Idiogram of Ricinus communis L.

The Journal of Heredity 69:191-196. 1978.

Idiogram of Ricinus communis L. HARRY S. PARIS, OVED SHIFRISS, AND GOJKO JELENKOVIC

CCORDING to Rieger et al.13, the term idiogram was coined by Navashin who defined it as a diagrammatic representation of chromosome morphology. Originally, idiograms were constructed on the basis of chromosome morphology at metaphase of mitosis 2'7>8>15. McClintock9 demonstrated the usefulness of the pachytene stage of meiosis for the study of chromosome morphology. As a result, idiograms of rye 6 , maize10, tomato12, and barley H are based on pachytene chromosomes. An idiogram of pachytene chromosomes can be presented either as a series of chromomere drawings or as a series of line drawings. The idiograms of rye 6 and barley14 are examples of chromomere drawings. The idiograms of maize10 and tomato12 are examples of line drawings. The castor plant, Ricinus communis L., has a somatic chromosome number of 20'. Jakob3 was the first to report that the ten pachytene bivalents of castor are easily distinguishable from one another. A more accurate description of the pachytene complement was later reported by Jelenkovic and Harrington4. The purpose of this paper is to present a detailed idiogram of castor pachytene chromosomes as well as a more complete description of these chromosomes. The idiogram is of the line drawing type, largely because the zones of heterochromatin and euchromatin are sharply differentiated. The idiogram was constructed for two reasons: first, to compare karyotypes of different races", and second, to correlate possible variations in chromosome morphology with the phenomenon of genetic instability that affects sex expression in this, species16>17.

A

Materials and Methods Plant material Two plants of race A39 were used in this study. A39 was derived from a cross between R7, a sex-reversal mutant of cultivar Gamadon, and M3, an inbred of cultivar U.S.3/415-9 (O. Shifriss, unpublished monograph). The two plants did not differ phenotypically, nor did they differ in their pachytene chromosomes. Both produced

an abundance of large male flower buds, making them very suitable for pachytene analysis. Jelenkovic and Harrington4 used another plant, temporarily designated 715-2, of this race in their pachytene study. Cytological techniques Male flower buds, usually collected late in the morning, were fixed in an ethanol: propionic acid 2:1 solution. The vials containing the buds and fixative were immersed in hot water (close to boiling) to speed up the fixation process, which usually lasted about 1.5 hours. The fixative was changed several times during this period. Fixation was judged to be complete when the calyx had become colorless. Buds were stored in fixative at 0°F. After fixation, the buds were placed in tap water in which cellulysin (Calbiochem, San Diego, California) and pectinase (ICN Life Sciences, Nutritional Biochemicals Division, Cleveland, Ohio) had been dissolved. The buds were kept in the enzyme solution at room temperature for 2 days at which time they were either used or placed in tap water and stored in a refrigerator up to 2 weeks. Temporary slides were made from buds that contained pollen mother cells in the pachytene stage. A small piece of each bud was smeared in several drops of propionic carmine and a coverslip was then applied. After 10-20 minutes of staining, the slide was gently heated several times over an alcohol burner to destain the cells. The specimen was then placed between several sheets of bibulous paper and squashed, using thumb pressure, then sealed with wax. Cells were observed under oil in Leitz Ortholux (lOOx/1.30 N.A.) and Zeiss Universal (100x/1.25 N.A.) microscopes equipped with phase contrast optics and photometric equipment. For photography, 4 x 5 inch Kodak Plus-X film was processed with Kodak D76 Developer and Kodak Fixer. Prints were made from the negatives on a Beseler enlarger and processed with Kodak Dektol, Kodak Fixer, and F-5 Kodabromide or Luminos Resin Coated 4 Paper. Chromosomes were measured from the prints using a Keuffel and Esser Map Measure. Idiogram construction

The senior author is a former graduate student and the junior authors are members of the Department of Horticulture and forestry, Cook College, Rutgers—The State University, New Brunswick, New Jersey 08903. Paper of the Journal Series, New Jersey Agricultural Experiment Station. Address reprint requests to Dr. Shifriss.

Measurements of chromosome arm lengths were obtained from 16 cells in which all chromosomes could be followed from end to end and the positions of all centromeres were discernable. Centromeres were not included in total length. Measurements of lengths and positions of chromosome landmarks were obtained not

191

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FIGURE 1 —Idiogram of the ten pachytene bivalents of race A39. Lengths of chromosome arms were obtained from the data presented in Table I. Centromeres are lined up by arrow. Long arms of chromosomes are to the left, short arms to the right. Thin lines represent euchromatic regions, thickened portions of lines represent heterochromatic regions, and spaces in the lines represent constrictions. Only one of the two satellites of chromosome 2 is represented. See text.

