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Mapping Maize Knobs Using Cytogenetic Techniques
Leslie R. Nelson1,2, Danielle N. Charley2,3, Takeo Angel Kato-Yamakake4, & Carolyn J.
Lawrence2,5
Knobs are highly condensed, heterochromatic structures on chromosomes that are best
observed in the pachytene stage of meiosis. In maize (Zea mays ssp. mays), knobs occupy
defined regions of each chromosome arm. Presence or absence of knobs in these regions among
various wild and cultivated accessions of maize and its close relatives has been determined and
represents one measure of diversity within the genus Zea. In addition, knobs have been used as
markers for various genetic and cytogenetic analyses. Here we aim to precisely map and analyze
maize knob positions as a part of a larger study to determine whether all knobs occupying the
same region of a given chromosome arm are related by descent or knob-occupying chromosomal
regions are permissive to knob formation from existing knob-associated repetitive elements
present throughout the genome.
Background:
Knobs are a region area where numerous organized 180 base pair (bp) and 350 bp repeats
are formed on a chromosome arm (Peacock 1981). According to McClintock, knobs are wellknown features of maize chromosomes. Huge blocks of heterochromatin create the knobs
(McClintock 1930). The location and size of the knobs vary among lines, see Figure 1
(Yamakake 1975 and McClintock, Barbara, T.A. Kato, and A. Blumenschein also reviewed in
1
University of New Mexico, Albuquerque, NM 87111 USA
Department of Genetics, Development & Cell Biology, Iowa State University, Ames, IA 50011 USA
3
Northern Arizona University, Flagstaff, 86001 AZ USA
4
Colegio Postgraduados Colpos, MEXICO
5
Corn Insects & Corn Genetics Research Unit, USDA-ARS, Ames, IA 50011 USA
2
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Kato 2004). Using fluorescence in-situ hybridization (FISH) and extended exposure times,
numerous knob repeats (also called 180 bp repeats) were shown to appear at the ends of every
maize chromsome as well as along the lengths of the chromosomes (Lamb, Meyer, Corcoran,
Kato, Han & Birchler 2007). Where positions of chromosomal elements are detectible in the
pachytene stage of meiosis, cytological maps are created (e.g., Koumbrais & Bass 2003 and
Amarillo & Bass 2004). Pachytene is an early stage of meiosis where chromatin has begun to
condense, but the chromosomes remain enough extended to visualize large structures, like knobs
and smaller heterochromatic regions called chromomeres. It is unclear if all knobs present in the
same (or approximately the same) chromosomal location across various cultivars of maize are
related by descent or the knobs were built up over evolutionary time from the knob repeats that
occur throughout the genome within regions permissive to their existence. Cytological
measurements by numerous researchers regarding knob locations have been performed over the
years, but the mechanism by which knobs in similar locations arose remains unclear. Data
recently collected by YTA Kato and analyzed as a part of this work (see Appendix I) show that
knob locations vary slightly, but it is not clear whether the actual locations vary slightly or the
preparation technique introduces error and the difference among locations of knobs in the same
region are an artifact. This work aims to determine whether knobs occupying approximately the
same chromosomal locations are homologous (i.e., related by descent) or represent convergence
on the same, permissive regions of the various chromosomes across maize lines.
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Figure 1. Idiogram of pachytene chromosomes of maize showing knob positions and different
chromosome segments with their relative lengths.
Methods and Materials:
L.R.N. participated in an intense week long Cytogenetic Workshop led by David F.
Weber and Thomas Peterson before the research component of the eight-week GWC research
internship began. Once that training was complete, solutions of acetocarmine and propiocarmine
were prepared in the lab as described by Kato (Yamakake 1975) and Weber (personal
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communication). Seed stocks used in the research were grown by the North Central Regional
Plant Introduction Station. Materials used to collect data included Cudu (also called Cudei) PI
222285 lot 85ncab01, Mandan Black PI 213806 lot 80ncai01, Missouri 17 (Mo17) PI 558532 lot
08ncai01, B73 PI 550473 lot n8ncai01, and a hybrid of B73 x Mo17 Ames 19097 lot 04ncai01
SD. Using a razor blade, the stalk was cut longitudinally and tassels were harvested without
sacrificing the plant. The tassels were harvested at an early stage (in the boot). Office tape was
wrapped around the stalk for stability and each plant utilized could later be used as a female.
Tassels were immediately immersed in a solution of 3 parts 95% ethanol to one part glacial
acetic acid. After two days of storage at room temperature, the solution was replaced with 70%
ethanol and samples were stored at 4 degrees C (Yamakake 1975). After extraction the Carmine
Smear Technique was followed (Weber, personal communication).
Spikelets were dissected using number 5 Dumoxel and 55 Dumostar forceps. Once
harvested, the spikelets were returned to 70% ethanol where anthers were dissected out. Pollen
mother cells found inside the anther were extruded and stained using either acetocarmine or
propiocarmine (Weber). A cover slip was placed over the cells and preparations were observed
using a Nikon Labophot microscope. The computer software SPOT was used for analysis.
