Microsatellite markers associated with quantitative trait loci controlling antibody response to Escherichia coli and Salmonella enteritidis in young broilers

A unique resource population was produced to facilitate detection of microsatellite markers associated with quantitative trait loci controlling antibody (Ab) response in broiler chickens. Three F1 males were produced by mating two lines divergently selected on Ab response to Escherichia coli vaccination. Each F1 male was mated with females from four genetic backgrounds: F1, high-Ab line (HH), low-Ab line and commercial line, producing three resource families, each with four progeny types. About 1700 chicks were immunized with E. coli and Salmonella enteritidis vaccines. Selective genotyping was conducted on the individuals with highest or lowest average Ab to E. coli and S. enteritidis within each progeny type in each sire family. Twelve markers were significantly associated with Ab to E. coli and six of them were also associated with Ab to S. enteritidis, mostly exhibiting a similar low effect (approximately 0.35 phenotypic SD) in all progeny types. Four markers exhibited a highly significant and much larger effect (approximately 1.7 SD), but only in progeny of females from the HH, suggesting that a backcross to the high parental line should be preferred over the commonly used F2 population. Results from two markers suggested a quantitative trait locus on chromosome 2 around 400 cM. The marker MCW0083, significant in two sire families, is closely linked to the bone morphogenetic protein 2 (BMP2) gene, known to be associated with the control of T-cell transformation in humans.     

Enhanced reactivation and mutagenesis after transfection of carcinogen-treated monkey kidney cells with UV-irradiated simian virus 40 (SV40) DNA

Monkey kidney cells, either untreated or pretreated with UV-light at 254 nm or mitomycin C, were transfected 24 hours later with the intact or UV-irradiated DNA from the thermosensitive tsB201 simian virus 40 mutant unable to grow at 41 degrees C. The survival of the viral progeny obtained from the UV-irradiated DNA is increased in pretreated cells compared to the survival of the viral progeny obtained in untreated cells. Irradiation of the viral DNA enhances the reversion frequency of the viral progeny towards a wild type phenotype able to grow at 41 degrees C. Pretreatment of the cells with UV or mitomycin C does not increase the reversion frequency.     

Nonparental progeny resulting from protoplast fusion inTrichoderma in the absence of parasexuality

The purpose of this study was to characterize a number of progeny from intra- and interstrain protoplast fusion within the genusTrichoderma. We wished to determine whether parasexuality or other genetic mechanisms occur in these fungi. When two different auxotrophs of the same strain were fused, rapidly growing prototrophic progeny were obtained in high frequencies. When single spore isolates of these strains were prepared, equal numbers of strains indistinguishable from the two parental auxotrophic strains were obtained, even though 10⁹–10¹⁰ conidia were tested per strain. Thus, progeny from intrastrain fusions all appeared to be balanced heterokaryons, and no evidence of recombination between the two parental strains was obtained. When 16 separate interstrain fusions were conducted, very different results were obtained, regardless of whether fusions were within or between species. Following interstrain fusions, presumptive somatic hybrids developed very slowly and in low numbers as compared with hybrids from intrastrain fusions. Most were weakly prototrophic. These slow-growing progeny were unstable and sectors developed from them. Such sectors themselves were unstable and gave rise to other progeny. Usually sectors were more strongly prototrophic and more rapid growing than the original progeny strain. Sectoring gave rise to a very wide range of morphotypes. Most of these morphotype variants were stable through conidiation; thus, these types did not occur as a consequence of heterokaryosis. Isozyme analysis was conducted on over 1000 progeny strains. Nearly all progeny were identical to one or the other parental isozyme phenotypes. A few progeny, when tested as soon as possible after fusion, exhibited the isozyme phenotypes of both parents, but such biparental banding patterns were rapidly lost upon subsequent reculturing. Isozyme banding patterns of multimeric enzymes never gave band patterns indicative of heterokaryosis or heterozygosis. Banding patterns indicative of heterozygous diploids or recombinants were never detected. Despite the extreme variation in morphotype and nutritional requirements among progeny, isozyme banding patterns of derived progeny from any fusion were invariably identical to one or the other parental strains. From these results, we conclude that protoplast fusion in the genusTrichoderma gives rise to great variability, but that the classical parasexual cycle is not required for variation to occur.     