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Paris et al.: Idiogram of the castor plant

193

only from chromosomes of these 16 cells but also from chromosomes of other cells, because individual chromosome landmarks were not visible in every cell. Lengths and positions of all chromosome landmarks are based on 10-15 samples of each. The lengths and positions of heterochromatic regions and constrictions other than centromeres are delineated by numbers. These numbers designate relative positions on each chromosome arm, from immediately adjacent to the centromere, 0.00, to the end of the arm, 1.00. Thicknesses of heterochromatic regions are relative estimates; they were not measured.

G H

Results The idiogram is presented in Figure 1. Data on mean length, rank in mean length, relative length, and arm ratio of the chromosomes are presented in Table I. The chromosomes are illustrated individually in Figure 2. Figure 3 illustrates a microsporocyte in which the pachytene chromosomes are relatively extended. Figure 4 illustrates a microsporocyte in which the pachytene chromosomes are relatively contracted. In both cells all bivalents are easily followed and distinguished from each other. The individual chromosomes are described as follows. Chromosome I is the longest and least heterochromatic chromosome of the complement. The two arms are of almost identical length. The arm containing two chromomeres adjacent to the centromere also contains a small chromomere approximately two thirds of its length from the centromere. This small chromomere is occasionally subdivided into two smaller chromomeres when the chromosome is not too contracted. Chromosome 2 is the main nucleolar organizer of race A39. The long arm contains one large and one small heterochromatic segment in the first one-fifth of its distance from the centromere. The short arm consists of five heterochromatic segments, the second from the centromere being distinctly the longest and thickest. The first and third segments are occasionally subdivided when the chromosome is not too contracted. Thefifth(terminal)

B' FIGURE 2—Photomicrographs and interpretive drawings of the ten pachytene bivalents. Chromosome 1—A, A'; chromosome 2—B,B'; chromosome 3—C,C; chromosome 4—D,D'; chromosome 5—E,E'; chromosome 6—F,F'; chromosome 7—G,G'i chromosome 8—H,H'; chromosome 9 — / , / ' ; chromosome 1 0 — / , / ' ; (X3000).

segment is separated from the fourth segment by a secondary constriction and is unpaired (seen as two satellites). The nucleolus is associated with this constriction. Another constriction separates the third segment from the fourth. Chromosome 3 is recognized by the pattern of distribution of heterochromatin in the proximal regions of its

Table I. Pachytene chromosome lengths in Ricinus communis, race A39 Relative length in percentage of Mean length and standard error, in microns Chromosome 1 2

2 4

5 6

1 * 9 10

long arm 15.23 16.62 13.34 13.46 10.81 13.59 16.20 16.03 10.70 9.02

± ± ± ± ± ± ± ± ± ±

1.03 1.50 0.86 0.81 0.83 1.04 1.16 1.12 0.70 0.52

short arm 15.15 ± 5.92 :t 9.28 :t 9 . 5 6 :t 10.30 :t 5.87 :t 3 . 9 6 :t 6.98 :t 6.16 dt 7.58 ±

0.85 0.43 0.88 0.66 0.87 0.24 0.19 0.32 0.36 0.40

phrnmfl-

total

Rank in rnc&n length

longest some

total length of all chromosomes

Arm ratio

30.38 i 1.81 22.54 : 1.84 22.62 d 1.70 23.02 i 1.44 21.11 i 1.29 19.46 i 1.22 20.16 i 1.32 23.01 it 1.38 16.86 d: 1.02 16.60 ± 0.82

I V IV II VI VIII VII III IX X

100 74 "74 76 «9 64 66 76 55 55

14.08 10.45 10.48 10.67 9.78 9.02 9.34 10.66 7.81 7.69

1.01 2.81 1.44 1.41 1.05 2.32 4.09 2.30 1.74 1.19

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The Journal of Heredity

arms. In the long arm, the heterochromatic region is often divided into three segments. Beyond this region lie two minute chromomeres and a tertiary constriction. In the short arm, the heterochromatic region is divided into three segments, the one immediately adjacent to the centromere being the longest. The proximal heterochromatic region of the short arm extends through a longer proportion of the arm than does its counterpart in the long arm. The short arm also contains a chromomere near its distal end. Chromosome 4 is the second longest chromosome of the complement. It is also the second least heterochromatic. The heterochromatic regions have distinctly less thickness than those of chromosome 3. The proximal heterochromatic region of the long arm is usually subdivided into two segments of equal size. In addition, a small chromomere is often found nearby. Chromosome 5 has two arms of almost identical length. The proximal heterochromatic region adjacent to the centromere in the long arm is divided into three segments, the middle one being the smallest. The proximal heterochromatic region adjacent to the centromere in the short arm is divided into two segments of equal size. A prominent chromomere is located near these segments. The two arms of chromosome 5 can be distinguished by identifying one or both of these patterns of distribution of heterochromatin. Chromosome 6 is one of the most distinctive chromosomes of the complement. The long arm has one large chromomere flanking the centromere and one smaller chromomere proximal to a tertiary constriction which is located about one-third of the way from the centromere. The short arm contains the thickest heterochromatic block of the entire complement. A euchromatic region separates this block from a triangular-shaped heterochromatic region at the distal end. Chromosome 7 occasionally participates along with chromosome 2 in organizing the nucleolus of A39. It is the only chromosome in which the centromere is difficult to distinguish; it is also the only one in which the centromere is not immediately flanked by heterochromatin on both sides. The long arm contains a large heterochromatic segment about one-eight of the distance from the centromere. The short arm consists of five heterochromatic segments which appear to increase in size gradually from the centromere. The nucleolar organizing region is located at or near the distal end of this arm. Chromosome 8 is almost equal to chromosome 4 in total length. The long arm has a chromomere adjacent to the centromere plus two very small ones a short distance away. These two chromomeres appear as a large chromomere when the chromosome is highly contracted. The short arm contains six separate segments of heterochromatin and a tertiary constriction. Chromosome 9 and chromosome 10 (see below) are the shortest chromosomes of the complement. The long arm contains three small chromomeres, including one flanking the centromere. The short arm contains two heterochromatic blocks close to the centromere and four tertiary constrictions. Chromosome 10 has more heterochromatin in the long arm than in the short arm. In the long arm, the hetero-

