Molecular cloning and characterization of the germline-restricted chromosome sequence in the zebra finch

The zebra finch (Taeniopygia guttata) germline-restricted chromosome (GRC) is the largest chromosome and has a unique system of transmission in germ cells. In the male, the GRC exists as a single heterochromatic chromosome in the germline and is eliminated from nuclei in late spermatogenesis. In the female, the GRC is bivalent and euchromatic and experiences recombination. These characteristics suggest a female-specific or female-beneficial function of the GRC. To shed light on the function of GRC, we cloned a portion of the GRC using random amplified polymorphic DNA–polymerase chain reaction and analyzed it using molecular genetic and cytogenetic methods. The GRC clone hybridized strongly to testis but not blood DNA in genomic Southern blots. In fluorescent in situ hybridization analysis on meiotic chromosomes from synaptonemal complex spreads, the probe showed hybridization across a large area of the GRC, suggesting that it contains repetitive sequences. We isolated a sequence homologous to the GRC from zebra finch chromosome 3 and a region of chicken chromosome 1 that is homologous to zebra finch chromosome 3; the phylogenetic analysis of these three sequences suggested that the GRC sequence and the zebra finch chromosome 3 sequence are most closely related. Thus, the GRC sequences likely originated from autosomal DNA and have evolved after the galliform–passeriform split. The present study provides a foundation for further study of the intriguing GRC

Broad-scale recombination pattern in the primitive bird Rhea americana (Ratites, Palaeognathae).

Birds have genomic and chromosomal features that make them an attractive group to analyze the evolution of recombination rate and the distribution of crossing over. Yet, analyses are biased towards certain species, especially domestic poultry and passerines. Here we analyze for the first time the recombination rate and crossover distribution in the primitive ratite bird, Rhea americana (Rheiformes, Palaeognathae). Using a cytogenetic approach for in situ mapping of crossovers we found that the total genetic map is 3050 cM with a global recombination rate of 2.1 cM/Mb for female rheas. In the five largest macrobivalents there were 3 or more crossovers in most bivalents. Recombination rates for macrobivalents ranges between 1.8-2.1 cM/Mb and the physical length of their synaptonemal complexes is highly predictive of their genetic lengths. The crossover rate at the pseudoautosomal region is 2.1 cM/Mb, similar to those of autosomal pairs 5 and 6 and only slightly higher compared to other macroautosomes. It is suggested that the presence of multiple crossovers on the largest macrobivalents is a feature common to many avian groups, irrespective of their position throughout phylogeny. These data provide new insights to analyze the heterogeneous recombination landscape of birds

Heterochromatic microbivalents in the Greater rhea.

<p>A. Pachytene nucleus immunostained for SMC, centromeres and the heterochromatin marker H3K9me3. Arrows point to some of the heterochromatic microbivalents and to the interstitial heterochromatin in SC 3. Bar: 10 μm. B-C. Notice that heterochromatin marks on the bi-armed microbivalents do not extend beyond the tip of the short arms. Bar: 1 μm.</p

Analysis of crossing over in rhea oocytes.

<p>A. In the graph each dot represents a single pachytene nucleus with n foci. The horizontal line marks the average number of foci found in 85 oocytes. B. Relationship between average SC length (μm) and the average number of MLH1 foci on the six largest autosomal bivalents. C. MLH1 foci on microbivalents. The numbers below the X-axis indicate the rank length of the SC. With the exception of microbivalents ranking 7th and 8th, most micro-SCs show a single focus. D. Distribution of MLH1 foci on individual SCs. For each SC, the X-axis indicates the relative positions of the MLH1 foci on the short (left) and long (right) arms. Each histogram represents the distribution of MLH1 foci along the six largest autosomal SCs and the ZW pair. The bin width in each histogram represents a fraction of the total length of each SC, and it is equivalent to 0.5 μm. “C” = centromere. Het: heterochromatin.</p

Number of MLH1 foci and crossover rates for individual and grouped SCs.

<p>Number of MLH1 foci and crossover rates for individual and grouped SCs.</p

Molecular cloning and characterization of the germline-restricted chromosome sequence in the zebra finch

The zebra finch (Taeniopygia guttata) germline-restricted chromosome (GRC) is the largest chromosome and has a unique system of transmission in germ cells. In the male, the GRC exists as a single heterochromatic chromosome in the germline and is eliminated from nuclei in late spermatogenesis. In the female, the GRC is bivalent and euchromatic and experiences recombination. These characteristics suggest a female-specific or female-beneficial function of the GRC. To shed light on the function of GRC, we cloned a portion of the GRC using random amplified polymorphic DNA–polymerase chain reaction and analyzed it using molecular genetic and cytogenetic methods. The GRC clone hybridized strongly to testis but not blood DNA in genomic Southern blots. In fluorescent in situ hybridization analysis on meiotic chromosomes from synaptonemal complex spreads, the probe showed hybridization across a large area of the GRC, suggesting that it contains repetitive sequences. We isolated a sequence homologous to the GRC from zebra finch chromosome 3 and a region of chicken chromosome 1 that is homologous to zebra finch chromosome 3; the phylogenetic analysis of these three sequences suggested that the GRC sequence and the zebra finch chromosome 3 sequence are most closely related. Thus, the GRC sequences likely originated from autosomal DNA and have evolved after the galliform–passeriform split. The present study provides a foundation for further study of the intriguing GRC