Retroelement genome painting: cytological visualization of retroelement expansions in the genera Zea and Tripsacum

Abstract

Divergence of abundant genomic elements among the Zea and Tripsacum genera was examined cytologically and a tool kit established for subsequent studies. The LTR regions from the CRM, Huck, Grande, Prem1, Prem2/Ji, Opie, Cinful-1, and Tekay retroelement families were used as FISH probes on mitotic chromosome spreads from a "trispecies" hybrid containing chromosomes from each of three species: Zea mays (2n = 20), Z. diploperennis (2n = 20), and Tripsacum dactyloides (2n = 36). Except for Tekay, which painted both Zea and Tripsacum chromosomes with nearly equal intensity, the retroelement probes hybridized strongly to the Zea chromosomes, allowing them to be distinguished from those of Tripsacum. Huck and Grande hybridized more intensely to maize than to Z. diploperennis chromosomes. Tripsacum genomic clones containing retroelement sequences were isolated that specifically paint Tripsacum chromosomes. The retroelement paints proved effective for distinguishing different genomes in interspecific hybrids and visualizing alien chromatin from T. dactyloides introgressed into maize lines. Other FISH probes (180-bp knob, TR-1, 5S, NOR, Cent4, CentC, rp1, rp3, and alpha-ZeinA) could be simultaneously visualized with the retroelement probes, emphasizing the value of the retroelement probes for cytogenetic studies of Zea and Tripsacum.

Figure 1.

Hybridization of abundant maize retroelements to Tripsacum and Zea. (A) Chromosomes from a maize × Z. diploperennis hybrid labeled with Opie (red), Huck (green), and the 180-bp knob repeat (white). The chromosomes of Z. diploperennis have small terminal knobs and are weakly labeled by the Opie and Huck elements. (B–F) Chromosomes from a “trispecies” hybrid containing T. dactyloides, Z. diploperennis, and Z. mays genomes are hybridized with pairs of abundant maize retroelements differently labeled in red and green. Chromosomes were counterstained with DAPI. The second row contains gray value images of only the red channel and the third row contains only the green channel with the probe in each case indicated.

Figure 2.

Selection of Tripsacum fosmids for chromosome painting. Ninety-six fosmids were dotted on a nylon membrane and hybridized to (A) maize Cot-100 DNA and to (B) genomic DNA from T. dactyloides. Circled dots represent fosmids that were tested as FISH probes. Fosmids circled in purple did not hybridize strongly to either maize or Tripsacum genomic DNA. Fosmids circled in blue hybridized to Tripsacum but not to maize DNA and red circles indicate fosmids that hybridized to maize but not to Tripsacum DNA. The “trispecies” hybrid chromosome spreads were hybridized with labeled fosmid DNA (red) and with Prem1 (C and D) or Huck (E and F). The fosmid used is indicated and the fosmid hybridization signals alone are presented in gray values below the merged images. TF-E12, although isolated from Tripsacum, hybridizes more strongly to Zea chromosomes. TF-B5, TF-C12, and TF-H7 hybridize more strongly to Tripsacum.

Figure 3.

Selection of small DNA probes that distinguish Zea and Tripsacum genomes. The “trispecies” hybrid chromosomes were hybridized with the labeled clone TC#5 (A) and with TC#25 (B) (red) and Huck (green). A′ and B′ show the gray value images for the TC probes. Genomic DNA from Z. diploperennis (1), T. dactyloides (2), S. bicolor (3), T. laxum (4), Z. mays (5), and Z. luxurians (6) was digested with HindIII, electrophoresed through an agarose gel, blotted to nylon membranes, and hybridized to ³²P-labeled TC#5 (C) or TC#25 (D). C′ and D′ show the ethidium bromide-stained gels.

Figure 4.

Divergence of centromeric elements between Tripsacum and Zea. The “trispecies” hybrid chromosome spreads were hybridized with FITC-labeled genomic DNA from T. dactyloides (green), 50× unlabeled genomic maize DNA, Texas Red-labeled Huck, Grande, Prem1, and maize 180-bp knob repeat (red) (A); CentC (green) and Grande (red) (B); and full-length CRM (green) and Grande (red) (C). A′ shows only the Tripsacum genomic DNA signal.

Figure 5.

Use of abundant retroelements and GISH to paint Tripsacum and Zea chromatin. (A–D) Chromosome preparations were hybridized with Oregon green-labeled Tripsacum genomic DNA (green), 50× unlabeled autoclaved maize genomic DNA, and a cocktail of maize retroelements including Prem1, Huck, Opie, and Grande as well as the 180-bp knob repeat (red). (A′–D′) Chromosomes were hybridized to TC#5, TC#25, TF-B5-3 (red), Huck, Grande, and Prem1 (green). Chromosomes were obtained from a “trispecies” hybrid (A), lines thought to contain an additional T. dactyloides chromosome (B and C), and a reciprocal translocation involving maize chromosome 2 and a Tripsacum chromosome (D). Arrows in B and C indicate the sites of hybridization to Tripsacum probes. (D) The red arrow indicates the T-2 translocation chromosome and the white arrow denotes the 2-T chromosome.

Figure 6.

Hybridization of maize repetitive sequences to “trispecies” hybrid chromosome spreads. The trispecies hybrid chromosome spreads were hybridized simultaneously to a chromosome-painting probe in green, to Huck in A–D and TF-B5-2 in E, and to various repetitive elements. Repetitive probes were (A) “TAGⁿ” oligonucleotide probe (red), (B) 5S ribosomal RNA (red), (C) TR-1 knob (red) and 180-bp knob (blue), (D) Cent4 (red), and (E) 17S ribosomal RNA (red) sequences. The gray value images below A–D show only the red channel from the images above them. E′ shows the NOR signal only and E″ shows the chromosomes stained with DAPI. (A–E) Arrows indicate hybridization signals. (E″) Arrowheads denote the distal DAPI signals that are separated from the remainder of the chromosome because of a partially uncondensed NOR from maize and Z. diploperennis as illustrated in E′. The site of hybridization of the NOR probe on the Tripsacum chromosome was smaller and did not have an uncondensed appearance.

Figure 7.

Hybridization of single-locus probes. The “trispecies” hybrid chromosome spreads were hybridized with probes that detect the maize rp1, rp3, or α-zeinA gene clusters (red) together with TC#5 (green). The gray value images show only the red channel and the arrows indicate hybridization sites.