Single Feature Polymorphisms Research Articles

Extracting genotype information of Arabidopsis thaliana recombinant inbred lines from transcript profiles established with high-density oligonucleotide arrays.

Polymorphic probes identified via a sequence-based approach are suitable to infer the genotypes of recombinant inbred lines from hybridisation intensities of GeneChip ® transcript profiling experiments. The sequences of the probes of the ATH1 GeneChip® exactly match transcript sequences of the Arabidopsis thaliana reference genome Col-0, whereas nucleotide differences and/or insertions/deletions may be observed for transcripts of other A.thaliana accessions. Individual probes of the GeneChip® that show sequence polymorphisms between different A.thaliana accessions may serve as single-feature polymorphism (SFP) markers, provided that the sequence changes cause differences in hybridisation intensity for the accessions of interest. A sequence-based approach identified features on the high-density oligonucleotide array that showed sequence polymorphisms between A.thaliana accessions Col-0 and C24. Hybridisation intensities of polymorphic probes were extracted from genome-wide transcript profiles of Col-0/C24 and C24/Col-0 recombinant inbred lines and assessed after standardisation via sliding window analyses to identify SFP markers. The genotypes of the recombinant inbred lines were determined with the SFP markers and the resulting data were integrated with information, which had been established previously with single nucleotide polymorphism and insertion/deletion markers, to enrich the linkage map of the Col-0/C24 and C24/Col-0 recombinant inbred populations. Congruence between the molecular marker map and the sequence maps of the A.thaliana Col-0 chromosomes proved the reliability of the genotype information which was deduced from the transcript profiles of the Col-0/C24 and C24/Col-0 recombinant inbred lines.

Microarray-based large scale detection of single feature polymorphism in Gossypium hirsutum L.

Microarrays offer an opportunity to explore the functional sequence polymorphism among different cultivars of many crop plants. The Affymetrix microarray expression data of five genotypes of Gossypium hirsutum L. at six different fibre developmental stages was used to identify single feature polymorphisms (SFPs). The background corrected and quantile-normalized log2 intensity values of all probes of triplicate data of each cotton variety were subjected to SFPs call by using SAM procedure in R language software. We detected a total of 37,473 SFPs among six pair genotype combinations of two superior (JKC777 and JKC725) and three inferior (JKC703, JKC737 and JKC783) using the expression data. The 224 SFPs covering 51 genes were randomly selected from the dataset of all six fibre developmental stages of JKC777 and JKC703 for validation by sequencing on a capillary sequencer. Of these 224 SFPs, 132 were found to be polymorphic and 92 monomorphic which indicate that the SFP prediction from the expression data in the present study confirmed a ~58.92% of true SFPs. We further identified that most of the SFPs are associated with genes involved in fatty acid, flavonoid, auxin biosynthesis etc. indicating that these pathways significantly involved in fibre development.

Detection of Single‐feature Polymorphisms (SFPs) between two tomato varieties and their application in defining the introgressions of resistance loci

AbstractThe objectives of the study were to (i) demonstrate that the hybridization data from microarrays can yield information on sequence variation between two inbred lines, an introgression line ‘Mogeor’ and its genetic background ‘Moneymaker’; (ii) characterize, by means of the identified SFPs, the introgressed genomic segments of ‘Mogeor’, carrying resistance genes; and (iii) deliver a set of genetically anchored SFPs potentially useful for breeding. In this work, the GeSNP software was used to identify SFPs in tomato using Affymetrix data from a previous experiment. Sequencing of 12 putative polymorphism‐containing amplicons yielded a SFP probe set validation rate of 90%. In total, 92 Gene Models putatively harbouring SFPs were identified, distributed as following: 61 Gene Models on chromosome 9, and one to eight on the remaining tomato chromosomes apart from chromosomes 7, 8 and 12. Newly discovered SFPs from microarray data can thus provide not only useful information for definition of introgressed genomic regions, but also identification of candidate genes and new markers for MAS.

