Knobs are highly condensed, heterochromatic structures on chromosomes that are best observed in the pachytene stage of meiosis. In maize (Zea
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
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.
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 well-known 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 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.
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 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.
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 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 1. Idiogram of pachytene chromosomes of maize showing knob positions and different chromosome segments with their relative lengths.
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.
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.
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 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 1-26-2 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.
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.
1. "Meiosis." 26 July 2010. http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/Meiosis.html
. (24 Feb.
2010)
2. Amarillo, FE, and H, Bass. “Two new loci on the Cytogenetic FISH 9 map”. MaizeGDB (2004)
3. 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
4. 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.
5. 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.
6. 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
7. McClintock, Barbara, T.A. Kato, and A. Blumenschein . Chromosome Constitution of Races of Maize. Chapingo:
Colegio De Postgraduados, 1981.
8. 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.
9. Weber, D.F. Personal communication. 07 June 2010
10. Yamakake, Takeo A. "Ctyological Studies of Maize and Teosinte in Relation to their Origin and Evolution." (1975):
75-76.
Table 1 shows both the short & long arm lengths with and without the knobs of Chromosome 4 .