Cytological Analysis of Crossover Frequency and Distribution in Male Meiosis of Cardueline Finches (Fringillidae, Aves).

Meiotic recombination is an important source of genetic diversity. Using immunolocalization of several meiotic proteins at the spreads of male pachytene cells, we estimated the number of recombination nodules per cell and their distribution along the macrochromosome 1 of the Common linnet, Eurasian bullfinch, Eurasian siskin, and European goldfinch. The macrochromosomes of the two former species have metapolycentromeres, composed of several centromeric domains. We detected significant interspecies differences in the mean numbers of recombination nodules per genome: 52.9 ± 2.8 in the linnet, 49.5 ± 3.5 in the bullfinch, 61.5 ± 6.3 in the siskin and 52.2 ± 2.7 in the goldfinch. Recombination patterns on macrochromosome 1 were similar across species, with more nodules localized near chromosome ends and fewer around centromeres. The distance from the proximal nodule to the centromere depended on the nodule count per chromosome arm, with more events leading to a closer location. However, species with different centromere types showed no difference in this regard. We propose that the deficiency of recombination sites near centromeres could be due to the sequential occurrence of crossovers starting from the chromosome ends and may not be attributed to any suppressive effect of the centromere itself.

Establishment of the Primary Avian Gonadal Somatic Cell Lines for Cytogenetic Studies.

The last decade was marked by a steep rise in avian studies at genomic and cellular levels. Cell lines are important tools for in vitro studies in cell biology and cytogenetics. We developed a simple method of primary somatic cell culture establishment from the ovaries of the great tits (Parus major) and testes of ten Passerine species, characterized the cellular composition of the ovary-derived lines using RT-PCR and immunolocalization of the tissue-specific markers and tested the efficiency of two methods of genetic transformation of the ovary-derived cell line. We found that the ovary-derived cell cultures of the great tit were composed of fibroblasts mainly, but also contained interstitial and granulosa cells. They were cultivated until the 10th passage without any noticeable decrease in their proliferative activity. The testis-derived cell cultures had lower proliferative potential. However, both ovary- and testis-derived cell cultures provided enough material for high quality mitotic metaphase chromosome preparations. The efficiency of its transduction with lentivirus containing a GFP reporter was very low, while electroporation with episomal vectors expressing GFP resulted in a high yield of GFP-positive cells. The proposed method could be used for the generation of high quality material for various cytogenetic and genomic studies.

Germline-Restricted Chromosomes and Autosomal Variants Revealed by Pachytene Karyotyping of 17 Avian Species.

Karyotypes of less than 10% of bird species are known. Using immunolocalization of the synaptonemal complex, the core structure of meiotic chromosomes at the pachytene stage, and centromere proteins, we describe male pachytene karyotypes of 17 species of birds. This method enables higher resolution than the conventional analyses of metaphase chromosomes. We provide the first descriptions of the karyotypes of 3 species (rook, Blyth's reed warbler, and European pied flycatcher), correct the published data on the karyotypes of 10 species, and confirm them for 4 species. All passerine species examined have highly conservative karyotypes, 2n = 80-82 with 7 pairs of macrochromosomes (including the ZZ sex chromosome pair which was not unambiguously distinguished from other macrochromosomes in most species) and 33-34 pairs of microchromosomes. In all of them, but not in the common cuckoo, we revealed single copies of the germline-restricted chromosomes varying in size and morphology even between closely related species. This indicates a fast evolution of this additional chromosome. The interspecies differences concern the sizes of the macrochromosomes, morphology of the microchromosomes, and sizes of the centromeres. The pachytene cells of the gouldian finch, brambling, and common linnet contain heteromorphic synaptonemal complexes indicating heterozygosity for inversions or centromere shifts. The European pied flycatcher, gouldian finch, and domestic canary have extended centromeres in several macro- and microchromosomes.

Mendelian nightmares: the germline-restricted chromosome of songbirds.

Germline-restricted chromosomes (GRCs) are accessory chromosomes that occur only in germ cells. They are eliminated from somatic cells through programmed DNA elimination during embryo development. GRCs have been observed in several unrelated animal taxa and show peculiar modes of non-Mendelian inheritance and within-individual elimination. Recent cytogenetic and phylogenomic evidence suggests that a GRC is present across the species-rich songbirds, but absent in non-passerine birds, implying that over half of all 10,500 bird species have extensive germline/soma genome differences. Here, we review recent insights gained from genomic, transcriptomic, and cytogenetic approaches with regard to the genetic content, phylogenetic distribution, and inheritance of the songbird GRC. While many questions remain unsolved in terms of GRC inheritance, elimination, and function, we discuss plausible scenarios and future directions for understanding this widespread form of programmed DNA elimination.

