• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of geneticsGeneticsCurrent IssueInformation for AuthorsEditorial BoardSubscribeSubmit a Manuscript
Genetics. Feb 2007; 175(2): 945–958.
PMCID: PMC1800609

Linkage Maps for the Pacific Abalone (Genus Haliotis) Based on Microsatellite DNA Markers


This study presents linkage maps for the Pacific abalone (Haliotis discus hannai) based on 180 microsatellite DNA markers. Linkage mapping was performed using three F1 outbred families, and a composite linkage map for each sex was generated by incorporating map information from the multiple families. A total of 160 markers are placed on the consolidated female map and 167 markers on the male map. The numbers of linkage groups in the composite female and male maps are 19 and 18, respectively; however, by aligning the two maps, 18 linkage groups are formed, which are consistent with the haploid chromosome number of H. discus hannai. The female map spans 888.1 cM (Kosambi) with an average spacing of 6.3 cM; the male map spans 702.4 cM with an average spacing of 4.7 cM. However, we encountered several linkage groups that show a high level of heterogeneity in recombination rate between families even within the same sex, which reduces the precision of the consolidated maps. Nevertheless, we suggest that the composite maps are of significant potential use as a scaffold to further extend the coverage of the H. discus hannai genome with additional markers.

WITH increased interest in genomics, significant progress has been made in constructing genetic maps for aquatic animals over the past decade. This has been done, mainly for teleost fishes, using information for quantitative trait loci (QTL) mapping to elevate aquaculture technology (Jackson et al. 1998; Danzmann et al. 1999; Sakamoto et al. 1999; Ozaki et al. 2001; Perry et al. 2001; Robinson et al. 2001; Cnaani et al. 2003; Somorjai et al. 2003; Moen et al. 2004; Reid et al. 2005; Yu and Guo 2006) and for comparative mapping to understand the evolutionary processes of organisms (Barbazuk et al. 2000; Postlethwait et al. 2000; Woods et al. 2000; Liu et al. 2002; Jaillon et al. 2004; Naruse et al. 2004; Kai et al. 2005; Gharbi et al. 2006). However, such accelerated progress in genomics has not been the case with a marine gastropod, the abalone (Haliotidae).

Abalone species contain >15 subgenera comprising ~70 taxa (Lindberg 1992) and support an important marine fishery and aquaculture worldwide. The so-called Pacific abalone includes Haliotis discus hannai, H. discus discus, H. madaka, and H. gigantea, among which H. discus hannai is the major abalone resource for coastal fisheries. Genetic mapping is of great interest in the aquaculture of H. discus hannai. There is a growing concern about the reduction of natural H. discus hannai resources largely because of overfishing and environmental deterioration (Kawamura 2002). This has prompted the need for domestication to ensure a stable supply of commercial products (Kijima 2005). The development of viable aquaculture systems for this species has been opposed by their slow growth rate, as it takes several years to cultivate abalone to reach a harvestable size. This problem would be surmountable by establishing superior strains with enhanced growth, through marker-assisted selection programs based on genetic maps on which QTL influencing growth performance are mapped. Previous breeding studies have indicated that the growth of H. discus hannai has a genetic basis (Hara 1992; Hara and Kikuchi 1992; Kawahara et al. 1997, 1999; Kobayashi et al. 2006). On the other hand, with reference to evolutionary biology, the comparative mapping strategy exploring syntenic alignments of genes/molecular markers will help resolve the evolutionary complexities among the four members of Pacific abalone. The systematics of Pacific abalone have not been well defined owing to incongruities between the morphological/ecological differences (Ino 1952) and the extent of genetic divergence (Hara and Fujio 1992; Lee and Vacquier 1995; An et al. 2005). A recent microsatellite analysis found several lines of evidence suggesting the existence of genetic boundaries among them (Sekino and Hara 2007). However, neither the taxonomic status nor the evolutionary process of the genomes has been resolved.

In this study, we aimed to construct genetic linkage maps for H. discus hannai. The karyotype of H. discus hannai consists of 18 pairs of chromosomes, comprising 10 pairs of metacentric and 8 pairs of submetacentric chromosomes (Arai et al. 1982). The genome size is estimated to be ~1.6 Gb (Sekino et al. 2006). Several classes of molecular markers are needed in linkage mapping to cover a wide range of the genome, and anonymous DNA markers such as amplified fragment length polymorphisms (AFLPs) and random amplified polymorphic DNA (RAPDs) could serve as an efficient tool to achieve extensive genome coverage, as actually shown in H. discus hannai (Liu et al. 2006). We consider, however, that microsatellite-based linkage maps are imperative to tackle the challenging issues described above, given the codominant property of microsatellites with a wealth of segregation information and the transferability across populations with which homologies of markers and thereby linkage groups among populations can readily be established.

This study presents H. discus hannai linkage maps constructed using 180 microsatellite markers, which, according to us, is the first report of microsatellite-based linkage maps among any species of Haliotidae. The use of backcross or F2 populations derived from inbred lines is a rather unrealistic option for linkage mapping in this species, owing not only to time constraints associated with line breeding but also to an underlying inbreeding depression resulting in deformity and low survival (Park et al. 2006). We, therefore, screened three F1 outbred crosses for linkage mapping. Linkage maps were initially constructed for each family and sex, and the individual map information was subsequently used to generate a composite linkage map for each sex. The use of multiple mapping families allowed the detection of large map differences in several linkage groups within sexes, possibly caused by chromosomal variations.


Abalone mapping families:

Experimental crossing of H. discus hannai was conducted at the Iwate Fisheries Technology Center (Kamaishi, Iwate, Japan; April 2004), using matured abalone derived from wild captives and a hatchery-raised strain, which had been subjected to selection for growth over four generations (Kobayashi et al. 2006). We used three families (F, L, and M) for linkage mapping. Families F and L were maternal half-sibs, which had different male parents (wild) and a common female parent (selected strain). Family M was produced by mating an individual from the selected strain (♀) with a wild-caught abalone (♂). These families were raised in separate aquarium tanks with a constant water temperature (20°). Juvenile abalones were sampled at 4 months of age and stored in 99% ethanol until DNA extraction. Genomic DNA of each parent and progeny (N = 96 for families F and L; N = 60 for family M) was extracted from a small piece of foot muscle tissue following phenol/chloroform procedures (Sambrook et al. 1989).

