Copyright © 2008 by the Genetics Society of America DOI: 10.1534/genetics.l08.087726
Note
Extraordinary Tertiary Constrictions of Tripsacum dactyloides
Chromosomes: Implications for Karyotype Evolution of
Polyploids Driven by Segmental Chromosome Losses
Dai-Hoe Koo and Jiming Jiang1
Department of Horticulture, University of Wisconsin, Madison, Wisconsin 53706
Manuscript received January 31, 2008 Accepted for publication April 2, 2008
ABSTRACT
Tripsacum dactyloides (2n — 2x — 36) is an ancient tetraploid species. Here we report that T. dactyloides chromosomes contain an extraordinary tertiary constriction, which causes a radical and distant separation of a terminal segment from the chromosome. The relationships between extraordinary tertiary constriction and segmental chromosome loss as well as karyotype evolution of polyploid species are discussed.
THE karyotypes of higher eukaryotic species are generally stable. Major karyotypic differences among closely related plant species are often caused by dramatic chromosomal rearrangements. For example, the chromosome number of a species can change due to a chromosomal translocation and loss of a derived chromosome that contains mainly inert genetic material (Darlington 1937). This mechanism was carefully studied using classical cytological methods in the Crepis species (Babcock 1947) and has recently been brilliantly illustrated using a comparative chromosome painting technique in Arabi-dopsis thalianaand its related Brassicaceae species (Lysak et al. 2006).
A polyploidization event and its post-diploidization process can result in dramatic karyotype changes because the loss of a chromosomal segment or even a complete chromosome may not be lethal for a newly formed polyploid plant. Chromosome rearrangements, such as intergenomic translocations, were reported in several classical polyploid species, including tobacco and wheat (Kenton et al. 1993; Jiang and Gill 1994). Maize {Zea mays) provides an excellent example of the complexity of karyotype evolution after a polyploidization event. Maize was derived from an ancient tetraploid (Gaut and Doebley 1997). This tetraploidization event occurred as recently as 4.8 million years ago (Swigonova etal. 2004). On the basis of the most recent physical mapping data, the two progenitor species of maize contained 2n = 20 chromosomes (Wei et al. 2007). It is unclear how the 40
1 Corresponding author: Department of Horticulture, 1575 Linden Dr., University of Wisconsin, Madison, WI 53706. E-mail: jjiangl Owisc.edu
chromosomes in the initial tetraploid have decreased to the current 20 chromosomes of maize, despite the extensive comparative mapping effort on maize and its related cereal species (Ahn and Tanksley 1993; Wilson etal. 1999; Wei et al. 2007).
Tripsacum and Zea are sister genera belonging to the subtribe Maydinae. Tripsacum consists of nine species, including both diploid (2n = 2x = 36) and tetraploid (2n = 4x = 72) species (Tantravahi 1968). Since the tetraploidization event of maize occurred before the divergence of maize and Tripsacum species (Gaut et al. 2000), the diploid Tripsacum species should also represent ancient tetraploids. Thus, karyotypic studies of Tripsacum species may provide clues for the evolutionary history of maize chromosome rearrangements after its tetraploidization event.
Tňpsacum dactyloides (2n = 36) is cytologically recognized as a diploid species. The 36 T dactyloides chromosomes form 18 bivalents in meiosis without meiotic irregularities (Anderson 1944). During the karyotypic analysis of T dactyloides cv. Pete, we often observed >36 chromosomes in somatic metaphase cells prepared from root tips. Metaphase cells with a clear count of 36 chromosomes were surprisingly rare. Most metaphase cells included one or several very small extra chromosomes (Figure la). These small chromosomes appeared to be the satellites associated with the secondary constrictions of nucleolus organizer (NOR) chromosomes, because the satellites can often be distantly separated from the rest of the NOR chromosomes via the secondary constrictions. We performed fluorescence in situ hybridization (FISH) using probe pTa71 that contains a 45S ribosomal RNA gene  (Gerlach and Bedbrook
Genetics 179: 1119-1123 (June 2008)
1120
D.-H. Koo and J. Jiang
Figure 1.—Tertiary constrictions associated with T. dactyloides chromosomes, (a) A somatic metaphase cell of T. dactybides. This metaphase cell contains >36 chromosomes if the 3 small chromosomes (arrows) are counted as independent chromosomes. The chromosomes were stained by 4',6-diamidino-2-phenylindole (DAPI) and pseudo-colored in red. (b) FISH mapping of 45S rDNA (green signals indicated by arrows), (c) Detection of centromere-specific histone CENH3 by immunofluorescence assay. Two chromosomal segments (arrows) are not independent chromosomes because they lack the CENH3 signals, (d) FISH mapping of telomeric DNA probe pAaT4 and the centromeric probe CentC. Two chromosomal segments (arrows) do not contain CentC signals and contain telomeric signals at only one of the two ends. Tertiary constrictions are associated with most chromosomes and some of the them are indicated by arrowheads. FISH and immunofluorescence assay followed published protocols (Jiang et al. 1996; Jin et al. 2004). Bars, 10 |xm.
