Silvana Creste

Detection of single sequence repeat polymorphisms in denaturing polyacrylamide sequencing gels by silver staining

Large-scale use of molecular markers in plant breeding is limited by the throughput capacity for genotyping. DNA polymorphisms can be detected in denaturing polyacrylamide gels indirectly by nucleotide labeling or directly by staining. Fluorescent-labeling or radiolabeling requires sophisticated infrastructure not always available in developing countries. We present an improved low-cost method for silver staining and compare it to 2 other methods for their ability to detect simple sequence repeat polymorphisms in denaturing polyacrylamide gels bound to glass plates. The 3 procedures differed in their requirement for an oxidation pretreatment, preexposure with formaldehyde during silver nitrate impregnation, inclusion of silver thiosulfate, and by their replacement of sodium carbonate for sodium hydroxide to establish alkaline conditions for silver ion reduction. All methods detected the same banding pattern and alleles. However, important differences in sensitivity, contrast, and background were observed. Two methods gave superior sensitivity, detecting down to 1 μL of loaded amplification products. Our improved method gave lower backgrounds and allowed reutilization of staining solutions. The use of thin (<1 mm) denaturing sequencing gels allows genotyping of 60–96 samples within 4 h. Use of smaller loading sample volumes and reutilization of staining solutions further reduced costs.

Genetic characterization of banana cultivars (Musa spp.) from Brazil using microsatellite markers

Microsatellite markers were used to characterize 35 banana (Musa spp.)genotypes cultivated in Brazil, including triploid cultivars and tetraploid hybrids. A total of 33 Musa-specific primers were tested, and 11 produced clear ,reproducible and discrete bands. The average number of alleles amplified per primer was 6.1, ranging from 4 to 8, with a total of 67 alleles identified. Phenetic analysis based on Jaccard similarity index derived from presence or absence of the alleles agreed with the morphological classification. Bootstrap analysis divided the genotypes into four clusters, according to genomic group and subgroup classification. The first cluster contained the majority of cultivars which have ‘A’ genome alone; while the second contained all triploid cultivars of the subgroup Prata (Pome) and their tetraploid hybrids. The third cluster contained cultivar ‘Maçã’ together with other genotypes considered for breeding purposes as similar to the Silk subgroup. These last two clusters formed a larger group including the majority of genotypes that resulted from hybridization between M. acuminata and M. balbisiana. The microsatellite loci were highly informative, with some pair of primers generating an unique finger printing for each genomic group and discriminating a genotype of doubtful classification, although somatic mutants from a subgroup were seldom distinguished from their original clone. Tetraploid hybrids exhibited distortion in the proportion of alleles donated by their triploid female parent. For a few primers, some genotypes exhibited a higher number of alleles than expected from their ploidy level, suggesting the occurrence of duplicated alleles or duplicated chromosomal regions.

Genetic diversity of Musa diploid and triploid accessions from the Brazilian banana breeding program estimated by microsatellite markers

Banana (Musa spp.) is one of the most consumed fruits worldwide. Production is based mainly on triploid cultivars, and most genetic improvement programs aim to generate tetraploid hybrids obtained from the crossing of established triploid cultivars with a diploid parent genotype, improved or wild, exhibiting the trait of interest, normally resistance to biotic factors. Microsatellites were used to investigate the genetic variability and relationships between 58 Musa genotypes, including 49 diploids and nine triploid cultivars maintained at the Musa germplasm collection of the Brazilian dessert banana breeding program. Thirty-three primer pairs developed for banana were tested, and nine amplified reproducible and discrete fragments, producing a total of 115 alleles. The average number of alleles amplified per primer was 12.8, ranging from 10 to 15. The diploid genotypes presented the largest genetic variability, demonstrated by the large number of alleles detected, and the low similarity between the clones. The phenetic analysis clustered the triploid cultivars in a separated group, with the exception of the ‘Nanica’ and ‘Gros Michel’ cultivars, which showed high similarity with the diploid cultivar ‘Mambee Thu’. It was not possible to separate the wild diploid genotypes from the cultivated ones, indicating a common origin of these genotypes. A high proportion of duplicated alleles and/or loci was observed for diploid and triploid genotypes. The information gathered about the similarity between diploid and triploid accessions will help to define potential crosses to maximize the recovery of the typical fruit qualities required in Brazil (AAB, Pome and Silk dessert banana).

