A comparative physical map reveals the pattern of chromosomal evolution between the turkey (Meleagris gallopavo) and chicken (Gallus gallus) genomes

Abstract

Background: A robust bacterial artificial chromosome (BAC)-based physical map is essential for many aspects of genomics research, including an understanding of chromosome evolution, high-resolution genome mapping, marker-assisted breeding, positional cloning of genes, and quantitative trait analysis. To facilitate turkey genetics research and better understand avian genome evolution, a BAC-based integrated physical, genetic, and comparative map was developed for this important agricultural species.

Results: The turkey genome physical map was constructed based on 74,013 BAC fingerprints (11.9 × coverage) from two independent libraries, and it was integrated with the turkey genetic map and chicken genome sequence using over 41,400 BAC assignments identified by 3,499 overgo hybridization probes along with > 43,000 BAC end sequences. The physical-comparative map consists of 74 BAC contigs, with an average contig size of 13.6 Mb. All but four of the turkey chromosomes were spanned on this map by three or fewer contigs, with 14 chromosomes spanned by a single contig and nine chromosomes spanned by two contigs. This map predicts 20 to 27 major rearrangements distinguishing turkey and chicken chromosomes, despite up to 40 million years of separate evolution between the two species. These data elucidate the chromosomal evolutionary pattern within the Phasianidae that led to the modern turkey and chicken karyotypes. The predominant rearrangement mode involves intra-chromosomal inversions, and there is a clear bias for these to result in centromere locations at or near telomeres in turkey chromosomes, in comparison to interstitial centromeres in the orthologous chicken chromosomes.

Conclusion: The BAC-based turkey-chicken comparative map provides novel insights into the evolution of avian genomes, a framework for assembly of turkey whole genome shotgun sequencing data, and tools for enhanced genetic improvement of these important agricultural and model species.

Figure 1

Turkey chromosome 2 (MGA2) rearrangements. Chromosome segments are shaded to indicate relative directionality: inversion 1, green and yellow; inversion 2, blue and brown. Arrows (red, inversion 1; blue, inversion 2) connect the inverted segment edges. Constrictions indicate the likely centromere locations. Selected overgo probe marker hybridizations from those supporting the rearrangements are shown. BES mate pairs whose spacing and strandedness support the two proposed rearrangements are listed. Inversion 1 is supported by markers RNT4 and 105G04T as well as 7 paired-end BES matches (Additional file 4: Table S4). Inversion 2 is supported by markers 100P13S and 103G13S, together with 6 paired-end BES spanning the breakpoint. DNA markers 02G07S and OTOR are located at the breakpoint of inversion 2 as landmarks.

Figure 2

FISH to pachytene chromosomes of turkey and chicken illustrating the MGA2/GGA3 inversions. Co-hybridization with CHORI-260 107N20 (red, WUGSC2.1/galGal3 BES coordinates 128288-325088) and 090K24 (green, 2711577-2958331) to turkey pachytene chromosomes gives overlapping signals (A) displayed by color-coded arrowheads, whereas with chicken pachytene chromosomes (B), 107N20 is telomere-adjacent and separated from 090K24 by about 2.5 Mb, as would be expected from the chicken sequence BES alignments. This is consistent with the p-arm inversion on MGA2 relative to GGA3 (Figure 1). Additional FISH with the TM1 centromere probe (unpublished observation) confirms that the MGA2 centromere is telomere-adjacent. Co-hybridization with CHORI-260 095D05 (red, WUGSC2.1/galGal3 BES coordinates 5361781-5538321) and 110E18 (green, 5654670-13311876) to turkey pachytene chromosomes (C) gives distinct, well-separated signals, whereas with chicken pachytene chromosomes (D), the 110E18 signal (green arrowheads) is split, as expected from its inconsistently spaced BES alignment, with one signal adjacent to the 095D05 hybridization (red arrowhead) and the other signal internal on MGA2. The p-arm of MGA2 (A, C) and GGA3 (B, D) is oriented toward the top of each image. These results support inversion of the turkey segment orthologous to the 5.6-11.6 Mb portion of GGA3, as shown in Figure 1. The chicken sequence coordinates of the BACs used as probes are indicated alongside the GGA3 bivalents shown in B and D. Scale bar = 1 μm.

Figure 3

Turkey chromosome p-arm inversions identified in MGA5, MGA7, MGA10 and MGA13. Relevant chromosome segments are shaded. Large segments are banded to indicate directionality, also shown by colored arrows flanking enlargements. Centromeres are shown by constrictions. A. MGA5. MGA5p is inverted with respect to GGA5p with an additional small inversion of fragment 2 (yellow). Centromere location in MGA5p is tentative (see Results and discussion). B. MGA7. GGA7p is inverted with respect to MGA7 (fragment 1, direction indicated by blue arrow) with movement of the centromere to a telomeric site in MGA7. C. MGA10. GGA8p is inverted with respect to MGA10 with movement of the centromere to a telomeric site in MGA10. In addition, fragment 3 (yellow) is also inverted (such that its direction is now the same in both species), and we place a turkey genome segment (fragment 1, red) orthologous to a 340 kb fragment from GGA8_random at a telomere-adjacent location on MGA10. D. MGA13. GGA11p is inverted with respect to MGA13 with movement of the centromere to at or near the telomere in MGA13.

Figure 4

FISH analysis using turkey BAC probes shows an inversion of 0.7 Mb between MGA16 and GGA14. CHORI-260 BAC 110H19 exhibits split signals (green arrows) in chicken, (A) leptotene stage nucleus and (C) pachytene bivalent FISH, as predicted by its widely spread and same strand BES matches to the chicken sequence (WUGSC2.1/galGal3 14315000-14844336). The FISH signal from CHORI-260 BAC 094E20 (red arrows, WUGSC2.1/galGal3 14715031-14890809) aligns consistently with an unbroken sequence mostly internal to the two 110H19 ends and overlapping with one of them on a (B) leptotene stage nucleus (same cell as A) and (D) pachytene (same bivalent as C). (E) Diagram of the inversion between MGA16 and GGA14 based on FISH analysis, overgo hybridization and BES alignments. Chicken sequence coordinates of the BACs are as shown. Asterisks: BES aligned too far apart and to the same DNA strand in the chicken sequence for CHORI-260 110H19 and 106P19. A, B: scale bar = 5 μm; C, D: scale bar = 1 μm.

Figure 5

FISH analysis of MGAZ/GGAZ illustrates a large Z chromosome inversion. CHORI-260 BACs 096G02 (green, WUGSC2.1/galGal3 coordinates 39290768-39535155) and 103I04 (red, 59681476-59892200) nearly co-localize on a metaphase turkey chromosome with the MGAZ centromere (A), but are well-separated on opposite arms on GGAZ (B), as would be expected from their chicken sequence coordinates. Shang et al. [42] located the GGAZ centromere at 42.2-42.5 Mb. (C) CHORI-260 BAC 099I24 (green, WUGSC2.1/galGal3 coordinates 64731310-64992979) labels the central portion of the second MGAZ arm (the two arms are of nearly identical length), as would be predicted based on the GGAZ sequence of Bellott et al. [35]. However, CHORI-260 098I08 (red, WUGSC2.1/galGal3 coordinates 49538900-49816462) nearly co-localizes with 099I24. These results are consistent with an inversion between the two Z chromosomes spanning at least 49-60 Mb. Overgo hybridization and BES alignment suggest a single ~19 Mb inversion from about 43.9 Mb to 62.8 Mb. Arrows point to the centromere for each chromosome. Scale bar = 2 μm.