Development of polymorphic microsatellite markers from barfin flounder (Verasper moseri) and their cross-species amplification

Barfin flounder (Verasper moseri) is a rare fish species in the world. Here, we reported 10 polymorphic microsatellite loci isolated from a dinucleotide-enriched genomic library of barfin flounder (Verasper moseri). The number of alleles, observed, and expected heterozygosity per locus in a test population ranged from 2 to 6, from 0.3333 to 1.0000, and from 0.4866 to 0.7774, respectively. One locus 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 microsatellite loci in additional five fish species was performed. These polymorphic microsatellite loci would be useful for investigating genetic population structure and construction of genetic linkage map in Verasper moseri. Gui-Dong Miao and Chang-Wei Shao Contributed equally to this work.     

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.     

Genetic linkage maps of barfin flounder (<Emphasis Type="Italic">Verasper moseri</Emphasis>) and spotted halibut (<Emphasis Type="Italic">Verasper variegatus</Emphasis>) based on AFLP and microsatellite markers

Barfin flounder (Verasper moseri) and spotted halibut (Verasper variegatus) are two economically important marine fish species for aquaculture in China, Korea and Japan. Construction of genetic linkage maps is an interesting issue for molecular marker-assisted selection (MAS) and for better understanding the genome structure. In the present study, we constructed genetic linkage maps for both fish species using AFLP and microsatellite markers based on an interspecific F₁ hybrid family (female V. moseri and male V. variegatus). The female genetic map comprised 98 markers (58 AFLP markers and 40 microsatellite markers), distributing in 27 linkage groups, and spanning 637 cM with an average resolution of 8.9 cM. Whereas the male genetic map consisted of 86 markers (48 AFLP and 38 microsatellite markers) in 24 linkage groups, covering a length of 625 cM with an average marker spacing of 10 cM. The expected genome length was 1,128 cM in female and 1,115 cM in male, and the estimated coverage of genome was 56% for both genetic maps. Moreover, five microsatellite markers were observed to be common to both genetic maps. This is the first time to report the genetic linkage maps of V. moseri and V. variegatus that could serve as the basis for genetic improvement and selective breeding, candidate genes cloning, and genome structure research.     

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.     

Development of 81 new polymorphic EST-derived microsatellite markers for the olive flounder,Paralichthys olivaceus

Expressed sequence tags (ESTs) can be used to identify microsatellite markers. We developed 81 polymorphic microsatellite markers from 4,940 ESTs of the olive flounder, Paralichthys olivaceus. Out of 100 EST-derived microsatellites for which PCR primers were designed, 81 loci were polymorphic in 30 individuals from a single natural population with 2–28 (mean 10.6) alleles per locus. The observed and expected heterozygosities of these loci were 0.033–1.000 and 0.033–0.965, respectively. Segregation analysis within a mapping family revealed non-amplifying null alleles at five loci. These new EST-derived microsatellite markers should be useful for population genetic analyses, pedigree tracing and constructing a linkage map for olive flounder.     

Isolation and characterization of 30 novel polymorphic microsatellite loci from Japanese halfbeak, Hyporhamphus sajori (Temminck et Schlegel, 1846)

The construction of genetic linkage map and evaluation of population genetic diversity both require large numbers of polymorphic molecular markers. In the study, 60 microsatellite loci were isolated from a dinucleotide-enriched genomic library of Japanese halfbeak (Hyporhamphus sajori). And 30 polymorphic microsatellite loci were found to be polymorphic between 2 and 11 alleles. The number of observed, expected heterozygosity and polymorphism information content per locus in 24 individuals ranged from 0.1667 to 1.000, 0.1828 to 0.9220, 0.1828 to 0.8945, respectively. Three loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction analysis and there was no significant linkage disequilibrium found between pairs of loci. As a result, 30 microsatellite loci probably should provide sufficient level of genetic diversity to investigate the fine-scale population structure, stock management and enhancement, genetic linkage map construction and molecular marker-assisted breeding in H. sajori.     

Isolation and characterization of polymorphic microsatellite loci from a repeat-enriched genomic library of stone flounder (Kareius bicoloratus) and cross-species amplification

Stone flounder (Kareius bicoloratus) is a rare fish species in China. Here, we reported 11 polymorphic microsatellite loci isolated from a repeat-enriched genomic library of stone flounder. The number of alleles, observed and expected heterozygosity per locus in 32 individuals ranged from 3 to 6, from 0.1613 to 0.8667 and from 0.1549 to 0.7932, respectively. Two loci 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 Kareius bicoloratus. Yong-Sheng Tian and Gui-Dong Miao are contributed equally.     

