Construction of a High-Density Microsatellite Genetic Linkage Map and Mapping of Sexual and Growth-Related Traits in Half-Smooth Tongue Sole (Cynoglossus semilaevis)

High-density genetic linkage maps of half-smooth tongue sole were developed with 1007 microsatellite markers, two SCAR markers and an F1 family containing 94. The female map was composed of 828 markers in 21 linkage groups, covering a total of 1447.3 cM, with an average interval 1.83 cM between markers. The male map consisted of 794 markers in 21 linkage groups, spanning 1497.5 cM, with an average interval of 1.96 cM. The female and male maps had 812 and 785 unique positions, respectively. The genome length of half-smooth tongue sole was estimated to be 1527.7 cM for the females and 1582.1 cM for the males. Based on estimations of the map lengths, the female and male maps covered 94.74 and 94.65% of the genome, respectively. The consensus map was composed of 1007 microsatellite markers and two SCAR markers in 21 linkage groups, covering a total of 1624 cM with an average interval of 1.67 cM. Furthermore, 159 sex-linked SSR markers were identified. Five sex-linked microsatellite markers were confirmed in their association with sex in a large number of individuals selected from different families. These sex-linked markers were mapped on the female map LG1f with zero recombination. Two QTLs that were identified for body weight, designated as We-1 and We-2, accounted for 26.39% and 10.60% of the phenotypic variation. Two QTLs for body width, designated Wi-1 and Wi-2, were mapped in LG4f and accounted for 14.33% and 12.83% of the phenotypic variation, respectively. Seven sex-related loci were mapped in LG1f, LG14f and LG1m by CIM, accounting for 12.5–25.2% of the trait variation. The results should prove to be very useful for improving growth traits using molecular MAS.     

Artificial Gynogenesis and Sex Determination in Half-Smooth Tongue Sole (Cynoglossus semilaevis)

Half-smooth tongue sole (Cynoglossus semilaevis) is an important cultured marine fish as well as a promising model fish for the study of sex determination mechanisms. In the present study, a protocol for artificial gynogenesis of half-smooth tongue sole was developed in order to identify the sex determination mechanism and to generate all-female stock. The optimal UV-irradiation dose for genetically inactivating sea perch spermatozoa was determined to be ≥30 mJ/cm². The optimal initiation time for cold shock of gynogenetic embryos was determined to be 5 min after fertilization, while the optimal temperature and treatment duration were determined to be 20–25 min at 5°C. Chromosomes from common diploids, gynogenetic haploids, and diploids were analyzed. WW chromosomes were discovered in some of the gynogenetic diploids. The microsatellite marker was applied to analyze gynogenetic diploid fry. Among the 30 gynogenetic diploid fry, 11 fry contained only one allele, while 19 contained two alleles, which had the same genotype as their mother. The female-specific DNA marker was observed in four individuals out of ten gynogenetic diploid fry. Ploidy analysis of 20 putative gynogenetic fry showed them all to be diploid. Thus, a protocol for the induction of artificial gynogenesis has been developed for the first time in half smooth tongue sole, and the sex determination mechanism in the tongue sole was determined to be female heterogametic with the ZW chromosome.     

Construction of Two BAC Libraries from Half-Smooth Tongue Sole Cynoglossus semilaevis and Identification of Clones Containing Candidate Sex-Determination Genes

Half-smooth tongue sole (Cynoglossus semilaevis) is an increasingly important aquaculture species in China. It is also a tractable model to study sex chromosome evolution and to further elucidate the mechanism of sex determination in teleosts. Two bacterial artificial chromosome (BAC) libraries for C. semilaevis, with large, high-quality inserts and deep coverage, were constructed in the BamHI and HindIII sites of the vector pECBAC1. The two libraries contain a total of 55,296 BAC clones arrayed in 144 384-well microtiter plates and correspond to 13.36 haploid genome equivalents. The combined libraries have a greater than 99% probability of containing any single-copy sequence. Screening high-density arrays of the libraries with probes for female-specific markers and sex-related genes generated between 4–46 primary positive clones per probe. Thus, the two BAC libraries of C. semilaevis provided a readily useable platform for genomics research, illustrated by the isolation of sex determination gene(s).     

