Genetic diversity in common carp stocks assayed by random-amplified polymorphic DNA markers

The common carp (Cyprinus carpio L.) is one of the major aquaculture species, contributing nearly 35% to the inland fish production in Karnataka, India. Stocks collected from Hungary (2), Indonesia and Vietnam were assessed alongside two local stocks in a series of culture performance trials with the objective of setting up a base population for developing a breeding programme. The present study deals with the genetic divergence and polymorphism in these six stocks using random‐amplified polymorphic DNA (RAPD) markers. A total of 180 decamer random primers were screened for polymerase chain reaction (PCR) amplification (OPA 1‐20, OPB1‐20, OPC1‐20, OPD1‐20, OPE1‐20, OPF1‐20, OPG1‐20, OPP1‐20 and OPM1‐20). Eight primers were selected for analysis of common carp genotypes (OPA‐7, OPA‐20, OPB‐17, OPF‐10, OP F‐9, OPG‐4, OPG‐9 and OPP‐16). Out of 492 bands recorded, 57.1% were polymorphic. Stepwise regression analysis was carried out to find best combination markers affecting body weight (P<0.001). The results demonstrate major differences in the genetic structures between different stocks. Dendrogram data showed grouping of individuals according to stocks and corresponding data variables revealed the per cent homology within the stock and also found markers correlating to the body weight.     

Genotypes,Antibiotic Resistance Profiles and Microarray‐Based Characterization of Methicillin‐Resistant Staphylococcus aureus Strains Isolated from Livestock and Veterinarians in Switzerland

Using different typing methods (MLST, spa‐, SCCmec‐ and agr‐typing), PFGE and DNA microarray‐based chip analysis, we characterized 20 MRSA strains isolated from livestock and veterinarians. PFGE analysis after macrorestriction with EagI provided seven different band patterns, which could be grouped into four clusters. One cluster consisted of all MRSA ST398 strains isolated from pigs, calves, mastitis milk and two veterinarians. One strain of ST398 from a veterinarian and the two strains of ST1 and ST8 formed the three other clusters. Antimicrobial susceptibility testing showed that 15 of 20 strains were resistant to ampicillin, cefoxitin, clindamycin, erythromycin, oxacillin, penicillin and tetracycline. All strains were susceptible to rifampin and vancomycin, 19 were susceptible to ciprofloxacin and 18 were susceptible to sulphamethoxazole/trimethoprim. Genes encoding different enterotoxins, leukotoxins and haemolysins were found in certain strains.     

河北省鼠疫菌株差异片段基因分型及其流行病学特征

目的对分离自河北省自然疫源地的115株鼠疫耶尔森菌(鼠疫菌)株进行基因分型,研究其基因型分布及流行特征。方法根据鼠疫菌基因组23个差异区段(different regions,DFR)设计引物,对115株鼠疫菌进行基因分型,应用PCR技术扩增鼠目的区段并做普通琼脂糖凝胶电泳,应用BioNumefics 5.0进行聚类分析。结果在河北省鼠疫疫源地分离到的115株鼠疫菌均属于1个基因型,即Genomovar11型。最近一次动物鼠疫流行(2005年)的生态型仍是鄂尔多斯高原型生态型。结论河北省分离到的115株鼠疫菌基因型为Genomovar11型。     

Prevalence and Characterization of Coagulase Positive Staphylococci Isolated from Salted Anchovy

A total of 100 salted anchovy samples were used to investigate the prevalence of S. aureus and other coagulase positive Staphylococci (CPS) as well as to determine the methicillin (MR) and antibiotic resistance (AR) profile, the presence of Panton-Valentine leukocidine (PVL) toxin gene (lukS/F-PV), slime factor properties (SFP), and the genotypic relatedness of the isolates. Agar disc diffusion assay (ADDA) and microdilution broth susceptibility test (MDBST) were applied to compare the specificity and sensitivity of the MR detection methods. A total of 41 CPS isolates were detected at the 10² and 10³ CFU/g levels in contrast to S. aureus. The 16S rRNA (genus specific) was detected in all the isolates in contrast to nuc (species-specific) and lukS/F-PV genes. A total of 16/41 isolates were found to be MR by using the three methods. Polymerase chain reaction (PCR) assay was a more sensitive and reliable method for the detection of MR isolates. The antibiotic resistance rates were 75.60, 73.17, 51.21, 31.70, 12.19, and 4.87% to penicillin, ampicillin, tetracycline, erythromycin, ciprofloxacin, and clindamycin, respectively. All the isolates were sensitive to gentamicin and vancomycin. The SFP were determined in all the isolates by using Congo Red agar, and 20 different genotypes were determined by using randomly amplified polymorphic DNA (RAPD)-PCR assay.     

