A Preliminary Genetic Linkage Map of Sinonovacula constricta(Lamarck, 1818) Based on Microsatellites Derived from RAD Sequencing

Sinonovacula constricta is one of the important economic aquaculture species in China. In this study, we constructed genetic linkage maps of S. constricta based on 300 microsatellite markers derived from RAD-seq using an F1 full-sib family. The female map contained 204 microsatellites assigned to 22 linkage groups, which covered 1529.5 cM with an average interval of 10.3 cM. The male consisted of 187 microsatellites in 19 linkage groups corresponding to the haploid chromosome number(n(28)19), which spanned 1429.3 cM with an average interval of 8.7 cM. The genome coverage was approximately 83.5% and 81.4%, respectively. An integrated map was constructed according to the common markers in parental linkage groups, which had a total length of 1683.8 cM with an average interval of 7.3 cM. The genome coverage of the integrated map was approximately 86.3%. The genetic linkage map would form the foundation for further studies on the quantitative trait loci(QTL), as well as accelerating the breeding process of this species.     

An AFLP Genetic Linkage Map of Pacific Abalone （Haliotis discus hannai）

A genetic linkage map of Pacific abalone （Haliotis discus hannai） was constructed using AFLP markers based on a two-way pseudo-testcross strategy in a full-sib family. With 33 primer combinations, a total of 455 markers （225 from the female parent and 230 from the male parent） segregated in a 1 ： 1 ratio, corresponding to DNA polymorphism： heterozygous in one parent and null in the other. The female framework map consisted of 174 markers distributed in 18 linkage groups, equivalent to the H. discus hannai haploid chromosome number, and spanning a total length of 2031.4 cM, with an average interval of 13.0 cM between adjacent markers. The male framework map consisted of 195 markers mapped on 19 linkage groups, spanning a total length of 2273.4cM, with an average spacing of 12.9cM between adjacent markers. The estimated coverage for the framework linkage maps was 81.2% for the female and 82.1% for the male, on the basis of two estimates of genome length. Fifty-two markers （11.4%） remained unlinked. The level of segregation distortion observed in this cross was 20.4%. These linkage maps will serve as a starting point for linkage studies in the Pacific abalone with potential application for marker-assisted selection in breeding programs.     

A genetic linkage map of marine shrimp Penaeus (Fenneropenaeus) chinensis based on AFLP, SSR, and RAPD markers

The Chinese shrimp Penaeus (Fenneropaeneus) chinensis is an important species in marine fishery and aquaculture in China. A female Chinese shrimp Penaeus (Fenneropaeneus) chinensis was captured from west coast of the Korean peninsula and mated with a “Yellow Sea No. 1” male to produce the first filial generation (F₁) 100 F₂ full-sib progeny from brother-sister crosses between F₁ families was used for the mapping study. A genetic linkage map of the Chinese shrimp was constructed, based on 354 markers, including 300 amplified fragment length polymorphism (AFLP) markers, 42 microsatellite (SSR) markers, and 12 randomly amplified polymorphism (RAPD) markers. Forty-seven linkage groups (LGs) were identified. The total map length was 4 580.5 cM, with an average spacing of 11.3 cM, covering 75.8% of the estimated genome size. The construction of this genetic linkage map was part of a genetic breeding program. This linkage map will contribute to the discovery of genes and quantitative trait loci (QTLs) in Chinese shrimp.     

Preliminary genetic linkage map of the abalone Haliotis diversicolor Reeve

Haliotis diversicolor Reeve is one of the most important mollusks cultured in South China. Preliminary genetic linkage maps were constructed with amplified fragment length polymorphism (AFLP) markers. A total of 2 596 AFLP markers were obtained from 28 primer combinations in two parents and 78 offsprings. Among them, 412 markers (15.9%) were polymorphic and segregated in the mapping family. Chi-square tests showed that 151 (84.4%) markers segregated according to the expected 1:1 Mendelian ratio (P<0.05) in the female parent, and 200 (85.8%) in the male parent. For the female map, 179 markers were used for linkage analysis and 90 markers were assigned to 17 linkage groups with an average interval length of 25.7 cm. For the male map, 233 markers were used and 94 were mapped into 18 linkage groups, with an average interval of 25.0 cm. The estimated genome length was 2 773.0 cm for the female and 2 817.1 cm for the male map. The observed length of the linkage map was 1 875.2 cm and 1 896.5 cm for the female and male maps, respectively. When doublets were considered, the map length increased to 2 152.8 cm for the female and 2 032.7 cm for the male map, corresponding to genome coverage of 77.6% and 72.2%, respectively.     

