Copyright (c) 2009 All rights reserved. http://works.bepress.com/peter_bundock Recent documents in Peter Bundock en-us Wed, 21 Oct 2009 23:18:54 PDT 3600 http://works.bepress.com/peter_bundock/147 http://works.bepress.com/peter_bundock/147 Tue, 20 Oct 2009 22:55:07 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/peter_bundock/146 http://works.bepress.com/peter_bundock/146 Tue, 29 Sep 2009 21:43:54 PDT Studies of genetic variability in crop plants are often based on polymorphism of DNA markers from anonymous genomic sites using highly polymorphic markers eg. SSRs. However variability at the level of the functional gene has been less studied even though it is a portion of this variability that is likely to create most of the genetically based phenotypic variability utilised by breeders in improvement programs. To determine the distribution, extent and type of variability between barley varieties at this level the asi gene of barley was analysed by sequencing a 2.3kb region comprising the promoter, coding and downstream regions from fourteen barley varieties. The asi gene is an intronless gene whose product functions as an inhibitor of a-amylase and subtilisin. The barley varieties studied were mostly cultivated varieties bred in Australia but included a wild barley (Hordeum spontaneum) and a North African landrace. As expected the wild barley variety had the largest number of SNPs and indels compared to the consensus sequence. However ten of the cultivated barley varieties had identical sequences over the 2.3kb of sequence except for the number of repeats in a microsatellite sequence in the promoter region. To compare the level of polymorphism displayed by the microsatellite with that based on SNP and indel haplotypes a further 36 barley varieties were genotyped for microsatellite length and selected sequence polymorphisms in the promoter and coding region. Peter C. Bundock http://works.bepress.com/peter_bundock/144 http://works.bepress.com/peter_bundock/144 Tue, 29 Sep 2009 21:38:26 PDT Studies of genetic variability in crop plants are often based on polymorphism of DNA markers from anonymous genomic sites using highly polymorphic markers eg. SSRs. However variability at the level of the functional gene has been less studied even though it is a portion of this variability that is likely to create most of the genetically based phenotypic variability utilised by breeders in improvement programs. To determine the distribution, extent and type of variability between barley varieties at this level the asi gene of barley was analysed by sequencing a 2.3kb region comprising the promoter, coding and downstream regions from fourteen barley varieties. The asi gene is an intronless gene whose product functions as an inhibitor of a-amylase and subtilisin. The barley varieties studied were mostly cultivated varieties bred in Australia but included a wild barley (Hordeum spontaneum) and a North African landrace. As expected the wild barley variety had the largest number of SNPs and indels compared to the consensus sequence. However ten of the cultivated barley varieties had identical sequences over the 2.3kb of sequence except for the number of repeats in a microsatellite sequence in the promoter region. To compare the level of polymorphism displayed by the microsatellite with that based on SNP and indel haplotypes a further 36 barley varieties were genotyped for microsatellite length and selected sequence polymorphisms in the promoter and coding region. Peter C. Bundock http://works.bepress.com/peter_bundock/143 http://works.bepress.com/peter_bundock/143 Tue, 29 Sep 2009 21:38:23 PDT Plant P450s belong to the cytochrome group that are membrane-bound enzymes, usually found in plant endoplasmic reticulum. This gene family is diverse in structure and function, which enables these enzymes to participate in numerous biosynthetic and degradative pathways. In plants, P450s are known to play important roles in production of hormones, pigments, oils, and defensive compounds. P450s are also involved in herbicide detoxification in cereal crops, including barley. In addition, the heme group of P450s is responsible for several catalytic reactivities of cytochrome P450 in plants. Purification of functional P450 enzymes has proven to be difficult due to their low abundance and lability (Chaple, 1998). However, in the last decade, molecular cloning techniques have been successfully used to isolate a large number of P450 genes from many species. Recently, sequencing of the complete Arabidopsis genome identified a total of 224 cytochrome P450 genes. However, the function of most of these genes is still unknown.In this paper, we describe our strategies to apply molecular cloning techniques to isolate P450 genes from barley. Two approaches have been utilised. The first approach is the application of Polymerase Chain Reaction based methods to clone gene fragments from genomic DNA. The second approach is the use of the International Triticeae EST Cooperative (ITEC) database (http://wheat.pw.usda.gov/genome/index.html) to search for P450 gene sequences. Expression techniques are being employed to investigate gene expression patterns of isolated barley P450 clones. Linh Nguyen http://works.bepress.com/peter_bundock/141 http://works.bepress.com/peter_bundock/141 Tue, 01 Sep 2009 23:27:49 PDT Peter C. Bundock http://works.bepress.com/peter_bundock/140 http://works.bepress.com/peter_bundock/140 Tue, 18 Aug 2009 23:21:26 PDT Gary A. Ablett http://works.bepress.com/peter_bundock/139 http://works.bepress.com/peter_bundock/139 Tue, 18 Aug 2009 23:21:24 PDT Robert J. Henry http://works.bepress.com/peter_bundock/138 http://works.bepress.com/peter_bundock/138 Tue, 18 Aug 2009 23:21:22 PDT Toni Pacey-Miller http://works.bepress.com/peter_bundock/137 http://works.bepress.com/peter_bundock/137 Tue, 18 Aug 2009 23:21:20 PDT Robert J. Henry http://works.bepress.com/peter_bundock/136 http://works.bepress.com/peter_bundock/136 Tue, 11 Aug 2009 23:52:30 PDT The blueprint to a new generation is contained within the seed. The processes in-volved in seed germination are however still somewhat a mystery on a genetic level. Byexamining the transcripts expressed at any time in the cell it is possible to determinewhich genes and their related proteins are being expressed at that precise moment intime. SAGE (Serial Analysis of Gene Expression) is a technique that allows rapid, de-tailed analysis of thousands of transcripts in a cell. The process of SAGE relies on twoprinciples. Firstly, a small sequence of nucleotides from the transcript, called a "tag"can effectively identify the original transcript from whence it came. Secondly, linkingthese tags allows rapid sequencing analysis of multiple transcripts. The abundanceof a particular tag relates directly to the expression level of the gene from which it isderived. The analysis of gene expression of different physiological states provides bothqualitative and quantitative information. This makes it an efficient tool for gene ex-pression studies in the post-genomics era. In this study the gene expression profile ofgerminating (malting) barley is being examined at seven intervals over a time courseof 120 hours post steeping. This will be compared to a baseline of dry ungerminatedseed. The identification of genes for improved malting quality can be identified andexamined using SAGE and ultimately used for commercial improvement. Toni Pacey-Miller