Intraspecific diversity of Edwardsiella ictaluri isolates from diseased freshwater catfish, Pangasianodon hypophthalmus (Sauvage), cultured in the Mekong Delta, Vietnam

A molecular epidemiology study was conducted on 90 Edwardsiella ictaluri isolates recovered from diseased farmed freshwater catfish, Pangasianodon hypophthalmus, cultured in the Mekong Delta, Vietnam. Thirteen isolates of E. ictaluri derived from diseased channel catfish, Ictalurus punctatus, cultured in the USA were included for comparison. All the E. ictaluri isolates tested were found to be biochemically indistinguishable. A repetitive (rep)‐PCR using the single (GTG)₅ primer was shown to possess limited discriminatory power, yielding two similar DNA profiles categorized as (GTG)₅‐PCR group 1 or 2 among the Vietnam isolates and (GTG)₅‐PCR group 1 within the USA isolates. Macrorestriction analysis identified 14 and 22 unique pulsotypes by XbaI and SpeI, respectively, among a subset of 59 E. ictaluri isolates. Numerical analysis of the combined macrorestriction profiles revealed three main groups: a distinct cluster formed exclusively of the USA isolates, and a major and minor cluster with outliers contained the Vietnam isolates. Antibiotic susceptibility and plasmid profiling supported the existence of the three groups. The results indicate that macrorestriction analysis may be regarded as a suitable typing method among the E. ictaluri species of limited intraspecific diversity. Furthermore, the findings suggest that E. ictaluri originating from Vietnam may constitute a distinct genetic group.     

A novel genotyping technique for distinguishing between Flavobacterium psychrophilum isolates virulent and avirulent to ayu, Plecoglossus altivelis altivelis (Temminck & Schlegel)

We developed a simple genotyping method for Flavobacterium psychrophilum for analysing two single nucleotide polymorphisms (SNPs) in the gyrA gene and to distinguish between isolates that are virulent and avirulent to ayu, Plecoglossus altivelis altivelis (Temminck & Schlegel). The genotyping method is an on/off switch assay and is based on the polymerase chain reaction technique with phosphorothioated primers. We classified 232 isolates from four families of fish (i.e. Plecoglossidae, Osmeridae, Cyprinidae and Salmonidae) into four genotypes (G‐C, A‐T, A‐C and G‐T). The G‐C type isolates exhibited strong pathogenicity to ayu, whereas the A‐T and G‐T types did not show any pathogenicity to this species. The A‐C type exhibited no or weak pathogenicity to ayu. These results indicate that genotyping F. psychrophilum isolates with two SNPs from gyrA can clearly distinguish between isolates potentially harmful to ayu (G‐C type) and those that are potentially not harmful or less harmful (A‐C, A‐T and G‐T type). The on/off switch assay provides a quick, simple, and very powerful DNA genotyping technique for F. psychrophilum isolates.     

Counting elusive animals: Comparing field and genetic census of the entire mountain gorilla population of Bwindi Impenetrable National Park, Uganda

Accurate population size estimates are an essential part of every effective management plan for conserving endangered species. However, censusing rare and elusive wild animals is challenging and often relies on counting indirect signs, such as nests or feces. Despite widespread use, the accuracy of such estimates has rarely been evaluated. Here we compare an estimate of population size derived solely from field data with that obtained from a combination of field and genetic data for the critically endangered population of mountain gorillas (Gorilla beringei beringei) in Bwindi Impenetrable National Park, Uganda. After genotyping DNA from 384 fecal samples at 16 microsatellite loci, the population size estimate was reduced by 10.1% to 302 individuals, compared with 336 gorillas estimated using the traditional nest-count based method alone. We found that both groups and lone silverbacks were double-counted in the field and that individuals constructed multiple nests with an overall rate of 7.8%, resulting in the overestimation of the population size in the absence of genetic data. Since the error associated with the traditional field method exceeded the estimated population growth of 5% in the last 4 years, future genetic censusing will be needed to determine how the population size is changing. This study illustrates that newly improved molecular methods allow fast, efficient and relatively affordable genotyping of several hundred samples, suggesting that genetic censusing can be widely applied to provide accurate and reliable population size estimates for a wide variety of species.     