Induction of Mitogynogenetic Diploids and Identification of WW Super-female Using Sex-Specific SSR Markers in Half-Smooth Tongue Sole (Cynoglossus semilaevis)

The half-smooth tongue sole (Cynoglossus semilaevis) is an important cultured marine fish as well as a promising model fish for the study of sex determination mechanisms. In the present study, a protocol for artificial induction of mitogynogenesis by hydrostatic pressure using heterologous sperm was developed in half-smooth tongue sole in order to assess homozygosity of gynogens and to identify WW super-female. The optimal initiation time for pressure shock of mitogynogenetic embryos was determined to be 21.5 min after insemination when water temperature is at 22–23°C, while the optimal pressure and treatment duration were determined to be 70 MPa for 4 min. About 1,500 mitogynogenetic diploid larvae were obtained. Ten tongue sole microsatellite markers were used for homozygosity analysis of 24 mitogynogenetic larvae. Among the 24 larvae, the percentage of homozygosity ranged from 73.91% to 87.50% with an average homozygosity of 80.54%. Sex-specific simple sequence repeat (SSR) markers, CseF-SSR1, were isolated and used for identifying WW super-female mitogynogens in the tongue sole. The amplification of genomic DNA using the sex-specific SSR marker produced one DNA band of 206 bp in ZZ males, two DNA bands of 206 and 218 bp in ZW females, and one DNA band of 218 bp in WW super-females. Four WW “super-female” gynogens were observed in 39 mitogynogenetic diploids, indicating a ZW sex determination mechanism in the tongue sole. Thus, a protocol for the induction of artificial mitogynogenesis has been developed for the first time in half-smooth tongue sole, and the WW super-female diploids were identified in the mitogynogens by sex-specific SSR markers. These findings lay the foundation and provide important tool for the elaboration of sex determination mechanism, generation of WW super-females, and development of clone line and breeding of all-female stock in the half-smooth tongue sole.     

Fosmid Library Construction and Initial Analysis of End Sequences in Female Half-smooth Tongue Sole (Cynoglossus semilaevis)

Half-smooth tongue sole (Cynoglossus semilaevis: Pleuronectiformes) is a commercially important cultured marine flatfish in China and forms an important fishery resource, but the research of its genome is underdeveloped. In this study, we constructed a female C. semilaevis fosmid library and analyzed the fosmid end sequences to provide a preliminary assessment of the genome. The library consists of 49,920 clones with an average insert size of about 39 kb, amounting to 3.23 genome equivalents. Fosmid stability assays indicate that female C. semilaevis DNA was stable during propagation in the fosmid system. Library screening with eight microsatellite markers yielded between two and five positive clones, and none of those tested was absent from the library. End-sequencing of both 5′ and 3′ ends of 1,152 individual clones generated 2,247 sequences after trimming, with an average sequence length of 855 bp. BLASTN searches of the nr and EST databases of GenBank and BLASTX searches of the nr database resulted in 259 (11.53%) and 287 (12.77%) significant hits (E < e ⁻⁵), respectively. Repetitive sequences analysis resulted in 5.23% of base pairs masked using both the Fugu and Danio databases, repetitive elements were composed of retroelements, DNA transposons, satellites, simple repeats, and low-complexity sequences. The fosmid library, in conjunction with the fosmid end sequences, will serve as a useful resource for large-scale genome sequencing, physical mapping, and positional cloning, and provide a better understanding of female C. semilaevis genome.     

Isolation of Female-Specific AFLP Markers and Molecular Identification of Genetic Sex in Half-Smooth Tongue Sole (<Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>)

The sex-specific molecular marker is a useful gene resource for studying sex- determining mechanisms and controlling fish sex. Artificially produced male and female half-smooth tongue sole (Cynoglossus semilaevis) were used to screen sex-specific amplified fragment length polymorphism (AFLPs) molecular markers. The phenotypic sex of 28 tongue soles was determined by histological sectioning of gonads. The AFLP analysis of 15 females and 13 males via 64 primer combinations produced a total of 4681 scorable bands, of which 42.11% and 43.39% of bands were polymorphic in females and males, respectively. Seven female-specific AFLP markers were identified and designated as CseF382, CseF575, CseF783, CseF464, CseF136, CseF618, and CseF305, respectively. One female-specific AFLP marker (CseF382) was amplified, recovered from the gels, cloned, and sequenced (accession no. DQ487760). This female-specific AFLP marker was converted into a single-locus polymerase-chain reaction (PCR) marker of a sequence-characterized amplified region (SCAR). A simple PCR method of using the specific primers was developed for identifying genetic sex of half-smooth tongue sole. PCR products demonstrated that the initial 15 females produced the female-specific band of about 350 bp, but the initial 13 male individuals failed to produce the band. We also investigated the applicability of the PCR primers in other tongue sole individuals. The same female-specific fragment of about 350 bp was found in the additional 59 female individuals, but not in the additional 58 male individuals. This AFLP-based molecular sexing technique may have great application potential in elucidation of sex determination mechanisms and sex control in half-smooth tongue sole.     

