Genes Genet. Syst. (2000) 75, p. 159–166 A linkage map of the turnip rosae (: Symphyta) based on random amplified polymorphic DNAs

Yoshikatsu Nishimori1, Jae Min Lee1,†, Megumi Sumitani1, Masatsugu Hatakeyama2, and Kugao Oishi1,2,* 1Graduate School of Science and Technology, Kobe University, Nada, Kobe 657-8501, Japan 2Department of Biology, Faculty of Science, Kobe University, Nada, Kobe 675-8501, Japan

(Received 3 April 2000, accepted 21 June 2000)

A linkage map was constructed for the sawfly, Athalia rosae (Hymenoptera), based on the segregation of random amplified polymorphic DNA (RAPD) markers and a visible mutation, yellow fat body (yfb). Forty haploid male progeny (20 yfb and 20 +) from a single diploid female parent (yfb/+) were examined. Sixty-one of the 180 arbitrary primers tested by polymerase chain reaction (PCR) produced one or more RAPD bands. A total of 79 RAPD markers were detected. Of these, seven showed significant deviation from the expected 1:1 ratio, and were therefore excluded from further analysis. The remaining 72 RAPD markers and the marker mutation, yfb, were subjected to linkage analysis. Sixty RAPD markers and the yfb marker were organized into 16 linkage groups, spanning a distance of 517.2 cM. Twelve RAPD markers showed no linkage relationship to any group. Thirteen gel-purified RAPD bands were cloned and sequenced to generate the sequence-tagged sites (STSs). A single locus was represented by two markers, with one of them having a short inter- nal deletion.

and Page, 1995) and four parasitic wasps, Bracon hebetor INTRODUCTION (Antolin et al., 1996), Nasonia vitripennis (Saul, 1993), Recent advances in the methodologies used to perform Aphelinus asychis (Kazmer et al., 1995), and Trichog- linkage analyses employing molecular markers have accel- ramma brassicae (Laurent et al., 1998), of which erated genetic linkage mapping in a number of organisms, all but one map (that for N. vitripennis) are based primarily including . The random amplified polymorphic on the segregation of RAPD markers. Hymenopteran in- DNA (RAPD)-PCR method (Williams et al., 1990) is con- sects are, indeed, amenable to linkage analyses using sidered to be one of the most convenient and useful of these RAPD markers due to their mode of reproduction. Most new methods (Heckel, 1993; Mazur and Tingey, 1995). hymenopteran species show haplo-diploidy: fertilized eggs RAPDs are detected by gel electrophoresis of the polymor- develop to diploid females while unfertilized eggs develop phic DNA fragments which are amplified in polymerase parthenogenetically to haploid males (Crozier, 1977). chain reactions (PCRs) using arbitrary primers. DNA Haploid sons inherit a single chromosome complement polymorphisms are basically caused by base substitutions from their diploid mother. Alleles at any given locus in a in the primer annealing sequences. Since this method heterozygous mother thus should theoretically segregate relies simply on the primers, and RAPDs are detected on at a 1:1 ratio in her haploid sons. the stained gels, no prior sequence information or special The turnip sawfly, Athalia rosae (Hymenoptera: apparatus are required. Symphyta: ), has unique features as an Genetic linkage maps have been constructed for five experimental organism (reviews: Oishi et al., 1993, 1995, hymenopteran insects, the honeybee, Apis mellifera (Hunt 1998). Mature oocytes dissected from the ovary can be activated easily by immersing them in distilled water, and nearly all of them develop to normal haploid male adults Edited by Masa-Toshi Yamamoto * Corresponding author. E-mail: [email protected] (Naito, 1982; Sawa and Oishi, 1989a). It is the only in- † Present address: National Institute of Bioscience and Human- sect species in which artificial fertilization by intracyto- Technology, Tsukuba 305- 8566, Japan. plasmic sperm injection (ICSI) has been achieved (Sawa 160 Y. NISHIMORI et al.

