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:Background: Heliconius butterflies are an excellent model system for studies of adaptive convergent and divergent phenotypic traits. Wing colour patterns are used as signals to both predators and potential mates and are inherited in a Mendelian manner. The underlying genetic mechanisms of pattern formation have been studied for many years and shed light on broad issues, such as the repeatability of evolution. In Heliconius melpomene, the yellow hindwing bar is controlled by the HmYb locus and several genes in this region show expression pattern differences across races. MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression that have key roles in many biological processes, including development. It seems likely that miRNAs could act as downstream regulators of genes involved in wing development, patterning and pigmentation. For this reason we characterised miRNAs in developing butterfly wings and examined differences in their expression between colour pattern races. Results: We sequenced small RNA libraries from two colour pattern races and detected 142 Heliconius miRNAs with homology to others found in miRBase. Several highly abundant miRNAs appeared to be differentially expressed between colour pattern races and this was tested further in different developing pupal wing stages using Northern blots. These revealed that differences in expression were due to developmental stage rather than colour pattern. Assembly of sequenced reads to the HmYb region identified miR-193 and miR-2788; located 2380bp apart in an intergenic region. A search for miRNAs in all available H. melpomene BAC sequences (~2.5Mb) did not reveal any other miRNA genes and no novel miRNAs were predicted. There were several regions where other small RNA sequences assembled to the HmYb region and appeared to be differentially expressed.These might represent other regulatory RNAs. Conclusions: Here we describe the first butterfly miRNAs and characterise their expression in developing wings. Some show differences in expression across developing pupal stages. Two miRNAs were located in the HmYb region. Future work will examine the expression of these miRNAs in different colour pattern races and identify miRNA targets among wing patterning genes. High-throughput sequencing of Heliconius melpomene endogenous small RNAs. Size fractionated small RNA from total RNA extracts of two different Heliconius melpomene races (Heliconius melpomene melpomene and Heliconius melpomene rosina) were isolated from wing tissue using miRVana kit. 100Âµg RNA from 11 individuals of different developmental stages was pooled for each race as follows: 4.1% larval stage <1; 2% larval stage 1-1.75; 2.9% larval stage 2-2.5; 22% larval stage 2.75-3; 19% larval stage > 3; 25% early pupae; 25% mid-melanin pupae. Sequences were ligated to adapters, purified again and reverse transcribed. After PCR amplification the sample was subjected to Solexa/Illumina high throughput pyrosequencing. Please see www.illumina.com for details of the sequencing technology.

Project description:Background: Heliconius butterflies are an excellent model system for studies of adaptive convergent and divergent phenotypic traits. Wing colour patterns are used as signals to both predators and potential mates and are inherited in a Mendelian manner. The underlying genetic mechanisms of pattern formation have been studied for many years and shed light on broad issues, such as the repeatability of evolution. In Heliconius melpomene, the yellow hindwing bar is controlled by the HmYb locus and several genes in this region show expression pattern differences across races. MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression that have key roles in many biological processes, including development. It seems likely that miRNAs could act as downstream regulators of genes involved in wing development, patterning and pigmentation. For this reason we characterised miRNAs in developing butterfly wings and examined differences in their expression between colour pattern races. Results: We sequenced small RNA libraries from two colour pattern races and detected 142 Heliconius miRNAs with homology to others found in miRBase. Several highly abundant miRNAs appeared to be differentially expressed between colour pattern races and this was tested further in different developing pupal wing stages using Northern blots. These revealed that differences in expression were due to developmental stage rather than colour pattern. Assembly of sequenced reads to the HmYb region identified miR-193 and miR-2788; located 2380bp apart in an intergenic region. A search for miRNAs in all available H. melpomene BAC sequences (~2.5Mb) did not reveal any other miRNA genes and no novel miRNAs were predicted. There were several regions where other small RNA sequences assembled to the HmYb region and appeared to be differentially expressed.These might represent other regulatory RNAs. Conclusions: Here we describe the first butterfly miRNAs and characterise their expression in developing wings. Some show differences in expression across developing pupal stages. Two miRNAs were located in the HmYb region. Future work will examine the expression of these miRNAs in different colour pattern races and identify miRNA targets among wing patterning genes.

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.

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The genetic basis of structural colour variation in mimetic Heliconius butterflies [erato]

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