A preliminary genetic map of Zhikong scallop (Chlamys farreri Jones et Preston 1904)

Zhikong scallop (Chlamys farreri Jones et Preston 1904) is one of the most important aquaculture species in China. The development of a genetic linkage map would provide a powerful tool for the genetic improvement of this species. Amplified fragment length polymorphism (AFLP) is a PCR‐based technique that has proven to be powerful in genome fingerprinting and mapping, and population analysis. Genetic maps of C. farreri were constructed using AFLP markers and a full‐sib family with 60 progeny. A total of 503 segregating AFLP markers were obtained, with 472 following the Mendelian segregation ratio of 1:1 and 31 markers showing significant (P<0.05) segregation distortion. The male map contained 166 informative AFLP markers in 23 linkage groups covering 2468 cM. The average distance between markers was 14.9 cM. The female genetic map consisted of 198 markers in 25 linkage groups spanning 3130 cM with an average inter‐marker spacing of 15.8 cM. DNA polymorphisms that segregated in a 3:1 ratio as well as the AFLP markers that were heterozygous in both parents were included to construct combined linkage genetic map. Five shared linkage groups, ranging from 61.1 to 162.5 cM, were identified between the male and female maps, covering 431 cM. Amplified fragment length polymorphism markers appeared to be evenly distributed within the linkage groups. Although preliminary, these maps provide a starting point for the mapping of the functional genes and quantitative trait loci in C. farreri.     

Genetic linkage map of the pearl oyster,Pinctada martensii (Dunker)

Genetic linkage maps were constructed with amplified fragment length polymorphism (AFLP) and microsatellite markers for the pearl oyster, Pinctada martensii (Dunker), the main bivalve used for marine pearl production in Asia. Twenty‐four AFLP and 84 microsatellite primer pairs were used for linkage analysis in a full‐sib family with two parents and 78 offspring. Of the 2357 AFLP fragments generated, 394 (16.7%) were polymorphic and segregating. Most (340 or 86.2%) of the markers segregated according to expected Mendelian ratios. Female and male linkage maps were constructed using 230 and 189 markers, including 15 and 10 microsatellites respectively. The female map consisted of 110 markers in 15 linkage groups, covering 1415.9 cM, with an average interval of 14.9 cM. The male map consisted of 98 markers in 16 linkage groups, with a total length of 1323.2 cM and an average interval of 16.1 cM. When unlinked doublets were considered, genome coverages were 78.5% for the female and 73.5% for the male map. Although preliminary, the genetic maps constructed here should be useful for future linkage and quantitative trait loci mapping efforts.     

A first‐generation genetic map of the Japanese scallop Patinopecten yessoensis‐based AFLP and microsatellite markers

We constructed genetic linkage maps of the Japanese scallop Patinopecten yessoensis using AFLP and microsatellite markers. With 32 AFLP primer combinations, a total of 413 markers (209 from the female parent and 204 from the male parent) segregated in a 1:1 ratio, corresponding to DNA polymorphisms which were heterozygous in one parent and null in the other. Among the six microsatellite markers we used, there were four polymorphic loci. Two segregated in the female parent, and the other two segregated in both parents. In the maternal parent, 161 framework markers were mapped in 20 linkage groups, with a total coverage of 2198.8 cM. In the paternal parent, 166 framework markers established a map with 21 linkage groups, spanning a genome length of 2137.6 cM. The AFLP markers on the maps were randomly distributed with an average spacing between markers of 14.7–15.6 cM. The estimated coverage for the framework maps are 77.9% both for the female and the male. These are the first linkage maps for P. yessoensis, which constitute a basis for further genome studies and provide a useful framework for consensus map construction by adding orthologous anchor markers developed in P. yessoensis.     

