Isolation of Microsatellite Loci in Sceloporus grammicus (Squamata, Phrynosomatidae)

The mesquite lizard (Sceloporus grammicus) exhibits multiple Robertsonian chromosomal rearrangements (mainly centric fissions) resulting in several cytotypes. In a transitional environment from oak-pine forests to a drier xeric habitat in central Mexico, two cytotypes (F5: 2n = 34 and FM2: 2n = 46) are known to hybridize. A partial genomic library was constructed from S. grammicus genomic DNA and then screened for microsatellites. Microsatellites are short tandem nucleotide repeats that have near universal occurrence in all eukaryotic genomes. Microsatellites exhibit variable length polymorphisms that can be characterized and utilized as genetic markers for population studies. Thirteen microsatellite arrays were isolated from the S. grammicus genomic library and PCR primers were designed in the flanking regions for the amplification of these alleles. These microsatellite loci would be the primary tool used to answer behavioral, ecological, chromosomal and evolutionary questions that influence the maintenance of this hybrid zone.

Isolation and characterization of S genome specific sequences from Aegilops sect. sitopsis species

Three S genome specific sequences were isolated from Aegilops sect. sitopsis species using different experimental approaches. Two clones, UTV86 and UTV39, were isolated from a partial genomic library obtained from DNA of Aegilops sharonensis, whereas a third clone, UTV5, was isolated from Aegilops speltoides. The three clones were characterized by sequencing, analysis of methylation, and sequence organization and abundance in some Aegilops and Triticum species. The clones UTV39 and UTV5 belong to the same family of tandem repeated sequences and showed high homology with a sequence already present in nucleotide databases. The UTV86 clone from Ae. sharonensis corresponded to an interspersed low frequency repeated sequence and did not show any significant homology with reported sequences. Southern hybridization experiments, using the cloned sequences as probes, detected polymorphism in the restriction patterns of all the five Aegilops species in section sitopsis. Aegilops speltoides showed the most divergent hybridization pattern. A close relationship was detected between the S genome of Ae. speltoides and the G genome of the wild Triticum timopheevii. In situ hybridization revealed a telomeric and (or) subtelomeric location of the sequences UTV39 and UTV5.Key words: Aegilops, genome-specific sequences, sitopsis, wheat evolution.

Microsatellites in the silkworm, Bombyx mori: Abundance, polymorphism, and strain characterization

We have isolated and characterized microsatellites (simple sequence repeat (SSR) loci) from the silkworm genome. The screening of a partial genomic library by the conventional hybridization method led to the isolation of 28 microsatellites harbouring clones. The abundance of (CA)n repeats in the silkworm genome was akin to those reported in the other organisms such as honey bee, pig, and human, but the (CT)n repeat motif is less common compared to bumble bee and honey bee genomes. Detailed analysis of 13 diverse silkworm strains with a representative of 15 microsatellite loci revealed a number of alleles ranging from 3 to 17 with heterozygosity values of 0.66-0.90. Along with strain-specific microsatellite markers, diapause and non-diapause strain-specific alleles were also identified. The repeat length did not show any relationship with the degree of polymorphism in the present study. The co-dominant inheritance of microsatellite markers was demonstrated in F1 offspring. A list of primer sequences that tag each locus is provided. The availability of microsatellite markers can be expected to enhance the power and resolution of genome analysis in silkworm.Key words: microsatellites, simple sequence repeats, polymorphisms, silkworm strains, Bombyx mori.

Rapid Detection of the Fusarium oxysporum Lineage Containing the Canary Island Date Palm Wilt Pathogen

Fusarium oxysporum f. sp. canariensis causes Fusarium wilt disease on the Canary Island date palm (Phoenix canariensis). To facilitate disease management, a polymerase chain reaction diagnostic method has been developed to rapidly detect the pathogen. A partial genomic library of F. oxysporum f. sp. canariensis isolate 95-913 was used to identify a DNA sequence diagnostic for a lineage containing all tested isolates of F. oxysporum f. sp. canariensis. Two oligonucleotide primers were designed and used to amplify a 567-bp fragment with F. oxysporum f. sp. canariensis DNAs. DNA from 61 outgroup isolates did not amplify using these primers. Once the primers were shown to amplify a 0.567-kb fragment from DNA of all the F. oxysporum f. sp. canariensis isolates tested, a rapid DNA extraction procedure was developed that led to the correct identification of 98% of the tested F. oxysporum f. sp. canariensis isolates.

