Copyright (c) 2009 All rights reserved. http://works.bepress.com/gary_ablett Recent documents in Gary A Ablett en-us Mon, 09 Nov 2009 23:22:20 PST 3600 http://works.bepress.com/gary_ablett/26 http://works.bepress.com/gary_ablett/26 Sun, 08 Nov 2009 21:02:25 PST L Slade Lee http://works.bepress.com/gary_ablett/25 http://works.bepress.com/gary_ablett/25 Tue, 20 Oct 2009 22:51:37 PDT Discovering single nucleotide polymorphisms (SNPs) in specific genes in a heterozygous polyploid plant species, such as sugarcane, is challenging because of the presence of a large number of homologues. To discover SNPs for mapping genes of interest, 454 sequencing of 307 polymerase chain reaction (PCR) amplicons (> 59 kb of sequence) was undertaken. One region of a four-gasket sequencing run, on a 454 Genome Sequencer FLX, was used for pooled PCR products amplified from each parent of a quantitative trait locus (QTL) mapping population (IJ76-514 × Q165). The sequencing yielded 96 755 (IJ76-514) and 86 241 (Q165) sequences with perfect matches to a PCR primer used in amplification, with an average sequence depth of approximately 300 and an average read length of 220 bases. Further analysis was carried out on amplicons whose sequences clustered into a single contig using an identity of 80% with the program cap3. In the more polymorphic sugarcane parent (Q165), 94% of amplicons (227/242) had evidence of a reliable SNP - an average of one every 35 bases. Significantly fewer SNPs were found in the pure Saccharum officinarum parent - with one SNP every 58 bases and SNPs in 86% (213/247) of amplicons. Using automatic SNP detection, 1632 SNPs were detected in Q165 sequences and 1013 in IJ76-514. From 225 candidate SNP sites tested, 209 (93%) were validated as polymorphic using the Sequenom MassARRAY system. Amplicon re-sequencing using the 454 system enables cost-effective SNP discovery that can be targeted to genes of interest and is able to perform in the highly challenging area of polyploid genomes. Peter C. Bundock http://works.bepress.com/gary_ablett/24 http://works.bepress.com/gary_ablett/24 Tue, 29 Sep 2009 20:32:01 PDT Timothy A. Holton http://works.bepress.com/gary_ablett/23 http://works.bepress.com/gary_ablett/23 Tue, 29 Sep 2009 20:32:00 PDT DNA sequence information is providing a growing resource for the identification of genetic markers for use in plants. The establishment of ITEC has made significant numbers of ESTs available for wheat and barley. EST databases have provided an important source for SSR or microsatellite markers from a range of plant species. SSR markers derived from ESTs may have advantages over those from traditional enriched genomic libraries. Wider transferability of these SSRs may be possible because of higher sequence conservation within ESTs. Mapping of the SSR provides a map location, in many cases, for a gene of known function. Single nucleotide polymorphism markers can also be designed from EST information. This provides a rich source of DNA polymorphism for use in genome mapping. We have designed molecular beacons to detect SNPs in barley. The relative value and potential application of SSR and SNP markers in barley will be discussed.Background: Microsatellite markers are valuable in genotyping and evaluation of genetic resources (WU and TANKSLEY 1993; SAGHAI MAROOF et al. 1994; GARLAND et al. 1999: MAGUIRE et al. 1999). ITMI initially focused on the mapping of genomes in the Triticeae using RFLP markers. More recently microsatellite or SSR markers have been more widely used because of their ease of analysis. These microsatellites have mostly been isolated from enriched genomic libraries. This process is laborious and expensive. The growing availability of DNA sequence information provides new opportunities for the development of molecular markers for use in the Triticeae. The recent completion of the sequence of the rice genome provides an important source of data. The International Triticeae EST Consortium (ITEC) provides a new source of species-specific sequence information from which markers can be derived. We have investigated the identification of microsatellite and single nucleotide polymorphism (SNP) markers in EST sequences. Two main options for the development of microsatellite markers are to source them directly from the target species or to identify them based upon information from a closely related species. Research at the Centre for Plant Conservation Genetics at Southern Cross University has addressed the isolation and characterisation of microsatellite loci from many plant species. Large numbers of loci have been identified in some species by producing enriched genomic libraries (ROSSETTO et al. 1999). In some cases such as sugarcane this has required optimisation of the microsatellite enrichment technique (CORDEIRO et al. 1999). Other species, such as pine trees have required screening of enriched libraries to allow efficient recovery of microsatellite loci (SCOTT et al. 2000a). Microsatellites derived from genomic libraries have been difficult to transfer between species in many cases, although exceptions have been found in some plant groups. For example, microsatellites appear to be widely transferable within the Myrtaceae, possibly due to limited sequence divergence in this family (ROSSETTO et al. 2000). Microsatellite transfer between Pinus species has also proven to be a useful option. Transfer of microsatellites within the cereals has been more limited (reference). One advantage of microsatellites derived from ESTs is the potential that they may be more transferable between species. Recently we demonstrated the potential to transfer microsatellite derived from grape ESTs to related species (SCOTT et al. 