Toni Pacey-Miller

Single-nucleotide polymorphism detection in plants using a single-stranded pyrosequencing protocol with a universal biotinylated primer

Analysis of variations in plant genomes is increasingly focused on single-nucleotide polymorphism (SNP) analysis, increasing the need for fast yet reliable, simple, and cost-effective techniques to handle the large number of these polymorphisms within large plant genomes. Pyrosequencing technology offers a technique that takes advantage of the interaction of four enzymes in a single-tube assay to measure DNA synthesis in real time. Pyrosequencing provides a DNA sequence and an advantage over alternative techniques in poorly characterized genomes such as those of most plant species. Here we compare the use of both single-stranded and double-stranded template Pyrosequencing on plant tissue for SNP identification. Different enzymatic strategies for double-stranded template preparation were compared. Preparation of double-stranded template from plant tissue required labor-intensive purification to allow double-stranded Pyrosequencing. A more cost-effective and less labor-intensive alternative to double-stranded template preparation in plants has been developed using a universal biotinylated primer to improve the efficiency of single-stranded Pyrosequencing. This provides an efficient high-throughput method for SNP analysis by Pyrosequencing.

DNA Banks and their role in facilitating the application of genomics to plant germplasm

Advances in genomics have provided technologies for high throughput analysis of plant genomes with potential for use in gene discovery in germplasm collections. The establishment of DNA banks facilitates this screening by making DNA from large numbers of plant accessions widely available. DNA banks require the development of appropriate policies for access and benefit sharing. Tools for automating sample and data handling are essential. Standard molecular methods for fingerprinting DNA accessions for international comparisons need to be determined. New screening technologies are required to take advantage of the emerging availability of large DNA collections. The Australian Plant DNA Bank aims to collect DNA from all Australian plant species and to sample the diversity within each species. DNA from all individuals of the species is being stored for rare species. Domesticated or economically important species from all countries are also being collected and stored. International networking of DNA banks will be a key step in linking genomics tools to global plant diversity.

Consistent production of cost-effective LongSAGE libraries

Serial analysis of gene expression (SAGE) and related techniques are gaining popularity as tools for exploring expression of plant genes but remain suboptimal because of smaller-than-expected average concatemer sizes. The presence of low-molecular-weight contaminants in high-molecular-weight concatemer fractions reduces the average size of cloned fragments, thereby limiting the viability of high-throughput sequencing methods. Implementation of an additional digestion step to promote formation of linear concatemer fragments appears to reduce the proportion of contaminants indirectly, but with variable results. We explored the effect of initial ditag polymerase chain reaction (PCR) quantity on the average size of cloned concatemers from the greater than 1000-bp fraction. The quantity of PCR material used was found to have a strong influence on the frequency of low-molecular-weight contaminants within this fraction, which has important implications for reducing costs associated with high-throughput sequencing of concatemer clones.