Rapid Identification Of Aspergillus Spp. Using A Pcr Based Melting Curve Method And Characterization Of A Novel Chitinase In Insect Resistant Maize Lines

Identification of fungal isolates is critical in studying Aspergillus flavus ecology and for developing methods to reduce aflatoxin contamination. In our efforts to track biocontrol applications of the atoxigenic A. flavus K49 (NRRL 30797), we have developed a rapid and accurate classification system for A. flavus based on PCR product melting temperatures (Tm). Using 18 primers and a total of 59 Aspergilli strains, including all 49 representatives of the Georgian peanut Vegetative Compatibility Groups (VCGs), a decision tree Tm flowchart was generated. The decision tree can classify all 59 strains using only 9 of the SSR primers and an average of 3.4 primers for each definitive classification. To confirm the effectiveness of the decision tree for strain identification, unknown samples isolated from experimental fields inoculated with various A. flavus strains in Stoneville, MS were analyzed. Ninety-six percent of the samples could be placed into a VCG using Tm(s) coupled with the decision tree. This dynamic system is an excellent tool for researchers studying biodiversity of A. flavus

Rapid Identification Of Aspergillus Spp. Using A Pcr Based Melting Curve Method And Characterization Of A Novel Chitinase In Insect Resistant Maize Lines

Identification of fungal isolates is critical in studying Aspergillus flavus ecology and for developing methods to reduce aflatoxin contamination. In our efforts to track biocontrol applications of the atoxigenic A. flavus K49 (NRRL 30797), we have developed a rapid and accurate classification system for A. flavus based on PCR product melting temperatures (Tm). Using 18 primers and a total of 59 Aspergilli strains, including all 49 representatives of the Georgian peanut Vegetative Compatibility Groups (VCGs), a decision tree Tm flowchart was generated. The decision tree can classify all 59 strains using only 9 of the SSR primers and an average of 3.4 primers for each definitive classification. To confirm the effectiveness of the decision tree for strain identification, unknown samples isolated from experimental fields inoculated with various A. flavus strains in Stoneville, MS were analyzed. Ninety-six percent of the samples could be placed into a VCG using Tm(s) coupled with the decision tree. This dynamic system is an excellent tool for researchers studying biodiversity of A. flavus

Agronomic and molecular characterization of Louisiana native Spartina alterniflora accessions

Coastal erosion and wetland deterioration are serious and widespread problems affecting Louisiana’s coastal zone. The application of agronomic and molecular techniques for improving crop species is well documented. However, these have not been routinely applied to species of ecological and environmental value. Spartina alterniflora is used extensively for shoreline protection and tidal marsh restoration because of its aggressive spreading habit and tolerance to salinity. The progress of marsh revegetation projects is limited by the costs and labor associated with vegetative propagation of Spartina. Hence, a breeding program was initiated to develop improved smooth cordgrass accessions with superior seed producing ability to accelerate coastal restoration projects by developing a seed-based propagation. The objectives were to: 1) evaluate the variation among the accessions collected from South Louisiana S. alterniflora native populations, and characterize accessions selected for use in genetic improvement; and 2) assess variability at the molecular level among selected plants used to establish a breeding program. One hundred twenty-six accessions of S. alterniflora were collected across South Louisiana in 1998. The accessions were characterized for location, date of collection, seed weight, and percent germination. Biplot and cluster analysis were used to analyze patterns of variation among accessions and locations. Vegetative and reproductive traits were evaluated during the growing seasons. Significant differences occurred among accessions for traits measured during vegetative stage and days to first panicle emergence. Date of collection contributed to overall variation among accessions, reflecting differences in maturity at the time of collection. Vegetative growth and differences in rust reaction allowed characterization of S. alterniflora accessions. Positive correlations were observed among vegetative traits, and negative correlations between those traits and rust reaction. Seven superior genotypes were selected for future population improvement. To assess genetic diversity within and between superior accessions, molecular and phenotypic characterizations were used to compare 40 selected genotypes, which were subjected to DNA fingerprinting using RAPD and AFLP. Cluster analysis results revealed considerable natural variation among the original collections for traits contributing to plant establishment from seed. The differences in clustering pattern demonstrate the usefulness of molecular markers in assessing genetic diversity more accurately

