Effects of an environmentally relevant concentration of diuron on oyster genitors during gametogenesis: responses of early molecular and cellular markers and physiological impacts.

Genitors of the Pacific oyster Crassostrea gigas were submitted during gametogenesis to a short pulse exposure to the herbicide diuron at a realistic environmental concentration. Histological analysis showed no effect of diuron on gametogenesis course, sex ratio and reproductive effort. A non-significant increase in testosterone and progesterone levels was observed in genitors exposed to the herbicide. At cell level, diuron exposure was shown to modulate the phagocytic activity of circulating hemocytes. The results of a transcriptional analysis showed that diuron affected the expression of genes belonging to functions known to play a major role during oyster gametogenesis such as gene transcription regulation, DNA replication and repair, DNA methylation and cytokinesis. Taking into account the results we previously obtained on the same genitors, this study showed a negative effect of diuron on oyster reproduction by inducing both structural and functional modifications of the DNA.

Study of genetic damage in the Japanese oyster induced by an environmentally-relevant exposure to diuron: evidence of vertical transmission of DNA damage.

Pesticides represent a major proportion of the chemical pollutants detected in French coastal waters and hence a significant environmental risk with regards to marine organisms. Commercially-raised bivalves are particularly exposed to pollutants, among them pesticides, as shellfish farming zones are subject to considerable pressure from agricultural activities on the mainland. The aims of this study were to determine (1) the genotoxic effects of diuron exposure on oyster genitors and (2) the possible transmission of damaged DNA to offspring and its repercussions on oyster fitness. To investigate these points, oysters were exposed to concentrations of diuron close to those detected in the Marennes-Oleron Basin (two 7-day exposure pulses at 0.4 and 0.6 µg L(-1)) during the gametogenesis period. Genomic abnormalities were characterized using two complementary approaches. The Comet assay was applied for the measurement of early and reversible primary DNA damage, whereas flow cytometry was used to assess the clastogenic and aneugenic effect of diuron exposure. Polar Organic Chemical Integrative Samplers (POCIS) were used in exposed and assay tanks to confirm the waterborne concentration of diuron reached during the experiment. The results obtained by the Comet assay clearly showed a higher level of DNA strand breaks in both the hemocytes and spermatozoa of diuron-exposed genitors. The transmission of damaged genetic material to gamete cells could be responsible for the genetic damage measured in offspring. Indeed, flow cytometry analyses showed the presence of DNA breakage and a significant decrease in DNA content in spat from diuron-exposed genitors. The transmission of DNA damage to the offspring could be involved in the negative effects observed on offspring development (decrease in hatching rate, higher level of larval abnormalities, delay in metamorphosis) and growth. In this study, the vertical transmission of DNA damage was so highlighted by subjecting oyster genitors to short exposures to diuron at medium environmental concentrations. The analysis of POCIS showed that oysters were exposed to integrated concentrations as low as 0.2 and 0.3 µg L(-1), emphasizing the relevance of the results obtained and the risk associated to chemical contamination for oyster recruitment and fitness.

Chromosomal organization of simple sequence repeats in the Pacific oyster (Crassostrea gigas): (GGAT)(4), (GT)(7) and (TA)(10) chromosome patterns.

Chromosome identification is essential in oyster genomic research. Fluorescence in situ hybridization (FISH) offers new opportunities for the identification of oyster chromosomes. It has been used to locate satellite DNAs, telomeres or ribosomal DNA sequences. However, regarding chromosome identification, no study has been conducted with simple sequence repeats (SSRs). FISH was used to probe the physical organization of three particular SSRs, (GGAT)(4), (GT)(7) and (TA)(10) onto metaphase chromosomes of the Pacific oyster, Crassostrea gigas. Hybridization signals were observed in all the SSR probes, but the distribution and intensity of signals varied according to the oligonucleotide repeat. The intercalary, centromeric and telomeric bands were observed along the chromosomes, and for each particular repeat every chromosome pair presented a similar pattern, allowing karyotypic analysis with all the SSRs tested. Our study is the first in mollusks to show the application of SSR in situ hybridization for chromosome identification and karyotyping. This technique can be a useful tool for oyster comparative studies and to understand genome organization in different oyster taxa.

A cytogenetic method for stacking gene pairs in common wheat.

