Valuation for usefulness of selected chromosomal markers for Bacillus anthracis identification. I. Valuation for markers SG-749, SG-450 and SG-300

The article presents results of valuation for B. anthracis-specificity and usefulness for its identification obtained for different chromosomal markers. In the first part of the study markers SG-749, SG-300 and SG-450 were analyzed. For the investigation RFLP-PCR and MSSCP techniques were used and different electrophoresis methods were tested. The results gave an information not only about specificity of tested markers but also about the possibility of shorten time necessary to obtain results of B. anthracis identification.     

Evaluation of usefulness for selected virulence markers for identifying pathogenic Yersinia enterocolitica strains. IV. Genes myfA and ureC

We check by polymerase chain reaction (PCR) the presence of gene ureC and myfA, encoding subunits of urease and Myf fimbriae, among clinical and food-originated strains of Yersinia to determine their usefulness as molecular virulence markers of Y. enterocolitica. The examinations were done on 130 clinical strains of Y. enterocolitica O:3/4 isolated in Poland from humans. All strains were obtained from stool and possessed the virulence plasmid pYV. In addition 40 isogenic, plasmid-cured strains were tested. The 52 strains including Y. enterocolitica (biotype 1A, 4, 2 and 1B), Y. pseudotuberculosis, Y. intermedia, Y. frederiksenii, Y. kristensenii, E. coli, Citrobatcer, Shigella and Salmonella were used as controls. The PCR assay resulted in detection of genes: ureC and myfA in genomic DNA of all 130 tested clinical strains of Y. enterocolitica pYV+, as well as in plasmid cured strains. Furthermore, ureC was found in all tested strains of Y. enterocolitica biotype A1 and in one strain of Y. intermedia and Y. kristensenii. In contrast to ureC, myfA was detected only in strains of Y. enterocolitica considered as pathogenic. Obtained results show, gene myfA seems to be the reliable virulence marker of Y. enterocolitica, whereas ureC is not recommended for identification of pathogenic strains of this species.     

Development and validation of molecular markers for grain cadmium in durum wheat

Durum wheat is capable of accumulating cadmium, a toxic heavy metal, in the grain at levels that have been deemed unsafe for human consumption. Previous studies have identified genetic variation as well as markers associated with Cd accumulation in durum wheat, which can be exploited to develop low Cd cultivars. Because the phenotyping for Cd content is very expensive, KASP markers were developed from molecular markers associated with grain Cd and tested for their usefulness for marker-assisted breeding. A total of 1278 unique genotypes from preliminary and advanced yield trials grown at multiple locations for 2 years were evaluated for grain Cd as well as screened for markers associated with Cd uptake. One marker on chromosome 5B was polymorphic in all crosses between high and low Cd parents and had r² values ranging from 0.38 to 0.85. Two other markers on the same chromosome predicted similar levels of variation in many trials; however, they were not polymorphic in all populations. The KASP markers accurately predicted up to 97% of the lines for Cd phenotype in different trials. This study identified two markers, Cad-5B and Ex_c1343_2570756, with an average prediction accuracy of 84–88%. These markers could be useful for marker-assisted selection for low grain Cd in durum wheat.     

Microsatellite markers for the study of cetacean populations

Microsatellites are one of the most important classes of nuclear genetic markers and offer many advantages for the study of marine mammals. Here we describe the isolation and characterization of 12 cetacean microsatellites which are then tested across 30 different cetacean species. For around half the species tested, five or more polymorphic loci were identified. Since many species were represented by only one or two specimens, this figure is likely to underestimate the usefulness of these markers. No relationship was found between microsatellite repeat length and proportion of species which gave polymorphic products.     