大豆引进种质抗胞囊线虫病、抗花叶病毒病和耐盐基因型鉴定及优异等位基因聚合种质筛选

我国从美国、俄罗斯、日本等26个国家或地区共引进大豆种质3218份, 仅对部分种质进行了大豆胞囊线虫病(Soybean cyst nematode, SCN)、大豆花叶病毒病(Soybean mosaic virus, SMV)和盐敏感性的抗性鉴定, 但基因型的系统分析尚未见报道。本研究针对大豆抗胞囊线虫病3个基因(rhg1、Rhg4、SCN3-11)和耐盐基因(GmSALT3)开发KASP标记5个, 结合与大豆花叶病毒抗性相关的1个SCAR标记(SCN11), 对1489份大豆引进种质进行基因型鉴定。结果表明, 具有优异等位基因的种质共1084份; 携带3个位点优异等位基因的种质19份, 包括抗胞囊线虫病3个位点(rhg1、Rhg4、SCN3-11)叠加(Peking型)种质3份, 聚合抗胞囊线虫病基因和抗花叶病毒病标记7份, 聚合抗胞囊线虫病和耐盐基因2份, 聚合抗胞囊线虫病、抗花叶病毒病和耐盐基因7份; 携带4个位点优异等位基因的种质9份, 包括聚合抗胞囊线虫病基因和抗花叶病毒病标记6份, 聚合抗胞囊线虫病和耐盐基因2份, 聚合抗胞囊线虫病、抗花叶病毒病和耐盐7份; 携带5个位点优异等位基因8份, 聚合了抗胞囊线虫病、抗花叶病毒病和耐盐优异等位变异。在这些携带优异等位变异的种质中, 44份已由前人证明具有相应的抗性。携带3个或3个以上优异等位基因的36份种质中, 有52.78%种质的一种或两种特性已被报道。在不携带抗性优异等位变异的种质中, 93份已证明有耐盐性或对SMV3号株系抗性, 这些种质可能存在新的抗性(等位)基因。本研究利用高通量分子标记筛选出的携带抗病、抗逆优异等位基因的种质为我国大豆资源表型鉴定、抗源的快速筛选及利用提供理论依据和新思路。     

An optimized high quality male DNA extraction from spermatophores in open thelycum shrimp species

The crucial step of most of the current genetic studies is the extraction of DNA of sufficient quantity and quality. Several genomic DNA isolation methods have been described to successfully obtain male DNA from shrimp species. However, all current protocols require invasive handling methods with males for DNA isolation. Using Aristeus antennatus as a model we tested a reliable non‐invasive differential DNA extraction method to male DNA isolation from spermatophores attached to female thelycum. The present protocol provides high quality and quantity DNA for polymerase chain reaction amplification and male genotyping. This new approach could be useful to experimental shrimp culture to select sires with relevant genetic patterns for selective breeding programs. More importantly, it can be applied to identify the mating pairs and male structure in wild populations of species as A. antennatus, where males are often difficult to capture. Our method could be also valuable for biological studies on other spermatophore‐using species, such as myriapods, arachnids and insects.     

Characterization of Puccinia graminis f. sp. tritici isolates derived from an unusual wheat stem rust outbreak in Germany in 2013

An unusual stem rust infestation occurred in German wheat fields in summer 2013. This study analysed 48 isolates derived from 17 Puccinia graminis f. sp. tritici (Pgt) samples and six races were identified: TKTTF, TKKTF, TKPTF, TKKTP, PKPTF and MMMTF. Infection type and genotypic data confirmed that none of these races belonged to the TTKS (Ug99) race group. German isolates of race TKTTF are phenotypically different to the ones responsible for the stem rust epidemic in Ethiopia in 2013–2014. Forty isolates were genotyped using a custom SNP array. Phylogenetic analysis showed that these 40 isolates represented two distinct lineages (clade IV and clade V). Thirty‐eight isolates clustered into clade IV, which previously was defined by Ethiopian isolates of race TKTTF. Race TKKTP is of special concern due to its combined virulence to stem rust resistance genes Sr24, SrTmp and Sr1RS^Amigo. The vulnerability to race TKKTP in US and international winter wheat was confirmed as 55% of North American and international cultivars and breeding lines resistant to race TTKSK (Ug99) became susceptible to TKKTP. Races identified in Germany in 2013 confirmed the presence of virulence to important resistance genes that are effective against race TTKSK. This information should be useful for breeders to select diverse and effective resistance genes in order to provide more durable stem rust resistance and reduce the use of fungicides.