Low frequency of extra-pair paternity in two colonies of the socially monogamous short-tailed shearwater Puffinus tenuirostris

The short-tailed shearwater is a colonially nesting, socially monogamous seabird. Little is known about mate fidelity and breeding behaviour in this species because breeding birds are nocturnal on land and spend much of their time within subterranean nesting burrows. Colonial breeding and extended sperm storage create opportunities for extra-pair copulations which may form a significant component of the mating strategy in this species. Multilocus DNA fingerprinting was used to examine the genetic relationship between nestlings and the male and female nest attendants in 83 burrows from two distinct breeding colonies. Genetic analyses identified nine nestlings, approximately equally distributed between the two colonies, that were not related to the attendant pair male in those burrows, implying extra-pair paternity through extra-pair copulations. These results are used retrospectively to discuss the characteristics of extra-pair copulations and extra-pair fertilizations and the implications for estimates of life-time reproductive success in the short-tailed shearwater.     

银合欢基因文库的构建和种子贮藏蛋白基因的分离

本文以λEMBL_3为载体,构建了银合欢(Leucaena leucocephala)的基因文库,所得重组子为3.5×10~6pfu。以大豆种子贮藏蛋白α′亚基基因为探针,从基因文库中分离得到了4个阳性克隆,并初步绘制了其中3个重组子的物理图谱。结果表明在这3个重组子的基因内部有一致的酶切位点。亚克隆的部分核苷酸序列与 GenBank 中的基因序列比较,表明与大豆种子贮藏蛋白α′亚基基因高度同源。     

Molecular genetic characterization of plant somatic hybrids

An efficient and easy method for genetic characterization of plant somatic hybrids is proposed. In a first qualitative approach, four somatic hybrids and their parental species (Nicotiana tabacum andN. plumbaginifolia) were characterized by DNA fingerprinting and Random Amplification of Polymorphic DNA (RAPD). After this, a quantitative estimation of the degree of parental contribution to the hybrids was carried out by means of a slot-blot analysis. Both qualitative methods, showed one hybrid identical toN. tabacum, two almost identical toN. plumbaginifolia, and a fourth similar to this parental species, but with someN. tabacum admixture. The quantitative method, for the same hybrids, gave 83%, 7%, 7%, and 37%N. tabacum DNA contribution, respectively.     

Molecular genetic analysis of the response of three soil microbial communities to the application of 2, 4-D

The responses of three different soil microbial communities to the experimental application of 2, 4-dichlorophenoxyacetic acid (2, 4-D) were evaluated with a variety of molecular genetic techniques. Two of the three soil communities had histories of prior direct exposure to 2, 4-D, and one had no prior direct application of any herbicide. Dominant 2, 4-D degrading strains isolated from these soils the previous year were screened for hybridization with three catabolic genes (tfdA, tfdAII, and tfdB) cloned from the well-studied 2, 4-D degradative plasmid, pJP4, revealing varying degrees of similarity with the three genes. Hybridization of total community DNA from the three soils with the tfd gene probes also indicated that pJP4-like tfd genes were not harboured by a significant percentage of the community. Community level response was evaluated by the comparison of different treatments by Random Amplified Polymorphic DNA (RAPD) fingerprints and by community DNA cross-hybridization. No differences between treatments within the same soil were detected in any of the RAPD fingerprints generated with 17 primers. Community DNA cross-hybridization also indicated that the application of 2, 4-D at the applied rates did not quantitatively affect the structure of the soil microbial communities present in the three soils during the time-frame studied.     

