Test of Mendelian segregation among 10 microsatellite loci in the fourth generation of bester (Huso huso L. ×Acipenser ruthenus L.)

Ten microsatellite loci were tested on Mendelian segregation in the bester – hybrid of beluga, Huso huso L. and sterlet, Acipenser ruthenus L. in the fourth generation. All studied loci showed disomic inheritance and Mendelian segregation of their alleles. The molecular approach by microsatellite DNA analysis represents a reliable tool for interspecific characterization of sturgeons and their hybrids (different alleles of several loci are present in sturgeon fishes, and interspecific hybrids posesses both maternal and paternal specific alleles). This is the first report of the Mendelian segregation testing in interspecific hybrid of acipenserid fishes using microsatellite DNA markers.     

Molecular identification of interspecific hybrids between Haliotis discus hannai Ino and Haliotis gigantea Gmelin using amplified fragment‐length polymorphism and microsatellite markers

Interspecific hybrids between Haliotis discus hannai Ino and Haliotis gigantea Gmelin were produced in this study. The hybridity of the interspecific hybrids was confirmed by using the methods of amplified fragment‐length polymorphism (AFLP) and microsatellite [simple sequence repeats (SSR)] markers. Five AFLP primer combinations were used to develop the AFLP profiles of H. discus hannai, H. gigantea and their reciprocal hybrids. AFLP analysis revealed that genetic variations of H. discus hannai and H. gigantea were relatively diverse and each species holds species‐specific bands. The AFLP profiles of reciprocal hybrids showed that all of the hybrids inherited bands specific to H. discus hannai and H. gigantea. Of a total of 20 microsatellite loci, which were selected from H. discus hannai microsatellite markers evaluated, eight loci were polymorphic in H. gigantea samples, with an average of 3.375 alleles per locus. Preliminary screening showed that, two of these eight microsatellite loci (Awb002 and Awb022) could be used as species‐specific markers to distinguish the hybrids and their parental species. Simple sequence repeats analysis showed that the reciprocal hybrids inherited one allele from each parent for both of the two SSR loci investigated. These data strongly suggest that the induced interspecific hybrid is a true hybrid between H. discus hannai and H. gigantea.     

Genetic and morphological evidence of hybridization between <Emphasis Type="Italic">Nematalosa japonica</Emphasis> and <Emphasis Type="Italic">N. come</Emphasis> (Clupeiformes: Clupeidae) off Okinawa Island,Ryukyu Archipelago,Japan

Two morphologically similar species of gizzard shad, Nematalosa japonica Regan and N. come (Richardson), sympatrically distributed off Okinawa Island, Japan, were examined using an allozyme locus (SOD*) and two nuclear polymerase chain reaction (PCR)-based DNA markers (ITS-1 and CaM), which provided diagnostic identification of each species. In addition, a multiplex PCR-based mitochondrial DNA (mtDNA) marker (16S) was used to characterize the distribution of mtDNA haplotypes among specimens. The species composition of sympatric and allopatric population samples from Tungkang, southern Taiwan, to Okinawa and the Shikoku Islands, Japan, were also examined. Gizzard shad with hybrid genotypes were detected in three populations from Okinawa Island, with hybrid frequency ranging from 1 to 67%. A backcross level of 2% was detected in the dominant hybrid frequencies of one population sample only. Morphological examination of hybrids showed intermediate forms, with hybrid indices of three meristic characters falling between those of the parental species (range 39–53; mean 45). Although principal component analysis showed differences between N. japonica and N. come based on the first principal component scores, hybrids were difficult to identify. Accordingly, satisfactory identification of species and hybrids could be achieved only using genetic tools. We also discuss the cause of hybridization and its relationship with recently conducted reclamation on Okinawa Island.     

