Motoyuki Hara

Loss of microsatellite and mitochondrial DNA variation in hatchery strains of Japanese flounder Paralichthys olivaceus

Abstract Genetic divergence within and between hatchery strains and wild populations of Japanese flounder Paralichthys olivaceus was assessed by means of microsatellite and mitochondrial DNA (mtDNA) sequencing analysis. All of the 11 microsatellite loci screened in this study showed marked polymorphisms. Sequences of the mtDNA control region of Japanese flounder were also highly variable: of approximately 443 base pairs (bp) sequenced, 132 variable sites comprised of 149 base-substitutions were found among the 490 individuals. Marked reductions of genetic variability in the hatchery strains compared with the wild populations were observed in terms of number of both microsatellite alleles and mtDNA haplotypes, and mtDNA haplotype diversity. Both molecular markers yielded high values of FST (ΦST) between the hatchery strains, and between the hatchery strains and wild populations. We conclude that, based on the reduced genetic variability observed in all the hatchery strains examined, bottleneck effects occurred when each strain was founded.

Microsatellite-based pedigree tracing in a Japanese flounder Paralichthys olivaceus hatchery strain: implications for hatchery management related to stock enhancement program

Effective population size in captive populations is affected by several factors such as the number of contributing broodstock to the next generation, sex ratio of parents, and variations in family size, which can be accurately evaluated by examination of the pedigree structure in the populations of interest. Here we present an example of microsatellite-based pedigree tracing in a hatchery strain of Japanese flounder Paralichthys olivaceus to be stocked into natural sea areas. We also detail the potential effect of selective operations (size selection) on the pedigree structure. The hatchery strain we screened was founded by using 18 wild captives (6 males and 12 females) through the use of the mesocosm spawning method, and the pedigree of the offspring including 113 individual larvae collected within 24 h after hatching, 216 individuals of 1 month old, and 407 individuals of 4 month old was unambiguously identified. The contribution of candidate broodstock to the next generation was highly skewed as the contribution to almost all of the offspring was monopolized by a single male, and a half of the females did not produce any offspring. The contribution of one family to the released fish selected for larger size (total length) was significantly high, while those of other two families were low (P<0.008).

Efficient detection of parentage in a cultured Japanese flounder Paralichthys olivaceus using microsatellite DNA marker

Abstract Parentage of offspring in a stock of a cultured Japanese flounder Paralichthys olivaceus was determined using four hyper-variable microsatellite DNA loci with many unique alleles. It was found that only 57% of the 14 broodstock parents actually contributed to the production of offspring in this experiment. The number of alleles per locus in the offspring was reduced 29% compared to the broodstock. The average heterozygosity of offspring (He=0.883) was significantly lower ( P

Application of Microsatellite Markers to Population Genetics Studies of Japanese Flounder Paralichthys olivaceus

Abstract: We examined population genetic structure by means of microsatellite analysis among 7 Japanese flounder (Paralichthys olivaceus) populations collected from coastal sea areas around Japan. As was expected, all of the 11 microsatellite loci examined were variable in all populations (number of alleles per locus, 15.2–18.2; average of expected heterozygosity, 0.74–0.78). Eleven population pairs in 21 possible pairwise comparisons showed significant genetic heterogeneity associated with allele frequency distributions or fixation index (FST). Modified Cavalli-Sforza chord distance (DA) and Neis standard genetic distance (DST) ranged from 0.051 to 0.090, and from 0.000 to 0.025, respectively. There was evidence that the populations assessed in this study were not drawn from a single panmictic population; however, it appears that Japanese flounder populations around Japan are not well-structured, as an estimate of the fixation index value among the 7 localities was very low (FST= 0.0025).

Genetic differences between hatchery stocks and natural populations in Pacific abalone (Haliotis discus) estimated using microsatellite dNA markers.

Genetic variations within and between nine hatchery stocks and seven natural populations of abalone including Ezo-abalone (Haliotis discus hannai) and Kuro-abalone (H. d. discus) were assayed with nine microsatellite markers. Marked reductions of genetic variability in the hatchery stocks were recognized in the allelic diversity and mean heterozygosity compared with the natural populations. Thirteen of 16 significant HWE deviations in hatchery stocks revealed heterozygotes excess, while all natural populations did not show such a tendency. Highly significant FST values were observed for all cases between the hatchery stocks, and between the hatchery stocks and natural populations. Genetic distance (DA) between each hatchery stock and the geographically proximal population (mean ± SD, 0.108 ± 0.035) were similar to those estimated for between the natural Ezo-abalone and Kuro-abalone (0.101 ± 0.021). The self-assignment test, which allocated individuals to their own stock with a high success rate, provided evidence of solid genetic differences among the nine hatchery stocks. These results suggests that the allelic composition and diversity in the natural populations was not necessarily reflected in the hatchery stocks owing to population bottleneck and genetic drift through seedling process, and thus the seedling and stocking practice of these hatchery stocks should take much notice of the results to conserve the genetic diversity of natural populations.

