Copyright (c) 2010 All rights reserved. http://works.bepress.com/frances_eliott Recent documents in Frances G Eliott en-us Thu, 26 Aug 2010 16:31:16 PDT 3600 http://works.bepress.com/frances_eliott/34 http://works.bepress.com/frances_eliott/34 Tue, 02 Mar 2010 13:54:38 PST Peter C. Bundock http://works.bepress.com/frances_eliott/33 http://works.bepress.com/frances_eliott/33 Tue, 02 Mar 2010 13:54:37 PST Karen S. Aitken http://works.bepress.com/frances_eliott/32 http://works.bepress.com/frances_eliott/32 Tue, 02 Mar 2010 13:54:36 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/frances_eliott/31 http://works.bepress.com/frances_eliott/31 Tue, 02 Mar 2010 13:54:34 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/frances_eliott/30 http://works.bepress.com/frances_eliott/30 Wed, 04 Nov 2009 18:55:57 PST 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/frances_eliott/29 http://works.bepress.com/frances_eliott/29 Mon, 03 Aug 2009 14:38:10 PDT Individual people respond to drugs differently in a genotype dependant manner. One of the key objectives of the human genome project was the acquisition of genomic information which would allow more targeted delivery of pharmaceutical products to consumers based on an individuals genotype, personalised medicine. The tools which have been developed in part to reach this goal are now being applied to agricultural species, including the cereals, and give greater power to genetic analysis. When used in combination, automated DNA Extraction, Next Generation Sequencing and high throughput genotyping technologies, allow a more tightly targeted approach to the resolution of genetic questions than ever before. It is now possible to quickly acquire genome wide data which differentiates between two or more key reference varieties which differ by important phenotypes. High throughput genotyping assays allow rapid analysis of populations which are either derived from the reference varieties or differ by traits. The same data can be used for cereal variety identification, an important quality control tool. These technologies have been deployed at the Centre for Plant Conservation Genetics and are aimed at a number of phenotypes, including starch properties, in barley, rice and wheat. Daniel LE Waters http://works.bepress.com/frances_eliott/28 http://works.bepress.com/frances_eliott/28 Mon, 03 Aug 2009 14:38:09 PDT Model organisms have well established genetic marker tools. However, genotyping of the wide range of agricultural and food species requires the discovery of genetic variations in each species that can be used to distinguish genotypes. Single Nucleotide Polymorphisms (SNPs) are the basis of most high throughput genetic analysis technologies. We have developed a generic approach to the discovery and analysis of SNPs in non-model organisms. SNP Characterisation and Mapping in Genomes (SCAMinG) has been developed from mutation detection based on enzymatic cleavage of DNA heteroduplexes as applied to naturally occurring genetic variation (ecoTILLinG). Our protocol involves application of high throughput capillary electrophoresis to provide efficient discovery of SNPs. SNPs discovered in this way are then available for analysis by other high throughput methods such as mass spectrometry using the mass array platform (Sequenom). These methods can be combined to map SNPs in the genome and to associate SNPs with key traits. The combined approach provides an efficient strategy for SNP genotyping in agricultural species. Frances G. Eliott http://works.bepress.com/frances_eliott/27 http://works.bepress.com/frances_eliott/27 Mon, 03 Aug 2009 14:38:08 PDT Little is known about the extent of allelic diversity of genes in the complex polyploid, sugarcane. Using sucrose phosphate synthase (SPS) Gene (SPS) Family III as an example, we have amplified and sequenced a 400 nt region from this gene from two sugarcane lines that are parents of a mapping population. Ten single nucleotide polymorphisms (SNPs) were identified within the 400 nt region of which seven were present in both lines. In the elite commercial cultivar Q165A, 10 sequence haplotypes were identified, with four haplotypes recovered at 9% or greater frequency. Based on SNP presence, two clusters of haplotypes were observed. In IJ76-514, a Saccharum officinarum accession, 8 haplotypes were identified with 4 haplotypes