Germline-Restricted Chromosome (GRC) in Female and Male Meiosis of the Great Tit (Parus major, Linnaeus, 1758).

All songbirds studied so far have a germline-restricted chromosome (GRC), which is present in the germ cells and absent in the somatic cells. It shows a wide variation in size, morphology, and genetic content between the songbird species. In this paper, we analyzed GRC behavior in female and male meiosis of the great tit, using immunolocalization of meiotic proteins and FISH with GRC-derived DNA probes. We found that, despite dozens of million years of independent evolution, the great tit GRC displays a striking similarity with the GRCs of two species of martins and two species of estrildid finches examined earlier. It was usually present in two copies in females forming recombining bivalent and in one copy in males forming a condensed heterochromatic body with dotted-like axial elements of the synaptonemal complex. We observed mosaicism for the GRC copy number in the female and male great tit. This indicates that one of the GRC copies might be passively lost during premeiotic germ cell divisions. After the meiotic prophase, the GRC was ejected from most male germ cells. The reverse and interspecies FISH with GRC-specific microdissected DNA probes indicates that GRCs of the great tit, pale martin, and zebra finch differ substantially in their genetic content despite similarities in the meiotic behavior.

Highly Conservative Pattern of Sex Chromosome Synapsis and Recombination in Neognathae Birds.

We analyzed the synapsis and recombination between Z and W chromosomes in the oocytes of nine neognath species: domestic chicken Gallus gallus domesticus, grey goose Anser anser, black tern Chlidonias niger, common tern Sterna hirundo, pale martin Riparia diluta, barn swallow Hirundo rustica, European pied flycatcher Ficedula hypoleuca, great tit Parus major and white wagtail Motacilla alba using immunolocalization of SYCP3, the main protein of the lateral elements of the synaptonemal complex, and MLH1, the mismatch repair protein marking mature recombination nodules. In all species examined, homologous synapsis occurs in a short region of variable size at the ends of Z and W chromosomes, where a single recombination nodule is located. The remaining parts of the sex chromosomes undergo synaptic adjustment and synapse non-homologously. In 25% of ZW bivalents of white wagtail, synapsis and recombination also occur at the secondary pairing region, which probably resulted from autosome-sex chromosome translocation. Using FISH with a paint probe specific to the germline-restricted chromosome (GRC) of the pale martin on the oocytes of the pale martin, barn swallow and great tit, we showed that both maternally inherited songbird chromosomes (GRC and W) share common sequences.

Homogeneously Staining Regions (HSR) in Chromosome 1 of the House Mouse: Synapsis and Recombination at Meiosis.

Amplified sequences constitute a large part of mammalian genomes. A chromosome 1 containing 2 large (up to 50 Mb) homogeneously staining regions (HSRs) separated by a small inverted euchromatic region is present in many natural populations of the house mouse (Mus musculus musculus). The HSRs are composed of a long-range repeat cluster, Sp100-rs, with a repeat length of 100 kb. In order to understand the organization and function of HSRs in meiotic chromosomes, we examined synapsis and recombination in male mice hetero- and homozygous for the HSR-carrying chromosome using FISH with an HSR-specific DNA probe and immunolocalization of the key meiotic proteins. In all homozygous and heterozygous pachytene nuclei, we observed fully synapsed linear homomorphic bivalents 1 marked by the HSR FISH probe. The synaptic adjustment in the heterozygotes was bilateral: the HSR-carrying homolog was shortened and the wild-type homolog was elongated. The adjustment was reversible: desynapsis at diplotene was accompanied by elongation of the HSRs. Immunolocalization of H3K9me2/3 indicated that the HSRs in the meiotic chromosome retained the epigenetic modification typical for C-heterochromatin in somatic cells. MLH1 foci, marking mature recombination nodules, were detected in the proximal HSR band in heterozygotes and in both HSR bands of homozygotes. Unequal crossing over within the long-range repeat cluster can cause variation in size of the HSRs, which has been detected in the natural populations of the house mouse.

Heterochiasmy and Sexual Dimorphism: The Case of the Barn Swallow (Hirundo rustica, Hirundinidae, Aves).