Detection of SNP and validation of a SFP InDel (deletion) in inverted repeat region of the Prunus species chloroplast genome

In order to control tree size, disease, precocity and stress most Prunus varieties are cultivated as composite plants grafted onto desirable rootstocks that impart all the afore-mentioned traits. Several Prunus rootstock breeding programs have been focused on the production of interspecific hybrids. The pedigree of most of these rootstocks remains unknown due to the lack of parental information necessitating the application of DNA-based knowledge in breeding programs. The amplification and sequencing of the chloroplast inverted repeat B (IRB) region spanning 25,960bp from P. cerasifera (myrobalan plum) Ehrh., P. amygdalus (almond) and P. persica (peach) using the ASAP method revealed a single nucleotide polymorphisms (SNP) in the rps19-rpl2 IRB region in myrobalan when compared to almond and peach. In addition, a prominent and an easily identifiable single feature polymorphism (SFP-InDel (deletion)) of 18 nucleotides was discovered in reference to the peach chloroplast genome in the ycf1 gene in the IRB region. In this work, it has been developed a highly useful polymorphic molecular marker to characterize the maternal parent in interspecific hybrids of Prunus rootstocks as a first step toward developing pedigree information. The ycf1 SFP-InDel (deletion) has been successfully used in several 3-way hybrids generated in the stone fruit rootstock breeding program for the characterization of new interspecific plant material. This SFP is expected to be highly utile in characterizing the maternal lineage of Prunus hybrids in other breeding programs.

Pigeons: A Novel GUI Software for Analysing and Parsing High Density Heterologous Oligonucleotide Microarray Probe Level Data.

Genomic DNA-based probe selection by using high density oligonucleotide arrays has recently been applied to heterologous species (Xspecies). With the advent of this new approach, researchers are able to study the genome and transcriptome of a non-model or an underutilised crop species through current state-of-the-art microarray platforms. However, a software package with a graphical user interface (GUI) to analyse and parse the oligonucleotide probe pair level data is still lacking when an experiment is designed on the basis of this cross species approach. A novel computer program called Pigeons has been developed for customised array data analysis to allow the user to import and analyse Affymetrix GeneChip® probe level data through XSpecies. One can determine empirical boundaries for removing poor probes based on genomic hybridisation of the test species to the Xspecies array, followed by making a species-specific Chip Description File (CDF) file for transcriptomics in the heterologous species, or Pigeons can be used to examine an experimental design to identify potential Single-Feature Polymorphisms (SFPs) at the DNA or RNA level. Pigeons is also focused around visualization and interactive analysis of the datasets. The software with its manual (the current release number version 1.2.1) is freely available at the website of the Nottingham Arabidopsis Stock Centre (NASC).

Single-feature polymorphism mapping of isogenic rice lines identifies the influence of terpene synthase on brown planthopper feeding preferences

BackgroundBph3, a major brown planthopper (BPH) resistance locus derived from the rice cultivar Rathu Heenati (RH), has been used as a stable donor of traits that improve highly susceptible aromatic rice varieties in Thailand. Map-based cloning was initiated using a set of isogenic lines (ILs) harboring the major Bph3 locus on chromosome 6. IL genomes were scanned with a 57 K Affymetrix Rice GeneChip to identify the gene responsible for Bph3.FindingsSingle-feature polymorphism (SFP) mapping was used to localize 84 candidate genes. An expression analysis of 15 selected candidate genes in the aromatic rice cultivar KDML105 (KD) and the ILs under normal conditions revealed two differentially expressed sequences. Following hopper feeding, only one candidate gene, Os04g27430, was differentially expressed. Os04g27430 encodes a putative sesquiterpene synthase (STPS) gene that was induced by BPH feeding in ILs. An antixenosis test in three selected ILs revealed a major role for STPS in insect preference during the first 120 hours of the rice-insect interaction. Functional SNPs in exon 5 that resulted in the deletion of seven amino acids in the susceptible rice line were identified. Moreover, three additional SNPs associated with three transcription binding sites were also identified, which might explain the differential response of Os04g27430 during the anti-feeding test.ConclusionOs04g27430 is the second known rice STPS induced by BPH. The gene may involve an antixenosis BPH resistance mechanism. The combination of the STPS and the Bph3 locus was more effective than Bph3 alone in the tested ILs.Electronic supplementary materialThe online version of this article (doi:10.1186/1939-8433-6-18) contains supplementary material, which is available to authorized users.