Transfer of Isolated Nuclei into Protoplasts of Trichoderma harzianum

Protoplasts released from young hyphae of Trichoderma harzianum contained 0 to 10 nuclei per protoplast, and most (about 80%) contained from 4 to 6 nuclei. Most protoplasts were larger than 3 μm in diameter. Nuclei were isolated from protoplasts of an auxotrophic mutant of T. harzianum and transferred into protoplasts obtained from another auxotroph of the same strain. This intrastrain nuclear transfer gave rise to numerous progeny which were stable, prototrophic, and heterokaryotic. Interstrain transfers in which nuclei from a wild-type prototroph of one strain were transferred into protoplasts from a lysine-deficient auxotroph of a second strain were also done. Heterokaryotic progeny were recovered from these interstrain transfers when the regenerating protoplasts were provided with a low concentration of lysine 48 h after the initial plating. Heterokaryotic progeny contained 11 to 17% of donor-type nuclei. Progeny homokaryotic for donor-type nuclei were obtained as single-spore isolates. These homokaryotic isolates expressed the isozyme pattern and colony morphology phenotype of the nuclear donor. When regenerating protoplasts were provided with lysine 10 days after the initial plating, only a single progeny was obtained. However, single-spore subprogeny of this nuclear transfer were prototrophic and exhibited a wide range of unstable morphological phenotypes.     

Fertility of triploid hybrids of Prussian carp (<Emphasis Type="Italic">Carassius gibelio</Emphasis>) with common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis> L.)

Fertility of backcross triploid hybrids containing one genome of Prussian carp and two genomes of common carp is investigated. The females of hybrids of Prussian carp and common carp (Prussian × common carp) are prolific and produce diploid gametes. Since males of such hybrids are sterile, their reproduction is realized by means of induced gynogenesis. Triploid progeny is obtained by backcrossing female Prussian × common carp with carp males. Among triploids obtained from hybrids F₁ and among hybrids of the first gynogenetic generation, there were no prolific specimens. However, in reproduction of diploid hybrids by means of gynogenesis during six generations, the female fertility in the backcross progeny is restored. From backcross triploid females (daughters of Prussian × common carp of the sixth gynogenetic generation), a viable triploid gynogenetic progeny and a tetraploid backcross (by carp) progeny are obtained. The obtained data may be considered as the experimental proof of the hypothesis of reticular speciation.     

Effects of genotype on pollen performance in Cucurbita pepo

Summary This study examines the assumption of the pollen competition hypothesis that genetic differences among microgametophytes lead to differences in pollen performance and result in non-random fertilization. In addition, we examined the assumption that pollen performance is genetically correlated with sporophyte vigor due to an overlap in gene expression between the two stages of the life cycle. The results from a pollen mixture experiment in which two cultivars of common zucchini were used show that the ability to sire seeds is nonrandom with respect to the cultivar of the pollen donor plant. The proportion of the progeny sired by the two cultivars is not independent of the region of the fruit where the seeds are produced. The progeny sired by the yellow cultivar outperformed the progeny sired by the green cultivar in a greenhouse study. In addition, the progeny sired by the yellow cultivar from the stylar region of the fruit germinated faster and had more leaf area than the progeny sired by the same cultivar from the peduncular end of the fruit. Thus, the most vigorous progeny are obtained from the stylar region of the fruit where the ovules are fertilized by the most vigorous microgametophytes.     

Genetic exchanges in the macrocysts of Dictyostelium discoideum.

Crosses were made between strains of Dictyostelium discoideum involving two drug resistance markers and the mating-type locus. Over 6000 progeny from 263 individual germinated macrocysts from four single-factor crosses, five two-factor crosses and one three-factor cross were characterized. In most cases the progeny from a single macrocyst were of one genotype, although in the population of macrocysts from any two-factor cross all possible parental and recombinant genotypes were recovered. There was no evidence of linkage between any of the markers examined. No selection against progeny carrying the methanol or the cycloheximide resistance markers was found in two-factor crosses, but selection against progeny carrying both resistance markers was found in the three-factor cross. Germination of macrocysts in all crosses was poor, only once exceeding 2.5% of the total macrocyst population. A variety of crosses and back-crosses with different parental strains indicated that germination might be influenced by both extrinsic (environmental) and multiple genetic factors. About 10% of the macrocysts yielded progeny spores that were ambivalent in their mating reactions. After extensive recloning these populations could be resolved to the normal matA (formerly A1) and mata (formerly A2) mating-types and might therefore have represented aneuploids. The results obtained with D. discoideum macrocysts differ from those obtained with other cellular slime moulds--Dictyostelium mucoroides, Dictyostelium giganteum and Polysphondylium pallidum--and are reminiscent of the results reported for germinated zygospores of Phycomyces blakesleeanus.     

