Fax +41 61 306 12 34 E-Mail karger@karger.ch www.karger.com Original Article Cytogenet Genome Res DOI: 10.1159/000314923 Precise Centromere Positioning on Chicken Chromosome 3 A. Zlotina   a S. Galkina   a A. Krasikova   a R.P.M.A. Crooijmans   b M.A.M. Groenen   b E. Gaginskaya   a S. Deryusheva   a   a  Saint-Petersburg State University, Saint-Petersburg, Russia; b  Animal Breeding and Genomics Centre, Wageningen University, Wageningen, The Netherlands Genome sequencing of the red jungle fowl (Gallus gal- lus) was completed at 6.6 ! coverage in 2004 [Interna- tional Chicken Genome Sequencing Consortium, 2004] followed by a new draft genome assembly (Gallus_gal- lus-2.1) in 2006 (http://www.ncbi.nlm.nih.gov). Although this new build improved the genome sequence assembly, the supercontigs on which this genome sequence is based are still interrupted by numerous gaps. Furthermore, many contigs still remain unanchored to specific chro- mosomes and instead are combined on chromosome ‘ChrUn’. On the current assembly, centromeres are indi- cated by 1.5-Mb gaps on macrochromosomes and by 0.5- Mb gaps on microchromosomes. Notwithstanding many attempts to clone centromer- ic repeats from the chicken genome, only 4 repetitive sequences have been described so far as centromere as- sociated sequences. These repeats are the chicken eryth- rocyte nuclear membrane (CNM) repeat found in peri- centromeric regions of microchromosomes and macro- chromosomes 6, 9 and W [Matzke et al., 1990; Wang et al., 2002; Krasikova et al., 2006], the XhoI repeat near the centromeric region of chromosome W [Solovei et al., 1998; Krasikova et al., 2006], the Hinf I repeat concen- trated in the pericentromeric region of chromosome 4 [Li and Leung, 2006], and the partially inverted tandem repeat (PIR) specific for the centromere of chromosome Key Words Centromere positioning  Chicken  CNM  FISH  GGA3  Immunostaining  Lampbrush chromosomes Abstract Despite the progress of the chicken (Gallus gallus) genome sequencing project, the centromeric sequences of most macrochromosomes remain unknown. This makes it difficult to determine centromere positions in the genome sequence assembly. Using giant lampbrush chromosomes from grow- ing oocytes, we analyzed in detail the pericentromeric re- gion of chicken chromosome 3. Without knowing the DNA sequence, the centromeres at the lampbrush stage are de- tectable by immunostaining with antibodies against cohe- sin subunits. Immunostaining for cohesin followed by FISH with 23 BAC clones, covering the region from 0 to 23 Mb on chicken chromosome 3 (GGA3), allowed us to map the GGA3 centromere between BAC clones WAG38P15 and WAG54M22 located at position 2.3 and 2.5 Mb, respectively. This corre- sponds to the gap between 2 supercontigs at the 2.4-Mb position in the current GGA3 sequence assembly (build 2.1). Furthermore, we have determined that the current putative centromeric gap at position 11.6–13.1 Mb corresponds in fact to a long cluster of tandem chicken erythrocyte nuclear membrane repeats (CNM). Copyright © 2010 S. Karger AG, Basel Accepted: January 13, 2010 by J. Smith Published online: July 3, 2010 Svetlana Galkina Saint-Petersburg State University Oranienbaumskoe sh., 2 Saint-Petersburg, 198504 (Russia) Tel. +7 812 450 7311, Fax +7 812 450 7310, E-Mail spbchromas  @  gmail.com © 2010 S. Karger AG, Basel 1424–8581/10/0000–0000$26.00/0 Accessible online at: www.karger.com/cgr