Complex Genetic Architecture of Cardiac Disease in a Wild Type Inbred Strain of Drosophila melanogaster

Natural populations of the fruit fly, Drosophila melanogaster, segregate genetic variation that leads to cardiac disease phenotypes. One nearly isogenic line from a North Carolina peach orchard, WE70, is shown to harbor two genetically distinct heart phenotypes: elevated incidence of arrhythmias, and a dramatically constricted heart diameter in both diastole and systole, with resemblance to restrictive cardiomyopathy in humans. Assuming the source to be rare variants of large effect, we performed Bulked Segregant Analysis using genomic DNA hybridization to Affymetrix chips to detect single feature polymorphisms, but found that the mutant phenotypes are more likely to have a polygenic basis. Further mapping efforts revealed a complex architecture wherein the constricted cardiomyopathy phenotype was observed in individual whole chromosome substitution lines, implying that variants on both major autosomes are sufficient to produce the phenotype. A panel of 170 Recombinant Inbred Lines (RIL) was generated, and a small subset of mutant lines selected, but these each complemented both whole chromosome substitutions, implying a non-additive (epistatic) contribution to the “disease” phenotype. Low coverage whole genome sequencing was also used to attempt to map chromosomal regions contributing to both the cardiomyopathy and arrhythmia, but a polygenic architecture had to be again inferred to be most likely. These results show that an apparently simple rare phenotype can have a complex genetic basis that would be refractory to mapping by deep sequencing in pedigrees. We present this as a cautionary tale regarding assumptions related to attempts to map new disease mutations on the assumption that probands carry a single causal mutation.

Introgression of novel traits from a wild wheat relative improves drought adaptation in wheat.

Root architecture traits are an important component for improving water stress adaptation. However, selection for aboveground traits under favorable environments in modern cultivars may have led to an inadvertent loss of genes and novel alleles beneficial for adapting to environments with limited water. In this study, we elucidate the physiological and molecular consequences of introgressing an alien chromosome segment (7DL) from a wild wheat relative species (Agropyron elongatum) into cultivated wheat (Triticum aestivum). The wheat translocation line had improved water stress adaptation and higher root and shoot biomass compared with the control genotypes, which showed significant drops in root and shoot biomass during stress. Enhanced access to water due to higher root biomass enabled the translocation line to maintain more favorable gas-exchange and carbon assimilation levels relative to the wild-type wheat genotypes during water stress. Transcriptome analysis identified candidate genes associated with root development. Two of these candidate genes mapped to the site of translocation on chromosome 7DL based on single-feature polymorphism analysis. A brassinosteroid signaling pathway was predicted to be involved in the novel root responses observed in the A. elongatum translocation line, based on the coexpression-based gene network generated by seeding the network with the candidate genes. We present an effective and highly integrated approach that combines root phenotyping, whole-plant physiology, and functional genomics to discover novel root traits and the underlying genes from a wild related species to improve drought adaptation in cultivated wheat.

SNP Detection by Using Affymetix Barley 1 Array in Barley

Nowadays the research for discovering SNPs is becoming one of the most important topics to understand the differences between genotypes of the same species. The objective of this study is to discover the SNPs using oligonucleotide microarrays in barley. Two methods are used for the prediction step: one is Robustified Projection Pursuit (RPP) method and another is based on a statistic test between probes of a probeset belonging different genotypes. Following the procedure described above, 124 single feature polymorphisms (SFPs), which can be used for identification of SNPs or insertion/deletions (INDELs), were detected among all genotypes. Sequencing of putative SFP-containing amplicons from the EST region in the genome yielded a validation rate of 85%, indicating the Affymetrix Barley 1 array could provide a satisfactory platform for identification of SNP in barley.