and Oishi, 1989b, c; Hatakeyama et al., 1994a, b, vated by placing them in distilled water. The carcass (the 2000b). We have been investigating the mechanisms of whole body less the ovaries, accessory organs and alimen- egg maturation and egg activation (Sawa and Oishi, tary canal) of the mother was frozen in liquid nitrogen and 1989a; Hatakeyama et al., 1990a, 1995, 2000a), and also stored at –80°C for DNA extraction. The male progeny the potential of polar body nuclei and mature and premature were reared and examined for and separated according to male gametes to participate in development (Hatakeyama their phenotype as to yfb at the mid-pupal stage. They et al., 1990b, 2000a). were further reared to the adult stage, aged for 1–3 days Thus, A. rosae provides a unique system in which to without food but with access to distilled water, frozen in pursue the events at and around fertilization. Elucidation liquid nitrogen, and stored individually at –80°C. of the molecular mechanisms requires knowledge of the genes responsible for such events. The importance of the Genomic DNA extraction and purification. Geno- genetic foundation, especially when an organism is being mic DNAs were extracted individually from the diploid developed as a model system, is obvious. Unfortunately, parental mother and from her haploid male progeny however at present little is known about A. rosae genetics. (which had been stored at –80°C) following the procedures There are only three visible marker mutations currently described by Sambrook et al. (1989) and by Hunt and Page available. (1995) with slight modifications. Briefly, the frozen indi- The present study is our first attempt aiming at con- viduals were each ground in a 1.5-ml microcentrifuge tube structing a genetic linkage map using molecular markers. with a plastic homogenizing pestle (Treff Lab), and then We screened 180 arbitrary 10-nucleotide primers and supplemented with 200 µl of CTAB homogenizing buffer detected 79 polymorphic DNAs (RAPDs). A total of 73 (1% hexadecyltrimethyl ammoniumbromide/750 mM markers, 72 RAPDs and a visible mutation, yfb, were used NaCl/50 mM Tris-HCl, pH 8.0/10 mM EDTA pH 8.0/100 for the genetic linkage analysis. Sixty-one markers µg/ml of proteinase K) and mixed. After incubation at formed 16 linkage groups spanning a distance of 517.2 60°C for 2 hr, 70 µl of 1.5 M NaCl/50 mM Tris-HCl, pH 8.0 cM. Some markers were cloned and sequenced to gener- was added, and gently mixed. Four hundred microliters ate sequence-tagged site (STS) landmarks. of phenol/chloroform/isoamyl alcohol (25:24:1) was then added to each microtube, mixed on a rotator at 3 rpm for more than an hour, and centrifuged at 10,000 rpm at room MATERIALS AND METHODS temperature. The supernatant was transferred to a fresh Sawfly. The turnip sawfly, Athalia rosae ruficornis microtube, supplemented with 1 µl of RNase A (10 mg/ml), Jakovlev (Abe, 1988), was reared in the laboratory under and incubated at 37°C for 1 hr. The phenol/chloroform/ a 16L-8D condition at 25°C. General biology, staging and isoamyl alcohol extraction was repeated two more times, methods of obtaining mature unfertilized eggs (oocytes) and followed by chloroform/isoamyl alcohol (24:1) extrac- have been described previously (Sawa et al., 1989; Sawa tion (400 µl) to remove phenol. The DNAs were each pre- and Oishi, 1989a; Hatakeyama et al., 1990a). Haplo-dip- cipitated by addition of 30 µl of 3 M sodium acetate (pH loid type of reproduction is the general mode in A. rosae as 5.0) and 600 µl of ice-cold ethanol, mixing, and centrifuga- in most other hymenopteran species. One distinct fea- tion at 10,000 rpm at room temperature. The precipi- ture of this species is that it is possible to activate mature tated DNAs were washed with 70% ethanol, and each eggs (oocytes) after dissecting them from the ovary simply sample of DNA was dissolved in 100 µl of TE (10 mM Tris- by immersing them in distilled water, and these eggs acti- HCl, pH 8.0/1 mM EDTA, pH 8.0), and stored at –20°C. vated in vitro then develop to normal haploid male adults Portions were adjusted with TE to a final concentration of (Naito, 1982; Sawa and Oishi, 1989a). 5 ng/µl and stored at 4°C for immediate usage. A single diploid parental mother, heterozygous for the marker mutation yfb (yfb/+), and her haploid male prog- PCR amplification. DNA polymorphisms were de- eny derived from in vitro parthenogenetically activated tected by the random amplified polymorphic DNA (RAPD)- eggs were examined. yfb (yellow fat body) is a mutation PCR method (Williams et al., 1990, 1993). Polymerase which produces a phenotype (yellow fat body coloration) chain reactions (PCRs) were based on the procedure that is distinguishable externally through the still unpig- described previously (Hunt and Page, 1995). Arbitrary mented cuticle in late-larval to mid-pupal stages, by con- 10-nucleotide-long primers (Operon 10-mer kits A through trast to the wild type greenish blue color (Sawa and Oishi, E, G through I and Q, Operon Technology) were used with 1989b). Individuals heterozygous for yfb show interme- the template A. rosae genomic DNAs to amplify DNA diate coloration. A single parental female (yfb/+) was fragments. Ten microliter PCR reaction solutions con- fed on diluted honey for 6 days (so that she would have a taining 10 ng of A. rosae genomic DNA, 0.2 µM primer, sufficient number of mature eggs) and was then given 100 µM dNTP (Toyobo), 1x PCR buffer (Toyobo), 2 mM

