Project description:Advances in genomic technologies are rapidly filling the gap in genomic information available from non-model species that are economically and ecologically important. Reduction in costs and higher throughput of data acquisition will progressively increase the application of genomics in population and quantitative genetics. In molecular breeding, the ability to rapidly develop large numbers of genetic markers and generate high-density genetic maps at low cost is critical because they provide the framework for identification of elements that regulate trait variation, connecting genetics and genomics to phenotype. To generate the first high-density, gene-based genetic map for Eucalyptus, we genotyped a hybrid segregating population with microarray-based markers. The genus Eucalyptus includes the most economically important species used for bioenergy pulp and paper production worldwide. A custom 25-mers oligonucleotide array was designed based on 20,726 expressed sequence tags consensus, and RNA from 28 biologically-replicated individuals allowed clear-cut detection of putative single feature polymorphisms (SFPs) segregating 1:1 and 3:1. A genotyping array confirmed the segregation in a larger portion of the family and a genetic map with 1064 gene-related markers is made available with an average of one marker every 1.2 cM. Interestingly, reanalyzing the data from the 28 genotypes using a mixed-model approach resulted in most of the mapped genes among the significant probesets. We also demonstrate that designing more probes per gene increases the chance of mapping those genes. Finally, in silico validatation shows that 87% of the markers map to expected location on the draft genome, an important characteristic of SFP markers. Simultaneously detecting and genotyping SFP markers in a subset of the mapping population instead of only the genitors was more efficient for mapping genes in outcrossing species than previously reported approaches. Mapping SFP markers require low genomic resources and can be done at relatively low cost. We demonstrate the possibility of mapping hundreds to thousands of genes that can ultimately be used in generating further genomic resources, such as genome assembly, or for more direct applications, such as QTL analysis. Two biological replication of 28 genotypes were hybridized to the custom microarray, labeled with Cy3 and Cy5. Genotypes are fullsib progenies of the cross E. urophylla X E. grandis. A loop design was employed for the hybridization using xylem tissue. In total the microarray comprised 103,000 probes representing 20,726 unigenes with an average of five probes per unigene. Twenty-six negative control probes were also included in the microarray.

Project description:Advances in genomic technologies are rapidly filling the gap in genomic information available from non-model species that are economically and ecologically important. Reduction in costs and higher throughput of data acquisition will progressively increase the application of genomics in population and quantitative genetics. In molecular breeding, the ability to rapidly develop large numbers of genetic markers and generate high-density genetic maps at low cost is critical because they provide the framework for identification of elements that regulate trait variation, connecting genetics and genomics to phenotype. To generate the first high-density, gene-based genetic map for Eucalyptus, we genotyped a hybrid segregating population with microarray-based markers. The genus Eucalyptus includes the most economically important species used for bioenergy pulp and paper production worldwide. A custom 25-mers oligonucleotide array was designed based on 20,726 expressed sequence tags consensus, and RNA from 28 biologically-replicated individuals allowed clear-cut detection of putative single feature polymorphisms (SFPs) segregating 1:1 and 3:1. A genotyping array confirmed the segregation in a larger portion of the family and a genetic map with 1064 gene-related markers is made available with an average of one marker every 1.2 cM. Interestingly, reanalyzing the data from the 28 genotypes using a mixed-model approach resulted in most of the mapped genes among the significant probesets. We also demonstrate that designing more probes per gene increases the chance of mapping those genes. Finally, in silico validatation shows that 87% of the markers map to expected location on the draft genome, an important characteristic of SFP markers. Simultaneously detecting and genotyping SFP markers in a subset of the mapping population instead of only the genitors was more efficient for mapping genes in outcrossing species than previously reported approaches. Mapping SFP markers require low genomic resources and can be done at relatively low cost. We demonstrate the possibility of mapping hundreds to thousands of genes that can ultimately be used in generating further genomic resources, such as genome assembly, or for more direct applications, such as QTL analysis. Two technical replication of 68 individuals were hybridized to the custom microarray, labeled with Cy3 and Cy5. Individuals are fullsib progenies of the cross E. urophylla X E. grandis. A loop design was employed for the hybridization using xylem tissue. In total the microarray comprised 43,803 probes representing 15,698 unigenes. These probes were pre-selected in a screening array based on their mendelian segregation and bimodal signal separation. Twenty-six negative control probes were also included in the microarray.

