Inheritance and usefulness of AFLP markers in channel catfish (Ictalurus punctatus), blue catfish (I. furcatus), and their F1, F2, and backcross hybrids

Eight primer combinations were used to investigate the application of amplified fragment length polymorphism (AFLP) markers in catfish for genetic analysis. Intraspecific polymorphism was low among channel catfish or blue catfish strains. Interspecific AFLP polymorphism was high between the channel catfish and blue catfish. Each primer combination generated from 70 to more than 200 bands, of which 38.6–75.7% were polymorphic between channel catfish and blue catfish. On average, more than 20 polymorphic bands per primer combination were produced as quality markers suitable for genetic analysis. All AFLP markers were transmitted into channel catfish?×?blue catfish F1 hybrids, except rare markers that were heterozygous in the parents and therefore were segregating in F1 hybrids. The two reciprocal channel catfish?×?blue catfish F1 hybrids (channel catfish female?×?blue catfish male; blue catfish female?×?channel catfish male) produced identical AFLP profiles. The AFLP markers were inherited and segregated in expected Mendelian ratios. At two loci, E8-b9 and E8-b2, markers were found at significantly lower frequencies than expected with F2 and backcross hybrids which had been selected for increased growth rates. The reproducibility of AFLP was excellent. These characteristics of the catfish AFLP markers make them highly useful for genetic analysis of catfish, especially for construction of genetic linkage and quantitative trait loci maps, and for marker-assisted selection.     

Inheritance and usefulness of AFLP markers in channel catfish (Ictalurus punctatus ), blue catfish ( I. furcatus) , and their F1, F2, and backcross hybrids

Eight primer combinations were used to investigate the application of amplified fragment length polymorphism (AFLP) markers in catfish for genetic analysis. Intraspecific polymorphism was low among channel catfish or blue catfish strains. Interspecific AFLP polymorphism was high between the channel catfish and blue catfish. Each primer combination generated from 70 to more than 200 bands, of which 38.6–75.7% were polymorphic between channel catfish and blue catfish. On average, more than 20 polymorphic bands per primer combination were produced as quality markers suitable for genetic analysis. All AFLP markers were transmitted into channel catfish × blue catfish F1 hybrids, except rare markers that were heterozygous in the parents and therefore were segregating in F1 hybrids. The two reciprocal channel catfish × blue catfish F1 hybrids (channel catfish female × blue catfish male; blue catfish female × channel catfish male) produced identical AFLP profiles. The AFLP markers were inherited and segregated in expected Mendelian ratios. At two loci, E8-b9 and E8-b2, markers were found at significantly lower frequencies than expected with F2 and backcross hybrids which had been selected for increased growth rates. The reproducibility of AFLP was excellent. These characteristics of the catfish AFLP markers make them highly useful for genetic analysis of catfish, especially for construction of genetic linkage and quantitative trait loci maps, and for marker-assisted selection. Received: 10 September 1997 / Accepted: 10 December 1997     

Cloning and characterization of microsatellite loci in channel catfish, Ictalurus punctatus

Short tandem repeat (microsatellite) loci were cloned from the channel catfish, Ictalurus punctatus , genome for use as molecular markers for genetic improvement of this important agricultural species. Plasmid clones containing catfish genomic DNA inserts were identified, by hybridization with tandem repeat DNA probes, and sequenced using automated laser fluorescence. A feral population of catfish displayed levels of heterozygosity greater than 0·7 for 13 of 22 loci and heterozygosity greater than 0·5 for 20 of 22 loci. Allelic polymorphism ranged from three to 17 alleles per locus in the feral population. Populations of domestic, farm-raised catfish and a research strain displayed levels of heterozygosity similar to the feral population. Non-invasive tissue sampling provided abundant material for the polymerase chain reaction-based genotype assay. The microsatellite loci will be useful in the molecular characterization and genetic improvement of channel catfish populations.     