Development of an Ultra-Dense Genetic Map of the Sunflower Genome Based on Single-Feature Polymorphisms

The development of ultra-dense genetic maps has the potential to facilitate detailed comparative genomic analyses and whole genome sequence assemblies. Here we describe the use of a custom Affymetrix GeneChip containing nearly 2.4 million features (25 bp sequences) targeting 86,023 unigenes from sunflower (Helianthus annuus L.) and related species to test for single-feature polymorphisms (SFPs) in a recombinant inbred line (RIL) mapping population derived from a cross between confectionery and oilseed sunflower lines (RHA280×RHA801). We then employed an existing genetic map derived from this same population to rigorously filter out low quality data and place 67,486 features corresponding to 22,481 unigenes on the sunflower genetic map. The resulting map contains a substantial fraction of all sunflower genes and will thus facilitate a number of downstream applications, including genome assembly and the identification of candidate genes underlying QTL or traits of interest.

Molecular mapping of the blast resistance gene Pi49 in the durably resistant rice cultivar Mowanggu

Rice blast, caused by the fungus Magnaporthe oryzae, is the most devastating fungal disease of rice. Mowanggu, a local japonica cultivar in Yunnan Province, China, confers broad-spectrum resistance to this pathogen. To identify the resistance gene(s) in Mowanggu, we obtained an F2 population and 280 F8 recombinant inbred lines (RILs) from a cross between Mowanggu and CO39, a highly susceptible indica cultivar. A linkage map with 145 simple sequence repeat (SSR) and single feature polymorphism markers over 12 chromosomes was constructed using the 280 RILs. The resistance evaluation of the F2 and F8 populations in both the growth chamber and in a natural rice blast nursery showed that a single dominant gene controls blast resistance in Mowanggu. Moreover, nine quantitative trait loci, which were responsible for different partial resistance components, were mapped on chromosomes 2, 3, 6, 8, 9, and 12, making contributions to the phenotypic variation ranging from 3.03 to 6.18 %. The dominant resistance gene, designated Pi49, was mapped on chromosome 11 with genetic distance of 1.01 and 1.89 cM from SSR markers K10 and K134, respectively. The physical distance between K10 and K134 is about 181 kb in the Nipponbare genome. The Pi49 gene accounted for the major phenotypic variation of disease severity in the growth chamber (where plants were inoculated with single blast isolates) and also accounted for most of the phenotypic variance of disease severity, lesion number, diseased leaf area, and lesion size in the blast nursery. Our study not only identified tightly linked markers for introgression of Pi49 into elite rice cultivars via marker-aided selection but also provides a starting point for map-based cloning of the new resistance gene.

An improved algorithm for the detection of genomic variation using short oligonucleotide expression microarrays

High-throughput microarray experiments often generate far more biological information than is required to test the experimental hypotheses. Many microarray analyses are considered finished after differential expression and additional analyses are typically not performed, leaving untapped biological information left undiscovered. This is especially true if the microarray experiment is from an ecological study of multiple populations. Comparisons across populations may also contain important genomic polymorphisms, and a subset of these polymorphisms may be identified with microarrays using techniques for the detection of single feature polymorphisms (SFP). SFPs are differences in microarray probe level intensities caused by genetic polymorphisms such as single-nucleotide polymorphisms and small insertions/deletions and not expression differences. In this study, we provide a new algorithm for the detection of SFPs, evaluate the algorithm using existing data from two publicly available Affymetrix Barley (Hordeum vulgare) microarray data sets and compare them to two previously published SFP detection algorithms. Results show that our algorithm provides more consistent and sensitive calling of SFPs with a lower false discovery rate. Simultaneous analysis of SFPs and differential expression is a low-cost method for the enhanced analysis of microarray data, enabling additional biological inferences to be made.

Development and application of a 6.5 million feature Affymetrix Genechip® for massively parallel discovery of single position polymorphisms in lettuce (Lactuca spp.).

