Meiotic pairing and gene expression disturbance in germ cells from an infertile boar with a balanced reciprocal autosome-autosome translocation.

Individuals carrying balanced constitutional reciprocal translocations generally have a normal phenotype, but often present reproductive disorders. The aim of our research was to analyze the meiotic process in an oligoasthenoteratospermic boar carrying an asymmetric reciprocal translocation involving chromosomes 1 and 14. Different multivalent structures (quadrivalent and trivalent plus univalent) were identified during chromosome pairing analysis. Some of these multivalents were characterized by the presence of unpaired autosomal segments with histone γH2AX accumulation sometimes associated with the XY body. Gene expression in spermatocytes was studied by RNA-DNA-FISH and microarray-based testis transcriptome analysis. Our results revealed a decrease in gene expression for chromosomes 1 and 14 and an up-regulated expression of X-chromosome genes for the translocated boar compared with normal individuals. We hypothesized that the observed meiotic arrest and reproductive failure in this boar might be due to silencing of crucial autosomal genes (MSUC) and disturbance of meiotic sex chromosome inactivation (MSCI). Further analysis revealed abnormal meiotic recombination (frequency and distribution) and the production of a high rate of unbalanced spermatozoa.

Meiotic Recombination Analyses in Pigs Carrying Different Balanced Structural Chromosomal Rearrangements.

Correct pairing, synapsis and recombination between homologous chromosomes are essential for normal meiosis. All these events are strongly regulated, and our knowledge of the mechanisms involved in this regulation is increasing rapidly. Chromosomal rearrangements are known to disturb these processes. In the present paper, synapsis and recombination (number and distribution of MLH1 foci) were studied in three boars (Sus scrofa domestica) carrying different chromosomal rearrangements. One (T34he) was heterozygote for the t(3;4)(p1.3;q1.5) reciprocal translocation, one (T34ho) was homozygote for that translocation, while the third (T34Inv) was heterozygote for both the translocation and a pericentric inversion inv(4)(p1.4;q2.3). All three boars were normal for synapsis and sperm production. This particular situation allowed us to rigorously study the impact of rearrangements on recombination. Overall, the rearrangements induced only minor modifications of the number of MLH1 foci (per spermatocyte or per chromosome) and of the length of synaptonemal complexes for chromosomes 3 and 4. The distribution of MLH1 foci in T34he was comparable to that of the controls. Conversely, the distributions of MLH1 foci on chromosome 4 were strongly modified in boar T34Inv (lack of crossover in the heterosynaptic region of the quadrivalent, and crossover displaced to the chromosome extremities), and also in boar T34ho (two recombination peaks on the q-arms compared with one of higher magnitude in the controls). Analyses of boars T34he and T34Inv showed that the interference was propagated through the breakpoints. A different result was obtained for boar T34ho, in which the breakpoints (transition between SSC3 and SSC4 chromatin on the bivalents) seemed to alter the transmission of the interference signal. Our results suggest that the number of crossovers and crossover interference could be regulated by partially different mechanisms.

Meiotic recombination analyses of individual chromosomes in male domestic pigs (Sus scrofa domestica).

For the first time in the domestic pig, meiotic recombination along the 18 porcine autosomes was directly studied by immunolocalization of MLH1 protein. In total, 7,848 synaptonemal complexes from 436 spermatocytes were analyzed, and 13,969 recombination sites were mapped. Individual chromosomes for 113 of the 436 cells (representing 2,034 synaptonemal complexes) were identified by immunostaining and fluorescence in situ hybridization (FISH). The average total length of autosomal synaptonemal complexes per cell was 190.3 µm, with 32.0 recombination sites (crossovers), on average, per cell. The number of crossovers and the lengths of the autosomal synaptonemal complexes showed significant intra- (i.e. between cells) and inter-individual variations. The distributions of recombination sites within each chromosomal category were similar: crossovers in metacentric and submetacentric chromosomes were concentrated in the telomeric regions of the p- and q-arms, whereas two hotspots were located near the centromere and in the telomeric region of acrocentrics. Lack of MLH1 foci was mainly observed in the smaller chromosomes, particularly chromosome 18 (SSC18) and the sex chromosomes. All autosomes displayed positive interference, with a large variability between the chromosomes.

