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.     

A microsatellite genetic linkage map of black rockfish (Sebastes schlegeli)

Ovoviviparous black rockfish (Sebastes schlegeli) is an important marine fish species for aquaculture and fisheries in China. Genetic information of this species is scarce because of the lack of microsatellite markers. In this study, a large number of microsatellite markers of black rockfish were isolated by constructing microsatellite-enriched libraries. Female- and male-specific genetic linkage maps were constructed using 435 microsatellite markers genotyped in a full-sib family of the fish species. The female linkage map contained 140 microsatellite markers, in which 23 linkage groups had a total genetic length of 1334.1 cM and average inter-marker space of 13.3 cM. The male linkage map contained 156 microsatellite markers, in which 25 linkage groups had a total genetic length of 1359.6 cM and average inter-marker distance of 12.4 cM. The genome coverage of the female and male linkage maps was 68.6% and 69.3%, respectively. The female-to-male ratio of the recombination rate was approximately 1.07:1 in adjacent microsatellite markers. This paper presents the first genetic linkage map of microsatellites in black rockfish. The collection of polymorphic markers and sex-specific linkage maps of black rockfish could be useful for further investigations on parental assignment, population genetics, quantitative trait loci mapping, and marker-assisted selection in related breeding programs.     

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.     

鲤鱼(Cyprinus carpio L.)体重和体长QTL的定位

利用265个AFLP标记、127个微卫星分子标记、37个EST-SSR标记和16个RAPD标记(共445个标记)对大头鲤/荷包红鲤抗寒品系的F2代雌核发育群体44个个体进行基因型检测,构建鲤鱼(Cyprinus carpio L.)遗传连锁图谱;利用软件WinQTLCart2.5采用复合区间作图法对体重和体长两个性状进行了QTL定位分析。结果共检测到与两个与体重性状相关的QTL,分别定位到LG24连锁群(qBWh-24-1)和LG20连锁群(qBWh-20-1)上,可解释的表型变异分别为7.98%和20.05%;检测到两个与体长性状相关的QTL,分别位于LG2连锁群(qFS-2-1)和LG20连锁群(qFS-20-1),可解释的表型变异分别为5.69%和12.69%,4个QTL的加性效应均为负值。     

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.     

鲤鱼（Cyprinuscarpio L．）体重和体长QTL的定位

利用265个AFLP标记、127个微卫星分子标记、37个EST-SSR标记和16个RAPD标记（共445个标记）对大头鲤／荷包红鲤抗寒品系的F2代雌核发育群体44个个体进行基因型检测，构建鲤鱼（CyprinuscarpioL.）遗传连锁图谱；利用软件WinQTLCart2.5采用复合区间作图法对体重和体长两个性状进行了QTL定位分析。结果共检测到与两个与体重性状相关的QTL，分别定位到LG24连锁群（qBWh-24-1）和LG20连锁群（qBWh-20-1）上，可解释的表型变异分别为7.98％和20.05％；检测到两个与体长性状相关的QTL，分别位于LG2连锁群（qFS-2-1）和LG20连锁群（qFS-20-1），可解释的表型变异分别为5.69％和12.69％，4个QTL的加性效应均为负值。     

Reproductive biology of yellowfin tuna T. albacares in the west-central Indian Ocean

Survey of yellowfin tuna in the west-central Indian Ocean was conducted on board of Chinese longliners during 2003, 2004 and 2005, which is a part of Chinese Tuna Fishery Scientific Observer Program (CTFSOP). The reproductive biology has been investigated. A total of 1 023 samples are collected including 417 ovaries and 606 testes. Spawning activities of yellowfin tuna have been studied for both male and female from January to June. The data showed that the average monthly sex ratio is 0.59, and the minimum length at sexual maturity is 101 cm for female and 110 cm for male respectively. Length at 50% sexual maturity is estimated at 113.77 cm for female and 120.20 cm for male, whereas maturation rate is 0.066 cm⁻¹ for female and 0.091 cm⁻¹ for male. Sex ratio by length class indicates that the proportion of male is higher than female’s along with size increasing; for instance, in the group of the body length longer than 145 cm, some females have their body length from 145 to 160 cm and males have their body length at 160 cm and even longer. Statistically, yellowfin tuna has a significant seasonal reproduction.     