Transfer of a dominant gene for powdery mildew resistance and DNA from Hordeum bulbosum into cultivated barley (H. vulgare)

Summary In an attempt to transfer traits of agronomic importance from H. bulbosum into H. vulgare we carried out crosses between four diploid barley cultivars and a tetraploid H. bulbosum. Eleven viable triploid F₁ plants were produced by means of embryo rescue techniques. Meiotic pairing between H. vulgare and H. bulbosum chromosomes was evidenced by the formation of trivalents at a mean frequency of 1.3 with a maximum of five per cell. The resulting triploid hybrids were backcrossed to diploid barley, and nine DC₁ plants were obtained. Three of the BC₁ plants exhibited H. bulbosum DNA or disease resistance. A species specific 611-bp DNA probe, pSc119.2, located in telomeres of the H. bulbosum genome, clearly detected five H. bulbosum DNA fragments of about 2.1, 2.4, 3.4, 4.0 and 4.8 kb in size present in one of the BC₁ plants (BC₁-5) in BamHI-digésted genomic Southern blots. Plant BC₁-5 also contained a heterozygous chromosomal interchange involving chromosomes 3 and 4 as identified by N-banding. One of the two translocated chromosomes had the H. bulbosum sequence in the telomeric region as detected using in situ hybridization with pSc119.2. Two other BC₁ plants (BC₁-1 and BC₁-2) were resistant to the powdery mildew isolates to which the barley cultivars were susceptible. Seventy-nine BC₂ plants from plant BC₁-2 segregated 32 mildew resistant to 47 susceptible, which fits a ratio of 11, indicating that the transferred resistance was conditioned by a single dominant gene. Reciprocal crosses showed a tendency towards gametoselection that was relative to the resistance. Mildew resistant plant BC₁-2 also had a 1-kb H. bulbosum DNA fragment identified with a ten-base random primer using polymerase chain reaction (PCR). Forty-three BC₁ plants, randomly sampled from the 79 BC₁ plants, also segregated 2320 for the presence versus absence of this 1-kb H. bulbosum DNA fragment, thereby fitting a 11 ratio and indicating that the PCR product originated from a single locus. The 1-kb DNA fragment and disease resistance were independently inherited as detected by PCR analysis of bulked DNA from 17 resistant and 17 susceptible plants as well as by trait segregation in the 43 individual plants. The progenies produced could serve as an important resistant source in plant breeding. This is the first conclusive report of the stable transfer of disease resistance and DNA from H. bulbosum to H. vulgare.     

The effects of non-homology in RAPD bands on similarity and multivariate statistical ordination in Brassica and Helianthus

 In order to estimate the impact of mis-coding non-homologous, co-migrating DNA bands as homologous, two sets of data were utilized. Analyses were conducted using three Helianthus species in which each co-migrating band had previously been confirmed. Comparisons of the similarities between these three Helianthus species using the original 177 RAPD bands and the corrected, homology verified, 197 RAPD band data set revealed that the triangular relationship among these three species was almost identical in both data sets. The non-homology errors in the Helianthanus data sets were found to be random. These random errors merely reduced the absolute similarities, but not the relative similarities nor the relationships among the taxa, in principal-coordinate-analysis ordination. Analyses of RAPDs for the classical Brassica U triangle were made by inserting random non-homologies for 5, 10, 15 and 20% of the original 220 RAPD bands. These analyses revealed a progressive decrease in similarities and less loading on the first two axes in principal coordinate analysis (PCO). However, the basic U triangle of relationships among these six Brassica species was maintained. It appears that if errors in homology of co-migrating DNA bands are random, this will have little effect on the relative similarities and on PCO ordination. This helps explain the successful use of RAPDs at the specific level. Received: 6 December 1997 / Accepted: 11 December 1997     

The potential of AFLPs in biosystematics: A first application inSolanum taxonomy(Solanaceae)

Using the AFLP technique highly informative DNA fingerprints were generated from 19 taxa ofSolanum sect.Petota (potatoes) and three taxa ofSolanum sect.Lycopersicum (tomatoes). Both phenetic and cladistic analyses were conducted from the individual genotypic level to the species level. An AFLP fingerprint, using a combination of suitable AFLP primers, generated 12 to 71 scorable fragments per genotype which was sufficient for taxonomic interpretation. The classifications based on the molecular markers were generally in agreement with current taxonomic opinions. Unexpectedly,S. microdontum was associated with ser.Megistacroloba rather than with ser.Tuberosa, andS. demissum (ser.Demissa) and species of ser.Acaulia appeared closely affiliated. AFLP is an efficient and reliable technique to generate biosystematic data and therefore a promising tool for evolutionary studies.     