Water stress reveals differential antioxidant responses of tolerant and non-tolerant sugarcane genotypes

The biochemical responses of the enzymatic antioxidant system of a drought-tolerant cultivar (IACSP 94-2094) and a commercial cultivar in Brazil (IACSP 95-5000) grown under two levels of soil water restriction (70% and 30% Soil Available Water Content) were investigated. IACSP 94-2094 exhibited one additional active superoxide dismutase (Cu/Zn-SOD VI) isoenzyme in comparison to IACSP 95-5000, possibly contributing to the heightened response of IACSP 94-2094 to the induced stress. The total glutathione reductase (GR) activity increased substantially in IACSP 94-2094 under conditions of severe water stress; however, the appearance of a new GR isoenzyme and the disappearance of another isoenzyme were found not to be related to the stress response because the cultivars from both treatment groups (control and water restrictions) exhibited identical changes. Catalase (CAT) activity seems to have a more direct role in H2O2 detoxification under water stress condition and the shift in isoenzymes in the tolerant cultivar might have contributed to this response, which may be dependent upon the location where the excessive H2O2 is being produced under stress. The improved performance of IACSP 94-2094 under drought stress was associated with a more efficient antioxidant system response, particularly under conditions of mild stress.

Lignification in Sugarcane: Biochemical Characterization, Gene Discovery, and Expression Analysis in Two Genotypes Contrasting for Lignin Content

Biochemical, histological, and transcriptional characterization of lignification identifies substantial differences in two sugarcane genotypes. Sugarcane (Saccharum spp.) is currently one of the most efficient crops in the production of first-generation biofuels. However, the bagasse represents an additional abundant lignocellulosic resource that has the potential to increase the ethanol production per plant. To achieve a more efficient conversion of bagasse into ethanol, a better understanding of the main factors affecting biomass recalcitrance is needed. Because several studies have shown a negative effect of lignin on saccharification yield, the characterization of lignin biosynthesis, structure, and deposition in sugarcane is an important goal. Here, we present, to our knowledge, the first systematic study of lignin deposition during sugarcane stem development, using histological, biochemical, and transcriptional data derived from two sugarcane genotypes with contrasting lignin contents. Lignin amount and composition were determined in rind (outer) and pith (inner) tissues throughout stem development. In addition, the phenolic metabolome was analyzed by ultra-high-performance liquid chromatography-mass spectrometry, which allowed the identification of 35 compounds related to the phenylpropanoid pathway and monolignol biosynthesis. Furthermore, the Sugarcane EST Database was extensively surveyed to identify lignin biosynthetic gene homologs, and the expression of all identified genes during stem development was determined by quantitative reverse transcription-polymerase chain reaction. Our data provide, to our knowledge, the first in-depth characterization of lignin biosynthesis in sugarcane and form the baseline for the rational metabolic engineering of sugarcane feedstock for bioenergy purposes.

Large-Scale Transcriptome Analysis of Two Sugarcane Genotypes Contrasting for Lignin Content

Sugarcane is an important crop worldwide for sugar and first generation ethanol production. Recently, the residue of sugarcane mills, named bagasse, has been considered a promising lignocellulosic biomass to produce the second-generation ethanol. Lignin is a major factor limiting the use of bagasse and other plant lignocellulosic materials to produce second-generation ethanol. Lignin biosynthesis pathway is a complex network and changes in the expression of genes of this pathway have in general led to diverse and undesirable impacts on plant structure and physiology. Despite its economic importance, sugarcane genome was still not sequenced. In this study a high-throughput transcriptome evaluation of two sugarcane genotypes contrasting for lignin content was carried out. We generated a set of 85,151 transcripts of sugarcane using RNA-seq and de novo assembling. More than 2,000 transcripts showed differential expression between the genotypes, including several genes involved in the lignin biosynthetic pathway. This information can give valuable knowledge on the lignin biosynthesis and its interactions with other metabolic pathways in the complex sugarcane genome.

