Isolation and characterization of 31 polymorphic microsatellite markers in barfin flounder (Verasper moseri) and the cross-species amplification in spotted halibut (Verasper variegatus)

The construction of a genetic linkage map and evaluation of population genetic diversity both require large numbers of polymorphic molecular markers. In the present study, we report 31 polymorphic microsatellite markers, of which 28 were isolated from two repeat-enriched libraries constructed from genomic DNA, and three were detected in two genes (MCH-R1 and MCH-R2) of barfin flounder (Verasper moseri). A total of 94 alleles were detected with an average of 3.0 alleles per locus. The number of alleles, observed and expected heterozygosity per locus ranged from two to six, from 0.30 to 1.00 and from 0.33 to 0.78, respectively. Three loci significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction (P < 0.0016) and no significant linkage disequilibrum between pairs of loci was found. Cross-species amplification of these markers was evaluated using the closely related species spotted halibut (Verasper variegatus). This study will potentially be useful for stock management, constructing of a genetic linkage map, mapping economically important quantitative trait loci (QTL), and studying the population genetic diversity of barfin flounder (Verasper moseri).     

Isolation and characterization of microsatellite DNA markers in the rare species barfin flounder (Verasper moseri) and its closely related species spotted halibut (V. variegatus)

Barfin flounder and spotted halibut have been selected in Japan as target species for stock enhancement due to the fact that the number of individuals of these species in the wild has been decreasing in the last three decades. Barfin flounder is now considered to be a rare species as its situation is critical. The first microsatellite DNA markers for barfin flounder and spotted halibut are described in this study. Cross‐amplification of barfin flounder markers was examined in spotted halibut species and vice versa.     

Histological studies on early oogenesis in barfin flounder (Verasper moseri)

There is much information on oogenesis from the resumption of the first meiotic division to oocyte maturation in many vertebrates; however, there have been very few studies on early oogenesis from oogonial proliferation to the initiation of meiosis. In the present study, we investigated the histological changes during early oogenesis in barfin flounder (Verasper moseri). In fish with a total length (TL) of 50mm (TL 50mm fish), active oogonial proliferation was observed. In TL 60mm fish, oocytes with synaptonemal complexes were observed. Before the initiation of active oogonial proliferation, somatic cells which surrounded a few oogonial germ cells, started to proliferate to form the oogonial cysts that accompanied oogonial proliferation. In TL 70mm fish, however, the cyst structure of the oocyte was gradually broken by the invagination of somatic cells, and finally the oocyte became a single cell surrounded by follicle cells. Upon comparison of nuclear size, DNA-synthesizing germ cells could be divided into two types: small nuclear cells and large nuclear cells. Based on histological observation, we propose that the small nuclear cells were in the mitotic prophase of oogonia and the large nuclear cells were in the meiotic prophase of oocytes, and that the nuclear size increases upon the initiation of meiosis.     

Isolation and characterization of polymorphic microsatellite loci from black sea bass (Centropristis striata) and cross-species amplification

Black sea bass (Centropristis striata) is an economically important serranid species. A number of 39 microsatellite loci were isolated from two enriched genomic library of C. striata. Eleven of these loci were polymorphic in a test population with alleles per locus ranging from 3 to 8, and observed and expected heterozygosities per locus from 0.26 to 1.00 and from 0.61 to 0.84, respectively. Four loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction and no significant linkage disequilibrium was found between pairs of loci. Cross-species amplification of these polymorphic microsatellite loci was performed in additional two related species. These polymorphic microsatellite loci would be useful for investigating genetic resource of C. striata and other related species.     

Isolation and characterization of novel polymorphic nuclear microsatellite markers from Ophrys fusca (Orchidaceae) and cross-species amplification

The present study reports the isolation and characterization of eight new polymorphic microsatellite loci from the sexually deceptive orchid Ophrys fusca. Microsatellites were isolated from two partially enriched genomic libraries using FIASCO (Fast Isolation by AFLP of Sequences COntaining repeats). Seventy-three loci were screened for primer design and primer pairs corresponding to eight different loci were selected for microsatellite characterization of two Portuguese populations. Total number of alleles per locus ranged from 10 to 32. All loci showed a high level of observed heterozygosity (H₀) ranging from 0.33 to 1 and were possible to amplify in 16 other species of Ophrys using the same primers. H. C. Cotrim and F. A. Monteiro have contributed equally to this work.     

