Simultaneous discrimination of the three genomes in hexaploid wheat by multicolor fluorescence in situ hybridization using total genomic and highly

repeated DNA probes

YASUHIKO M U K A I ' AND YUMIKO NAKAHARA Laboratory of Plant Molecular Genetics, Division of Natural Science, Osaka Kyoiku University,

4-698-1 Asahigaoka, Kashiwara, Osaka 582, Japan

AND

MAKI YAMAMOTO ' Department of Life Science, Kansai Women's Jiozior College, 3-11-1 Asahigaoka, Kashiwara, Osaka 582, Japan

Corresponding Editor: R. Appels

Received February 1 5, 1993

Accepted March 3, 1993

MUKAI, Y., NAKAHARA Y., and YAMAMOTO, M. 1993. Simultaneous discrimination of the three genomes in hexaploid wheat by multicolor fluorescence in situ hybridization using total genomic and highly repeated DNA probes. Genome, 36: 489-494.

Common wheat, Triticum aestivum, is an allohexaploid species consisting of three different genomes (A, B, and D). The three genomes were simultaneously discriminated with different colors. Biotinylated total genomic DNA of the diploid A genome progenitor Triticum urartu, digoxigenin-labeled total genomic DNA of the diploid D genome progenitor Aegilops squarrosa, and nonlabeled total genomic DNA of one of the possible B genome progenitors Ae. speltoides were hybridized in situ to metaphase chromosome spreads of Triticum aestivum cv. Chinese Spring. For detection, only two fluorochromes, fluorescein and rhodamine, were used. The A, B, and D genomes were simultaneously detected by their yellow, brown, and orange fluorescence, respectively. The genomic fluorescence in situ hybridization pattern of chromosome 4A of cv. Chinese Spring wheat showed that the distal 32% of the long arm was derived from a B genome chromosome. Furthermore, by using two highly repeated sequence probes, pSc119.2 and pAsl, and two fluorochromes simultaneously, we were able to identify all B and D genome chromosomes and chromosomes lA, 4A, and 5A of wheat.

Key words: common wheat, in situ hybridization, multicolor fluorescence.

MUKAI, Y., NAKAHARA Y., et YAMAMOTO, M. 1993. Simultaneous discrimination of the three genomes in hexaploid wheat by multicolor fluorescence in situ hybridization using total genomic and highly repeated DNA probes. Genome, 36 : 489-494.

Le ble commun, Triticum aestivum, est une espkce allohexaploi'de comportant trois genomes differents (A, B et D). Les trois genomes ont ete discrimines simultanement par des couleurs differentes. L'ADN ginomique total biotinyli du proginiteur diploi'de du genome A, le Triticum urartu, I'ADN ginomique total marque j. la digoxigenine du proginiteur diploi'de du ginome D, I'Aegilops squarrosa, et I'ADN genomique total non- marque de I'un des progeniteurs possibles du ginome B, I'Ae, speltoicles, ont i t6 hybridis in situ B des etale- ments de chromosomes en mitaphase de Triticurn aestivum cv. Chinese Spring. Pour la ditection de deux fluo- rochromes seulement, la fluoresciine et la rhodamine ont kte utilisees. Les genomes A, B et D ont it6 simultanement ditectes par leur fluorescence respective : jaune, brun et oranger. La fluorescence ginomique du pro- fil d'hybridation in situ du chromosome 4A du ble Chinese Spring a montrk que 32% de la portion distale du bras long ktaient derives d'un chromosome de ginome B. De plus, en utilisant deux sondes de siquences hautement rkpetitives, les pSc119,2 et pAsl, et deux fluorochromes simultanes, tous les chromosomes des ginomes B et D ont pu etre identifiis ainsi que les chromosomes 1 A, 4A et 5A du ble.

Mots cle's : bli commun, hybridation in situ, fluorescence multicolore. [Traduit par la ridaction]

Introduction Common wheat (Triticum aestivum L., 2n = 42, genome

formula AABBDD) is an allohexaploid comprised of three genetically related genomes (A, B, and D) and originated as a hybrid of emmer wheat (Triticum turgidurn L., 2n = 28, AABB) and Aegilops squarrosa L. (2n = 14, DD). Tetraploid wheat received its A genome from diploid wheat (Triticum urartu Thum., 2n = 14, AA). However, the origin of the B genome is still under discussion. At present, accumulating evidence suggests that Ae. speltoides Tausch. is most likely the B genome donor to polyploid wheat.