Microsatellite analysis:

The source of microsatellite markers used in this study was as follows: two markers suffixed with Hdd in Sekino and Hara (2001), six markers with Hd in Hara and Sekino (2005), 111 markers with Afa and Awb in Sekino et al. (2005, 2006), and 15 markers with Ahdh in Sekino and Hara (2007). These markers turned out to be informative in at least one of the three mapping families on the basis of our preliminary marker screening. In addition to the 134 markers previously reported, we analyzed 46 additional microsatellites for linkage analysis. Of these, 40 markers were developed through reanalyses of (CA)n- and (CT)n-enriched libraries constructed in the five studies cited above. The remaining six were developed from the nucleotide sequences of H. discus hannai or H. discus discus microsatellites previously posted on the GenBank/EMBL/DDBJ database, for which no microsatellite primer pair has been released. Here, we denote these novel markers as number symbols with the prefix Eab. Details for these markers are available in supplemental Table 1 at http://www.genetics.org/supplemental/.

Either a 5′-fluorescent-labeled microsatellite primer (dye primer) or a 5′-KS-tailed microsatellite primer (KS sequence: 5′-cgaggtcgacggtatcg-3′) in combination with a 5′-fluorescent-labeled KS primer (see 5′-tailed primer method in Oetting et al. 1995; Boutin-Ganache et al. 2001) was used to amplify microsatellite alleles in polymerase chain reaction (PCR) examinations. In both cases, Cy5-fluorescent dye was conjugated to the 5′ end of the primers (Sigma Genosys, Hokkaido, Japan) so that amplified alleles could be detected on an ALFexpress/ALFexpress II DNA sequencer (GE Healthcare Bio-Sciences, Piscataway, NJ). PCR assays were performed as described in Sekino et al. (2005) for the dye-primer method and in Sekino et al. (2006) for the 5′-tailed primer method.

Segregation and linkage analysis:

Departure of allelic segregation patterns from Mendelian expectations was assessed using a chi-square goodness-of-fit test. Subsequent linkage analyses were made for each sex separately. Genotype configurations of markers in each family were categorized into four expected segregation types when null-allele segregation was allowed: 1:1:1:1-ratio type (♀ × ♂: AB × CD or AB × AC), 1:2:1 type (AB × AB), 1:1 ♀ type (AB × AA or CC), and 1:1 ♂ type (AA or CC × AB). The expected 1:1- and 1:2:1-type markers were used as backcross (BC)-type and F2-type markers, respectively. We partitioned segregation data from expected 1:1:1:1-type markers into 1:1 ♀- and 1:1 ♂-type data (BC type) to perform linkage analysis for each sex (Jacobs et al. 1995; Viruel et al. 1995). All the statistical analyses described below were made using JoinMap version 3.0 software (Van Ooijen and Voorrips 2001) with the cross-pollinating (CP) coding scheme, which handles F1 outbred population data containing various genotype configurations (in this case, BC and F2 type) with phase unknown. Linkage between markers was examined by estimating LOD scores for recombination rate (θ) on the basis of the maximum likelihood method with the EM algorithm. JoinMap first calculates the G2-statistic for independence of segregation; then the obtained G2 is multiplied by a constant of 0.5 × log10e to convert the G2-value into the normal LOD scale. The statistical power of this approach in determining marker linkages is not influenced by segregation distortion (Maliepaard et al. 1998; Van Ooijen and Voorrips 2001). Significance of marker linkage was determined at a LOD threshold of 3.5, but a less-stringent threshold value (LOD = 2.5 or 2.0) was applied when incongruence of linkage grouping between families occurred, considering the haploid chromosome number reported for H. discus hannai (n = 18, see below). A threshold θ of 0.6 was set to detect suspect linkage possibly resulting from allele-coding errors. After assigning the markers into respective linkage groups, heterogeneity in θ for each pair of markers was tested between families using a G-test, where the observed number of recombinants and nonrecombinants in the individual families was compared with those estimated from the θ averaged between families.

Linkage maps:

Markers were linearly aligned in each linkage group, converting the recombination rates into the Kosambi map distance (centimorgans). Although families F and L had a common female parent, we constructed the female maps of the two families separately to investigate map differences within an individual between different crossing experiments, which may be caused by statistical errors and/or environmental factors. The position of markers was explored on the basis of the sequential buildup of the map (Stam 1993). First, the most informative pair of markers was selected, followed by sequential addition of other markers. The “ripple” was performed each time after adding one marker. The best-fitting position of an added marker was searched on the basis of the goodness-of-fit test (chi-square) for the resulting map. When a marker generated a negative map distance in the map or a large “jump” value in goodness-of-fit, which is the normalized difference in chi-square value before and after adding the marker (Van Ooijen and Voorrips 2001), the marker was removed, and map calculation was continued to construct a first-round map. After the first-round marker ordering, the previously removed markers were added to the first-round map and again subjected to the goodness-of-fit testing. In this manner, the marker ordering was continued up to the third round until an optimum order of markers was found. A consistent threshold value for the jump was set at 5.00. The “fixed-order” command was used when a difference of marker orders appeared between females and males or between families within sexes. The individual maps were visualized using MapChart version 2.2 (Voorrips 2002).

After visualizing the individual linkage maps, we constructed a composite map for each sex to summarize the individual maps. When none of the families yielded a significant difference in θ for all possible combinations of markers in a linkage group, a LOD weighted average of θ-values was calculated, from which a composite map for the linkage group was constructed (Stam 1993). In the absence of marker combination with a significant difference in θ between two of the three families in a linkage group, the values of θ from the two families were averaged in the same manner as described above. The resulting map was then used as a framework of the linkage group, to which only markers that were informative in the remaining one family were added. In other cases, family F, which had the highest number of informative markers in both females and males among the three families (see below), was defined as a framework to which information from the other families was added.

On the basis of the synthesized maps, expected genome length was obtained by using the following two methods. First, the average spacing s between markers, which is calculated by dividing the total observed map length by the number of marker intervals, was estimated, followed by adding twice the s-value to the observed map length of each linkage group (EG1, Fishman et al. 2001). Second, the observed map length of each linkage group was multiplied by (m + 1)/(m − 1), where m is the number of markers that were placed at different positions on the linkage group (EG2, method 4 in Chakravarti et al. 1991).