Synthetic Biology

Synthetic biology gives us a new hope because it combines various disciplines, such as genetics, chemistry, biology, molecular sciences, and other disciplines, and gives rise to a novel interdisciplinary science. We can foresee the creation of the new world of vegetation, animals, and humans with the interdisciplinary system of biological sciences. These articles are contributed by renowned experts in their fields. The field of synthetic biology is growing exponentially and opening up new avenues in multidisciplinary approaches by bringing together theoretical and applied aspects of science

Candida infections : detection and epidemiology

Despite the fact that the yeast Candida is the number 4 cause of bloodstream infections in the United States and ranks number 8 in Europe, adequate detection methods are lacking. Furthermore, relatively little is known about the epidemiology of Candida. Our aim was to improve the detection and identification of Candida infections and to study the epidemiology of Candida infections in Europe. We describe the development of a Nucleic Acid Sequence-Based Amplification (NASBAim) assay for the detection of Candida species in blood and blood cultures. The results were encouraging: when the NASBA assay was used to detect Candida in blood cultures which were negative in the automated blood culture system, the number of positive blood cultures increased with 62%. Furthermore, we demonstrated that a substantial increase in detection rate can already be obtained with a 2 day pre-culture step. Also, Candida RNA was detected in the blood of a patient, whereas no yeast was detected by the automated blood culturing system. In another patient the NASBA assay detected the infection two days earlier than the blood culture system. These results indicate that improved detection of Candida infections (detection rate as well as speed) is possible. Improved detection will lead to a reduced morbidity and mortality. Another study described in this thesis shows that Amplified Fragment Length Polymorphism (AFLPim) analysis is an excellent method for the identification of Candida species. The different species show very distinct clusters. The potential of storing AFLP patterns in general accessible databases will greatly enhance the chances of a correct identification. The second objective of this thesis was to study the epidemiology of Candida albicans infections in Europe. It appeared that a relatively high number of isolates which were involved in pneumonia produced (phospho)lipases, a putative virulence factor. Also, a significantly higher number of these isolates were among the higher producers of this enzyme. A similar trend was observed for the production of proteinases: all isolates obtained from pneumonia were positive in the proteinase assay, and 96% of these isolates were high producers. These results suggest that isolates involved in pneumonia are more virulent than isolates obtained from the other types of infection that were studied. Another interesting epidemiological finding is described. By using AFLP as a fingerprinting method, we typed a large collection of European C. albicans isolates. It was discovered, that isolates from Portugal and Spain all belonged to the same AFLP cluster (cluster 1), whereas isolates from the United Kingdom and all but one isolate from Germany belonged to another cluster (cluster 2). Isolates from France, Italy, Switzerland, and Turkey were represented in both clusters. These results indicate the presence of an Iberian and a Northern European clone. It can be concluded that our first objective, improved detection and identification of Candida infections, was feasible. The second objective, to study the epidemiology of C. albicans infections in Europe, resulted in two interesting discoveries which can be the onset of extensive epidemiological studies

The development and application of molecular markers for linkage mapping and quantitative trait loci analysis of important agronomic traits in oil palm (Elaeis guineensis Jacq.)

Oil palm (Elaeis guineensis) produces over five times more oil/year/hectare than oil seed rape and accounted for 33% of world vegetable oil production in 2011. Being a cross-pollinated perennial tree crop with long breeding cycles (typically 12 years) and a large planting area requirement (usually 143 palms/hectare), utilization of molecular technology could greatly improve the efficiency of oil palm breeding. In the present study, various approaches were used to develop molecular markers for genetic linkage mapping and QTL analysis, with the ultimate goal of marker-assisted selection in oil palm. Firstly, Representational Difference Analysis (RDA) and Amplified Fragment Length Polymorphism (AFLP) were coupled with Bulked Segregant Analysis (BSA) to try to identify marker(s) closely linked to the important shell-thickness gene. A novel combination of RDA with Roche 454 pyrosequencing enabled a more comprehensive study of the enrichment profiles compared to Sanger sequencing. Identification of >35% redundant sequences, repetitive sequences and organelle DNA suggested that subtractive hybridization and target enrichment of RDA were inefficient here, with the lack of elimination of common sequences masking the real difference products. The use of the AFLP method identified 29 primer pairs that yielded 49 putative shell-thickness related-polymorphic bands. A detailed analysis will need to be carried out to fully evaluate and validate these markers. The use of the relatively new Diversity Array Technology “Genotyping-By-Sequencing” (DArTSeq) platform through genotyping of two closely-related tenera self-pollinated F2 populations, 768 (n=44) and 769 (n=57), generated a total of 11,675 DArTSeq polymorphic markers of good quality. These markers were used in the construction of the first reported DArTSeq based high-density linkage maps for oil palm. Both genetic maps consist of 16 major independent linkage groups (total map length of 1874.8 and 1720.6 cM, with an average marker density of one marker every 1.33 and 1.62 cM, respectively), corresponding well with the 16 homologous chromosome pairs of oil palm (2n = 2x = 32; 14/16 chromosomes were confirmed by known location SSR markers). Preliminary quantitative trait loci (QTL) mapping of the yield and vegetative growth traits detected four significant and 34 putative as well as two significant and 30 putative QTLs for these small 768 and 769 populations, respectively. No common significant QTL were detected between the two closely-related controlled crosses which could have allowed combination of QTL across the two populations. Saturation of the shell-thickness (Sh) region with all available DArTSeq markers, as well as map integration around the Sh regions for both populations, identified 32 Single Nucleotide Polymorphism (SNP) and DArT markers mapped within a 5 cM flanking region of the Sh gene. Homology search of the DArTSeq marker sequence tag (64 bp) against the recently published oil palm genome assembly confirmed that 23 out of the 32 (72%) DArTSeq markers were located on the p5_sc00060 scaffold in which the SHELL gene was identified. The identified shell-thickness markers could be useful as molecular screening tools. This study demonstrated the potential and feasibility of using genomic resources available for genetic improvement of oil palm breeding programmes

