Heredity 78(1997) 645—654 Received23 September 1996 Inheritance and linkage of isozymes in Yponomeuta padellus (Lepidoptera, Yponomeutidae) LEON E. L. RAIJMANN*t, WIL E. VAN GINKELt, DAVID G. HECKELt & STEPH B. J. MENKEN tlnstitute for Systematics and Population Biology, University of Amsterdam, P0 Box 94766, 1090 GTAmsterdam, The Netherlands and cDepartment of Biological Sciences, Clemson University, Clemson, SC 29634-1903, USA. Inheritanceand genetic linkage of 29 allozyme loci were studied by single-pair crosses of Yponomeuta padellus (Lepidoptera, Yponomeutidae). All loci segregated as Mendelian genes with codominant alleles except for a null allele at the hbdh locus. The three loci est-2, 6pgdh, and fudh were sex-linked and occurred in that order along the Z chromosome. Autosomal linkage analysis was facilitated by the lack of crossing-over in females characteristic of Lepi- doptera, because linkage in female-informative crosses is all-or-none and observation of 'forbidden recombinants' provides conclusive evidence that two loci are not syntenic. Convinc- ing evidence was found for linkage of the autosomal loci me and mpi. This linkage group and aat-1, Ca,hbdh,hk-1, pgi and pgm were assigned to seven separate autosomes. Weaker evidence from male-informative families supported four additional linked pairs. Previous cyto- logical studies have shown that the sex chromosomes in heterogametic females are associated in a trivalent, consisting of a W chromosome translocated to an autosome (Av), paired with the Z chromosome and the homologous autosome A. Any locus located on this autosome and its homologous segment on the A" chromosome should show strict segregation of one allele to Sons and the other to daughters. However, none of the female-informative loci in this study satisfied that criterion. Use of the method of forbidden recombinants and its utility in the study of chromosomal evolution in Yponomeuta are discussed. Keywords:achiasmaticmeiosis, linkage, mapping of allozymes, sex chromosome trivalent. group of five morphologically similar species includ- Introduction ing Y padellus (Menken, 1980a). Secondly, Menken Thegenus Yponomeuta (Lepidoptera, Yponomeuti- (1980b) made an in-depth study of the inheritance dae) or small ermine moths has been used as a of 10 enzyme systems in Y cagnagellus, This paper is model system to study the evolution of insect—plant the first of a series of such studies in Y padellus. relationships (Menken et al., 1992; and references We have three main goals in this work. The first is therein). Allozyme variation has been analysed to establish which allozymes are sex-linked in Y intensively in host-associated populations of the padellus. In a recent review, Sperling (1994) pointed oligophagous Y padellus (Menken, 1982; Raijmann, out that in many groups of Lepidoptera, character- 1996), in particular in relation to the study of host istics affecting reproductive isolation and host race race formation (Raijmann & Menken, 1992). formation appear to be predominantly sex-linked. Although there is extensive information about popu- However, he identified Yponomeuta as a group to lation genetics based on allozyme data in Y padellus, which this generalization did not apply (see Van information on the transmission genetics of allo- Drongelen & Van Loon, 1980; Hendrikse, 1988). To zymes in this species is still absent. Thus far, two investigate further the validity of these claims, we studies have been carried out to study the inherit- would like to identify as many sex-linked marker loci ance of allozyme markers in Yponomeuta. First, the as possible. inheritance of five mainly diagnostic loci has been Our second goal is to begin construction of a reported within the so-called 'padellus-complex', a linkage map for the autosomes of Y padellus. Comparative linkage mapping in Lepidoptera is in *Correspondence E-mail: [email protected] its infancy compared to genome mapping in 1997The Genetical Society of Great Britain. 645 646 L. E. L. RAIJMANN ETAL. mammals (O'Brien et a!., 1988) and in certain plant Electrophoresis and enzyme staining families (Bennetzen & Freeling, 1993), but mapping Samplepreparations and starch gel electrophoresis a common set of anchor loci such as allozymes is a were carried out between 0°C and 4°C. Wings and reasonable first step towards that goal (Heckel, legs were removed, and individual moths were 1993). The general lack of crossing-over in female homogenized in 125 iL of grinding buffer containing Lepidoptera (Robinson, 1971) greatly facilitates 10 mM Tris, 10 mrvi maleic acid, 1 mi EDTA, 1 m autosomal linkage mapping because syntenic pairs of MgC12, 0.05 m NADP and pH adjusted to 7.4 with loci produce no recombinants in female-informative NaOH. Extracts were centrifuged at 9615 g at 4°C crosses: linkage, being all-or-none in such crosses, is for 5 mm and the clear supernatant was either easy to detect or