Isolation and characterization of 12 polymorphic microsatellite markers from ladyfish (Elops saurus Linnaeus)

Ladyfish (Elops saurus Linnaeus) is an economically important marine fish species. 76 microsatellite loci were isolated from an enriched genomic library of Elops saurus. Twelve of these markers were polymorphic in a test population with alleles per locus ranging from three to nine. The number of observed, expected heterozygosity and polymorphism information content (PIC) per locus in 20 individuals ranged from 0.2000 to 1.0000, 0.1897–0.8846, 0.1769–0.8476, respectively. One markers significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction analysis and there was no significant linkage disequilibrium found between pairs of markers. As a result, 12 microsatellite markers probably should provide sufficient level of genetic diversity to investigate the fine-scale population structure, stock management and enhancement, genetic linkage map construction and molecular marker-assisted breeding in Elops saurus Linnaeus.     

Characterization of ten polymorphic microsatellite markers for the protected Spanish moon moth <Emphasis Type="Italic">Graellsia isabelae</Emphasis> (Lepidoptera: Saturniidae)

Ten polymorphic microsatellite loci were developed for Graellsia isabelae. Polymorphism was assessed for 20 individuals from a Spanish population (Els-Ports-de-Beseit, Catalonia) and 39 more individuals from one population in the French Alps and six other Spanish localities. Overall, the number of alleles per locus ranged from 5 to 24. Els-Ports-de-Beseit showed an average number of alleles per locus of 9.80 (SD = 4.32), observed heterozygosity was 0.71 (SD = 0.226), and expected heterozygosity was 0.788 (SD = 0.146). Genotypic frequencies conformed to Hardy–Weinberg equilibrium at the Catalonian population, and no evidence for linkage disequilibrium was observed. Multilocus genotypes resulting from this set of markers will be useful to determine genetic diversity and differentiation within and among populations of this highly protected moth. Several loci amplified and resulted polymorphic in two related species: two loci in Actias neidhoeferi, and three loci in A. luna.     

Molecular Cloning of Growth Hormone Complementary DNA in Barfin Flounder (Verasper moseri)

The olive flounder (family Paralichthidae; Paralichthys olivaceus) growth hormone (ofGH) appears to be the most derived among known growth hormones, with the deletion of 14 consecutive amino acids in the carboxy-terminal region. To ascertain if this deletion is common to all flounders, growth hormone complementary DNA of the barfin flounder (bfGH) (family Pleuronectidae; Verasper moseri) has been cloned. It was amplified by polymerase chain reaction using single-strand cDNA from the pituitary gland. Excluding the poly(A) tail, the bfGH cDNA is 919 nucleotides long and contains a 609-bp open reading frame encoding a putative signal peptide of 17 amino acids and a mature protein of 186 amino acids. Northern blot analysis detected 1.0 kb of bfGH messenger RNA in the pituitary gland, which is a reasonable value considering the poly(A) tail. The deduced amino acid sequence of bfGH has 78% identity with the sequence of ofGH. A major difference is the presence of a 14 amino acid segment (140–153) in bfGH, as in other growth hormones, suggesting that this deletion in the olive flounder occurred after the divergence of the Pleuronectoidae. Received May 7, 1999; accepted July 13, 1999.     

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: Isolation and characterization of microsatellite DNA loci from the southern flounder, Paralichthys lethostigma

Paucity of polymorphic molecular markers in southern flounder (Paralichthys lethostigma) has been a major limitation in genetic improvement of this important economic fish. Hence, we constructed a repeat-enriched genomic library from P. lethostigma. A total of 39 new microsatellites were identified, for which 33 primer pairs were designed. After validating and scoring, 10 of these loci were polymorphic in a test population with the range of alleles from two to nine per locus. The observed and expected heterozygosities ranged from 0.2500 to 0.9000 and from 0.4469 to 0.8514, respectively. These polymorphic microsatellites will be useful for genetic diversity analysis and linkage map construction for P. lethostigma.     

Isolation and characterization of polymorphic microsatellite loci from fat greenling (Hexagrammos otakii)

Fat greenling (Hexagrammos otakii) is an economocally important marine fish species. Thirtyfive microsatellite loci were isolated from two enriched genomic library of Hexagrammos otakii. Ten of these loci were polymorphic in a test population with alleles per locus ranging from two to six, and observed and expected heterozygosities per locus from 0.2581 to 1.0000 and from 0.2892 to 0.7726, respectively. Four loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction and no significant linkage disequilibrium was found between pairs of loci. These polymorphic microsatellite loci would be useful for investigating genetic diversity of Hexagrammos otakii.     

Development and characterization of 10 novel polymorphic microsatellite loci in Homatula potanini (Günther, 1896)

Homatula potanini is a genus Paracobitis which belongs to the family Cobitidae, subfamily Nemacheilinae. We first isolated ten novel microsatellite DNA loci using the FIASCO protocol. The number of observed alleles per locus ranged from 5 to 14. The observed heterozygosity and expected heterozygosity ranged from 0.3750 to 0.9375 and 0.4028 to 0.8819 respectively. The polymorphic information content ranged from 0.3515 to 0.8556 (M = 0.6170). No significant linkage disequilibrium was detected among the loci. In the present study, we have identified 10 new markers for H.  potanini. These loci should provide sufficient levels in the evaluation of genetic diversity and designing effective conservation programs of H.  potanini.     