Cloning and Characterization of tesk1, a Novel Spermatogenesis-Related Gene,in the Tongue Sole (Cynoglossus semilaevis)

Testis-specific protein kinase 1 (Tesk1) is a serine/threonine kinase with unique structural features. In the present study, we cloned and characterized the tesk1 gene of tongue sole, Cynoglossus semilaevis. The full-length tesk1 cDNA consists of 1,672 nucleotides, encoding a 331 amino acid polypeptide with a characteristic structure composed of an N-terminal kinase domain and a C-terminal proline-rich domain. The tesk1 genomic sequence contains eight exons and seven introns. Real-time quantitative PCR revealed that tesk1 mRNA is expressed predominantly in the testis, though the level of expression varied throughout development. We used in situ hybridization to show that tesk1 mRNA is expressed in the spermatids of males and pseudo-males, but not in triploid males. Our results suggest that tongue sole Tesk1 may play a role in spermatogenesis.     

分子标记在绿豆遗传连锁图谱构建和基因定位研究中的应用

绿豆(Vigna radiata(L.)Wilczek)作为一种医食两用作物,不仅是重要的食物资源,在改善土壤环境、提高农民收入等方面也发挥着重要作用。然而,相对于大宗作物而言,绿豆基础研究薄弱,基因组研究更是落后。近年来,分子标记技术迅速发展,在绿豆基因组学研究中发挥了重要的作用。国内外利用分子标记技术已构建了超过20张绿豆遗传连锁图谱。一些优良基因尤其是与抗性相关的基因被鉴定或精细定位,为绿豆分子标记辅助选择打下基础,加快了抗性新品种的培育进程。本研究通过对分子标记技术在绿豆遗传连锁图谱构建、重要功能基因的定位等方面的应用进行综述,以期为绿豆遗传育种研究及功能基因组学分析提供参考。     

Genetic Linkage Map Construction and QTL Mapping of Salt Tolerance Traits in Zoysiagrass (Zoysia japonica)

Zoysiagrass (Zoysia Willd.) is an important warm season turfgrass that is grown in many parts of the world. Salt tolerance is an important trait in zoysiagrass breeding programs. In this study, a genetic linkage map was constructed using sequence-related amplified polymorphism markers and random amplified polymorphic DNA markers based on an F₁ population comprising 120 progeny derived from a cross between Zoysia japonica Z105 (salt-tolerant accession) and Z061 (salt-sensitive accession). The linkage map covered 1211 cM with an average marker distance of 5.0 cM and contained 24 linkage groups with 242 marker loci (217 sequence-related amplified polymorphism markers and 25 random amplified polymorphic DNA markers). Quantitative trait loci affecting the salt tolerance of zoysiagrass were identified using the constructed genetic linkage map. Two significant quantitative trait loci (qLF-1 and qLF-2) for leaf firing percentage were detected; qLF-1 at 36.3 cM on linkage group LG4 with a logarithm of odds value of 3.27, which explained 13.1% of the total variation of leaf firing and qLF-2 at 42.3 cM on LG5 with a logarithm of odds value of 2.88, which explained 29.7% of the total variation of leaf firing. A significant quantitative trait locus (qSCW-1) for reduced percentage of dry shoot clipping weight was detected at 44.1 cM on LG5 with a logarithm of odds value of 4.0, which explained 65.6% of the total variation. This study provides important information for further functional analysis of salt-tolerance genes in zoysiagrass. Molecular markers linked with quantitative trait loci for salt tolerance will be useful in zoysiagrass breeding programs using marker-assisted selection.     

大豆遗传图谱的构建和分析

利用大豆栽培品种科丰1号和南农1138－2杂交得到的重组近交系NJRIKY,通过RFLP,SSR,RAPD和AFLP4种分子标记的遗传连锁分析,构建了包含24个连锁群,由792个遗传标记组成的大豆较高密度连锁图谱,该图谱覆盖2320．7cM,平均图距2．9cM,SSR标记的多态性较高,在基因组中的位置相对稳定,可以作为锚定标记,有利于连锁群的归并和不同图谱的比较整合;而AFLP标记对于增加图谱密度效率较高,但其容易出现聚集现象,从而造成连锁群上有很大的空隙（gap),另外,在连锁群中有21．7％的分子标记出现偏分离,该图谱为基因定位,比较基因组学和重要农艺性状的QTL定位等研究打下了基础。     

