Cytogenetic Characterization and AFLP-Based Genetic Linkage Mapping for the Butterfly Bicyclus anynana, Covering All 28 Karyotyped Chromosomes

Abstract

BackgroundThe chromosome characteristics of the butterfly Bicyclus anynana, have received little attention, despite the scientific importance of this species. This study presents the characterization of chromosomes in this species by means of cytogenetic analysis and linkage mapping.Methodology/Principal FindingsPhysical genomic features in the butterfly B. anynana were examined by karyotype analysis and construction of a linkage map. Lepidoptera possess a female heterogametic W-Z sex chromosome system. The WZ-bivalent in pachytene oocytes of B. anynana consists of an abnormally small, heterochromatic W-chromosome with the Z-chromosome wrapped around it. Accordingly, the W-body in interphase nuclei is much smaller than usual in Lepidoptera. This suggests an intermediate stage in the process of secondary loss of the W-chromosome to a ZZ/Z sex determination system. Two nucleoli are present in the pachytene stage associated with an autosome and the WZ-bivalent respectively. Chromosome counts confirmed a haploid number of n = 28. Linkage mapping had to take account of absence of crossing-over in females, and of our use of a full-sib crossing design. We developed a new method to determine and exclude the non-recombinant uninformative female inherited component in offspring. The linkage map was constructed using a novel approach that uses exclusively JOINMAP-software for Lepidoptera linkage mapping. This approach simplifies the mapping procedure, avoids over-estimation of mapping distance and increases the reliability of relative marker positions. A total of 347 AFLP markers, 9 microsatellites and one single-copy nuclear gene covered all 28 chromosomes, with a mapping distance of 1354 cM. Conserved synteny of Tpi on the Z-chromosome in Lepidoptera was confirmed for B. anynana. The results are discussed in relation to other mapping studies in Lepidoptera.Conclusions/SignificanceThis study adds to the knowledge of chromosome structure and evolution of an intensively studied organism. On a broader scale it provides an insight in Lepidoptera sex chromosome evolution and it proposes a simpler and more reliable method of linkage mapping than used for Lepidoptera to date.

Linkage map of the peppered moth, Biston betularia (Lepidoptera, Geometridae): a model of industrial melanism

“…Interpretation of 3:1 segregating both informative (BI) dominant AFLP markers (mother and father heterozygous) is problematic in Lepidoptera because heterozygotes cannot be distinguished from the dominant homozygotes in the offspring and, moreover, they include the nonrecombinant maternal component. A number of approaches have been used to deal with these dominant BI markers in Lepidoptera (Jiggins et al, 2005;Kapan et al, 2006;Yasukochi et al, 2006;Van't Hof et al, 2008), each predicted to give a reasonable approximation of the actual marker order. We opted not to compromise the reliability of the linkage map and use only markers that are fully informative in all offspring.…”

“…The subsequent selective PCR amplification (selamp) used the same reaction mix as preamp except that 1.5 ml diluted preamp product replaced restriction-ligation as the template and the primers used were 100 nM fluorescent Eco þ 3 and 300 nM Mse þ 2 or Hha þ 2. Preamp and selamp thermal cycling profiles are described in Van't Hof et al (2008). The codes of the AFLP markers in Figures 1 and 2 refer to the primers and fragment length, with lowercase m, h and e for Mse, Hha and Eco primer cores, respectively, followed by the extensions in uppercase and the fragment size (for example, mAGeACG178).…”

“…This sample is concentrated on species within the Papilionoidea (Heliconius spp. (Jiggins et al, 2005;Kapan et al, 2006;The Heliconius Genome Consortium, 2012), Bicyclus anynana ( Van't Hof et al, 2008;Beldade et al, 2009), Colias hybrid (Wang and Porter, 2004), Papilio hybrid (Winter and Porter, 2010)) and one other macrolepidopteran, Bombyx mori (Bombycoidea) (Yasukochi et al, 2006;Yamamoto et al, 2008). Linkage maps are also available for two other distantly related superfamilies, Pyraloidea (Ostrinia nubilalis; Dopman et al, 2004) and Yponomeutoidea (Plutella xylostella (Heckel et al, 1999;Baxter et al, 2011)).…”

“…The gene composition of the Z chromosome is conserved in Lepidoptera, although not as strongly as some of the autosomes (Beldade et al, 2009;Baxter et al, 2011;Kroemer et al, 2011). A number of genes, such as kettin, tpi, 6-PGD, period, apterous and Ldh, have been assigned to the Z chromosomes of various Lepidoptera (Johnson and Turner, 1979;Gotter et al, 1999;Suzuki et al, 1999;Dopman et al, 2004;Narita et al, 2006;Pringle et al, 2007;Putnam et al, 2007;Van't Hof et al, 2008;Kroemer et al, 2011;Yoshido et al, 2011) and are now established lepidopteran Z-genes. The silkworm (Bombyx mori) whole-genome sequence assembly (The International Silkworm Genome Consortium, 2008) includes a large number of additional Z-linked genes, many of which have since also been found on the Z chromosome in other Lepidoptera (Beldade et al, 2009;Baxter et al, 2011;Kroemer et al, 2011).…”

“…Not a single gene has been found on the W chromosome in Heliconius spp., B. anynana, or P. xylostella, in spite of comprehensive gene-based surveys (Pringle et al, 2007;Beldade et al, 2009;Baxter et al, 2011). An exceptionally small W chromosome may partly explain this paucity of genes in B. anynana ( Van't Hof et al, 2008), but there is no such association for the B. mori W chromosome that is almost entirely composed of repetitive sequences originating from retrotransposons (Abe et al, 2005). Some records of W-linked genes are disputed, such as the dark morph locus in Papilio glaucus (Andolfatto et al, 2003) and an egg size determining gene, Esd, in the silkworm (Kawamura, 1988; but see Fujii et al, 2010).…”