牙鲆遗传作图及生长性状QTL定位

采用牙鲆日本群体和韩国群体杂交的92个F₁个体作为分离群体, 利用微卫星标记和Joinmap 4.0作图软件构建了牙鲆遗传连锁图谱。共有221个SSR标记用于连锁图谱构建, 雌性图谱中, 共178个微卫星标记定位到22个连锁群上, 观测总长度为(G _oa )599.0 cM, 覆盖率(C _oa )达76.27%。雄性图谱中, 共194个微卫星标记定位到23个连锁群上, G _oa 为693.4 cM, C _oa 为78.82%。对全长、体质量、体高3组数据进行主成分分析处理, 得到可解释3个性状的89.6%特征的一组数据, 命名为牙鲆生长性状GT。用WinQTLCart 2.5软件的复合区间作图,在已构建的遗传连锁图谱上对牙鲆生长性状GT进行QTL定位, 取LOD经验值2.5为QTL存在的阈值; 对微卫星标记进行性状—标记之间的回归分析。本研究共定位3个与牙鲆生长性状GT相关的QTLs, qGT-f4 qGT-m20 qGT-f20,可解释表型变异率分别为27.60%, 13.74%, 10.27%。在性状—标记之间的回归分析中, 得到22个与生长性状GT相关(P<0.05)的微卫星标记, 单个标记可解释表型变异率介于3.70%~10.42%, 其中6个微卫星标记scaffold558_51720、scaffold558_26183、scaffold903_69232、scaffold485_47120 、scaffold1262_77386、scaffold809_65154与生长性状GT之间呈极显著相关(P><0.01), 可解释表型变异率分别为10.42%、7.31%、10.07%、10.07%、8.39%和11.26%。     

AFLP-based genetic linkage map of marine shrimp Penaeus (Fenneropenaeus) chinensis

This paper presents the genetic linkage map of the Chinese shrimp Penaeus (Fenneropenaeus) chinensis constructed with 472 AFLP markers. A hundred F₁ progeny from an intercross between a female from the new variety “Yellow Sea No. 1” and wild caught male used for the mapping study. Two separate maps were constructed for each parent. The female linkage map consisted of 197 marker loci forming 35 linkage groups and spanned a total length of 2191.1 cM, with an average marker space of 13.5 cM. The male map consisted of 194 marker loci mapped to 36 linkage groups and spanned a total length of 1737.3 cM, with an average marker spacing of 11.0 cM. The level of segregation distortion observed in this study was 12.2%. The estimated genome length of P. chinensis was 3150.3 cM for the female and 2549.3 cM for the male, respectively. The observed genome coverage was 69.6% for the female and 68.1% for the male map. The linkage maps constructed in this study provide basic information for further linkage studies on Chinese shrimp, and more importantly, the construction of the maps are part of the work of the genetic breeding programs which will be used for growth discovered in the QTL analysis of P. chinensis.     

黄颡鱼遗传图谱构建及生长相关性状的QTL定位

以野生(♂)和人工养殖(♀)黄颡鱼杂交的100个F1个体为作图群体,用SSR、SRAP和TRAP3种DNA分子标记技术构建黄颡鱼的遗传连锁图谱。图谱整合了13个SSR标记,89个SRAP标记,26个TRAP标记。其中雌性框架图谱包括16个连锁群,图谱的长度为585.5cM;雄性框架图谱包括15个连锁群,图谱的长度为752.3cM;共享框架图谱包括5个连锁群,图谱的长度为231.3cM。用该连锁图谱对黄颡鱼的5个生长相关性状进行QTL扫描,在雌性图谱上检测到1个头宽的QTL,定位于第七连锁群上,LOD值为3.2,可解释的表型变异为13%。在雄性图谱上分别检测到1个体高和体长的QTL,均定位于第一连锁群上。体高QTL的LOD值为2.4,可解释的表型变异为12%。全长QTL的LOD值为2.1,可解释的表型变异为11%。3个QTL均可用于黄颡鱼的生长性状的标记辅助育种。     

Quantitative trait locus mapping of growth‐related traits in inter‐specific F1 hybrid grouper (Epinephelus fuscoguttatus × E. lanceolatus) in a tropical climate