Segregation and linkage analysis:

A total of 180 microsatellite markers proved to be informative in at least one of the three mapping families, of which 69 were commonly shared with polymorphisms across all the parents. The number of segregating markers was essentially similar among the families, ranging from 158 (family M) to 167 (family F) (Table 1). Markers with an expected segregation ratio of 1:1:1:1, which exploit maximally the codominant property of microsatellite markers, accounted for 65% or more of the markers in each family. Segregation distortion was found at 21 markers in family F, at 3 in family L, and at 2 in family M (P < 0.01). When a more stringent significance level adjusted for the haploid chromosome number (n = 18) was applied, 4 markers in family F, 2 in family L, and 1 in family M still showed significant segregation distortions (P < 0.0028, Table 1).

Number of segregating microsatellite markers in three mapping families of Pacific abalone Haliotis discus hannai

Converting 1:1:1:1-type segregation into BC-type segregation for both sexes revealed that 134–148 markers in females and 129–147 markers in males were available for linkage analysis (Table 2). Each family showed >10 cases of null-allele segregation (details not shown). The presence of null alleles sometimes reduced the segregation information of markers, depending on the genotype configurations of parents. For example, null-allele segregation was inferred in family L (♀) at the Afa121 marker (♀ × ♂: AØ × BC, where Ø denotes null allele), where the segregation pattern was completely determined for all alleles. In family F (maternal half-sib of family L), the genotype of the male parent at this marker was AA; therefore, the genotype configuration of the parents was expected to be ♀ × ♂: AØ × AA. However, no segregation information for any allele was derived in this case.

Number of markers in linkage groups (LGs) in each family for each sex (LOD threshold = 3.5)

Linkages between markers were examined, and the markers were assigned to linkage groups (LGs) without any suspect linkage (Table 2). Within female parents, the markers fell into 20 LGs in families F and L and 21 LGs in family M (LOD = 3.5). There were 2, 5, and 9 markers showing no significant linkage with other markers in families F, L, and M, respectively. Markers allocated to LG4 in families F and L formed 2 LGs in family M; the 2 LGs in family M joined when a more relaxed threshold LOD (2.5) was applied. For male parents, the threshold LOD (3.5) produced 18 LGs in families F and L. In family M, however, a total of 21 LGs appeared, where 3 LGs in families F and L (3, 7, and 14) were divided into 2 LGs. One unlinked marker remained in each of families F and L, whereas in family M 5 markers were not linked to other markers. Of the 21 LGs in family M, 2 LGs that were homologous to LG14 in families F and L were combined at a relaxed threshold LOD (2.0). In general, family M produced more numbers of LGs with many unlinked markers in both sexes most likely because of the smaller sample size (N = 60) compared with families F and L (N = 96 each).

Construction of linkage maps:

On the basis of the results of linkage grouping, we constructed an individual linkage map for each parent. As we expected, two female maps originating from the same female parent (families F and L) were similar with minor map differences, excepting LG16 and -17 (Figure 1). The markers on LG16 were highly clustered in both families F and L, and the marker orders were not consistent among the three families. As well, markers on LG17 in family F were clustered; furthermore, in family L all the marker pairs scored θ as zero. These observations were in contrast to the spaced marker alignment in family M. Such clustering of markers in certain LGs and families was also found in males, notably in LG10, -12, and -16 (Figure 1).

Figure 1.
Clustered marker alignment observed in four linkage groups. Distance was estimated on the basis of the Kosambi mapping function. All marker pairs in LG10 in family M (♂), a smaller group of LG12 (LG12A in Table 2) in family L (♀), and ...

We constructed composite maps for females and males (Figure 2). Families used for the development of consolidated maps are shown in Table 3. In four LGs (10♂, 12♂, 16♀♂, and 17♀), as described above, we found a large map difference between families even within sexes; therefore it was not possible to integrate the individual maps with the weighted averaging method. In addition, we considered that in these LGs, using family F as a framework would result in an overestimation of map differences between sexes. For these LGs, we therefore used the following compromise solutions to minimize risks of overestimation of map differences between sexes. For LG10♂ and 12♂, an individual male map, which had a less-conflicting marker arrangement with the consolidated female map, was selected as a framework map (family M for LG10♂, family L for LG12♂). For LG16 in both sexes, a pair of female and male maps, between which the maps were relatively conserved, was selected as a framework (family M in both sexes). For LG17♀, the individual map from family M showing a less-conflicting marker arrangement with the consolidated male map was used as a framework.

Figure 2. Figure 2. Figure 2.
Composite linkage maps for Haliotis discus hannai constructed using three F1 outbred families. For the source of markers, see text. A total of 160 markers are placed on the female map and 167 markers on the male map. The position of the Afa098 marker ...
Summary of composite linkage maps for Pacific abalone H. discus hannai

The composite female map consists of 19 LGs. A total of 160 markers are placed on the map, spanning 888.1 cM with an average spacing of 6.3 cM. The number of markers per LG ranges from 3 to 14, and the map length of LGs is within a range of 2.1 cM (LG18) and 97.5 cM (LG1). In this composite female map, two LGs of the individual female maps (LG12A and -12B in Table 2, see also Figure 1) were merged into one (LG12 in Figure 2). This bridging was possible using one marker (Afa093) as a link. In family F, the Afa093 marker with two other markers (Awb041 and Eab904) were assigned to LG12A; however, in family M, the Afa093 marker was assigned to LG12B, leaving the Awb041 and Eab904 markers on LG12A. For LG18 (Figure 2), the position of the Afa098 marker is unsettled because this marker segregated only in family M and only the Afa014 marker was shared among families. We used conservatively the map length of the shorter one, that is, the LG18 map where the Afa093 marker is placed between the Afa014 and Afa144 markers, to calculate the total map length. The male map comprises 18 LGs (Table 3 and Figure 2), which is in agreement with the haploid chromosome number of H. discus hannai, spanning 702.4 cM. A total of 167 markers are placed on the map with an average spacing of 4.7 cM. The expected genome length (EG1 and EG2) based on the composite maps was estimated to be 1126.7 cM (EG1) and 1186.7 cM (EG2) in female and, in male, 866.7 cM (EG1) and 931.4 cM (EG2) (Table 3). Genome coverage of the composite map is approximated as 78.6% (EG1) and 74.5% (EG2) in female and 81.0% (EG1) and 75.7% (EG2) in male.