Genetic Introgression Between Arctic Charr (Salvelinus Alpinus) and Brook Trout (Salvelinus Fontinalis) in Bavarian Hatchery Stocks Inferred from Nuclear and Mitochondrial DNA Markers

Nuclear insulin-like growth factor 2 gene (IGF-2), growth hormone 1 gene (GH-1) and internal transcribed spacer 1 (ITS-1) of the ribosomal DNA as well as the mitochondrial NADH-3 and NADH-4 dehydrogenase genes (ND-3/4) exhibited species-specific restriction fragment patterns and three microsatellite loci (Sfo18, Ssa85 and Ssa197) had non-overlapping allele size ranges in Arctic charr and brook trout and were used as diagnostic markers for testing genetic purity of hatchery stocks and wild populations of Arctic charr and brook trout in Bavaria, Germany. Screening of four wild populations (three in Arctic charr and one in brook trout) revealed only a single hybrid (back-cross to brook trout) individual in L. Starnberg. In contrast, in three (out of five) hatchery stocks of Arctic charr and in both hatchery stocks of brook trout hybrids were detected with the frequency from 3 to 100%. Three hatchery stocks (SS2, SA and BS1) represent a hybrid swarm because they contained a very high proportion of hybrids (from 83 to 100%) and most or all hybrid individuals had alien alleles at only one or a few of six unlinked diagnostic loci, indicating that post-F₁ hybrids represent the majority of individuals in these stocks and introgression has taken place. Release or escape of introgressed individuals from hatcheries into natural water bodies should be avoided in order to protect the biological diversity and genetic integrity of native fish populations.     

Authentication of flying-fish-meal content of processed food using PCR-RFLP

Nucleotide sequence and polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis of the 3′-portion of the mitochondrial 16S RNA gene (rDNA) coding sequence were used to differentiate between flying fishes and other fishes that are frequently used in ago-noyaki production. In this study, we successfully distinguished between flying fishes and the other fishes by combining amplified DNA fragments with universally designed primers and digesting the PCR products with AfaI and MfeI restriction endonucleases. PCR-RFLP of 15 ago-noyaki, 2 noyaki, and 8 other processed flying-fish products were analyzed to authenticate flying-fish content among processed seafood products. After digestion of the PCR products with both enzymes, we found that all ago-noyaki and processed flying fish products contained either two or three DNA fragments of ~200, 300, and 530 bp, and noyaki samples (which do not contain flying fish) contained only one fragment of ~530 bp. Here, we present a new procedure to confirm the content of flying-fish meal in ago-noyaki.     

Species identification of Alaska pollock,Gadus spp., and Micromesistius spp. in cod roe products using a PCR-based method

Fish species identification techniques for authentic food labeling were developed using species-specific PCR primers for cod roe products. A salted, seasoned fish roe product, karashimentaiko (chilli cod roe), is produced from the eggs of Alaska pollock, Theragra chalcogramma, according to the fair trade competition agreement authorized by the Fair Trade Commission of the Japanese government. To examine whether Alaska pollock ovaries or those of other fish species are being used as raw materials for the fish roe products, we developed species identification techniques using PCR amplification of a 255-bp fragment encoding the mitochondrial ATP synthase Fo subunit 6 (ATP6) gene with a species-specific primer set for Alaska pollock mitochondrial DNA. We also designed two species-specific primer sets corresponding to the mitochondrial ATP6 and cytochrome b (cytb) for Gadus spp. and Micromesistius spp. by PCR amplification of 332- and 223-bp fragments, respectively. We examined the species specificity of these PCR-based methods among nine commercially important Gadidae species.     

Genetic relationship between broodstocks of olive flounder, Paralichthys olivaceus (Temminck and Schlegel) using microsatellite markers

