Several DNA-based transposable elements are known to be present in vertebrate genomes, but few of them have been demonstrated to be active. The Tol2 element of the medaka fish is one such element and, therefore, is potentially useful for developing a gene tagging system and other molecular biological tools applicable to vertebrates. Towards this goal, analyses of the element at the molecular, cellular and population levels are in progress. Results so far obtained are described here.
The tetraploid species Avena agadiriana that was first described in 1985 is distributed on the Atlantic coastal strip south of Casablanca in Morocco. Five accessions of this species (M55, M59, M60, M71 and M74) were compared by using FISH and RFLP analysis of 18S-5.8S-26S rDNA. The FISH data indicated that three pairs of major hybridization sites of the rDNA were located on satellite chromosomes in accessions M55, M59, M60 and M71. Accession M74, however, had only two pairs of major sites of hybridization. A pair of the major rDNA sites in M71 was very small and closely located at the terminal region of Nor-ST (Nucleolar organizing region of subtelocentrics) chromosomes. RFLP analysis of the rDNA sequence fragments identified differences among M55, M71 and M74, whilst M59 and M60 were the same with regard to the four restriction enzyme fragments utilized. M74 always lost single rDNA fragments in four restriction enzyme digests. The RFLP data made it possible to distinguish M55 from M59 and M60 in the northern Haut-Atlas Mountains group. A unique 20 kb EcoRI fragment characterized M71. Thus, a combination of FISH and RFLP analysis of rDNA was a good tool for inferring intraspecific evolutionary relationship of A. agadiriana
Raphanus sativus var. hortensis f. raphanistroides (wild radish: Brassicaceae) is an insect-pollinated wild plant that grows mainly on beaches in East Asia. Starch gel electrophoresis was used to investigate the allozyme diversity and genetic structure of 25 Japanese and 9 Korean populations of this plant. Although the Korean populations were small, isolated, and patchily distributed, they maintained a high level of genetic diversity; the average percentage of polymorphic loci was 63.1%, the mean number of alleles per locus was 2.27, and the average heterozygosity was 0.278. The corresponding estimates for these parameters in the Japanese populations were 53.3%, 2.26, and 0.278. These estimates are considerably higher than those from species with similar life history and ecological characteristics, but they are lower than those from R. raphanistrum, the wild radish that grows in Europe and the U.S.A. The combination of an insect-pollinated, outcrossing breeding system, large population sizes, gene flow from cultivated radish population, and a propensity for high fecundity may explain the high level of genetic diversity within wild populations.
The hyper-variable segments (323∼327 bp) of the mitochondrial D-loop for 169 Carassius auratus fishes in Japan were amplified by the polymerase chain reaction and the amplified products were sequenced directly and compared. A dendrogram showing three major clusters was generated with the sequence data for 37 haplotypes at 66 polymorphic sites. One cluster (cluster I) exclusively consisted of the gengorobuna, which was regarded as an independent (sub) species. The triploid ginbuna belonged to two remaining clusters, mainly in the diploid ginbuna cluster (cluster III) and partially in the goldfish cluster (cluster II). This finding suggests that the triploid ginbuna has been derived from two different maternal lineages. The triploid ginbuna was considered to have come into existence during the last ice age on the basis of this phylogenetic data. No geographic differentiation was observed with respect to the triploid ginbuna sampled at three different localities in Japan; the Shibuta River in Kanagawa, Lake Imba in Chiba and Lake Biwa in Shiga. The phylogenetic tree also demonstrated a monophyletic relationship amongst the nigorobuna, the nagabuna and the ginbuna, sharing cluster III. The nigorobuna and nagabuna populations have most likely arisen from geographic and temporal variations within the ginbuna populations. We also discuss the evolutionary origin of the triploid in view of its paternal ancestors.