Comparative study of mannose-binding C-type lectin isolated from channel catfish (Ictalurus punctatus) and blue catfish (Ictalurus furcatus)

Mannose-binding C-type lectin (MBL) was isolated from channel catfish (Ictalurus punctatus) NWAC 102 and 103 strains, blue catfish (Ictalurus furcatus) D+B and Rio Grande strains, hybrid catfish (channel catfish female NWAC 103 x blue catfish male D+B) sera, and purified by affinity chromatography from channel catfish Norris strain serum. Reduction of purified channel catfish MBL with 2-ME yielded a single band of 62 kDa by SDS-PAGE and Western blot analysis using guinea pig anti-MBL IgG as primary antibody. Channel catfish NWAC 102 strain, channel catfish NWAC 103 strain and hybrid catfish sera had molecular masses of 63 kDa for MBL. Blue catfish (D+B strain) serum MBL had a molecular mass of 66 kDa. Rio Grande blue catfish serum MBL had a molecular mass of 65 kDa. Amino acid composition analysis (mol%) of the affinity-purified channel catfish MBL found a high content of serine present. Functional binding studies of channel catfish and blue catfish MBLs binding to Edwardsiella ictaluri were done using a dot-immunoblot ELISA method. A dot-immunoblot ELISA binding assay was done to compare nine different strains and species of channel catfish and blue catfish for their levels of serum MBL. Blue catfish had higher levels of MBL than did the various strains of channel catfish tested. MBL could be used as a genetic marker for selection of disease resistance in the different strains of catfish used in aquaculture. This study describes the presence of serum MBL in catfish and evidence for a C-type lectin complement pathway of innate immunity.     

Effects of sperm concentration and egg number on fertilization efficiency with channel catfish (Ictalurus punctatus) eggs and blue catfish (I. furcatus) spermatozoa

The mean sperm concentration of 10 blue catfish (Ictalurus furcatus ) was 1.03 x 10(10) per gram of testis. Testis weighed 3.9 and 17.2 g, with a mean of 6.6 g per fish. Fertilization rate of channel catfish (Ictalurus punctatus ) eggs fertilized with 5.00 x 10(4) to 1.20 x 10(7) blue catfish spermatozoa per egg was 17 to 87%, with an overall mean of 65%. Sperm concentrations of 5.0 x 10(4)/egg exhibited a lower, 16.6% (P < 0.05) fertilization rate than higher sperm concentrations (1.25 x 10(5) to 1.20 x 10(8)/egg). Batches of 450, 2,000, 5,000, 8,000 and 11,000 eggs were similarly fertilized with various sperm concentrations. Mean fertility rate ranged from 25 to 67%, with an overall mean of 53%. The largest egg mass produced the lowest (P < 0.05) fertilization rate. A combination of 450 eggs per batch and 5.0 x 10(5) to 1.20 x (8) sperm per egg produced the highest rate of fertilization (67 to 87%).     

Respiratory-cardiovascular physiology and chloroethane gill flux in the channel catfish, Ictalurus punctatus

A fish respirometer-metabolism chamber was used to obtain in vivo respiratory-cardiovascular and chloroethane gill flux data on transected channel catfish (Ictalurus punctatus). Methods used for spinal transection, attachment of an oral membrane (respiratory mast), placement and attachment of blood cannulas and urine catheters are described. Respiratory physiology, cardiac output and chemical extraction efficiencies for 1,1,2,2-tetrachloroethane (TCE), pentachloroethane (PCE), and hexachloroethane (HCE) were determined on 419–990 g catfish. The overall mean values (± s.d.) for ventilation volume (Q_v), effective respiratory volume (Q_w), oxygen consumption (Vo₂ and percentage utilization of oxygen (U) were 17-3 ±4–71 h^?1 kg^?1, 9·8±l·71 h^?1 kg^?1, 71·6±12·5mg h^?1 kg^?1, and 49± 10%, respectively, while cardiac output calculated via the Fick Method was 2·4±0·61 h^?1 kg^?1. Additional measurements were made on ventilation rate (V_r), total plasma protein, haematocrit (Hct), and urine volume; while both arterial and venous blood were analysed for pH, oxygen partial pressure (P02), carbon dioxide partial pressure (Pco₂), total oxygen (To₂), total carbon dioxide (Tco₂) and total ammonia (TAMM). Physiological measurements taken at 24 h were not significantly different from those taken at 48 h and indicated no deterioration of the in vivo preparation. All of these values agreed well with literature values on UTitransected channel catfish, except for Hct which was lower for cannulated animals used in this study. Overall, these data provide strong support for the use of transected channel catfish for in vivo collection of physiological and chemical gill flux data. The mean initial chemical extraction efficiencies for TCE, PCE and HCE were 41, 61 and 73%, respectively. Chemical clearances (Cl_X) for these same three chemicals were 5·9, 9·3 and 10·8 1 h^?1 kg^?1, respectively. The approximate 1: 1 relationship between effective respiratory volume (Q_w) and chemical clearance (Cl_x) indicated that branchial uptake of PCE and HCE was water flow-limited. Chemical gill flux observed for channel catfish and chloroethanes was similar to that observed for rainbow trout in previous studies and provided further support for the flow-limited model of chemical flux across fish gills.     