For the first generation of a selective breeding programme, it is important to minimize the possibility of inbreeding. This mostly occurs by mating between closely related individuals, while proper mating can provide an opportunity to establish the base families with wide genetic variation from which selection for subsequent generations can be more effective. Genotyping with microsatellite‐based DNA markers can help us determine the genetic distances between the base populations. The genetic markers further facilitate the identification of the correct parents of the offspring (parentage assignments) reared together with many other families after hatching. We established a genetic analysis system with microsatellite DNA markers and analysed the genetic distances of three farmed stocks and a group of fish collected from wild populations using eight microsatellite markers. The averaged heterozygosity of the farming stocks was 0.826 and that of the wild population was 0.868. The hatchery strains had an average of 8.6 alleles per marker, which was less than a wild population that carried an average of 14.3 alleles per marker. Significant Hardy–Weinberg disequilibrium (HWDE) was observed in two farming stocks (P<0.05). Despite relatively low inbreeding coefficiency of the hatchery populations, the frequency of a few alleles was highly represented over others. It suggests that the hatchery stocks to some extent have experienced inbreeding or they originated from closely related individuals. We will develop a selective program using the DNA markers and will widen the usage of the DNA‐based genetic analysis system to other fish species.     

The effect of domestication on a brown trout (Salmo trutta m fario) broodstock in Hungary

Molecular markers (PCR–RFLP and microsatellite) were used to investigate the genetic background of the only brown trout (Salmo trutta m fario) broodstock in Hungary which due to the hydrogeography of the country should theoretically belong to the Danubian lineage. PCR–RFLP (mitochondrial DNA control region and lactate dehydrogenase and somatolactin genes) as well as microsatellite (BFRO002, OMM1064, Ssa408uos, SsoSL417, SsoSL438) markers were used to distinguish between Danubian and Atlantic lineages of brown trout in the Lillafüred broodstock. Altogether 435 fish were tagged during the experimental period. Due to mortalities, fin clips were collected from 401 individuals. According to the genetic analysis of the mitochondrial DNA, the Danubian haplotype is present in only one individual (0.2 %) of the broodstock. Analysis of the nuclear markers revealed that alleles characteristic of both the Atlantic and the Danubian lineages are found in the population. However, Atlantic alleles dominate throughout the broodstock which is in Hardy–Weinberg equilibrium according to the investigated markers. Results indicate that the original broodstock that was introduced to the farm following its construction in 1933 was of the Atlantic lineage. Although later fish from a local stream were collected and added to the broodstock, the number of these was limited and they were almost exclusively males. Fish from this farm that are stocked by anglers can have a significant genetic impact on trout populations of natural streams.     

利用AFLP技术筛选锯缘青蟹性别差异DNA片段

采用高盐和酚氯仿异戊醇 (PCI)结合法提取DNA ,利用AFLP技术 ,应用 5 2个引物组合 ,检测了锯缘青蟹 (Scyllaser rata)雌雄基因组DNA的多态性 ,筛选与锯缘青蟹性别相关的分子标记。实验中共扩增出 4 312条带 ,筛选出候选差异DNA片段 74 8条。这些差异DNA片段的获得 ,为研究锯缘青蟹性别的分子标记奠定了基础     

AFLP and microsatellites as genetic tags to identify cultured gilthead seabream escapees: data from a simulated floating cage breaking event

Genetic discrimination using DNA fingerprinting is rapidly developing for cultured stock and wild fish populations. Microsatellites and AFLPs are being widely used in aquaculture to assign fish or processed fish products, to their claimed origin, paternity or strain. In the present study, 147 AFLP and 4 microsatellite markers were used as genetic tags in gilthead seabream, Sparus auratus. Specimens from two different hatchery broodstocks (one of Atlantic and one of Mediterranean origin) and wild fishes from a natural population were fingerprinted. Putative offspring from these broodstocks were computer-generated, and the confidence in the parentage assignment of their genetic profiles to the hatchery broodstock assessed. The virtual offspring were then mixed with specimens from a natural population to simulate an accidental escape from a floating cage. The risk of false paternity inclusion was evaluated to test the ability to identify either Atlantic or Mediterranean hatchery offspring among wild fish. The method proved to be reliable, and could therefore be used to forecast the impact of fish farm escapees.     