Reproductive Biology of Yeilowfin Tuna T. albacares in the West-Central Indian Ocean

Survey of yellowfin tuna in the west-central Indian Ocean was conducted on board of Chinese Iongliners during 2003, 2004 and 2005, which is a part of Chinese Tuna Fishery Scientific Observer Program (CTFSOP). The reproductive biology has been investigated. A total of 1023 samples are collected including 417 ovaries and 606 testes. Spawning activities of yellowfin tuna have been studied for both male and female from January to June. The data showed that the average monthly sex ratio is 0.59, and the minimum length at sexual maturity is 101 cm for female and 110 cm for male respectively. Length at 50% sexual maturity is esti- mated at 113.77 cm for female and 120.20 cm for male, whereas maturation rate is 0.066 cm-1 for female and 0.091 cm-1 for male. Sex ratio by length class indicates that the proportion of male is higher than female's along with size increasing; for instance, in the group of the body length longer than 145 cm, some females have their body length from 145 to 160 cm and males have their body length at 160 cm and even longer. Statistically, yellowfin tuna has a significant seasonal reproduction.     

Isolation, cloning and sequencing of AFLP markers related to disease-resistance traits in Fenneropenaeus chinensis

Amplified fragment length polymorphism (AFLP) technique was used to analyze the fingerprinting of four successive generations ofFenneropenaeus chinensis to reveal their disease-resistance traits. Some loci showed quite different genetic frequencies due to artificial selection, which implied that these fragments were putative markers related to the disease-resistance trait. We developed a simple and effective method to further characterize these AFLP fragments. Specific AFLP bands were cut directly from polyacrylamide gels, re-amplified, cloned and sequenced. Eight putative genetic markers were sequenced and their sizes ranged from 63 to 209 bp. The sequences were submitted to dbGSS (database of Genome Sequence Survey); and the BLAST analysis showed low similarity to the function genes, indicating these markers were tightly linked to a disease-resistance trait but were not functional genes. This research was supported by special funds from the National Key Basic Research Program (G1999012007) and the National High-Tech Research and Development Program of China (863 Program. 2001AA620105).     

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.     

Genetics and phylogeny of genus Coilia in China based on AFLP markers

The taxonomy of Coilia has been extensively studied in China, and yet phylogenetic relationships among component taxa remain controversial. We used a PCR-based fingerprinting technique, amplified fragment length polymorphism (AFLP) to characterize and identify all four species of Coilia in China. We examined the genetic relationships of the four species of Coilia and a subspecies of Coilia nasus with AFLP. A total of 180 AFLP loci were generated from six primer combinations, of which 76.11% were polymorphic. The mean genetic distance between pairs of taxa ranged from 0.047 to 0.596. The neighbor-joining tree and UPGMA dendrogram resolved the investigated species into three separate lineages: (1) C. mystus, (2) C. grayii and (3) C. brachygnathus, C. nasus, and C. nasus taihuensis. Phylogenetic analysis of the AFLP data is inconsistent with current morphological taxonomic systems. The AFLP data indicated a close relationship among C. brachygnathus, C. nasus taihuensis, and C. nasus. Therefore, the two species described under Coilia (C. brachygnathus and C. nasus taihuensis) are treated as synonyms of C. nasus.     

Studies on the breeding habits of the minke whale (Balaenoptera acutorostrata) in the Yellow Sea

Minke whales caught in the Yellow Sea range from 4.16 m to 8.60 m in length with an average length of 6.93 m for the female and 6.14 m for the male. Sex ratio of the female is 74.3%. The pregnancy ratio of all sexually matured females is 65.8%. The minimum size of the pregnant whale observed is 6.60 m in length. Females having a body length of more than 7 m long are all sexually matured. According to the studies on the embryos obtained from the females caught in the northern Yellow Sea, there is only one breeding season each year. Mating mostly occurs from July through September, and the length of pregnancy is ten to eleven months with the peak of calving from May through July. Most females breed one calf each time, only two cases with twins. Sex ratio of female fetus is 56.6%, and the newly born calf measures 250–270 cm in body length. This paper was published in Chinese inOceanologia et Limnologia Sinica 13 (4): 338–345.     

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.     

BAC end sequencing of Pacific white shrimp <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>: a glimpse into the genome of Penaeid shrimp

Little is known about the genome of Pacific white shrimp (Litopenaeus vannamei). To address this, we conducted BAC (bacterial artificial chromosome) end sequencing of L. vannamei. We selected and sequenced 7 812 BAC clones from the BAC library LvHE from the two ends of the inserts by Sanger sequencing. After trimming and quality filtering, 11 279 BAC end sequences (BESs) including 4 609 paired- ends BESs were obtained. The total length of the BESs was 4 340 753 bp, representing 0.18% of the L. vannamei haploid genome. The lengths of the BESs ranged from 100 bp to 660 bp with an average length of 385 bp. Analysis of the BESs indicated that the L. vannamei genome is AT-rich and that the primary repeats patterns were simple sequence repeats (SSRs) and low complexity sequences. Dinucleotide and hexanucleotide repeats were the most common SSR types in the BESs. The most abundant transposable element was gypsy, which may contribute to the generation of the large genome size of L. vannamei. We successfully annotated 4 519 BESs by BLAST searching, including genes involved in immunity and sex determination. Our results provide an important resource for functional gene studies, map construction and integration, and complete genome assembly for 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。     