recovered at 13% or greater frequency. Again, two clusters of haplotypes were observed. The results suggest that there may be two SPS Gene Family III genes per genome in sugarcane, each with different numbers of different alleles. This suggestion is supported by sequencing results in an elite parental sorghum line, 403463-2-1, in which 4 haplotypes, corresponding to two broad types, were also identified. Primers were designed to the sugarcane SNPs and screened over bulked DNA from high and low Sucrose-containing progeny from a cross between Q165A and IJ76-514. The SNP frequency did not vary in the two bulked DNA samples, suggesting that these SNPs from this SPS gene family are not associated with variation in sucrose content. Using an ecotilling approach, two of the SPS Gene Family III haplotypes were mapped to two different linkage groups in homology group 1 in Q165A. Both haplotypes mapped near QTLs for increased sucrose content but were not themselves associated with any sugar-related trait. C Lynne McIntyre http://works.bepress.com/frances_eliott/26 http://works.bepress.com/frances_eliott/26 Wed, 29 Jul 2009 20:01:40 PDT Single nucleotide polymorphism (SNP) analysis in polyploid genomes such as sugarcane requires the application of tools that allow quantitative analysis of mixed alleles even when analysing DNA from a single genotype. We have developed several techniques and adapted them to analysis of sugarcane SNP. Association of SNP or the numbers of homeologous alleles defined by a SNP is required to associate allele dose with phenotype in sugarcane. Protocols for ecotilling using an ABI Genetic Analyzer platform for capillary electrophoresis have been optimised for use in SNP discovery. Sequenom analysis allows efficient quantitative analysis of known SNP in association studies. Novel chemistries for RT PCR based upon Locked Nucleic Acid (LNA) strand displacement probes provide new options for analysis of sugarcane SNP. Giovanni M. Cordeiro http://works.bepress.com/frances_eliott/25 http://works.bepress.com/frances_eliott/25 Wed, 29 Jul 2009 20:01:39 PDT The sugarcane genome poses the challenge of being highly polyploid and requiring ingenuity to overcome impediments of straightforward genome analyses methods that are often taken for granted in simpler genomes and inbred species. We have developed and evaluated a number of technologies to accurately locate and score the level of frequency of a SNP at any individual locus. Giovanni M. Cordeiro http://works.bepress.com/frances_eliott/24 http://works.bepress.com/frances_eliott/24 Wed, 29 Jul 2009 20:01:38 PDT Giovanni M. Cordeiro http://works.bepress.com/frances_eliott/23 http://works.bepress.com/frances_eliott/23 Wed, 29 Jul 2009 20:01:37 PDT Giovanni M. Cordeiro http://works.bepress.com/frances_eliott/22 http://works.bepress.com/frances_eliott/22 Wed, 29 Jul 2009 20:01:36 PDT Sugarcane cultivars are polyploid, aneuploid, interspecific hybrids between the domesticated species S. officinarum, and a wild relative S. spontaneum. Their chromosome number ranges from 100-130 with approximately 10% contributed by S. spontaneum and ploidy number ranging between 8 and 10. This chromosomal allele complexity limits mapping to dominant single dose DNA markers with multiple dose alleles offering a greater challenge. Verifying haplotypes by SNP marker patterns may offer a solution for this challenge. EST alignments are a rich source for mining SNPs, and the use of pyrosequencing has proved to be a reliable method for measuring the SNP base ratios allowing quantitative SNP allelotyping in sugarcane. Measuring the base ratios at the SNP loci would discriminate single from multiple dose alleles. Verifying the haplotype compositions will genotype the alleles which may provide some information on the phenotypic, genotypic relationship in sugarcane. We have identified SNP haplotypes in isogenes of sucrose phosphate synthase, the sucrose biosynthetic enzyme of sugarcane and are examining these in segregating progeny displaying variation in sucrose accumulation. Ouzi Amouyal http://works.bepress.com/frances_eliott/21 http://works.bepress.com/frances_eliott/21 Wed, 29 Jul 2009 20:01:34 PDT The highly complex and polyploid genome of sugarcane provides challenges to nearly all technologies developed to analyse diploid genomes. In cultivated clones, chromosome