' ~ u t h o r to whom all correspondence should be addressed. P r ~ r i ~ e d i n Cariada / I rnpr~rnC nu Cnriada

Fluorescence in situ hybridization (FISH) is a powerful tool for chromosome mapping and for analyzing genome organization and evolution. In the field of plant molecu- lar cytogenetics, this technique was used to detect parental genomes in hybrids (Schwarzacher et al. 1989; Anamthawat-Jonsson et al. 1990; Leitch et al. 1990) or in allopolyploids (Bennett et al. 1992). Furthermore, FISH was used to detect alien segments in translocations (Heslop-Harrison et al. 1990; Mukai et al. 1993), to visu- alize gene expression (Yamamoto and Mukai 1990), and to determine the chromosomal location of specific sequences (Yamamoto and Mukai 1990; Leitch et al. 1991).

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GENOME, VOL. 36. 1993

FIG. 1. Multicolor in situ hybridization in cv. Chinese Spring wheat with total genomic DNA probes from the diploid prog- enitors. (A) A complete metaphase cell of double ditelo 1A. (B) A prophase nucleus. (C) Portion of a metaphase cell of double ditelo 1A. Arrows indicate telosomes 1AS and IAL. (D) Portion of a chromosome spread. The arrow indicates breakpoint on the long arm of 4A chromosome. Scale bar = 10 p.

Since a variety of probe-labeling procedures are avail- able, simultaneous detection is now possible. Two or more sequences can be detected in the same cell by using fluorochromes of different color (Lichter et al. 1990). Recently, Reid et al. (1992) succeeded in the simultaneous visualization of seven different DNA probes by FISH using combinatorial fluorescence and digital imaging microscopy. The multicolor FISH tech- nique should be especially useful in developing plant molecular cytogenetics. Leitch et al. (1991) demonstrated the simultaneous detection and localization of two highly repeated DNA sequences in rye chromosomes. Here we report simultaneous detection of three genomes of an allohexaploid wheat using multicolor FISH with total genomic probes and highly repeated sequences.

Materials and methods Plant materials and cytological preparations

Triticum aestivum cv. Chinese Spring and double ditelo- somic lines of cv. Chinese Spring were used for FISH experi- ments. Chromosomal preparation for FISH was as described by Mukai et al. (1990).

DNA probes Total genomic DNA was isolated from young plants of

T, urartu, Ae, speltoides, and Ae. squarrosa using the proce- dure described by Rogers and Bendich (1988). Clone pScll9 includes highly repeated sequences from rye, Secale cereale L. (Bedbrook et al. 1980), and consists of three subunits des- ignated 1, 2, and 3 (Mclntyre et al. 1990). Clone pAsl con- tains a 1-kb insert of Ae. squarrosa repeated sequences (Rayburn and Gill 1987). These probes were used for the

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MUKAI ET AL. 49 1

FIG. 2. Fluorescence in situ hybridization with both the rye repetitive DNA probe (pSc119.2) and the Ae. squarrosa repetitive DNA probe (pAsl) in cv. Chinese Spring wheat. (A) Biotinylated pSc119.2 probe was detected with FITC on the prophase cell spread. (B) Digoxigenin-labeled pAsl probe was detected with rhodamine. (C) Simultaneous visualization of two probes. The photograph was taken by double exposures. (D) Simultaneous detection of pSc119.2 and pAsl probes on the metaphase plate. (E) Portion of a chromosome spread of a metaphase plate. Scale bar = 10 pm.

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GENOME, VOL. 36, 1993

FIG. 3. Idiogram of the chromosomes of cv. Chinese Spring wheat showing locations of the pScll9 (black band) and the pAs 1 (hatched band) sequences.

identification of wheat chromosomes (Rayburn and Gill 1985; Rayburn and Gill 1986).

Labeling of DNA probes Total genomic DNA was sheared by passing it several

times through an 18-gauge hypodermic needle. Total genomic DNA of T. urartu was labeled with biotin-16-dUTP (Boehringer Mannheim) and total genomic DNA of Ae. squarrosa was

labeled with digoxigenin- 11-dUTP (Boehringer Mannheim) by nick translation or random primer method. The insert of pScll9 (pSc119.2) and the whole plasmid of pAsl were labeled with biotin- 16-dUTP and digoxigenin- 1 1 -dUTP, respectively, by the random primer method.