Development of nuclear DNA markers to characterize genetically diverse groups of <Emphasis Type="Italic">Misgurnus anguillicaudatus</Emphasis> and its closely related species

Repetitive DNA sequences, ManDra and ManBgl, were isolated from the DraI and BglII digests of the genomic DNA of Misgurnus anguillicaudatus, respectively. A primer set of ManDra distinguished two genetically different groups (A and B) of M. anguillicaudatus by specific electrophoretograms. A primer set of ManBgl amplified the DNA of M. anguillicaudatus and M. mizolepis. The individuals of M. anguillicaudatus were divided into two groups depending on the fragment sizes, in which the groups A and B (B-1 and B-2) showed 400 and 460 bp, respectively. M. mizolepis was distinguished by a different pattern (400-, 460-, and 510-bp fragments). PCR–RFLP analyses of recombination activating gene 1 gave a clear difference between A or B-2 (443-bp fragment) and B-1 groups (296- and 147-bp fragments). Clonal lineages and hybrids between B-1 and B-2 groups could be identified by appearance of three fragments (443, 296, and 147 bp). The combined analyses using the above three nuclear markers discriminated among nuclear genomes of genetic groups (A, B-1 and B-2) of M. anguillicaudatus and M. mizolepis. In several localities, natural hybridizations between the group B-1 and B-2 loaches and introgressions of clonal mitochondrial genomes into the group B-1 loaches were detected.     

Development of simple sequence repeat markers in Pyropia yezoensis (Bangiales,Rhodophyta) by high‐throughput sequencing technology

Pyropia yezoensis is one of the most important economical seaweeds across the world and major efforts are underway to increase the production. However, its genome is relatively unexplored. In this study, genome sequence of wild‐type strain LS was determined through paired‐end sequencing of small‐size fractionated genomic library. In total, 283,606 contigs were assembled and 20,620 SSR‐containing sequences were identified. Most of the SSRs contained tri‐ and di‐nucleotide motifs (95.42% and 2.34% respectively), of which GCC was the most abundant (15.7%). Furthermore, 1,253 pairs of primer sets were designed and 124 of them were selected randomly for validation. The results showed that 120 pairs were successful in PCR, and the remaining four failed to generate PCR product at various conditions. Among the 120 pairs of which the PCR products were subsequently sequenced by Sanger sequencing, 104 pairs amplified SSRs with the same motif and repeat times, three pairs with the same motif but different repeat times and 13 pairs with different motifs. Moreover, 21 primer pairs amplified polymorphic products between LS and an improved strain HT, and tri‐nucleotide SSRs showed higher polymorphism frequency when compared to di‐nucleotide SSRs. These SSR markers may enrich the current molecular resources in P. yezoensis.     

Parentage assignment in hybrid abalones (Haliotis rufescens × Haliotis discus hannai) based on microsatellite DNA markers

Parentage analysis in aquaculture determines genealogical relationships between broodstock and progeny when the parents are unknown. Thus, parentage analysis is a useful tool to establish pedigree reports in molecular‐assisted selection programs. Here, we evaluated 10 heterologous microsatellite markers for parentage assignment in abalone hybrids produced from 43 abalone broodstocks of red abalone (Haliotis rufescens) and Japanese abalone (H. discus hannai). The allele frequencies, exclusion probabilities and broodstock contributions were calculated using CERVUS, PAPA and GERUD software. The polymorphic information content (PIC) values showed that most of the microsatellite loci were highly informative (>0.7) and more than 90% of parentage assignment was possible with a minimum of 5–6 microsatellite markers. Parentage assignment for hybrid and pure‐red progeny showed a better performance than pure‐Japanese progeny. This result could be due to the high level of allele loss in the parental genotypes. In addition, results indicated that only two sires contributed over 80% and 90% of red and hybrid progenies, respectively. This study gives a new molecular tool to support marker‐assisted selection in abalone hybrids produced in Chile.     