Location of <Emphasis Type="Italic">Vibrio anguillarum</Emphasis> resistance-associated trait loci in half-smooth tongue sole <Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis> at its microsatellite linkage map

A cultured female half-smooth tongue sole (Cynoglossus semilaevis) was crossed with a wild male, yielding the first filial generation of pseudo-testcrossing from which 200 fish were randomly selected to locate the Vibrio anguillarum resistance trait in half-smooth tongue sole at its microsatellite linkage map. In total, 129 microsatellites were arrayed into 18 linkage groups, ≥4 each. The map reconstructed was 852.85 cM in length with an average spacing of 7.68 cM, covering 72.07% of that expected (1 183.35 cM). The V. anguillarum resistance trait was a composite rather than a unit trait, which was tentatively partitioned into Survival time in Hours After V. anguillarum Infection (SHAVI) and Immunity of V. Anguillarum Infection (IVAI). Above a logarithm of the odds (LOD) threshold of 2.5, 18 loci relative to SHAVI and 3 relative to IVAI were identified. The 3 loci relative to IVAI explained 18.78%, 5.87% and 6.50% of the total phenotypic variation in immunity. The microsatellites bounding the 3 quantitative trait loci (QTLs) of IVAI may in future aid to the selection of V. anguillarum-immune half-smooth tongue sole varieties, and facilitate cloning the gene(s) controlling such immunity.     

Genetic mapping and QTL analysis for body weight in Jian carp (Cyprinus carpio var. Jian) compared with mirror carp (Cyprinus carpio L.)

We report the genetic linkage map of Jian carp(C yprinus carpio var. Jian). An F1 population comprising 94 Jian carp individuals was mapped using 254 microsatellite markers. The genetic map spanned 1 381.592 c M and comprised 44 linkage groups,with an average marker distance of 6.58 c M. We identified eight quantitative trait loci(QTLs) for body weight(BW) in seven linkage groups,explaining 12.6% to 17.3% of the phenotypic variance. Comparative mapping was performed between Jian carp and mirror carp( Cyprinus carpio L.),which both have 50 chromosomes. One hundred and ninety-eight Jian carp marker loci were found in common with the mirror carp map,with 186(93.94%) showing synteny. All 44 Jian carp linkage groups could be one-to-one aligned to the 44 mirror carp linkage groups,mostly sharing two or more common loci. Three QTLs for BW in Jian carp were conserved in mirror carp. QTL comparison suggested that the QTL confidence interval in mirror carp was more precise than the homologous interval in Jian carp,which was contained within the QTL interval in Jian carp. The syntenic relationship and consensus QTLs between the two varieties provide a foundation for genomic research and genetic breeding in common carp.     

Development and validation of 89 novel expressed sequence tag-derived microsatellite markers in blood clam, <Emphasis Type="Italic">Tegillarca granosa</Emphasis>

Blood clam, Tegillarca granosa, is an important shellfish in Chinese mariculture industry. Investigative research in this species, such as genetic linkage mapping, requires a large panel of molecular markers. In present study, a total of 89 polymorphic microsatellite markers were developed in T. granosa using the sequence database of Life Sciences Technology 454 next generation sequencing technology. All 89 loci were characterized in 20 individual clams from a natural population inhabiting Yueqing Gulf, Zhejiang Province, China. The number of alleles per polymorphic locus varied between 2 and 15, while the observed heterozygosity, expected heterozygosity and polymorphic information content varied between 0.000 and 1.000, 0.102 and 0.921, and 0.048 and 0.886, respectively. Of the 89 loci identified, 32 loci deviated significantly from Hardy-Weinberg equilibrium following Bonferroni correction. Thirty nine markers, which were shown to be polymorphic in a full-sibling family, were tested in Mendelian segregations. As expected, 32 loci were co-dominantly segregated in a Mendelian fashion. These novel developed microsatellite markers represent useful research tools for investigation of population genetic structure and genetic diversity in this species.     