Chromosome lengths were measured in micros (µm) and were subsequently converted to
centiMcClintock (cCM) using a math conversion that reports the percentage relative distance of
loci from the centromere.
Results:
Many images were taken of the various stages of meiosis. The best cell found was a
Mo17 meiocyte preparation. In this image (Figures 2 and 3) a single chromosome lay away from
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the nucleolus and other chromosomes allowing for identification and measurement of cytological
landmarks. The total length of the chromosome measured 61 µm. The short arm measured 24 µm
and the long arm measured 37 µm. The arm ratio is inferred to be 1.54, which is in keeping with
published arm ratios for maize chromosome 4 (Weber, personal communication). In addition,
chromomeres referred to as “snake eyes” appear toward the end of the short arm and a darker
region on its long arm were observed. Both are diagnostic for chromosome 4 (Weber, personal
communication).
Figure 2. A Mo17 meiocyte stained with acetocarmine. Annotations show the length of
chromosome 4. The large ball to the left of the measured chromosome is the nucleolus. All
other chromosomes are together above the to the left of the nucleolus.
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Figure 3. The same Mo17 meiocyte stained with acetocarmine. Labelled in the image are
chromosome 4’s centromere, the “snake eyes” chromomeres on the short arm, and the darker
region on the long arm.
Discussion:
Of the many meiocytes observed, one had a chromosome that could be recognized as
chromosome 4. As shown in Figure 4, no knobs are expected to occur on chromosome 4 of mo17
(i.e., there are no blue regions on chromosome 4 when the 180 bp knob repeat is labeled
fluorescently). As shown in Figure 4, in Mo17, only chromosome 8 is shows knob repeat
labeling (Kato, A; Lamb, JC; Birchler, J.2004). Nonetheless, in this work chromosome 4’s
centromere, a dark region on the long arm, and the “snake eye” chromomeres were located.
Because in Figure 4 intense red labeling appears on the short arm of chromosome 4, we
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hypothesize that the “snake eyes” may represent either the microsatellite 1-26-2 clone and a
pMTY9ER telomere-associated sequence used as a red probe in that preparation.
To proceed with this work, more data should be collected including repetitive
measurements of cytological locations of knobs within the same germplasm to determine to what
degree preparation techniques introduce error into cytological positions. Next, the degree of
error within a line would be compared to the variations in cytological positions of knobs across
diverse materials. If the error for position within a material is statistically significantly lower
within a line than between lines, we would infer that there is a region of the genome permissive
to knob occurrence (knobs are a convergent phenomenon). Alternatively, if the range of knob
locations is similar both within and between lines, the question of descent versus convergent
evolution would remain unanswered by these analyses.
Figure 4. Somatic chromosome karotyping of B73 and Mo17 probed with the FISH mixture
described by Kato et al., 2004. Knob 180-bp repeat (blue), 5S 2-3-3 (yellow, 2L), NOR-173 clone
(green, 6S), CentC (green), subtelomeric 4-12-1 clone (green), Cent4 (red, 4C), microsatellite 126-2 clone (red), pMTY9ER telomere-associated sequence (red), and TR-1 (white). Scale bar=
10 µm. B73 and Mo17 stocks were obtained by Jim Birchler from Ed Coe.
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Acknowledgements:
•
We thank Dave Weber, Thomas Peterson, Trent Moore, Mark Millard, and the NCRPIS
maize crew for help with this work as well as the Iowa State University George
Washington Carver Research Internship program personnel for their support. This work
is funded by the National Science Foundation and USDA-ARS.
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References:
"Meiosis." 26 July 2010.
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/Meiosis.html. (24 Feb. 2010)
Amarillo, FE, and H, Bass. “Two new loci on the Cytogenetic FISH 9 map”. MaizeGDB (2004)
Creighton HB and B McClintock B A Correlation of Cytological and Genetical Crossing-Over in
Zea mays. Proc Natl Acad Sci 17. (1931) 492-497
Kato, A, JC Lamb, and JA Birchler. "Chromosomes painting using repetitive DNA sequences as
probes for somatic chromosomes identification in maize." Proc Natl Acad Sci 101.
(2004): 13554-13559.
Koumbaris, G and H Bass. “A new single-locus cytogenetic mapping system for maize:
overcoming FISH detections limits with marker-selected sorghum BAC clones.” Plant J
35. (2003) 647-659.
Lamb, JC, JM Meyer, B Corcoran, A Kato, F Han, and JA Birchler. "Distinct chromosomal
distributions of highly repetitive sequences in maize." (2007): 34-49
McClintock, Barbara, T.A. Kato, and A. Blumenschein . Chromosome Constitution of Races of
Maize. Chapingo: Colegio De Postgraduados, 1981.
Peacock, WJ, ES Dennis, MM Rhoads, and AJ Pryor. "Highly repeated DNA sequence limited to
knob heterochromatin in maize." Proc Natl Acad Sci 78. (1981): 4490-4494.
Weber, D.F. Personal communication. 07 June 2010
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Yamakake, Takeo A. "Ctyological Studies of Maize and Teosinte in Relation to their Origin and
Evolution." (1975): 75-76.
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Appendix I