Linkage maps of microsatellite DNA markers for the Pacific oyster Crassostrea gigas

We constructed male and female consensus linkage maps for the Pacific oyster Crassostrea gigas, using a total of 102 microsatellite DNA markers typed in 11-day-old larvae from three families. We identified 11 and 12 linkage groups in the male and female consensus maps, respectively. Alignment of these separate maps, however, suggests 10 linkage groups, which agrees with the haploid chromosome number. The male linkage map comprises 88 loci and spans 616.1 cM, while the female map comprises 86 loci and spans 770.5 cM. The male and the female maps share 74 loci; 2 markers remain unlinked. The estimated coverages for the consensus linkage maps are 79% for the male and 70-75% for the female, on the basis of two estimates of genome length. Ninety-five percent of the genome is expected to lie within 16 and 21 cM of markers on the male and female maps, respectively, while 95% of simulated minimum distances to the male and female maps are within 10.1 and 13.6 cM, respectively. Females have significantly more recombination than males, across 118 pairs of linked markers in common to the parents of the three families. Significant differences in recombination and orders of markers are also evident among same-sex parents of different families as well as sibling parents of opposite sex. These observations suggest that polymorphism for chromosomal rearrangements may exist in natural populations, which could have profound implications for interpreting the evolutionary genetics of the oyster. These are the first linkage maps for a bivalve mollusc that use microsatellite DNA markers, which should enable them to be transferred to other families and to be useful for further genetic analyses such as QTL mapping.     

Development of 15 polymorphic genic microsatellite DNA markers of Pacific abalone Haliotis discus hannai

Fifteen polymorphic genic microsatellite DNA markers were developed based on the expressed sequence tags of Pacific abalone Haliotis discus hannai we ourselves generated, which were used to type 32 individuals representing a cultured population. The number of alleles each locus ranged from two to 12. The observed and expected heterozygosities ranged from 0.094 to 0.969 and from 0.091 to 0.878, respectively. Among 15 loci, four were found to deviate significantly from Hardy–Weinberg equilibrium (P_HW < 0.001). No linkage disequilibrium was found between these loci. It is certain that these markers will facilitate the management and exploitation of the genetic resource of Pacific abalone.     

Isolation and characterization of 125 microsatellite DNA markers in the blacklip abalone,Haliotis rubra

Australian abalone species are subject to wild harvest and aquaculture production. This study characterized 125 polymorphic microsatellite markers in the blacklip abalone, Haliotis rubra, and evaluated cross‐species amplification in Haliotis laevigata and Haliotis coccoradiata. Segregation analysis of a mapping family revealed non‐amplifying polymerase chain reaction null alleles at 34 loci. Cross‐species amplification was achieved for 89 loci.     

Development and characterization of 60 microsatellite markers in the abalone Haliotis diversicolor

The abalone, Haliotis diversicolor, is one of the most important mariculture species in southern China. We developed 60 new polymorphic microsatellite markers for H. diversicolor and characterized them in 30 individuals from a cultured population in Sanya, China. All 60 markers were found to be polymorphic. The number of alleles ranged from two to nine per locus, with an average of 4.12/locus. The expected and observed heterozygosities ranged from 0.10 to 0.88 and from 0.07 to 0.87, respectively. Forty-four loci were in Hardy-Weinberg equilibrium. These 44 microsatellite markers should be useful for genome mapping and population genetic studies.     

Development of gene-targeted SNP markers for genomic mapping in Pacific abalone Haliotis discus hannai Ino

Useful and novel DNA markers are needed for aquaculture genetics and breeding. In this study, we report the discovery and development of gene-targeted single nucleotide polymorphisms (SNPs) for genomic mapping in the Pacific abalone Haliotis discus hannai Ino. Single EST or EST-contigs from 66 genes that had positive BLASTx matches (E-value ≤ 1e-8) were used for polymerase chain reaction (PCR) amplification. PCR products from the two parents of one mapping family were directly sequenced, and 83 SNP loci were found from 17 genes. Allele-specific PCR (AS-PCR) was developed and optimized for genotyping of 11 SNP loci in 120 progeny of the mapping family. Nine of the loci conformed to the expected Mendelian ratio of 1:1 based on the χ² test (P > 0.05) and could potentially be used for linkage map construction. Our data also indicate that the sequencing of two parents may be a practical strategy for the discovery of informative SNPs for linkage mapping in a particular mapping population.     

