The structure of gene networks that constrain and promote diversification in Heliconius melpomene
Ontology highlight
ABSTRACT: Comparison of whole genome and tiled gene expression between different colour pattern races of H. melpomene in developing wings. A total of 96 ds-cDNA samples labelled with Cy3 were hybridized onto 8 custom-designed Roche NimbleGen 12x135K format microarrays (Roche NimbleGen) for a total of 96 identical subarrays with 135,000 probes of 60 bp each and each hybridising a separate sample. The number of samples corresponded to two races, four stages and four tissue sections (forewing proximal, forewing distal, hindwing and eye), all of them with three independent replicates.
Project description:We investigated gene expression levels in Heliconius erato butterflies with divergent wing patterns across a 656KB genomic interval linked to the red color pattern wing polymorphism. This included comparison of expression between two H. erato color pattern populations (H. e. petiverana and a H.e. etylus x H. himera hybrid) across three sections of the forewing that differed in pigmentation (the basal, mid, and distal wing sections) and five different stages of pupal development (Day 1, 3, 5 pupae and ommochrome and melanin pigmentation stages). These results allowed us to determine whether certain genes in this interval were differentially expressed between the wing pattern elements, and, therefore, potentially responsible for adaptive color pattern variation in these butterflies. Forewings from a total of 29 individuals, covering three biological replicates of five developmental time points for each of the two H. erato distinct phenotypes were dissected, with the only exception being that there were only two replicates of the day 1 hybrid phenotype. Individuals were reared at 25˚C and dissected at the following stages: a) day 1 = 12 hr after pupation; b) day 3 = 60 hr after pupation; c) day 5 = 108 hr after pupation; d) early ommochrome = ~ 156 hr after pupation when red scales in forewing partially mature, showing a pale orange color; and e) early melanin = ~ 180 hr after pupation melanic scales begin to turn black and are present primarily at the center of the wing. Using wing veins as landmarks, each forewing was cut into three sections corresponding to the color pattern boundaries: basal (F1), middle (F2), and apical (F3). A eight custom-designed Roche NimbleGen 12x135K format microarrays with probes spanning a 656,307bp genomic region (Roche NimbleGen Inc., Madison, Wisconsin, United States) were used to hybridize double stranded cDNA from 87 tissue samples. Repetitive sequence elements found more than five times across all currently available H. erato genomic sequences, including the probed region as well as additional genomic BAC sequences, were masked from the tiling region. The remaining unmasked non-repetitive genomic sequences were tiled using 60 bp probes staggered every 13 bp on average, with slight modifications to ensure probe quality, for a total of 48,547 probes. Microarry design and printing was performed by Roche NimbleGen. cDNA labeling, hybridization, and array scanning was performed by the City of Hope Microarray Facility (Duarte, California, United States). In addition to the probes from the red color pattern intervals, the arrays also include 40,763 probes across two other genomic intervals not addressed in this study, 45,046 probes representing 12450 transcripts from a recent transcriptome assembly at 1-6X coverage, and 3248 random probes. Results from the transcriptome and other color pattern intervals will be published separately, however, we analyzed all probes together for array normalization and quality control.
Project description:This experiment aims to compare the transcriptomic response of three potato potato cultivars to salt stress. Plants were grown in hydroponic conditions (0.5MS +0.05MES) with long day light cycle (16h/8h). Roots were collected at ZT10 (time point 6h) and the following day at ZT4 (time point 24h). Three biological replicates were used and each biological replicate is composed of the roots of three plants. Tissues were flash-frozen in liquid nitrogen and subsequently ground with a mortar. RNA was extracted with FavorPrep™ Plant Total RNA Mini Kit from FAVORGEN. DNA was degraded in the column with RNase-free DNase I (Roche).
Project description:A murine model that mimic the decidualization and regression observed in human was used to investigate the molecular mechanisms underlying the dynamic processes in endometrium. Ovariectomized mice were treated sequentially with steroid hormones and then, to induce decidualization, oil was injected into the uterine lumen. A process similar to menstruation was induced by hormone-withdrawal. The uterine tissues were collected at 4 time-points after the induction of decidualization. Experiment Overall Design: The uterine tissues were collected at 36 (T1), 48 (T2), 60 (T3) and 84 hours (T4) after the last progesterone injection. There are 4 experimental animals for each time-point, and the RNAs collected from animals within the same time-point group were pooled together for Affymetrix microarray analysis using U74Av2 Chips.
