Heterochromatin condensation and evolution of unique satellite-DNA families in two parasitic wasp species: Diadromus pulchellus and Eupelmus vuilleti (Hymenoptera)

Large quantities of satellite DNA families (15%-25% of the genome) were found in the DNA of two species of parasitic wasps, Diadromus pulchellus and Eupelmus vuilleti. In both species the satellite DNA was found to consist wholly or largely of a single family unique to that species. Several clones of each family were obtained and sequenced. Palindromes in each consensus sequence suggest the formation in vivo of hairpin structures that may play a role in the mode of heterochromatin condensation in these insects. The ancestral repeating motifs were determined from the consensus sequences. Plausible scenarios are presented for the evolution of the two satellite DNAs. The occurrence of only one family of satellite DNAs in both species may indicate that, in male haploids, such families have shorter persistence times than necessary for the origins of new duplicated sequences.     

Dispersal and parasitizing abilities of Eupelmus vuilleti (Hymenoptera: Eupelmidae) within a column of cowpea seeds

Eupelmus vuilleti (Crawford) is an ectoparasitoid of the seed-eating beetle Bruchidius atrolineatus (Pic), which is an important pest of stored cowpea, Vigna unguiculata Walp, seeds in West Africa. Herein, we investigated the dispersal abilities of females within columns of seeds to assess the potential of E. vuilleti as a biological control agent of bruchids in cowpea granaries. The influence of host presence together with the 2 abiotic factors light and gravity on parasitoid movement and parasitization efficiency were analyzed. E. vuilleti females were able to travel through large seed masses and parasitize hosts located at the end of the seed column opposed to their introduction zone. Parasitoid movement was stimulated by light. E. vuilleti females exhibited a negative geotropism. Females introduced at the bottom of the seed column dispersed more and parasitized more hosts than females introduced at the top. Host presence had some influence on the dispersal of the parasitoids within the seed column at a host density of 10 infested seeds for 16,000-18,000 uninfested seeds. This depended on female introduction zone because gravity was the major factor influencing dispersal. The possible applications of these results for biological control of bruchids in cowpea granaries are discussed.     

The S-locus of Nicotiana alata: genomic organization and sequence analysis of two S-RNase alleles

Genomic clones encoding the S ₂- and S ₆-RNases of Nicotiana alata Link and Otto, which are the allelic stylar products of the self-incompatibility (S) locus, were isolated and sequenced. Analysis of genomic DNA by pulsed-field gel electrophoresis and Southern blotting indicates the presence of only a single S-RNase gene in the N. alata genome. The sequences of the open-reading frames in the genomic and corresponding cDNA clones were identical. The organization of the genes was similar to that of other S-RNase genes from solanaceous plants. No sequence similarity was found between the DNA flanking the S ₂- and S ₆-RNase genes, despite extensive similarities between the coding regions. The DNA flanking the S ₆-RNase gene contained sequences that were moderately abundant in the genome. These repeat sequences are also present in other members of the Nicotianae.     

Potential impact of the native hyperparasitoid Conura albifrons (Hymenoptera: Chalcididae) on the exotic biological control agent Diadromus pulchellus (Hymenoptera: Ichneumonidae)

The solitary pupal parasitoid Diadromus pulchellus was released in 2010 as a classical biological control agent against leek moth, Acrolepiopsis assectella, an important new pest of onion crops, Allium spp. in Eastern North America. Post-release monitoring using sentinel leek moth pupae revealed that the facultative hyperparasitoid Conura albifrons was attacking leek moth and potentially D. pulchellus as well. We used laboratory choice and no-choice tests to assess the potential impact of C. albifrons on leek moth and D. pulchellus. C. albifrons is capable of developing in newly formed leek moth pupae and leek moth pupae containing first instar D. pulchellus, as well as in fully developed D. pulchellus pupae. Survivorship of both leek moth and D. pulchellus exposed to C. albifrons was significantly lower than that of unexposed controls. In choice trials, prior host experience significantly influenced host choice by C. albifrons. These results suggest that C. albifrons could impact the establishment of D. pulchellus through both competition and intraguild predation, and that the impact has the potential to change as the relative frequency of the two hosts shifts in field populations.     