Genetic and virulence characterization of Flavobacterium columnare from channel catfish (Ictalurus punctatus)

Aim: To develop a method for conducting pulsed-field gel electrophoresis (PFGE) on Flavobacterium columnare, to use PFGE to characterize F. columnare channel catfish isolates, and to determine whether variation in pathogenic potential exists in F. columnare isolates from channel catfish. Methods and Results: On the basis of PFGE-derived profiles, similarity dendrograms constructed for more than 30 F. columnare isolates showed two major genetic groups with more than 60% similarity. Channel catfish fingerlings challenged with PFGE group A isolates by bath immersion had significantly higher average mortalities (>60%) than fish challenged with PFGE group B isolates (<9%). However, abrasion and skin mucus removal made channel catfish fingerlings susceptible to disease caused by group B isolates following immersion exposure. Conclusion: Our results suggest that two genetic divisions of F. columnare channel catfish isolates exist, and that isolates in PFGE group A isolates tend to be more pathogenic to immunocompetent channel catfish fingerlings than PFGE group B isolates. Significance and Impact of the Study: PFGE is a potentially useful tool for determining whether F. columnare isolates are more likely to be primary or secondary pathogens. Pathogenesis research for columnaris disease in catfish should focus on pathogenic isolates from PFGE group A.     

不同水温下氟甲砜霉素在斑点叉尾鮰体内的药代动力学研究

研究不同水温(18℃和28℃)条件下,单剂量(10mg/kgb·w)强饲氟甲砜霉素,在斑点叉尾鮰(Ictaluruspunc-tatus)体内药代动力学特征.采用高效液相色谱紫外检测法可以同时检测血浆中氟甲砜霉素及其代谢物氟甲砜霉素的浓度.用3p97药代动力学软件处理药时数据.结果表明:在不同水温条件下氟甲砜霉素在斑点叉尾鮰体内的药时数据均符合一室开放式模型.药时规律符合理论方程C血浆=71921(e-0.036t-e-0.18t)和C血浆=91061(e-0.081t-e-0.301t).18℃和28℃的条件下,主要药代动力学参数:吸收半衰期T1/2ka分别为31845h和21301h,消除半衰期T1/2ke分别为191118h和81519h,达峰时间Tpeak分别为111136h和51953h,最大血药浓度Cmax分别为41074μg/mL和41226μg/mL,曲线下面积AUC分别为1741547(μg/mL)/h和811279(μg/mL)/h,平均驻留时间MRT分别为271581h和121290h,相对表观分布容积V/F(c)分别为11580L/kg和115121L/kg.采用氟甲砜霉素防治斑点叉尾鮰细菌性疾病,建议在18℃左右口服10mg/kg体重剂量的氟甲砜霉素,2d给药1次;在28℃左右口服10mg/kg体重剂量的氟甲砜霉素,1d给药1次.试验过程中在斑点叉尾鮰血浆样品中未检测到氟甲砜霉素的主要代谢物氟甲砜霉素胺.       

Use of RAPD markers to screen somatic hybrids between Solanum tuberosum and S. brevidens

Summary The identification of somatic hybrids between Solanum tuberosum and S. brevidens can be carried out using polymerase chain reaction (PCR) and arbitrary 10-mer primers to generate random amplified polymorphic DNA (RAPD) markers. Five commercial primers have been tested. Each primer directed the amplification of a genome-specific fingerprint for the fusion parents and S. brevidens. The size of the amplified DNA fragments ranged from 100 to 1800 base pairs. The somatic hybrids showed a combination of the parental banding profiles with four of the five primers surveyed, whereas regenerants from one of the parents had the same or a similar banding pattern to that of the parent. Thus RAPD markers provide a quick, simple and preliminary screening method for putative somatic hybrids.Abbreviations EDTA ethylenediaminetetraacetic acid, - PCR polymerase chain reaction - RAPD random amplified polymorphic DNA - RFLP restriction fragment length polymorphisms - TBE Tris-borate-EDTA buffer - Tris trizma base     

Identification of genetic markers for Korean native cattle (Hanwoo) by RAPD analysis