BackgroundHigh-resolution genetic maps are needed in many crops to help characterize the genetic diversity that determines agriculturally important traits. Hybridization to microarrays to detect single feature polymorphisms is a powerful technique for marker discovery and genotyping because of its highly parallel nature. However, microarrays designed for gene expression analysis rarely provide sufficient gene coverage for optimal detection of nucleotide polymorphisms, which limits utility in species with low rates of polymorphism such as lettuce (Lactuca sativa).ResultsWe developed a 6.5 million feature Affymetrix GeneChip® for efficient polymorphism discovery and genotyping, as well as for analysis of gene expression in lettuce. Probes on the microarray were designed from 26,809 unigenes from cultivated lettuce and an additional 8,819 unigenes from four related species (L. serriola, L. saligna, L. virosa and L. perennis). Where possible, probes were tiled with a 2 bp stagger, alternating on each DNA strand; providing an average of 187 probes covering approximately 600 bp for each of over 35,000 unigenes; resulting in up to 13 fold redundancy in coverage per nucleotide. We developed protocols for hybridization of genomic DNA to the GeneChip® and refined custom algorithms that utilized coverage from multiple, high quality probes to detect single position polymorphisms in 2 bp sliding windows across each unigene. This allowed us to detect greater than 18,000 polymorphisms between the parental lines of our core mapping population, as well as numerous polymorphisms between cultivated lettuce and wild species in the lettuce genepool. Using marker data from our diversity panel comprised of 52 accessions from the five species listed above, we were able to separate accessions by species using both phylogenetic and principal component analyses. Additionally, we estimated the diversity between different types of cultivated lettuce and distinguished morphological types.ConclusionBy hybridizing genomic DNA to a custom oligonucleotide array designed for maximum gene coverage, we were able to identify polymorphisms using two approaches for pair-wise comparisons, as well as a highly parallel method that compared all 52 genotypes simultaneously.

High Resolution Mapping of Candidate Alleles for Desiccation Resistance in Drosophila melanogaster under Selection

The ability to counter periods of low humidity is an important determinant of distribution range in Drosophila. Climate specialists with low physiological tolerance to desiccation stress are restricted to the tropics and may lack the ability to further increase resistance through evolution. Although the physiological adaptations to desiccation stress are well studied in Drosophila and other ectotherms, factors underlying evolutionary responses remain unknown because of a paucity of genetic data. We address this issue by mapping evolutionary shifts in D. melanogaster under selection for desiccation resistance. Genomic DNA from five independent replicate selected, and control lines were hybridized to high density Affymetrix Drosophila tiling arrays resulting in the detection of 691 single feature polymorphisms (SFPs) differing between the treatments. While randomly distributed throughout the genome, the SFPs formed specific clusters according to gene ontology. These included genes involved in ion transport and respiratory system development that provide candidates for evolutionary changes involving excretory and respiratory water balance. Changes to genes related to neuronal control of cell signaling, development, and gene regulation provide candidates to explore novel biological processes in stress resistance. Sequencing revealed the nucleotide shifts in a subset of the SFPs and highlighted larger regions of genomic diversity surrounding SFPs. The association between natural desiccation resistance and a 463-bp region of the 5' promoter region of the Dys gene undergoing allele frequency changes in response to selection in the experimental evolution lines was tested in an independent population from Coffs Harbour, Australia. The allele frequencies of 23 SNPs common to the two populations were inferred from the parents of the 10% most and 10% least resistant Coffs Harbour flies. The frequencies of the selected alleles were higher at all sites, with three sites significantly associated with the resistant Coffs Harbour flies. This study illustrates how rapid mapping can be used for discovering natural molecular variants associated with survival to low humidity and provides a wealth of candidate alleles to explore the genetic basis of physiological differences among resistant and susceptible Drosophila populations and species.

Predictions of single-nucleotide polymorphism differentiation between two populations in terms of mutual information

Mutual information (I) provides a robust measure of genetic differentiation for the purposes of estimating dispersal between populations. At present, however, there is little predictive theory for I. The growing importance in population biology of analyses of single-nucleotide and other single-feature polymorphisms (SFPs) is a potent reason for developing an analytic theory for I with respect to a single locus. This study represents a first step towards such a theory. We present theoretical predictions of I between two populations with respect to a single haploid biallelic locus. Dynamical and steady-state forecasts of I are derived from a Wright-Fisher model with symmetrical mutation between alleles and symmetrical dispersal between populations. Analytical predictions of a simple Taylor approximation to I are in good agreement with numerical simulations of I and with data on I from SFP analyses of dispersal experiments on Drosophila fly populations. The theory presented here also provides a basis for the future inclusion of selection effects and extension to multiallelic loci.