Growth‐related traits are the main target of genetic breeding programmes in grouper aquaculture. We constructed genetic linkage maps for tiger grouper (Epinephelus fuscoguttatus) and giant grouper (E. lanceolatus) using 399 simple sequence repeat markers and performed a quantitative trait locus (QTL) analysis to identify the genomic regions responsible for growth‐related traits in F₁ hybrid grouper (E. fuscoguttatus × E. lanceolatus). The tiger grouper (female) linkage map contained 330 markers assigned to 24 linkage groups (LGs) and spanned 1,202.0 cM. The giant grouper (male) linkage map contained 231 markers distributed in 24 LGs and spanned 953.7 cM. Six QTLs affecting growth‐related traits with 5% genome‐wide significance were detected on different LGs. Four QTLs were identified for total length and body weight on Efu_LG8, 10, 13 and 19 on the tiger grouper map, which explained 6.6%–12.0% of the phenotypic variance. An epistatic QTL with a reciprocal association was observed between Efu_LG8 and 10. Two QTLs were identified for body weight on Ela_LG3 and 10 on the giant grouper map, which explained 6.9% of the phenotypic variance. Two‐way analysis of variance indicated that the QTL on Efu_LG13 interacts with the QTLs on Ela_LG3 and 10 with large effects on body weight. Furthermore, these six QTLs showed different features among the winter, summer and rainy seasons, suggesting that environmental factors and fish age affected these QTLs. These findings will be useful to understand the genetic structure of growth and conduct genetic breeding in grouper species.     

三疣梭子蟹莱州湾、舟山野生个体定向交配产生F2代家系的AFLP分析

以三疣梭子蟹莱州湾、舟山野生群体杂交（莱州湾♀×舟山♂）产生F2代家系63个个体及F1代亲本为实验群体，采用AFLP分子标记对其进行遗传分析。35对选择性引物共扩增出1742个位点，其中分离位点为417个，不分离位点为1325个，多态位点比例为23．9％。在分离位点中，符合1：1孟德尔分离比例的位点为226个，占总分离位点数的54．2％；偏离1：1孟德尔分离比例的位点为31个，占总分离位点数7．4％。符合3：1孟德尔分离比例的位点为128个，占总分离位点数的30．7％；偏离3：1孟德尔分离比例的位点为32个，占总分离位点数的7．7％。家系内基因多样性指数为0．130，Shannon指数为0．193。遗传距离分析显示，子代个体与母本Nei’s遗传距离介于0．064～0．111之间，与父本Nei’s遗传距离介于0．065～0．107之间。其中，63号个体与48号个体分别与父、母本亲缘关系最远。本实验中，偏分离位点比例属正常范围，杂交F2代家系遗传多样性偏低。本研究结果为三疣梭子蟹野生群体及家系遗传多样性分析，遗传连锁图谱构建等方面提供一定的技术支持。     

A genetic linkage map of the Japanese flounder, Paralichthys olivaceus

We report the first genetic linkage map of the Japanese flounder (Paralichthys olivaceus) constructed with 111 microsatellite markers and 352 AFLP fragments. The parental male linkage map consisted of 25 linkage groups while the female map consisted of 27 groups, with an average resolution of 8 and 6.6 cM, respectively. We have identified linkage among 96% of the markers and the total map length was estimated to be around 1000–1200 cM. This study reports the first low-density linkage map for the Japanese flounder and describes differences in sex recombination. Recombination rates were higher in male flounder compared to the female (7.4:1), a rare condition among vertebrates. This map is a starting point for the mapping of single loci and quantitative traits in flatfish species.     

A first genetic linkage map of bluegill sunfish (<Emphasis Type="Italic">Lepomis macrochirus</Emphasis>) using AFLP markers