The development and application of molecular markers for linkage mapping and quantitative trait loci analysis of important agronomic traits in oil palm (Elaeis guineensis Jacq.)

Oil palm (Elaeis guineensis) produces over five times more oil/year/hectare than oil seed rape and accounted for 33% of world vegetable oil production in 2011. Being a cross-pollinated perennial tree crop with long breeding cycles (typically 12 years) and a large planting area requirement (usually 143 palms/hectare), utilization of molecular technology could greatly improve the efficiency of oil palm breeding. In the present study, various approaches were used to develop molecular markers for genetic linkage mapping and QTL analysis, with the ultimate goal of marker-assisted selection in oil palm. Firstly, Representational Difference Analysis (RDA) and Amplified Fragment Length Polymorphism (AFLP) were coupled with Bulked Segregant Analysis (BSA) to try to identify marker(s) closely linked to the important shell-thickness gene. A novel combination of RDA with Roche 454 pyrosequencing enabled a more comprehensive study of the enrichment profiles compared to Sanger sequencing. Identification of >35% redundant sequences, repetitive sequences and organelle DNA suggested that subtractive hybridization and target enrichment of RDA were inefficient here, with the lack of elimination of common sequences masking the real difference products. The use of the AFLP method identified 29 primer pairs that yielded 49 putative shell-thickness related-polymorphic bands. A detailed analysis will need to be carried out to fully evaluate and validate these markers. The use of the relatively new Diversity Array Technology “Genotyping-By-Sequencing” (DArTSeq) platform through genotyping of two closely-related tenera self-pollinated F2 populations, 768 (n=44) and 769 (n=57), generated a total of 11,675 DArTSeq polymorphic markers of good quality. These markers were used in the construction of the first reported DArTSeq based high-density linkage maps for oil palm. Both genetic maps consist of 16 major independent linkage groups (total map length of 1874.8 and 1720.6 cM, with an average marker density of one marker every 1.33 and 1.62 cM, respectively), corresponding well with the 16 homologous chromosome pairs of oil palm (2n = 2x = 32; 14/16 chromosomes were confirmed by known location SSR markers). Preliminary quantitative trait loci (QTL) mapping of the yield and vegetative growth traits detected four significant and 34 putative as well as two significant and 30 putative QTLs for these small 768 and 769 populations, respectively. No common significant QTL were detected between the two closely-related controlled crosses which could have allowed combination of QTL across the two populations. Saturation of the shell-thickness (Sh) region with all available DArTSeq markers, as well as map integration around the Sh regions for both populations, identified 32 Single Nucleotide Polymorphism (SNP) and DArT markers mapped within a 5 cM flanking region of the Sh gene. Homology search of the DArTSeq marker sequence tag (64 bp) against the recently published oil palm genome assembly confirmed that 23 out of the 32 (72%) DArTSeq markers were located on the p5_sc00060 scaffold in which the SHELL gene was identified. The identified shell-thickness markers could be useful as molecular screening tools. This study demonstrated the potential and feasibility of using genomic resources available for genetic improvement of oil palm breeding programmes