New polymorphic microsatellite markers in Pacific abalone Haliotis discus hannai and their application to genetic characterization of wild and aquaculture populations

Seven new microsatellite markers were developed for the Pacific abalone (Haliotis discus hannai, Haliotidae), and allelic variability was compared between a wild population and a hatchery population in Yeosu, Korea. All loci amplified readily and demonstrated allelic variability, with the number of alleles ranging from 6 to 15 in the wild population and from 3 to 12 in farmed populations. Average observed and expected heterozygosities were estimated at 0.65 and 0.77 in the hatchery samples, and 0.79 and 0.87 in the wild samples. These results indicated lower genetic variability in the hatchery population, as compared with the wild population and significant genetic differentiation between the wild population and the hatchery samples (F _ST=0.055, p<0.001). These microsatellite loci may be valuable for future population genetic studies and for tracking hatchery samples used in stock enhancement programs.     

Development of 11 polymorphic microsatellite loci in the small abalone (Haliotis diversicolor Reeve)

Eleven polymorphic microsatellite loci for Haliotis diversicolor were isolated and characterized from (CT)(n) - and (AC)(n) -enriched library. They were tested in 24 individuals from a natural population. All of them were polymorphic, with the number of alleles varying between three and 10. The observed heterozygosities and expected heterozygosities ranged from 0.083 to 0.913 and from 0.159 to 0.856, respectively. The 11 isolated microsatellite loci, except SA-JMU6 and SA-JMU12, followed Hardy-Weinberg expectations. Seven sets of primers also amplify in closely related species, H. ovina and H. asinine. These 11 polymorphic microsatellites will be useful for analysing the population structure and genetic diversity in H. diversicolor.     

Genetic heterogeneity of the tropical abalone (Haliotis asinina) revealed by RAPD and microsatellite analyses

Genetic heterogeneity of the tropical abalone, Haliotis asinina was examined using randomly amplified polymorphic DNA (RAPD) and microsatellite analyses. One hundred and thirteen polymorphic RAPD fragments were generated. The percentage of polymorphic bands of H. asinina across overall primers was 85.20%. The average genetic distance of natural samples within the Gulf of Thailand (HACAME and HASAME) was 0.0219. Larger distance was observed when those samples were compared with HATRAW from the Andaman Sea (0.2309 and 0.2314). Geographic heterogeneity and F(ST) analyses revealed population differentiation between H. asinina from the Gulf of Thailand and the Andaman Sea (p < 0.0001). Three microsatellite loci (CUHas1, CUHas4 and CUHas5) indicated relatively high genetic diversity in H. asinina (total number of alleles = 26, 5, 23 and observed heterozygosity = 0.84, 0.42 and 0.33, respectively). Significant population differentiation was also found between samples from different coastal regions (p < 0.0001). Therefore, the gene pool of natural H. asinina in coastal Thai waters can be genetically divided to 2 different populations; the Gulf of Thailand (A) and the Andaman Sea (B).     

Linkage mapping of melanoma (MLM) using 172 microsatellite markers

The incidence of malignant melanoma is currently increasing faster than any other cancer and in 5-12% of cases occurs in a familial context in which the disease cosegregates as an autosomal dominant trait. To identify the location of genes that predipose individuals to familial melanoma (MLM), we have carried out linkage analysis in three large Australian melanoma pedigrees using 172 microsatellite markers spread across all autosomes. Three additional smaller families were typed for 70 of the same markers. In five of the six families we found lod scores between 1.0 and 2.3, which may provide evidence for the location of melanoma genes in proximity to some of these markers. If this turns out to be the case, these data potentially demonstrate that MLM is genetically heterogeneous since there was no marker for which all families gave significantly high LODs. These data provide the foundation for an exclusion map for melanoma and, more importantly, high-light areas of the genome for others to substantiate the potential positions of some of the genes that may be responsible for susceptibility to MLM.     

