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).     

草鱼种质退化相关SRAP分子标记的筛选

采用相关序列扩增多态性(sequence-realted amplified polymorphism,SRAP)技术分析野生草鱼和家养草鱼,并筛选与草鱼种质退化相关的分子遗传标记。共进行88对引物组合的检测,产生标记数目共计905个。依据标记在群体中出现的频率和变化规律,共筛选出21个可能与种质相关的特异性标记,对这些特异性标记进行测序并将测序结果进行BLAST分析,发现测得片段中有8个片段能在GeneBank中找到同源性较高的序列,而其他片段与数据库中序列的相似性较低。     

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.     

Genetic analysis of floating Enteromorpha prolifera in the Yellow Sea with AFLP marker

Extremely large accumulation of green algae Enteromorpha prolifera floated along China’coastal region of the Yellow Sea ever since the summer of 2008.Amplified Fragment Length Polymorphism (AFLP) analysis was applied to assess the genetic diversity and relationships among E. prolifera samples collected from 9 affected areas of the Yellow Sea.Two hundred reproducible fragments were generated with 8 AFLP primer combinations,of which 194 (97%) were polymorphic. The average Nei’s genetic diversity, the coefficiency of genetic differentiation (Gst), and the average gene flow estimated from Gst in the 9 populations were 0.4018, 0.6404 and 0.2807 respectively. Cluster analysis based on the unweighed pair group method with arithmetic averages (UPGMA) showed that the genetic relationships within one population or among different populations were all related to their collecting locations and sampling time. Large genetic differentiation was detected among the populations.The E. prolifera originated from different areas and were undergoing a course of mixing.     

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.     

Application of AFLP markers for population genetic study on half-smooth tongue sole Cynoglossus semilaevis

The genetic diversity of wild and hatchery populations of half-smooth tongue sole Cynoglossus semilaevis, based on observation of amplified fragment length polymorphism (AFLP) was described. Two hundred individuals from four wild populations, Laizhou (LZ), Weihai (WH), Qingdao (QD), Rizhao (RZ), and one hatchery population, Mingbo (MB), were screened using eight different AFLP primer combinations. A total of 384 loci were screened in the five studied populations. 48.4%, 51.3%, 50.7%, 49.3% and 45.8% of these loci were polymorphic among the individuals tested in the LZ, WH, QD, RZ and MB populations, respectively. The number of polymorphic loci detected by single primer combinations ranged from 17 to 35. The average heterozygosity of the LZ, WH, QD, RZ and MB populations was 0.072, 0.093, 0.092, 0.090 and 0.063, respectively. The WH population showed the highest genetic diversity in terms of total number of AFLP bands, total number of polymorphic bands, average heterozygosity and percentage of low frequency (0-0.2) polymorphic loci among all the populations, while the LZ population was the lowest among the wild populations. Compared with the wild populations, the hatchery population showed a low genetic viability.     

Design of Vibrio 16S rRNA Gene Specific Primers and Their Application in the Analysis of Seawater Vibrio Community

The pathogenic species of genus Vibrio cause vibriosis, one of the most prevalent diseases of maricultured animals and seafood consumers. Monitoring their kinetics in the chain of seafood production, processing and consumption is of great importance for food and mariculture safety. In order to enrich Vibrio-representing 16S ribosomal RNA gene （rDNA） fragments and identify these bacteria further real-timely and synchronously among bacterial flora in the chain, a pair of primers that selectively amplify Vibrio 16S rDNA fragments were designed with their specificities and coverage testified in the analysis of seawater Vibrio community. The specificities and coverage of two primers, VF169 and VR744, were determined theoretically among bacterial 16S rDNAs available in GenBank by using BLAST program and practically by amplifying Vibrio 16S rDNA fragments from seawater DNA. More than 88.3% of sequences in GenBank, which showed identical matches with VR744, belong to Vibrio genus. A total of 33 clones were randomly selected and sequenced. All of the sequences showed their highest similarities to and clustered around those of diverse known Vibrio species. The primers designed are capable of retrieving a wide range of Vibrio 16S rDNA fragments specifically among bacterial flora in seawater, the most important natural environment of seafood cultivation.     

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.     

3种柔鱼线粒体基因与核核糖体基因片段序列比较分析

为准确检测柔鱼（Ommαstrephesbartram川、茎柔鱼（ Dosidicus gigas）与阿根廷滑柔鱼（ IIIex argentinus ） 的种间遗传差异,对线粒体16SrRNA、细胞色素b（Cytb）与编码核糖体大亚基的基因（28SrDNA）片段序列进 行测定。经比对获得同源片段序列的长度分别为444、430、464坤,其中16SrRNA与28SrDNA基因片段上分别存在3处和47处碱基插入/缺失。核昔酸组成分析表明;3种柔鱼在3个基因片段上的核音酸组成差异不显著, 在线粒体2个基因片段上的A＋T含量（16SrRNA;69.90%、72.01%、74.66%; Cytb; 63.61%、68.91%、71.65% ） 均明显高于C＋C含量（16SrRNA;30.10%、27.99%、25.34%; Cytb; 36.39%、31.09%、28.35% ）,而在28SrDNA 基因片段上的A＋T含量（37.16%、36.74%、38.29% ）明显低于C＋C含量（62.84%、63.26%、61.71 %）0 3种柔鱼 在28SrDNA基因片段上检测到的核昔酸替代率最低,为6.68%,而蛋白质编码基因Cytb核昔酸替代率最高,为 20.93%,核营酸替代均发生在密码子第3位点上,而且未引起氨基酸替代。基于邻接法、最大简约法与最大似然 法重建的系统树显示,柔鱼与茎柔鱼的亲缘关系较近。根据C严b基因片段序列分析,柔鱼与茎柔鱼和阿根廷滑柔鱼的分歧时间分别为653-790万a和765 - 925万a,种间分化事件发生在中新世至上新世间。     