The potential for non-reciprocal Robertsonian translocations of wheat (Triticum aestivum L.) to assist in the stacking of genes was assessed from a study of their cytological and genetic behaviour. To obtain translocations, a double monosomic (3B+5A; 2n=40=19ii+2i) was crossed reciprocally with a contrasting disomic. Individuals inheriting a broken monosome were identified from the loss of one arm-specific DNA marker coupled with retention of a marker for the opposite arm. No double breaks (potential translocations) were found in 180 cross progeny recovered from pollen of the double monosomic but two instances (loss of 5AL plus 3BS; loss of 5AL plus 3BL) were found in 251 progeny recovered from ovules. Meiotic pairing and multi-color genome-specific fluorescence in situ hybridization (mcGISH) showed that each plant with a double break contained one translocated chromosome between the A and B genomes that had rejoined at the centromere and that formed a trivalent (19ii+ liii) in about 83% of PMC. Most trivalents (approximately 92%) aligned at metaphase in a 'V' configuration(alternate disjunction) while the rest aligned in linear 'I'(adjacent disjunction) or ambiguous 'L' configurations. Genetic analysis of a testcross of these 'fusion monosomics' showed that this preferential co-orientation of the trivalent influenced the assortment of the chromosome arms involved. Loci that were located in the hemizygous ends of the 'V' trivalent showed strong quasi-linkage in that most ovules from the female testcross carried relevant DNA markers either from both standard chromosomes or from neither. This shows that, in most cases, the two standard chromosomes assorted to the same pole while the fused monosome segregated to the opposite pole. For heterozygous loci (present both on the fusion monosome and the standard chromosomes) assortment was either independent or showed partial linkage to the hemizygous arm depending on the reported recombination distance from centromere. Marker assortment was further distorted in male testcrosses and in doubled haploids (made from the fusion monosomics by the maize method) by the strong selective advantage of pollen or haploids that inherited the standard chromosomes rather than the deficiencies. This genetic data shows that under the combined influence of alternate disjunction and natural selection, progeny of fusion monosomics will revert to the standard disomic arrangement, fixing the gene content of both hemizygous arms in the process. Thus, any pair of genes could be targeted for joint fixation by isolating the fusion monosome that will link them temporarily in a segregating population.

Genome discrimination in progeny of interspecific hybrids between Brassica napus and Raphanus raphanistrum.

Genomic in situ hybridization (GISH) applied to the F1 interspecific hybrid between oilseed rape (Brassica napus, AACC, 2n = 38) and wild radish (Raphanus raphanistrum, RrRr, 2n = 18) showed the predicted 19 chromosomes from B. napus and 9 chromosomes from R. raphanistrum. The very low female fertility of these interspecific hybrids when backcrossed to R. raphanistrum led to only two descendants. Their chromosome number varied between 45 and 48. Both of these progenies showed only 9 chromosomes from R. raphanistrum and 36-39 chromosomes from B. napus. These results indicate the efficiency and limits of GISH as a suitable tool to assess and interpret the behavior of chromosomes after such interspecific crosses. The unexpected chromosome combination is discussed.

Characterization of six wheat x Thinopyrum intermedium derivatives by GISH, RFLP, and multicolor GISH.

Restriction fragment length polymorphism (RFLP) analysis and multicolor genomic in situ hybridization (GISH) are useful tools to precisely characterize genetic stocks derived from crosses of wheat (Triticum aestivum) with Thinopyrum intermedium and Thinopyrum elongatum. The wheat x Th. intermedium derived stocks designated Z1, Z2, Z3, Z4, Z5, and Z6 were initially screened by multicolor GISH using Aegilops speltoides genomic DNA for blocking and various combinations of genomic DNA from Th. intermedium, Triticum urartu, and Aegilops tauschii for probes. The probing (GISH) results indicated that lines Z1 and Z3 were alien disomic addition lines with chromosome numbers of 2n = 44. Z2 was a substitution line in which chromosome 2D was substituted by a pair of Th. intermedium chromosomes; this was confirmed by RFLP and muticolour GISH. Z4 (2n = 44) contained two pairs of wheat--Th. intermedium translocated chromosomes; one pair involved A-genome chromosomes, the other involved D- and A- genome chromosomes. Z5 (2n = 44) contained one pair of wheat--Th. intermedium translocated chromosomes involving the D- and A-genome chromosomes of wheat. Z6 (2n = 44) contained one pair of chromosomes derived from Th. intermedium plus another pair of translocated chromosomes involving B-genome chromosomes of wheat Line Z2 was of special interest because it has some resistance to infection by Fusarium graminearum.

Genomic in situ hybridization (GISH) discriminates between the A and the B genomes in diploid and tetraploid Setaria species.

Genomic in situ hybridization (GISH) was used to investigate genomic relationships between different Setaria species of the foxtail millet gene pool (S. italica) and one interspecific F1 hybrid. The GISH patterns obtained on the two diploid species S. viridis (genome A) and S. adhaerans (genome B), and on their F1 hybrid showed clear differentiation between these two genomes except at the nucleolar organizing regions. Similar GISH patterns allowed differentiation of S. italica from S. adhaerans. However, GISH patterns did not distinguish between the genomes of S. italica and its putative wild ancestor S. viridis. GISH was also applied to polyploid Setaria species and enabled confirmation of the assumed allotetraploid nature of S. faberii and demonstration that both S. verticillata and S. verticillata var. ambigua were also allotetraploids. All these tetraploid species contained two sets of 18 chromosomes each, one from genome A and the other from genome B. Only one polyploid species, S. pumila, was shown to bear an unknown genomic composition that is not closely related either to genome A or to genome B.