The quantification of the haemagglutinin content of influenza whole virus and Tween-ether split vaccines

Monovalent whole virus and Tween-ether split vaccines prepared from influenza A/Bangkok, A/Brazil and B/Singapore were assayed for haemagglutinin content using single radial immunodiffusion (SRID), quantitative sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunization of guinea pigs. When SRID was performed with split vaccines, haemagglutinin values were consistently recorded which were in the range of 50 to 25% of the values obtained before disruption of virions. When, however, disruption was conducted in the presence of excess detergent, thus preventing aggregate formation of solubilized haemagglutinin, test values comparable with those of whole virus vaccines were obtained. In agreement with these results, immunization experiments revealed that whole virus and corresponding split vaccines exhibited comparable immunogenicity in guinea pigs. Additionally it could be calculated from SDS-PAGE and densitometer tracings, obtained by scanning the gels after staining with either Coomassie blue or FITC-Con A, that 90 to 95% of whole virus HA2 was recovered in Tween-ether split vaccines. On the basis of these findings we conclude that precise quantification of Tween-ether split vaccines is not possible by the SRID test alone. As aggregate formation of solubilized haemagglutinin occurs, we suggest that either a physico-chemical method including a disaggregation procedure, such as SDS treatment, or immunological evaluation of the original whole virus preparation before disruption of virions should be applied as an additional criterion for quantification of influenza Tween-ether split vaccines.     

AP-PCR assay of DNA alterations in the progeny of male mice exposed to low-level gamma-radiation

By comparative analysis of fingerprints of arbitrarily primed polymerase chain reaction (AP-PCR) products, DNA alterations in somatic cells of the progeny (F1 generation) of male mice chronically exposed to low-doses of gamma-radiation was investigated. Male BALB/c mice exposed to 10-50 cGy were mated with unirradiated females 15 days after irradiation. DNA was isolated from biopsies taken from tail tips of 2-month-old progeny. Preliminary AP-PCRs were carried out with 17 primers representing core sequences of micro- and/or minisatellites or their flanking oligonucleotides. Best quantitatively reproduced AP-PCR fingerprints of genomic DNA were obtained with one of these primers, a 20-mer oligonucleotide flanking the micro-satellite locus Atplb2 on mouse chromosome 11. Comparative analysis of individual fingerprints of AP-PCR products obtained on DNA templates from the progeny of irradiated and intact males revealed an increased variability of micro-satellite-associated sequences and an increased frequency of "non-parental bands" in DNA-fingerprints from the progeny of males chronically exposed to gamma-radiation 15 days before mating (at the postmeiotic stage of spermatogenesis). The results show that increased micro-satellite instability can be initiated by irradiation of the male parent to subsequently arise or be transmitted to the soma of the F1 generations.     

The development of F1 progeny from mature egg cells after terahertz radiation of parental drosophila

The effects of terahertz radiation (0.1–2.2 THz) on the dynamics of reaching the imago stage by drosophila of the F1 progeny obtained from the crossing of irradiated and unirradiated parental individuals in different combinations were studied. A shift in the maximum emergence peak for an earlier period and a shortening of the period of reaching the adult state were found in the progeny of both sexes obtained from irradiated females. The development up to the imago stage significantly differs in the progeny of irradiated males and irradiated females in a number of parameters. It was suggested that the effect of terahertz radiation on the dynamics of the onset of the imago stage can be associated with one or different mechanisms that change the expression of the genes and signaling pathways that control the development of drosophila.     

Agrobacterium-Mediated Transformation of Switchgrass and Inheritance of the Transgenes

Switchgrass (Panicum virgatum L.) has been developed into an important biofuel crop. Embryogenic calli induced from caryopses or inflorescences of the lowland switchgrass cultivar Alamo were used for Agrobacterium-mediated transformation. A chimeric hygromycin phosphotransferase gene (hph) was used as the selectable marker and hygromycin as the selection agent. Embryogenic calli were infected with Agrobacterium tumefaciens strain EHA105. Calli resistant to hygromycin were obtained after 5 to 8 weeks of selection. Soil-grown transgenic switchgrass plants were obtained 4 to 5 months after Agrobacterium infection. The transgenic nature of the regenerated plants was demonstrated by PCR, Southern blot hybridization analysis, and GUS staining. T1 progeny were obtained after reciprocal crosses between transgenic and untransformed control plants. Molecular analyses of the T1 progeny revealed various patterns of segregation. Transgene silencing was observed in the progeny with multiple inserts. Interestingly, reversal of the expression of the silenced transgene was found in segregating progeny with a single insert.     