The comparison between allozyme and RAPD makers for the population genetic structure analysis of scallop Chlamys farreri

1INTRODUCTION Since the first use in human genetic research in1966,allozyme has become the most popular genetic marker for population genetics studies for almost all groups of animals and plants(Lewontin and Hubby,1966;Murphy et al.,1996).In the past seve…     

Genetic Variation and Differentiation in Wide Ranging Populations of Razor Clam （Sinonovacula constricta） Inferred from AFLP Markers

The genetic variation and differentiation of the razor clam Sinonovacula constricts distributed along the coast of China were studied through amplified fragment length polymorphism(AFLP)analysis. Six primer combinations generated 193 fragments. The He values varied from 0. 322 to 0. 463 and the percentage of polymorphic loci ranged from 74. 1% to 98. 4%, which indicates a high level of genetic diversity. Cluster analysis by Nei's pairwise distance grouped all specimens by geographical origins. AMOVA consistently showed that genetic variation among populations was 8. 71%, and most of the variation came from the genetic variation within populations(91.29%). Genetic differentiation among the six populations was moderate; pairwise FST ranged from 0. 0282 to0. 1480, which indicated that S. Constricts populations along the coast of China are genetically connected. Among all the six populations, the Beihai population is the mostly differentiated from the others, suggesting that Hainan Island and Leizhou Peninsula act as barriers to gene flow. All populations abide isolation by distance model as indicated by Mantel test, except for ZS(Zhoushan)and YQ(Yueqing)populations. Information obtained in this study will provide guidelines for conservation and fishery management of this species in the future.     

红鳍笛鲷微卫星DNA标记的开发与遗传多样性分析

以三亚外海的红鳍笛鲷(Lutjanus erythropterus)自然群体为研究对象,利用磁珠富集法开发19个适用于红鳍笛鲷的微卫星DNA标记,其中16个为多态性标记,等位基因数为2～11,期望杂合度为0.242～0.896,观测杂合度为0.156～1.000,Ler 14、Ler 29、Ler 49等3个位点偏离哈迪-温伯格平衡(P<0.0031),Ler9-Ler18、Ler12-Ler19、Ler46-Ler47、Ler46-Ler49、Ler18-Ler50、Ler14-Ler51等6对位点间检测到明显的连锁(P<0.05),Ler29、Ler13、Ler49、Ler19、Ler24、Ler25、Ler14、Ler51、Ler18、Ler9等10个位点属于高度多态位点[多态信息含量(PIC)>0.5],Ler47、Ler12、Ler15、Ler46等4个为中度多态位点(0.25>PIC>0.5)。     

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.     

Mitochondrial DNA control region diversity and population structure of Pacific herring (Clupea pallasii) in the Yellow Sea and the Sea of Japan

To investigate the genetic variation and population structure of Pacific herring in the Yellow Sea and the genetic differentiation between the Yellow Sea and the Sea of Japan,fragments of 479-bp mitochondrial DNA control region were sequenced for 110 individuals collected from three different periods in the Yellow Sea and one locality in the Sea of Japan.High haplotype diversity and moderate nucleotide diversity were observed in Pacific herring.AMOVA and exact test of population differentiation showed no significant genetic differentiations among the three populations of the Yellow Sea and suggested the populations can be treated as a single panmictic stock in the Yellow Sea.However,a large and significant genetic differentiation(WST50.11;P50.00) was detected between the populations in the Yellow Sea and the Sea of Japan.The high sea water temperature in the Tsushima Strait was thought a barrier to block the gene exchange between populations of the two sea areas.The neutrality tests and mismatch distribution indicated recent population expansion in Pacific herring.     

Preliminary Analysis of Population Genetic Diversity of Cultivated Laminaria japonica 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.     

Large-Scale Screening of Growth-Related Variants in Chinese Tongue Sole(Cynoglossus semilaevis)

Growth is the most valuable economic trait for improving aquaculture fish species, since fast growth can reduce labor cost and make more economic benefits. However, the knowledge about how many and which genes are related to the growth of Chinese tongue sole is limited. High-throughput sequencing screening of variants is a fast, economical and accurate assay to identify genes related to growth in crops, livestock and other aquaculture products. In the present study, genome-wide resequencing of 30 female Chinese tongue sole individuals from large and small groups to screen growth trait-related variations(SNPs, InDels) was carried out. In total, 6545735 SNPs and 1016745 InDels were detected, while 31 genes related to growth traits were identified. Their functions were mainly involved in muscle architecture, post-embryonic development, neurosensory development and hormone regulation. Furthermore, 18 of the 31 genes related to growth trait are located on W chromosome, indicating that W chromosome plays an important role in regulating the body size of female tongue soles. The markers and genes identified in our study can be applied to selective breeding of tongue sole and thus will promote the aquaculture industry and increase economic efficiency.     

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.