Jorge A. da Silva

Use of physiological parameters as fast tools to screen for drought tolerance in sugarcane

Drought is one of the major limitations to plant productivity worldwide. Identifying suitable screening tools and quantifiable traits would facilitate the crop improvement process for drought tolerance. In the present study, we evaluated the ability of four relatively physiological parameters (variable-to-maximum chlorophyll a fluorescence ratio, F v/F m; estimated leaf chlorophyll content via SPAD index; leaf temperature, LT; and, leaf relative water content, RWC) to distinguish between drought tolerant and susceptible sugarcane genotypes subjected to a 90-d drought cycle. Eight field-grown genotypes were studied. By 45 d after the onset of treatments, the F v/F m, SPAD index and RWC of drought-stressed plants had declined significantly in all genotypes compared to values at the onset of well-watered treatments. However, the reductions were more severe in leaves of susceptible genotypes. Under drought stress, the tolerant genotypes as a group, maintained higher F v/F m (8%), SPAD index (15%), and RWC (16%) than susceptible genotypes. In general, LT of drought-stressed plants was higher (~4oC) than that of well-watered plants but the relative increase was greater among drought susceptible genotypes. Under drought stress, LT of tolerant genotypes was on average 2.2oC lower than that of susceptible genotypes. The results are consistent with the tolerant-susceptible classification of these genotypes and indicate that these tools can be reliable in screening for drought tolerance, with F v/F m, SPAD index and LT having the added advantage of being nondestructive and easily and quickly assessed.

Saccharum spontaneum L. ‘SES 208’ genetic linkage map combining RFLP- and PCR-based markers

A 527 marker linkage map ofSaccharum spontaneum L. ‘SES 208’ (2n = 64) was established by analyzing 208 single-dose (SD) arbitrarily primed PCR polymorphisms, 234 SD RFLPs, 41 double-dose (DD) and one triple-dose (TD) polymorphisms. A map hypothesis constructed using these markers (minimum LOD = 4.00,θ = 0.25 M) had 64 linkage groups with 13 SD, nine DD, and one TD markers unlinked. Eight chromosome homology groups were identified by using DD fragments as well as SD RFLPs that identified more than one linkage group. Linkages in repulsion phase were absent from the map, as found in two previous genetic studies of this species. Together, these data demonstrate that SES 208 displayed polysomic segregation, a genetic behavior typical of autopolyploid species. As with previous studies, it was concluded that SES 208 behaved like an auto-octoploid, which was also in agreement with the number of homology groups observed. Aχ2 was used to test whether the 527 markers were randomly distributed throughout the genome: both arbitrarily primed PCR markers and RFLPs had a distribution that was statistically indistinguishable from random. The integrated arbitrarily primed PCR-RFLP map had a predicted genomic coverage of 93% (considering only 442 SD polymorphisms) and an average interval between markers of 6 cM. SD markers were used to estimate the genome size of SES 208 at ca. 33 00 cM.

Yield components as indicators of drought tolerance of sugarcane

Water deficit is one of the major factors limiting the production of sugarcane (Saccharum officinarum L.). A study of the effects of limited water condition on yield components and their relationship with productivity can aid breeding programs in selecting for high yielding genotypes under this condition. The objective of this study was to investigate the relationships among the parameters stalk number, stalk height, stalk diameter, and stalk weight with cane yield in sugarcane growing in a field under moderate water stress during its grand growth period, in order to provide information to help breeders in adopting traits for selecting drought tolerant varieties. Seventy-eight genotypes plus two controls, one drought-tolerant and one drought-susceptible, were grown under a moderate water deficit condition in the field in 2005/2006 at Weslaco, TX. Productivity and yield components were measured. Under stress, the tolerant control (TCP93-4245) showed higher productivity, stalk number, stalk height and stalk weight than the susceptible one (TCP87-3388). However, the susceptible control showed higher stalk diameter. Linear association was found between productivity and its yield components, but stalk diameter showed to be fairly unstable among genotypes. Stalk height showed significant correlation with stalk number, stalk diameter and stalk weight. Stalk diameter also showed positive correlation with stalk weight. Therefore, during the selection procedure, when one of these traits is enhanced by drought tolerance, the correlated trait should also increase, making it feasible to select genotypes with high productivity, stalk number, stalk height, and stalk weight under water deficit.

Improving sugarcane for biofuel: engineering for an even better feedstock

Sugarcane is a proven biofuel feedstock and accounts for about 40% of the biofuel production worldwide. It has a more favorable energy input/output ratio than that of corn, the other major biofuel feedstock. The rich resource of genetic diversity and the plasticity of autopolyploid genomes offer a wealth of opportunities for the application of genomics and technologies to address fundamental questions in sugarcane towards maximizing biomass production. In a workshop on sugarcane engineering held at Rutgers University, we identified research areas and emerging technologies that could have significant impact on sugarcane improvement. Traditional plant physiological studies and standardized phenotypic characterization of sugarcane are essential for dissecting the developmental processes and patterns of gene expression in this complex polyploid species. Breeder friendly DNA markers associated with target traits will enhance selection efficiency and shorten the long breeding cycles. Integration of cold tolerance from Saccharum spontaneum and Miscanthus has the potential to expand the geographical range of sugarcane production from tropical and subtropical regions to temperate zones. The Flex‐stock and mix‐stock concepts could be solutions for sustaining local biorefineries where no single biofuel feedstock could provide consistent year‐round supplies. The ever increasing capacities of genomics and biotechnologies pave the way for fully exploring these potentials to optimize sugarcane for biofuel production.

