Egizia Falistocco

Linkage mapping in apomictic and sexual Kentucky bluegrass (Poa pratensis L.) genotypes using a two way pseudo-testcross strategy based on AFLP and SAMPL markers

Abstract The high versatility of the mode of reproduction and the retention of a pollen recognition system are the factors responsible for the extreme complexity of the genome in Poa pratensis L. Two genetic maps, one of an apomictic and one of a sexual genotype, were constructed using a two-way pseudo-testcross strategy and multiplex PCR-based molecular markers (AFLP and SAMPL). Due to the high ploidy level and the uncertainty of chromosome pairing-behavior at meiosis, only parent-specific single-dose markers (SDMs) that segregated 1:1 in an F1 mapping population (161 out of 299 SAMPLs, and 70 out of 275 AFLPs) were used for linkage analysis. A total of 41 paternal (33 SAMPLs and 8 AFLPs) and 47 maternal (33 SAMPLs and 14 AFLPs) SDMs, tested to be linked in coupling phase, were mapped to 7+7 linkage groups covering 367 and 338.4 cM, respectively. The comparison between the two marker systems revealed that SAMPL markers were statistically more efficient than AFLP ones in detecting parent-specific SDMs (75% vs 32.4%). There were no significant differences in the percentages of distorted marker alleles detected by the two marker systems (27.8% of SAMPLs vs 21.3% of AFLPs). The pairwise comparison of co-segregational groups for linkage detection between marker loci suggested that at least some of the P. pratensis chromosomes pair preferentially at meiosis-I.

Monitoring of mutagens in urban air samples

This research was designed to examine the presence of mutagenic/carcinogenic compounds in urban airborne particulates sampled with the inhalable PM-10 high volume sampler in two different streets of Brescia, a heavily industrialized town in northern Italy, using the Tradescantia/micronucleus test and a bacterial mutagenicity test (Kado test, a more sensitive version of the Ames test). In addition, the Tradescantia/micronucleus test was used for in situ monitoring of gaseous pollutants in other urban areas of Brescia and in two car tunnels, one with heavy car traffic in Perugia, a town in central Italy, and one in Brescia with moderate traffic. The Tradescantia-micronucleus test carried out on extracts of airborne particulates gave positive results only for the sample collected in the traffic-congested street where also higher bacterial mutagenicity was found. The in situ monitoring of the urban areas with the Tradescantia/micronucleus test always gave negative results. Monitoring carried out in the two car tunnels showed a significant increase in micronuclei frequency only in flowers exposed in the smaller and more polluted tunnel.

Mutagenicity of extracts of lake drinking water treated with different disinfectants in bacterial and plant tests

Abstract Raw water and drinking water samples collected from five treatment plants supplied by a northern Italian lake in two periods of the year (summer and winter) were studied for their mutagenicity. The water samples were concentrated on silica C 18 cartridges and the adsorbates were tested at increasing doses with a bacterial short-term mutagenicity test (Ames test with Salmonella typhimurium TA98 and TA100 strains), which reveals the gene-mutation-inducing ability of pollutants, and with a plant genotoxicity bioassay ( Tradescantia /micronucleus test), which determines clastogenicity (chromosome-breaking ability). Raw water samples from all treatment plants were found to contain bacterial direct-acting mutagens detectable mainly with TA98 strain. The analyses of drinking water samples after water treatment showed some interesting results: TA98 mutagenicity was reduced when ozone was used together with chlorine dioxide, but TA100 mutagenicity was increased, though only in the summer sample; mutagenicity detectable with both strains was always reduced after chlorine dioxide disinfection; on the contrary, in all treatment plants using NaClO TA98 mutagenicity of winter samples increased. Raw lake water induced a high number of micronuclei in the Tradescantia /micronucleus test, showing a strong clastogenicity. This activity was higher in the NaClO-treated samples, and lower with the other disinfectants. Therefore, disinfection of lake water with ozone and/or chlorine dioxide seems to be a suitable alternative to the use of NaClO for controlling the formation of nonvolatile mutagens. The concentration method coupled with the two mutagenicity tests was found to be a simple, rapid and relatively inexpensive system for monitoring treatment plants and studying the influence of different disinfection systems on water mutagenicity.