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.

Segregation and Linkage Analysis of 75 Novel Microsatellite DNA Markers in Pair Crosses of Japanese Abalone (Haliotis discus hannai) Using the 5′-Tailed Primer Method

We present novel microsatellite markers of the Japanese abalone (Haliotis discus hannai) for general mapping studies in this species. A total of 75 microsatellite markers were developed, and the allele-transmission patterns of these markers were studied in three families generated by pair crosses. For allele scoring, we employed the 5′-tailed primer polymerase chain reaction (PCR) technique, which substantially reduces the cost for fluorescent labeling of primers. Of the 225 possible marker-family combinations (75 markers × 3 families), 18 cases of informative null-allele segregation were inferred. When such null-allele segregations were allowed, more than 70% of the 75 markers in the families turned out to be markers with an expected segregation ratio of 1:1:1:1, allowing maximal exploitation of the codominant nature of microsatellite markers. There were 16 instances of segregation distortion at the 5% significance level. The test for independence of segregation assigned the 75 markers into 17 linkage groups, which is in close agreement with the haploid chromosome number of H. discus hannai (n = 18). Six markers could not be placed into any linkage group. We suggest that these markers could help construct a H. discus hannai linkage map.

Genetic difference between Ezo-awabi Haliotis discus hannai and Kuro-awabi H. discus discus populations: Microsatellite-based population analysis in Japanese abalone

This study documents the genetic population relationships between Ezo-awabi Haliotis discus hannai and Kuro-awabi H. discus discus by means of the microsatellite technique with an emphasis on the extent of genetic difference between Ezo-and Kuro-awabi populations. Eight markers were employed to screen five populations each collected from Ezo-and Kuro-awabi habitats. All eight loci showed polymorphisms in all populations (number of alleles per locus, 11.1–14.1; averaged expected heterozygosity, 0.64–0.70). The number of loci that yielded significant genetic heterogeneities (allele frequency distribution and/or pairwise θ) between Ezo- and Kuro-awabi populations was larger than that generated in comparisons between populations within Ezo- and Kuro-awabi habitats. According to the bootstrap neighbor-joining trees constructed on the basis of two genetic distance measures (DA and DST), the 10 populations fell into two clusters of populations (Ezo-awabi and Kuro-awabi habitat groups), and the results of hierarchical AMOVA testing also supported the neighborjoining clustering. The outcomes presented here suggest that the microsatellite markers used in this study are potentially an efficient means to differentiate Ezo- and Kuro-awabi populations.

Parentage testing for hatchery-produced abalone Haliotis discus hannai based on microsatellite markers: preliminary evaluation of early growth of selected strains in mixed family farming

This study dealt with the detection of parentage in a mixed family tank of abalone Haliotis discus hannai using microsatellite DNA markers. As a preliminary investigation, the early growth rate between selected and non-selected abalone families, which were reared together in the mixed family tank from settlement stage to juvenile stage (7 months of age), was also compared. The selected families were derived from seven parents (three females and four males) selected for larger size in shell length at about 1-year-old, and the non-selected families originated from five wild captives (three females and two males). Parentage analysis based on eight microsatellite markers unambiguously allocated the 170 juveniles sampled from the single tank to the 17 parental pairs. The family size was highly heterogeneous among families, as two males in the selected families and one male in the non-selected families dominated the contribution to the offspring pool (>80%). The mean shell length of the selected families was approximately 23% larger than that of the non-selected families (Student’s t-test, P<0.001). This study demonstrated that the use of microsatellite markers is effective for parentage determination in the mixed family farming, commonly used in abalone hatcheries, and selective operations for larger size could improve the growth of the next generation.

Analysis of Microsatellite DNA Polymorphisms in Rockfish Sebastes thompsoni and Application to Population Genetics Studies

Abstract: Population differentiation and relationships among 6 natural rockfish populations collected from northern coastal seas around Japan were assayed using microsatellite DNA loci. Seven loci examined were polymorphic in all populations. The number of alleles per locus ranged from 6.7 to 9.3, and the average of observed and expected heterozygosity ranged from 0.63 to 0.68, and from 0.66 to 0.69, respectively. The observed genotype frequencies at each locus were almost in agreement with Hardy-Weinberg expectations with two exceptions (P < .05). The allele frequencies of 16 population pairs were significantly different (P < .05). Genetic distance (DA) between 6 populations ranged from 0.03 to 0.08. According to a neighbor-joining tree generated from the DA values, the 6 populations fell into 3 clusters. These clusters were correlated with the geographical positions of each population; larval dispersions due to water current were also found to have an effect on these results.