Genetic variability among sugarcane genotypes based on polymorphisms in sucrose metabolism and drought tolerance genes

Target region amplification polymorphism (TRAP) markers were used to estimate the genetic similarity (GS) among 53 sugarcane varieties and five species of the Saccharum complex. Seven fixed primers designed from candidate genes involved in sucrose metabolism and three from those involved in drought response metabolism were used in combination with three arbitrary primers. The clustering of the genotypes for sucrose metabolism and drought response were similar, but the GS based on Jaccard’s coefficient changed. The GS based on polymorphism in sucrose genes estimated in a set of 46 Brazilian varieties, all of which belong to the three Brazilian breeding programs, ranged from 0.52 to 0.9, and that based on drought data ranged from 0.44 to 0.95. The results suggest that genetic variability in the evaluated genes was lower in the sucrose metabolism genes than in the drought response metabolism ones.

Challenges, Opportunities and Recent Advances in Sugarcane Breeding

Katia C. Scortecci1, Silvana Creste2, Tercilio Calsa Jr.3, Mauro A. Xavier2, Marcos G. A. Landell2, Antonio Figueira4 and Vagner A. Benedito5* 1Department of Cell Biology and Genetics, Universidade Federal do Rio Grande do Norte (UFRN), Natal, RN 2Centro de Cana – Instituto Agronomico de Campinas (IAC), Ribeirao Preto, SP 3Department of Genetics, Universidade Federal de Pernambuco (UFPE), Recife, PE 4Plant Breeding Laboratory , Centro de Energia Nuclear na Agricultura (CENA), Universidade de Sao Paulo (USP), Piracicaba, SP 5Laboratory of Plant Functional Genetics, Genetics and Developmental Biology Program, Plant & Soil Sciences Division, West Virginia University (WVU), Morgantown, WV 1,2,3,4Brazil 5USA

Expression Profile of Sugarcane Transcription Factor Genes Involved in Lignin Biosynthesis

Cell wall recalcitrance, which is conferred in part by lignin, is the main bottleneck in lignocellulosic ethanol production. Transcription factors (TFs) have been suggested as targets to reduce or modify lignin. Here we analysed the expression profile of nine sugarcane TFs, their relationships with genes of the monolignol biosynthesis pathway, and their effects on lignin content and composition. Our assays compared two sugarcane genotypes with different lignin contents. To identify differences between tissue types and between the top and bottom of the plant, the culm was divided into intermediary and mature internodes, and the internodes were separated into pith and rind. The expression profiles obtained for the nine TFs were rather complex, showing that not only the genotype but also the tissue type and developmental stage influenced the results. Pearson correlation analysis indicated that ShMYB58/63 was positively correlated with the syringyl/guaiacyl ratio. In addition, a Bayesian network showed predicted interactions between the TFs and genes for lignin biosynthesis that were previously reported in the literature, as well as novel interactions such as those between ShMYB58/63 and ShF5H. These findings suggest that in sugarcane culm, the differential lignin deposition between tissue types (rind and pith) and at different developmental stages is under transcriptional regulation.

Water stress alters lignin content and related gene expression in two sugarcane genotypes.

The lignin deposition in the stem of two sugarcane genotypes was assessed on exposure to water stress. The lignin content and the morphoanatomical characterization of the stem indicated that IACSP94-2094 plants are more lignified than those of IACSP95-5000 genotype, under normal water supply conditions, which was especially associated with higher lignin contents in the rind of mature internodes. Water deficit had negative impact on the biomass production, mostly with IACSP94-2094 plants, possibly due to stress severity or higher susceptibility of that genotype during the stem-lengthening phase. Water deficit led to significant alterations in the expression levels of lignin biosynthesis genes and led to an approximate 60% increase of lignin content in the rind of young internodes in both genotypes. It is concluded that the young rind region was more directly affected by water stress and, depending on the genotype, a higher lignin accumulation may occur in the stem, thus implying lower quality biomass for bioethanol production.