Genomic organization, sequence interrelationship, and physical localization using in situ hybridization of two tandemly repeated DNA sequences in the genus Olea

Two tandemly repeated DNA sequences, the 81-bp family and pOS218, have been isolated from a Sau3AI Olea europaea ssp. sativa partial genomic library. Sequencing of the 81-bp element showed the monomer to be between 78 and 84 bases long and to contain 51-58% adenine and thymidine residues. Comparison between the monomers revealed heterogeneity of the sequence primary structure. The clone pOS218 is 218 bases long, and sequence comparison between the two elements revealed that an internal region of the pOS218 repeated DNA sequence had 79% homology to the 81 bp repeat sequence. A breakage-reunion mechanism, involving the CAAAA sequence, could be responsible for the derivation of pOS218 from the 81 bp family element. By using double target in situ hybridization, co-localization of the two sequences on Olea chromosomes was observed. The sequences were present at DAPI stained heterochromatic regions, as major or minor sites having a subtelomeric or interstitial location. Methylation studies using two sets of isoschizomers, Sau3AI-MboI and MspI-HpaII, demonstrated that most cytosine residues in the GATC sites and the internal cytosine in the CCGG sites of both elements were methylated in O. europaea ssp. sativa. No major difference in methylation was apparent between DNA extracted from young leaves or from callus of O. europaea ssp.sativa. Both elements are also present in Olea chrysophylla, Olea oleaster, and Olea africana, but are absent from other Oleaceae genera, including Phillyrea, Forsythia, Ligustrum, Parasyringa, and Jasminum.Key words: in situ hybridization, methylation, Oleaceae, phylogenetic relationships, repeated sequences.

Characterization of a microsatellite locus in the parasitoid wasp Aphelinus abdominalis (Hymenoptera: Aphelinidae)

Primers for DNA amplification using the polymerase chain reaction (PCR) were synthesized for a microsatellite locus isolated from a partial genomic library of the aphid parasitoid Aphelinus abdominalis (Dalman). Screening for genetic polymorphism at this locus in two laboratory strains of this wasp revealed the presence of two alleles different in the number of (GT) and (GGC) repeats. The relative frequencies of the two alleles were not significantly different between the two strains or between diploid females and haploid males. Heterozygosity at this microsatellite locus was estimated to be 0.40 which is within the range in other hymenopterous species. Given that A. abdominalis is a good candidate for augmentative release programmes in greenhouses against aphids, we suggest that microsatellite markers may have application in discriminating among aphelinid sibling species and strains. The markers provide a means for studying the performance and impact of selected parasitoid lines on pest dynamics in field release experiments.

Isolation and characterization of a developmentally regulated homeobox sequence in the Mexican axolotl Ambystoma mexicanum

A homeobox-containing genomic DNA fragment was isolated from the Mexican axolotl. This clone was obtained from a partial genomic library enriched for sequences that cross-hybridized with the Drosophila Antp homeobox under low stringency hybridization conditions. DNA sequence analysis revealed that this sequence (Ahox1) was 66% homologous to the Antp homeobox sequence and was most closely related to the mouse Hox-1.6 (84% identity) and Drosophila lab (79% identity) homeobox sequences. Several cross-hybridizing fragments to Ahox1 were detected in both mouse and axolotl genomic DNA. This sequence was also shown to be conserved in other Ambystoma species. Northern blot analysis revealed that genes containing this sequence are developmentally regulated. Transcripts hybridizing to the Ahox1 homeobox probe were detected during the neurula and tail bud stages of development.Key words: axolotl, homeobox, mouse, Drosophila, gene expression.