Variation of genome size and organization within hexaploid Festuca arundinacea

SummaryCytophotometric, karyological, and biochemical analyses were carried out in the meristems of seedlings obtained from seeds collected from 35 natural populations of hexaploid Festuca amndinacea in Italy. Highly significant differences between populations were observed in the amount of nuclear DNA (up to 32.3%). These changes are linked to variations in the amount of heterochromatin and in the frequency of repeated DNA sequences, and particularly of a fraction of them. Differences between populations in the arm ratios and total length of the chromosomes were also observed. The genome sizes of the populations are correlated positively with the mean temperature during the year and with that of the coldest month at the stations, and correlate negatively with their latitudes. The intraspecific genome changes observed are discussed in relation to other pertinent data to be found in the literature and in relation to their possible role in environmental adaptation.

Genomic relationships between Medicago murex Willd. and Medicago lesinsii E. small. investigated by in situ hybridization

Abstract.Medicago murex Willd. is an annual species (2n = 14) widespread in the wild and of remarkable interest for pastures in regions with a mediterranean climate. It is considered closely related to Medicago lesinsii E. Small (2n = 16) but, up to now, there is no evidence demonstrating their genetic affinity. This research was undertaken to investigate the genomic relationships between M. murex and M. lesinsii by using genomic in situ hybridization (GISH). In this study GISH experiments were performed using both species as sources of chromosomes and genomic probes. To better evaluate the results of the hybridization, the labelled DNA of each species was hybridized to chromosomes of the same species and to chromosomes of the diploid Medicago littoralis (2n = 16). Strong hybridization signals were found on chromosomes of M. murex and M. lesinsii after GISH. Differences in the hybridization strength were not observed when slides from interspecific hybridization were compared with the control preparations. These results suggest that consistent divergences of the DNA sequences did not occur after the separation of the two species. Instead very reduced cross hybridization was found on chromosome spreads of M. littoralis hybridized with the DNA of M. lesinsii or M. murex. The distribution of the ribosomal genes (rDNA) investigated by fluorescent in situ hybridization (FISH) appeared similar in both M. murex and M. lesinsii. The GISH technique may be a valuable approach to obtain information on evolution of the 2n = 14 species and on the origin of the polyploids Medicago rugosa (2n = 30) and Medicago scutellata (2n = 30). The first attempt to investigate the genomic composition of M. scutellata using a genomic probe is reported in this paper.

Cytological, morphological and molecular analyses of controlled progenies from meiotic mutants of alfalfa producing unreduced gametes

A program of sexual polyploidization was carried out in alfalfa using plants from wild diploid species that produced male or female unreduced gametes. Sixteen progenies from 2x-4x and 2x-2x crosses were examined with a combination of morphological, cytological and molecular analyses. The chromosome counts revealed diploid, tetraploid and aneuploid plants. Plants with B chromosomes were also detected. The leaf area of the plants was a useful characteristic for distinguishing tetraploid from diploid plants obtained by unilateral or bilateral sexual polyploidization. Leaf shape and leaf margin were not correlated with the ploidy levels. Plants with supernumerary chromosomes displayed obovate or elliptic leaves which differed markedly from the range of forms typical of diploid and tetraploid alfalfa plants. RAPD markers were investigated in all progeny plants to determine maternal and paternal amplification products. Three alfalfa-specific primers proved to be effective in revealing the hybrid origin of the plants. A combination of cytological, morphological and molecular analyses is essential for a detailed genetic characterization of progenies in programs of sexual polyploidization.