Polymorphic microsatellite DNA markers for Banksia hookeriana (Proteaceae)

We developed 11 polymorphic microsatellite DNA markers for an Australian native shrub Banksia hookeriana (Proteaceae). The number of alleles per locus in 37 individuals varied from three to 17, observed and expected heterozygosities ranged from 0.297 to 0.838 and from 0.279 to 0.900, respectively. Two loci (BH‐B5 and BH‐B107) showed significant deviation from Hardy–Weinberg equilibrium (P < 0.05), and null alleles may be present at these two loci. All loci showed independent inheritance.     

Polymorphic microsatellite DNA markers for Banksia attenuata (Proteaceae)

We developed 11 polymorphic microsatellite DNA markers for an Australian native shrub Banksia attenuata. The number of alleles per locus in 50 individuals varied from five to 18, observed and expected heterozygosities ranged from 0.300 to 0.740 and from 0.537 to 0.918, respectively. Six loci showed no significant deviation from Hardy–Weinberg equilibrium (P > 0.05), and null alleles appear to exist at locus BA‐B1. All loci showed independent inheritance.     

Species identification of the tropical abalone (Haliotis asinina, Haliotis ovina, and Haliotis varia) in Thailand using RAPD and SCAR markers

A randomly amplified polymorphic DNA (RAPD) analysis was used to identify the species- and population-specific markers of abalone; Haliotis asinina, H. ovina, and H. varia in Thai waters. Fifteen species-specific and six population-specific RAPD markers were identified. In addition, an 1650 bp band (UBC195) that was restricted to H. ovina from the Gulf of Thailand (east) was also found. All of the specific RAPD markers were cloned and sequenced. Twenty pairs of primers were designed and specificity-tested (N = 12 and 4 for target and non-target species, respectively). Seven primer pairs (CUHA1, 2, 4, 11, 12, 13, and 14) were specifically amplified by H. asinina DNA, whereas a single pair of primers showed specificity with H. ovina (CUHO3) and H. varia (CUHV1), respectively. Four primer pairs, including CUHA2, CUHA12, CUHO3, and CUHV1, were further examined against 216 individuals of abalone (N = 111, 73, and 32, respectively). Results indicated the species-specific nature of all of them, except CUHO3, with the sensitivity of detection of 100 pg and 20 pg of the target DNA template for CUHA2 and CUHA12 and CUHV1, respectively. The species-origin of the frozen, ethanol-preserved, dried, and boiled H. asinina specimens could also be successfully identified by CUHA2.     

Polymorphic microsatellite DNA markers for Daviesia triflora (Papilionaceae)

We developed 11 polymorphic microsatellite DNA markers for an Australian native plant, Daviesia triflora. The number of alleles per locus in 40 individuals varied from four to 19, and observed and expected heterozygosities ranged from 0.450 to 0.925 and from 0.497 to 0.899, respectively. Nine loci showed no significant deviation from Hardy–Weinberg equilibrium (P > 0.05), and null alleles appear to exist at loci DT‐A102 and DT‐B103. All loci showed independent inheritance.     

Parentage determination of mandarin fish (Siniperca chuatsi) based on microsatellite DNA markers

In this study, ten microsatellite loci were chosen to estimate the parentage of 260 progeny in five mandarin fish (Siniperca chuatsi) full-sib families. Simulation based on allele frequency date demonstrated the combined exclusion power would be over 97% if the number of loci was up to nine. Based on the information from these nine loci, 98% of progeny were unambiguously allocated to their putative parental pairs in the parentage analysis. The assignment success rate by the real data set was lower than that predicted by the simulations, with 94% of progeny assigned correctly. The discrepancies might be caused by a scoring error or allelic dropout caused by poor quality genomic DNA. Moreover, 69 progeny were selected randomly for the double-blind test, the result indicated that 95% of the progeny can be correctly assigned to their families. This study provided a microsatellite-based approach for parentage assignment in S. chuatsi, and that will be useful for investigation of genetic background and molecular marker-assisted selective breeding in this species.     

Characterization of 12 single nucleotide polymorphisms (SNPs) in Pacific abalone, Haliotis discus hannai

We report here for the first time 12 polymorphic single nucleotide polymorphisms (SNPs) in a commercially important gastropod, Pacific abalone (Haliotis discus hannai) that were identified by searching expressed sequence tag database. These SNP loci (seven nuclear and five mitochondrial SNPs) were polymorphic among 37 wild abalone individuals, based on a four-primer allele-specific polymerase chain reaction analysis. All loci had two alleles and the minor allele frequency ranged from 0.027 to 0.473. For the seven nuclear SNPs, the expected and observed heterozygosities ranged from 0.053 to 0.499 and from 0.054 to 0.811, respectively.     