Genetic variation and identification of promising sour cherries inferred from microsatellite markers

The aim of this study was to identify the group of highly polymorphic microsatellite markers for identification of promising sour cherries. From among 30 tested microsatellite (SSR) markers, 19 were selected to profile genetic variation in sour cherries due to high polymorphisms. Results indicated a high level of polymorphism of the accessions based on these markers. Totally 148 alleles were generated at 19 SSR loci which 122 alleles were polymorphic. The number of total alleles per locus ranged from 2 to 15 with an average of 7.78 and polymorphism percentage varied from 50 to 100% with an average of 78.76%. Also, PIC varied from 0.47 to 0.89 with an average of 0.79 and heterozygosity ranged from 0.35 to 0.55 with a mean of 0.45. According to these results, these markers specially PMS3, PS12A02, PceGA34, BPPCT021, EMPA004, EMPA018, and Pchgms3 produced good and various levels of amplifications and showed high heterozygosity levels. By the way, the genetic similarity showed a high diversity among the sour cherries. Cluster analysis separated improved cultivars from promising sour cherries, and the PCoA supported the cluster analysis results. Since the studied sour cherries were superior to the improved cultivars and were separated from them in most groups, these sour cherries can be considered as distinct genotypes for further evaluations in the framework of breeding programs and new cultivar identification in cherries. Results also confirmed that the set of microsatellite markers employed in this study demonstrated usefulness of microsatellite markers for the identification of sour cherry genotypes.     

Identification of RAPD markers linked to A and B genome sequences in Musa L.

Plantains and bananas (Musa spp. sect. eumusa) originated from intra- and interspecific hybridization between two wild diploid species, M. acuminata Colla. and M. balbisiana Colla., which contributed the A and B genomes, respectively. Polyploidy and hybridization have given rise to a number of diploid, triploid, and tetraploid clones with different permutations of the A and B genomes. Thus, dessert and highland bananas are classified mainly as AAA, plantains are AAB, and cooking bananas are ABB. Classification of Musa into genomic groups has been based on morphological characteristics. This study aimed to identify RAPD (random amplified polymorphic DNA) markers for the A and B genomes. Eighty 10-mer Operon primers were used to amplify DNA from M. acuminata subsp. burmannicoides clone 'Calcutta 4' (AA genomes) and M. balbisiana clone 'Honduras' (BB genomes). Three primers (A17, A18, and D10) that produced unique genome-specific fragments in the two species were identified. These primers were tested in a sample of 40 genotypes representing various genome combinations. The RAPD markers were able to elucidate the genome composition of all the genotypes. The results showed that RAPD analysis can provide a quick and reliable system for genome identification in Musa that could facilitate genome characterization and manipulations in breeding lines.     

Molecular markers with potential to replace phage typing for Salmonella enterica serovar typhimurium

Using Amplified Fragment Length Polymorphism (AFLP) analysis of isolates from 23 phage types, we isolated 11 molecular markers that are potentially useful for molecular typing of Salmonella enterica serovar typhimurium. We tested these and 11 previously studied markers for their ability to discriminate among isolates and for correlation of their distribution with phage types. The Simpson's index of discriminatory power for the molecular markers is 0.96. One hundred and twenty one isolates from 33 phage types tested were divided into 51 types which are further grouped into 24 patterns. Eight patterns can unambiguously identify 8 phage types and a further 12 correlated with phage type distribution, showing the usefulness of these markers for molecular phage typing.     

Characterization of Iberian pig genotypes using AFLP markers

The use of the AFLP (amplified fragment length polymorphism) technique for the characterization of highly inbred Iberian pig breed genotypes and the detection of strain-specific polymorphisms is demonstrated. Twelve different primer combinations were used on individual DNA samples from animals belonging to two black hairless Iberian pig strains, Guadyerbas and Coronado. These amplification reactions allowed the detection of more than 1700 amplification products of which 26 were identified as strain-specific markers, present in all individuals of one strain and absent in the other. Comparison of male and female amplification products within one strain also allowed the identification of 8 male-specific amplified bands. AFLP showed a great power of marker detection due to a high multiplex ratio and high reproducibility. Comparison of similarity and co-ancestry coefficient matrices also showed the usefulness of AFLP markers to estimate genetic relationships between individuals pigs.     

New approach for isolation of VNTR markers.

