GISH and FISH pattern on mitotic (A, B, E, F, and G) and meiotic metaphase chromosomes (C and D) of TA5536 (A, C, D, and E), disomic addition line DA1H^t (B), z5A recombinant chromosomes (rec1–rec3) (F), telosomic chromosome tz5AL, isochromosome iz5AL, and translocation chromosomes (T1–T3) (G). Total genomic DNA from E. trachycaulus and the centromeric clone pAet6-J9 were labeled with fluorescein-12-dUTP (except for the pAet6-J9 FISH pattern on T2 in G) and visualized by yellow-green fluorescence. Chromosomes were counterstained with propidium iodide and fluoresced red. (A) The total genomic DNA from E. trachycaulus generated a striped GISH pattern on a pair of z5A chromosomes in TA5536. The four yellow-green chromosome segments originated from chromosome 1H^t of E. trachycaulus and the four red segments, including the centromere, originated from wheat chromosome 5A. (B) Clone pAet6-J9 hybridized strongly to all 42 wheat centromeres in line DA1H^t, but very weakly to the centromeres of chromosomes 1H^t (arrowheads). (C and D) GISH on the meiotic metaphase I cells in pollen mother cells from anthers of F₁ plants of dDt5A/TA5536 with the chromosome constitution 20″ + z5A′ + 5AS′ + 5AL′. Chromosome z5A was univalent (arrowhead) in C and was paired in the distal region of the long arm with the 5AL telosome (arrowhead) in the form of a rod bivalent in D. (E) (Middle) Three FISH sites on chromosome z5A probed with clone pAet6-J9; (left) The sequential GISH result on the same chromosome; and (right) a merged image from the first left and middle images. The chromosome, the GISH signals, and the FISH signals were pseudocolored blue, green, and magenta, respectively. The merged image shows that the top FISH site mapped to the centromeric wheat segment (W1), the second site mapped to the second wheat segment (between W2 and W3), and the third site mapped to the distal region of the last Elymus segment (E4) (arrowhead). (F) GISH pattern on z5A chromosomes and three recombinant chromosomes derived from either selfed or backcrossed derivatives of dDt5A/TA5536. The chromosomes rec1, rec2, and rec3 had the first three (E1, E2, E3), first two (E1, E2), and the last Elymus segment(s) (E4), respectively. Rec3 is a telosomic chromosome. (G) tz5AL and iz5AL show a telosomic chromosome and an isochromosome, respectively; T1 shows a translocation chromosome with the short arm from an unknown wheat chromosome and the long arm from z5AL; T2 (GISH) shows a translocation chromosome with three Elymus bands (one in the short arm and two in the long arm). T2 (pAet6-J9) shows a sequential FISH pattern using clone pAet6-J9 labeled with rhodamine-6-dUTP and visualized by red fluorescence, identifying T2 as a small metacentric chromosome, with the GISH band in the short arm derived from the last Elymus segment of z5A and the two in the long arm corresponding to the middle two Elymus segments of z5A. T3 shows a translocation chromosome with five Elymus bands, two in the short arm corresponding to the last two Elymus segments of z5A, and the long arm of T3 is identical to the long arm of z5A. Bars, 10 μm.

Proposed origin of chromosome z5A by chromosome breakage and fusion of the broken chromatin blocks. (Left) Wheat chromosome 5A, (middle) z5A, and (right) telosome 1H^tS, from which the four Elymus segments present in z5A were derived. The centromeres of wheat and Elymus are shown in red and blue, respectively. The original centromere of wheat chromosome 5A was split into two portions in z5A. The major part is the functional centromere, located within the W1 segment, and the smaller part is located in W2. The third and smallest centromere was derived from part of the centromere of chromosome 1H^t (E4). Marker analysis indicated that the entire short arm of 1H^t was not present in z5A. GISH and meiotic pairing analyses showed that the E1 segment was derived from the distal region of 1H^tS and W4 was derived from the distal region of 5AL. Because the marker order in segments E1 and E4 is conserved relative to wheat, but reversed in segments E2 and E3, either a paracentric inversion in the short arm preexisted in 1H^t or fusion of the broken chromatin blocks occurred, as indicated.

Schematic summary of Southern hybridization results and the molecular marker positions on the z5A chromosome. (Top) Schematics for telosomic chromosomes 1H^tS and 1H^tL, z5A, three recombinant chromosomes (rec1–rec3), and isochromosome iz5AL. A scaled-up z5A chromosome is shown on the right. The Elymus short-arm chromatin is in black, the Elymus long-arm chromatin is in gray, and wheat chromatin is in white. The Elymus centromere is shown in blue and the wheat centromere in red. A schematic of the consensus map of wheat group 1 chromosome is on the left. Only tested markers are listed. The “+” and “−” indicate “presence” and “absence” of markers, respectively. The marker order in Elymus segments E1 and E4 is conserved relative to wheat, whereas the order is reversed in segments E2 and E3. None of the group 1 long-arm markers hybridized to z5A.

Hypothetical scheme leading to changes in chromosome number after interspecific hybridization, whole-genome duplication, and chromosomal rearrangements. Species A and B with n = 2 chromosomes have differentiated cytoplasms (indicated by open and shaded outer circles) and nuclear genomes. Species A has a fertility factor F1 on chromosome A1 that is essential for restoring fertility to species-A-specific cytoplasm and species B has another fertility factor, F2 on chromosome B2, that is essential for gametophytic or sporophytic viability. Interspecific hybridization between species A and B followed by whole-genome duplication created genomic stress that resulted in chromosomal repatterning and the formation of the novel chromosome Z3 by nonhomologous recombination, harboring both fertility factors F1 and F2. Plants with such a chromosomal constitution have strong selective advantage and will form species D with n = 3 chromosomes.