To the best of our knowledge, Wang et al. 2013) represent the first published report of Cas9-mediated genome editing in Lepidoptera and set three important precedents. The first example of Cas9 genome editing in a species other than B. mori was described by Li et al. 2015a) that caused deletions in three genes in the swallowtail butterfly Papilio xuthus. As we describe below, we also successfully scored protein-coding hits similar to Zhu et al. 2015), although our efficiency rates remain similarly low.
Genotyping
Future Prospects
B€ottcher R et al (2014) Efficient chromosomal gene modification with CRISPR/cas9 and PCR-based homologous recombination donors in cultured Drosophila cells. Ling L et al (2015) MiR-2 family targets awd and fng to regulate wing morphogenesis in Bombyx mori. Liu Y et al (2014) Highly efficient multiplex-directed mutagenesis and genomic structure variation in Bombyx moricells using CRISPR/Cas9.
Ma S et al (2014) CRISPR/Cas9-mediated multiplex genome editing and heritable mutagenesis of BmKu70in Bombyx mori. Markert MJ et al (2016) Genomic access to monarch migration using TALEN- and CRISPR/Cas9-mediated targeted mutagenesis. Perry M et al (2016) Molecular logic behind three-way stochastic choices that expand butterfly color vision.
Terenius O et al (2011) RNA interference in Lepidoptera: a review of successful and unsuccessful studies and implications for experimental design. Zhang Z et al (2015) Functional analysis of Bombyx Wnt1 during embryogenesis using the CRISPR/Cas9 system.
Phenotypic Effects of Major Loci: The Red Locus Optix
Mimicry patterns in Heliconius butterflies have contributed significantly to our understanding of the genetic basis for adaptation over the past 40 years. There are three parental races that contribute variation to the hybrid zone, pictured in top row H. The three main loci control wing patterns, D controls elements of the red/orange pattern, A controls the shape of the forewing band (two spots or one), and Yb produces yellow front wing belt.
Recent molecular analysis has confirmed that these phenotypes are indeed recombinants between closely linked elements located in non-coding DNA near optix (Wallbank et al. 2016). So there are at least three very closely connected elements that independently control different red spots on the wing. The locus is homologous to Yb, although it seems likely that the supergene includes several functional loci (Joron et al. 2006).8 Nijhout (1990).9 Papa et al. 1985) conclude that YlandSd are linked, but that YlandLy is independent is separated. SdandLya is now known to be the same locus, so it is unclear whether Ylis is not linked.
Phenotypic Effects of Major Loci: The Yellow Locus Cortex
Phenotypic Effects of Major Loci: The Shape Locus WntA
Phenotypic Effects of Other Loci
Quantitative Analysis
Non-genetic Effects and Plasticity
First, most of the variation in wing pattern among hybrid butterflies can be explained by genetic variation at only a small fraction of key loci. Second, in the wild there is very little phenotypic variation in wing pattern between individuals occurring across a wide range of altitudes and habitats—except in genetically diverse wing pattern races. In summary, while plasticity may play a role in many aspects of Heliconius biology, such as behavioral learning, there is no evidence that it plays a role in the evolution of wing pattern.
A Distribution of Effect Sizes?
The major locus control of Heliconius patterns appears to fit the predictions of the “Nicholson two-step model” (Huber et al. Turner 1981; Baxter et al. 2009), with a few major loci and additional modifiers of small effect. Turner has recognized this difficulty, but suggested that either multiple rounds of 'two-step' evolution would have to occur, or that changes at just one of the loci would be sufficient to confer a fitness advantage (Turner1977). Another discrepancy between theory and empirical data is that data from crossing experiments refer to the phenotypic effects of genetic loci, and not to individual mutations (Baxter et al.2009).
As pointed out by Fisher (1930) and more recently in the dissection of large-effect QTL in other organisms (Stam and Laurie 1996; Linnen et al. 2013), large-effect loci can result from the accumulation of many mutations at a single locus. Testing the "two-step model" therefore becomes a much more challenging problem of disentangling the order and magnitude of effect of individual mutations at a single locus.
This represents a clear case of one-step evolution with 'major consequences', so there are certainly at least some cases involving major changes (The Heliconius Genome Consortium, 2012). Overall, the 'robust' adaptive landscape of mimicry is therefore likely to favor adaptation via large steps, as described under a two-step theory, and this may provide part of the explanation for the large effect loci involved in Heliconius mimicry.
Supergenes and Polymorphism
The genetic architecture of 3–4 large loci is ancestral because it is shared by all other species in the genus studied (Huber et al.2015), thus inH. Effectively, there is a block of approximately 400 kb of DNA sequence that is inherited in complete association with different wing pattern shapes (Joron et al. 2011). Similar inversions have been seen in complex polymorphisms in other species – notably a behavioral and plumage polymorphism in the white-throated sparrow, a social polymorphism in fire ants and a behavioral polymorphism in the collared wader (Thompson and Jiggins 2014; Huynh et al. .2011; Wang et al. 2013; Küpper et al. 2015; Lamichhaney et al. 2015).
Perhaps most similar to the Heliconius numata case is Papilio polytes, in which a highly localized inversion around the Dsxgene controls a wing pattern mimic polymorphism (Kunte et al.2014; Nishikawa et al.2015). The second aspect of a supergene that provides mimicry is a strong dominance pattern (Llaurens et al. 2015). Surprisingly, a heterozygous genotype is distinct but appears to be stabilized due to its effective mimicry of a different species (Le Poul et al. 2014).