distilled water for another day. The mother was then dis- MgCl2 and 0.25 U of rTaq DNA polymerase (Toyobo) were sected, and mature eggs (oocytes) were artificially acti- prepared. A drop of mineral oil was overlaid on the sur- Linkage map of Athalia rosae 161 face of the solution. Amplification was done using a ther- line lysis method (Sambrook et al., 1989). mal cycler (Astec PC-700) with the following conditions: Inserted DNA fragments were sequenced by direct se- 45 cycles of 94°C for 1 min, 35°C for 1 min, and 72°C for 2 quencing using a thermo sequenase cycle sequencing kit min. (Amersham-Pharmacia). DNA fragments were amplified by PCR with the fluorescence labeled M13 forward and Gel electrophoresis and detection of RAPD markers. reverse primers using the conditions: 30 cycles of 95°C for PCR amplification products (10 µl each) were subjected to 30 sec, 50°C for 30 sec and 70°C for 1 min, and sequenced 1.4% TAE (Tris-acetate/EDTA) agarose gel electrophore- using a DNA sequencer (Li-Cor, 4200L). sis based on the procedures described in Sambrook et al. Primers for specifically amplifying the polymorphic (1989). Gels were run in 1x TAE at 100 V for 45 min. DNA fragments were designed by using Internet-based The gels were stained with ethidium bromide solution, software, Primer3 from the Center for Genome Research, viewed and photographed on the UV transilluminator. the Whitehead Institute for Biomedical Research, Cam- The criteria for the RAPD markers were: (1) the bands bridge, Massachusetts, USA. The length and the Tm of were present in the parental mother, and (2) the corre- the designed primers were from 18 to 27 bases and from sponding bands in the male progeny showed the presence/ 57 to 63°C, respectively. Overlapping of the initial absence type of polymorphism (Hunt and Page, 1992). primer annealing sites was avoided. Whether the poly- The RAPD markers were named by the designation of morphic DNA bands were specifically amplified with the primer provided by the supplier (Operon Technology), fol- newly designed primers was determined using the PCR lowed by a dash and the approximate size of the amplified conditions: 35 cycles of 94°C for 1 min, 55°C for 1 min and fragment in base pairs. 72°C for 2 min, followed by 1% TAE agarose gel electro- phoresis. Linkage analysis. Linkage analyses were done with MAPMAKER V2.0 software (Lander et al., 1987). The RESULTS AND DISCUSSION data files were first prepared with the “haploid” data type. In each RAPD marker, polymorphic DNA bands DNAs from a single diploid parental mother heterozy- were coded as follows: “A” for band present, “B” for band gous for a marker mutation, yfb/+, and 40 of her haploid absent, and “–” for band difficult to evaluate. Putative male progeny (20 yfb and 20 wild type) derived from eggs linkage groups were estimated by pair-wise comparisons parthenogenetically activated in vitro were each subjected of markers with statistical threshold, LOD score, of either to RAPD-PCR analysis. About 10 µg of genomic DNA 3.0 or 2.5, and with minimum recombinant fraction of was obtained per individual, which is an amount sufficient 0.34. The Kosambi mapping function in which the recom- for 1000 PCR reactions. The quality of each DNA sample, binant fraction was converted to map distances in cM evaluated by 1% agarose gel electrophoresis, was judged units was used. The code was altered from “A” to “B”, good when a single high molecular weight band was and “B” to “A” for the markers which were not linked to observed. Polymorphic DNAs were amplified by PCR the groups, and again putative linkage groups were using commercially available arbitrary 10-mer primers. estimated. Then, three-point analyses were performed Of the 180 primers examined, 61 produced a total of 79 on each linkage group to generate the most likely order of RAPD markers (Table 1), 117 produced no polymorphic the linked markers. DNA band and two did not produce any amplified DNA. All of the 79 RAPD markers showed the band pres- Establishment of sequence-tagged sites (STSs). ence/absence type of polymorphism, and segregation Selected polymorphic DNA bands were cloned by using a among the 40 haploid males was generally quite good TOPO TA cloning kit with TOP 10F’ cells (Invitrogen). (Table 1). Figure 1 shows one such example. In this RAPD markers were amplified by PCR as above but with case, the parental diploid female had the RAPD marker the volume of reaction solution doubled. The PCR prod- band D04-0950. Three of the 20 wild type haploid male ucts were subjected to 1% TAE low melting-point agarose progeny and 16 of the 20 yfb haploid male progeny had the (SeaPlaque, FMC) gel electrophoresis and stained with same band, clearly indicating the linkage between the yfb ethidium bromide. The polymorphic DNA bands were cut marker gene and the D04-0950 marker. out from the gels, and the pieces of gels were allowed to Of the 79 RAPD markers, seven showed a significant melt at 65°C. The DNA fragments in the gel solution deviation from the expected 1:1 ratio (Table 1), and were were cloned into the TA cloning vector using a TOPO TA thus not included in the further analysis. A total of 73 cloning kit (Invitrogen) according to the supplier’s markers, 72 RAPDs and the single marker mutation (yfb), protocol. The vector plasmids were introduced into the were used for linkage analysis. Fifty-seven markers (56 TOP 10F’ competent cells (Invitrogen) and transformants RAPD markers and the yfb) were organized into 18 link- were selected as described in Sambrook et al. (1989). age groups at LOD 3.0. Sixteen RAPD markers were Plasmid DNAs were extracted and purified by the alka- without linkage to any group. In the honey bee, part of 162 Y. NISHIMORI et al.