Single feature polymorphisms (SFPs) for drought tolerance in pigeonpea (Cajanus spp.)

Single feature polymorphisms (SFPs) are microarray-based molecular markers that are detected by hybridization of DNA or cRNA to oligonucleotide probes. With an objective to identify the potential polymorphic markers for drought tolerance in pigeonpea [Cajanus cajan (L.) Millspaugh], an important legume crop for the semi-arid tropics but deficient in genomic resources, Affymetrix Genome Arrays of soybean (Glycine max), a closely related species of pigeonpea were used on cRNA of six parental genotypes of three mapping populations of pigeonpea segregating for agronomic traits like drought tolerance and pod borer (Helicoverpa armigiera) resistance. By using robustified projection pursuit method on 15 pair-wise comparisons for the six parental genotypes, 5,692 SFPs were identified. Number of SFPs varied from 780 (ICPL 8755 × ICPL 227) to 854 (ICPL 151 × ICPL 87) per parental combination of the mapping populations. Randomly selected 179 SFPs were used for validation by Sanger sequencing and good quality sequence data were obtained for 99 genes of which 75 genes showed sequence polymorphisms. While associating the sequence polymorphisms with SFPs detected, true positives were observed for 52.6% SFPs detected. In terms of parental combinations of the mapping populations, occurrence of true positives was 34.48% for ICPL 151 × ICPL 87, 41.86% for ICPL 8755 × ICPL 227, and 81.58% for ICP 28 × ICPW 94. In addition, a set of 139 candidate genes that may be associated with drought tolerance has been identified based on gene ontology analysis of the homologous pigeonpea genes to the soybean genes that detected SFPs between the parents of the mapping populations segregating for drought tolerance.Electronic supplementary materialThe online version of this article (doi:10.1007/s10142-011-0227-2) contains supplementary material, which is available to authorized users.

Molecular Mapping of the New Blast Resistance Genes Pi47 and Pi48 in the Durably Resistant Local Rice Cultivar Xiangzi 3150

The indica rice cultivar Xiangzi 3150 (XZ3150) confers a high level of resistance to 95% of the isolates of Magnaporthe oryzae (the agent of rice blast disease) collected in Hunan Province, China. To identify the resistance (R) gene(s) controlling the high level of resistance in this cultivar, we developed 286 F(9) recombinant inbred lines (RILs) from a cross between XZ3150 and the highly susceptible cultivar CO39. Inoculation of the RILs and an F(2) population from a cross between the two cultivars with the avirulent isolate 193-1-1 in the growth chamber indicated the presence of two dominant R genes in XZ3150. A linkage map with 134 polymorphic simple sequence repeat and single feature polymorphism markers was constructed with the genotype data of the 286 RILs. Composite interval mapping (CIM) using the results of 193-1-1 inoculation showed that two major R genes, designated Pi47 and Pi48, were located between RM206 and RM224 on chromosome 11, and between RM5364 and RM7102 on chromosome 12, respectively. Interestingly, the CIM analysis of the four resistant components of the RILs to the field blast population revealed that Pi47 and Pi48 were also the major genetic factors responsible for the field resistance in XZ3150. The DNA markers linked to the new R genes identified in this study should be useful for further fine mapping, gene cloning, and marker-aided breeding of blast-resistant rice cultivars.

A high-density transcript linkage map with 1,845 expressed genes positioned by microarray-based Single Feature Polymorphisms (SFP) in Eucalyptus

