QTL Mapping for Resistance to Iridovirus in Asian Seabass Using Genotyping-by-Sequencing

Identifying quantitative trait loci (QTL) for viral disease resistance is of particular importance in selective breeding programs of fish species. Genetic markers linked to QTL can be useful in marker-assisted selection (MAS) for elites resistant to specific pathogens. Here, we conducted a genome scan for QTL associated with Singapore grouper iridovirus (SGIV) resistance in an Asian seabass (Lates calcarifer) family, using a high-density linkage map generated with genotyping-by-sequencing. One genome-wide significant and three suggestive QTL were detected at LG21, LG6, LG13, and LG15, respectively. The phenotypic variation explained (PVE) by the four QTL ranged from 7.5 to 15.6%. The position of the most significant QTL at LG21 was located between 31.88 and 36.81 cM. The SNP marker (SNP130416) nearest to the peak of this QTL was significantly associated with SGIV resistance in an unrelated multifamily population. One candidate gene, MECOM, close to the peak of this QTL region, was predicted. Evidence of alternative splicing was observed for MECOM and one specific category of splicing variants was differentially expressed at 5 days post-SGIV infection. The QTL detected in this study are valuable resources and can be used in the selective breeding programs of Asian seabass with regard to resistance to SGIV.     

Tracing Asian Seabass Individuals to Single Fish Farms Using Microsatellites

Traceability through physical labels is well established, but it is not highly reliable as physical labels can be easily changed or lost. Application of DNA markers to the traceability of food plays an increasingly important role for consumer protection and confidence building. In this study, we tested the efficiency of 16 polymorphic microsatellites and their combinations for tracing 368 fish to four populations where they originated. Using the maximum likelihood and Bayesian methods, three most efficient microsatellites were required to assign over 95% of fish to the correct populations. Selection of markers based on the assignment score estimated with the software WHICHLOCI was most effective in choosing markers for individual assignment, followed by the selection based on the allele number of individual markers. By combining rapid DNA extraction, and high-throughput genotyping of selected microsatellites, it is possible to conduct routine genetic traceability with high accuracy in Asian seabass.     

Mapping Quantitative Trait Loci for Omega-3 Fatty Acids in Asian Seabass

Omega-3 fatty acids are essential fatty acids for human health. Therefore, increasing both percentage of omega-3 and a better fatty acid profile in fish fillets is one of the breeding goals in aquaculture. However, it is difficult to increase the omega-3 content in fish fillets, as the phenotypic selection of these traits is not easily feasible. To facilitate the genetic improvement of the Asian seabass for optimal fatty acid profiles, a genome-wide scan for quantitative trait loci (QTL) affecting fatty acid level in the flesh of the Asian seabass was performed on an F₂ family containing 314 offspring. All family members were genotyped using 123 informative microsatellites and 22 SNPs. High percentages of n-3 polyunsaturated fatty acids (PUFA), especially C22:6 (DHA 16.48?±?3.09 %) and C20:5 (EPA 7.19?±?0.86 %) were detected in the flesh. One significant and 54 suggestive QTL for different fatty acids and a water content trait were detected on the whole genome. QTL for C18:0b was located on linkage groups (LG) 5. QTL for total n-3 PUFA content in flesh were mapped onto LG6 and LG23 with the phenotypic variance explained ranging from 3.8 to 6.3 %. Four QTL for C22:6 were detected on LG6, LG23, and LG24, explaining 3.9 to 4.9 % of the phenotypic variance, respectively. Mapping of QTL for contents of different fatty acids is the first step towards improving the omega-3 content in the fillets of fish by using marker-assisted selection and is important for understanding the biology of fatty acid deposition.     

Molecular Genetic Analysis of Essential Tremor

Essential tremor (ET) is the most common extrapyramidal disorder of the central nervous system with autosomal dominant transmission in the majority of cases and age-dependent penetrance of the mutant gene. In a number of cases, it shares some phenotypic features with autosomal dominant idiopathic torsion dystonia (locusDYT1on chromosome 9q32–34) and is genetically heterogeneous: distinct variants of ET were mapped to chromosomes 3q13 (ETM1) and 2p22–25 (ETM2). We performed studies of candidate loci in a group of Slavonic (11 patients) and Tajik (19 patients) families with ET. Mutational analysis of the DYT1 gene in probands did not reveal the major deletion 946–948delGAG characteristic of idiopathic torsion dystonia, which allows one to genetically distinguish the studied hereditary forms of ET and torsion dystonia. Based on analysis of genetic linkage in informative Tajik pedigrees with ET, linkage to locus ETM1 on chromosome 3q13 was established in four families. Maximum pairwise Lod score was 2.46 at recombination fraction of = 0.00; maximum combined multipoint Lod score was 3.35 for marker D3S3515 and a common mutant haplotype for markers D3S3620, D3S3576, and D3S3720 allowed us to locate a mutant gene in a relatively narrow chromosome region spanning 2 cM. In one informative pedigree with ET, both candidate loci ETM1 and ETM2 were definitely excluded on the basis of negative Lod scores obtained by linkage estimations, which testifies to the existence of another distinct gene for autosomal dominant ET.     