numbers vary in excess of 100, and the ploidy number between loci can vary from 8 and 14 within an individual. This restricts mapping to simplex (single dose) DNA markers; and makes it impossible to determine potential ‘active’ gene haplotypes. SNP markers offer the best solution to overcome many of the difficulties, although it is in itself a challenge to develop for this complex genome. The recent release of a large scale sugarcane EST sequencing project into the public databases has provided a means of identifying potential SNPs for development into markers that can be used for mapping, cultivar identification and potentially, gene haplotype discrimination. We outline the methods we have used to measure SNP base ratios to allow quantitative SNP allelotyping in sugarcane that will allow mapping of single dose markers and potentially identify gene haplotypes linked to phenotypes Giovanni M. Cordeiro http://works.bepress.com/frances_eliott/19 http://works.bepress.com/frances_eliott/19 Sun, 12 Jul 2009 16:42:58 PDT Giovanni M. Cordeiro http://works.bepress.com/frances_eliott/18 http://works.bepress.com/frances_eliott/18 Sun, 12 Jul 2009 16:42:57 PDT Genetic linkage maps of sugarcane have to date been largely based on one or a combination of RAPD, RFLP, SSR and AFLP markers. However high throughput and potentially low-cost genotyping based on single nucleotide polymorphisms (SNPs) along with the possibility of perfect markers has made SNPs based on expressed sequences such as ESTs an attractive option for marker development in sugarcane. To test the feasibility of SNP marker based genotyping in sugarcane we have developed a system for SNP detection based on Ecotilling using CEL I polymorphism detection. Partial CEL I digests of labelled PCR products generated from the DNA of individuals can be used for both discovery and determining segregation patterns. However genotyping with this approach is not high throughput or cheap but to this end SNPs segregating as single dose in progeny sub-samples can be genotyped cheaply and with high-throughput using mass spectrometry (Sequenom). The strategy of utilising Ecotilling for SNP confirmation and determination of segregation pattern followed by mass spectrometry based genotyping on full progeny sets has been tested and confirmed using members of the sucrose-phosphate synthase gene family in sugarcane. In addition to genotyping for mapping, mass spectrometry should also be a useful method of allele frequency determination for LD based association studies. Peter C. Bundock http://works.bepress.com/frances_eliott/15 http://works.bepress.com/frances_eliott/15 Sun, 12 Jul 2009 16:42:55 PDT Pollen contamination continues to be a problem for many operational controlled-pollination programs for hybrid trees. In the case of the Slash x Caribbean pine hybrid in Queensland, productivity losses may occur if pure Slash is deployed on sites optimal for hybrids. We report a DNA marker assay that distinguishes between representatives of Slash and Caribbean pine breeding populations. Furthermore, interspecific hybrid F1s were distinguishable from individuals believed to be derived from self fertilisation or outcrossing to other Slash pine. Unlike earlier reported DNA assays for verification of hybrid pines, this assay was definitive for the reference population. The multi-locus assay was optimised for a single-tube PCR reaction, offering estimated 75% savings in the turnaround time and costs compared with individual PCR amplification of the six loci. The PEE displayed more variation at these loci than PCH despite the PCH having been sampled over a more extensive geographical range than the PEE. Frances G. Eliott http://works.bepress.com/frances_eliott/14 http://works.bepress.com/frances_eliott/14 Sun, 12 Jul 2009 16:42:54 PDT Frances G. Eliott http://works.bepress.com/frances_eliott/13 http://works.bepress.com/frances_eliott/13 Sun, 12 Jul 2009 16:42:53 PDT Commercial sugarcane cultivars (Saccharum spp. hybrids) are both polyploid and aneuploid with chromosome numbers in excess of 100; these chromosomes can be assigned to 8 homology groups. To determine the utility of single nucleotide polymorphisms (SNPs) as a means of improving our understanding of the complex sugarcane genome, we developed markers to a suite of SNPs identified in a list of sugarcane ESTs. Analysis of 69 EST