Isolation, cloning and sequencing of AFLP markers related to disease-resistance traits in Fenneropenaeus chinensis

Amplified fragment length polymorphisms （AFLP） technique was used to analyze the fingerprinting of four successive generations of Fenneropenaeus chinensis to reveal their disease-resistance traits. Some loci showed quite different genetic frequencies due to artificial selection, which implied that these fragments were putative markers related to the disease-resistance trait. We developed a simple and effective method to further characterize these AFLP fragments. Specific AFLP bands were cut directly from polyacrylamide gels, re-amplified, cloned and sequenced. Eight putative genetic markers were sequenced and their sizes ranged from 63 to 209 bp. The sequences were submitted to dbGSS （database of Genome Sequence Survey）; and the BLAST analysis showed low similarity to the function genes, indicating these markers were tightly linked to a disease-resistance trait but were not functional genes.     

Preliminary analysis of population genetic diversity of cultivated <Emphasis Type="Italic">Laminaria japonica</Emphasis> sporophyte via AFLP technique

The amplified fragment length polymorphic DNA (AFLP) technique was adopted to estimate the population genetic polymorphism among 30 sporophytes of Laminaria japonica collected from a cultivating farm in Rongcheng, China. Three methods were used for genomic DNA extraction from Laminaria japonica sporophyte and only the products obtained using the improved genomic DNA extraction kit method proved qualified for AFLP analysis. The parameters of the method were optimized. Samples of forty milligrams and the cell lysis time of 120 min were suggested to replace the parameters recommended by the manufacturer. Thirty individuals of Laminaria japonica from the same cultivating site were investigated using one pair of selective primers. A total of 21 loci were obtained and 17 of them were polymorphic. The mean percent age of polymorphic loci of this population was 80.95%. The Nei’s gene diversity (H) within this population was 0.3028 and the average Shannon’s Information index (I) was 0.4498. A genetic distance matrix among different individuals was constructed as well. Through this study, an applicable AFLP genetic analysis working system for Laminaria japonica sporophyte was established. The results of this research also revealed a high level of genetic diversity within the studied population.     

Characterization of genic microsatellite markers derived from expressed sequence tags in Pacific abalone (Haliotis discus hannai)

Simple sequence repeat (SSR) markers were developed from the expressed sequence tags (ESTs) of Pacific abalone (Haliotis discus hannai). Repeat motifs were found in 4.95% of the ESTs at a frequency of one repeat every 10.04 kb of EST sequences, after redundancy elimination. Seventeen polymorphic EST-SSRs were developed. The number of alleles per locus varied from 2–17, with an average of 6.8 alleles per locus. The expected and observed heterozygosities ranged from 0.159 to 0.928 and from 0.132 to 0.922, respectively. Twelve of the 17 loci (70.6%) were successfully amplified in H. diversicolor. Seventeen loci segregated in three families, with three showing the presence of null alleles (17.6%). The adequate level of variability and low frequency of null alleles observed in H. discus hannai, together with the high rate of transportability across Haliotis species, make this set of EST-SSR markers an important tool for comparative mapping, marker-assisted selection, and evolutionary studies, not only in the Pacific abalone, but also in related species.     

Analysis of the genetic diversity and differentiation of <Emphasis Type="Italic">Fenneropenaeus penicillatus</Emphasis> populations using AFLP technology

Fenneropenaeus penicillatus (redtail shrimp) is an important marine commercial animal in China. Recently, its resources have been depleted rapidly as a result of, for example, over-exploitation and environmental degradation of spawning grounds. Therefore, we analyzed the genetic diversity and differentiation of nine wild populations of F. penicillatus of China (Ningde, Lianjiang, Putian, Xiamen, Quanzhou, Zhangpu, Dongshan, Nanao, and Shenzhen populations) by amplified fragment length polymorphism (AFLP) technology, to provide genetic information necessary for resource protection, rejuvenation, artificial breeding, and sustainable use of the resource. Eight AFLP primer pairs were used for amplification, and 508 bands were detected among the populations. The results show that the percentage of polymorphic loci (P) ranged from 41.34% to 63.58%; the Nei’s gene diversity (H) of the populations was 0.119 4-0.230 5; and Shannon’s Information Index (I) was 0.184 1-0.342 5. These genetic data indicate that the genetic diversity of F. penicillatus was high. The genetic differentiation coefficient (GST=0.216 2) and gene flow (Nm=1.812 4) show that there was a high level of genetic differentiation and a moderate level of gene flow among populations. More studies on the genetic differentiation mechanism of F. penicillatus along the south-eastern coast of China need to be conducted to find more effective scientific protection strategies for the conservation of F. penicillatus genetic resources.