Genetic Parentage Analysis Confirms a Polygynandrous Breeding System in the European Grayling (Thymallus thymallus)

Knowing the breeding system of a species is important in order to understand individual variation in reproductive success. Large variation in reproductive success and thus reproductive skew strongly impacts on the effective number of breeders and thus the long-term effective population size (N_e). Fishes, in particular species belonging to the salmonid family, exhibit a wide diversity of breeding systems. In general, however, breeding systems are rarely studied in detail in the wild. Here we examine the breeding system of the spring-spawning European grayling Thymallus thymallus from a small Norwegian stream using parentage assignment based on the genotyping of 19 polymorphic microsatellite loci. In total 895 individual grayling fry and 154 mature grayling (57 females and 97 males) were genotyped. A total of 466 offspring were assigned a father, a mother, or a parent pair with a confidence of 90% or higher. Successfully reproducing males had on average 11.9 ± 13.3 (SD) offspring with on average 2.1 ± 1.2 partners, whereas successful females had on average 9.5 ± 12.8 offspring and 2.3 ± 1.5 partners. Parents with more partners also produced more offspring. Thus the grayling breeding system within this small stream revealed a polygynandrous breeding system, similar to what has been observed for many other salmonid fish species. The present study thus unambiguously corroborates a polygynadrous breeding system in the European grayling. This knowledge is critical for managing populations of this species, which has suffered significant local population declines throughout its range over the last several decades.     

A RasGTP-Induced Conformational Change in C-RAF Is Essential for Accurate Molecular Recognition

The dysregulation of Ras-RAF signaling is associated with many types of human cancer. However, the kinetic and dynamic features of the mutual molecular recognition of Ras and RAF remain unknown. Here, we developed a technique for imaging single-pair fluorescence resonance energy transfer in living cells, and coupled this technique to single-molecule kinetic analysis to investigate how C-RAF (a subtype of RAF) molecules distinguish the active form of Ras (RasGTP) from the inactive form (RasGDP). Functional fragments of C-RAF containing the Ras-binding domains did not detect the switch in Ras activity in living cells as efficiently as did C-RAF. Single-molecule analysis showed that RasGDP associates with closed-conformation C-RAF, whereas the association of C-RAF with RasGTP immediately triggers the open RAF conformation, which induces an effective interaction between C-RAF and RasGTP. Spontaneous conformational changes from closed C-RAF to the open form rarely occur in quiescent cells. The conformational change in C-RAF is so important to Ras-RAF molecular recognition that C-RAF mutants lacking the conformational change cannot distinguish between RasGDP and RasGTP. The manipulation of the conformation of an effector molecule is a newly identified function of RasGTP.     

Evidence for female-biased dispersal in the protandrous hermaphroditic Asian Seabass, Lates calcarifer

Movement of individuals influences individual reproductive success, fitness, genetic diversity and relationships among individuals within populations and gene exchange among populations. Competition between males or females for mating opportunities and/or local resources predicts a female bias in taxa with monogamous mating systems and a male-biased dispersal in polygynous species. In birds and mammals, the patterns of dispersal between sexes are well explored, while dispersal patterns in protandrous hermaphroditic fish species have not been studied. We collected 549 adult individuals of Asian seabass (Lates calcarifer) from four locations in the South China Sea. To assess the difference in patterns of dispersal between sexes, we genotyped all individuals with 18 microsatellites. Significant genetic differentiation was detected among and within sampling locations. The parameters of population structure (F(ST)), relatedness (r) and the mean assignment index (mAIC), in combination with data on tagging-recapture, supplied strong evidences for female-biased dispersal in the Asian seabass. This result contradicts our initial hypothesis of no sex difference in dispersal. We suggest that inbreeding avoidance of females, female mate choice under the condition of low mate competition among males, and male resource competition create a female-biased dispersal. The bigger body size of females may be a cause of the female-biased movement. Studies of dispersal using data from DNA markers and tagging-recapture in hermaphroditic fish species could enhance our understanding of patterns of dispersal in fish.     