Development, inheritance and cross-species amplification of microsatellite markers from Acacia mangium

Microsatellite markers were developed in Acacia mangium Willd. to provide highly variable co-dominant markers for linkage mapping and studies of the breeding system. After an enrichment procedure 40% of colonies contained microsatellites in contrast with less than 1% from a non-enriched library. The majority of microsatellite sequences were AC repeats. Co-dominant segregation of alleles in two full-sib crosses of A. mangium was demonstrated at 33 microsatellite loci. The markers were highly variable relative to restriction fragment lengths polymorphisms (RFLPs). In the two pedigrees 53% of microsatellite loci were fully informative compared with 15% of RFLPs. Based on alleles detected among four parental genotypes, the microsatellites consisting of dinucleotide repeats were more polymorphic than those with tri- and tetra-nucleotide repeats. The microsatellite markers were not as transferable across species in the genus Acacia as RFLPs. Two thirds of the primers developed in A. mangium (subgenus Phyllodineae, section Juliflorae) amplified DNA from other species within the same section but failed to amplify in species from the subgenus Acacia. The availability of multiallelic, PCR-based, co-dominant microsatellite loci makes possible efficient studies of gene flow and breeding systems in A. mangium, a species with low allozyme variation. Received: 30 December 1999 / Accepted: 10 May 2000     

Isolation and characterization of polymorphic microsatellite loci from a dinucleotide-enriched genomic library of starry flounder (Platichthys stellatus) and cross-species amplification

Starry flounder (Platichthys stellatus) is a rare fish species in China. Here, we reported 12 polymorphic microsatellite loci isolated from a dinucleotide-enriched genomic library of starry flounder (P. stellatus). The number of alleles, observed and expected heterozygosity per locus in 30 individuals ranged from two to six, from 0.2500 to 1.0000 and from 0.4512 to 0.7667, respectively. One locus significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction and no significant linkage disequilibrium between pairs of loci was found. Cross-species amplification of these microsatellite loci in additional three fish species was performed. These polymorphic microsatellite loci would be useful for investigating genetic population structure and construction of genetic linkage map in P. stellatus. Guidong Miao and Changwei Shao have contributed equally.     

Twelve new polymorphic microsatellite markers from the loggerhead sea turtle (Caretta caretta) and cross-species amplification on other marine turtle species

We describe 12 new polymorphic dinucleotide microsatellite loci and multiplex Polymerase Chain Reaction conditions from the loggerhead sea turtle Caretta caretta. Levels of polymorphism were assessed in 50 individuals from the nesting population of the Cape Verde Islands. Number of alleles ranged from 3 to 13 (average of 7.33) and the values of observed heterozygosities from 0.32 to 0.80 (average of 0.61). Cross-species amplification on three other marine turtles, Chelonia mydas, Eretmochelys imbricata and Dermochelys coriacea, revealed polymorphism and variability at eight, eleven and three loci, respectively.     

Sequence and organization of the complete mitochondrial genomes of spotted halibut (Verasper variegatus) and barfin flounder (Verasper moseri).

In this work, the mitochondrial genomes for spotted halibut (Verasper variegatus) and barfin flounder (Verasper moseri) were completely sequenced. The entire mitochondrial genome sequences of the spotted halibut and barfin flounder were 17,273 and 17,588 bp in length, respectively. The organization of the two mitochondrial genomes was similar to those reported from other fish mitochondrial genomes containing 37 genes (2 rRNAs, 22 tRNAs and 13 protein-coding genes) and two non-coding regions (control region (CR) and WANCY region). In the CR, the termination associated sequence (ETAS), six central conserved block (CSB-A,B,C,D,E,F), three conserved sequence blocks (CSB1-3) and a region of 61-bp tandem repeat cluster at the end of CSB-3 were identified by similarity comparison with fishes and other vertebrates. The tandem repeat sequences show polymorphism among the different individuals of the two species. The complete mitochondrial genomes of spotted halibut and barfin flounder should be useful for evolutionary studies of flatfishes and other vertebrate species.     

PERMANENT GENETIC RESOURCES: Development of microsatellite markers in Rhinanthus angustifolius and cross-species amplification

Fifteen polymorphic microsatellite loci were developed from an enriched genomic library of the annual plant Rhinanthus angustifolius and characterized using 36 individuals. These markers provided high polymorphism ranging from two to 15 alleles per locus. Four loci showed significant departure from Hardy-Weinberg equilibrium, probably because of the occurrence of null alleles. No significant linkage disequilibrium was detected between pairs of loci. Tests of cross-species transferability were performed on four congeners with a success rate of 100% in Rhinanthus minor, 93% in R. mediterraneus and R. glacialis, and 80% in R. alectorolophus. These microsatellite loci will be useful tools to study mating system, gene flow and hybridization in the genus Rhinanthus.     

