R. Lozano

Cloning and characterization of a fish centromeric satellite DNA

A highly repetitive DNA sequence family from the genome of Sparus aurata has been cloned and characterized. The family is composed of repeat units of 186 bp in length, and it accounts for 2% of the fish genome. Data from Southern blots and in situ hybridization demonstrate that repeating units are tandemly arranged at the centromeres of all the chromosomes in this species. The repetitive sequence is AT rich (67%) and is characterized by short stretches of consecutive AT base pairs and by short direct and inverted repeats. Sequence analysis of six cloned monomers of the family reveals some variation among clones at random positions and also distinguishes two subfamilies of repeats that differ in a highly divergent block of 31 bp. These two subfamilies do not seem to be located in separate domains but occur together in the centromere of each chromosome pair. The presence of this repeat family in the genome of other Sparidae species, some of which are relatively distant from S. aurata, indicates that this repetitive sequence could be an important component of the centromere in this fish family.

Chromosomal structure of populations of Scilla autumnalis in the Iberian Peninsula

Four cytological races of Scilla autumnalis, Liliaceae, were found in a study of 31 populations from Spain and Portugal. Two are diploids with 2n = 14, designated AA and B7B7, which differ by 70 per cent in DNA content, one is an allotetraploid AAB7B7 and one an autotetraploid of the B7 genome. Races AA and AAB7B7 are Iberian endemics, B7B7 is widespread throughout the Mediterranean, while the autotetraploid is the common European race. In Iberia, the races are parapatric and distribution is not related to climatic conditions. The populations are chromosomally heterogeneous. Polymorphisms include B-chromosomes (five types) and supernumerary segments. Large euchromatic segments occur on the homoeologues Al and Bl in both diploids and the allotetraploid, converting these acrocentrics to metacentrics. A wide spectrum of non-polymorphic numerical and structural variants was also found. The chromosomal structure of this species complex is discussed.

Cytogenetic analysis of gilthead seabream Sparus aurata (Pisces, Perciformes), a deletion affecting the NOR in a hatchery stock

We have cytogenetically characterized a hatchery stock of gilthead seabream, Sparus aurata. The study included larvae, juveniles and adults. In S. aurata (diploid chromosome number 2n = 48), a pair of NORs is located at the ends of the short arms of the first submetacentric pair of chromosomes. In this stock we discovered a polymorphism which affects the NORs, and, by means of several cytogenetic and molecular techniques, we demonstrate that this polymorphism is due to the complete deletion of one of the two NORs in a high number of individuals. The significance of these cytogenetic characteristics for this species are discussed since they may be the source of aquaculture problems.

rDNA site number polymorphism and NOR inactivation in natural populations ofAllium schoenoprasum

Nucleolar-organiser activity has been studied by silver staining and byin situ hybridization with an rDNA probe in two populations ofAllium schoenoprasum. One population is monomorphic with NORs and rDNA sites terminal on the short arm of pair 8 in all individuals. The other populations is monomorphic for pair 8 NORs but is also polymorphic for NORs on the long arm of pair 7. All plants in this population carry ribosomal cistrons on both chromosomes of pair 7 but 0, 1 or 2 of these sites can be active in rRNA synthesis. Cis-acting nucleolar-suppression affects the pair 7 locus. We suggest that there has been progressive reduction in the number of NORs during the evolution ofA. schoenoprasum.

Asphodelus tenuifolius andA. fistulosus (Liliaceae) are morphologically, genetically, and biologically different species

The biological analysis of six populations ofAsphodelus tenuifolius and 12 populations ofA. fistulosus has confirmed that they are separate species. Both their floral structures (length of the tepals, stamens, anthers and style) and also their pollen size are clearly different.A. tenuifolius has only the 2n = 28 chromosome race, whileA. fistulosus has 2n = 28 and 2n = 56.A. tenuifolius is genetically less variable thanA. fistulosus and they have different electrophoretic mobilities. Gene duplication phenomena exist in the 2n = 28 level of both species.

