Collins A. Kimbeng

Molecular Diversity Among Members of the Saccharum Complex Assessed Using TRAP Markers Based on Lignin-Related Genes

In addition to the cultivation of sugarcane for sugar, the crop is considered seriously as an important bioenergy grass crop for its high biomass production ability. But, lignin is a serious bottleneck in the bioconversion of lignocellulosic biomass to ethanol. Hence, genetic relationships among 64 genotypes within the Saccharum complex were studied with respect to lignin-related genes using target region amplified polymorphic (TRAP) primers derived from caffeic acid O-methyltransferase (COMT), cinnamoyl alcohol dehydrogenase (CAD), cinnamoyl coA reductase (CCR), and ferrulate 5-hydroxylase (F5H) genes. While the average polymorphism detected by the TRAP markers was 43%, the markers derived from F5H gene (34%) were less polymorphic in comparison to those derived from COMT (46%), CCR (44%), and CAD (46%) genes. The lignin gene-based TRAP markers differentiated members of the Saccharum complex broadly according to previously established genetic relationships in the order of Miscanthus > Erianthus > Saccharum spontaneum > Saccharum robustum/Saccharum barberi/Saccharum sinense > Saccharum officinarum/cultivars. Principal coordinate analysis showed that 29% of the total variation was explained by the genotypes with respect to the lignin-related genes. The association of genetic variation revealed in this study with the biomass composition-related genes of the genotypes within a species will be helpful to design breeding strategies to develop superior energy cane cultivars with improved biomass quality of the sugarcane.

Molecular Markers Associated with Resistance to Aspergillus flavus in Maize Grain: QTL and Discriminant Analyses

ABSTRACT Aflatoxin contamination of maize (Zea mays L.) grain caused by Aspergillus flavus is a serious health hazard to animals and humans. Resistance to infection by A. flavus is poorly understood. The objectives of this investigation were to identify potential candidate markers associated with resistance in maize kernels and pollen grains to A. flavus using a mapping population derived from a cross between Mp313E (resistant) and SC212m (susceptible) inbred lines. The parents, F1, and F2 plants, were planted in the field in 2005. Each F2 plant was self-pollinated to produce F2:3 seed. Fresh pollen collected from parental lines, F1, and each F2 plant was germinated on a growth medium in the presence of A. flavus conidia. Selfed seeds from parents, F1, and F2 plants, were challenged with A. flavus conidial suspension and incubated using a medium-free method. Percent kernels uninfected (PKU) and number of pollen grains germinated (NPG) were recorded. A linkage map was constructed with JoinMap 3.0 using DNA profiles of all F2 individuals produced from amplified fragment length polymorphism (AFLP) and target region amplification polymorphism (TRAP) markers. Interval mapping and multiple-QTL model (MQM) mapping analyses were performed using MapQTL 4.0 software. Three marker-QTL associations were observed for log-transformed PKU. Potential markers associated with this trait were also identified via discriminant analysis (DA). The markers identified via DA pointed to the same genomic regions as identified via the QTL-mapping strategy. For log-transformed NPG, five marker-QTL associations were detected. One QTL was associated with a TRAP marker. The DA confirmed the existence of three QTL. The QTL detected for NPG were different from the QTL detected for PKU. Thus, resistances of pollen and kernels to A. flavus appeared to be controlled by different genetic systems/mechanisms. Correlation between pollen germination and percent kernel infection was negligible (r = 0.067), suggesting that the two traits can be improved independently.

Identification of cold-responsive genes in energycane for their use in genetic diversity analysis and future functional marker development.

Breeding for cold tolerance in sugarcane will allow its cultivation as a dedicated biomass crop in cold environments. Development of functional markers to facilitate marker-assisted breeding requires identification of cold stress tolerance genes. Using suppression subtractive hybridization, 465 cold-responsive genes were isolated from the cold-tolerant energycane Ho02-144. Predicted gene interactions network indicated several associated pathways that may coordinately regulate cold tolerance responses in energycane. Expression analysis of a select set of genes, representing signaling and transcription factors, genes involved in polyamine and antioxidant biosynthesis, protein degradation and in the repair of damaged proteins in the cytosol, showed their time-dependent regulation under cold-stress. Comparative expression profiles of these genes between Ho02-144 and a cold-sensitive clone (L79-1002) showed that almost all genes were induced immediately upon imposition of cold stress and maintained their expression in Ho02-144 whereas they were either downregulated or their upregulation was very low in L79-1002. Simple sequence repeat markers derived from 260 cold-responsive genes showed allelic diversity among the cold-sensitive commercial hybrids that were distinct from the Saccharum spontaneum clones. Future efforts will target sequence polymorphism information of these genes in our ongoing QTL and association mapping studies to identify functional markers associated with cold tolerance in sugar/energycane.

Historical Use of Cultivars as Parents in Florida and Louisiana Sugarcane Breeding Programs.

Sugarcane (Saccharum L. spp. hybrids) growers depend on breeding programs for new, high-yielding cultivars that have resistance to abiotic and biotic stresses, so breeders continually seek out widely adapted, high yielding germplasm to be used as parents for their programs. Cultivars are sometimes used for this purpose, but their use may be minimized to prevent genetic diversity erosion. The purpose of this study was to determine the importance of cultivars as parents in three USA (one in Florida and two in Louisiana) sugarcane breeding programs by quantifying the percentage of cultivars that had these parental groupings based on published registrations and crossing records. The percentage of cultivars with at least one commercial parent for each program was 81.8%, 77.5%, and 64.3% for the Houma (Ho), Louisiana, Canal Point (CP), Florida and Louisiana State University (LSU) programs, respectively, but cultivars were recently used as parents in only 11.8% (Ho), 16.39% (CP), and 34.3% (LSU) of crosses. The results indicate that the CP and Ho programs should consider increasing the use of cultivars as parents in their breeding programs to increase the probability of selecting potential commercial genotypes, but this should be balanced with high diversity crosses to avoid the loss of diversity.

Identification of Genomic Regions Controlling Leaf Scald Resistance in Sugarcane Using a Bi-parental Mapping Population and Selective Genotyping by Sequencing

Leaf scald, caused by Xanthomonas albilineans, is a major sugarcane disease worldwide. The disease is managed primarily with resistant cultivars obtained through classical breeding. However, erratic symptom expression hinders the reliability and reproducibility of selection for resistance. The development and use of molecular markers associated with incompatible/compatible reactions could overcome this limitation. The aim of the present work was to find leaf scald resistance-associated molecular markers in sugarcane to facilitate marker-assisted breeding. A genetic linkage map was constructed by selective genotyping of 89 pseudo F2 progenies of a cross between LCP 85-384 (resistant) and L 99-226 (susceptible) using 1,948 single dose (SD) markers generated from SSR, eSSR, and SNPs. Of these, 1,437 SD markers were mapped onto 294 linkage groups, which covered 19,464 cM with 120 and 138 LGs assigned to the resistant and susceptible parent, respectively. Composite interval mapping identified 8 QTLs associated with the disease response with LOD scores ranging from 3.0 to 7.6 and explained 5.23 to 16.93% of the phenotypic variance. Comparative genomics analysis with Sorghum bicolor allowed us to pinpoint three SNP markers that explained 16% phenotypic variance. In addition, representative stress-responsive genes close to the major effect QTLs showed upregulation in their expression in response to the bacterial infection in leaf/meristem tissue.