Table 1. Segregation of RAPD markers in haploid male progeny from a single diploid mother

RAPD marker* Number of progeny with band Deviation from RAPD marker* Number of progeny with band Deviation from expected ratio*** expected ratio*** present absent uncertain** (Chi-square) present absent uncertain** (Chi-square)

A02 -0800 18 22 0 E03 -1400 18 22 0 -1100 18 22 0 -1300 22 18 0 A09 -1400# 13 27 0 4.9 E04 -0550 24 16 0 -1100 24 16 0 E07 -0400 19 21 0 A10 -1400 23 17 0 E08 -0650 18 22 0 A15 -1500 19 19 2 -0560 22 18 0 A16 -1500# 30 10 0 10.0 E13 -0780 19 20 1 A18 -2200 20 19 1 E16 -0600 25 15 0 -1700# 28 11 1 7.2 E18 -1200## 21 19 0 -1500 19 20 1 G10 -0600 21 19 0 -1400# 13 26 1 4.3 G13 -1100 15 25 0 B11 -1300 21 18 1 G15 -1500 18 22 0 B12 -0900 22 18 0 G16 -1700 25 15 0 B18 -0480 22 17 1 G18 -1800 24 16 0 B20 -1800# 30 9 1 11.3 H03 -1500 18 21 1 -0800 22 17 1 H04 -1100 22 18 0 C01 -0600 17 23 0 H12 -0800 25 15 0 C06 -0700 17 22 1 H20 -1000## 16 24 0 C08 -0900 20 17 3 I04 -0560## 16 24 0 -0600 16 21 3 I08 -0850 22 18 0 C09 -0770## 22 17 1 I11 -0400 18 22 0 C10 -1200 14 26 0 -0350 22 18 0 C12 -1600 25 13 2 I16 -0950 18 21 1 C14 -1800 18 20 2 I19 -0800 19 20 1 C15 -1300 24 15 1 Q01 -1100## 18 22 0 C16 -3000# 11 29 0 8.1 -0800 21 19 0 C19 -0550 19 17 4 Q03 -1500 15 24 1 D02 -0950 17 23 0 Q05 -1400 21 19 0 D03 -0500## 16 23 1 Q08 -1600## 22 18 0 D04 -0950 19 21 0 Q10 -1700## 25 15 0 D10 -1800 19 18 3 Q11 -0700 15 25 0 D11 -1200 16 23 1 Q12 -1300## 18 21 1 -0900 20 19 1 Q13 -2500 22 18 0 D12 -1700## 16 24 0 -1600 23 17 0 -1600 24 16 0 Q14 -2500 26 14 0 -1500 20 20 0 -1600 14 26 0 D15 -0800 15 24 1 Q15 -0900# 13 27 0 4.9 D18 -1200 16 23 1 Q16 -0800## 22 18 0 -1100 23 16 1 E02 -1200## 20 20 0 -0560 24 16 0