Elsewhere we have reported an efficient method for isolating VNTR (Variable Number of Tandem Repeats) markers. Several of the VNTR markers isolated in those experiments were sequenced, and a DNA sequence of 9 bp (GNNGTGGG) emerged as an apparent consensus sequence for VNTR markers. To confirm this result and to develop more VNTR markers, we synthesized nine different 18-base-long oligonucleotides whose sequences each included GNNGTGGG. When 102 cosmid clones selected by these oligonucleotides were tested for polymorphism, 34 (33%) of them showed multiallelic VNTR polymorphisms (average heterozygosity 68%). This procedure represents a new and efficient approach for isolating additional VNTR markers and supports the idea that the GNNGTGGG sequence may play an important role in the generation of the multiallelic systems within the human genome.     

A consensus list of microsatellite markers for olive genotyping

Cultivar identification is a primary concern for olive growers, breeders, and scientists. This study was aimed at examining the SSR markers retrieved from the literature and currently used in olive study, in order to select those most effective in characterizing the olive accessions and to make possible the comparison of data obtained by different laboratories. Olive microsatellite profiles were assessed by four independent laboratories, which analyzed 37 pre-selected SSR loci on a set of 21 cultivars. These SSR markers were initially tested for their reproducibility, power of discrimination and number of amplified loci/alleles. Independent segregation was tested for each pair of SSRs in a controlled cross and the allelic error rate was quantified. Some of them were finally selected as the most informative and reliable. Most of the alleles were sequenced and their sizes were determined. Profiles of the reference cultivars and a list of alleles with their sizes obtained by sequencing are reported. Several genetic parameters have been analysed on a larger set of cultivars allowing for a deeper characterization of the selected loci. Results of this study provide a list of recommended markers and protocols for olive genotyping as well as the allelic profile of a set of reference cultivars that would be useful for the establishment of a universal database of olive accessions.     

Development of species-specific SCAR markers for identification of three medicinal species of Phyllanthus

Phyllanthus amarus Schum. & Thonn. has been widely used in traditional medicine in Thailand as an antipyretic, a diuretic, to treat liver diseases and viral infections. Two closely related species, P. debilis L. and P. urinaria Klein ex Willd., with different and less effective medicinal properties, are less commonly used. These three species are similar in morphology and often occur in overlapping populations in nature. The latter two species can easily be mistaken for P. amarus and collected for medicinal uses, which can lead to undesirable results. DNA fingerprints of these species were obtained using RAPD-PCR techniques. RAPD markers specific for each species were identified. Primers for highly specific sequence-characterized-amplified-regions (SCAR) were then designed from nucleotide sequences of specific RAPD markers. These primers efficiently amplified SCAR markers of 408, 501 and 319 bp unique to P. amarus, P. debilis and P. urinaria respectively. This method of plant identification was rapid and highly specific when tested against DNA of several closely related species and was able to amplify specific markers from mixed DNA samples.     

Development of species-specific SCAR markers for identification of three medicinal species of Phyllanthus

Phyllanthus amarus Schum.& Thonn.has been widely used in traditional medicine in Thailand as an antipyretic.a diuretic.to treat liver diseases and viml infections.Two closely related species,P. debills L.and P.urinaria KIein ex Willd.,with different and less effective medicinal properties,are less commonly used.These three species are similar in morphology and often Occur in overlapping populations in nature.The latter two species can easily be mistaken for P.amarus and collected for medicinal uses, which can lead to undesirable results.DNA fingerprints of these species were obtained using RAPD-PCR techniques.RAPD markers specific for each species were identified.Primers for highly specific sequence-characterized-amplified-regions (SCAR) were then designed from nucleotide sequences of specific RAPD markers.These primers efficiently amplified SCAR markers of 408,501 and 319 bp unique to P.amarus,P.debilis and P.urinaria respectively.This method of plant identification Was rapid and highly specific when tested against DNA of several closely related species and was able to amplify specific markers from mixed DNA samples.     