Cytogenetic analysis of somatic and germinal cells from 38,XX/38,XY phenotypically normal boars.

Many chromosomal abnormalities have been reported to date in pigs. Most of them have been balanced structural rearrangements, especially reciprocal translocations. A few cases of XY/XX chimerism have also been diagnosed within the national systematic chromosomal control program of young purebred boars carried out in France. Until now, this kind of chromosomal abnormality has been mainly reported in intersex individuals. We investigated 38,XY/38,XX boars presenting apparently normal phenotypes to evaluate the potential effects of this particular chromosomal constitution on their reproductive performance. To do this, we analyzed (1) the chromosomal constitution of cells from different organs in one boar; (2) the aneuploidy rates for chromosomes X, Y, and 13 in sperm nuclei sampled from seven XY/XX boars. 2n = 38,XX cells were identified in different nonhematopoietic tissues including testis (frequency, <8%). Similar aneuploidy rates were observed in the sperm nuclei of XY/XX and normal individuals (controls). Altogether, these results suggest that the presence of XX cells had no or only a very limited effect on the reproduction abilities of the analyzed boars.

The Medicago genome provides insight into the evolution of rhizobial symbioses.

Legumes (Fabaceae or Leguminosae) are unique among cultivated plants for their ability to carry out endosymbiotic nitrogen fixation with rhizobial bacteria, a process that takes place in a specialized structure known as the nodule. Legumes belong to one of the two main groups of eurosids, the Fabidae, which includes most species capable of endosymbiotic nitrogen fixation. Legumes comprise several evolutionary lineages derived from a common ancestor 60 million years ago (Myr ago). Papilionoids are the largest clade, dating nearly to the origin of legumes and containing most cultivated species. Medicago truncatula is a long-established model for the study of legume biology. Here we describe the draft sequence of the M. truncatula euchromatin based on a recently completed BAC assembly supplemented with Illumina shotgun sequence, together capturing ∼94% of all M. truncatula genes. A whole-genome duplication (WGD) approximately 58 Myr ago had a major role in shaping the M. truncatula genome and thereby contributed to the evolution of endosymbiotic nitrogen fixation. Subsequent to the WGD, the M. truncatula genome experienced higher levels of rearrangement than two other sequenced legumes, Glycine max and Lotus japonicus. M. truncatula is a close relative of alfalfa (Medicago sativa), a widely cultivated crop with limited genomics tools and complex autotetraploid genetics. As such, the M. truncatula genome sequence provides significant opportunities to expand alfalfa's genomic toolbox.

Studies of male and female meiosis in inv(4)(p1.4;q2.3) pig carriers.

Inversions are well-known structural chromosomal rearrangements in humans and pigs. Such rearrangements generally have no effect on the carrier's phenotype. However, the presence of an inversion may impair spermatogenesis and lead to the production of recombinant gametes, responsible for early miscarriages, stillbirth, or congenital abnormalities. This is the first report on meiotic segregation and pairing behavior of the inv(4)(p1.4;q2.3) pericentric inversion in pigs. Despite the very large size of the inverted fragment (76% of the chromosome), SpermFISH results showed that only 4.08% of the gametes produced by male heterozygotes were unbalanced. This low proportion could be explained by the particular behavior of normal and inverted SSC4 chromosomes during the initial stages of meiosis. Indeed, immunohistochemistry analyses revealed that heterosynapsis occurred in 92% of the cells, whereas synaptic adjustment was detected in a few spermatocytes only. Unexpectedly, the proportion of unbalanced gametes produced by female heterozygotes, estimated by FISH on metaphase II oocytes, was also very low (3.69%) and comparable to that in males. According to previous results for male and female meiotic processes, different proportions of recombinant gametes in the two genders would have been expected. Complementary studies should be carried out to further document the meiotic behavior of inversions in pigs.

Intraspecies genomic diversity and natural population structure of the meat-borne lactic acid bacterium Lactobacillus sakei.