Isolation and characterization of 10 polymorphic microsatellite loci from small yellow croaker (Pseudosciaena polyactis)

Small yellow croaker (Pseudosciaena polyactis) is an economically important marine fish species. About 43 microsatellite loci were isolated from two enriched genomic library of Pseudosciaena polyactis. Ten of these loci were polymorphic in a test population with alleles per locus ranging from two to six, and observed and expected heterozygosities per locus from 0.3750 to 0.8750 and from 0.3112 to 0.8121, respectively. No loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction and no significant linkage disequilibrium was found between pairs of loci. These polymorphic microsatellite loci would be useful for investigating genetic diversity of Pseudosciaena polyactis.     

Novel microsatellite markers for Atlantic sturgeon (Acipenser oxyrinchus) population delineation and broodstock management

Twenty‐one new disomic, polymorphic microsatellite DNA markers were isolated in Atlantic sturgeon, Acipenser oxyrinchus oxyrinchus. These markers yielded a total of 220 alleles in a survey of 16 fish; two to 21 alleles/locus were observed. Each locus segregated in a Mendelian fashion when tested in a family, and a set of 14 of the loci distinguished between siblings and half‐siblings. Average observed heterozygosity ranged from 18.8 to 100.0%, and no linkage disequilibrium was detected. These loci should detect sufficient genetic diversity to allow kinship analysis for broodstock management, gene marking for stocking assessment and life history studies, and delineation of fine‐scale population structure.     

Polymorphic microsatellite DNA markers in the mudflat topshell Diloma subrostrata (Gastropoda,Trochidae)

Very little information is available on the intraspecific genetic structure of topshell populations. Here, we report the characterization of five polymorphic microsatellite loci in the New Zealand mudflat topshell, Diloma subrostrata. The number of alleles per locus ranged from two to 23, observed and expected heterozygosities did not deviate from Hardy–Weinberg equilibrium (P < 0.05) and no linkage disequilibrium was detected between locus pairs (P < 0.05). We are currently using these markers to investigate genetic population structure of D. subrostrata in New Zealand.     

Microsatellite loci developed for black-crowned night-heron (Nycticorax nycticorax) and their cross-amplifications in Ardeidae

The present study reports isolation and characterization of 11 polymorphic microsatellite markers for Nycticorax nycticorax. A total of 82 alleles were detected with an average of 7.5 alleles per locus. The observed heterozygosity (H _O) ranged from 0.25 to 1.00, and the expected heterozygosity (H _E) ranged from 0.51 to 0.88. Analyses revealed no evidence for Linkage disequilibrium between any two loci, and only one locus was significantly deviated from HWE with the estimation of exact P values by the Markov chain method (P < 0.001). The 11 loci were successfully amplified in 11 other Ardeidae species. These results demonstrate these markers can be used for the study of intra- and inter-specific genetic diversity.     

Isolation and characterization of polymorphic microsatellites in Iris ensata (Iridaceae)

• Premise of the study: Microsatellite primers were developed in Iris ensata (Iridaceae) to provide polymorphic markers for further studies into population genetics. • Methods and Results: Thirteen polymorphic microsatellite loci were isolated from I. ensata. These loci were successfully amplified in two natural populations of I. ensata from eastern China (Longwangshan, Zhejiang Province) and northeastern China (Jinchuan, Jilin Province). There was no significant linkage disequilibrium found for any pair of loci. These loci contained between two and 12 alleles per locus across all 48 individuals of I. ensata. The number of alleles per locus varied from two to 10 at the population level and the observed and expected heterozygosities ranged from 0.167 to 0.958 and from 0.284 to 0.853, respectively. • Conclusions: These loci showed high levels of polymorphism and could be used to study the population genetic structure, genetic relationships, and phylogeography of I. ensata.     

Characterization of polymorphic microsatellite markers,isolated from ginger (Zingiber officinale Rosc.)

The present study reports isolation and characterization of eight polymorphic microsatellite markers for Zingiber officinale Rosc. (Ginger). A total of 34 alleles were detected across the 20 accessions, with an average of 4.3 alleles per locus. Values for observed and expected heterozygosities ranged from 0 to 1.0 and from 0.23 to 0.67, respectively. The heterozygote deficits were observed at three loci. At the significance threshold (P < 0.05) of the eight loci, seven were found to have deviated from Hardy–Weinberg equilibrium, whereas significant linkage disequilibria were observed between 10 pairs of loci. Our data indicate the existence of moderate level of genetic diversity among the ginger accessions genotyped with eight markers.