*** Markers are named by the primer designation (Operon) followed by a dash and the approximate size (bp) of the amplified fragment. Markers which were not used in the map construction because of a significant deviation in segregation are indicated by #, and those which showed no linkage are indicated by ##. *** Progeny which failed to show any DNA amplification following PCR. *** Chi-square values are shown only for the markers which showed siginificant deviation (P < 0.05) from the expected 1:1 ratio.

the linkage analysis was performed using values of LOD I. less than 3.0, and the map was constructed combining the Compared to the haploid chromosome number, n = 8, the results of analyses at varied LOD scores (Hunt and Page, present results are clearly insufficient. That 12 out of 72 1995). Following the same principle, we finally con- RAPD markers are “orphans” also indicates that a signifi- structed a genetic map with 61 markers (60 RAPD cant fraction of the genome was not covered by the present markers and one visible marker) organized into 16 groups, map. Still, the present results are definitely a beginning. spanning 517.2 cM (Fig. 2). Four of the 16 RAPD In such an extensively studied species as the honey bee, “orphan” markers mentioned above were incorporated into the presently available genetic map is composed of 26 link- the linkage group, but the others still showed no linkage age groups, while the haploid chromosome number is 16 relationship. The yfb gene was mapped in linkage group (Hunt and Page, 1995). The usefulness of a genetic map Linkage map of Athalia rosae 163

Fig. 1. (Top). An example of the segregation of a RAPD marker amplified by using a random 10-mer primer, D04. M: molecular size marker. Representative marker sizes in bp are shown to the left. N: negative control. P: Diploid heterozygous yfb/+ female parent. Lanes 1–20: Haploid hemizygous + male progeny. Lanes 21–40: Haploid hemizygous yfb male progeny. A 950-bp band (arrow) showed polymorphism. (Bottom). Production of the D04-0950 band using STS primers (Table 2) following the cloning and sequencing.

Fig. 2. A linkage map of A. rosae based on segregation of RAPD markers. RAPD markers are designated by the primer name (random 10-mer primer, Operon Technology), followed by a dash and an approximate size in bp of the amplified fragment of DNA. Corrected map intervals in cM are shown to the left of each linkage group. The markers which were cloned, sequenced and converted to sequence- tagged sites (STSs), are boxed. depends on how complete the map is. Obtaining a more et al., 1998), and combine the results. complete map is simply a matter of labor, which should When one uses molecular markers to construct a genetic not be too extensive with the aid of molecular markers. map, it is desirable to have a number of markers cloned We may also make use of other methods such as amplified and sequenced, and make them available as sequence- fragment length polymorphism (Vos et al., 1995; Reineke tagged sites (STSs). One may then establish physical 164 Y. NISHIMORI et al.

relationships among clones and also select the same which mapped to the same position (Fig. 2). genomic region from any new library. Thirteen RAPD If the genetic linkage map is related to the chromo- markers were selected and cloned. They were sequenced, somes, the usefulness becomes even greater. With and primer sets to specifically amplify the markers were molecular markers available, this can be done most eas- designed (Fig. 2, Table 2). In no case did the homology ily, at least in theory, by using the fluorescence in situ hy- search reveal similarity with any reported sequences in bridization (FISH) technique. In insects without poly- the database. Two RAPD markers, E03-1400 (1400 bp) tene chromosomes, this has been done only for multiple- and E03-1300 (1300 bp), were amplified with the same copy genes or sequences (e. g., Brown and Knudson, 1997; primer, and were mapped at the same locus or closely Sahara et al., 1999). Our preliminary results indicate linked loci (linkage group II, Fig. 2). Individual male that a single-copy gene can be localized on the A. rosae progeny either had the 1400 bp band or the 1300 bp band, mitotic metaphase chromosome (Results will be presented but not both. The parental mother had both bands. elsewhere). We employed a complete cDNA (about 6 kb) Upon cloning and sequencing, it was revealed that E03- for vitellogenin, a typical egg yolk protein (Nose et al., 1400 is 1369 bp long and E03-1300 is 1288 bp long, with 1997), as a probe. A single twin-spot was detected on one the same sequence except that the latter had an 81-bp- of the middle-sized chromosomes. We do not yet have a long internal deletion (Fig. 3). We further confirmed the genomic clone for the vitellogenin gene in A. rosae. reproducibility of RAPDs using newly designed STS Information on the genomic DNA sequences available for primers. Three RAPD markers (D04-0950, E03-1400 and the vitellogenin gene in other insects (Trewitt et al., 1992; E03-1300) were selected and amplified with the genomic Yano et al., 1994; Hiremath and Lehtoma, 1997) indicates, DNAs and STS primers. In all cases, the bands corre- however, that the gene is somewhere around 10 kb in sponding to the original RAPD bands were amplified. length including introns. If the results hold for other Results for D04-0950 are shown in Fig. 1. It is most prob- genes, we have a good start now with the established STSs able that marker E08-0560 (linkage group XII) also car- available. ries a short internal deletion in comparison to E08-0650,