Genetic linkage maps were constructed for bluegill sunfish, Lepomis macrochirus, using AFLP in a F₁ inter-population hybrid family based on a double-pseudo testcross strategy. Sixty-four primer combinations produced 4,010 loci, of which 222 maternal loci and 216 paternal loci segregated at a 1:1 Mendelian ratio, respectively. The female and male framework maps consisted of 176 and 177 markers ordered into 31 and 33 genetic linkage groups, spanning 1628.2 and 1525.3 cM, with an average marker spacing of 10.71 and 10.59 cM, respectively. Genome coverage was estimated to be 69.5 and 69.3% for the female and male framework maps, respectively. On the maternal genetic linkage map, the maximum length and marker number of the linkage groups were 122.9 cM and 14, respectively. For the paternal map, the maximum length and marker number of the linkage groups were 345.3 cM and 19, respectively, which were much greater than those on the maternal genetic linkage map. The other genetic linkage map parameters of the paternal genetic linkage map were similar to those in the maternal genetic linkage map. For both the female and male maps, the number of linkage groups was greater than the haploid chromosome number of bluegill (2n = 48), indicating some linkage groups may distribute on the same chromosome. This genetic linkage mapping is the first step toward to the QTL mapping of traits important to cultured breeding in bluegill.     

Centromere-linkage in the turbot (Scophthalmus maximus) through half-tetrad analysis in diploid meiogynogenetics

Seventy nine microsatellite markers selected across all linkage groups (LG) from a previous turbot genetic map were studied in a diploid meiogynogenetic family for centromere mapping using half-tetrad analysis. Significant deviations from Mendelian segregation were observed at 25% loci analyzed. The clustering of distorted loci at specific LGs, suggested the existence of genes of different deleterious effects. The lack of Mendelian segregation distortion at 1 day and 10 days post-hatching larvae at these loci precluded an explanation based on aberrant meiotic segregation. Heterozygote frequency distribution in gynogenetic offspring showed close to 50% values above 0.667, which suggested high chiasma interference in turbot. Complete interference appeared as the best fitting function when estimating centromere position. However, Kosambi and Haldane functions performed better at specific LGs as a consequence of the variable crossover pattern of centromere-distant markers among LG. Great concordance between half-tetrad data and the positions previously reported in the turbot map was observed. Most centromeres were localized with an error around or below 5 cM and closely linked markers exist now in 8 LGs. Centromere location was mostly in accordance with previous karyotypic information.     

Characterization of 95 novel microsatellite markers for Zhikong scallop Chlamys farreri using FIASCO-colony hybridization and EST database mining

ABSTRACT:   In order to construct a simple sequence repeat (SSR)-based genetic linkage map and to promote molecular marker-assisted selection (MAS) in scallop breeding, the methods of Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO)-colony hybridization and expressed sequence tag (EST) database mining were modified and used to develop 95 novel microsatellite markers for Zhikong scallop. The SSR-enriched library constructed by the FIASCO method consisted of 830 clones, and 295 (35.5%) positive clones were identified after colony hybridization. One hundred and fifty clones were randomly sequenced and the results showed all clones contained at least one microsatellite. Of 91 primer pairs designed, 72 were amplified scorable polymerase chain reaction (PCR) products and 70 were polymorphic with the allele number range of 3–16 alleles/locus (average 7.0 alleles/locus). When EST database mining was performed, 66 microsatellites containing ESTs were identified from 3467 sequences deposited in GenBank. Based on cluster analysis of length and GC content of the flanking regions, 47 primer pairs were designed and 23 scorable EST SSRs were obtained. Compared with genomic SSRs developed in this study, EST SSRs showed lower genetic variability with an average of 4.2 alleles/locus. The results in the present study demonstrate that modified FIASCO-colony hybridization is an efficient and low-cost method for the isolation of large numbers of microsatellite markers for scallop species.     

应用SSR和SRAP标记构建青虾遗传连锁图谱

采用SSR和SRAP标记结合拟测交策略构建青虾(Macrobrachium nipponense)遗传连锁图谱。共有175个标记(含27个SSR、148个SRAP标记)分布在53个连锁群上。每个连锁群含2~8个标记,其中不少于3个标记的连锁群有35个,连锁对18个,平均每个连锁群的标记数为3.3个;连锁群长度在6.7~91.2 cM之间,相邻标记间最大间隔为49.0 cM,最小为1.4 cM,平均间隔为13.1 cM。青虾框架图谱长度为997.2 cM,图谱观察总长度为2 270.5cM,根据估算,青虾遗传连锁图谱预期长度为4 380.6 cM,图谱的覆盖率为51.83%。本研究构建了青虾遗传连锁图谱,该图谱也是淡水虾蟹类第一张遗传连锁图谱,可为青虾QTL定位、基因克隆、遗传选育等提供指导,并为进一步构建高密度的青虾遗传连锁图谱奠定了基础。     