Molecular structure and chromosomal localization of major repetitive DNA families in the chickpea (Cicer arietinum L.) genome

Three major repetitive DNA sequences were isolated from a genomic library of chickpea (Cicer arietinum L.) and characterized with respect to their genomic organization and chromosomal localization. All repetitive elements are genus-specific and mostly located in the AT-rich pericentric heterochromatin. Two families are organized as satellite DNAs with repeat lengths of 162–168 bp (CaSat1) and 100 bp (CaSat2). CaSat1 is mainly located adjacent to the 18S rDNA clusters on chromosomes A and B, whereas CaSat2 is a major component of the pericentric heterochromatin on all chromosomes. The high abundance of these sequences in closely related species of the genus Cicer as well as their variation in structure and copy number among the annual species provide useful tools for taxonomic studies. The retrotransposon-like sequences of the third family (CaRep) display a more complex organization and are represented by two independent sets of clones (CaRep1 and CaRep2) with homology to different regions of Ty3-gypsy-like retrotransposons. They are distributed over the pericentric heterochromatin block on all chromosomes with extensions into euchromatic regions. Conserved structures within different crossability groups of related Cicer species suggest independent amplification or transposition events during the evolution of the annual species of the genus.     

Whole-genome amplification-based GenomiPhi for multiple genomic analysis of individual early porcine embryos

The multiple displacement amplification (MDA) method, which relies on isothermal DNA amplification using the DNA polymerase of the bacteriophage phi29, was recently developed for high-performance, whole-genome amplification (WGA). The objective of the present study was to determine whether a target sequence could be successfully amplified by conventional PCR when the genomic DNA of a single Day-7 porcine blastocyst (derived from SCNT of a gene-engineered fibroblast) was amplified by the MDA method and used as a template. The yield of double-stranded DNA was 103.5 ± 16.0 ng/embryo (range, 75-125), as assessed by a PocoGreen assay. However, non-specific products (20 ± 5 ng/tube) were also generated, even in the negative control. Thus, ∼81% of the 103.5 ng (84 ng) of amplified DNA was estimated to be porcine sequences (2.2 × 10³-fold enrichment). In addition, PCR confirmed the presence of transgenes, as well as endogenous α-1,3-galactosyltransferase and homeobox Nanog genes in all embryos. Sequencing of the amplified products verified the fidelity of this system. In conclusion, the MDA-mediated WGA, which was simple, inexpensive, and did not require a thermal cycler, could be a powerful tool for multiple genomic analyses of individual early porcine embryos.     

Microarray-based survey of repetitive genomic sequences in Vicia spp.

A modified DNA microarray-based technique was devised for preliminary screening of short fragment genomic DNA libraries from three Vicia species (V. melanops, V. narbonensis, and V. sativa) to isolate representative highly abundant DNA sequences that show different distribution patterns among related legume species. The microarrays were sequentially hybridized with labeled genomic DNAs of thirteen Vicia and seven other Fabaceae species and scored for hybridization signals of individual clones. The clones were then assigned to one of the following groups characterized by hybridization to: (1) all tested species, (2) most of the Vicia and Pisum species, (3) only a few Vicia species, and (4) preferentially a single Vicia species. Several clones from each group, 65 in total, were sequenced. All Group I clones were identified as rDNA genes or fragments of chloroplast genome, whereas the majority of Group II clones showed significant homologies to retroelement sequences. Clones in Groups III and IV contained novel dispersed repeats with copy numbers 10²–10⁶/1C and two genus-specific tandem repeats. One of these belongs to the VicTR-B repeat family, and the other clone (S12) contains an amplified portion of the rDNA intergenic spacer. In situ hybridization using V. sativa metaphase chromosomes revealed the presence of the S12 sequences not only within rDNA genes, but also at several additional loci. The newly identified repeats, as well as the retroelement-like sequences, were characterized with respect to their abundance within individual genomes. Correlations between the repeat distributions and the current taxonomic classification of these species are discussed.     