Heterogeneity in recombination rate:

We further detailed the extent of differences in recombination rate (θ) between families within and between sexes. We made comparisons for each linkage group and then summed up the number of pairwise comparisons with a significant difference in θ (P < 0.01) over the linkage groups. There were 268–552 comparable marker pairs depending on the parental combinations. The common female parent of families F and L yielded significant differences in θ in 5.8% of the comparisons (32 of 552 comparisons); however, the majority of these significant differences (23/32) were found in one linkage group (LG16). Excepting this comparison, the proportion of significant difference in θ varied from 19.1 to 38.4% in the “within-sexes” category and from 23.3 to 45.0% in the “between-sexes” category. When the percentage of marker pairs with significant heterogeneity in θ was compared between the two categories, there was little difference between them (two-tailed Mann–Whitney test, U = 11, P = 0.066).

Heterogeneity testing for θ between sexes, which was performed exclusively on the basis of 69 markers that were informative in all the parents, revealed that almost all the marker pairs in LG4 (7 segregating markers, 21 pairs) and LG9 (6 markers, 15 pairs) generated higher values of θ in females (θ-values in LG4 and -9 are given in supplemental Table 2 at http://www.genetics.org/supplemental/). Nonparametric analysis did not provide a significant difference in distributions of θ within sexes in LG4 at the threshold P of 0.01 (Friedman's test: ♀, χ2 = 4.5, P = 0.11; ♂, χ2 = 2.5, P = 0.29), and in LG9 (♀, χ2 = 4.9, P = 0.09; ♂, χ2 = 7.0, P = 0.03). However, the difference was significant in all combinations between females and males (Wilcoxon's signed-rank test: LG4, |z| = 3.7–4.0, P < 0.0005; LG9, |z| = 3.1–3.3, P < 0.005). In other LGs, such sex-specific difference in θ was not evident (data not shown). Obviously, the use of markers commonly segregating in all the parents limits the number of marker combinations to calculate values of θ, from which the results obtained are rather conservative. In practice, we excluded from the analyses four LGs (6, 13, 15, and 18), where no pair of markers segregating from all the parents was found, and five LGs (3, 5, 7, 10, and 14), where ≤3 pairs of commonly segregating markers were available.


Abalone linkage maps:

Little progress has so far been made in constructing linkage maps for molluscan species despite the aquacultural significance and much interest from evolutionary biology, except for a few species such as the eastern oyster (Crassostrea virginica) (AFLPs, Yu and Guo 2003) and the Pacific oyster (C. gigas) (AFLPs, Li and Guo 2004; microsatellites, Hubert and Hedgecock 2004). Among abalone species, the only example is an AFLP-based linkage map for H. discus hannai, to which several RAPDs (10 markers) and microsatellites (9 markers) were added (Liu et al. 2006). We report here the first microsatellite-based linkage maps for an abalone species, using 180 microsatellite markers, which overcome the disadvantages of AFLP markers such as the difficulty in determination of inheritance mode owing to the dominant/recessive nature and the limited portability (Danzmann and Gharbi 2001; see also Sebastian et al. 2000).

We constructed a consolidated linkage map of H. discus hannai for each sex. A disagreement of the number of linkage groups between the composite female and male maps was found (♀, 19; ♂, 18), but aligning the two separate maps produces 18 linkage groups, consistent with the haploid chromosome number of this species. Thus, the results are more reasonable than the AFLP-based linkage maps for H. discus hannai, where 22 linkage groups in female and 19 in male were presumed (Liu et al. 2006). However, several linkage groups, such as LG15 and -18, are sparsely populated with a few markers. This uneven distribution of markers across linkage groups, together with the considerably shorter map lengths (♀, 888.1 cM; ♂, 702.4 cM) compared with the AFLP-based maps (♀, 1773.6 cM; ♂, 1365.9 cM) (Liu et al. 2006), suggests the paucity of markers; therefore appreciable gaps remain in the microsatellite-based maps. Moreover, in several linkage groups there was a large map difference between families even within sexes. Thus, it should be noted that the estimated map lengths and marker arrangements in some cases provide only a provisional solution.

Sex-related difference in recombination rate:

Since the discovery of heterogeneous fractions of meiotic recombination between sexes in Drosophila (Morgan 1912), sex-specific differences in recombination rates have been found in a diverse range of organisms, for example, in plants (Burt et al. 1991; De Vicente and Tanksley 1991; Graner et al. 1991; Sewell et al. 1999), mammals (Donis-Keller et al. 1987; Mikawa et al. 1999; Neff et al. 1999; Lynn et al. 2005), and aquatic animals such as teleost fishes (Sakamoto et al. 2000; Kondo et al. 2001; Singer et al. 2002; Woram et al. 2004; Kai et al. 2005; Lee et al. 2005; Gharbi et al. 2006). Although limited information is available for molluscan species, higher recombination rates in females have been reported (eastern oyster, Yu and Guo 2003; Pacific oyster, Li and Guo 2004; Hubert and Hedgecock 2004), and in H. discus hannai as well (Liu et al. 2006). In our results, overall, heterogeneities in recombination rates would be attributable largely to individual differences, rather than to sex-specific differences. Nevertheless, we found two linkage groups (4 and 9) where the recombination rates are higher in females than in males. Using the markers segregating from all the parents decreases the number of comparable marker pairs, which reduces the amount of recombination information. Sex-related differences in recombination rates are known to be region dependent within chromosomes (Lynn et al. 2005). Altogether, underlying sex-specific differences in recombination rates may be masked in the comparisons. Interestingly, Liu et al (2006) reported that they identified a sex-determination locus of H. discus hannai in their AFLP-based map, which has a linkage with a microsatellite marker (Awb076). This marker is placed on LG3 in this study, implying that LG3 corresponds to a sex-related chromosome. If this holds true, unlike the case of the medaka (Oryzias latipes) (Kondo et al. 2001), the suppressed recombination of the (putative) sex-related chromosome in males, if any, would not be extensive, given the relatively conserved marker arrangement and map length between sexes (Figure 2).

The phenomenon of sex-related differences in recombination events is affected by various complex factors (Lindahl 1991), and neither the cytogenetic difference between sexes nor the sex-determination mechanism is currently well understood in H. discus hannai; the question of sex specificity in the rate of recombination remains to be resolved in future comprehensive studies.

