Alignment of the genomes of Brachypodium distachyon and temperate cereals and grasses using bacterial artificial chromosome landing with fluorescence in situ hybridization

Abstract

As part of an initiative to develop Brachypodium distachyon as a genomic "bridge" species between rice and the temperate cereals and grasses, a BAC library has been constructed for the two diploid (2n = 2x = 10) genotypes, ABR1 and ABR5. The library consists of 9100 clones, with an approximate average insert size of 88 kb, representing 2.22 genome equivalents. To validate the usefulness of this species for comparative genomics and gene discovery in its larger genome relatives, the library was screened by PCR using primers designed on previously mapped rice and Poaceae sequences. Screening indicated a degree of synteny between these species and B. distachyon, which was confirmed by fluorescent in situ hybridization of the marker-selected BACs (BAC landing) to the 10 chromosome arms of the karyotype, with most of the BACs hybridizing as single loci on known chromosomes. Contiguous BACs colocalized on individual chromosomes, thereby confirming the conservation of genome synteny and proving that B. distachyon has utility as a temperate grass model species alternative to rice.

Figure 1.

DNA from B. distachyon BAC clones was NotI restricted and the fragments were separated by PFGE. BACs were obtained by partial restriction of B. distachyon ABR5 genomic DNA with HindIII, followed by PFGE and cloning of the size-separated genomic DNA from a gel slice in the region of 100–130 kb (size selection 2 in Table 3). The average insert size of the BAC inserts in this size selection experiment is estimated to be 92.3 kb. M, λ-ladders (New England Biolabs and Roche); V, the position of the 7.0-kb pBeloBAC11 NotI vector fragment.

Figure 2.

Identification of B. distachyon ABR1 (2n = 2x = 10) chromosomes by dual-color FISH with BAC clones. (A) 1p, ABR1-63-E11 (red); 1q, ABR1-26-H1 (green). (B) 2p, ABR1-41-E10 (green); 2q, ABR5-1-H3 (red). (C) 3p, ABR5-33-F12 (red); 3q, ABR1-56-H6 (green). (D) 4p, ABR5-33-F2 (green); 4q, ABR5-32-C1 (red). (E) Multicolor FISH with three different BAC clones and 25S rDNA (yellow): 1q (subterminal), ABR1-41-A8 (green); 1q (interstitial), ABR1-59-F9 (red); 4q, ABR1-47-F4 (green); 5p (NOR), 25S rDNA. (F) Multicolor FISH with one BAC clone (red), 5S rDNA (green), and 25S rDNA (yellow). 4q (interstitial), 5S rDNA; 5p (NOR), 25S rDNA; 5q (subterminal), ABR1-63-E2. Color symbols in A–E describe the localization of landmarks in particular chromosome arms of ABR1 (p, short arm; q, long arm). (G) Ideogram of B. distachyon ABR1 (n = 5) chromosomes showing physical localization of all mapped BAC clones and the two auxiliary landmarks (5S and 25S rDNA). Asterisks indicate the clones from B. distachyon ABR5 library. Blue fluorescence, DAPI. Bar, 5 μm.

Figure 3.

High-resolution FISH mapping of closely linked on mitotic metaphase preparations (A and D) pairs of B. distachyon BAC clones on B. distachyon (2n = 2x = 10) zygotene (B) and pachytene (E) chromosomes. (A and B) ABR1-26-H1 (green) and ABR1-41-A8 (red) apparently colocalizing (yellow) in the distal part of the long arm of chromosome pair 1 at mitotic metaphase (A), while, on the zygotene chromosomes, ABR1-41-A8 is clearly more distal than ABR1-26-H1. (D) ABR1-56-H6 (red) and ABR1-54-D7 (green) at mitotic metaphase colocalizing (yellow) in the distal part of the long arm of chromosome pair 3 at mitotic metaphase. (E) BAC–FISH of the same clones to the pachytene chromosome reveals that ABR1-54-D7 (green) is located more distally than ABR1-56-H6. (C and F) Diagrams showing different resolution of BAC–FISH mapping on A and B and D and E. Blue fluorescence, DAPI. Bar, 5 μm.

Figure 4.

Comparative analysis of (A, D, and G) B. distachyon ABR1 (2n = 2x = 10), (B, E, and H) ABR114 (2n = 2x = 20), (C, F, and I) ABR113 (2n = 4x = 30), and (L) Triticale (2n = 6x = 42) genomes by FISH of the B. distachyon ABR1/5 BAC clones. (A–C) ABR1-32-C1 (green), 5S rDNA (red). (D–F) ABR1-32-C1 (green); ABR5-33-F2 (red). Color symbols on A and D describe the localization of landmarks on particular chromosome arms. (G–I) ABR1-63-E6 (green). (L) ABR1-41-H4 (green). (J and K) Selected chromosomes from putative ancestral diploid species (B. distachyon ABR1 and ABR114) and an interspecific hybrid (ABR113) hybridizing with the BAC clones or a 5S ribosomal DNA probe. The chromosomes were extracted from (A and D) B. distachyon ABR1, (B and E) ABR114, and (C and F) ABR113. Bars: (A–I) 5 μm; (L) 10 μm.