Molecular phylogeny of the grass genus Brachypodium P. Beauv. based on RFLP and RAPD analysis

Nuclear genome analysis using RFLPs and RAPDs has been assessed within different species of the genus Brachypodium P. Beauv. and representatives of other grasses in order to determine the characteristics of the Brachypodium genome and to establish its evolutionary position in relation to other Pooideae. Distinctive features of the Brachypodium genome are its small size, the low amount of repetitive DNA, the lack of restriction fragment length polymorphisms within the genus for the assayed probe/enzyme combinations, and the genomic variability demonstrated at species level by random DNA amplification. These molecular studies confirm Brachypodium as an isolated ancient genus best placed in its own tribe (Brachypodieae). Its relationships to other tribes Bromeae, Triticeae, Poeae are resolved, Brachypodieae being the earliest tribe to diverge from this core of pooids. Within the genus two major Old World clades are distinguishable: an annual clade, represented only by B. distachyon; and a perennial clade, represented by all the other species studied (except B. mexicanum). The perennial American species B. mexicanum appears equally attached to these two clades. RFLP data were found to be useful in obtaining phylogenies at generic and higher rank levels, whereas the highly variable RAPD data were more suitable for resolving interspecific and intraspecific evolutionary pathways.     

DNA insertions as a component of the evolution of unique satellite DNA families in two genera of parasitoid wasps: Diadromus and Eupelmus (Hymenoptera)

In the two parasitoid wasps, Diadromus collaris and Eupelmus orientalis, the satellite DNAs were each found to consist wholly or largely of a single family (5%-7% of the genome). Several clones of each family were obtained and sequenced. The repeat unit in each species is characterized by both the repetition of a basic motif and the presence of an inserted sequence. Sequence comparisons with satellite DNA from D. pulchellus and E. vuilleti provide plausible scenarios for the evolution of the satellite DNA in each genus. Palindromes and A-rich tracts in each consensus sequence suggest the formation, in vivo, of hairpin structures and bend centers that may play a role in heterochromatin condensation in insects. The insertions in the repeat units of each species also contain these structural features, suggesting that maintenance of these insertions requires constraints similar to those pertaining to the rest of the satellite- DNA unit.      

DNA heterogeneity and genetic control of tumorigenesis in nicotiana tumorous and nontumorous genotypes

The possible relevance of changes in amounts of highly repetitive DNA sequences for plant differentiation and dedifferentiation processes has been suggested in several cases. Data are lacking however on (1) the genetic control of these phenomena and (2) cause-effect relationships between DNA amplification and specific ontogenetic patterns. The present study was carried out on a Nicotiana genetic system consisting of the tumorous amphidiploid N glauca X N langsdorffii, a nontumorous mutant of it, their F₁, and a backcross to the tumorous parent. Backcross segregation ratios were shown to be compatible with a “single gene” hypothesis, the F₁ plant being nontumorous but showing a low percentage of tumors induced by wounds, 6-azauracil or X-rays. In vitro studies of excised pith tissue grown on Linsmaier and Skoog medium for different periods of time showed the presence, confirmed by cytological analyses, of amplification of highly repetitive sequences only in the nontumorous stock, as judged by reassociation experiments in the first 24–96 hours of culture. CsCl analytical ultracentrifugation of those sequences showed the appearance in the same stock of a heavy DNA satellite (density = 1.721 gm/ml), whose presence was also confirmed by derivative melting curves. Amplification seemed to be essential for the initiation of cell division, which was completely inhibited in the nontumorous genotype and partially influenced in the F₁ by incorporation during the critical period (24–96 hours of the primary explant) of 5-bromo-2′-deoxy-uridine. The results are discussed in terms of an hypothesis of an integrated gene-controlled, hormone-mediated regulatory system of cell proliferation involving changes in target repetitive DNA sequences.     