Preliminary analysis of microsatellite markers derived from sugarcane expressed sequence tags (ESTs)

Expressed sequence tags (ESTs) in the sugarcane (Saccharum spp) database (SUCEST) were electronically searched and 402 microsatellites identified. Various dinucleotide and trinucleotide simple sequence repeat (SSR) motifs were found, with these being more frequently observed in ESTs obtained from flower cDNA libraries. PCR primers were designed for 20 of these SSRs and were tested on eight sugarcane genotypes, the sequences of these primers and a list of known sugarcane genes containing SSR motifs being presented in this paper. Polymorphisms were evident both at the cultivar level and between Saccharum species. These results show that EST-derived SSRs in Saccharum species are useful because they are polymorphic and transferable. The large number of microsatellites that will eventually be available from the SUCEST database (containing 295,000 submitted reads) will have many potential applications in linkage mapping and the planning of crosses.

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 a = 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.

Economics of sugar-based ethanol production and related policy issues.

The feasibility of integrating ethanol production into an existing sugar mill was analyzed by a stochastic spreadsheet model. As the price of corn continues to rise, ethanol producers will eventually need to look at other feedstock alternatives. Sugarcane has been proven to work well in the production of ethanol in Brazil. The results indicated existing U.S. sugar mills could economically switch to ethanol production. As imports into the United States threaten to undermine the U.S. sugar program, sugarcane producers have a viable alternative. At the very least, the alternative exists to diversify their income streams with ethanol production.

Photosynthetic Capacity and Water Use Efficiency in Sugarcane Genotypes Subject to Water Deficit During Early Growth Phase

The objective of this study was to compare the gas exchange, photosynthetic capacity and water potential of sugarcane genotypes cultivated under water deficit conditions imposed during the initial growth phase. Experiments were performed in a greenhouse using two sugarcane genotypes namely: HoCP93-776 (drought susceptible) and TCP02-4587 (drought tolerant). Sixty days after planting, two different water treatments were applied (i.e., with or without water deficit). At 0,30 and 60 days after the treatment, gas exchange variables were evaluated for their relationship with water use, intrinsic instantaneous water use efficiency and instantaneous carboxylation efficiency. The SPAD index, photosynthetic pigments, water potential and relative water content in the leaves were also analyzed. The genotype HoCP93-776 was more sensitive to drought treatment as indicated by the significantly lower values of SPAD index, photosynthetic pigments, water potential (Ψw) and relative water content (RWC) variables. The genotype TCP02-4587 had higher water potential, stomatal control efficiency, water use efficiency (WUE), intrinsic instantaneous water use efficiency (WUEintr), instantaneous carboxylation efficiency and photosynthetic capacity. The highest air vapor pressure deficit during the drought conditions could be due to the stomatal closing in the HoCP93-776, which contributed to its lower photosynthetic capacity.

Cold Responsive Gene Expression Profiling of Sugarcane and Saccharum spontaneum with Functional Analysis of a Cold Inducible Saccharum Homolog of NOD26-Like Intrinsic Protein to Salt and Water Stress

Transcriptome analysis of sugarcane hybrid CP72-1210 (cold susceptible) and Saccharum spontaneum TUS05-05 (cold tolerant) using Sugarcane Assembled Sequences (SAS) from SUCEST-FUN Database showed that a total of 35,340 and 34,698 SAS genes, respectively, were expressed before and after chilling stress. The analysis revealed that more than 600 genes are differentially expressed in each genotype after chilling stress. Blast2Go annotation revealed that the major difference in gene expression profiles between CP72-1210 and TUS05-05 after chilling stress are present in the genes related to the transmembrane transporter activity. To further investigate the relevance of transmembrane transporter activity against abiotic stress tolerance, a S. spontaneum homolog of a NOD26-like major intrinsic protein gene (SspNIP2) was selected for functional analysis, of which expression was induced after chilling stress in the cold tolerant TUS05-05. Quantitative real-time PCR showed that SspNIP2 expression was increased ~2.5 fold at 30 minutes after cold treatment and stayed induced throughout the 24 hours of cold treatment. The amino acid sequence analysis of the cloned SspNIP2 confirmed the presence of six transmembrane domains and two NPA (Asn-Pro-Ala) motifs, signature features of major intrinsic protein families. Amino acid analysis confirmed that four amino acids, comprising the ar/R (aromatic residue/arginine) region responsible for the substrate specificity among MIPs, are conserved among monocot silicon transporters and SspNIP2. Salinity stress test on SspNIP2 transgenic tobacco plants resulted in more vigorous transgenic lines than the non-transgenic tobacco plants, suggesting some degree of tolerance to salt stress conferred by SspNIP2. SspNIP2-transgenic plants, exposed to 2 weeks of water stress without irrigation, developed various degrees of water stress symptom. The water stress test confirmed that the SspNIP2 transgenic lines had lower evapotranspiration rates than non-transgenic lines, suggesting that SspNIP2 transgenic lines showed a slight tolerance to the early water stress compared to wild type plants.

Genetics of Polyploids

Large gains in the knowledge of plant genetics have been made during the last decade due to the development and application of DNA markers. The use of DNA markers has allowed the construction of linkage maps, assessment of genetic variability, and gene tagging in a variety of species (Sobral and Honeycutt, 1994). Densely populated DNA marker linkage maps have enabled map-based gene cloning and marker-assisted selection in some plants. Furthermore, DNA markers have contributed information to the fields of ecology, population genetics, and evolution (see the relevant chapters in Parts I and II).