2000b). We are currently investigating transfer of sugarcane microsatellites derived from ESTs to sorghum and other related species compared with those from the International Consortium for Sugarcane Biotechnology (ICSB).Microsatellites: The ITEC database was searched for microsatellite sequences and the loci identified are being evaluated for wheat and barley. Preliminary results with wheat indicate low levels of polymorphism for the microsatellites in wheat sequences. This may be due to the types of cDNA libraries represented currently in the ITEC database. We have evaluated 29 SSRs from barley EST sequences. These barley SSRs have also been evaluated in wheat. Most barley SSRs performed well, in contrast to a similar set of wheat SSRs derived from ESTs.Single nucleotide polymorphisms: Single nucleotide polymorphisms have been detected by analysis of the Genbank DNA database. Comparison of EST sequences from a cDNA library derived from the variety Alexis and the sequences in the database allowed the detection of possible SNP loci. In other cases, over 50 SNP primer pairs have been designed and used to amplify from 10 barley genotypes to reveal potential SNPs. The initial application of these results has been in the design of variety specific assays for use in identification of barley genotypes.Molecular beacons: The molecular beacon technique was demonstrated to work effectively in the analysis of a transgene in barley (KOTA et al. 1999). This work is being extended to the application of molecular beacons to the detection of SNPs in barley. Variety specific molecular beacons are likely to have wide application in industry. Robert J. Henry http://works.bepress.com/gary_ablett/22 http://works.bepress.com/gary_ablett/22 Thu, 03 Sep 2009 21:19:40 PDT Kamel Chabane http://works.bepress.com/gary_ablett/21 http://works.bepress.com/gary_ablett/21 Tue, 01 Sep 2009 23:21:51 PDT Gary A. Ablett http://works.bepress.com/gary_ablett/20 http://works.bepress.com/gary_ablett/20 Tue, 01 Sep 2009 23:21:50 PDT DNA based varietal analysis has now moved from being a research tool to become a routine analytical method (Henry 2001). Cost effective DNA marker analysis requires high throughput facilities with a high level of automation. Microsatellite or simple sequence repeat markers (SSR's) provide a reliable method for routine analysis. Large numbers of microsatellites have been developed for barley and large sets of marker data have been collected on Australian and international barley varieties. More recently single nucleotide polymorphism (SNP) markers have become a useful alternative. A wide array of techniques is now available for SNP analysis. Detection of admixtures of barley is possible without the need to analyse individual grains. Issues associated with the transition from research to routine applications will be discussed.DNA analysis of grain samples has often required germination of the sample so that DNA can be extracted from the grain. We now use ground grain samples directly as a source of DNA for analysis. Microsatellite analysis of DNA from a bulk sample has been shown to detect minor contaminants. Detection of alleles for the main variety establish the identity of the sample and minor peaks corresponding to alleles from contaminating varieties can reveal contamination levels of 1% or less. This avoids the need to test large numbers of samples individually as have been required for techniques such as protein electrophoresis.We have employed DNA fingerprinting techniques to distinguish contamination of Harrington grain by other varieties, namely Stirling, Fitzgerald and Gairdner. Unlike morphological and protein-based tests, DNA techniques rely on a biochemical which is invariant in all tissues. The advantage of this is that the assays are not influenced by crop growth conditions, or seed treatment and storage environment. Also with the huge numbers of DNA markers available, DNA-based techniques particularly those involving microsatellite markers, are rapidly becoming the most widely accepted tools for plant variety identification.Microsatellite markers have been developed that successfully distinguish Stirling, Fitzgerald and Gairdner from Harrington. PCR with primers to a microsatellite produced a band with Fitzgerald, and a band with Stirling, which are both markedly different from the bands produced by Harrington and Gairdner. A second microsatellite can also be used to distinguish Stirling, Fitzgerald and Gairdner from Harrington. These band sizes are all highly reproducible, and comparison of both markers can be used to distinguish each of these varieties from the other three. Additional markers are available to distinguished these varieties from 22 barley cultivars tested so far.Results from defined mixes of Stirling, Fitzgerald, and Gairdner with Harrington suggest we can detect as little as 1% contamination by Fitzgerald, 2.5 % contamination by Stirling, and 5% contamination by Gairdner. Further development is needed to produce a reliable assay for detecting lower levels of Gairdner. Initial screening of 57 samples supplied to us suggested all have less than 5% contamination with Stirling or Fitzgerald. Some samples had peaks corresponding to 2.5% contamination with Stirling. The 24 samples tested had no major contamination (>10%) with Gairdner. Robert J. Henry http://works.bepress.com/gary_ablett/19 http://works.bepress.com/gary_ablett/19 Tue, 18 Aug 2009 23:14:32 PDT Gary A. Ablett http://works.bepress.com/gary_ablett/18 http://works.bepress.com/gary_ablett/18 Tue, 11 Aug 2009 23:35:07 PDT Gary A. Ablett http://works.bepress.com/gary_ablett/16 http://works.bepress.com/gary_ablett/16 Tue, 11 Aug 2009 23:35:06 PDT Robert J. Henry