The effects of periodic warming on the survival and fecundity of Diadromus pulchellus during long-term storage

Biological control strategies capitalise on natural mechanisms such as predation and parasitism to reduce the need for chemical applications to control insect pests. In Canada, the parasitic wasp Diadromus pulchellus Wesmael (Hymenoptera: Ichneumonidae) is being investigated for its use in the biological control of an invasive crop pest, the leek moth, Acrolepiopsis assectella (Zeller) (Lepidoptera: Acrolepiidae). Large numbers of insects will be needed for releases to ensure that populations of D. pulchellus establish quickly and impact leek moth populations. Since the current culture is not producing the number of insects required for large-scale releases, the accumulation and storage of D. pulchellus might be a viable option to obtain ideal numbers. Currently, little is known about the optimal conditions for the long-term storage and release of D. pulchellus, which overwinter in nature as adults. Using insects from the active culture, the effect of intermittent, short-term warming on cold-stored adults was evaluated for survivorship and fecundity. In accordance with previous findings, females survived cold storage more readily than males. However, the warming regimes employed had no significant impacts on overall survivorship. Cold-stored females had reduced fecundity compared to females maintained in the culture, though no significant differences were noted between the treatments. In addition, the offspring sex-ratio for all treatments was male skewed. Thus, the warming procedures utilised provided no advantages over current techniques for the long-term storage of D. pulchellus intended for release.     

Polypeptides of Acrolepiopsis assectella cocoon (Lepidoptera: Yponomeutoidea): an external host-acceptance kairomone for the parasitoid Diadromus pulchellus (Hymenoptera: Ichneumonidae)

Contact kairomones are essential for host-acceptance behaviour by female parasitoids. In the solitary endoparasitoid wasp, Diadromus pulchellus, this behaviour depends mainly on compound(s) in the cocoon of their host, Acrolepiopsis assectella pupae. Extracts of empty cocoons and polypeptides extracted from cocoons were tested in acceptance behaviour assays using cotton fibre lures bearing extracts. Extractions with solvents of increasing polarity indicated that the active compounds were polar, while SDS-PAGE showed that four glycopolypeptides contained enough information to trigger host-acceptance behaviour in female wasps. This kairomonal activity was found to be due to the protein moieties, and was independent of any glycosylation. These four glycopolypeptides might be two variants of two soluble sericin-like polypeptides differing in their degree of glycosylation.     

Methylation status of Sillago japonica satellite DNA examined by bisulfite modification

A member of Sillago japonica satellite DNA contained internal subrepeats in its 174 bp unit. S. Japonica genomic DNA isolated from liver tissue was subjected to bisulfite modification, and the DNA sequences of about 40 bp flanked by both subrepeats were amplified by polymerase chain reaction (PCR). This protocol, combination of bisulfite reaction and PCR, converts cytosines in the genomic DNA to thymines in the amplified DNA, whereas 5-methylcytosines in the genomic DNA remain as cytosines. Sequence analysis of the amplified DNA fragments revealed that most of the cytosine residues at CpG were methylated in this region.     

Interspecific Relations Between Two Sympatric Species of Hymenoptera, Dinarmus basalis (Rond) and Eupelmus vuilleti (Crw), Ectoparasitoids of the Bruchid Callosobruchus maculatus (F)

Dinarmus basalis (Rond) and Epelmus vuilleti (Crw) are two Hymenopteran species, which are solitary ectoparasitoids of bruchid larvae. In the presence of seeds of Vigna unguiculata (Walp) containing hosts parasitized by E. vuilleti, a high percentage of D. basalis females avoided multiparasitism whatever the age of the eggs or the larvae present on the host. The least avoidance was observed when the hosts were parasitized by E. vuilleti 30 min beforehand. This avoidance behavior is adaptive and is related to the low survival chances of the D. basalis larvae when they are in interspecific competition with E. vuilleti larvae. The analysis of the behavior of D. basalis demonstrated that the avoidance of multiparasitism could be due to the perception of two signals; an external signal deposited on the surface of the seeds during the E. vuilleti oviposition phase and an internal signal due to the presence of the eggs and larvae at the surface of the hosts. E. vuilleti females did not avoid multiparasitism and multiparasitized the hosts bearing D. basalis eggs or larvae. The behavior of E. vuilleti females was not disturbed by the presence of its competitor. Under these conditions of interspecific competition, the survival chances of E. vuilleti larvae were very high whatever the age of its competitor D. basalis. The two species of parasitoids could move in a column containing healthy seeds of V. unguiculata and patches with seeds containing parasitized or unparasitized larvae. The distribution of D. basalis females introduced into these columns depended on the host quality. They avoided the patches containing the hosts parasitized E. vuilleti and were found in the patches with healthy hosts. The behavior of E. vuilleti females was very different; the distribution of the females and the parasitism and multiparasitism rates were not affected by the quality of the hosts present in the patches. The adaptive significance of the behaviors of these two species was analyzed in relation to the survival chances of their offspring.     