Heterogeneities in recombination rate within sexes:

We used three mapping families to construct linkage maps. The merit of using multiple families for map construction is not only to increase the map density of consolidated maps but also to allow assessment of heterogeneities in the recombination rate between families (Burr et al. 1988; Ellis et al. 1992; Sakamoto et al. 2000; Hubert and Hedgecock 2004; Woram et al. 2004). Furthermore, the experimental design of this study encompasses two maternal half-sib families, from which we were able to evaluate variations in the recombination rate within an individual (female parent in this case) between different crossing experiments. In most linkage groups, recombination rates estimated for the female parent of the half-sib families did not contradict each other. There were significant heterogeneities in recombination rate at 5.8% of possible pairs of markers (32/552), but most of the significant differences (23/32) were observed in one linkage group (LG16), in which markers were highly clustered in both families with unconformable marker order between families (Figure 1). Exclusion of LG16 leaves 2.2% of significant cases in the heterogeneity testing (9/416), which could occur by chance to a greater extent (α0.01). The clustered arrangements of markers in LG16 are in contrast to more spaced marker alignment in the female map of family M, and clustering behavior of markers was also evident in LG10♂, 12♂, 16♂, and 17♀ (Figure 1). Mapping imprecision caused by sampling variations associated with sample sizes and statistical treatments (Liu 1998; Lespinasse et al. 2000), or environmental differences between crossing experiments (Levine 1955; Simchen and Stamberg 1969; Beavis and Grant 1991), may be responsible for the within-individual map differences among the closely clustered markers (i.e., LG16 and -17 in the female maps of families F and L). However, these kinds of variance would not generate the large map differences observed in the other cases. Concentrated occurrences of scoring errors at markers in particular linkage groups are also very unlikely. Chromosomal rearrangements could play a role in suppressing recombination events, resulting in clustering of markers (Ellis et al. 1992; Kianian and Quiros 1992; Vallejos et al. 1992). Because in this study the suppressed recombination was found to be independent of the sex, chromosomal variations (genome structure variations) could be a probable cause for the clustering behaviors. A clustered arrangement of markers was not found in the AFLP-based maps for H. discus hannai (Liu et al. 2006), owing to the fact that we analyzed multiple families without which any variation in recombination rate within sexes cannot be detected. Chromosomal variations could also account for the differences in recombination rate among individuals (Liu 1998). It is interesting that extensive differences in recombination rate within sexes were unveiled in another mollusk (the Pacific oyster), caused most probably by chromosomal variations among members in wild populations, from which the ancestry of mapping families originated (Hubert and Hedgecock 2004). More mapping data as well as cytogenetic data should be accumulated to test this hypothesis. Selective force, on the other hand, may have acted as a cause of the aberrant alignments of markers, especially in LG16 in family F, where the parental maps were compressed in both sexes. In LG16 in family F, there were just two markers with segregation distortion at the corrected significance level (P < 0.0028). However, applying a less-stringent threshold probability (0.01) increases the number of distorted markers by 16 markers, which are almost all the markers involved in this linkage group.

Future applications and research:

One of the most important purposes of this study was to develop baseline linkage maps for H. discus hannai, for which the genomic structure is poorly understood. Owing to the variations in the rates of recombination, however, in several instances we had to make approximations for marker alignments and map distances. Although such a compromise reduces the precision of resulting maps, it offers a scaffold that can be applied for further genomic studies. From an evolutionary interest, the linkage maps have potential to bring out the patterns of genome evolution among the Pacific abalones (H. discus hannai, H. discus discus, H. madaka, and H. gigantea). The maps, despite the need for increased density of markers, are also potentially useful for identifying genes influencing commercially important traits of Pacific abalones such as growth, resistance to amyotrophia (Hara et al. 2004), and color variation on the shell (Kobayashi et al. 2004, 2005).

Concomitant with future application of the linkage maps, several topics remain to be addressed. Because there was a large difference in recombination rates within sexes, in particular linkage groups, more families should be screened to develop a consensus marker alignment and map length. Molecular markers with limited transferability among populations, such as AFLPs, would be of less use to deal with this issue unless sequencing analyses to establish the homologies of fragments are done. In this context, our finding that H. discus hannai has a potential to generate a high variability in the rate of recombination even within sexes is illuminating, as sequence-specific markers with the portability from population to population, for example, microsatellites and EST-derived SNPs, will enable a more detailed understanding of the map differences among individuals. The position of the centromere should be determined to enhance our understanding of the recombination mode along with the chromosomes. In H. discus hannai, this could be achieved efficiently with the gene–centromere mapping technique using artificial triploids (Fujino et al. 1997; Zhang et al. 1998) or gynogenetic diploids (Li and Kijima 2005).


We are grateful to Toshimasa Kobayashi for performing crossing experiments of abalone. We also thank Akiyuki Ozaki for helpful discussion during the preparation of this manuscript. Two anonymous reviewers provided constructive comments to improve the manuscript.