Centromere mapping in the Pacific abalone (Haliotis discus hannai) through half-tetrad analysis in gynogenetic diploid families

Centromere mapping is an essential prerequisite for our understanding of the composition and structure of genomes. For centromere mapping, in two meiogynogenetic families of the Pacific abalone (Haliotis discus hannai), we screened 97 microsatellite markers that cover all linkage groups from a currently available abalone linkage map. Microsatellite analysis showed that no unique paternal allele was found in all gynogenetic progeny, which confirmed 100% success of induction of gynogenesis. In the control crosses, all 97 microsatellite loci were compatible with Mendelian inheritance, while in meiogynogenetic progeny, 5.2% of the microsatellite loci showed segregation distortions from an expected 1:1 ratio of two homozygote classes. The second division segregation frequency of the microsatellites ranged from 0.037 to 0.950 with a mean of 0.399, indicating the existence of interference. Heterogeneity among linkage groups in the crossover distribution was observed. Centromere location was mostly in accordance with the abalone karyotype, but differences in marker order between linkage and centromere maps occurred. Information on the positions of centromeres in relation to the microsatellite loci will represent a contribution towards assembly of genetic maps in the commercially important abalone species.     

Semen cryopreservation of small abalone (Haliotis diversicolor supertexa)

Methods for cryopreserving spermatozoa and maximizing fertilization rate in Taiwan small abalone, Haliotis diversicolor supertexa, were developed. The gametes (spermatozoa and eggs) of small abalone were viable 3 h post-spawning, with fertilization, and development rate decreasing with time. A minimum of 10(2) cell/ml sperm concentration and a contact time of 2 min between gametes is recommended for artificial insemination of small abalone eggs. Eight cryoprotectants, dimethyl sulfoxide (DMSO), dimethyl acetamide (DMA), ethylene glycol (EG), propylene glycol (PG), butylene glycol (BG), polyethylene glycol, glycerol and methanol, were tested at concentrations between 5 and 25% to evaluate their effect on motility of spermatozoa exposed to cryoprotectant for up to 60 min at 25 degrees C before freezing. The least toxic cryoprotectant, 10% DMSO, was added to artificial seawater (ASW) to formulate the extender for freezing. Semen was diluted 1:1 with the extender, inserted into 1.5 ml microtubes and frozen using a cooling rate between -3.5 and -20 degrees C/min to various transition temperatures (0, -30, -60, -90 and -120 degrees C), followed by transfer and storage in liquid nitrogen (-196 degrees C). The microtubes were thawed from +45 to +145 degrees C/min. Spermatozoa, cooled to -90 degrees C at a cooling rate of -12 or -15 degrees C/min and then immersed in liquid nitrogen, had the best post-thaw motility. Post-thaw sperm motility was markedly reduced compared to fresh sperm. More frozen-thawed spermatozoa are required to achieve fertilization rates comparable to those achieved using fresh spermatozoa.     

Anonymous and EST-based microsatellite DNA markers that transfer broadly across the fig tree genus (Ficus, Moraceae)

? Premise of the study: We developed a set of microsatellite markers for broad utility across the species-rich pantropical tree genus Ficus (fig trees). The markers were developed to study population structure, hybridization, and gene flow in neotropical species. ? Methods and Results: We developed seven novel primer sets from expressed sequence tag (EST) libraries of F. citrifolia and F. popenoei (subgen. Urostigma sect. Americana) and optimized five previously developed anonymous loci for cross-species amplification. The markers were successfully tested on four species from the basal subgenus Pharmacosycea sect. Pharmacosycea (F. insipida, F. maxima, F. tonduzii, and F. yoponensis) and seven species of the derived subgenus Urostigma (F. citrifolia, F. colubrinae, F. costaricana, F. nymphaeifolia, F. obtusifolia, F. pertusa, and F. popenoei). The 12 markers amplified consistently and displayed polymorphism in all the species. ? Conclusions: This set of microsatellite markers is transferable across the phylogenetic breadth of Ficus, and should therefore be useful for studies of population structure and gene flow in approximately 750 fig species worldwide.