基于遗传连锁图谱筛选虾夷扇贝性别相关AFLP分子标记的方法

基于近年来他人构建的遗传连锁图谱,对2011–2013年2–5月期间采集的雌性、雄性及雌雄同体虾夷扇贝进行性别相关AFLP分子标记筛选。从雄性和雌性遗传连锁图谱上选取合计18对引物组合,经3批次试验,有8对引物扩增出性别相关的条带,其中Eb Mc、Ej Mf和Ei Mk重复性好,有4对引物扩增出雌雄同体特异性条带,5对引物扩增出雌性特异性条带。结果表明,基于高密度遗传连锁图谱筛选性别相关分子标记的方法简单可行,雌雄同体的基因组DNA与雌性和雄性存在显著差异,可能是一个单独的群体。     

Application of amplified fragment length polymorphism markers to assess molecular polymorphisms in gynogenetic haploid embryos of turbot (Scophthalmus maximus)

Among the variety of cultured marine species, the turbot Scophthalmus maximus is a fish of growing importance in European aquaculture. In this paper, an advanced application of AFLPs to estimate the genetic diversity of haploid gynogenetic families with the aim of obtaining a preliminary genetic map is presented. Ten EcoRI/TaqI primer combinations were tested in four families comprising diploid mothers and their haploid progenies. The amplified fragment length polymorphism (AFLP) analysis revealed an average of 6.8 polymorphic bands per primer combination and a total number of 88 polymorphisms out of 579 fragments. Among various primer pairs, seven combinations were selected in relation to the quality of profiles and number of polymorphic fragments, to be used in the determination of genetic linkage relationship between AFLP markers within the largest haploid family. Co‐migration of non‐homologous fragments was also investigated in one primer combination adding a fourth selective nucleotide to the three used in the classic TaqI AFLP protocol. Surprisingly, a rate of 38.7% of non‐homologous fragments co‐migrating with monomorphic bands was identified, due to the combined effect of homoplasy and the protocol used. Additional polymorphic markers discovered by this protocol were included in the linkage map. The turbot AFLP linkage map comprises 52 AFLP markers distributed in 12 linkage groups. On the basis of this map, turbot expected total genome length sums up to 1225.6 cM. The results confirm the usefulness of AFLPs in revealing genome segregation in haploid turbot progeny.     

Production of a base population and its responses to F1 selection in the bay scallop, Argopecten irradians irradians Lamarck (1819)

A base population of the bay scallop, Argopecten irradians irradians Lamarck, was produced by crossing two cultured bay scallop populations. After 1 year of rearing, the top 10% truncation selection of the top 10% ( i =1.755) was carried out in the base population of about 1300 adults. A control parental group with a an identical number to the select parental group was randomly selected from the entire population before isolation of the select parental group. The result showed that, at the larval stage, the growth rate of larvae in the selected line was significantly higher than that of the control ( P <0.05), and that the genetic gain was 6.78%. Owing to the lower density of control at the spat stage, the mean shell length of the control line was larger than that of the select line at day 100. When the same density was adjusted between two lines in the grow-out stage (from day 100 to 160), the daily growth rate of the selected line was significantly higher than that of the control line ( P <0.05). Survival of the select line was significantly larger than that of the control line in the grow-out stage. In conclusion, the results obtained from this experiment indicate that selective breeding from a base population with a high genetic diversity established by mass spawning between different populations appears to be a promising method of genetic improvement in bay scallop, A. irradians irradians Lamarck.     

