FISH and DAPI staining of the synaptonemal complex of the Nile tilapia (Oreochromis niloticus) allow orientation of the unpaired region of bivalent 1 observed during early pachytene

Abstract

Bivalent 1 of the synaptonemal complex (SC) in XY male Oreochromis niloticusshows an unpaired terminal region in early pachytene. This appears to be related to recombination suppression around a sex determination locus. To allow more detailed analysis of this, and unpaired regions in the karyotype of other Oreochromis species, we developed techniques for FISH on SC preparations, combined with DAPI staining. DAPI staining identified presumptive centromeres in SC bivalents, which appeared to correspond to the positions observed in the mitotic karyotype (the kinetochores could only be identified sporadically in silver stained EM SC images). Furthermore, two BAC clones containing Dmo (dmrt4) and OniY227 markers that hybridize to known positions in chromosome pair 1 in mitotic spreads (near the centromere, FLpter 0.25, and the putative sex determination locus, FLpter 0.57, respectively) were used as FISH probes on SCs to verify that the presumptive centromere identified by DAPI staining was located in the expected position. Visualization of both the centromere and FISH signals on bivalent 1 allowed the unpaired region to be positioned at Flpter 0.80 to 1.00, demonstrating that the unpaired region is located in the distal part of the long arm(s). Finally, differences between mitotic and meiotic measurements are discussed. (Foresti et al., 1993; Carrasco et al., 1999; Campos-Ramos et al., 2001;Griffin et al., 2002;Campos-Ramos et al., 2003). The exact relationship between these unpaired regions and sex determining loci in this genus is not clear: for example, a terminal region of the largest bivalent shows delayed pairing in XY O. niloticus but not in XX or YY genotypes (Carrasco et al., 1999), however sex-linked LG1 markers in this species have been mapped by FISH onto a small pair of chromosomes (Lee et al., 2003; Mota-Velasco, unpublished observations; Cnaani et al., 2008). Unpairing in both the largest bivalent and a small bivalent have been observed in WZ O. aureus at pachytene (Campos-Ramos et al., 2001), and linkage studies suggest two unlinked genes affect sex determination in this species, with the dominant one (WZ/ZZ, in LG3) mapping to the largest pair of chromosomes (Lee et al., 2004; Cnaani et al., 2008).The kinetochore (centromere) has been visualised in some TEM synaptonemal complex preparations in O. niloticus (Carrasco et al., 1999), allowing orientation of the unpaired region with respect to the chromosome, but this has not been achieved consistently. In this study, we set out to develop a technique that would allow both identification of the centromere and FISH on pachytene stage chromosomes, using male O. niloticus, with the objective of being able to simultaneously visualise unpaired 4 regions, centromeres and FISH markers at pachytene, to further the study of the role of delayed meiotic pairing in the evolution of sex determination in this genus. 5 Materials and Methods Experimental fishPhenotypic male and female O. niloticus (originating from Lake Manzallah, Egypt) used in this experi...

Chromosome Evolution in African Cichlid Fish: Contributions from the Physical Mapping of Repeated DNAs

“…The chromosome signals generated after hybridization of BAC-C4E09 allowed the identification of several chromosome elements in the karyotype of O. niloticus , including pairs 1-9, 11, 15-19, and 22. The largest chromosome pair of O. niloticus has been referred to as pair 1 in other studies because the arrangement of the karyotype was based on chromosome size [Oliveira and Wright, 1998;Ferreira and Martins, 2008;Ocalewicz et al, 2009;Valente et al, 2009]. BAC-C4E09 showed strong signals in the short arm of the largest m/sm chromosome pair (pair 1) of the non-tilapiine species, in the telomeric region of the st/a chromosome pair 12 of H. obliquidens ( fig.…”

“…However, Cnaani et al [2008] proposed that this larger chromosome pair in Nile tilapia may reflect prior evolutionary history of this pair as differentiated sex chromosomes, and that the sex determination locus is now on a small chromosome. Several repetitive sequences are distributed along the large chromosome pair of O. niloticus [Ferreira and Martins, 2008;Valente et al, 2009, and references cited therein] with different patterns of hybridization, due the difference in the copy number of this repetitive sequence between the supposed X and Y chromosomes [Harvey et al, 2003;Ocalewicz et al, 2009]. The presence of ROn-1 and the repeated sequences inserted in the BAC-C4E09 clone in the largest chromosome of African cichlids suggest that the accumulation and variable copy number of these repeated sequences in this region were integral to the early differentiation of the homologous sex chromosome.…”

Telomeres in Fishes

“…The position of the current and relict centromeres and ITS sites in O. karongae suggests that the 3 fusions all occurred in different orientations [Mota-Velasco et al, 2010]. Two ITSs have also been observed in chromosome 1 of O. karongae that has been corroborated to share the homology with the largest chromosome pair in the Nile tilapia [Ocalewicz et al, 2009b;Mota-Velasco et al, 2010]. In the Erythrinidae family, 2n = 54 is the most frequently observed chromosome number.…”

Repetitive DNA and Meiotic Behavior of Sex Chromosomes in <i>Gymnotus pantanal</i> (Gymnotiformes, Gymnotidae)

“…The X and Y allosomes have a differentiated behavior in the pachytene stage, in which a large portion of these chromosomes do not exhibit pairing during prophase I. Physical mapping with probes of the DMRT4 and OniY227 genes, both of which are related to sex determination, has verified their presence in these chromosomes in the O. niloticus , but their location is not in the asynaptic region [Ocalewicz et al, 2009]. The description of sex systems based only on mitotic chromosomes has led to errors regarding its actual existence, such as the XX/XY system described for Leporinus lacustris , which was not supported by the ultrastructural analysis of meiotic chromosomes through the synaptonemic complex [Mestriner et al, 1995].…”