In contrast, among the derived alleles, dominance patterns follow the typical color hierarchy seen in other Heliconius species (Le Poul et al.2014). These patterns suggest that dominance is a property of the alleles themselves, and not of the genetic background.
Conclusions
This will be a fascinating system in which to explore the mechanisms underlying the development of dominance. Colosimo PF et al (2005) Widespread parallel evolution in sticklebacks through recurrent fixation of ectodysplasin alleles. Ferguson L et al (2010) Characterization of a hotspot for mimicry: assembly of the butterfly wing transcriptome into a genomic sequence at the HmYb/Sb locus.
Huber B et al (2015) Conservatism and novelty in the genetic architecture of adaptation in Heliconius butterflies. Lamichhaney S et al (2015) Structural genomic variation underlies alternative reproductive strategies in the ruff (Philomachus pugnax). Le Poul Y, Whibley A, Chouteau M, Prunier F, Llaurens V, Joron M (2014) Evolution of dominance mechanisms in the butterfly mimicry supergene.
Martin A et al (2012) Diversification of complex butterfly wing patterns by recurrent regulatory evolution of a Wnt ligand. Sheppard PM, Turner JRG, Brown KS, Benson WW, Singer MC (1985) Genetics and evolution of Muellerian mimicry in Heliconius butterflies.
Research Background
It is hypothesized that a supergenic unit is created by recombination events and fixed by the inhibitory effects of a chromosomal inversion on recombination (Nijhout2003; Joron et al.2011), although the mechanism underlying this hypothesis has remained unclear. It is assumed that these pigmentation processes involved in Batesian mimicry of P. polytes must be downstream of Hgen. To comprehensively elucidate the evolutionary processes of this mimic, it is important to elucidate the H locus and its structure and function.
Recently, Kunte et al. 2014) and our group independently identified the Hgene locus and revealed its structure (Nishikawa et al. 2015).
Papilio Genome Projects Reveal the H Locus and Chromosomal Inversion Near dsx
Thus, the putative Hlocus region located on chromosome-25 is believed to be only one long and unique heterozygous site among the entire autosomal chromosomes, which structure is maintained by reduced recombination due to the chromosomal inversion.
Linkage Mapping of the H Locus
The association between the region and mimic phenotype in natural populations was further investigated using single nucleotide polymorphisms (SNPs) in 54 wild-caught females (Nishikawa et al. 2015). It is noteworthy that the Hlocus revealed by linkage mapping completely coincides with the long heterozygous region revealed by whole genome sequencing. This means that a genetic locus responsible for some polymorphic trait with a long heterozygous region can only be identified by genome sequencing without linkage mapping.
Detailed Structure of a Long Heterozygous Region Linked to the H Locus
The left breakpoint that binds to Prosper lies approximately 700 bp downstream of the sixth exon of the dsxinh chromosome but approximately 14.6 kbp upstream of the first exon of the dsxinH chromosome. The correct breakpoint that binds to Sir2 is at approximately 8.9 kb upstream of the first exon of dsxinh chromosome but at approximately 1.1 kb downstream of dsxinH chromosome. Compared with dsxinh chromosome (dsx_h), dsxinH chromosome (dsx_H) was longer in the second, fourth, fifth and sixth introns and sixth exon.
Just outside both breakpoints, in contrast, greater than 99% homology between the h and H sequences was performed (Fig. 10.2d). These structures implied that many mutations and numerous insertion and deletion events may have accumulated in the inverted region of H after the inversion and were maintained by suppression of recombination between two chromosomes.
Dimorphic Dsx Structure Associated with the H and h Alleles
These observations suggest that no special isoform of dsx_H appears to be involved in the mimetic wing coloration, although further evidence is needed to show this possibility. In males showing purely non-mimetic phenotype, we found only one isoform of dsx that skips exons 3 and 4 included in all female isoforms, implying the importance of exons 3 and 4 for the mimicry. However, in these regions of three female isoforms, there was only one amino acid (the C-terminal end of F1) that specifically changed indsx_H, as described above.
The male-specific isoform of dsx_H was barely expressed in prepupal to pupal wings, suggesting that male dsx_His is not involved in the mimetic phenotype (Fig. 10.4).
Expression Profiles of Genes Around the Inverted Region of H Locus
A newly arisen geneU3X was found only in the heterozygous region of the H chromosome in the whole genome of P. We found that there appeared to be no significant differences in expression level of each isoform (F1, F2 and F3) of dsx between mimetic and non-mimetic wings at P1–2, P4–5 and P10.5 stages (Nishikawa et al. 2015). However, the expression level of dsx_H in mimetic female wings was quite higher than in non-mimetic wings at early pupal stages, but dsx_h did not show such expression profiles.
RNA-seq analyzes support the findings that dsx_H was dominantly expressed in Hh female wings (Nishikawa et al. 2015). Differentiated expression level between dsx_h and dsx_H becomes significant on female wings at the P2 stage, when the pattern of wing pigmentation can be determined (Nishikawa et al.2013) (Fig.10.4). In the report by Kunte et al. 2014), however, the expression pattern of dsx_H was upregulated at the late pupal stage, which was different with our result.
Some of the nucleotide changes in regulatory regions or intron regions between dsx_Handdsx_h may be responsible for the specific regulation of dsx_Hin female arms. The above results suggest that not only amino acid substitution but also regulatory changes for femratx_Hare may be involved in the mimetic phenotype.
Functional Analysis of dsx