Mutagenicity and clastogenicity of gas stove emissions in bacterial and plant tests

The aim of this research was to study the gaseous and particulate emissions of genotoxic substances during cooking with two types of methane stoves (a new one and an old one). The particulates were sampled both with a cascade impactor air sampler and an impinger with ice trap and analyzed by two bacterial mutagenicity tests (Ames and Kado tests) and by HPLC for polycyclic aromatic hydrocarbons (PAH). Gaseous emissions were studied in situ using the Ames test, a clastogenicity plant test (Tradescantia‐micronucleus test), and in an automated system for chemical analyses. Clear indirect mutagenicity was found only with the Kado test (TA98‐S9) in extracts of particulates emitted from the old methane stove and collected with the impinger. Similar mutagenicity (TA98+S9) was also found for the finest fraction of particulates (<0.5 um) collected from both stoves. Gaseous emissions of both stoves caused clastogenicity in the in situ experiments with the Tradescantia‐micronucleus test. The physico‐chemical analyses of the emissions showed also the presence of very fine particulates and trace amounts of PAH. The exposure of these genotoxins could be particularly important for occupationally exposed individuals in homes and businesses and for susceptible subjects living indoors for long periods (infants, children, the sick, and the elderly). Environ. Mol. Mutagen. 31:402–408, 1998.

Cytogenetic Investigations and Karyological Relationships of two Medicago: M. Sativa L. (Alfalfa) and M. Arborea L.

SUMMARYA karyological survey with the purpose of comparing the chromosome complement of two tetraploid species, Medicago sativa (alfalfa) and M. arborea, was undertaken as the two species will be used in a programme of breeding including somatic hybridization. The study of the karyotypes has shown that the two species could be easly separated on the basis of chromosome morphology. This will allow the indendification of heterokaryons in somatic hybrids. The karyological study allowed for some interesting hypotheses on the origin of the two species.

Cytogenetic characterization of cultivated globe artichoke (Cynara cardunculus var. scolymus) and cardoon (C. cardunculus var. altilis)

Cynara cardunculus L. is a typical Mediterranean species comprising two important cultivated types, the globe artichoke (Cynara cardunculus var. scolymus) which is grown for its edible heads and the cultivated or leafy cardoon (C. cardunculus var. altilis) appreciated for its fleshy stems and leaf stalks. It includes also a third form, the wild cardoon (C. cardunculus var. sylvestris) which is considered the ancestor of the cultivated forms. Despite progress in the evolutionary field, advanced chromosome studies on C. cardunculus are almost nonexistent. The objective of this study was to fill in this gap by providing a refined cytogenetic characterization of the cultivated scolymus and altilis varieties. The karyomorphological analysis showed that artichoke and cardoon share an identical karyotype. All chromosomes are metacentric but are markedly differentiated with respect to their length, therefore they could be separated into three groups of different size: large (L), medium (M) and small (S). As a first step towards the physical mapping of artichoke and cardoon chromosomes, the FISH technique was applied to localize the position of 18S-5.8S-25S rRNA genes (45S rDNA). The fluorescent signals obtained by the FISH experiments constituted reliable landmarks for the identification of two pairs of M chromosomes and two pairs of S chromosomes. The overall results represent a significant advance in C. cardunculus cytogenetics and suggest further investigation of the wild sylvestris variety in order to acquire more exhaustive information on the evolutionary pathway of the species.

Chromosome investigations in annual Medicago species (Fabaceae) with emphasis on the origin of the polyploid Medicago rugosa and M. scutellata

Abstract Chromosome number variations play an important role in the genus Medicago. In addition to polyploidy there are cases of dysploidy as evidenced by two basic numbers, x = 8 and x = 7, the latter limited to five annual species having 2n = 14. Annuals are diploid with the exception of Medicago scutellata and Medicago rugosa which have 2n = 30 and are considered the result of crosses between the 2n = 16 and 2n = 14 species. However, this hypothesis has never been tested. This study was carried out to investigate the 2n = 14 and 2n = 30 karyotypes and verify the allopolyploid origin of M. scutellata and M. rugosa. Fluorescence in situ hybridization (FISH) of rDNA probes and genomic in situ hybridization (GISH) were performed. FISH showed that all five diploids with 2n = 14 have one pair of 45S and one pair of 5S rDNA sites. M. scutellata displayed four sites of 45S and four sites of 5S rDNA, while in M. rugosa only one pair of each of these sites was found. GISH did not produce signals useful to identify the presumed progenitors with 14 chromosomes. This result suggests alternative evolutionary pathways, such as the formation of tetraploids (2n = 32) and subsequent dysploidy events leading to the chromosome number reduction.