Table 2. Primer sequences for the established sequence-tagged sites RAPD Linkage STS primer sequence Database marker group (5' - 3') accession number

A02-1100 III Forward ＡＧＣＧＣＣＧＡＣＴＴＡＴＴＣＡＴＣＡＣ AB040472 Reverse ＡＴＴＴＣＴＣＧＴＣＡＴＣＧＡＡＴＣＧＣ A02-0800 VII Forward ＴＡＧＣＴＧＴＴＧＴＴＴＴＴＧＣＧＧＧ AB040473 Reverse ＧＴＧＡＧＡＡＧＧＣＣＡＡＧＡＴＣＣＣＴ A10-1400 VIII Forward ＴＣＴＣＣＴＧＡＧＴＴＴＴＣＴＡＴＴＣＴＴＧＧＡＡ AB040474 Reverse ＡＡＴＴＧＴＴＣＣＧＣＣＡＴＴＣＴＴＴＴ A15-1500 V Forward ＡＡＣＣＴＧＴＣＡＣＣＡＴＣＣＣＴＧＡＧ AB040475 Reverse ＴＧＴＴＴＣＣＧＡＣＧＴＣＡＴＧＧＴＡＡ B20-0800 VIII Forward ＡＡＡＡＴＣＡＡＧＡＧＣＧＴＴＣＧＧ AB040476 Reverse ＡＧＡＡＴＣＧＣＧＧＣＡＣＡＴＡＡＡＴＣ C01-0600 XVI Forward ＴＴＧＡＴＣＣＧＣＴＧＧＡＡＡＡＡＴＴＣ AB040477 Reverse ＧＧＡＴＣＣＧＧＡＧＡＣＣＴＧＡＡＡＡＴ C14-1800 VI Forward ＣＡＣＧＴＴＣＡＴＡＴＡＴＧＴＴＣＧＣＴＡＴＴＣＡ AB040478 Reverse ＡＧＴＡＣＡＴＡＡＧＣＧＡＧＧＣＡＧＧＣ C19-0550 V Forward ＧＧＧＡＴＧＴＣＣＣＧＡＧＴＣＣＡＣ AB040479 Reverse ＣＴＡＧＣＧＧＣＴＣＧＣＴＧＡＡＡＣ D04-0950 I Forward ＧＴＧＡＣＡＡＣＡＧＴＴＧＧＣＧＡＡＡＡ AB040480 Reverse ＣＴＧＧＣＴＡＴＣＴＣＡＧＣＣＣＧＴＡＧ E02-0560 IV Forward ＧＡＴＡＴＧＣＴＣＧＣＡＧＴＣＡＡＴＴＣＣ AB040481 Reverse ＧＡＣＧＴＧＧＴＴＣＧＴＣＧＴＡＣＴＣＣ E03-1400 II Forward ＴＧＣＴＡＡＣＴＴＴＴＡＡＴＣＧＣＡＡＧＡＧＡ AB040482 Reverse ＡＧＴＣＡＧＴＡＣＧＧＣＧＧＡＧＡＡＴＧ E03-1300 II Forward ＴＧＣＴＡＡＣＴＴＴＴＡＡＴＣＧＣＡＡＧＡＧＡ AB040483 Reverse ＡＧＴＣＡＧＴＡＣＧＧＣＧＧＡＧＡＡＴＧ E08-0650 XII Forward ＣＡＧＣＣＧＣＡＡＧＡＧＴＴＡＧＧＡＡＡ AB040484 Reverse ＡＡＡＧＴＡＧＧＴＧＣＡＡＣＡＡＴＧＡＣＧＡ Linkage map of Athalia rosae 165

Fig. 3. Alignment of the sequences of markers E03-1400 and E03-1300. The sequences match exactly except that the latter has an 81- bp-long internal deletion. Arrows indicate the sequence of the initial 10-mer primer. Dashed arrows indicate the STS primers.

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