中华鳖多态性微卫星标记跨物种扩增研究

微卫星是一种优秀的分子标记,并广泛应用于生物和医学的各个领域。但微卫星标记的开发成本较高,因而,利用近缘种已开发的微卫星标记已成趋势。利用本实验室已开发的21个中华鳖微卫星对鼋、斑鳖、山瑞鳖、刺鳖、佛罗里达鳖、两爪鳖、黄喉拟水龟和乌龟等8个物种进行跨物种检验。结果表明,4个微卫星位点(CST21、CST42、CST50和CST52)的引物能在所有8只个体中稳定扩增;而CST16在所有个体中都不能扩增出目的片段。跨物种扩增山瑞鳖结果最好,21个位点中有20个位点能稳定扩增;而乌龟扩增效果最差,只有6个位点能被扩增出目的产物。总之,中华鳖的21个微卫星位点多数可以在鳖科内各属间进行跨物种扩增,部分可以在跨科物种间扩增。     

Development of polymorphic microsatellite markers in barfin flounder (<Emphasis Type="Italic">Verasper moseri</Emphasis>) and spotted halibut (<Emphasis Type="Italic">Verasper variegatus</Emphasis>) by the cross-species amplification

Barfin flounder (Verasper moseri) and spotted halibut (Verasper variegatus) are two commercially important flatfish species in the Northeast Asia. In the present study, we reported polymorphic microsatellite markers in V. moseri and V. variegatus by the cross-species amplification of microsatellite primers developed previously in two other related marine fish species. A total of 244 polymorphic microsatellite markers were selected for cross-species amplification in V. moseri and V. variegatus, of which 182 markers deriving from Atlantic halibut (Hippoglossus hippoglossus) and 62 markers deriving from Japanese flounder (Paralichthys olivaceus). A sample of 10 individuals were detected. As a result, a total of 67 loci showed polymorphisms in V. moseri, and 62 loci showed polymorphisms in V. variegatus, with the observed number of alleles per locus ranging from two to five in V. moseri, and from two to seven in V. variegatus, respectively. This paper provided more candidate microsatellite markers which could be useful for construction of genetic linkage maps, evaluation of population genetic structure and stock management of V. moseri and V. variegatus.     

Development of 52 new polymorphic microsatellite markers for the olive flounder, Paralichthys olivaceus

We characterized 52 new microsatellite markers isolated from (GT)(n) and (CT)(n) microsatellite-enriched genomic libraries of the olive flounder (Paralichthys olivaceus). All markers were polymorphic, with eight to 30 (mean 15.1) alleles detected in 30 individuals from a single natural population. Observed heterozygosity ranged from 0.20 to 1.0. Segregation analysis within a mapping family revealed non-amplifying null alleles at six loci. These results indicate that these new microsatellite markers will be useful for population genetic, parentage, and genome mapping studies.     

Isolation and characterization of new microsatellite markers from the Japanese flounder (Paralichthys olivaceus)

The isolation and characterization of eight polymorphic microsatellite loci from a Japanese flounder partial genomic library are reported. The eight markers isolated in this study were highly polymorphic and their positions on the linkage genome map of the Japanese flounder were determined. Therefore, they are useful for ecological studies of wild populations. These markers are more effective than other markers with no information of chromosomal locations.     

New polymorphic microsatellite markers for the summer flounder, Paralichthys dentatus

Given the ecological and commercial importance of the summer flounder (Paralichthys dentatus), there is a surprising paucity of information on the molecular genetics of this species. Some studies published to date are concentrated on the reproduction biology. To address this shortcoming, a microsatellite-enriched genomic DNA library of P. dentatus was generated and screened by sequencing. Twelve dinucleotide microsatellite loci were characterized by genotyping 24 samples. The observed number of alleles ranged from three to thirteen with an average of 8.25, while the effective number of alleles ranged from 2.21 to 8.28 with an average of 5.06. The observed and expected heterozygosities ranged from 0.0833 to 0.9583 and from 0.5594 to 0.8980, respectively. Significant deviations from Hardy–Weinberg expectations were detected at three loci and linkage disequilibrium between two loci was significant after applying Bonferroni correction. In cross-species amplification, three species showed at least two polymorphic loci. The 12 highly polymorphic microsatellite markers represent a powerful molecular tool, which will allow for detailed population genetic analyses on this important marine fish.     

Development of polymorphic microsatellite markers for the Trichoglossus haematodus and cross-species amplification in Trichoglossus moluccanus

Molecular Biology Reports - Trichoglossus haematodus is the most popular parrots globally and one of the most bred species in Korea's zoos. However, despite its popularity, there are limited...