Copyright (c) 2009 All rights reserved. http://works.bepress.com/frances_eliott Recent documents in Frances G Eliott en-us Thu, 05 Nov 2009 23:22:18 PST 3600 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