1979). The 45S rDNA was mapped to the terminal regions of two small chromosomes that were clearly not associated with any satellites (Figure lb).
We wondered if the small chromosomes were B chromosomes since B chromosomes were previously reported in several Tripsacum species (Tantravahi 1968). We performed immunofluorescence assay using an antibody against the centromere-specific histone CENH3. This anti-CENH3 antibody was developed in rice (Nagaki et al. 2004) and recognizes the CENH3 protein associated with both A and B chromosomes in maize (Jin et al. 2005). We did not observe immunofluorescence signals on the small "extra chromosomes" in T. dactyloides, suggesting that these chromosomal segments without CENH3 signals are not independent chromosomes (Figure lc).
The FISH and immunoassay results show that the small chromosomal segments without CENH3 signals were likely separated from chromosomes by tertiary constrictions (TCs). Indeed, TCs were frequently observed on many metaphase chromosomes (Figure Id) and the distance between the two chromosomal segments separated by a TC was highly variable among different chromosomes and different metaphase cells and in different preparations. Some TCs were extraordinary because the connection between the two chromosome segments were hardly recognized in the metaphase cells (Figure Id). Such extraordinary TCs have not been previously reported. We also performed FISH analysis using the telomeric DNA probe pAaT4 (Richards and Ausubel 1988) and the CentC repeat that is specific to the centromeres of both maize (Ananiev etal. 1998) and
Note
1121
Figure 2.—FISH mapping of knob repeat and retroelement TF-B5-3 on the chromosomal segments separated by tertiary constrictions, (a) FISH analysis of probe pTd-8 containing the 180-bp maize knob repeat (pink) and the centromeric repeat CentC (turquoise). A chromosomal segment (arrowhead) is covered almost completely by the knob signals. Two other chromosomal segments (arrows) are not fully covered by the signals from the knob repeat, (b) DAPI staining of a metaphase cell, (c) FISH analysis of knob repeat (green) and retrotransposon probe TF-B5-3 (red). (d) Merged image of b and c. TF-B5-3 signals are visible on two chromosomal segments (arrows) separated by tertiary constrictions. Bars, 10 |xm.
T. dactyloides chromosomes (Lamb and Birchler 2006). Some chromosomal segments did not show CentC signals and contained telomeric signals at only one of the two ends (Figure Id). These results confirmed that these chromosomal segments are not independent chromosomes. To reveal the DNA composition of the chromosomal segments separated by the TCs, we performed FISH analysis using probe pTd-8 containing the 180-bp maize knob repeat cloned from T. dactyloides. A large block of the knob repeat was found either at one end or at both ends of every T. dactyloides chromosome (Figure 2a). The chromosomal segments separated by TCs were always associated with a block of the knob repeat. The 18 dif-
ferent T. dactyloides chromosomes can be grouped into two types on the basis of the location of the knobs: type I with knobs on both arms and type II with a knob only on one arm (Figure 3). The 45S rDNA is located on a pair of the type II chromosomes.