Lactobacillus sakei is a food-borne bacterium naturally found in meat and fish products. A study was performed to examine the intraspecies diversity among 73 isolates sourced from laboratory collections in several different countries. Pulsed-field gel electrophoresis analysis demonstrated a 25% variation in genome size between isolates, ranging from 1,815 kb to 2,310 kb. The relatedness between isolates was then determined using a PCR-based method that detects the possession of 60 chromosomal genes belonging to the flexible gene pool. Ten different strain clusters were identified that had noticeable differences in their average genome size reflecting the natural population structure. The results show that many different genotypes may be isolated from similar types of meat products, suggesting a complex ecological habitat in which intraspecies diversity may be required for successful adaptation. Finally, proteomic analysis revealed a slight difference between the migration patterns of highly abundant GapA isoforms of the two prevailing L. sakei subspecies (sakei and carnosus). This analysis was used to affiliate the genotypic clusters with the corresponding subspecies. These findings reveal for the first time the extent of intraspecies genomic diversity in L. sakei. Consequently, identification of molecular subtypes may in the future prove valuable for a better understanding of microbial ecosystems in food products.

Distribution of microsatellites in the genome of Medicago truncatula: a resource of genetic markers that integrate genetic and physical maps.

Microsatellites are tandemly repeated short DNA sequences that are favored as molecular-genetic markers due to their high polymorphism index. Plant genomes characterized to date exhibit taxon-specific differences in frequency, genomic location, and motif structure of microsatellites, indicating that extant microsatellites originated recently and turn over quickly. With the goal of using microsatellite markers to integrate the physical and genetic maps of Medicago truncatula, we surveyed the frequency and distribution of perfect microsatellites in 77 Mbp of gene-rich BAC sequences, 27 Mbp of nonredundant transcript sequences, 20 Mbp of random whole genome shotgun sequences, and 49 Mbp of BAC-end sequences. Microsatellites are predominantly located in gene-rich regions of the genome, with a density of one long (i.e., > or = 20 nt) microsatellite every 12 kbp, while the frequency of individual motifs varied according to the genome fraction under analysis. A total of 1,236 microsatellites were analyzed for polymorphism between parents of our reference intraspecific mapping population, revealing that motifs (AT)n, (AG)n, (AC)n, and (AAT)n exhibit the highest allelic diversity. A total of 378 genetic markers could be integrated with sequenced BAC clones, anchoring 274 physical contigs that represent 174 Mbp of the genome and composing an estimated 70% of the euchromatic gene space.

The complete genome sequence of the meat-borne lactic acid bacterium Lactobacillus sakei 23K.

Lactobacillus sakei is a psychotrophic lactic acid bacterium found naturally on fresh meat and fish. This microorganism is widely used in the manufacture of fermented meats and has biotechnological potential in biopreservation and food safety. We have explored the 1,884,661-base-pair (bp) circular chromosome of strain 23K encoding 1,883 predicted genes. Genome sequencing revealed a specialized metabolic repertoire, including purine nucleoside scavenging that may contribute to an ability to successfully compete on raw meat products. Many genes appear responsible for robustness during the rigors of food processing--particularly resilience against changing redox and oxygen levels. Genes potentially responsible for biofilm formation and cellular aggregation that may assist the organism to colonize meat surfaces were also identified. This genome project is an initial step for investigating new biotechnological approaches to meat and fish processing and for exploring fundamental aspects of bacterial adaptation to these specific environments.

Physical and genetic map of the Lactobacillus sakei 23K chromosome.

The Lactobacillus sakei 23K chromosome was analysed by pulsed-field gel electrophoresis after digestion with the restriction enzymes AscI, NotI and SfiI. The chromosome size was estimated to be 1845+/-80 kb. The use of I-CeuI, specific for rrn genes encoding 23S rRNAs, showed that seven rrn loci were present, on 40% of the chromosome. The seven rrn clusters were mapped and their orientation was determined, allowing the position of the replication origin to be estimated. Partial I-CeuI digestions were used to construct a backbone and the different restriction fragments obtained with AscI, NotI and SfiI were assembled to a physical map by Southern hybridization. Eleven L. sakei gene clusters previously identified were mapped, as well as 25 new loci located randomly on the chromosome and 11 regions flanking the rrn gene clusters. A total of 47 clusters were thus mapped on L. sakei chromosome. The new loci were sequenced, allowing the identification of 73 complete or incomplete coding sequences. Among these 73 new genes of L. sakei, the function of 36 could be deduced from their similarity to known genes described in databases. However, 10 genes had no homologues, 10 encoded proteins similar to proteins of unknown function and 17 were similar to hypothetical proteins.