Stable integration and conditional expression of electroporated transgenes in chicken embryos

The in ovo electroporation in chicken embryos has widely been used as a powerful tool to study roles of genes during embryogenesis. However, the conventional electroporation technique fails to retain the expression of transgenes for more than several days because transgenes are not integrated into the genome. To overcome this shortcoming, we have developed a transposon-mediated gene transfer, a novel technique in chicken manipulations. It was previously reported that the transposon Tol2, originally found in medaka fish, facilitates an integration of a transgene into the genome when co-acting with Tol2 transposase. In this study, we co-electroporated a plasmid containing a CAGGS-EGFP cassette cloned in the Tol2 construct along with a transposase-encoding plasmid into early presomitic mesoderm or optic vesicles of chicken embryos. This resulted in persistent expression of EGFP at least until embryonic day 8 (E8) and E12 in somite-derived tissues and developing retina, respectively. The integration of the transgene was confirmed by genomic Southern blotting using chicken cultured cells. We further combined this transposon-mediated gene transfer with the tetracycline-dependent conditional expression system that we also developed recently. With this combined method, expression of a stably integrated transgene could be experimentally induced upon tetracycline administration at relatively late stages such as E6, where a variety of organogenesis are underway. Thus, the techniques proposed in this study provide a novel approach to study the mechanisms of late organogenesis, for which chickens are most suitable model animals.     

Upregulation of PHLDA2 in Dicer knockdown HEK293 cells

It has been reported that RNAi-dependent chromatin silencing in vertebrates is not restricted to the centromeres. To address whether RNAi machinery could regulate the chromatin structure of imprinted genes, we knocked down Dicer in HEK293 cells and found that the expression of PHLDA2, one of the several genes in the imprinted gene domain of 11p15.5, was specifically upregulated. This was accompanied by a shift towards more activated chromatin at PHLDA2 locus as indicated by change in H3K9 acetylation, however, the methylation state at this locus was not affected. Furthermore, we found that PHLDA2 was downregulated in growth-arrested HEK293 cells induced by either serum deprivation or contact inhibition. This suggests that PHLDA2 upregulation might be a direct result of Dicer depletion rather than the consequence of growth arrest induced by Dicer knockdown. Considering the reports that there is consistent placental outgrowth in PHLDA2 knockout mice and that PHLDA2 overexpression in mice causes growth inhibition, we speculate that PHLDA2 may be a candidate for contributing to the reduced growth rate of Dicer-deficient cells and the very early embryonic lethality in Dicer knockout mice.     

Genetic structure and mating system in the palila, an endangered Hawaiian honeycreeper, as assessed by DNA fingerprinting

We conducted DNA fingerprinting analyses to ascertain the mating system and population genetic structure of the palila, an endangered Hawaiian honeycreeper, which occupies a fragmented range on the Mauna Kea volcano of the island of Hawai'i. DNA fingerprinting of twelve complete families from the Pu'u La'au population revealed no evidence of extrapair fertilization or intraspecific brood parasitism. Band-sharing coefficients from fingerprints produced with two probes revealed that the large Pu'u La'au population on the southwest slope of Mauna Kea, and a smaller, geographically separate population on the east slope (at Kanakaleonui) had relatively high and virtually identical levels of minisatellite variability (mean S of 0.27 for each population based on combined data of M13 and Jeffreys 33.15 probes). The two populations also had nearly identical allele frequencies based on their mean corrected similarity, S_ij, of 0.98. These data suggest that the two populations have not been fragmented long and/or have sufficient current gene flow to ameliorate any affects of genetic drift. We conclude that present levels of inbreeding are low within both populations, and that proposed translocations of individuals from Pu'u La'au to Kanakaleonui appear appropriate from a genetic standpoint.