  • An, H.-S., Y.-J. Jee, K.-S. Min, B.-L. Kim and S.-J. Han, 2005. Phylogenetic analysis of six species of Pacific abalone (Haliotidae) based on DNA sequences of 16s rRNA and cytochrome c oxidase subunit I mitochondrial genes. Mar. Biotechnol. 7: 373–380. [PubMed]
  • Arai, K., H. Tsubaki, Y. Ishitani and K. Fujino, 1982. Chromosomes of Haliotis discus hannai INO and H. discus REEVE. Bull. Jpn. Soc. Sci. Fish. 48: 1689–1691.
  • Barbazuk, W. B., I. Korf, C. Kadavi, J. Heyen, S. Tate et al., 2000. The syntenic relationship of the zebrafish and human genomes. Genome Res. 10: 1351–1358. [PMC free article] [PubMed]
  • Beavis, W. D., and D. Grant, 1991. A linkage map based on information from four F2 populations of maize (Zea mays L.). Theor. Appl. Genet. 82: 636–644. [PubMed]
  • Boutin-Ganache, I., M. Raposo, M. Raymond and C. F. Deschepper, 2001. M13-tailed primers improve the readability and usability of microsatellite analyses performed with two different allele-sizing methods. BioTechniques 31: 24–28. [PubMed]
  • Burr, B., F. A. Burr, K. H. Thompson, M. C. Albertson and C. W. Stuber, 1988. Gene mapping with recombinant inbreds in maize. Genetics 118: 519–526. [PMC free article] [PubMed]
  • Burt, A., G. Bell and P. H. Harvey, 1991. Sex differences in recombination. J. Evol. Biol. 4: 259–277.
  • Chakravarti, A., L. K. Lasher and J. E. Reefer, 1991. A maximum likelihood method for estimating genome length using genetic linkage data. Genetics 128: 175–182. [PMC free article] [PubMed]
  • Cnaani, A., E. M. Hallerman, M. Ron, J. I. Weller, M. Indelman et al., 2003. Detection of a chromosomal region with two quantitative trait loci, affecting cold tolerance and fish size, in an F2 tilapia hybrid. Aquaculture 223: 117–128.
  • Danzmann, R. G., and K. Gharbi, 2001. Gene mapping in fishes: a means to an end. Genetica 111: 3–23. [PubMed]
  • Danzmann, R. G., T. R. Jackson and M. M. Ferguson, 1999. Epistasis in allelic expression at upper temperature tolerance QTL in rainbow trout. Aquaculture 173: 45–58.
  • De Vicente, M. C., and S. D. Tanksley, 1991. Genome-wide reduction in recombination of backcross progeny derived from male versus female gametes in an interspecific cross of tomato. Theor. Appl. Genet. 83: 173–178. [PubMed]
  • Donis-Keller, H., P. Green, C. Helms, S. Cartinhour, B. Weiffenbach et al., 1987. A genetic linkage map of the human genome. Cell 51: 319–337. [PubMed]
  • Ellis, T. H. N., L. Turner, R. P. Hellens, D. Lee, C. L. Harker et al., 1992. Linkage maps in pea. Genetics 130: 649–663. [PMC free article] [PubMed]
  • Fishman, L., A. J. Kelly, E. Morgan and J. H. Willis, 2001. A genetic map in the Mimulus guttatus species complex reveals transmission ratio distortion due to heterospecific interactions. Genetics 159: 1701–1716. [PMC free article] [PubMed]
  • Fujino, K., S. Okumura and H. Inayoshi, 1997. Temperature tolerance differences among normal diploid and triploid Pacific abalone. Bull. Jpn. Soc. Sci. Fish. 53: 15–22.
  • Gharbi, K., A. Gautier, R. G. Danzmann, S. Gharbi, T. Sakamoto et al., 2006. A linkage map for Brown trout (Salmo trutta): chromosome homeologies and comparative genome organization with other salmonid fish. Genetics 172: 2405–2419. [PMC free article] [PubMed]
  • Graner, A., A. Jahoor, J. Schondelmaier, H. Siedler, K. Pillen et al., 1991. Construction of an RFLP map of barley. Theor. Appl. Genet. 83: 250–256. [PubMed]
  • Hara, M., 1992. Breeding of abalone–cross and selection. Fish Genet. Breed. Sci. 18: 1–12 (in Japanese).
  • Hara, M., and Y. Fujio, 1992. Genetic difference among abalone species. Fish Genet. Breed. Sci. 17: 55–61 (in Japanese).
  • Hara, M., and S. Kikuchi, 1992. Increasing the growth rate of abalone, Haliotis discus hannai, using selection techniques. NOAA Tech. Rep. NMFS 106: 21–26.
  • Hara, M., and M. Sekino, 2005. Genetic difference between Ezo-awabi Haliotis discus hannai and Kuro-awabi H. discus discus populations: microsatellite-based population analysis in Japanese abalone. Fish. Sci. 71: 754–766.
  • Hara, M., M. Sekino, A. Kumagai and T. Yoshinaga, 2004. The identification of genetic resistance to amyotrophia in Japanese abalone, Haliotis discus discus. J. Shellfish Res. 23: 1157–1161.
  • Hubert, S., and D. Hedgecock, 2004. Linkage maps of microsatellite DNA markers for the Pacific oyster Crassostrea gigas. Genetics 168: 351–362. [PMC free article] [PubMed]
  • Ino, T., 1952. Biological studies on the propagation of Japanese abalone (genus Haliotis). Bull. Tokai Reg. Fish. Res. Lab. 5: 1–102 (in Japanese).
  • Jackson, T. R., M. M. Ferguson, R. G. Danzmann, A. G. Fishback, P. E. Ihssen et al., 1998. Identification of two QTL influencing upper temperature tolerance in three rainbow trout (Oncorhynchus mykiss) half-sib families. Heredity 80: 143–151.
  • Jacobs, J. M. E., H. J. Van Eck, P. Arens, B. Verkerk-Bakker, B. Te Lintel Hekkert et al., 1995. A genetic map of potato (Solanum tuberosum) integrating molecular markers, including transposons, and classical markers. Theor. Appl. Genet. 91: 289–300. [PubMed]
  • Jaillon, O., J. M. Aury, F. Brunet, J. L. Petit, N. Stange-Thomann et al., 2004. Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype. Nature 431: 946–957. [PubMed]
  • Kai, W., K. Kikuchi, M. Fujita, H. Suetake, A. Fujiwara et al., 2005. A genetic linkage map for the tiger pufferfish, Takifugu rubripes. Genetics 171: 227–238. [PMC free article] [PubMed]
  • Kawahara, I., T. Noro, M. Omori, M. Hasekura and A. Kijima, 1997. Genetic progress for growth in different selected populations of abalone, Haliotis discus hannai, at different hatcheries. Fish Genet. Breed. Sci. 25: 81–90 (in Japanese with English abstract).
