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Additional copies of CBX2 in the genomes of males of mammals lacking SRY, the Amami spiny rat (Tokudaia osimensis) and the Tokunoshima spiny rat (Tokudaia tokunoshimensis)

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Abstract

Tokudaia osimensis (the Amami spiny rat) and Tokudaia tokunoshimensis (the Tokunoshima spiny rat) have a sex chromosome composition of XO/XO, no Y chromosome. The mammalian sex-determining gene, SRY, is also absent in these species, which indicates that these spiny rats exhibit a novel sex-determining mechanism that is independent of SRY. To identify a candidate gene that controls this mechanism, the copy numbers and chromosomal locations of 10 genes with important functions in gonadal differentiation were determined: ATRX, CBX2 (M33), DMRT1, FGF9, NR0B1 (DAX1), NR5A1 (Ad4BP/SF1), RSPO1, SOX9, WNT4, and WT1. Multiple bands were detected for NR0B1 in Southern blot analysis, which suggested the presence of multiple copies of the gene in the genomes of these two species. CBX2 was localized to two loci in both sexes of the two species by fluorescence in situ hybridization mapping: 3q24 and 6p11.2 in T. osimensis and 10q25–q26 and 14q12–q13.1 in T. tokunoshimensis. Quantification of copy numbers in the two species by quantitative real-time PCR indicated that there were two or three more copies of CBX2 per haploid genome in males (T. osimensis, n = 3; T. tokunoshimensis, n = 2) than in females (T. osimensis, n = 4; T. tokunoshimensis, n = 2), whereas NR0B1 was present as a single copy in both. The results suggest that additional copies of CBX2 in males might be involved in a novel sex-determining mechanism in species that lack SRY.

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Abbreviations

AP:

Alkaline phosphatase

ASA:

American Standards Association

BSA:

Bovine serum albumin

BrdU:

Bromodeoxyuridine

cDNA:

Complementary DNA

CDP:

Cytidine diphosphate

DIG:

Digoxygenine

dUTP:

Deoxyuridine 5-triphosphate

FISH:

Fluorescence in situ hybridization

IgG:

Immunoglobulin G

IUCN:

International Union for Conservation of Nature

kb:

Kilo base pairs

KO:

Knockout

MEM:

Minimal essential medium

qRT-PCR:

Quantitative real-time PCR

RACE:

Rapid amplification of cDNA ends

SD:

Standard deviation

SDS:

Sodium dodecyl sulfate

SSC:

Saline sodium citrate

UTR:

Untranslated region

UV:

Ultraviolet

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Acknowledgments

The authors thank T. Hashimoto for capturing animals, C. Nishida-Umehara and K. Matsubara for help with cell culture and FISH, K. Morohashi for providing the mouse cDNA clones, and Y. Katoh-Fukui for helpful advises. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas (no. 16086201) from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and a grant from the Naito Foundation, Japan.

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Authors and Affiliations

  1. Graduate School of Life Science, Hokkaido University, Kita 10 Nishi 8, Kita-ku, Sapporo, Hokkaido, 060-0810, Japan

    Asato Kuroiwa, Sanae Handa, Chigusa Nishiyama & Eriko Chiba

  2. Forestry and Forest Products Research Institute, Tsukuba, Ibaraki, 305-8687, Japan

    Fumio Yamada

  3. Naha Nature Conservation Office, Ministry of the Environment, Yamashita-cho 5-21, Naha, Okinawa, 900-0027, Japan

    Shintaro Abe

  4. Laboratory of Animal Genetics, Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8601, Japan

    Yoichi Matsuda

  5. Laboratory of Animal Cytogenetics, Faculty of Science, Hokkaido University, Kita 10 Nishi 8, Kita-ku, Sapporo, Hokkaido, 060-0810, Japan

    Asato Kuroiwa

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  1. Asato Kuroiwa
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  2. Sanae Handa
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Correspondence to Asato Kuroiwa.

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Responsible Editor: Fengtang Yang.

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Kuroiwa, A., Handa, S., Nishiyama, C. et al. Additional copies of CBX2 in the genomes of males of mammals lacking SRY, the Amami spiny rat (Tokudaia osimensis) and the Tokunoshima spiny rat (Tokudaia tokunoshimensis). Chromosome Res 19, 635–644 (2011). https://doi.org/10.1007/s10577-011-9223-6

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  • DOI: https://doi.org/10.1007/s10577-011-9223-6

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