Construction of a Genetic Linkage Map and Genetic Analysis of Domestication Related Traits in Mungbean (Vigna radiata)

The genetic differences between mungbean and its presumed wild ancestor were analyzed for domestication related traits by QTL mapping. A genetic linkage map of mungbean was constructed using 430 SSR and EST-SSR markers from mungbean and its related species, and all these markers were mapped onto 11 linkage groups spanning a total of 727.6 cM. The present mungbean map is the first map where the number of linkage groups coincided with the haploid chromosome number of mungbean. In total 105 QTLs and genes for 38 domestication related traits were identified. Compared with the situation in other Vigna crops, many linkage groups have played an important role in the domestication of mungbean. In particular the QTLs with high contribution were distributed on seven out of 11 linkage groups. In addition, a large number of QTLs with small contribution were found. The accumulation of many mutations with large and/or small contribution has contributed to the differentiation between wild and cultivated mungbean. The useful QTLs for seed size, pod dehiscence and pod maturity that have not been found in other Asian Vigna species were identified in mungbean, and these QTLs may play the important role as new gene resources for other Asian Vigna species. The results provide the foundation that will be useful for improvement of mungbean and related legumes.     

梨遗传连锁图谱的构建及其与苹果图谱的比较

以‘丰水’为母本、‘砀山酥梨’为父本杂交所得的F1代104株单体为作图群体,利用SSR分子标记进行遗传连锁分析,应用Jionmap 3.0作图软件,构建了一张包含104个SSR分子标记,分属于18个连锁群的梨遗传连锁图谱,覆盖梨基因组总长831.8cM,平均图距为8.0cM。根据定位到该图谱上的SSR标记与苹果‘Fiesta’图谱进行比较,25个共有的SSR标记将该图谱和苹果图谱各连锁群连接起来,这些标记不仅呈现良好的共线性而且它们之间的相对遗传距离也很相近。研究认为,SSR标记作为锚定引物,可以与不同物种的遗传图谱相比较整合,为不同物种之间遗传信息的转移提供参考依据;同时该研究为梨树相关性状的基因定位、分离以及克隆奠定了基础。     

甘蓝型油菜分子标记连锁图谱的构建及显性细胞核雄性不育基因的图谱定位

在显性细胞核雄性不育系Rs1046A和双低油菜品种Samourai构建的回交分离群体中,运用AFLP和SSR两种标记技术构建了一个甘蓝型油菜(Brassica napus L.)的分子标记遗传连锁图谱。该图谱共包含138个AFL．P标记、83个SSR标记和1个形态标记,分布于18个主要连锁群、2个三联体和1个连锁对上,图谱总长度为2646cM,偏分离标记的比例为11．7％。显性细胞核雄性不育基因Ms被定位到第10连锁群(LG10)上。同时,偏分离标记聚集于第8连锁群(LG8)和第16连锁群(LGl6)的末端,形成了十分明显的偏分离标记密集区域。研究结果对于油菜核不育两型系的分子标记辅助选择育种具有重要意义,同时也为克隆和分离核不育基因以及研究核不育的分子机理打下了良好的基础。     

茄子分子遗传图谱的构建及果实性状的QTL定位

以茄子(Solanum melongena)材料09-101-M和10TL-102-F4-1的重组自交系(RIL)为作图群体,构建总长度为991.7c M、共包含16个连锁群157个位点、平均图距为6.32 c M的遗传图谱。应用复合区间作图法(CIM),共检测到18个与茄子果实性状相关的QTLs,其中10个为主效QTLs,8个QTLs在两年两点的实验中能够被重复检测到。在所有QTLs中,控制果重的QTL fw1.1的效应值最大,为23.8%–31.6%,被定位在LG01(E09)上E25M34–E33M57b区域内;果长、果径与果重显著相关,且控制果长、果径与果重的QTL位于同一连锁群的相同区域。     

Polymorphic dinucleotide microsatellites in tongue sole (Cynoglossus semilaevis)

The tongue sole, Cynoglossus semilaevis, is a rare marine flatfish distributed in Chinese coastal waters. From a (GT)_n‐enriched genomic library, 57 microsatellites were isolated and characterized. Seventeen of these loci were polymorphic in a test population with alleles ranging from three to 13, and observed and expected heterozygosities from 0.1613 to 1.0000 and from 0.2126 to 0.8983, respectively. Five loci deviated from the Hardy–Weinberg equilibrium in the sampled population, and linkage disequilibrium between two loci was significant after applying Bonferroni correction. Three additional fish species assessed for cross‐species amplification revealed that only one locus was also polymorphic in one species. These polymorphic microsatellite loci should provide sufficient level of genetic diversity to evaluate the breeding strategy and investigate the fine‐scale population structure in C. semilaevis.     

