription factors that are likely to be regulators of sucrose accumulation. The data also indicate that aquaporins, as well as lignin biosynthesis and cell wall metabolism genes, are strongly related to sucrose accumulation. Moreover, sucrose-associated genes were shown to be directly responsive to short term sucrose stimuli, confirming their role in sugar-related pathways. Conclusion - Gene expression analysis of sugarcane populations contrasting for sucrose content indicated a possible overlap with drought and cell wall metabolism processes and suggested signaling and transcriptional regulators to be used as molecular markers in breeding programs. Transgenic research is necessary to further clarify the role of the genes and define targets useful for sugarcane improvement programs based on transgenic plants.