The effects of pollen composition on fitness components in a neotropical herb

Summary Experimental pollinations of Costus allenii (Zingiberaceae) were conducted to assess the effects of pollen composition on fitness. Plants were selfed, outcrossed with the first nearest neighbor, and outcrossed with pollen mixtures obtained from the nearest 2, 3, and 5 plants. Cross type had a significant effect on seed production, seed weight and total-plant dry weight. Progeny from crosses with 3, and 5 parents grew significantly larger than selfed progeny, or those from 1-parent crosses. Competition experiments indicated the superiority of progeny from 3-, and 5-parent crosses over progeny from 1-parent crosses, but no differences in competitive ability were observed between progeny from 3-, and 5-parent crosses. Relative fitness, based on 1) seed production, 2) percent germination, and 3) dry weight, varied significantly among crosses, and was greatest for crosses with 3 parents and lowest for selfs. The relative fitness of progeny from 5-parent crosses was lower than that of all other outcrossed classes. We suggest that the significant effect of pollen composition on fitness results from variation in the genetic similarity of seed and pollen parents, which is a function of spatial distribution and population structure.     

Significance of specific-locus germ-cell mutations in mice.

On the basis of a historical-control incidence of 39 mutants in 688 921 progeny (5.7 mutants/10(5) animals or 0.82 mutations/locus/10(5) gametes) proposals are made for the numbers of test progeny required when screening for possible mutagens using the specific-locus test in mice (7 loci). It is recommended that 25,000 control progeny should be included in each test, to establish homogeneity with the historical controls. This would also be the number of progeny required from treated males, unless significantly positive results had been obtained with smaller numbers. It would appear that the greater sensitivity of post-spermatogonial stages could more than compensate for practical difficulties in sampling these stages, rather than spermatogonia, in screening tests.     

Evolution of H3N2 influenza virus in a guinea pig model

Studies of influenza virus evolution under controlled experimental conditions can provide a better understanding of the consequences of evolutionary processes with and without immunological pressure. Characterization of evolved strains assists in the development of predictive algorithms for both the selection of subtypes represented in the seasonal influenza vaccine and the design of novel immune refocused vaccines. To obtain data on the evolution of influenza in a controlled setting, naïve and immunized Guinea pigs were infected with influenza A/Wyoming/2003 (H3N2). Virus progeny from nasal wash samples were assessed for variation in the dominant and other epitopes by sequencing the hemagglutinin (HA) gene to quantify evolutionary changes. Viral RNA from the nasal washes from infection of naïve and immune animals contained 6% and 24.5% HA variant sequences, respectively. Analysis of mutations relative to antigenic epitopes indicated that adaptive immunity played a key role in virus evolution. HA mutations in immunized animals were associated with loss of glycosylation and changes in charge and hydrophobicity in and near residues within known epitopes. Four regions of HA-1 (75–85, 125–135, 165–170, 225–230) contained residues of highest variability. These sites are adjacent to or within known epitopes and appear to play an important role in antigenic variation. Recognition of the role of these sites during evolution will lead to a better understanding of the nature of evolution which help in the prediction of future strains for selection of seasonal vaccines and the design of novel vaccines intended to stimulated broadened cross-reactive protection to conserved sites outside of dominant epitopes.     

The reduction of animal usage by the application of indirect haemagglutination in the potency testing of diphtheria toxoid in combined vaccines

Eight Adsorbed Diphtheria-Tetanus vaccines and 13 Diphtheria-Tetanus-Pertussis vaccines made by four different manufacturers were tested for the potency of the diphtheria components in guinea-pigs by the method of British Pharmacopoeia (1973). Two-hundred-and-ten guinea-pig sera consisting of ten sera related to each vaccine sample thus obtained were titrated for diphtheria antitoxin by indirect haemagglutination (IHA) and the conventional toxin neutralization (TN) tests. Statistical analysis of the results showed a good correlation between the titres obtained with the two tests. The potencies of the diphtheria components of various vaccines calculated from the antitoxin content of the respective guinea-pig sera titrated by the IHA test correlated significantly with the potencies obtained from the antitoxin content titrated by the routinely used TN test. The use of IHA in place of the TN test thus offers as an alternative that permits a reduction in animal usage.     