contigs showed a median of 9 SNPs per EST and an average of 1 SNP per 50 bp of coding sequence. The quantitative presence of each base at 58 SNP loci within 19 contiguous sequence sets was accurately and reliably determined for 9 sugarcane genotypes, including both commercial cultivars and ancestral species, through the use of quantitative light emission technology in pyrophosphate sequencing. Across the 9 genotypes tested, 47 SNP loci were polymorphic and 11 monomorphic. Base frequency at individual SNP loci was found to vary approximately twofold between Australian sugarcane cultivars and more widely between cultivars and wild species. Base quantity was shown to segregate as expected in the IJ76-514 × Q165 sugarcane mapping population, indicating that SNPs that occur on one or two sugarcane chromosomes have the potential to be mapped. The use of SNP base frequencies from five of the developed markers was able to clearly distinguish all genotypes in the population. The use of SNP base frequencies from a further six markers within an EST contig was able to help establish the likely copy number of the locus in two genotypes tested. This is the first instance of a technology that has been able to provide an insight into the copy number of a specific gene locus in hybrid sugarcane. The identification of specific and numerous haplotypes/alleles present in a genotype by pyrophosphate sequencing or alternative techniques ultimately will provide the basis for identifying associations between specific alleles and phenotype and between allele dosage and phenotype in sugarcane. Giovanni M. Cordeiro http://works.bepress.com/frances_eliott/11 http://works.bepress.com/frances_eliott/11 Sun, 12 Jul 2009 16:42:52 PDT DNA markers of various kinds (AFLPs, SSRs, SNPs etc.) are being used for many crop species as a means to improve genetic gains in breeding programs by marker assisted selection (MAS – selecting parent plants based on the presence of a marker rather than measuring the trait of interest each generation). For the purposes of MAS, DNA markers should ideally be reliable and cheap to assay. Point mutations or single nucleotide polymorphisms (SNPs) as they are now more commonly known are an excellent source of markers because of their occurrence throughout the genome. Various methods have been developed to assay SNPs for genotyping in both plants and animals. The Sequenom MASS Array system provides a high throughput method of assaying SNPs (genotyping) that is both reliable and cheap.The Sequenom MASS Array system has been used at Southern Cross to assay SNPs in sugarcane genes. A particular difficulty when working at the DNA level with sugarcane is its highly polyploid nature (multiple copies of each chromosome – compared with only two in diploids), which has meant that it has been difficult to detect low dose and in particular single dose SNP markers due to the low signal from the single dose allele. However with the Sequenom system the proportion of the homologous chromosomes which carry a particular SNP allele can be estimated. This method has been used in two projects within the CRC-SIIB marker program. About 300 genes are being targeted based on independent evidence from other CRC projects that these genes may influence important sugarcane traits. In one project a large number of sugarcane clones have been genotyped using a large number of SNPs. The results will be used to determine if there are associations between SNP alleles and the traits of interest that have been scored across this broad range of sugarcane clones. In a second project SNPs have been genotyped across the parents of a genetic linkage mapping population. SNPs that appeared to be single dose were tested across a subset of the progeny of the parents of the mapping population and if segregation was observed in this subset, the entire mapping population of 220 individuals was genotyped. SNPs genotyped in this way have been added to the linkage map being constructed by CRC-SIIB at CSIRO which can be used for the detection of trait associations within the mapping population. The number of SNPs has to date been limited due to a lack of knowledge about SNP sites in the parents of the sugarcane mapping population. However a much larger number of SNPs are expected to be found in this cross using new methods of DNA sequencing. Peter C. Bundock