Vasa Identifies Germ Cells and Critical Stages of Oogenesis in the Asian Seabass

Germ cells produce sperm and eggs for reproduction and fertility. The Asian seabass (Lates calcarifer), a protandrous marine fish, undergoes male-female sex reversal and thus offers an excellent model to study the role of germ cells in sex differentiation and sex reversal. Here we report the cloning and expression of vasa as a first germ cell marker in this organism. A 2241-bp cDNA was cloned by PCR using degenerate primers of conserved sequences and gene-specific primers. This cDNA contains a polyadenylation signal and a full open reading frame for 645 amino acid residues, which was designated as Lcvasa for the seabass vasa, as its predicted protein is homologous to Vasa proteins. The Lcvasa RNA is maternally supplied and specific to gonads in adulthood. By chromogenic and fluorescent in situ hybridization we revealed germ cell-specific Lcvasa expression in both the testis and ovary. Importantly, Lcvasa shows dynamic patterns of temporospatial expression and subcellular distribution during gametogenesis. At different stages of oogenesis, for example, Lcvasa undergoes nuclear-cytoplasmic redistribution and becomes concentrated preferentially in the Balbiani body of stage-II~III oocytes. Thus, the vasa RNA identifies both female and male germ cells in the Asian seabass, and its expression and distribution delineate critical stages of gametogenesis.     

Genome-Wide Association Study Identifies Loci Associated with Resistance to Viral Nervous Necrosis Disease in Asian Seabass

Viral nervous necrosis disease (VNN), caused by nervous necrosis virus (NNV), is one major threat to mariculture. Identifying loci and understanding the mechanisms associated with resistance to VNN are important in selective breeding programs. We performed a genome-wide association study (GWAS) using genotyping-by-sequencing (GBS) to study the genomic architecture of resistance to NNV infection in Asian seabass. We genotyped 986 individuals from 43 families produced by 15 founders with 44498 bi-allelic genetic variants using GBS. The GWAS identified three genome-wide significant loci on chromosomes 16, 19, and 20, respectively, and six suggestive loci on chromosomes 1, 8, 14, 15, 21, and 24, respectively, associated with resistance to NNV infection measured as binary and quantitative traits. Using the 500 most significant markers in combination with a training population of 800 samples could reach a genomic prediction accuracy of 0.7. Candidate genes significantly associated with resistance to NNV, including lysine-specific demethylase 2A, beta-defensin 1, and cystatin-B, which play important roles in immune responses against virus infection, were identified. Almost all the candidate genes were differentially expressed in different tissues against NNV infection. The significant genetic variants can be used in genomic selection and help understand the mechanism of resistance to VNN. Future studies should use populations of large effective size and whole genome resequencing to identify more useful genetic variants.     

菊花分子育种研究进展

对菊花分子育种的研究起源于20世纪90年代初,导入外源基因的主要目的是改变菊花的株型、花色,提高抗虫性、抗病性,增强对寒冷、干旱、盐碱的耐性。近年来,选用不同类型的启动子构建新型载体的研究也为改善菊花育种工作提供了参考。这些都预示着菊花分子育种的研究逐渐走向全面和成熟。     

Characterization of Microsatellites in the IGF-2 and GH Genes of Asian Seabass (Lates calcarifer)

Four microsatellites were identified by screening the DNA sequences of Asian seabass (Lates calcarifer) deposited to GenBank. Two markers each are located in the growth hormone gene (GH) and in the insulin-like growth factor II gene (IGF-2), respectively. The markers were characterized by genotyping 34 Asian seabass individuals. All 4 microsatellites showed polymorphism: the number of alleles per locus ranged from 2 to 11 (average, 5.0), while the expected heterozygosity ranged from 0.51 to 0.85 (average, 0.63) at the 4 loci. Cross-priming with all 4 primer pairs was tested in species belonging to 5 different genera, but no bands were amplified. These microsatellites are the first genomic DNA markers characterized in L. calcarifer; thus they may be valuable for research and aquaculture production of this species. Received April 10, 2000; accepted July 13, 2000.     

Molecular Marker Based Coefficient of Parentage Analysis for Establishing Distinctness in Indian Rice (Oryza sativa L) Varieties

Molecular characterization of 32 Indian rice varieties of different agro-climatic zones resulted in mean heterozygosity value of 0.622, 0.819 and 0.890 over polymorphic loci and marker index value of 1.00, 6.75 and 4.16, respectively for RAPD, ISSR and STMS primers. The three marker systems resulted in 201 polymorphic bands (94.36%) out of a total of 213 bands. The probability of a chance identical match between two varieties was very low (2.08x10¹⁰) in combined molecular marker analysis as compared to individual marker system (RAPD, 7.5 x 10^-4; ISSR, 1.5 x 10^-3 and STMS, 3.9 x 10^-6). The combined average genetic similarity for molecular markers and coefficient of parentage (COP) across all 496 pairwise combinations revealed a non-significant relationship (r = 0.215).     