In order to develop the specific genetic marker for Korean native cattle (Hanwoo), randomly amplified polymorphic DNA (RAPD) analysis of 6 different cattle breeds was attempted by using 38 decamer primers. In comparison of RAPD patterns, two distinctive DNA bands specific for Hanwoo were detected. One was 296 bp of DNA fragment found to be specific only for female Hanwoo when primer GTCCACACGG was employed. In individual analysis of this RAPD marker was observed only in female individuals with the possibility of 85.3%. The other was 521 bp of RAPD marker amplified using TCGGCGATAG and AGCCAGCGAA primers, which showed 83.0% of genetic frequency in 85 male and 68 female individuals tested. Nucleotide sequencing of these genetic markers revealed that 296 bp marker has a short microsatellite-like sequence, ACCACCACAC, and a tandem repeat sequence of microsatellite GAAAAATG in the determined sequence. Two distinctive tandem repeats of microsatellite sequences, AAC and GAAGA, were also appeared in 521 bp DNA marker. In BLAST search, any gene having high homology with these markers was not found     

Plasma corticosteroid dynamics in channel catfish, Ictalurus punctatus (Rafinesque), during and after oxygen depletion

Plasma corticosteroid concentrations in channel catfish, Ictalurus punctatus , (normally 1.0 ± 0.3 μg/100 ml) increased significantly (to 5.9 ± 1.2μg/100 ml) in response to acute oxygen depletion and then returned to control levels within 30 min after the dissolved oxygen concentration was increased; however, a secondary increase in plasma corticosteroid levels was observed 6 h after exposure. Corticosteroid levels also increased in fish exposed to dissolved oxygen concentration of <0.2 mg/1 for three days. Methylene blue was not effective in preventing interrenal response to low dissolved oxygen. No diurnal plasma corticosteroid rhythm was observed in fish exposed to diurnal chemical rhythms of culture ponds.     

Histophathology and electron microscopy of channel catfish virus in infected channel catfish,Ictalurus punctatus (Rafinesque)*

A histologic and electron microscopic study was made on selected organs from channel catfish Ictalurus punctatus (Rafinesque) fingerlings that were experimentally infected with channel catfish virus (CCV). Histopathology was characterized by necrosis and haemorrhage in kidney and liver, and haemorrhage in the spleen and gastrointestinal tract. Virus replication occurred in nuclei of cells in the kidney, liver and spleen. Intranuclear inclusion bodies consisting of geometric crystalline arrays and lamellar structures were associated with virus replication.     

Novel polymorphisms in Stearoyl-CoA Desaturase (SCD) and Fatty Acid Desaturase 2 (FADS2) in channel catfish (Ictalurus punctatus,Rafinesque, 1818)

The Channel catfish is one of the most important freshwater fish species in North America. Conventionally their selection for genetic improvement is focused in growth related traits but not on quality traits as flavor and fatty acid deposition. The aim of this study was the sequencing of Stearoyl-CoA Desaturase (SCD) and Fatty Acid Desaturase 2 (FADS2) genes, two of main genes in the biosynthesis of long chain polyunsaturated fatty acids, for novel variation discovering as a preliminary of marker assisted broader assessment. In SCD gene, two novel synonym polymorphisms (Exon 1 and 3) and a non-synonymous Serine coding polymorphism in exon 3, were found. In FADS2, two synonymous polymorphisms (Exon 1 and 3) and multiple polymorphisms in non-coding regions were discovered . Further association analysis of these novel variations could reveal punctual effect on fatty acid deposition traits and their utility for marker assisted selection purposes.     

中国舞毒蛾六个地理种群的RAPD分析及SCAR标记构建

舞毒蛾Lymantria dispar L.是世界性农林害虫, 包含不同的亚种, 其中亚洲舞毒蛾的雌蛾具有较强的飞行能力, 已成为国际性的重要检疫性有害生物。然而, 不同舞毒蛾亚种及种群间形态难辨, 因此采用传统的手段鉴别舞毒蛾亚种种群是很困难的。本研究首先采用RAPD标记分析了中国舞毒蛾6个地理种群的遗传多态性。结果表明, 所检测的舞毒蛾种群的遗传分化系数G_st为0.7571, 由此推算出的平均有效迁移数（基因流参数）N_em为0.1604, 说明不同舞毒蛾种群间的遗传分化程度较高, 缺乏广泛的基因流动。本研究在RAPD遗传分析基础之上, 筛选出了4个舞毒蛾种群的特异性遗传位点, 然后对这些特异性位点进行了克隆测序、 序列分析和位点特异性引物设计。结果表明, 其中2个舞毒蛾种群的位点特异性引物可产生序列特征性扩增区域（SCAR）标记。经验证, 这些标记可被用来鉴别特定的舞毒蛾地理种群, 因此有助于对这些舞毒蛾地理种群的分布与扩散进行监测。     