Identification of quantitative trait loci for growth‐related traits in the swimming crab Portunus trituberculatus

The swimming crab (Portunus trituberculatus) is an economically important species in Asian aquaculture. Implementing growth‐related traits of P. trituberculatus into genetic breeding programmes is an ongoing effort. We used a previously published genetic linkage map of P. trituberculatus, containing 55 simple sequence repeat (SSR) markers and 172 amplified fragment length polymorphism (AFLP) techniques to map quantitative trait loci (QTL) for growth‐related traits in a single full sibling F₂ family. Ten growth‐related traits were measured for QTL mapping. Composite interval mapping identified 9 QTL on the female map and 16 on the male map. Individual QTL with additive effects explained 11–38% of the phenotypic variance for various traits using the female parent's map, and from 1% to 21% using the male parent's map. Two QTL explaining a large percentage of variation in body weight were detected on chromosome 17 on the female map, and on chromosome 16 on the male map, and contributed 38% and 18% of the phenotypic variance respectively. This is the first study to report the detection and positioning of major QTL affecting growth in a true crab species (Brachyura). The mapping of growth‐related QTL in this study raises the possibility of improving the growth of P. trituberculatus through marker‐assisted selection.     

海湾扇贝杂交家系与自交家系生长和存活的比较

2002年春季,利用国内现有的海湾扇贝两个不同遗传背景的养殖群体A和B的种贝为材料,采用自体受精和异体受精两种交配策略,建立了海湾扇贝的自交系和杂交系。实验由AA(A♀×A♂)、BB(B♀×B♂)、AB(A♀×B♂)、BA(B♀×A♂)四个组组成。杂交使两个表型性状———生长和存活都得到了改良。就生长而言,杂交组比自交组快,杂种优势在幼虫期为35.34%、养成阶段为21.17%;杂交组AB的生长速度比自交组AA的提高了35.53%(幼虫期)和43.32%(养成阶段),杂交组BA的生长速度比自交组BB的提高了35.13%(幼虫期)和12.34%(养成阶段)。就存活而言,两个杂交组的存活率都高于相应的自交组,杂种优势的平均值在幼虫期为19.93%,养成阶段为31.46%;杂交组AB的存活率比自交组AA的提高了25.23%(幼虫期)和49.44%(养成阶段),杂交组BA的存活率比自交组BB的提高了12.36%(幼虫期)和21.29%(养成阶段)。A、B两个群体间存在的遗传差异是它们能够获得杂种优势和性状得到改良的基础。     

Crosses between two subspecies of bay scallop Argopecten irradians and heterosis for yield traits at harvest

In order to improve the yield of bay scallop Argopecten irradians by exploiting geographic subspecies heterosis, crosses between and within two subspecies, the northern A. i. irradians (Lamarck) and the southern A. i. concentricus (Say), were created and the offspring were cultured under the same environmental conditions. The two parental populations were collected from Bohai Bay in northern China and Beibu Bay in southern China, which were distinct in particular shell colours. The results indicated that the shell colour of bay scallop was inheritable and could be used as a morphological mark to distinguish hybrid and purebred individuals. At harvest, hybrid offspring always produced a significantly higher yield than purebred offspring. Positive heterosis was achieved for shell length, shell height, shell width, total weight and adductor weight, which was 5.02, 5.22, 3.88, 7.53 and 9.47 respectively. Hybrid gain was 10.86% for shell length, 10.48% for shell height, 9.77% for shell width, 34.90% for total weight and 41.69% for adductor weight, respectively, and all were significantly >5%. The present study reveals that it is effective for improving yield by hybridization between different geographic subspecies in the bay scallop.     

利用地下海水进行海湾扇贝越冬的效果研究

把5cm以上海湾扇贝亲贝按43-50个／m^3密度吊养于水泥沉淀池,利用附近井水调温：使池水温度达4℃以上。越冬两个半月,成活率84％以上,而自然海区越冬贝成活率仅70％．升温促熟后．亲贝产卵孵化率60％以上,而自然海区越冬贝仅为15．1％。地下海水越冬贝的单位水体出苗量为505万粒／m^3和544万粒／m^3,而自然海区越冬贝出苗量仅有209万粒／m^3．证明利用地下海水调温,在沉淀池进行海湾扇贝亲贝越冬,效果明显,完全可行．