Characterization of the chromatin of some liliaceous species after digestion with restriction endonucleases and sequential Giemsa, fluorochrome and silver staining

A good C-banding pattern or a modified C-banding pattern is obtained after digestion with some restriction endonucleases (REs) on the fixed metaphase chromosomes of four liliaceous species. The C bands of Allium subvillosum and Muscari comosum are resistant to digestion with some REs whose recognition sequences are either GC- or AT-rich. The telomeric C-bands of A. cepa are resistant not only to some endonucleases whose recognition sequences are absent in the DNA satellite existing in these regions, but also to some endonucleases for which there exist numerous recognition sequences. However, the GC-rich C-band associated to the NOR of Scilla autumnalis is entirely digested by Hae III, and some of the faint GC-rich C-bands of the three NORs of A. subvillosum are digested by Hae III, Alu I or Rsa I. The action of endonucleases on plant chromosomes in general, and particularly on heterochromatin and NORs, together with the usefulness of these enzymes in cytogenetic analyses of plants, are discussed.

A comparative analysis of NORs in diploid and triploid salmonids: implications with respect to the diploidization process occurring in this fish group

In order to study the way in which the process of genome diploidization involving salmonid fishes affects the ribosomal genes, chromosome location of active nucleolus organizer regions (NORs) was determined in diploid and triploid individuals of two species of salmonids — rainbow trout and coho salmon. These species belong to an aquaculturally important fish group of tetraploid ancestral origin. Diploids of both species show only one NOR-bearing chromosome pair, while new NOR sites, not detected in diploids, are observed in triploid karyotypes. However, the number of active NORs has not increased significantly with respect to the expected for the triploid level because some of the standard diploid NORs are repressed in triploids. Active NORs appear as CMA3-positive and DAPI-negative clusters in diploid and triploid karyotypes of both species, except the M3 type of rainbow trout which was observed only in triploid individuals. The data obtained suggest: (i) that diploidization of NORs still remains incomplete in the species of this group, and (ii) that the response of NOR-bearing chromosomes to fluorochromes can depend on changes between the inactivated and activated NOR-states associated with the triploidization. The variations in the rDNA of the NOR-type M3, which occur during the diploidization process, can account for the absence of response to fluorochrome staining. Other structural and evolutionary aspects regarding NORs are also discussed.

A cytogenetical and molecular analysis of the ribosomal cistrons of Allium sphaerocephalon L. (Liliaceae)

Allium sphaerocephalon is a species with a high number of secondary constrictions and with intra- and interindividual variation in their number. We analysed whether all the secondary constrictions are involved in nucleolus formation, whether the same variability exists in the number of NOR, and the mechanism(s) responsible for this variability. Our cytological data have shown that (i) all the secondary constrictions may be involved in the nucleolus organization, (ii) the variability in number is in accordance with the numbers of NOR, and (iii) the variability could be due to unequal exchanges among ribosomal genes on homologous and non-homologous chromosomes. Ribosomal DNA sequence homogeneity detected in this species supports this conclusion.

Genetic variability in Muscari comosum L. (Liliaceae) IV Geographical distribution and adaptive role of the polymorphic variants of chromosome 2

Muscari comosum L. (Liliaceae) has a chromosomal polymorphism for a pericentric inversion and a supernumerary chromosome segment probably due to an unequal interchange or insertional translocation. Both arrangements are widely distributed throughout the species range and the mean genetic distance among populations is D=0.131±0.075. There are no correlations between genetic distance and geographic distance or latitude. Only appreciable decreases in the frequencies of the inversion are detected in populations with ecologically marginal characteristics. There is a permanent and extended association between chromosomal inversion and an enzymatic locus (ADH). An excess of individuals heterozygous for the inversion was found and female productivity of heterozygotes is higher than that of corresponding homozygotes. A low rate of inversion heterozygosity in populations with ecologically marginal characteristics could be explained by natural selection. With respect to the adaptive role of the segment, although no homozygotes are found and may be selected against, heterozygotes could have heterotic effects.

Restriction endonuclease digestion of meiotic chromosomes of Allium subvillosum L.

Meiotic chromosomes of the liliaceous plant Allium subvillosum were characterized by means of digestion with the restriction endonucleases (REs) Hae III and Msp I followed in some cases by treatments with proteinase K or nuclease S1. Both REs are capable of digesting euchromatin, giving a C-like banding pattern. Something similar can be observed when chromosomes are digested with the two restriction endonucleases followed by treatments with proteinase K. By contrast, heterochromatic regions can be digested only after sequential treatments using Hae III plus nuclease S1. These results are discussed in relation to the structural organization of plant meiotic chromosomes as well as the special conformation of plant heterochromatin.