to rule out. stamped on cellulose acetate gels or soaked onto Our third goal is to seek genetic evidence to Whatman 3 MM chromatography paper wicks for corroborate cytogenetic investigations in the genus. insertion into 11 per cent w/v Connaught starch gels. Nilsson et a!. (1988) studied the first meiotic division To analyse fumarate hydratase and sucrase, we in males and females of several Yponomeuta species, carried out cellulose acetate electrophoresis follow- including Y padellus. They found that the sex ing the procedure of Hebert & Beaton (1989), using chromosomes in the heterogametic females were Titan III cellulose acetate plates (Helena Labora- associated in a sex chromosome trivalent (written as tories, Beaumont, TX, U.S.A.). All other enzymes AAWZ) consisting of a W chromosome translocated were studied on starch gels, prepared according to to an autosome to form an A" chromosome. This A"' Ayala et a!. (1972). The clarity of both phosphoglu- chromosome was paired with the homologous auto- comutase and 6-phosphogluconic dehydrogenase was some and the Z chromosome. In the homogametic much improved by adding 0.06 mrvi NADP to the males the two Z chromosomes were paired in the heated gel mixture and in the cathode vessel prior to usual bivalent, thus the male karyotype could be electrophoresis. Buffer systems, staining methods, represented as 2n =3OAA+ZZ,and the female subunit structure, and number of alleles for all karyotype as 2n =29AA+ AAWZ. Transmission enzyme systems are listed in Table 1. Sucrase was genetics studies would give us the opportunity to test stained with a solution of 0.2 M Na2HPO4 adjusted allozyme loci for their occurrence on the trans- with citric acid to pH =7.0,15 mg mL' sucrose, located autosome. 1 mg mL1 glucose oxidase, 1.5 mg mL MTT and In this paper, single-pair crosses of Y padellus 0.5 mg mL PMS with a 1 per cent agar overlay. were used to analyse the inheritance of 22 enzyme Multiple loci were numbered sequentially from the systems in support of these three objectives. The high level of genetic polymorphism in the source most cathodally migrating protein product. Allele designation generally follows Menken (1982) and population provided enough variability to address our questions, without the need of first creating Arduino & Bullini (1985). inbred strains. Our results to date suggest specific strategies for building on this information, to exploit Linkageanalysis the favourable features of the genetic system of Forthe initial steps in data analysis, we used version Lepidoptera in comparative genome studies within 3.50 of the LINKAGE-i program (Suiter et al., 1983). Yponomeuta, and to investigate the evolutionary The error-checking and equal-segregation tests of dynamics of host race formation. LINKAGE-i are appropriate to our data, but its method of estimating recombination rates depends Materialsand methods critically on the assumption of crossing-over in both sexes,whjch is not valid here. LINKAGE-i also makes Over300 fifth instar larvae were collected from a Y certain assumptions about gametic phase which may padellus population infesting Crataegusspp.in be too strong when phase is unknown. Thus for Leiden, The Netherlands. These larvae were raised analysing linkage, we used only part of the LINKAGE-i to adulthood in the laboratory and undamaged output, as described below. Estimation of recombi- specimens were selected for the breeding experi- nation fractions among the sex-linked loci was ments. Immediately after emergence, one male and straightforward, because hemizygosity of females one female were placed in a glass jar together with a rendered the situation formally equivalent to a back- young twig of Crataegusspp.as oviposition substrate, cross. The remaining analysis was restricted to auto- and an eppendorf tube with sugar-water (5 per cent somal loci. w/v glucose) as a substitute for nectar. A total of 51 The LINKAGE-i printout was examined by hand, to such single-pair crosses was set up. separate the locus pairs into four types: informative The Genetical Society of Great Britain, Heredity, 78, 645—654. Table 1 Enzyme systems, buffers and staining methods surveyed in Yponomeutapadellus Locus Buffer Staining Subunit Alleles . Enzyme system EC number abbreviation systemt methods structure 2. Aconitate hydratase 4.2.1.3 acon-1 1 — 100 acon-2 Monomeric 100 98 Alcohol dehydrogenase 1.1.1.1 ad/i-i II 2 Dimeric 100 91 adh-2 Dimeric 100 92 Aspartate aminotransferase 2.6.1.1 aat-i 3 Dimeric 100 92 aat-2 100 Carbonate dehydratase 4.2.1.1 ca III 1 Dimeric 100 90 . Esterase 3.1.1.2 est-i Iv 4 Dimeric 100 94 est-2 Monomeric 100 96 Fucose dehydrogenase 1.1.1.122 fudh I 4 Monomeric 105 97 94 Fumarate hydratase 4.2.1.2 fli VI 5 Tetrameric 108 100 92 ti" Glucose-6-phosphate dehydrogenase 1.1.1.49 g6pdh I 4 Monomeric 100 98 Glutamic-pyruvic transaminase 2.6.1.2 gpt III 2 — 100 Glyceraldehyde-3-phosphate dehydrogenase 1.2.