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Fluorescence In Situ Hybridization Analysis Reveals Multiple Loci of Knob-associated DNA Elements in One-knob and Knobless Maize Lines

Sami S.M. Adawy, Robert M. Stupar and Jiming Jiang

Department of Horticulture, University of Wisconsin-Madison, Madison, Wisconsin (SSMA,RMS,JJ), and Agriculture Genetic Engineering Research Institute, Agricultural Research Center, Giza 12619, Egypt (SSMA)

Correspondence to: Jiming Jiang, Department of Horticulture, University of Wisconsin-Madison, Madison, WI 53706. E-mail: jjiang1{at}wisc.edu


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Fluorescence in situ hybridization analyses were conducted to examine the presence or absence of the 180- and 350-bp knob-associated tandem repeats in maize strains previously defined as "one-knob" or "knobless." Multiple loci were found to hybridize to these two repeats in all maize lines analyzed. Our results show that the number of 180- and 350-bp repeat loci do not correlate with the number of knobs in maize and that these tandem repeats are not independently sufficient to confer knob heterochromatin, even when present at megabase sizes. (J Histochem Cytochem 52:1113–1116, 2004)

Key Words: fluorescence in situ • hybridization • fiber-fluorescence in situ • hybridization • tandem repeat • knob • heterochromatin

KNOBS, first described by McClintock (1929)Go in maize, are heavily stained heterochromatic features that are often used as cytological landmarks in genetic and cytogenetic research. The discovery of the knob structures in maize spawned several decades of research focusing on these cytological features. Important biological and genetic characteristics, such as flowering time, recombination frequency, and meiotic drive, have been correlated with the presence of knob loci in maize (Rhoades 1978Go; Buckler et al. 1999Go). Several researchers have surveyed the distribution of knobs among different maize strains using cytological analysis of meiotic pachytene chromosomes (Brown 1949Go; Ibrahim 1960Go; McClintock et al. 1981Go; Crughtai and Steffensen 1987Go). Maize knobs have been found to be highly polymorphic in both size and number across different strains, including some maize strains in which knobs are absent, a condition termed "knobless."

Two families of tandemly repeated DNA sequences have been identified within the maize knobs. Peacock et al. (1981)Go showed that a 180-bp tandem repeat is the main component in maize knobs. Ananiev et al. (1998a)Go more recently reported a 350-bp tandem repeat, TR-1, that is also associated with knobs. A correlation between the size of the knob and the copy number of the 180-bp repeat was previously demonstrated by radioactive DNA in situ hybridization (Dennis and Peacock 1984Go), such that highly repetitive loci associate with large knobs and less repetitive loci associate with smaller knobs. Fluorescence in situ hybridization (FISH) using the 180- and 350-bp repeats in two different maize lines has shown that these sequences may also be located in chromosomal regions without apparent knob formation (Ananiev et al. 1998aGo; Chen et al. 2000Go).

To further investigate the relationship between the locations of tandem repeats and knob formation, we conducted FISH analysis of the 180- and 350-bp repeats in six different maize accessions (Table 1). These accessions are inbred lines or varieties that were previously well characterized as either knobless or having one knob locus based on acetocarmine staining of meiotic pachytene chromosomes (Brown 1949Go; Crughtai and Steffensen 1987Go). Therefore, if the 180- and 350-bp repeats are only located in chromosomal regions associated with knobs, we would expect that these two repeats hybridize to either zero or one locus on the metaphase chromosomes of these maize accessions.


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Table 1

Maize accessions used in the FISH study

 
C103 was classified as a one-knob variety (Crughtai and Steffensen, 1987Go). We observed one pair of chromosomes hybridized with the 350-bp repeat and two pairs of chromosomes hybridized with the 180-bp repeat (Figure 1A). Similarly, Mo17 was also classified as a one-knob line (Crughtai and Steffensen, 1987Go). We observed one pair of chromosomes hybridized with the 350-bp repeat (Figure 1B). Another pair of chromosomes showed a very large hybridization site to the 180-bp repeat. At least two other pairs of chromosomes showed weak hybridization to the 180-bp repeat, although the number of chromosomes with weak hybridization signals was not consistent in different cells.



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Figure 1

FISH and fiber-FISH analyses of the 180- and 350-bp repeats in one-knob and knobless maize lines. The 180-bp repeat is detected by a green color and the 350-bp repeat by a red color. (A) C103. (B) Mo17. (C) Parker's Flint accession PI 255979. (D) Parker's Flint accession Ames 21974. (E) Tama Flint. (F) Wilber's Flint. (G) Two fiber-FISH signals derived from the 350-bp repeat locus of Mo17. (H) Interspersion of fiber-FISH signals derived from the 350-bp repeat (red) and the 180-bp repeat (green). Bars: B,E,F = 5 µm; A,C,D = 10 µm; H = 20 µm; G = 50 µm.

 
Three knobless lines, Parker's Flint, Wilbur's Flint, and Tama Flint, were used in FISH analysis. We analyzed two different accessions of Parker's Flint. Both accessions contain one pair of chromosomes that hybridize to the 350-bp repeat. The 180-bp repeat probe generated several strong hybridization sites as well as a number of weak sites in the two Parker's Flint accessions (Figures 1C and 1D). Both Wilbur's Flint and Tama Flint showed one pair of chromosomes that hybridized to the 350-bp repeat and several chromosomes that hybridized to the180-bp repeat (Figures 1E and 1F).