Structural organization of interspersed repetitive elements present in the DNA of Mus musculus

Two-dimensional displays of the restriction fragments from the DNA of Mus musculus revealed a complex species-specific pattern produced from nonsatellite repetitive sequences. The patterns have been used as a guide in the direct purification of a group of broadly interspersed repeated DNA sequences (characterized by a 1350-bp Eco-Bam fragment) that have been studied by molecular cloning, restriction mapping and genomic Southern blotting. These studies show that the cloned representatives originate from an abundant group of sequences that share homology with about 2% of the mouse genome. The sequences do not appear to share homology with mouse-interspersed-family-1 (MIF-1) nor with the major AT-rich satellite sequences of mouse. They appear to be part of a group of larger repetitive elements that is both broadly interspersed and heavily methylated in normal mouse tissue.     

Nucleotide sequence of a major satellite DNA element isolated from a saltwater fishSillago japonica

A member of satellite repetitive DNA was isolated and sequenced from a saltwater fishSillago japonica (Percoidei). This sequence consists of several oligo-dA/dT tracts and two inverted repeats which resemble each other. Dot blot hybridization analysis using a satellite DNA clone pSJ2 among the species in the suborder Percoidei revealed that the pSJ2 sequence was amplified at least after the family Sillaginidae had been derived.     

Viability of diploid males in the parasitic wasp,Diadromus pulchellus (Hym.: Ichneumonidae)

In the HymenopteraDiadromus pulchellus diploid males have been observed in samples collected in the wild and bred in the laboratory. Using a yellow body color mutant strain, a protocol of crosses, involving inbred individuals, allows the routine production of such diploid males. These males result from the development of fertilized ova and emerge normally. Their preimaginal viability is similar to those of other individuals, and their imaginal viability does not differ from that of haploid males. Diploid males present normal external morphology and neither mosaicism nor intersexuality was observed. However, they are bigger than haploid males and have head size and wing length similar to those of females. The significance of diploid male viability in hymenopteran populations is discussed.     

Molecular cloning and restriction enzyme analysis of a long repetitive DNA sequence in rice

Rice long repetitive DNA (9–20 kbp) reassociating at Cot 50 M.s was cloned in pBR325. Out of several recombinants (Cam^r Amp^r Tet^s), only a few were selected randomly for further characterization. The insert size in all these clones was 3–4 kbp. Restriction enzyme analysis showed the absence ofEcoRI andBclI sites, presence of a singlePstI andPvuII site and multiple sites forAluI in 3 clones namely pRLl, pRL7 and pRL10. TheBamHI-PstI fragment of about 0.4 kbp in the pRL7 insert DNA (pRL7-0.4 kbp) was subcloned in M13mpl8 and partially sequenced using Sanger’s dideoxynucleotide chain termination method. Dot matrix comparison of this sequence with rice rDNA sequences revealed low homology with the 25 S rDNA sequence of rice, however, hybridisation did not indicate any homology.     

苔藓植物DNA提取方法研究

提取高质量的 DNA是对苔藓植物遗传多样性进行研究的基础。该文以苔藓植物为试材 ,用 5种方法 ,即快速提取法、改良 CTAB法、CTAB法、SDS法及高盐法 (第一种为自行设计 ,第二种是对原有方法的改进 )对苔藓植物 DNA提取方法进行了比较研究。结果表明 ,快速提取法和改良 CTAB法是 2种适合于苔藓植物 DNA提取的方法。这 2种方法提取的 DNA浓度和纯度均比较高 ,凝胶电泳显示无明显降解现象 ,适宜作为 PCR扩增的模板 ,并成功地进行了 RAPD扩增。