  • Kawahara, I., T. Noro, M. Omori and A. Kijima, 1999. Estimation of heritability for juvenile growth rate in the abalone, Haliotis discus hannai INO. Fish Genet. Breed. Sci. 28: 95–103 (in Japanese with English abstract).
  • Kawamura, T., 2002. Abalone species: current status of abalone resources and research trend. Kaiyo Monthly 34: 467–469 (in Japanese).
  • Kianian, S. F., and C. F. Quiros, 1992. Generation of a Brassica oleracea composite RFLP map: linkage arrangements among various populations and evolutionary implications. Theor. Appl. Genet. 84: 544–554. [PubMed]
  • Kijima, A., 2005. Current status and future perspective of breeding studies on abalone. J. Anim. Genet. 32: 101–112 (in Japanese).
  • Kobayashi, T., I. Kawahara, O. Hasakura and A. Kijima, 2004. Genetic control of bluish shell color variation in the Pacific abalone, Haliotis discus hannai. J. Shellfish Res. 23: 1153–1156.
  • Kobayashi, T., M. Hara, S. Kikuchi, S. Sakamoto and A. Kijima, 2005. Genetic control of whitish shell color variation in the Pacific abalone, Haliotis discus hannai. Fish Genet. Breed. Sci. 34: 143–147 (in Japanese with English abstract).
  • Kobayashi, T., M. Hara, M. Kobayashi and M. Sekino, 2006. Evaluation of growth performance of Pacific abalone Halitotis discus hannai selected for juvenile size for 4 generations. Aquacult. Sci. 54: 209–215 (in Japanese with English abstract).
  • Kondo, M., E. Nagao, H. Mitani and A. Shima, 2001. Differences in recombination frequencies during female and male meioses of the sex chromosomes of the medaka, Oryzias latipes. Genet. Res. 78: 23–30. [PubMed]
  • Lee, Y.-H., and V. D. Vacquier, 1995. Evolution and systematics in Haliotidae (Mollusca: Gastropoda): inference from DNA sequences of sperm lysin. Mar. Biol. 124: 267–278.
  • Lee, B.-Y., W.-J. Lee, J. T. Streelman, K. L. Carleton, A. E. Howe et al., 2005. A second-generation genetic linkage map of tilapia (Oreochromis spp.). Genetics 170: 237–244. [PMC free article] [PubMed]
  • Lespinasse, D., M. Rodier-Goud, L. Grivet, A. Leconte, H. Legnate et al., 2000. A saturated genetic linkage map of rubber tree (Hevea spp.) based on RFLP, AFLP, microsatellite, and isozyme markers. Theor. Appl. Genet. 100: 127–138.
  • Levine, R. P., 1955. Chromosome structure and the mechanism of crossing over. Proc. Natl. Acad. Sci. USA 41: 727–730. [PMC free article] [PubMed]
  • Li, L., and X. Guo, 2004. AFLP-based genetic linkage maps of the Pacific oyster Crassostrea gigas Thunberg. Mar. Biotechnol. 6: 26–36. [PubMed]
  • Li, Q., and A. Kijima, 2005. Segregation of microsatellite alleles in gynogenetic diploid Pacific abalone (Haliotis discus hannai). Mar. Biotechnol. 7: 669–676. [PubMed]
  • Lindahl, K. F., 1991. His and hers recombinational hotspots. Trends Genet. 7: 273–276. [PubMed]
  • Liu, B.-H., 1998. Statistical Genomics: Linkage, Mapping, and QTL Analysis. CRC Press, Boca Raton, FL.
  • Liu, T. X., Y. Zhou, J. P. Kanki, M. Deng, J. Rhodes et al., 2002. Evolutionary conservation of zebrafish linkage group 14 with frequently deleted regions of human chromosome 5 in myeloid malignancies. Proc. Natl. Acad. Sci. USA 99: 6136–6141. [PMC free article] [PubMed]
  • Liu, X., X. Liu, X. Guo, Q. Gao, H. Zhao et al., 2006. A preliminary genetic linkage map of the Pacific abalone Haliotis discus hannai INO. Mar. Biotechnol. 8: 386–397. [PubMed]
  • Lindberg, D. R., 1992. Evolution, distribution, and systematics of Haliotidae, pp. 3–18 in Abalone of the World: Biology, Fisheries, and Culture, edited by S. A. Shepherd, M. J. Tegner and S. A. Guzmán Del Próo. Blackwell Science, Oxford.
  • Lynn, A., S. Schrump, J. Cherry, T. Hassold and P. Hunt, 2005. Sex, not genotype, determines recombination levels in mice. Am. J. Hum. Genet. 77: 670–675. [PMC free article] [PubMed]
  • Maliepaard, C., F. H. Alston, G. Van Arkel, L. M. Brown, E. Chevreau et al., 1998. Aligning male and female linkage maps of apple (Malus pumila Mill.) using multi-allelic markers. Theor. Appl. Genet. 97: 60–73.
  • Mikawa, S., T. Akita, N. Hisamatsu, Y. Inage, Y. Ito et al., 1999. A linkage map of 243 DNA markers in an intercross of Gottingen miniature and Meishan pigs. Anim. Genet. 30: 407–417. [PubMed]
  • Moen, T., J. J. Agresti, A. Cnaani, H. Moses, T. R. Famula et al., 2004. A genome scan of a four-way tilapia cross supports the existence of a quantitative trait locus for cold tolerance on linkage group 23. Aquacult. Res. 35: 893–904.
  • Morgan, T. H., 1912. Complete linkage in the second chromosome of the male of Drosophila. Science 36: 719–720.
  • Naruse, K., M. Tanaka, K. Mita, A. Shima, J. Postlethwait et al., 2004. A medaka gene map: the trace of ancestral vertebrate proto-chromosomes revealed by comparative gene mapping. Genome Res. 14: 820–828. [PMC free article] [PubMed]
  • Neff, M. W., K. W. Broman, C. S. Mellersh, K. Ray, G. M. Acland et al., 1999. A second-generation genetic linkage map of the domestic dog, Canis familiaris. Genetics 151: 803–820. [PMC free article] [PubMed]
  • Oetting, W. S., H. K. Lee, D. J. Flanders, G. L. Wiesner, T. A. Sellers et al., 1995. Linkage analysis with multiplexed short tandem repeat polymorphisms using infrared fluorescence and M13 tailed primers. Genomics 30: 450–458. [PubMed]
  • Ozaki, A., T. Sakamoto, S. Khoo, K. Nakamura, M. R. M. Coimbra et al., 2001. Quantitative trait loci (QTLs) associated with resistance/susceptibility to infectious pancreatic necrosis virus (IPNV) in rainbow trout (Oncorhynchus mykiss). Mol. Genet. Genomics 265: 23–31. [PubMed]