The <Emphasis Type="Italic">FTO</Emphasis> Gene Is Associated with Growth and Omega-3/-6 Ratio in Asian Seabass

Polymorphisms in the FTO gene are associated with obesity and body mass index in humans and livestock. Little information of whether FTO plays an important role in aquaculture fish species is available. We cloned and characterized the FTO gene in an economically important food fish species: Asian seabass (Lates calcarifer). The full-length cDNA of the gene is 3679 bp, containing an ORF of 1935 bp encoding 644 amino acids, a 216 bp 5′ UTR and a 1538 bp 3′ UTR. The gene consisted of nine exons and eight introns and was 117,679 bp in length. Phylogenetic analysis revealed that the gene in Asian seabass was closely related to those of Japanese flounder and Nile tilapia. Analysis of its expressions using qRT-PCR showed that it was expressed ubiquitously, but was higher in the liver, stomach and intestine. Comparative analysis of the genomic sequences of part of intron 1 of the gene among 10 unrelated individuals identified two SNPs. Analysis of associations between SNPs and traits (i.e. growth, oil content, omega-3 and -6 contents) in an F₂ family demonstrated that the two SNPs were significantly associated with growth, oil content, omega-3 content and omega-3/-6 ratio. Altogether, our data suggest that the gene or/and its linked genes play an important role in growth and fatty acid synthesis, and that the SNPs associated with traits may be used as markers for selecting quicker growth and higher omega-3/-6 ratio at the fingerling stage.     

燕麦分子育种研究进展

燕麦是一种主要生长在温带冷凉地区的粮饲兼用作物,近年来燕麦的营养价值和降低胆固醇特性的发现使人们认识到燕麦及其制品是一种健康食品,促进了燕麦产业发展,对燕麦品种培育提出了更高要求。在此背景下,现代生物技术与常规育种技术结合是满足这一需求的重要途经。本文综述了国内外燕麦分子育种方面的研究进展:(1)我国燕麦种质资源的收集与遗传多样性研究。我国的燕麦种质资源收集工作起始于20世纪50年代,迄今收集并保存了5282份燕麦种质资源。对这些资源的遗传多样性分析研究表明,国内的燕麦种质资源中内蒙古和山西的资源多样性最高;(2)利用各种分子标记构建燕麦遗传连锁图谱研究;(3)一些重要农艺性状的QTL定位研究以及全基因组关联分析研究。包括产量、含油量、β-葡聚糖含量、蛋白质含量、抽穗期、抗病基因、抗冻性等重要农艺性状;(4)标记辅助基因组选择技术在燕麦中的应用;(5)燕麦遗传工程育种研究进展。同时,本文将国内的主要研究进展与国外相关的最新研究成果进行了比较,并对当前分子育种中存在的问题及今后的研究方向进行了探讨,希望能为今后通过生物技术手段培育燕麦新品种提供一定的参考。     

T-cadherin Is Essential for Adiponectin-mediated Revascularization

Adipose tissue secretes protein factors that have systemic actions on cardiovascular tissues. Previous studies have shown that ablation of the adipocyte-secreted protein adiponectin leads to endothelial dysfunction, whereas its overexpression promotes wound healing. However, the receptor(s) mediating the protective effects of adiponectin on the vasculature is not known. Here we examined the role of membrane protein T-cadherin, which localizes adiponectin to the vascular endothelium, in the revascularization response to chronic ischemia. T-cadherin-deficient mice were analyzed in a model of hind limb ischemia where blood flow is surgically disrupted in one limb and recovery is monitored over 28 days by laser Doppler perfusion imaging. In this model, T-cadherin-deficient mice phenocopy adiponectin-deficient mice such that both strains display an impaired blood flow recovery compared with wild-type controls. Delivery of exogenous adiponectin rescued the impaired revascularization phenotype in adiponectin-deficient mice but not in T-cadherin-deficient mice. In cultured endothelial cells, T-cadherin deficiency by siRNA knockdown prevented the ability of adiponectin to promote cellular migration and proliferation. These data highlight a previously unrecognized role for T-cadherin in limb revascularization and show that it is essential for mediating the vascular actions of adiponectin.