基于黄瓜基因组重测序的InDel标记开发及其应用

基于黄瓜基因组重测序结果,搜索InDel位点,按照每隔1～3M个碱基对的距离选择并设计遍布全基因组的代表性InDel引物134对,以16份黄瓜典型种质检测其有效性。结果显示,134对引物均获得扩增产物,其中具有多态性的引物116对,占引物总数的86.6%。116对引物充分揭示出16份种质的多样性和特异性。本研究所开发的InDel分子标记将为黄瓜种质资源和分子遗传育种研究提供有力的遗传工具。     

Phenotyping human leukemic T-Cell lines: Enzyme markers; surface antigens,and cytogenetics

We have compared phenotypic markers for a series of established human leukemic T-cell lines collected from different laboratories. Cell lines were tested first for genetic markers using polymorphic enzymes and then for expression of T lymphoid cell surface differentiation antigens using monoclonal antibodies. Chromosomal analysis was used as an additional method for identification of selected cell lines. On the basis of enzyme markers, it was possible to assign each of the cell lines examined to one of nine different groups. With two exceptions, surface antigen phenotypes for each of 12 cell lines were clearly distinctive. Thus, some groups of cell lines indistinguishable by enzyme markers could be further subdivided by surface antigen phenotyping. However, significant quantitative variation in expression of individual antigens was observed. In addition, surface antigen expression was not uniform in different subcultures of one cell line studied in detail. These results indicate that leukemic T-cell lines cannot be used generally as simple models of surface antigen expression in normal T-cell differentiation.     

A search for genetic markers associated with egg production in the ostrich (Struthio camelus)

The aim of the current study was to search for genetic markers, microsatellite loci associated with laying performance in ostriches. The material consisted of two groups of ostrich hens characterized by high or low laying performance (over 75 and less than 25 eggs per season, respectively). The investigation covered 30 microsatellite loci characteristic for the ostrich (the CAU group) and led to identification of significant differences in allele and genotype frequencies between the two groups of hens considered. Out of a total of 30 microsatellite loci examined, 28 showed different alleles in relation to analyzed performance groups. In hens of high laying performance (HP group, n = 12), specific alleles occurred in 23 microsatellite loci (40 alleles of 243 identified), while in those of low egg production (LP group, n = 12), they occurred in 22 (51 alleles of 243 identified). The results indicate the usefulness of the microsatellite loci as the potential genetic markers associated with laying performance that can be applied for genetic improvement of ostrich flocks.     

Evaluation of the usefulness of selected virulence markers for identification of virulent Yersinia enterocolitica strains. II. Genotypic markers associated with the pYV plasmid

Pathogenic strains of Yersinia enterocolitica bear virulence associated plasmid pYV. Unfortunately plasmid pYV is easily lost by these bacteria incubated at elevated temperatures (37 degrees C) or long stored at room temperatures. This sometimes makes difficult the detection of the virulence plasmid, especially by its isolation or biochemical tests. On the other hand, observations done by some authors suggest that polymerase chain reaction (PCR) could be useful for demonstration of the pYV plasmid of Yersinia strains. Accordingly to this observation the aim of the presented study was to check the usefulness of plasmid-localised genes virF and yadA, detected by PCR, for the identification of the virulent strains of Y. enterocolitica. In the presented study one hundred and fifty two clinical strains of Y. enterocolitica belonging to serogroup O3 were investigated by the PCR for the presence of genes virF and yadA. Bacterial strains were first tested for the presence of pYV plasmid. In addition the phenotypic features: calcium dependence, Congo red binding and autoagglutination were determined. In this way the virulence plasmid was found in 130 of 152 examined strains. For PCR studies also forty plasmid-cured strains of Y. enterocolitica and 32 non-Y. enterocolitica, Enterobacteriaceae strains were included. The obtained results show that the tested genes were present only in Yersinia strains possessing the pYV plasmid and no one non-specific PCR product was observed. The detection level of these genes in nested PCR permits to detect pathogenic Y. enterocolitica in suspension composed of 1 x 10(3) CFU/ml of pYV+ bacilli and 3 x 10(9) CFU/ml plasmid-cured, isogenic bacteria. In the study it was shown that genes virF and yadA were useful virulence markers, which could be helpful in clinical studies for the detection of the virulence plasmid in Y. enterocolitica strains long stored or incubated at elevated temperatures.     