The single location of the 350-bp repeat was ideal for fiber-FISH analysis. We analyzed the 350-bp locus in C103 and Mo17, and the microscopic sizes of the fiber-FISH signals were converted into kilobases using a 3.21 kb/µm conversion rate (Cheng et al. 2002Go). The 350-bp repeat array was estimated to be 842 ± 191 kb in C103 based on measurements of 15 fibers and 1282 ± 136 kb in Mo17 based on 7 fibers. Figure 1G shows two Mo17 signals located in the same microscopic field. The FISH signals derived from both C103 and Mo17 DNA fibers were largely contiguous, but small gaps with consistent locations were observed along the fibers. This indicates that the 350-bp repeat arrays are interrupted by other DNA sequences, most likely invasion of retrotransposons (Ananiev et al. 1998bGo).

In Wilbur's Flint, the 350-bp locus overlapped with weak FISH signals derived from the 180-bp repeat. Two-color fiber-FISH revealed short, interspersed 180-bp repeat signals within the 350-bp repeat arrays (Figure 1H). Interspersion of these two repeats was also reported in a 12-kb cosmid clone derived from maize chromosome 9 (Ananiev et al. 1998aGo). Short stretches of DNA sequences within the two repeats show some level of homology (Ananiev et al. 1998aGo; Hsu et al. 2003Go). It was suggested that the 350-bp repeat may have evolved from a 180-bp ancestral repeat (Ananiev et al. 1998aGo; Hsu et al. 2003Go). Interspersion of the fiber-FISH signals derived from these two repeats supports a common evolutionary origin for the two repeat families.

Previous studies showed that the 180- and 350-bp repeats are the main components of maize knobs (Peacock et al. 1981Go; Ananiev et al. 1998aGo,bGo). A correlation between knob size and the 180-bp repeat content has previously been documented using in situ hybridization (Dennis and Peacock 1984Go). Chen et al. (2000)Go observed that some maize 180-bp repeat loci do not associate with knob-like heterochromatin. We demonstrate that all four knobless maize accessions display multiple loci containing these two repeats, suggesting that these repeats do not necessarily confer the heterochromatic knob structure. In Mo17, the size of the FISH signals derived from the 350-bp repeat is significantly smaller than the major pair of FISH signals derived from the 180-bp repeat (Figure 1B). We assume that the single knob in this line is associated with the large 180-bp locus. Thus, the 350-bp array, which contains >1 Mb of DNA, is not associated with a distinct knob feature. These results suggest that the presence of high-copy 180- and 350-bp tandem repeats is not sufficient to induce a knob-like heterochromatic structure. The extreme condensation of knob heterochromatin may be conditioned by a combination of tandem repeats and other unknown factors.


    Footnotes
 
Received for publication April 4, 2004; accepted April 7, 2004


    Literature Cited
 Top
 Summary
 Literature Cited
 

Ananiev EV, Phillips RL, Rines HW (1998a) A knob-associated tandem repeat in maize capable of forming fold-back DNA segments: are chromosome knobs megatransposons? Proc Natl Acad Sci USA 95:10785–10790[Abstract/Free Full Text]

Ananiev EV, Phillips RL, Rines HW (1998b) Complex structure of knob DNA on maize chromosome 9: retrotransposon invasion into heterochromatin. Genetics 149:2025–2037[Abstract/Free Full Text]

Brown WL (1949) Numbers and distribution of chromosome knobs in United States maize. Genetics 34:524–536[Free Full Text]

Buckler ES, Phelps-Durr TL, Buckler CSK, Dawe RK, Doebley JF, Holtsford TP (1999) Meiotic drive of chromosomal knobs reshaped the maize genome. Genetics 153:415–426[Abstract/Free Full Text]

Chen CC, Chen CM, Hsu FC, Wang CJ, Yang JT, Kao YY (2000) The pachytene chromosomes of maize as revealed by fluorescence in situ hybridization with repetitive DNA sequences. Theor Appl Genet 101:30–36[CrossRef]

Cheng Z, Buell CR, Wing RA, Jiang J (2002) Resolution of fluorescence in-situ hybridization on rice mitotic prometaphase chromosomes, meiotic pachytene chromosomes and extended DNA fibers. Chromosome Res 10:379–387[CrossRef][Medline]

Crughtai SR, Steffensen DM (1987) Heterochromatic knob composition of commercial inbred lines of maize. Maydica 32:171–187

Dennis ES, Peacock WJ (1984) Knob heterochromatin homology in maize and its relatives. J Mol Evol 20:341–350[Medline]

Hsu FC, Wang CJ, Chen CM, Hu HY, Chen CC (2003) Molecular characterization of a family of tandemly repeated DNA sequences, TR-1, in heterochromatic knobs of maize and its relatives. Genetics 164:1087–1097[Abstract/Free Full Text]

Ibrahim MA (1960) A survey of chromosome knobs in maize varieties. Genetics 45:811–817[Free Full Text]

McClintock B (1929) Chromosome morphology in Zea mays. Science 69:629

McClintock B, Kato Y, Blumenshein A (1981) Chromosome Constitution of Races of Maize. Chapingo, Mexico, Colegio de Postgraduados

Peacock WJ, Dennis ES, Roades MM, Pryor A (1981) Highly repeated DNA sequence limited to knob heterochromatin in maize. Proc Natl Acad Sci USA 78:4490–4494[Abstract]

Rhoades MM (1978) Genetic effects of heterochromatin in maize. In Walden BD, ed. Maize Breeding and Genetics. New York, Wiley and Sons, 641–672





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