Genetic polymorphism of plus-tree clones and their seed progeny in the scotch pine clone plantation

The genetic variability at 12 allozyme (ten were found to be polymorphic) loci was studied in the archive-clone plantation of 23 plus-trees of Pinus sylvestris and their seed progeny in the southeast of Ukraine. More than half of the clones possessed four to eight heterozygous loci, while their seed progeny was characterized by a lower degree of variation as compared with maternal trees. Seed progeny was obtained from high outcrossing rate (t _m = 95%). The clone progeny was characterized by a high rate of distortions of allele segregation in megagametophytes and a high percentage of significant deviations in distribution of genotypes of seed embryos from that theoretically expected in accordance with the Hardy-Weinberg ratio.     

Cytological and molecular evaluation of the reproductive behavior of Tripsacum andersonii and a female fertile derivative (Poaceae)

Research was conducted to characterize the reproductive behavior of the highly sterile Tripsacum andersonii Gray and its viable progeny through breeding, cytological, and molecular studies. Four progeny were obtained from open-pollinated seeds of clones (M-34445, M-34450 and M-34455) of T. andersonii maintained at the USDA-ARS National Germplasm Repository, Miami, Florida. One of the progeny had 64 chromosomes, which is typical of T. andersonii, and probably resulted from apomictic reproduction. Karyotypes of the other three progeny indicated a tetraploid Tripsacum genomic constitution (2n = 4x = 72) plus a haploid set of Zea (1n = 1x = 10) chromosomes. Two of these progeny were completely sterile, whereas one (95-51) produced ～5% seed set when crossed with diploid (2n = 36) T. dactyloides (L.)L. The partially fertile 95-51 produced four progeny, one with 2n = 72 (elimination of 10 Zea chromosomes), two with 2n = 82 (apomictic reproduction) and one with 2n = 100 (sexual polyploidization). Polymerase Chain Reaction - Random Amplified Polymerase DNA analysis verified that T. andersonii accessions from seven countries were genetically uniform, and that its progeny were derived through apomixis and sexual polyploidization. This analysis also confirmed that chromosome elimination, apomixis, and sexual polyploidization reproductive behaviors occur in the T. andersonii derivative 95-51.     

Significance of specific-locus germ-cell mutations in mice

On the basis of a historical-control incidence of 39 mutants in 688 921 progeny (5.7 mutants /10⁵ animals or 0.82 mutations/locus/10⁵ gametes) proposals are made for the numbers of test progeny required when screening for possible mutagens using the specific-locus test in mice (7 loci).It is recommended that 25 000 control progeny should be included in each test, to establish homogeneity with the historical controls. This would also be the number of progeny required from treated males, unless significantly positive results had been obtained with smaller numbers.It would appear that the greater sensitivity of post-spermatogonial stages could more than compensate for practical difficulties in sampling these stages, rather than spermatogonia, in screening tests.     

Homologues of the maize rust resistance gene Rp1-D are genetically associated with a major rust resistance QTL in sorghum

As part of a comparative mapping study between sugarcane and sorghum, a sugarcane cDNA clone with homology to the maize Rp1-D rust resistance gene was mapped in sorghum. The cDNA probe hybridised to multiple loci, including one on sorghum linkage group (LG) E in a region where a major rust resistance QTL had been previously mapped. Partial sorghum Rp1-D homologues were isolated from genomic DNA of rust-resistant and -susceptible progeny selected from a sorghum mapping population. Sequencing of the Rp1-D homologues revealed five discrete sequence classes: three from resistant progeny and two from susceptible progeny. PCR primers specific to each sequence class were used to amplify products from the progeny and confirmed that the five sequence classes mapped to the same locus on LG E. Cluster analysis of these sorghum sequences and available sugarcane, maize and sorghum Rp1-D homologue sequences showed that the maize Rp1-D sequence and the partial sugarcane Rp1-D homologue were clustered with one of the sorghum resistant progeny sequence classes, while previously published sorghum Rp1-D homologue sequences clustered with the susceptible progeny sequence classes. Full-length sequence information was obtained for one member of a resistant progeny sequence class ( Rp1-SO) and compared with the maize Rp1-D sequence and a previously identified sorghum Rp1 homologue ( Rph1-2). There was considerable similarity between the two sorghum sequences and less similarity between the sorghum and maize sequences. These results suggest a conservation of function and gene sequence homology at the Rp1 loci of maize and sorghum and provide a basis for convenient PCR-based screening tools for putative rust resistance alleles in sorghum.