A Genetic Linkage Map of Sole (Solea solea): A Tool for Evolutionary and Comparative Analyses of Exploited (Flat)Fishes

Linkage maps based on markers derived from genes are essential evolutionary tools for commercial marine fish to help identify genomic regions associated with complex traits and subject to selective forces at play during exploitation or selective breeding. Additionally, they allow the use of genomic information from other related species for which more detailed information is available. Sole (solea solea L.) is a commercially important flatfish species in the North Sea, subject to overexploitation and showing evidence of fisheries-induced evolutionary changes in growth- and maturation-related traits. Sole would definitely benefit from a linkage map to better understand how evolution has shaped its genome structure. This study presents a linkage map of sole based on 423 single nucleotide polymorphisms derived from expressed sequence tags and 8 neutral microsatellite markers. The total map length is 1233.8 cM and consists of 38 linkage groups with a size varying between 0 to 92.1 cM. Being derived from expressed sequence tags allowed us to align the map with the genome of four model fish species, namely medaka (Oryzias latipes), Nile tilapia (Oreochromis niloticus), three-spined stickleback (Gasterosteus aculeatus) and green spotted pufferfish (Tetraodon nigroviridis). This comparison revealed multiple conserved syntenic regions with all four species, and suggested that the linkage groups represent 21 putative sole chromosomes. The map was also compared to the linkage map of turbot (Scophthalmus maximus), another commercially important flatfish species and closely related to sole. For all putative sole chromosomes (except one) a turbot homolog was detected, confirming the even higher degree of synteny between these two flatfish species.     

RAPD和SSR两种标记构建的中国对虾遗传连锁图谱

利用RAPD和SSR分子标记结合拟测交策略,对中国对虾(Fenneropenaeuschinensis)“黄海1号”雌虾与野生雄虾作为亲本进行单对杂交产生的F1代,采用RAPD和SSR两种分子标记技术初步构建了中国对虾雌、雄遗传连锁图谱。对460个RAPD引物和44对SSR引物进行筛选,共选出61个RAPD引物和20对SSR引物,用于对父母本和82个F1个体进行遗传分析。共得到母本分离标记146个(RAPD标记128个,微卫星标记18个)和父本分离标记127个(RAPD标记109个,微卫星标记18个)。雌性图谱包括8个连锁群、9个三联体和14个连锁对,标记间平均间隔为11·28cM,图谱共覆盖1173cM,覆盖率为59·36%;雄性图谱包括10个连锁群、12个三联体和7个连锁对,标记间平均间隔为12·05cM,图谱共覆盖1144·6cM,覆盖率为62·01%。中国对虾遗传图谱的构建为其分子标记辅助育种、比较基因组作图及数量性状位点的定位与克隆奠定了基础。     

用AFLP标记快速构建遗传连锁图谱并定位一个新基因tms5

报导了一个分子标记连锁图的快速构建方法.通过对水稻(Oryza sativa L.)"安农S-1"和"南京11"的F2分离群体的AFLP分析找到了142个AFLP标记,用这142个AFLP标记以及已定位的25个SSR标记和5个RFLP标记构建了水稻12个染色体的分子标记连锁图,该图覆盖水稻基因组的1 537.4 cM,相邻标记间的平均间距为9.0 cM,这是在国内建立的第一张AFLP标记连锁图.在建立连锁图谱的同时把一个新基因tms5 (水稻温敏核不育基因)定位在第2染色体上.     

用AFLP标记快速构建遗传连锁图谱并定位一个新基因tms5

报导了一个分子标记连锁图的快速构建方法。通过对水稻(Oryza sativa L．)“安农S-1”和“南京11”的F2分离群体的AFLP分析找到了142个AFLP标记,用这142个AFLP标记以及已定位的25个SSR标记和5个RFIP标记构建了水稻12个染色体的分子标记连锁图,该图覆盖水稻基因组的1537．4cM,相邻标记间的平均间距为9．0cM,这是在国内建立的第一张AFLP标记连锁图。在建立连锁图谱的同时把一个新基因tms5(水稻温敏核不育基因)定位在第2染色体上。