鲮微卫星引物的鉴定及其适用性检验

利用4种鲤科鱼类的14对微卫星引物对西江流域一批野生鲮进行PCR扩增。将各扩增条带进行克隆测序,发现引物MFW1、MFW2、MFW15、MFW17、Cc7、Cc11、Bgon22扩增出的产物含有微卫星重复序列。进一步对鲮的微卫星位点MFW1、MFW2、Cc7重新设计引物,并将其分别命名为Cm1、Cm2、Cm3,新引物对鲮的扩增特异性增强。采用引物Cm1、Cm2、Cm3、Cc11、MFW15、MFW17、Bgon22对西江流域一批野生鲮进行引物适用性检验。结果表明,除引物MFW15、Cc11无多态性外,其余5对引物(Cm1、Cm2、Cm3、MFW17、Bgon22)在取样群体中扩增图谱带型丰富,随引物不同,各标记在群体中检测到的等位基因数为2到16个。各微卫星座位的期望杂合度(He)及观察杂合度(Ho)范围分别为0~0.9038和0~1,平均分别为0.6881(0.1819SD)和0.7772(0.1931SD)。座位连锁分析显示Cm1与Bgon22之间存在显著性水平连锁关系(P<0.05),其余各座位之间未检测到明显的连锁关系(P>0.05)。研究群体的遗传多样性指数平均为0.6823,多态性水平相对较高。以上结果表明,筛选获得的7个微卫星座位适于对鲮进行遗传多样性分析。     

Microsatellite DNA markers of Ezo abalone (Haliotis discus hannai): a preliminary assessment of natural populations sampled from heavily stocked areas

Reproductive success of released animals becomes a growing interest in the context of stock enhancement program and conservation biology. In the case of the Ezo abalone (Pacific abalone; Haliotis discus hannai), limited information is available about the extent of interbreeding between natural and released hatchery populations. This study aimed to develop microsatellite DNA markers from Ezo abalone to address the issue. We developed 38 microsatellite markers that worked well in three full-sib families established from wild caught abalone. Of these, nine markers, with which alleles could be scored easily and accurately, were applied to two population samples derived from coastal areas where intensive releases of hatchery-produced abalone seedlings have been carried out. Multilocus estimates of genetic population differentiation calculated based on the nine markers (F_ST analogue θ and R_ST analogue ρ) rejected a null hypothesis of genetic homogeneity between the populations (θ=0.048, P<0.00001; ρ=0.029, P=0.0051). Although both populations conformed to Hardy Weinberg's equilibrium at almost all of the nine loci, the results of simulation analysis for variance of relatedness coefficient provided evidence of nonrandom mating in each population, possibly caused by the cumulative effect of stocking on the genetic make-up. The population analysis presented here is preliminary; but we consider that the microsatellite markers are potentially an efficient means to examine the reproductive contribution of released abalone to natural resources.     

Microsatellite loci in tench: isolation and variability in a test population

Because of their high variability and rapid evolution, microsatellites became increasingly important in genetic research, e.g. population structure and differentiation studies, gene mapping and parentage analysis. However, such loci have not been isolated in tench so far. Applying a PCR based method of generating microsatellite enriched DNA fragment libraries we were able to identify nine loci (MTT-1 to MTT-9). The variability of these microsatellite loci was determined in 50 tench individuals originating from a wild population of Lake Döllnsee, Germany. Three loci were found to be monomorphic. The remaining six loci segregated for two to nine alleles. The observed heterozygosities at polymorphic loci were high (0.500–0.959) with only one exception: locus MTT-8 (0.167). These polymorphic microsatellite loci showed a much higher level of genetic variability than the allozyme loci previously studied in the same individuals. Thus, they seem to be more suitable for genetic studies of tench. On the other hand, it remains to be checked in other populations if the three loci that did not show any variation in this population are generally monomorphic in this species.     

Water quality affects SDH activity, protein content and RNA:DNA ratios in fish (Catla catla, Labeo rohita and Oreochromis mossambicus) raised in ponds of a sewage-fed fish farm

