Copyright (c) 2010 All rights reserved. http://works.bepress.com/peter_bundock Recent documents in Peter Bundock en-us Thu, 26 Aug 2010 16:57:52 PDT 3600 http://works.bepress.com/peter_bundock/161 http://works.bepress.com/peter_bundock/161 Sun, 04 Apr 2010 00:09:09 PDT Wild barley (Hordeum spontaneum) represents a significant genetic resource for crop improvement in barley (Hordeum vulgare) and for the study of the evolution and domestication of plant populations. The Isa gene from barley has a putative role in plant defense. We identified 16 Single Nucleotide Polymorphisms (SNPs) in the coding region of the Isa locus of 189 wild barley accessions from 8 sites that were characterized for 16 ecogeographical variables. The pattern of SNPs suggested a large number of recombination events within this gene and 7 amino acid substitutions were present in the coding region. Highly significant correlations were found between diversity at the Isa locus and key water variables - evaporation, rainfall, humidity and latitude. The association is evident at both a local and regional level. These results are consistent with the possibility that inhibition of the plant’s own α-amylase by BASI (isa encoded protein) may be a novel plant defense function. Study of the promoter and coding region of this gene indicate evolution of function at both levels. James K. Cronin http://works.bepress.com/peter_bundock/160 http://works.bepress.com/peter_bundock/160 Wed, 03 Mar 2010 17:43:01 PST Eucalyptus globulus Labill. ssp. globulus is an important tree species for the pulp and paper industry, and several breeding programmes throughout the world are striving to improve key traits such as growth and wood density. This study aimed to detect quantitative trait loci (QTL) for growth, wood density, relative bark thickness and early flowering in a single full-sib E. globulus family grown across seven sites. Growth was measured a number of times over a 6-year period, enabling temporal stability of growth QTL to be studied. Ten putative QTL (LOD>2.0) were detected in the single family, which was of moderate size. Based on permutations of the trait data, six of these QTL were significant at the experimentwise significance level of 0.1 for at least one of the four models implemented in analysis to remove site effects. For wood density, two putative QTL explained 20% of the variance for the trait, indicating that a small number of QTL might explain a reasonable proportion of the trait variance. One of these QTL was found to be independent of QTL for growth whereas the second QTL co-segregated with a QTL for relative incremental growth. The marker nearest to this QTL was associated with fast growth but low wood density. A putative growth QTL at year 6 was found to be relatively stable across ages. In addition, it was found that residuals from models based on measurements from across all families across all sites in the trial detected QTL with greater experimentwise significance. Peter C. Bundock http://works.bepress.com/peter_bundock/159 http://works.bepress.com/peter_bundock/159 Wed, 03 Mar 2010 17:18:34 PST The construction of linkage maps based on RAPD markers using F1 intraprovenance cross in Eucalyptus globulus subsp. globulus is reported. Twenty-one microsatellite loci originating from E. globulus and four other Eucalyptus species were added to the RAPD maps. Linkages between microsatellites previous reported for E. grandis/E. urophylla were found to be conserved in E. globulus allowing confident assignment of homology for several linkage groups between maps of these species. Homology was also identifiable between most linkage groups of the two E. globulus parents based on microsatellites and RAPD loci segregating from both parents. At a LOD score threshold of 4.9 the male parent has 13 linkage groups covering 1013 cM with 101 framework markers ordered at LOD 3.0. The female parent has 11 linkage groups covering 701cM with 97 framework markers. On the female map there were more regions of segregation distortion than expected and genetic mechanisms to explain distorted segregation are discussed. Several linkages that arise between pairs of E. globulus linkage groups as the LOD score is reduced are supported by interspecific homologies identified using microsatellite loci. Peter C. Bundock http://works.bepress.com/peter_bundock/158 http://works.bepress.com/peter_bundock/158 Tue, 23 Feb 2010 22:05:28 PST The sequencing of the sugarcane genome to produce a complete assembled sequence will be an important platform for sugarcane research. However, because of the complexity of this genome, assembly of the sequence from the short sequence reads that can now be cost effectively collected remains a significant challenge. Genome sequence may produce more immediate outcomes before complete assembly of the genome is achieved. Deep sequencing of gene rich fractions of the