Twenty-three polymorphic markers, including 14 for LG 12 (Ppsm), were developed for nine-spined sticklebacks (Table 1). On average, 10.6 alleles per locus (range = 2-47) were detected across the two populations (Table 1). The mean number of observed alleles and average expected heterozygosities were 10.3 and 0.66 in the Baltic Sea and 2.0 and 0.27 in Pyöreälampi, respectively (Additional file 1). Out of the 14 Ppsm loci, 10 were homozygous in all females and heterozygous in all males in the Pyöreälampi population (Figure 1, Additional file 1). Consequently, the number of observed alleles and expected and observed heterozygosities were significantly higher in males than females in this population (Wilcoxon signed rank test, P < 0.01 for allele number, P < 0.001 for heterozygosities). In the Baltic Sea population, observed heterozygosity was higher in males than females (P < 0.01; Figure 1), but no significant differences were found in the number of alleles and expected heterozygosity (P > 0.05). While no loci showed significant F _IS in females, negative F _IS was detected in males at 11 Ppsm loci in the Baltic Sea and at 13 Ppsm loci in Pyöreälampi (Figure 1, Additional file 1). Linkage disequilibrium was observed in all pairs of polymorphic Ppsm loci in Pyöreälampi, although 47 out of the 91 comparisons were not significant in the Baltic Sea (Additional file 2). In particular, linkage disequilibrium was not detected in several combinations including highly polymorphic loci (e.g. Ppsm3-5 and Ppsm14). The trend test identified significant associations between phenotypic sex and Ppsm loci in both populations (Additional file 3). All polymorphic Ppsm loci showed an association with sex in Pyöreälampi, whereas significant association was not observed for four Ppsm loci (Ppsm1, Ppsm3, Ppsm4 and Ppsm14) in the Baltic Sea (Additional file 3).

The analyses of genotypic frequencies at Ppsm loci identified several alleles that were observed only in males (Table 2). All males from Pyöreälampi possessed both male-specific and nonspecific alleles at each of the polymorphic loci (Table 2). No polymorphism was detected in the male-specific alleles in this population. Male-specific alleles were found at nine loci in all males of the Baltic Sea (Table 2). Although a male-specific allele was not detected at Ppsm5, the same allele (229) was observed in most (23 out of 24) males and a few (2 out of 24) females. F _ST values between females and males were significant for 13 Ppsm loci in both populations (Figure 1). For these loci, F _ST values were larger in the Pyöreälampi than in the Baltic Sea.

A total of 161 alleles were observed at 14 loci, with an average of 11.5 alleles per locus (range = 2-31) in three-spined sticklebacks (Additional files 4 and 5). Linkage disequilibrium was observed in only eight out of the 91 comparisons (Additional file 6). Expected heterozygosity ranged from 0.39 to 0.93 among the loci with a mean of 0.70 (Additional file 5). Several loci showed different levels of observed heterozygosity between females and males (Figure 2, Additional file 5). Although no loci showed significant F _IS in females, 12 loci exhibited significant F _IS in males (Figure 2, Additional file 5). Out of these 12 loci, five were heterozygous in all males, showing negative F _IS. At these loci, which were located in two chromosomal regions (i.e. 3.2-4.0 Mb and 9.4-11.8 Mb), male-specific alleles were detected in all males in heterozygous states (Table 3). In contrast, although two to 20 alleles were observed for each locus, no heterozygous males were found at seven loci that showed positive F _IS (Figure 2, Additional file 5). No male-specific alleles were identified at these loci (Table 3). MICRO-CHECKER analyses indicated the presence of null alleles at the seven loci in males, but not in females. These loci were located sequentially in two separate chromosomal regions (i.e. 5.1-7.4 Mb and 14.7-19.6 Mb) of LG 19 (Figure 2, Additional file 4).

Significant associations between phenotypic sex and loci were detected for the five loci that possessed male-specific alleles (Additional file 7). F _ST between females and males was significant for these, but not for the other loci (Figure 2).

Our results indicate that 13 markers (Ppsm2-14) are linked to each other, and are associated with phenotypic sex in nine-spined sticklebacks. These indications were more robust in a population with low genetic diversity (Pyöreälampi) than in one with high genetic diversity (Baltic Sea). Indeed, all males of Pyöreälampi possessed one particular male-specific allele for each of the 13 loci. Given the male heterogametic inheritance of this species 28 , these alleles should be located on the Y chromosome. Accordingly, only one Y chromosome haplotype was identified in this population. In contrast, three out of the 13 loci did not exhibit an association with phenotypic sex in the Baltic Sea population. Since these three loci were highly polymorphic, this could be due to their high mutation rates. Even in this highly variable population, the male-specific allele was monomorphic at seven loci. However, polymorphisms were detected in the remaining alleles, which are putatively located on the X chromosome. The different level of polymorphism between the sex chromosomes is explainable by the fact that the effective population size of the Y chromosome is one-third of that of the X chromosome 29 . In general, our results indicate that sex chromosomes are highly differentiated throughout most of their length in nine-spined sticklebacks.