BackgroundTechnological advances are progressively increasing the application of genomics to a wider array of economically and ecologically important species. High-density maps enriched for transcribed genes facilitate the discovery of connections between genes and phenotypes. We report the construction of a high-density linkage map of expressed genes for the heterozygous genome of Eucalyptus using Single Feature Polymorphism (SFP) markers.ResultsSFP discovery and mapping was achieved using pseudo-testcross screening and selective mapping to simultaneously optimize linkage mapping and microarray costs. SFP genotyping was carried out by hybridizing complementary RNA prepared from 4.5 year-old trees xylem to an SFP array containing 103,000 25-mer oligonucleotide probes representing 20,726 unigenes derived from a modest size expressed sequence tags collection. An SFP-mapping microarray with 43,777 selected candidate SFP probes representing 15,698 genes was subsequently designed and used to genotype SFPs in a larger subset of the segregating population drawn by selective mapping. A total of 1,845 genes were mapped, with 884 of them ordered with high likelihood support on a framework map anchored to 180 microsatellites with average density of 1.2 cM. Using more probes per unigene increased by two-fold the likelihood of detecting segregating SFPs eventually resulting in more genes mapped. In silico validation showed that 87% of the SFPs map to the expected location on the 4.5X draft sequence of the Eucalyptus grandis genome.ConclusionsThe Eucalyptus 1,845 gene map is the most highly enriched map for transcriptional information for any forest tree species to date. It represents a major improvement on the number of genes previously positioned on Eucalyptus maps and provides an initial glimpse at the gene space for this global tree genome. A general protocol is proposed to build high-density transcript linkage maps in less characterized plant species by SFP genotyping with a concurrent objective of reducing microarray costs. HIgh-density gene-rich maps represent a powerful resource to assist gene discovery endeavors when used in combination with QTL and association mapping and should be especially valuable to assist the assembly of reference genome sequences soon to come for several plant and animal species.

Bulk Segregant Analysis Using Single Nucleotide Polymorphism Microarrays

Bulk segregant analysis (BSA) using microarrays, and extreme array mapping (XAM) have recently been used to rapidly identify genomic regions associated with phenotypes in multiple species. These experiments, however, require the identification of single feature polymorphisms (SFP) between the cross parents for each new combination of genotypes, which raises the cost of experiments. The availability of the genomic polymorphism data in Arabidopsis thaliana, coupled with the efficient designs of Single Nucleotide Polymorphism (SNP) genotyping arrays removes the requirement for SFP detection and lowers the per array cost, thereby lowering the overall cost per experiment. To demonstrate that these approaches would be functional on SNP arrays and determine confidence intervals, we analyzed hybridizations of natural accessions to the Arabidopsis ATSNPTILE array and simulated BSA or XAM given a variety of gene models, populations, and bulk selection parameters. Our results show a striking degree of correlation between the genotyping output of both methods, which suggests that the benefit of SFP genotyping in context of BSA can be had with the cheaper, more efficient SNP arrays. As a final proof of concept, we hybridized the DNA from bulks of an F2 mapping population of a Sulfur and Selenium ionomics mutant to both the Arabidopsis ATTILE1R and ATSNPTILE arrays, which produced almost identical results. We have produced R scripts that prompt the user for the required parameters and perform the BSA analysis using the ATSNPTILE1 array and have provided them as supplemental data files.

Comparative physical mapping between wheat chromosome arm 2BL and rice chromosome 4

Physical maps of chromosomes provide a framework for organizing and integrating diverse genetic information. DNA microarrays are a valuable technique for physical mapping and can also be used to facilitate the discovery of single feature polymorphisms (SFPs). Wheat chromosome arm 2BL was physically mapped using a Wheat Genome Array onto near-isogenic lines (NILs) with the aid of wheat-rice synteny and mapped wheat EST information. Using high variance probe set (HVP) analysis, 314 HVPs constituting genes present on 2BL were identified. The 314 HVPs were grouped into 3 categories: HVPs that match only rice chromosome 4 (298 HVPs), those that match only wheat ESTs mapped on 2BL (1), and those that match both rice chromosome 4 and wheat ESTs mapped on 2BL (15). All HVPs were converted into gene sets, which represented either unique rice gene models or mapped wheat ESTs that matched identified HVPs. Comparative physical maps were constructed for 16 wheat gene sets and 271 rice gene sets. Of the 271 rice gene sets, 257 were mapped to the 18-35Mb regions on rice chromosome 4. Based on HVP analysis and sequence similarity between the gene models in the rice chromosomes and mapped wheat ESTs, the outermost rice gene model that limits the translocation breakpoint to orthologous regions was identified.