Molecular cloning and functional characterization of a novel delayed rectifier potassium channel from channel catfish (Ictalurus punctatus): expression in taste buds

The gustatory system of channel catfish is widely studied for its sensitivity to amino acids. As a first step in identifying the molecular components that play a role in taste transduction in catfish, we cloned the full-length cDNA for Kv2-catfish, a novel K(+) channel that is expressed in taste buds. The deduced amino acid sequence is 816 residues, and shares a 56-59% sequence identity with Kv2.1 and Kv2.2, the other members of the vertebrate Kv2 subfamily of voltage-gated K(+) channels. The Kv2-catfish RNA was expressed in taste buds, brain, skeletal muscle, kidney, intestine and gills, and its gene is represented as a single copy in the catfish genome. Recombinant channels expressed in XENOPUS: oocytes were selective for K(+), and were inhibited by tetraethylammonium applied to the extracellular side of the membrane during two-electrode voltage clamp analysis with a 50% inhibitory constant of 6.1 mM. The channels showed voltage-dependent activation, and did not inactivate within 200 ms. Functionally, Kv2-catfish is a voltage-gated, delayed rectifier K(+) channel, and its primary structure is the most divergent sequence identified among the vertebrate members of the Kv2 subfamily of K(+) channels, being related equally well to Kv2.1 and Kv2.2.     

REVEALING GENETIC MARKERS IN GELIDIUM VAGUM (RHODOPHYTA) THROUGH THE RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) TECHNIQUE1

The recently developed random amplified polymorphic DNA technique was evaluated as a method for characterizing isolates of the agarophyte Gelidium vagum Okamura. Reaction conditions for single primer polymerase chain reaction were optimized to obtain a high degree of reproducibility of the amplified bands generated from purified G. vagum DNA. A total of 165 primers, including both (A + T)- and (G + C)-rich sequences, was screened for DNA amplification using template DNA from a single Gelidium isolate. None of the 45 (A + T)-rich primers was positive (i.e. band-producing). Of the (G + C)-rich primers, 47 were positive, generating a total of 322 prominent amplification products for DNA from 13 different G. vagum isolates. Polymorphic DNA loci were detected by 37 of the primers. Unweighted pair-group arithmetic average cluster analysis (UPGMA) of these loci was used to group the G. vagum isolates and thereby determine which were most similar. G. latifolium, used as an out-group for the UPGMA analysis, showed a high degree of dissimilarity.     

SNP discovery in wild and domesticated populations of blue catfish,Ictalurus furcatus,using genotyping‐by‐sequencing and subsequent SNP validation

Blue catfish, Ictalurus furcatus, are valued in the United States as a trophy fishery for their capacity to reach large sizes, sometimes exceeding 45 kg. Additionally, blue catfish × channel catfish (I. punctatus) hybrid food fish production has recently increased the demand for blue catfish broodstock. However, there has been little study of the genetic impacts and interaction of farmed, introduced and stocked populations of blue catfish. We utilized genotyping‐by‐sequencing (GBS) to capture and genotype SNP markers on 190 individuals from five wild and domesticated populations (Mississippi River, Missouri, D&B, Rio Grande and Texas). Stringent filtering of SNP‐calling parameters resulted in 4275 SNP loci represented across all five populations. Population genetics and structure analyses revealed potential shared ancestry and admixture between populations. We utilized the Sequenom MassARRAY to validate two multiplex panels of SNPs selected from the GBS data. Selection criteria included SNPs shared between populations, SNPs specific to populations, number of reads per individual and number of individuals genotyped by GBS. Putative SNPs were validated in the discovery population and in two additional populations not used in the GBS analysis. A total of 64 SNPs were genotyped successfully in 191 individuals from nine populations. Our results should guide the development of highly informative, flexible genotyping multiplexes for blue catfish from the larger GBS SNP set as well as provide an example of a rapid, low‐cost approach to generate and genotype informative marker loci in aquatic species with minimal previous genetic information.