Based on karyotype analyses, the Y chromosome of nine-spined sticklebacks is characterized by a much larger size than the other chromosomes 10 28 . Because none of the autosomes or autosomal arms are missing, the additional segment observed in the Y chromosome is assumed to have originated from a tandem duplication of the ancient Y chromosome or a duplication of an autosomal segment followed by insertion into the Y chromosome 28 . Although we examined allelic variation along the sex chromosomes with several markers - for which primers were developed in conserved sequences of divergent species - no loci showed the patterns of multilocus amplification which can be an indication of the Y chromosome duplication. Thus, an extra segment of the Y chromosome is more likely to be derived from a duplication of an autosome than that of the Y chromosome.

In three-spined sticklebacks, an allozyme of isocitrate dehydrogenase (IDH) has been found to be associated with phenotypic sex 30 31 . In accordance with this observation, a linkage mapping analysis showed that the sex determining region is located near the IDH gene of LG 19 8 . In our study, male-specific alleles, which should be located on the Y chromosome, were identified at five loci. Indeed, three of these loci were closely linked to the IDH gene (11.3 Mb). However, the remaining two loci were found in a chromosomal region (3.2-4.0 Mb) far from the IDH gene, implying that there is extensive differentiation. In addition, male-specific null alleles were identified at several loci located in two different chromosomal regions. This lack of heterozygous males suggests that amplification of Y chromosomal alleles is absent at these loci. This could be due to complete deletion of the microsatellite loci or polymorphisms in the primer binding sites of these loci in the Y chromosome. Overall, Y specific alleles and null alleles were identified over 16.4 Mb of a chromosomal region (3.2-19.6 Mb), which corresponds to 81% of the X chromosome. A large deletion of the Y chromosome was also suggested by a previous study using fluorescence in situ hybridization 26 . Indeed, the deletion was assumed to be present in one of the regions where Y chromosomal null alleles were identified (i.e. 14.7-19.6 Mb). Other Y chromosomal null alleles were found in the region where multiple inversions appear to have occurred 26 . It has also been demonstrated by sequence analysis that the Y chromosome exhibits substantial nucleotide divergence from the homologous region on the X chromosome mainly due to multiple insertions and deletions 8 . Together with these studies, our results suggest that the Y chromosome is highly degenerate in three-spined sticklebacks. Since closely related stickleback species exhibit the rapid turnover of sex chromosomes and sex determination systems, their sex chromosomes are thought to be at the early stages of evolution, as is generally observed in lower vertebrates 8 10 . Our study uncovered high levels but distinct patterns of sex chromosome differentiation in closely related stickleback species, which diverged about 13 million years ago 32 . In contrast to three-spined sticklebacks, we did not detect signatures of Y chromosome degeneration in the nine-spined stickleback despite the apparent sex chromosome heteromorphy in this species 28 . Therefore, it is unlikely that the Y chromosome of the nine-spined stickleback has degenerated as much as that of the three-spined stickleback.

Sexually antagonistic selection is expected to facilitate the suppression of recombination between sex chromosomes, and thus assumed to be the primary driving force behind sex chromosome differentiation 14 . Additionally, chromosome rearrangements, such as inversions, translocations and centric fusions involving an autosome and a sex chromosome, are known to reduce or suppress crossing over in the regions around the breakpoints in heterozygotes with the standard arrangement 14 . Such rearrangements can create linkage between sexually antagonistic genes and sex chromosomes, and thereby can be favored by natural selection 14 33 . As such, the high genetic divergence between the sex chromosomes of nine-spined and three-spined sticklebacks could be a result of both sexually antagonistic selection and chromosome rearrangements. Further research needs to be focused on the role of sexually antagonistic selection in sex chromosome differentiation in these species. In addition, it should be interesting to investigate intraspecific variation of sex chromosome differentiation and rearrangements among genetically divergent populations.

To facilitate comparative genomic analyses, we devised a simple method for the development of microsatellite markers at target chromosomes and genomic regions using the sequenced genomes of other species. Our approach is similar to cross-species transfer of microsatellite markers, which can be performed without sequence information from a target species, but is different in the sense that conserved microsatellite flanking regions in divergent species are used as primer binding sites. Thus, it is possible to minimize amplification failures, which are a general concern in the cross-species utility of microsatellite markers 34 . In fact, when primers are designed using three-spined stickleback sequences alone, amplification success in nine-spined sticklebacks is low (28.6%) 22 . Moreover, our approach has an advantage over traditional cross-species amplification approaches because microsatellite markers can be developed for specific chromosomes and genomic regions based on the sequenced genome of other species. Furthermore, since primers are developed for conserved sequences in divergent species, these markers should be useful also in other stickleback species. Therefore, our approach for the development of microsatellite markers can facilitate comparative genomic analyses of stickleback species. In light of the rapidly increasing numbers of sequenced genomes and genomic resources, molecular tools for species lacking sequenced genomes can be developed using available genomic information of other species, as demonstrated in this study.