genome from many genotypes will define genetic diversity in sugarcane genes and provide very large numbers of genetic markers. We have obtained random shot gun sequences from the whole genome of several important genotypes and will use those as references to determine the success of genome enrichment systems. The genotype R570 will be used as a source of BAC clones for extensive sequencing of the sugarcane genome. Q165 and IJ76-514 are parents of populations with extensive maps. SP80-3280 is the subject of large scale sequencing. LA purple and Mandalay allow analysis of Saccharum officinarum and S. spontaneum genomes separately. Robert J. Henry http://works.bepress.com/peter_bundock/157 http://works.bepress.com/peter_bundock/157 Tue, 23 Feb 2010 22:05:27 PST Peter C. Bundock http://works.bepress.com/peter_bundock/156 http://works.bepress.com/peter_bundock/156 Tue, 23 Feb 2010 22:05:25 PST Robert J. Henry http://works.bepress.com/peter_bundock/155 http://works.bepress.com/peter_bundock/155 Tue, 23 Feb 2010 22:05:25 PST Peter C. Bundock http://works.bepress.com/peter_bundock/154 http://works.bepress.com/peter_bundock/154 Tue, 23 Feb 2010 22:05:24 PST Karen S. Aitken http://works.bepress.com/peter_bundock/152 http://works.bepress.com/peter_bundock/152 Tue, 23 Feb 2010 22:05:21 PST Nicole F. Rice http://works.bepress.com/peter_bundock/151 http://works.bepress.com/peter_bundock/151 Tue, 23 Feb 2010 22:05:20 PST Sugarcane is genetically complex due to polyploidy, aneuploidy and hybridisation which have led to a large but variable number of copies of each chromosome and a large number of chromosomes overall in the genomes of commercial varieties. To map genes of interest, ‘single dose’ SNPs are desirable because they are fully informative across the mapping population. However discovery of these SNPs requires considerable depth of sequencing to find the single copy alleles and confirm the polymorphisms. To achieve this depth, 454 sequencing of pooled PCR amplicons was utilised on the parents of a QTL mapping population. Three hundred pooled amplicons from each parent were sequenced yielding 96,755 and 86,241 sequences from the two parents, with average sequence depth of approximately 300 and average read length of 220 bases. In the more polymorphic parent, 94% of amplicons analysed (227/242) had evidence of a reliable SNP – an average of a SNP every 35 bases. Candidate single dose SNPs were validated and genotyped for mapping across the progeny using the Sequenom MassARRAY (MALDI-TOF mass spectrometer) system. From 225 candidate SNP sites tested, 209 (93%) were validated as polymorphic using the Sequenom system. Genotyping across the mapping population was carried out for 197 SNPs. Amplicon re-sequencing using the 454 system enables cost effective SNP discovery that can be targeted to genes of interest. This approach should be useful for the detection of SNPs in polyploid species generally - for linkage mapping, association studies and for population & ecological genetics. Peter C. Bundock http://works.bepress.com/peter_bundock/150 http://works.bepress.com/peter_bundock/150 Tue, 23 Feb 2010 22:05:19 PST As part of a marker program for QTL discovery, 313 sugarcane genes were targeted for the development of SNP markers. However, discovering useful SNPs for mapping these candidates from EST sequences in the public domain was found to be inefficient. As an alternative approach we designed primers to amplify regions of more than 200 of these genes for re-sequencing using 454 Life Sciences Genome Sequencer™ FLX. A region of a four gasket 454 sequencing run was used for the pooled amplicons from each of two mapping population parents. The sequencing yielded 96,755 and 86,241 sequences with perfect matches to a PCR primer used in amplification for the female (IJ76-514) and male (Q165) parents respectively. More than 94% of amplicons from Q165 had a supported SNP, with one SNP every 35 bases and a total of 1,632 SNPs discovered. For IJ76-514, a pure Saccharum officinarum clone, there were significantly fewer SNPs (1,013), with one SNP every 58 bases. More than 200 of these discovered SNPs have been assayed on the Sequenom Mass ARRAY® system with a high proportion being validated. Amplicon re-sequencing using the 454 system resulted in cost effective SNP discovery in a majority of the candidate genes. To map selected genes in sugarcane it is preferable that polymorphisms used for marker development be present on one homeolog only (single dose). Due to the sequence depth attained it has been possible to target assay design to SNPs of low frequency enabling enrichment for single dose markers. Peter C. Bundock http://works.bepress.com/peter_bundock/149 http://works.bepress.com/peter_bundock/149 Sat, 12 Dec 2009 11:59:04 PST Peter C. Bundock 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/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