Sex chromosomes and associated rDNA form a heterochromatic network in the polytene nuclei of Bactrocera oleae (Diptera: Tephritidae)

Abstract

The olive fruit fly, Bactrocera oleae, has a diploid set of 2n = 12 chromosomes including a pair of sex chromosomes, XX in females and XY in males, but polytene nuclei show only five polytene chromosomes, obviously formed by five autosome pairs. Here we examined the fate of the sex chromosomes in the polytene complements of this species using fluorescence in situ hybridization (FISH) with the X and Y chromosome-derived probes, prepared by laser microdissection of the respective chromosomes from mitotic metaphases. Specificity of the probes was verified by FISH in preparations of mitotic chromosomes. In polytene nuclei, both probes hybridized strongly to a granular heterochromatic network, indicating thus underreplication of the sex chromosomes. The X chromosome probe (in both female and male nuclei) highlighted most of the granular mass, whereas the Y chromosome probe (in male nuclei) identified a small compact body of this heterochromatic network. Additional hybridization signals of the X probe were observed in the centromeric region of polytene chromosome II and in the telomeres of six polytene arms. We also examined distribution of the major ribosomal DNA (rDNA) using FISH with an 18S rDNA probe in both mitotic and polytene chromosome complements of B. oleae. In mitotic metaphases, the probe hybridized exclusively to the sex chromosomes. The probe signals localized a discrete rDNA site at the end of the short arm of the X chromosome, whereas they appeared dispersed over the entire dot-like Y chromosome. In polytene nuclei, the rDNA was found associated with the heterochromatic network representing the sex chromosomes. Only in nuclei with preserved nucleolar structure, the probe signals were scattered in the restricted area of the nucleolus. Thus, our study clearly shows that the granular heterochromatic network of polytene nuclei in B. oleae is formed by the underreplicated sex chromosomes and associated rDNA.

Chromosomal localization and partial sequencing of the 18S and 28S ribosomal genes from Bradysia hygida (Diptera: Sciaridae)

“…Although research related to rRNA in diptera is scarce, we found that in many species of culicidae (Rafael et al, 2006) and tephritidae (Drosopoulou et al, 2012), the rRNA genes are usually localized in sex chromosomes; however, in some cases (e.g., Rhagoletis pomonella), it can be found in autosomal chromosomes (Procunier and Smith, 1993).…”

2D and 3D Chromosome Painting in Malaria Mosquitoes

“…Other protocols require the pooling of many (typically 10-30) chromosome fragments in order to successfully amplify the sample 7,11,28,40 . Although pooling of multiple chromosomes is possible using our method, the increased likelihood of sample contamination emphasizes the importance of beginning the experiment with as few chromosomes as possible.…”

“…This method is also applied to studying the 3D dynamics of chromosome territories in development 9 and pathology 10 . Differentially labeled chromosome paints are used for the visualization of individual mitotic 11,12 , meiotic 13,14 , or interphase non-polytene 15,16 and polytene 9,11,17 chromosomes. A simple and robust protocol to obtain chromosome paints would be very useful in expanding this technique to non-model organisms, such as mosquitoes.…”

“…When coupled with whole genome amplification (WGA), microdissection results in powerful downstream applications including FISH 11,12 and next-generation genome sequencing [25][26][27] . Previously, the technique required the use of specialized microdissection needles that had to be manually controlled by an experienced user 28 .…”

“…Therefore microdissection, amplification, and subsequent analysis of a single chromosome are far more challenging, especially in organisms with small genomes like in Drosophila or Anopheles. Although paints have been developed from single microdissected chromosomes of a human 33 and a wasp 45 , a successful FISH experiment required multiple (at least [10][11][12][13][14][15] microdissected mitotic chromosomes of a fruit fly 11 . However, the ability to microdissect and amplify a single chromosome would be important for (i) reducing the chance of contamination with material from a different chromosome, (ii) minimizing the number of chromosomal preparations needed for microdissection, (iii) lowering nucleotide and structural polymorphism of the microdissected sample in both FISH and sequencing downstream applications.…”