Sequence Polymorphism Discovery in Wheat Microsatellite Flanking Regions using Pyrophosphate Sequencing

Ablett, Gary A; Hill, Helen; Henry, Robert J.

doi:10.1007/s11032-006-6262-3

Cited by 24 publications

(14 citation statements)

References 10 publications

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“…Significant resources have been devoted to the development of SNPs as high‐throughput markers and also to SNP discovery. Extensive SNP discovery projects have been undertaken in many species, including humans (Sachidanandam et al ., 2001), model species such as Arabidopsis thaliana (Jander et al ., 2002) and Drosophila melanogaster (Hoskins et al ., 2001), and in crop plants, such as barley (Rostoks et al ., 2005a), maize (Ching et al ., 2002), rice (Shen et al ., 2004; McNally et al ., 2006), soybean (Zhu et al ., 2003) and wheat (Ablett et al ., 2006; Ravel et al ., 2006). In species in which no genome sequence is available, large‐scale SNP discovery in genes has generally relied on sequence information in libraries of expressed sequence tags (ESTs) for either direct discovery (Somers et al ., 2003; Bundock et al ., 2006) or as the basis for primer design for re‐sequencing (Rostoks et al ., 2005b; Choi et al ., 2007).…”

Section: Introductionmentioning

confidence: 99%

Targeted single nucleotide polymorphism (SNP) discovery in a highly polyploid plant species using 454 sequencing

Bundock

Eliott

Ablett

et al. 2009

Plant Biotechnology Journal

133

100

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SummaryDiscovering 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 CAP 3. 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.

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Section: Introductionmentioning

confidence: 99%

Targeted single nucleotide polymorphism (SNP) discovery in a highly polyploid plant species using 454 sequencing

Bundock

Eliott

Ablett

et al. 2009

Plant Biotechnology Journal

133

100

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“…Several diagnostic applications across many organisms have been developed using this method. In bread wheat pyrosequencing is used for instance to genotype SNPs (Huang and Röder 2005;Ablett et al 2006) or to carry out quantitative transcriptome profilings (Salentijn et al 2009). Also, 454 shotgun sequencing is a pyrosequencing-based approach which can be used for genome sequencing and, subsequently, to identify SNPs.…”

Section: Pyrosequencing and Breeding Applicationsmentioning

confidence: 99%

Discrimination of alleles and copy numbers at the Q locus in hexaploid wheat using quantitative pyrosequencing

et al. 2011

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Speltoid spikes are characterized by pyramidal spike morphology featuring an elongated rachis and tenacious glumes. Speltoids are considered undesirable spike aberrants in wheat breeding leading to increased heterogeneity within a cultivar candidate. As a consequence, the presence of speltoids may result in rejection of a cultivar candidate during official field trials or denial of cultivar certification during seed multiplication. A reliable method is, thus, required to assess the occurrence of speltoids, early on in a wheat breeding program. The domestication gene Q located on the long arm of wheat chromosome 5A is known to suppress the speltoid phenotype in wheat. Here, a quantitative pyrosequencing assay was developed to distinguish between normal wheat plants, which possess two copies of the Q allele, and aberrants, which are either aneuploids lacking the correct number of chromosome 5A copies or plants which carry the primitive q allele. An accurate and reproducible determination of the Q gene copy number was achieved for different wheat genotypes based on homoeologous sequence quantification with two primer combinations at the Q locus. Single plants with one to four copies of the Q allele could be detected by quantitative pyrosequencing which corresponded to the occurrence of speltoid (1 Q allele), normal (2 Q alleles), and compact (more than 2 Q alleles) spikes. Q and q specific alleles could be differentiated at SNP position 2299 of the Q gene. This SNP is assumed to be related to the emergence of freethreshing wheat forms. To our knowledge this is the first report for detection of aneuploids and differentiation of Q alleles in bread wheat using pyrosequencing technology. In future, quantitative pyrosequencing assay can be applied in wheat breeding programs to carry out marker-assisted selection against the presence of speltoid spike aberrants.

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“…Analysis of the smallest unit of DNA sequence difference, the single nucleotide polymorphism (SNP), is currently the preferred approach because of the low cost and high level of automation of the assays (Sobrino et al 2005). Earlier genetic markers used in cereals are able to be converted to SNP assays (Ablett et al 2006;Bundock et al 2006) to take advantage of these technologies.…”

Section: High-throughput Low-cost Genomic Testsmentioning

confidence: 99%

Genomics as a Tool for Cereal Chemistry

Henry

2007

Cereal Chem

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Genomics is providing a new set of tools for cereal chemistry. Analysis of the DNA of the major cereals is increasing the understanding of the basis of grain quality at the gene level. Differences in the sequence of the gene in different cultivars or differences in the level of expression of the gene may be used to explain differences in processing or end‐use quality characteristics. For example, quantitative analysis of the levels of expression of genes at different stages during seed development in wheat or germination in barley can be used to define the genetic basis of differences in wheat and barley quality. Rice quality traits such as fragrance and gelatinization temperature (cooking temperature) can be explained by DNA sequence differences in specific genes identified using genomics approaches. Rapid and reliable species and cultivar identification based on DNA analysis methods developed using genomics tools can be applied to grain and to food products. This technology has special advantages in the analysis of complex mixtures of cereals. Technologies for very high‐throughput and very low‐cost analysis of large numbers of samples are now available. These may be applied by cereal chemists at many levels: selection in cereal breeding, optimizing processing, and analysis of the identity and composition of grain or food samples.

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Sequence Polymorphism Discovery in Wheat Microsatellite Flanking Regions using Pyrophosphate Sequencing

Cited by 24 publications

References 10 publications

Targeted single nucleotide polymorphism (SNP) discovery in a highly polyploid plant species using 454 sequencing

Targeted single nucleotide polymorphism (SNP) discovery in a highly polyploid plant species using 454 sequencing

Discrimination of alleles and copy numbers at the Q locus in hexaploid wheat using quantitative pyrosequencing

Genomics as a Tool for Cereal Chemistry

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