The cytoplasm of Triticum timopheevi causes cytoplasmic male sterility (CMS) in common wheat (T. aestivum) cv. ‘Chinese Spring’ (CS), and that of Aegilops kotschyi causes CMS in spelt wheat (T. spelta) var. duhamelianum (Sp). CS has fertility-restoring (Rf) genes against the latter cytoplasm and Sp has the ones against the former. To know the genetic system concerning to CMS, we crossed 66 F8 recombinant inbred lines (RILs) derived from a cross between CS and Sp as males to the alloplasmic lines of CS and Sp having the cytoplasms of T. timopheevi and Ae. kotschyi, respectively. The fertilities of respective F1 plants derived from the crosses were examined for QTL analysis. The major QTLs detected in both systems were located on the short arm of chromosome 1B. One minor QTL on chromosome 2B was also commonly detected in both of the systems, while other minor QTLs against T. timopheevi cytoplasm were distributed on the chromosomes 2A, 4B, and 6A.
Nucleotide sequence polymorphisms of the intron of the chloroplast trnK (UUU) gene, including a matK gene, were investigated within two wild Fagopyrum species, F. leptopodum and F. statice, to assess the degree and pattern of the inter- and intranspecific differences in coding and noncoding chloroplast DNA regions in higher plants. Ten and five accessions were used for F. leptopodum and F. statice, respectively. The length of the trnK intron region in these species ranged from 2494 to 2506 bp. In the trnK intron, the net nucleotide substitution number per site (Da) between the two species was 0.00109, lower than the nucleotide diversity (π), 0.00195 for F. leptopodum and 0.00144 for F. statice, suggesting a low level of interspecific divergence. This result seems to be due to the phylogenetic pattern that both species are interspersed with each other, which was revealed by the phylogenetic analyses based on the nucleotide substitutions and indels. In the matK gene region (1524 bp), seven and two nucleotide substitutions were found within F. leptopodum and F. statice, respectively. All of the nine nucleotide substitutions (eight of which were nonsynonymous) within and between F. leptopodum and F. statice were clustered in the 5' part of the matK gene region, and no variation was found in the 3' part. This suggests that most of the 3' part is occupied by the conserved domains that are important for the binding activity of the gene product to the precursor mRNA, and therefore implies that the 3' part is more functionally constrained than the 5' part.
Phylogenetic relationships among cultivated landraces and natural populations of wild subspecies of Tartary buckwheat were investigated at the individual level by constructing a phylogenetic tree based on amplified fragment length polymorphism (AFLP) markers. Seven individuals from seven cultivated landraces and 35 individuals from 21 natural populations of wild subspecies were utilized for AFLP analyses. Three groups were recognized: (1) all cultivated landraces and wild subspecies from northern Pakistan, central and eastern Tibet, and northwestern Yunnan, (2) wild subspecies from central and southern Sichuan, (3) wild subspecies from northern Sichuan and eastern Tibet. It was concluded that cultivated Tartary buckwheat probably originated in eastern Tibet or northwestern Yunnan in China.
A 314-bp tandemly repeated DNA sequence, named pAc074, was characterized in Allium cepa by fluorescence in situ hybridization (FISH) analyses using random amplified fragment as probe. The nucleotide sequences of the clone pAc074 is partially homologous to the satellite DNA sequences, ACSAT1, ACSAT2, and ACSAT3, of A. cepa with 81%, 81% and 78% similarity, respectively. Our sequential C-banding and FISH with pAc074 probe also clearly showed a close relation between C-heterochromatin at telomeric region and pAc074 sequences on all the chromosomes except on chromosome 6. On the long arm of chromosome 7, pAc074 sequences appeared as interstitial band which did not correspond to C-heterochromatin bands. Instead, the C-heterochromatin bands corresponded with the 5S rDNA signals. This is the first evidence of simultaneous banding of the 5S rDNA and C-band in A. cepa.
A large insert genomic bacterial artificial chromosome (BAC) library was constructed from the inbred Hd-rR strain of the medaka, Oryzias latipes. Approximately 92, 000 clones were gridded on high-density replica filters. Insert analysis of randomly selected clones indicated a mean insert size of 210 kb and predicted a 24 times coverage of the medaka genome. The library was hybridized with a single locus DNA fragment, and the resulting positive clones were characterized and shown to be compatible with a 24-fold redundant library. This first large insert genomic library of the medaka should increase the speed of genomic analyses for this fish species.