Development and Validation of 89 Novel Expressed Sequence Tag-Derived Microsatellite Markers in Blood Clam,Tegillarca granosa

Blood clam, Tegillarca granosa, is an important shellfish in Chinese mariculture industry. Investigative research in this species, such as genetic linkage mapping, requires a large panel of molecular markers. In present study, a total of 89 polymorphic microsatellite markers were developed in T. granosa using the sequence database of Life Sciences Technology 454 next generation sequencing technology. All 89 loci were characterized in 20 individual clams from a natural population inhabiting Yueqing Gulf, Zhejiang Province, China. The number of alleles per polymorphic locus varied between 2 and 15, while the observed heterozygosity, expected heterozygosity and polymorphic information content varied between 0.000 and 1.000, 0.102 and 0.921, and 0.048 and 0.886, respectively. Of the 89 loci identified, 32 loci deviated significantly from Hardy-Weinberg equilibrium following Bonferroni correction. Thirty nine markers, which were shown to be polymorphic in a full-sibling family, were tested in Mendelian segregations. As expected, 32 loci were co-dominantly segregated in a Mendelian fashion. These novel developed microsatellite markers represent useful research tools for investigation of population genetic structure and genetic diversity in this species.     

野生二粒小麦 Triticum dicoccoides抗杀锈病基因 YrH52分子定位的初步研究(英文)

主要对以色列野生二粒小麦赫尔蒙种群中分离获得的一个抗条锈病基因进行了分子定位研究 ,将源于赫尔蒙山具抗杀锈病的种系 T.dicoccides H52与普通的栽培种 Langdon进行杂交并创建了 F2 代遗传图。研究发现 H52种系抗条锈病的能力由一种显性基因控制 ,将其暂定名为 Yr H52。从 1 2 0个微卫星标记中 ,已经检测到来自亲本 91 %的多态性 ,而且从其中 56个微卫星分子标记中产生了 79个分离的位点 ,有 9个位点显示出了与 Yr H 52基因连锁 ,其重组率 0 .0 2～ 0 .3 5,遗传距离 2 .0 0～ 4 3 .3 7cm之间 ,L OD值 3 .56～ 54.2 2。由 1 0个微卫星位点和 Yr H52构建的染色体 1 B遗传图 ,其图距全长为 1 0 1 .5cm。Yr H52基因位于 Xgwm2 64 a和 Xgwm2 64 c之间 ,且与 Xgwm2 64 a、Xgwm1 8紧密连锁 ,两侧依次分别与 Xgwm1 3 1 a、Xgwm63 6b、Xgwm2 64 c、Xgwm4 0 3 a、Xgwm1 53、Xgwm550 a和 Xgwm1 2 4连锁。同时 ,Yr H52也与 REL P标记物 N or1紧密连锁 ,图距 1 .4 cm,L OD2 9.62。这显然与野生二粒小麦另一个抗条锈病基因 Yr1 5不同 ,研究证明 Yr1 5与 N or1图距是 1 1 .0 cm。     

流沙湾卵鳎微卫星标记的遗传多样性分析

为分析湛江流沙湾海域优势渔种卵鳎的遗传多样性,应用微卫星标记技术,选用15对微卫星引物,以等位基因数、基因杂合度、多态信息含量、固定指数等遗传参数为指标,评估卵鳎群体内的遗传多态性。结果表明:共检测到90个等位基因,等位基因数从1~12不等,平均为6.0;有效等位基因数从1.0~8.4,平均为4.0,多态性位点比例为53%,显示其具有中等杂合子水平,其中8个多态位点的期望杂合度(He)为0.670~0.881,平均为0.800,观测杂合度(Ho)为0.353~1.000,平均为0.773,多态信息含量(PIC)值为0.616~0.870,平均为0.773,群体内固定指数F为-0.199~0.564,平均为0.046;流沙湾卵鳎群体具有高度遗传多样性。     

Development of a SCAR marker for male gametophyte of Gracilariopsis lemaneiformis based on AFLP technique

The red alga Gracilariopsis lemaneiformis （Bory） is an economically valuable macroalgae. As a means to identify the sex of immature Gracilariopsis lemaneiformis, the amplified fragment length polymorphism （AFLP） technique was used to search for possible sex- or phase-related markers in male gametophytes, female gametophytes, and tetrasporophytes, respectively. Seven AFLP selective amplification primers were used in this study. The primer combination E-TG/M-CCA detected a specific band linked to male gametophytes. The DNA fragment was recovered and a 402-bp fragment was sequenced. However, no DNA sequence match was found in public databases. Sequence characterized amplified region （SCAR） primers were designed from the sequence to test the repeatability of the relationship to the sex, using 69 male gametophytes, 139 female gametophytes, and 47 tetrasporophytes. The test results demonstrate a good linkage and repeatability of the SCAR marker to sex. The SCAR primers developed in this study could reduce the time required for sex identification of Gracilariopsis lemaneiformis by four to six months. This can reduce both the time investment and number of specimens required in breeding experiments.     