Fluorescence in situ hybridization Chromosomal DNA was denatured in 70% formamide - 2X

SSC for 2 rnin at 68°C and dehydrated in an ethanol series at -20°C. For genomic in situ hybridization, the hybridization mixture (100 pL for total volume) consists of 50% formamide, 10% dextran sulfate, 2X SSC, 250ng of biotin-labeled T. urartu genomic DNA, 250 ng of digoxigenin-labeled Ae. squarrosa genomic DNA, 2 ~g of unlabeled sheared Ae. speltoides genomic DNA, and 20 pg of sonicated salmon sperm DNA. For in situ hybridization using repeated sequences the hybridization mixture (100 pL) consists of 50% formamide, 10% dextran sulfate, 2X SSC, 50 ng of biotin- labeled pSc 1 19.2, 200 ng of digoxigenin-labeled pAsl, and 20 pg of sonicated salmon sperm DNA. The mixture was denatured for 10 min at 100°C and immediately quenched in ice for at least 10 min. A 10-CLL aliquot of the mixture was applied to each slide. Hybridization took place overnight in a moist chamber at 37°C. After hybridization, the slides were washed in 2X SSC at room temperature for 5 min, 50% for- mamide - 2X SSC at 37°C for 15 min, 2X SSC at room tem- perature for 15 min, 1X SSC at room temperature for 15 min, and 4X SSC at room temperature for 5 min. The detection of biotin with fluorescein isothiocyanate (FITC) conjugated avidin (Boehringer Mannheim) and digoxigenin with rhodamine- conjugated sheep anti-digoxigenin Fab fragment (Boehringer Mannheim) was made simultaneously. Slides were incubated in 10 pg/mL FITC-conjugated avidin and 20 pg/mL rhodamine- conjugated anti-digoxigenin in detection buffer containing 4X SSC - 1% BSA for 1 h at 37°C. After incubation, the slides were washed in 4X SSC for 10 min, 0.1% Triton X-100 in 4X SSC for 10 min, 4X SSC for 10 min, and 2X SSC for 5 min, all at room temperature. The slides were immediately mounted in antifade solution (90% glycerol and 0.1% para- phenylene diamine) without counterstaining. Slides were examined with a Nikon fluorescence microscope with a B2 (blue) or G (green) excitation filter. Photographs were taken on Kodak EKTAR film ASA 1000.

Results We analyzed the cv. Chinese Spring double ditelosomic

stocks by FISH using total genomic DNA to determine which genome the chromosomes belong. Figures 1A and l C show fluorescent microphotographs of mitotic meta- phases of the double ditelo 1A line of cv. Chinese Spring wheat. The hybridization sites of the A genome probe were detected by yellow fluorescence, while those of the D genome probe were detected by orange fluorescence. The two 1AS and 1AL telosomes clearly fluoresce yel- low. The B genome chromosomes were neither labeled yellow nor orange but appeared faint brown as a result of cross-hybridization of the A and B genome probes. The satellited chromosomes that belong to the B genome were always labeled brown. We were also able to detect the chromosomes of each genome in prophase cells (Fig. 1B).

The genomic FISH pattern of the 4A chromosome is shown in Fig. 1D. The short arm and the proximal 68% of the long arm was labeled yellow, whereas the remain- ing distal 32% of the long arm was labeled brown. The distal part of chromosome 4A was derived from the B genome by translocation. No other translocation was

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MUKAl ET AL. 49 3

found in cv. Chinese Spring wheat by multicolor FISH using total genomic DNA probes.

Figures 2A and 2B show the results of simultaneous in situ hybridization and detection of biotin-labeled pSc 1 19.2 and digoxigenin-labeled pAs 1 to prophase spreads of cv. Chinese Spring wheat. The biotinylated pSc 1 19.2 was detected with avidin-FITC (B2 excitation filter, 450-490 nm; emission filter, 520 nm) (Fig. 2A), while the digoxigenin-labeled pAsl was detected with anti-digoxigenin antibody coupled to rhodamine (G exci- tation filter, 510-560 nm; emission filter, 590 nm) (Fig. 2B). Rhodamine fluorescence was also faintly visi- ble by B2 excitation. As shown in Fig. 2C, both biotin- and digoxigenin-labeled probes are clearly visualized on a single photograph by double exposure.