The spatial and seasonal variability of the respiratory enzyme succinate dehydrogenase and the protein content were examined in different tissues of fish cultured in three ponds along the effluent gradient of a sewage-fed fish farm. Indian major carp Catla catla (150-230 g) and Labeo rohita (60-190 g) cultured in both the middle and last points of the sewage effluent (stocking pond 1) and (stocking pond 4) and Oreochromis mossambicus (50-160 g), a naturally growing fish of the inlet (facultative pond) and the out let of the sewage effluent (stocking pond 4) were procured every month during the period of January-December, 2005 and were subjected to determination of succinate dehydrogenase activity, total protein, DNA and RNA contents from gill, liver and muscle tissue respectively. The SDH activity of all three test fishes (Catla catla, Labeo rohita and Oreochromis mossambicus) was reduced significantly (ANOVA; P < 0.05) when cultured in SP-4 compared to SP-1 in the case of Catla catla and Labeo rohita and in facultative pond in the case of Oreochromis mossambicus.Conspicuous differences in the SDH activity of fish between the last and first stocking pond or the facultative pond were clearly due to the result of the differences in water quality. There was a direct relationship between SDH activity in gill tissue of any of the fish investigated and ammonia-N concentration of water or water pH. This shows that the respiratory activity of these fishes was strongly affected by the ammonia and pH of water. In other words, this suggests that as the distance from the point source increases, there was a substantial improvement of water quality in the ponds located along the sewage effluent gradient. Evidently, there is a progressive pattern of growth, survival and physiological health of fish and abundance of favorable diversity of food organisms with rich biodiversity.     

微卫星位点AciG-35在中华鲟野生个体中不同等位基因的序列变异研究

本研究利用PCR扩增和序列测定得到了1个特殊4碱基重复微卫星位点AciG-35在中华鲟野生个体中的34个等位基因序列。根据序列测定的结果,初步研究了该位点的核心重复区序列以及两端侧翼序列的变异情况。该位点不同等位基因的核心序列和侧翼序列都表现出一定程度的变异,其中核心重复序列主要表现为点突变,侧翼序列主要表现为各种碱基的替换、插入和缺失以及片段的插入缺失。此外,还发现了AciG-35引物在中华鲟中的多位点扩增现象。研究结果对于准确解释微卫星分子标记用于群体遗传分析时的数据结果具有重要意义,对在微卫星标记的应用过程中可能存在的一些问题一并进行了探讨。     

多重RT-PCR体系检测4种虾病毒的方法

根据多重RT-PCR的技术原理,利用对虾传染性表皮与造血组织坏死症病毒、白斑综合征病毒、黄头病毒和桃拉综合征病毒的基因序列分别设计了4对特异引物,建立多重RT-PCR体系用于虾4种病毒的检测。多重RT-PCR体系能特异地扩增出IHHNV、WSSV、YHV和TSV的目的片段:TSV特异性扩增片段508 bp,WSSV 特异性扩增片段435 bp,IHHNV 特异性扩增片段301 bp 和YHV。特异性扩增片段614 bp。结果表明,多重PCR虾病毒检测系统具有较高的特异性和敏感性,并对其它对虾病原呈阴性。IHHNV、TSV、WSSV和YHV模板在多重PCR虾病毒检测体系中的检测下限分别为0.1,1,0.02和0.2 pg。病毒感染病料检测试验中,该检测体系的检测结果与单纯PCR的检测结果呈现出较好的吻合度。     

Microsatellite marker isolation and cultured strain identification in <Emphasis Type="Italic">Carassius auratus gibelio</Emphasis>

Microsatellites have become the preferred molecular markers for strain selection and genetic breeding in fish. In this study a total of 105 microsatellites were isolated and identified in gibel carp (Carassius auratus gibelio) by microsatellite sequence searches in GenBank and other databases and by screening and sequencing of positive clones from the genomic library enriched for AG and GATA repeats. Moreover, nineteen microsatellites were randomly selected to design locus-specific primer pairs, and these were successfully used to identify and discriminate different cultured strains of gibel carp including strains A, D, L, and F. Three different types of microsatellite pattern were distinguished by the number and length of fragments amplified from the 19 primer pairs, and some microsatellite primer pairs were found to produce different microsatellite patterns among strains and strain-specific fragments. In addition, some duplicated alleles were also detected in two microsatellite patterns. Therefore, the current study provides direct molecular markers to discriminate among different cultured strains for selective breeding and aquaculture practice of gibel carp.