Heterochiasmy, a sex-based difference in recombination rate, has been detected in many species of animals and plants. Several hypotheses about evolutionary causes of heterochiasmy were proposed. However, there is a shortage of empirical data. In this paper, we compared recombination related traits in females and males of the barn swallow Hirundo rustica (Linnaeus, 1758), the species under strong sexual selection, with those in the pale martin Riparia diluta (Sharpe and Wyatt, 1893), a related and ecologically similar species with the same karyotype (2N = 78), but without obvious sexual dimorphism. Recombination traits were examined in pachytene chromosome spreads prepared from spermatocytes and oocytes. Synaptonemal complexes and mature recombination nodules were visualized with antibodies to SYCP3 and MLH1 proteins, correspondingly. Recombination rate was significantly higher (p = 0.0001) in barn swallow females (55.6 ± 6.3 recombination nodules per autosomal genome), caused by the higher number of nodules at the macrochromosomes, than in males (49.0 ± 4.5). They also showed more even distribution of recombination nodules along the macrochromosomes. At the same time, in the pale martin, sexual differences in recombination rate and distributions were rather small. We speculate that an elevated recombination rate in the female barn swallows might have evolved as a compensatory reaction to runaway sexual selection in males.

Germline-restricted chromosome (GRC) in the sand martin and the pale martin (Hirundinidae, Aves): synapsis, recombination and copy number variation.

All songbirds studied to date have an additional Germline Restricted Chromosome (GRC), which is not present in somatic cells. GRCs show a wide variation in genetic content and little homology between species. To check how this divergence affected the meiotic behavior of the GRC, we examined synapsis, recombination and copy number variation for GRCs in the closely related sand and pale martins (Riparia riparia and R. diluta) in comparison with distantly related estrildid finches. Using immunolocalization of meiotic proteins and FISH with GRC-specific DNA probes, we found a striking similarity in the meiotic behavior of GRCs between martins and estrildid finches despite the millions of years of independent evolution. GRCs are usually present in two copies in female and in one copy in male pachytene cells. However, we detected polymorphism in female and mosaicism in male martins for the number of GRCs. In martin and zebra finch females, two GRCs synapse along their whole length, but recombine predominately at their ends. We suggest that the shared features of the meiotic behavior of GRCs have been supported by natural selection in favor of a preferential segregation of GRCs to the eggs.

Interbreed variation in meiotic recombination rate and distribution in the domestic chicken Gallus gallus.

The efficiency of natural and artificial selection is critically dependent on the recombination rate. However, interbreed and individual variation in recombination rate in poultry remains unknown. Conventional methods of analysis of recombination such as genetic linkage analysis, sperm genotyping and chiasma count at lampbrush chromosomes are expensive and time-consuming. In this study, we analyzed the number and distribution of recombination nodules in spermatocytes of the roosters of six chicken breeds using immunolocalization of key proteins involved in chromosome pairing and recombination. We revealed significant effects of breed ( R 2 = 0.17 ; p < 0.001 ) and individual ( R 2 = 0.28 ; p < 0.001 ) on variation in the number of recombination nodules. Both interbreed and individual variations in recombination rate were almost entirely determined by variation in recombination density on macrochromosomes, because almost all microchromosomes in each breed had one recombination nodule. Despite interbreed differences in the density of recombination nodules, the patterns of their distribution along homologous chromosomes were similar. The breeds examined in this study showed a correspondence between the age of the breed and its recombination rate. Those with high recombination rates (Pervomai, Russian White and Brahma) are relatively young breeds created by crossing several local breeds. The breeds displaying low recombination rate are ancient local breeds: Cochin (Indo-China), Brown Leghorn (Tuscany, Italy) and Russian Crested (the European part of Russia).

Germline-restricted chromosome (GRC) is widespread among songbirds.

An unusual supernumerary chromosome has been reported for two related avian species, the zebra and Bengalese finches. This large, germline-restricted chromosome (GRC) is eliminated from somatic cells and spermatids and transmitted via oocytes only. Its origin, distribution among avian lineages, and function were mostly unknown so far. Using immunolocalization of key meiotic proteins, we found that GRCs of varying size and genetic content are present in all 16 songbird species investigated and absent from germline genomes of all eight examined bird species from other avian orders. Results of fluorescent in situ hybridization of microdissected GRC probes and their sequencing indicate that GRCs show little homology between songbird species and contain a variety of repetitive elements and unique sequences with paralogs in the somatic genome. Our data suggest that the GRC evolved in the common ancestor of all songbirds and underwent significant changes in the extant descendant lineages.