Mature nine-spined sticklebacks were collected from the Baltic Sea (60°12' N, 25°11' E) and from Pyöreälampi pond (66°16' N, 29°26' E) in 2008. These populations were selected to cover different levels of genetic diversity (average heterozygosity at 12 microsatellite loci, H _E = 0.590 in Baltic Sea and H _E = 0.004 in Pyöreälampi) 35 , which can influence the identification of sex chromosome differentiation using microsatellite markers. Three-spined sticklebacks were sampled from the Baltic Sea (60°12' N, 25°11' E) in 2008. Phenotypic sex was determined by examining gonads after the fish had been anesthetised with an overdose of MS-222 (tricane methanesulphonate). All procedures were performed under license from the Animal Experiment Board in Finland (ELLA; STH379A).

Using the three-spined stickleback genome 36 , microsatellites were surveyed in regions randomly chosen over a wide range of LG 12 (Table 1), which corresponds to the sex chromosomes in nine-spined sticklebacks 10 27 . To design primers for nine-spined sticklebacks, conserved regions were searched in microsatellite flanking regions by aligning sequences of the three-spined stickleback and medaka 37 . Based on the location of microsatellites and conserved regions, primer sequences were designed manually, targeting appropriate melting temperature and GC content (Table 1). In addition to LG 12, some primers were developed for other linkage groups to verify variability of marker loci on sex chromosomes and autosomes (Table 1). For three-spined sticklebacks, 14 microsatellites covering a wide range of the sex chromosomes of this species (LG 19) were selected based on the sequenced genome 36 (Additional file 4). Of these, nine markers were previously reported 38 39 , and new primers were developed for the remaining five loci with the genome sequences using WebSat 40 . Since the genome sequences were obtained from a female specimen 25 , the novel primers were assumed to be designed based on the X chromosome sequences. All novel microsatellite primers were deposited in the National Center for Biotechnology Information (NCBI) Probe Database [PUIDs: 10552794-10552816, 10701232-10701236].

Total DNA was extracted from fin clips using a silica-fine based purification 41 following proteinase K digestion. Each forward primer was labelled with a fluorescent dye (FAM, HEX or TET), and the 5'-end of each reverse primer was modified with a GTTT-tail 42 . For efficient screening, PCRs were conducted using the Qiagen Multiplex PCR Kit (Qiagen) in 10 μl reaction volumes containing 1× Multiplex PCR Master Mix, 0.5× Q-Solution, 2 pmol of each primer and 10-20 ng of template DNA. The reactions were performed by the following cycle: an initial activation step at 95°C for 15 min, followed by 30 s at 94°C, 90 s at 53°C and 60 s at 72°C for 30 cycles with a final extension at 60°C for 5 min. PCR products were visualized with a MegaBACE 1000 automated sequencer (Amersham Biosciences) and their sizes were determined with ET-ROX 550 size standard (Amersham Biosciences). Polymorphism was initially screened using 24 individuals (12 females and 12 males) of nine-spined or three-spined sticklebacks from the Baltic Sea. For the identified polymorphic loci, a total of 48 individuals (24 females and 24 males for nine-spined sticklebacks and 25 females and 23 males for three-spined sticklebacks) were genotyped in each population. Alleles were scored using Fragment Profiler 1.2 (Amersham Biosciences) with visual inspection and manual corrections of alleles.

Locus specific heterozygosity and F _IS were calculated using FSTAT 2.9.3 43 . The significance of F _IS was assessed by 10 000 permutations. For marker loci located on sex chromosomes, F _IS is expected to be lower in heterogametic males than in homogametic females if alleles are not shared between the sex chromosomes. Linkage disequilibrium was tested between pairs of loci in each population. An association between phenotypic sex and alleles at marker loci was investigated using the trend test 44 45 as implemented in PowerMarker 3.25 46 . To identify Y chromosome specific alleles, genotypic frequencies and allele distributions were compared between males and females. In addition, to evaluate genetic differentiation between sex chromosomes, F _ST was estimated between females and males using the method of Weir and Cockerham 47 as implemented in GENEPOP 4.0 48 . Statistical significance of F _ST values was determined using 10 000 permutations. In three-spined sticklebacks, some polymorphic loci appeared to be homozygous in all males (see Results). For these loci, the presence of sex-specific null alleles was tested using MICRO-CHECKER 49 . Since a large proportion of microsatellites are conserved between three-spined and nine-spined sticklebacks 22 27 , microsatellite locations on LG 12 in nine-spined sticklebacks were estimated based on the three-spined stickleback genome. Based on the fact that the orientation of supercontig 3 of LG 19 in Ensembl is inverted as compared to the genetic map 26 , marker locations on LG 19 were determined by reversing the sequence of this supercontig according to Ross and Peichel 26 (Additional file 4). Sequential Bonferroni corrections 50 were applied for all multiple comparisons to minimize type I errors.