Construction of a 7-fold BAC library and cytogenetic mapping of 10 genes in the giant panda (Ailuropoda melanoleuca)

Abstract

Background: The giant panda, one of the most primitive carnivores, is an endangered animal. Although it has been the subject of many interesting studies during recent years, little is known about its genome. In order to promote research on this genome, a bacterial artificial chromosome (BAC) library of the giant panda was constructed in this study.

Results: This BAC library contains 198,844 clones with an average insert size of 108 kb, which represents approximately seven equivalents of the giant panda haploid genome. Screening the library with 15 genes and 8 microsatellite markers demonstrates that it is representative and has good genome coverage. Furthermore, ten BAC clones harbouring AGXT, GHR, FSHR, IRBP, SOX14, TTR, BDNF, NT-4, LH and ZFX1 were mapped to 8 pairs of giant panda chromosomes by fluorescence in situ hybridization (FISH).

Conclusion: This is the first large-insert genomic DNA library for the giant panda, and will contribute to understanding this endangered species in the areas of genome sequencing, physical mapping, gene cloning and comparative genomic studies. We also identified the physical locations of ten genes on their relative chromosomes by FISH, providing a preliminary framework for further development of a high resolution cytogenetic map of the giant panda.

Figure 1

Characterization of the giant panda BAC library. A. Analysis of the size of BAC clones by PFG electrophoresis. M: Mid range Marker II for PFGE (Bio-labs, New England). Lanes 1–19: NotI digested DNA of randomly picked recombinant BAC clones. PFGE conditions were set with Bio-rad CHEF III: 6.0 v/cm for 12 h, linear pulse time ramping from 0.1 to 40 s. B. BAC insert size distribution in the library. Insert sizes were determined from 261 BAC clones. The horizontal axis shows the size range in kb while the vertical axis displays the number of clones corresponding to each size range. Insert sizes are reported in a cumulative histogram.

Figure 2

Physical locations of ten genes on G-banded ideogram of giant panda. The G-banded ideogram was drawn with software Video TesT-karyotype3.1 (Video TesT Ltd, Russia, 2003). The names of the bands have not been listed because no international standard karyotype has yet been established for the giant panda.

Figure 3

Representative scheme of chromosome locations of AGXT (a+b), BDNF (c+d), LH (e+f), and NT-4 (g+h) in giant panda identified by FISH. a, c, e, g show G-banded metaphase chromosomes spread before FISH; b, d, f, h show the same chromosomes spread after FISH. Arrow shows the specific signals.