Project description:Heliconius butterfly wing pattern diversity offers a unique opportunity to investigate how natural genetic variation can drive the evolution of complex adaptive phenotypes. Here we took a large-scale transcriptomic approach to identify the network of genes involved in Heliconius wing pattern development and variation. This included applying 147 microarrays representing the Heliconius transcriptome to assay shifts in gene expression across pupal development among several wing pattern morphs of Heliconius erato. We focused in particular on genes differentially expressed relative to the gene optix, which controls red pattern elements in wings. We combined expression results from three hindwing morphs from Peru and from dissected basal to apical wing elements in two forewing morphs to uncover two main classes of genes. First we looked for candidate upstream regulators of optix by determining transcripts expressed differently across basal to apical sections of the forewing prior to optix expression. Second, we assessed how optix regulates downstream gene expression by targeting transcripts with differential expression similar to optix, where expression differs among red wing pattern elements of both the forewing and hindwing. This study is an analysis of two distinct datasets generated using the same microarray platform. One dataset involved comparative analysis of forewing sections of different color morphs, while the other compared whole hindwings with different color patterns. For the forewing analysis we compared proximal, medial, and distal wing sections of two color pattern morphs: H. erato petiverana and a hybrid H. himera x H. erato etylus. The proximal section in H. erato petiverana is black and the hybrid form orange/red, the medial section is red in H. erato petiverana and pale yellow in the hybrid form, and the distal section is black in both races. For the hindwing analysis, we compared hindwing color pattern gene expression in three races that meet in a hybrid zone in Peru. H. erato emma has a rayed hindwing, H. erato favorinus has a yellow-barred hindwing, and H. erato amphritrite has a black hindwing. Wings were dissected at five time intervals: 1, 3, and 5 days after pupation, when orange/red ommochrome pigments were beginning to be expressed (~7 days after pupation), and when black melanin pigments were starting to pepper the center of the wings (~8 days after pupation). In the forewings, Days 1, 3, and 5 were at 12, 36, and 60 hours post-pupation. In the hindwings these stages were sampled at 24, 48, and 72 hours. Samples hybridized to microarrays included three replicates each of each race, stage, and wing section for forewings (3 replicates x 2 morphs x 3 wing sections x 5 stages, with one replicate wing missing for Day 1 H. e. petiverana = 87 samples) and four replicates of each stage and race for hindwings (4 replicates x 3 races x 5 stages = 60 samples). Total RNA was extracted and converted to cDNA. Cy3-labeling of samples, hybridization, and array scanning was performed according to NimbleGen protocols (2008): for the forewings this was performed at the City of Hope Functional Genomics Core, while the hindwings were run separately at NCSU.Samples were hybridized to NimbleGen HD2 12-plex arrays. These arrays include 12 identical subarrays with 135,000 60 bp probes each, each hybridizing a separate sample. Samples were distributed across arrays to prevent repeat conditions as much as possible and to space similar conditions in different regions of the slide. The array design involved two classes of probes. First there was a tiling component involving 89,310 probes tiled across three genomic intervals. Results from the tiling data were used for the initial discovery of the optix gene and are not the focus of the present study. The second component involved a representation of a set of 12,450 transcript contigs at 1-6X coverage for a total of 40,046 probes, with a mean coverage of 3-4 probes per contig. The number of probes for each contig depended on the ability to create suitable probes according to NimbleGen probe selection criteria and was limited by the small size of some transcripts and the minimum spacing criterion of 15 bp apart. Sequences of low complexity and high repeats with the rest of the genome (>5X representation), determined by comparison against 1.6 MB of genomic sequence available at the time, were avoided for designing probes. An additional 3,248 random probes were placed on the array for quality control.
Project description:A microarray containing 62,876 unigenes selected from CitEST database and prepared by Nimblegen Systems was used for identifying candidate resistance genes against P. parasitica at 48 hours after inoculation Four resistant and four susceptible F1 hybrids were selected from the population derived from the cross between Citrus sunki Hort. ex. Tan. and Poncirus trifoliate (L.) Raf cv. Rubidoux, respectively susceptible and resistant to P. parasitica. It was proposed that differentially expressed genes between resistant and susceptible hybrids and their parents provide essential candidates for identifying transcripts involved in disease resistance A total of 62,876 unigenes (18,712 unigenes of Citrus sinensis; 31,583 unigenes of Citrus reticulata and 12,581 unigenes of Poncirus trifoliate) selected from CitEST database assembled from the EST submitted to NCBI (Genebank accession number EY649559 to EY842485) were used to construct genome-wide oligonucleotide cDNA microarrays by Roche NimbleGen Systems using a multi-step approach to select probes with optimal predicted hybridization characteristics. Three probes were selected per unigene, comprising a probe set, and each probe set is represented on the final array by two replicates. All probes were designed as ‘‘perfect match’’ (PM) oligonucleotides (oligos).