We noted that some of the chromosomal segments separated by TCs were not completely covered by the FISH signals from the knob repeat (Figure 2a), indicating that the knob repeats account for only part of these chromosomal segments. We performed FISH using probe TF-B5-3 that contains a retrotransposon distributed almost uniformly in the T. dactyloides genome (Lamb and Birchler 2006). This probe produced very
1122
D.-H. Koo and J. Jiang
Typel
■   i
Figure 3.—An ideogram of the karyotype and a model of segmental chromosome loss in T. dactyloides. Type II chromosome is the product of a terminal deletion of a type I chromosome. The small arrow points to the deletion breakpoint that is possibly linked to a tertiary constriction.
faint or no signals on the knob regions of T. dactyloides chromosomes (Figure 2c). The FISH results confirmed that most observed chromosomal segments separated by TCs contain additional chromatin in addition to the knob repeats (Figure 2, b-d).
Since TCs were never observed on the short arms of type II chromosomes, it appears that the type II chromosomes evolved from the type I chromosomes by loss of a terminal segment (Figure 3). We propose that type II chromosomes are products of terminal deletions of type I chromosomes and that the breakpoints of such terminal deletions are associated with the extraordinary TCs located on the type I chromosomes (Figure 3). This hypothesis is supported by the fact that the sizes of the short arms of the type II chromosomes match well with the sizes of the arms of type I chromosomes without the terminal segments separated by the TCs (Figure Id and Figure 3). The heterochromatic knobs in T. dactyloides and maize contain the same 180-bp repeat (Lamb and Birchler 2006). It is interesting to note that the large knobs in maize are located mainly in interstitial chromosomal regions (Buckler et al. 1999). Both terminal and interstitial knobs were observed on chromosomes of Zea diploperennis (2n = 20) (Lamb et al. 2007). Chromosomal mapping of knobs in other Zea and Tripsacum species will reveal the impact of loss of terminal knobs on karyotype evolution of both maize and T. dactyloides.
Although the proposed terminal deletions in T. dactyloides can result in loss of the telomeric heterochro-matin as well as subtelomeric euchromatin, the ancient polyploid nature of the T. dactyloides genome may prevent the lethality of such terminal deletions. In addition, all Tripsacum species are perennials with well-developed underground rhizomes that result in asexual propagation (Tantravahi 1968). Asexual propagation would favor retention of chromosomal mutations that may
negatively affect transmission of the chromosome in meiosis but have other selective advantages for the plant. Leitch and Bennett (2004) recently showed that many polyploid species contain less DNA than the combined DNA of the parental species. The loss of DNA, or genome downsizing, following polyploidization may be a widespread phenomenon in nature (Leitch and Bennett 2004). How the genome of a polyploid species becomes downsized is unknown. It may be caused by one or a combination of several documented molecular mechanisms, including elimination of specific DNA sequences (Liu et al. 1998; Ozkan et al. 2001) or retrotransposon-based illegitimate recombinations (Devos et al. 2002). The karyotyping results of T. dactyloides show that segmental chromosome loss is a potential mechanism that can result not only in the downsizing of the genome but also in major changes to the karyotype of a polyploid species.
This  research  was  supported  by  grant DBI-0421671  from  the National Science Foundation.
LITERATURE CITED
Ahn, S., and S. D. Tanksley, 1993 Comparative linkage maps of the rice and maize genomes. Proc. Natl. Acad. Sei. USA 90: 7980-7984.
Ananiev, E. V., R L. Phillips and H. W. Rines, 1998 Chromosome-specific molecular organization of maize (Zea mays L.) centro-meric regions. Proc. Natl. Acad. Sei. USA 95: 13073-13078.
Anderson, E., 1944 Cytological observations on Tripsacum dactyloides. Ann. Mo. Bot. Gard. 31: 317-323.
Babcock, E. B., 1947 The Genus Crepis. University of California Press, Berkeley, CA .
Buckler, E. S., T. L. Phelps-Durr, C S. K. Buckler, R K. Dawe,J. E Doebley et al., 1999 Meiotic drive of chromosomal knobs reshaped the maize genome. Genetics 153: 415—426.
Darlington, C. D., 1937 Recent Advances in Cytology. P. Blakiston's Son & Co., Philadelphia.
Devos, K. M.,J. K. M. Brown and J. L. Bennetzen, 2002 Genome size reduction through illegitimate recombination counteracts genome expansion in Arabidopsis. Genome Res. 12: 1075-1079.
Gaut, B. S., and J. F. Doebley, 1997 DNA sequence evidence for the segmental allotetraploid origin of maize. Proc. Natl. Acad. Sei. USA 94: 6809-6814.