  • Park, C., Q. Li, T. Kobayashi and A. Kijima, 2006. Inbreeding depression traits in Pacific abalone Haliotis discus hannai by factorial mating experiments. Fish. Sci. 72: 774–780.
  • Perry, G. M. L., R. G. Danzmann, M. M. Ferguson and J. P. Gibson, 2001. Quantitative trait loci for upper thermal tolerance in outbred strains of rainbow trout (Oncorhynchus mykiss). Heredity 86: 333–341. [PubMed]
  • Postlethwait, J. H., I. G. Woods, P. Ngo-Hazelett, Y.-L. Yan, P. D. Kelly et al., 2000. Zebrafish comparative genomics and the origins of vertebrate chromosomes. Genome Res. 10: 1890–1902. [PubMed]
  • Reid, D. P., A. Szanto, B. Glebe, R. G. Danzmann and M. M. Ferguson, 2005. QTL for body weight and condition factor in Atlantic salmon (Salmo salar): comparative analysis with rainbow trout (Oncorhynchus mykiss) and Arctic charr (Salvelinus alpinus). Heredity 94: 166–172. [PubMed]
  • Robinson, B. D., P. A. Wheeler, K. Sundin, P. Sikka and G. H. Thorgaard, 2001. Composite interval mapping reveals a major locus influencing embryonic development rate in rainbow trout (Oncorhynchus mykiss). J. Hered. 92: 16–22. [PubMed]
  • Sakamoto, T., R. G. Danzmann, N. Okamoto, M. M. Ferguson and P. E. Ihssen, 1999. Linkage analysis of quantitative trait loci associated with spawning time in rainbow trout (Oncorhynchus mykiss). Aquaculture 173: 33–43.
  • Sakamoto, T., R. G. Danzmann, K. Gharbi, P. Howard, A. Ozaki et al., 2000. A microsatellite linkage map of rainbow trout (Oncorhynchus mykiss) characterized by large sex-specific differences in recombination rates. Genetics 155: 1331–1345. [PMC free article] [PubMed]
  • Sambrook, J., E. F. Fritsch and T. Manitaris, 1989. Molecular Cloning: A Laboratory Manual, Ed. 2. Cold Spring Harbor Laboratory Press, Plainview, NY.
  • Sebastian, R. L., E. C. Howell, G. J. King, D. F. Marshall and M. J. Kearsey, 2000. An integrated AFLP and RFLP Brassica oleracea linkage map from two morphologically distinct doubled-haploid mapping populations. Theor. Appl. Genet. 100: 75–81.
  • Sekino, M., and M. Hara, 2001. Microsatellite DNA loci in Pacific abalone Haliotis discus discus (Mollusca, Gastropoda, Haliotidae). Mol. Ecol. Notes 1: 8–10.
  • Sekino, M., and M. Hara, 2007. Individual assignment tests proved genetic boundaries in a species complex of Pacific abalone (genus Haliotis). Conserv. Genet. (in press).
  • Sekino, M., T. Saido, T. Fujita, T. Kobayashi and H. Takami, 2005. Microsatellite DNA markers of Ezo abalone (Haliotis discus hannai): a preliminary assessment of natural populations sampled from heavily stocked areas. Aquaculture 243: 33–47.
  • Sekino, M., T. Kobayashi and M. Hara, 2006. Segregation and linkage analysis of 75 novel microsatellite markers in pair crosses of Japanese abalone (Haliotis discus hannai) using the 5′-tailed primer method. Mar. Biotechnol. 8: 453–466. [PubMed]
  • Sewell, M. M., B. K. Sherman and D. B. Nealea, 1999. A consensus map for loblolly pine (Pinus taeda L.). I. Construction and integration of individual linkage maps from two outbred three-generation pedigrees. Genetics 151: 321–330. [PMC free article] [PubMed]
  • Simchen, G., and J. Stamberg, 1969. Fine and coarse controls of genetic recombination. Nature 222: 329–332. [PubMed]
  • Singer, A., H. Perlman, Y. Yan, C. Walker, G. Corley-Smith et al., 2002. Sex-specific recombination rates in zebrafish (Danio rerio). Genetics 160: 649–657. [PMC free article] [PubMed]
  • Somorjai, I. M. L., R. G. Danzmann and M. M. Ferguson, 2003. Distribution of temperature tolerance quantitative trait loci in Arctic charr (Salvelinus alpinus) and inferred homologies in rainbow trout (Oncorhynchus mykiss). Genetics 165: 1443–1456. [PMC free article] [PubMed]
  • Stam, P., 1993. Construction of integrated genetic linkage maps by means of a new computer package: JOINMAP. Plant J. 3: 739–744.
  • Vallejos, C. E., N. S. Sakiyama and C. D. Chase, 1992. A molecular marker-based linkage map of Phaseolus vulgaris L. Genetics 131: 733–740. [PMC free article] [PubMed]
  • Van Ooijen, J. W., and R. E. Voorrips, 2001. JoinMap 3.0, Software for the Calculation of Genetic Linkage Maps. Plant Research International, Wageningen, The Netherlands.
  • Viruel, M. A., R. Messeguer, M. C. De Vicente, J. Garcia-Mas, P. Puigdoménech et al., 1995. A linkage map with RFLP and isozyme markers for almond. Theor. Appl. Genet. 91: 964–971. [PubMed]
  • Voorrips, R. E., 2002. MapChart: software for the graphical presentation of linkage maps and QTLs. J. Hered. 93: 77–78. [PubMed]
  • Woods, I. G., P. D. Kelly, F. Chu, P. Ngo-Hazelett, Y. L. Yan et al., 2000. A comparative map of the zebrafish genome. Genome Res. 10: 1903–1914. [PMC free article] [PubMed]
  • Woram, R. A., C. Mcgowan, J. A. Stout, K. Gharbi, M. M. Ferguson et al., 2004. A genetic linkage map for Arctic char (Salvelinus alpinus): evidence for higher recombination rates and segregation distortion in hybrid versus pure strain mapping parents. Genome 47: 304–315. [PubMed]
  • Yu, Z., and X. Guo, 2003. Genetic linkage map of the eastern oyster Crassostrea virginica Gmelin. Biol. Bull. 204: 327–338. [PubMed]
  • Yu, Z., and X. Guo, 2006. Identification and mapping of disease-resistance QTLs in the eastern oyster, Crassostrea virginica Gmelin. Aquaculture 254: 160–170.
  • Zhang, G., Z. Wang, Y. Chang, J. Song, J. Ding et al., 1998. Triploid induction in Pacific abalone Haliotis discus hannai INO by 6-dimethylaminopurine and the performance of triploid juveniles. J. Shellfish Res. 17: 783–788.

Articles from Genetics are provided here courtesy of Genetics Society of America
PubReader format: click here to try


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...