Development of genomic microsatellite multiplex PCR using dye-labeled universal primer and its validation in pedigree analysis of Pacific oyster (<Emphasis Type="Italic">Crassostrea gigas</Emphasis>)

There is an increasing requirement for traceability of aquaculture products, both for consumer protection and for food safety. There are high error rates in the conventional traceability systems depending on physical labels. Genetic traceability technique depending on DNA-based tracking system can overcome this problem. Genealogy information is essential for genetic traceability, and microsatellite DNA marker is a good choice for pedigree analysis. As increasing genotyping throughput of microsatellites, microsatellite multiplex PCR has become a fast and cost-effective technique. As a commercially important cultured aquatic species, Pacific oyster Crassostrea gigas has the highest global production. The objective of this study was to develop microsatellite multiplex PCR panels with dye-labeled universal primer for pedigree analysis in C. gigas, and these multiplex PCRs were validated using 12 full-sib families with known pedigrees. Here we developed six informative multiplex PCRs using 18 genomic microsatellites in C. gigas. Each multiplex panel contained a single universal primer M13(?21) used as a tail on each locus-specific forward primer and a single universal primer M13(?21) labeled with fluorophores. The polymorphisms of the markers were moderate, with an average of 10.3 alleles per locus and average polymorphic information content of 0.740. The observed heterozygosity per locus ranged from 0.492 to 0.822. Cervus simulations revealed that the six panels would still be of great value when massive families were analysed. Pedigree analysis of real offspring demonstrated that 100% of the offspring were unambiguously allocated to their parents when two multiplex PCRs were used. The six sets of multiplex PCRs can be an important tool for tracing cultured individuals, population genetic analysis, and selective breeding program in C. gigas.     

Structure of mitochondrial DNA control region of Pholis fangi and its phylogenetic implication

In this study, the entire mitochondrial DNA （mtDNA） control region （CR） of Pholis fangi was amplified via polymerase chain reaction followed by direct sequencing. The length of the mtDNA CR consensus sequence of P. fangi was 853 bp in length. In accordance with the recognition sites as were previously reported in fish species, the mtDNA CR sequence of P. fangi can be divided into 3 domains, i.e., the extended terminal associated sequence （ETAS）, the central conserved sequence block （CSB）, and the CSB domain. In addition, the following structures were identified in the mtDNA CR sequence of P. fangi：2 ETASs in the ETAS domain （TAS and cTAS）, 6 CSBs in the central CSB domain （CSB-F to CSB-A）, and 3 CSBs in the CSB domain （CSB-1 to CSB-3）. These demonstrated that the structure of the mtDNA CR of P. fangi was substantially different from those of most other fish species. The mtDNA CR sequence of P. fangi contained one conserved region from 656 bp to 815 bp. Similar to most other fish species, P. fangi has no tandem repeat sequences in its mtDNA CR sequence. Phylogenetic analysis based on the complete mtDNA CR sequences showed that there were no genetic differences within P. fangi populations of the same geographical origin and between P. fangi populations of different geographical origins.     

Measurement of single-fish target strength in the South China Sea

We measured the target strength (TS) of three commercial fish species: whitespotted spinefoot (Siganus canaliculatus), black porgy (Acanthopagrus schlegelii), and creek red bream (Lutjanus argentimaculatus), in the South China Sea. The TS of caged or tethered fish (n=76 total) was measured using a Simrad EY60 portable scientific echosounder at 120 kHz. We evaluated the relationship between TS and total length (TL, cm) for the three species. This is the first attempt to use split-beam acoustics to measure single-fish TS in the South China Sea by Chinese researchers. Our results will improve the accuracy and precision of acoustic abundance estimates of commercially important species and further the development of underwater acoustic survey techniques in fisheries in the South China Sea.     