Gaut, B. S., M. L. d'Ennequin, A. S. Peek and M. C. Sawkins, 2000 Maize as a model for the evolution of plant nuclear genomes. Proc. Natl. Acad. Sei. USA 97: 7008-7015.
Gerlach, W. L., and J. R Bedbrook, 1979 Cloning and characterization of ribosomal RNA genes from wheat and barley. Nucleic Acids Res. 7: 1869-1885.
Jiang, J., and B. S. Gill, 1994 Different species-specific chromosome translocations in Triticum timopheevii and T turgidum support diphy-letic origin of polyploid wheats. Chromosome Res. 2: 59-64.
Jiang, J., S. H. Hulbert, B. S. Gill and D. C Ward, 1996 Interphase fluorescence in situ hybridization mapping: a physical mapping strategy for plant species with large complex genomes. Mol. Gen. Genet. 252: 497-502.
Jin, W. W., J. R Melo, K Nagaki, P. B. Talbert, S. Henikoff et al.,
2004    Maize centromeres: organization and functional adaptation in the genetic background of oat. Plant Cell 16: 571-581.
Jin, W. W., J. C. Lamb, J. M. Vega, R K. Da we, J. A. Birchler et al.,
2005    Molecular and functional dissection of the maize B centromere. Plant Cell 17: 1412-1423.
Kenton, A., A. S. Parokonny, Y. Y. Gleba and M. D. Bennett, 1993 Characterization of the Nicotiana tabacum L. genome by molecular cytogenetics. Mol. Gen. Genet. 240: 159-169.
Lamb, J. C, and J. A. Birchler, 2006 Retroelement genome painting: cytological visualization of retroelement expansions in the genera Zea and Tripsacum. Genetics 173: 1007-1021.
Note
1123
Lamb, J. C, J. M. Meyer, B. Corcoran, A. Kato, F. P. Han et al, 2007 Distinct chromosomal distributions of highly repetitive sequences in maize. Chromosome Res. 15: 33-49.
Leitch, I. J., and M. D. Bennett, 2004 Genome downsizing in polyploid plants. Biol. J. Linn. Soc. 82: 651-663.
Liu, B.J. M. Vega, G. Segal, S. Abbo, H. Rodová et al, 1998 Rapid genomic changes in newly synthesized amphiploids of Triticum and Aegilops. I. Changes in low-copy noncoding DNA sequences. Genome 41: 272-277.
Lysak, M. A., A. Berr, A. Pečínka, R. Schmidt, K McBreen et al, 2006 Mechanisms of chromosome number reduction in Arabi-dopsis thaliana and related Brassicaceae species. Proc. Natl. Acad. Sei. USA 103: 5224-5229.
Nagaki, K, Z. K Cheng, S. Ouyang, P. B. Talbert, M. Kim et al, 2004 Sequencing of a rice centromere uncovers active genes. Nat. Genet. 36: 138-145.
Ozkan, H., A. A. Levy and M. Feldman, 2001 Allopolyploidy-induced rapid genome evolution in the wheat (Aegilops-Triticum) group. Plant Cell 13: 1735-1747.
Richards, E. J., and F M. Ausubel, 1988 Isolation of a higher eu-karyotic telomere from Arabidopsis thaliana. Cell 53: 127-136.
Swigonova, Z.J. S. Lai J. X. Ma, W. Ramakrishna, V Llaca et al, 2004 Close split of sorghum and maize genome progenitors. Genome Res. 14: 1916-1923.
Tantravahi, R. V, 1968 Cytology and crossability relationships of Tripsacum. Ph.D. Thesis, Bussey Institute, Harvard University, Cambridge, MA.
Wei, F, E. Coe, W. Nelson, A. K Bharti, F. Engler et al, 2007 Physical and genetic structure of the maize genome reflects its complex evolutionary history. PLoS Genet. 3: 1254-1263.
Wilson, W. A., S. E. Harrington, W. L. Woodman, M. Lee, M. E. Sorrells et al, 1999 Inferences on the genome structure of progenitor maize through comparative analysis of rice, maize and the domesticated panicoids. Genetics 153: 453-473.
Communicating editor: A. H. Paterson