The order of two repeated sequences on the chromo- somes of cv. Chinese Spring wheat can be determined by using two-color detection of probes labeled with biotin and digoxigenin (Figs. 2D and 2E). In cv. Chinese Spring wheat, the pSc119.2 probe is known to hybridize to I1 chromosomes (4A, 2D, 3D, 5D, and all seven B genome chromosomes; Rayburn and Gill 1985). The present FISH study using pSc119.2 as a probe revealed signals on 13 chromosomes in addition to 5A and 4D. Since all B genome chromosomes were observed to have multiple pSc 1 19.2 sites, individual identification is possi- ble. Rayburn and Gill (1987) reported that the pAsl probe preferentially labeled D genome chromosomes. The heavily red-fluoresced chromosomes belong to the D genome (Fig. 2E). Smaller sites of hybridizations can also be observed in some A and B genome chromosomes, i.e., lA, 4A, l B , 3B, 6B, and 7B. Simultaneous visual- ization of hybridization signals provides the precise posi- tion and order of these sequences, as shown in Fig. 3. By using pSc 119.2 and pAsl as probes and two fluoro- chromes we were able to identify 17 of the 21 chromo- some pairs of wheat.

Discussion This is the first report of simultaneous visualization of

three genomes on metaphase spread of hexaploid wheat by multicolor FISH using total genomic DNA as probes. The A, B, and D genome chromosomes of common wheat fluoresced yellow, brown, and orange, respec- tively. The preferential hybridization of the T urartu genomic probe to the A genome of hexaploid wheat and that of the Ae. squarrosa probe to the D genome indi- cated that their genomic integrity has been substantially retained in the allohexaploid species.

Bennett et al. (1992), by using genomic in situ hybridization (GISH), demonstrated the allopolyploid origin of Milium montianum (2n = 22) and the homology between eight large chromosomes of this species and M. vernale (2n = 8). Moreover, GISH in combination with multicolor FISH is a powerful tool for investigating genome homology between polyploid species and their diploid progenitors.

In the present study one translocation between differ- ent genomes was detected. In hexaploid wheat, a cyclical translocation involving chromosome arms 4AL, 5AL, and 7BS was proposed by Naranjo et al. (1987) based on meiotic pairing analysis. Liu et al. (1992) further con-

these arms by isozyme and RFLP analysis. There is much evidence showing the existence of a segment derived from the original 7BS on the present 4AL arm (Kobrehel and Feillet 1975; Benito and Perez de la Vega 1979; Naranjo et al. 1987; Chao et al. 1989). However, the size of the translocated segment and its breakpoint has not been determined. Recently, based on the cytologi- cally based physical maps of molecular markers, Werner et al. (1992) demonstrated that at least 20% of the distal region of 4AL is derived from the 7BS terminus. Multicolor FISH analysis clearly showed that the distal 32% of the 4AL arm of cv. Chinese Spring wheat was derived from a B genome chromosome. The long arm of chromosome 4A of cv. Chinese Spring has a size of 7.5 pm (Gill et al. 1991). Thus the 7BS segment trans- ferred to 4AL has a size of about 2.4 pm.

Visualizing multiple targets simultaneously in a single hybridization experiment is of major importance for the physical mapping of genes along a plant chromosome (Leitch et al. 1991). The present study showed that using the two repeated sequences pSc 1 19.2 and pAs 1, we were able to identify 17 chromosome pairs of hexaploid wheat. These sites are also very useful as cytological landmarks. By using these clones for chromosome identi- fication, another sequence labeled by a different reporter molecule can be allocated to a single metaphase plate without further chromosome identification such as band- ing analysis.

Multicolor FISH also allows the development of more detailed maps of specific chromosome regions. The number of probes on a particular chromosome of interest could be ordered along a single chromosome. The combination experiment where two probes are detected by the use of two fluorophores allows us to confirm map order for all probes.

Acknowledgements This work was supported in part by a Grant-in-Aid for

Scientific Research (No. 04305009) from the Ministry of Education, Science and Culture, Japan, and a grant from Sapporo Bioscience Foundation, Japan.

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