Sequences characterization of microsatellite DNA sequences in Pacific abalone (Haliotis discus hannai)

The microsatellite-enriched library was constructed using magnetic bead hybridization selection method, and the microsatellite DNA sequences were analyzed in Pacific abalone Haliotis discus hannai. Three hundred and fifty white colonies were screened using PCR-based technique, and 84 clones were identified to potentially contain microsatellite repeat motif. The 84 clones were sequenced, and 42 microsatellites and 4 minisatellites with a minimum of five repeats were found (13.1% of white colonies screened). Besides the motif of CA contained in the oligoprobe, we also found other 16 types of microsatellite repeats including a dinucleotide repeat, two tetranucleotide repeats, twelve pentanucleotide repeats and a hexanucleotide repeat. According to Weber(1990), the microsatellite sequences obtained could be categorized structurally into perfect repeats (73.3%), imperfect repeats(13.3%), and compound repeats (13.4%). Among the microsatellite repeats, relatively short arrays (＜ 20 repeats) were most abundant,accounting for 75.0%. The largest length of microsatellites was 48 repeats, and the average number of repeats was 13.4. The data on the composition and length distribution of microsatellites obtained in the present study can be useful for choosing the repeat motifs for microsatetlite isolation in other abalone species.     

Genetic variation of natural and cultured stocks of Paralichthys olivaceus by allozyme and RAPD

Population genetics of the left-eyed flounder, Paralichthys olivaceus, including natural and cultured stocks distributed in the coastal waters near Qingdao of eastern maritime China, was analyzed in allozyme and RAPD. The results showed that among total 29 gene loci of 15 isozymes, 9 and 7 were po- lymorphic in natural and cultured stocks, respectively. The status of genetic diversity in P. olivaceus is low in terms of polymorphic loci in chi-square test and genetic departure index of Hardy-Weinberg equi- librium. More alleles in IDHP, CAT, GDH and Ldh-C allozymes were found in the fish, which could be used as markers in assortive breeding and distinguishing stock, population or species evolution. Total 88 and 86 RAPD bands ranging from 200 to 2 500 bp were recognized individually in average of 7.8–8.0 bands per primer. The genetic diversity in cultured stock is lower than that in natural ones showing an ob- viously decreasing genetic divergence. Therefore, effective countermeasures must be taken to protect ge- netic resources of marine cultured fishes. The 2 markers have their own pros and cons. Combining the 2 markers to investigate the genetic variation of populations is suggested. The results provide basic data of this flounder and they are useful for studying genetic improvement and genetic resources of the fish.     

Development of Genomic Microsatellite Multiplex PCR Using Dye-Labeled Universal Primer and Its Validation in Pedigree Analysis of Pacific Oyster(Crassostrea gigas)

There is an increasing requirement for traceability of aquaculture products, both for consumer protection and for food safety. There are high error rates in the conventional traceability systems depending on physical labels. Genetic traceability technique depending on DNA-based tracking system can overcome this problem. Genealogy information is essential for genetic traceability, and microsatellite DNA marker is a good choice for pedigree analysis. As increasing genotyping throughput of microsatellites, microsatellite multiplex PCR has become a fast and cost-effective technique. As a commercially important cultured aquatic species, Pacific oyster Crassostrea gigas has the highest global production. The objective of this study was to develop microsatellite multiplex PCR panels with dye-labeled universal primer for pedigree analysis in C. gigas, and these multiplex PCRs were validated using 12 full-sib families with known pedigrees. Here we developed six informative multiplex PCRs using 18 genomic microsatellites in C. gigas. Each multiplex panel contained a single universal primer M13(-21) used as a tail on each locus-specific forward primer and a single universal primer M13(-21) labeled with fluorophores. The polymorphisms of the markers were moderate, with an average of 10.3 alleles per locus and average polymorphic information content of 0.740. The observed heterozygosity per locus ranged from 0.492 to 0.822. Cervus simulations revealed that the six panels would still be of great value when massive families were analysed. Pedigree analysis of real offspring demonstrated that 100% of the offspring were unambiguously allocated to their parents when two multiplex PCRs were used. The six sets of multiplex PCRs can be an important tool for tracing cultured individuals, population genetic analysis, and selective breeding program in C. gigas.