Project description:The expression of genes in P. infestans isolates 06_3928A and NL07434 was monitored from 2 to 4 days time course of a potato infection. Genes encoding known and putative effector proteins were found induced at two and/or three days post-inoculation. We used a custom chip GPL8093 from Roche Nimblegen’s proprietary Maskless Array Synthesis (MAS) technology uses digital light processing and rapid, high-yield photochemistry to synthesize long oligo, high-density DNA microarrays with extreme flexibility. Each GPL8093 chip measures the expression level of 18155 genes from Phytophthora infestans with 60-mer probe pairs (PM/MM) per gene. Total RNA samples recovered from mycelia in two medias (rye sucrose agar and V8 agar) and infected potato leaves from a time couse infection (2, 3 and 4 days post incoulation). Experiments included two biological repllicates from each sample. We carried out total RNA extractions (mycelia and potato infected material) for two P. infestans isolates 06_3928A and NL07434. cDNA synthesis was performed Nimblegen.
Project description:Cxcl14+/- mice were mated with Cxcl14+/- mice, the embryos of pregnant females were collected on E13.5. After genotyping, the maternal part and fetal part of Cxcl14-/- and Cxcl14+/+ placentas were dissect out to extract total RNA respectively (n = 3). Total RNA was extracted using PureYield™ RNA Midiprep System (Promega).The maternal part of placenta that contained mesometrial lymphoid aggregate of pregnancy (MLAp) and decidua basalis (DB); the fetal part ofplacenta consisted of spongiotrophoblast and labyrinthin layer.Microarray hybridization was performed using a Mouse NimbleGen cDNA Microarray Kit (Roche), at CapitalBio Co., Ltd.(Beijing,China). Primary data were scaned using NimbleGen MS200 (Roche), and then extracted using NimbleScan system (Roche). Cxcl14+/- mice were mated with Cxcl14+/- mice, the embryos of pregnant females were collected on E13.5. After genotyping, the maternal part and fetal part of Cxcl14-/- and Cxcl14+/+ placentas were dissect out to extract total RNA respectively (n = 3). Total RNA was extracted using PureYield™ RNA Midiprep System (Promega).The maternal part of placenta that contained mesometrial lymphoid aggregate of pregnancy (MLAp) and decidua basalis (DB); the fetal part ofplacenta consisted of spongiotrophoblast and labyrinthin layer.Microarray hybridization was performed using a Mouse NimbleGen cDNA Microarray Kit (Roche), at CapitalBio Co., Ltd.(Beijing,China). Primary data were scaned using NimbleGen MS200 (Roche), and then extracted using NimbleScan system (Roche).
Project description:Growth in 0.3 M NaCl M63 minimal medium affects the transcriptome of Dickeya dadantii 3937.in comparison to that of cells grown in M63 medium. NimbleGen design name 2005-05-31_Ogg_Erwinia_24mer, NimbleGen design ID 2181 A 1:2 expression design for 4597 genes from Dickeya dadantii 3937 with 20 24-mer probe pairs (PM/MM) per gene. Each probe is replicated 2 times. The design includes random GC and other control probes. Protocol: High-density DNA array prepared with Maskless Array Synthesizer (MAS) technology. See manufacturer's website at http://www.nimblegen.com/.
Project description:Investigation of whole genome gene expression level changes in zebrafish liver under naphthalene treatedï¼?high-concentration group and low-concentration groupï¼?, compared to the solvent control group revealed that 76 genes and 88 genes were differentially expressed respectively in the fish caged at the low-concentration and high-concentration. KEGG pathway and GO analysis of the differentially expressed genes, showed significant enrichment in several meaningful categories. Healthy 5-month-old adult zebrafish (AB strain) maintenance and chemical exposure to 84μg/L and 840μg/L naphthalene and 0.005% Dimethyl sulfoxide as the solvent control were performed according to published research protocols. Prior to exposure, the fish were acclimatized in 50L aerated fresh water in glass tanks for 2 weeks, under controlled environmental conditions with the water temperature maintained at 26±0.5â?? for 16-h light and 8-h dark photoperiod. After 21 d of treatment, adult zebrafish livers were removed by dissection, and immediately transferred to RNA-later Stabilization Reagent(Qiagen,76106) , prior to storage at 4â?? for histopathological and microarray analysis. NimbleGen Gene Expression 12X135K zebrafish microarrays and One-Color DNA labeling Kit (NimbleGen, WI) were used for genome-wide expression analysis of naphthalene-treated zebrafish.
Project description:Mice with colonic tumors (chemically induced by AOM/DSS) were treated with Nutlin or control vehicle solution to analyze p53 target gene expression in vivo.