Project description:In order to pinpoint the most differentially expressed genes between Eucalyptus grandis leaf blades and vascular (xylem) tissues as well as between E. grandis and Eucalyptus globulus xylem tissues, a total number of nine 50mer-oligoprobes covering the length of each one of 21,432 unique sequences derived from the Genolyptus EST dataset were synthesized “on-chip” in duplicate, randomly distributed in two blocks of each slide. Probes were also synthesized from ten cDNA sequences encoding known human proteins as negative controls, totaling 21,442 sequences. Leaves and xylem samples were taken from two E. grandis clonal trees, i.e., both derived from the same matrix tree and harboring the same genotype. Two additional xylem samples were collected from two other E. grandis clonal trees of a different genotype, as well as from two E. globulus clonal trees. Therefore, ten cDNA samples and ten identical chips were produced at Roche NimbleGen for the microarray assays, with a total number of 385,956 features per slide. Besides the discovery of differentially expressed genes between leaf and xylem, we wanted to test the validity of the assumed “technical” and “biological duplicates” since all trees were field-grown and four years-old in age. A ten chip study using total RNA recovered from mature leaf and vascular (xylem) tissues of Eucalyptus grandis and xylem from Eucalyptus globulus trees. Two clonal trees of E. grandis (E.grandis_Clone A_Ramet 1 and E.grandis_Clone A_Ramet 2), derived from a single matrix tree and therefore genomically identical, were the source of two samples of leaf RNA and two samples of xylem RNA, individually hybridized to four chips after cDNA synthesis/Cy3 labeling. Two other clonal trees of E. grandis (E.grandis_Clone B_Ramet 1 and E.grandis_Clone B_Ramet 2), derived from a different matrix tree, were the source of two additional samples of xylem RNA individually hybridized to four chips after cDNA synthesis/Cy3 labeling. Likewise, two pairs of clonal trees of E. globulus (E.globulus_Clone A_Ramet 1 and E.globulus_Clone A_Ramet 2/ E.globulus_Clone B_Ramet 1 and E.globulus_Clone B_Ramet 2), derived from two distinct matrix trees, were the source of four additional samples of xylem RNA, individually hybridized to four chips after cDNA synthesis/Cy3 labeling. Each chip measures the expression level of 21,432 genes from Eucalyptus sp. and ten human genes (negative controls) with nine 50-mer probe pairs (PM/MM) per gene in two separate blocks per chip (technical duplicate), totalizing 18 hybridization signal values per gene per chip.

Project description:Technological 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. SFP 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. The 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.

Project description:miR-93/106b and their host gene minichromosome maintenance complex component 7 (MCM7) reside at chr7q22, a region frequently rearranged in leiomyomas. We explored the expression of miR-93/106b in leiomyoma and paired myometrium (N=62) from untreated and patients exposed to hormonal therapies (GnRHa, Depo-Provera and oral contraceptives) from African Americans and Caucasians, and their regulatory functions in isolated paired (N=15) leiomyoma and myometrial smooth muscle cells (LSMC and MSMC) and leiomyosarcoma cell line (SKLM-S1). At tissue level leiomyomas expressed significantly lower levels of miR-93 and elevated MCM7 as compared to myometrium with limited racial influence or hormonal exposure on their expression. Assessing the regulatory function of miR-93/106b through doxycycline-inducible lentiviral transduction in microarray analysis, tissue factor (F3) and IL-8 were identified as their possible targets. At tissue level leiomyomas expressed a significantly lower level of F3 and an elevated IL-8 which exhibited an inverse relationship with miR-93, but with limited racial or hormonal influences. Gain-of-function of miR-93/106b in LSMC, MSMC and SKLM-S1 dose-dependently repressed F3 and IL-8 through direct interactions with their respective 3M-bM-^@M-^YUTRs and indirectly through F3 repression inhibited IL8, CTGF and PAI-1 expression, confirmed by using siRNA silencing or factor Vlla (FVIIa) activation of F3, as well as reducing the rate of proliferation, while increasing caspase 3/7 activity. We concluded that differential expression of miR-93/106b and their direct and/or indirect regulatory functions on F3, IL-8, CTGF and PAI-1 expression, with key roles in inflammation and tissue turnover may be of significance in the outcome of leiomyoma growth and associated symptoms. Total RNA isolated from TF324 cells transfected with DOX-inducible lentiviral construct carrying miR-106b~25 cluster with and without Dox treatments for 6 days was subjected to gene expression profiling using Sentirx Beadchip Array HumanHT-12_v4.

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Gene expression and single feature polymorphisms in xylem tissue on an Eucalyptus pseudo-testcross population

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