ISSR as new markers for genetic characterization and evaluation of relationships among phytoplankton

In order to increase the molecular tools and markers needed for the identification of phytoplankton species, the inter simple sequence repeat (ISSR) fingerprinting was adapted to micro-algae and its use in genetic analysis was demonstrated. Twelve strains, 6 Alexandrium, 4 Pseudo-nitzschia, 1 Skeletonema and 1 Tetraselmis were analysed for the first time with ISSR amplifications. The patterns were highly polymorphic and very reproducible. The 6 primers gave 223 polymorphic markers that clearly and easily distinguished all 12 strains (mainly toxic ones) and gave 187 polymorphic markers among the Alexandrium and the Pseudo-nitzschia species. ISSR amplifications also indicated a large occurrence of simple sequence repeat (SSR) in phytoplankton genomes, especially in Pseudo-nitzschia, and show their usefulness to cluster intra and inter species. ISSR markers were found to be good markers for genetic characterization and diversity study and led to consider them as new tools for the survey of phytoplankton.     

Histological markers of solid cell nests of the thyroid. With some emphasis on their expression in thyroid ultimobranchial-related tumors

It was recently demonstrated that C cells, mucosubstances, and the intermediate filament epidermal keratin are present in solid cell nests (SCN) of the thyroid. The reliability and usefulness of these markers in detecting these ultimobranchial nests have, so far, not been comparatively investigated. In the light of this, a study for the presence of these tracers in SCN at different stages of postnatal life was undertaken. The existence of carcinoembryonic antigen (CEA) in this gut-derived tissue was also searched for. Epidermal keratin was present in the epidermoid-like cells from all SCN studied; calcitonin-immunoreactive C cells and mucosubstances did not always occur. These findings reveal that this cytokeratin would remain as a potential marker to detect and trace back the ultimobranchial tissue component of the thyroid in earlier stages of development. The presence of CEA in practically all SCN surveyed would further support the view that they originate in the gut. This antigen would also be of great value for histological identification of these nests. The usefulness of these markers for the interpretation of the histogenesis of some ultimobranchial-related thyroid neoplastic growths, specifically the medullary and mucoepidermoid carcinomas, is briefly discussed.     

B1 insertions as easy markers for mouse population studies

Few simple, easy-to-score PCR markers are available for studying genetic variation in wild mice populations belonging to Mus musculus at the population and subspecific levels. In this study, we show the abundant B1 family of short interspersed DNA elements (SINEs) is a very promising source of such markers. Thirteen B1 sequences from different regions of the genome were retrieved on the basis of their high degree of homology to a mouse consensus sequence, and the presence of these elements was screened for in wild derived mice representing M. spretus, macedonicus and spicilegus and the different subspecies of M. musculus. At five of these loci, varying degrees of insertion polymorphism were found in M. m. domesticus mice. These insertions were almost totally absent in the mice representing the other subspecies and species. Six other B1 elements were fixed in all the Mus species tested. At these loci, polymorphism associated with three restriction sites in the B1 consensus sequence was found in M. musculus. Most of these polymorphisms appear to be ancestral as they are shared by at least one of the other Mus species tested. Both insertion and restriction polymorphism revealed differences between five inbred laboratory strains considered to be of mainly domesticus origin, and at the six restriction loci a surprising number of these strains carried restriction variants that were either not found or very infrequent in domesticus. This suggests that in this particular group of loci, alleles of far Eastern origin are more frequent than expected.     

DNA markers for sex: Molecular evidence for gender dimorphism in dioecious Mercurialis annua L.

Male specific Random Amplified Polymorphic DNA (RAPD) markers, OPB01-1562 and OPC07-303, were identified and sequenced in dioecious Mercurialis annua. Sequence Characterized Amplified Region (SCAR) primers were designed. Several internal segments of OPB01-1562 were amplified as male specific SCAR markers. These markers were PCR amplified from strong, intermediate and weak male subtypes selected according to their resistance to feminization by cytokinin. Nucleotide sequence of OPB01-1562 isolated from three male subtypes were near identical. The OPB01-1562 and derived SCAR markers were absent in females as well as hexaploid Mercurialis male and monoecious individuals. The gender relationship of the markers was maintained in all ecotypes tested. There were 2 internal fragments of OPB01-1562, which were PCR amplified from all genotypes of diploid and hexaploid Mercurialis. It is argued that identification of gender specific DNA suggests a dimorphic differentiation of the genome of dioecious Mercurialis annua.