10

FIGURE 3—Photomicrograph and interpretive drawing of microsporocyte with extended, relatively uncontracted pachytene chromosomes (X3000).

chromatic region adjacent to the centromere is composed of four segments, the two central ones being the thickest. A small chromomere is often seen nearby and is followed by a very prominent tertiary constriction. In the short arm, the heterochromatic region adjacent to the centromere is usually subdivided into two segments, the proximal being the thicker of the two. A distinct chromomere is located nearby. This chromosome probably contains other chromomeres and constrictions whose positions are not defined here.

Discussion The method of depicting the pachytene chromosomes of castor is similar to that used in maize10 and tomato12. Unlike the maize and tomato idiograms, however, the positions and lengths of all chromosome landmarks have been quantified in castor. Quantification may prove useful

Paris et al.: Idiogram of the castor plant by similar rankings obtained in three other races". The largest discrepancy in ranking between the previous report and this one is in chromosome 8. This discrepancy was probably caused by initial confusion of this chromosome with chromosome 9. Arm ratios Based on the nomenclature of Levan et al. 5 , there are five metacentric (m), four submetacentric (sm), and one subterminal (st) chromosomes in the castor complement. The ratio m:sm:st reported earlier was 5:2:3, due to slightly higher arm ratios of chromosomes 6 and 8. The largest discrepancy in arm ratio between the two reports is in chromosome 1. The ratio reported earlier was 1.27; we have found it to be 1.01. It is difficult to account for this difference. Data for arm length of chromosome 1 in three other races of castor showed that the arm ratio was approximately 1.0 in these races11. Nucleolar organizers Three nucleolar organizing chromosomes, 2, 7, and 9, were reported earlier. We were not able to confirm that chromosome 9 is a nucleolar organizer. As viewed at pachytene, the short arms of chromosomes 2 and 7 are the only parts of the genome that contain nucleolar organizing regions. Landmarks

to FIGURE 4—Photomicrograph and interpretive drawing of microsporocyte with short, highly contracted pachytene chromosomes (x3000).

in studies of some cryptic changes in chromosome morphology. Differences between the previous report on castor pachytene chromosomes4 and this one are summarized as follows. Chromosome length The two reports differ in ranking of the chromosomes. We agree that chromosome 1 is the longest chromosome and that chromosome 9 and chromosome 10 are the shortest chromosomes. However, the ranking of chromosomes 2 through 8 is different. There are two reasons for this. First, the lengths of these seven chromosomes are within 4 /xm of one another. Second, as can be seen by comparing Figures 3 and 4, great differences in chromosome lengths exist between cells. In order to avoid unfairly weighing one chromosome against another, the data on chromosome length presented here were obtained only from cells in which all chromosomes could be measured. This precaution was not taken in the earlier report. The rank in mean length presented here is supported

New landmarks are reported here for most of the chromosomes. One of the major findings is that the short arm of chromosome 2 can be resolved into five easily recognizable heterochromatic segments. Based on new landmarks, it is now much easier to distinguish chromosomes 3, 4, and 5 from one another. Perhaps one of the most unusual of the new landmarks is the long tertiary constriction in the long arm of chromosome 10. There may still be more chromosome landmarks that have not been described. None of these, however, are any larger than the smallest ones presented in the idiogram. These very small landmarks may be of little or no value for distinguishing the chromosomes or detecting slight changes in chromosome morphology because the smaller any of these landmarks is, the less often it will appear. The reasons for a landmark not appearing include overlapping of the chromosome in the region at which the landmark resides and a high degree of contraction of the chromosome. Even if there is no overlapping and the chromosome is relatively uncontracted the landmark may not appear. Though the cause is not well understood, the presence in some cells and the absence in others of a particular chromosome landmark may simply be a phenotypic variation in the architecture of the chromosome.

Summary An idiogram is presented for the ten pachytene bivalents of Ricinus communis L., race A39, showing chromosome length, centromere position, as well as lengths and positions of heterochromatic regions and constrictions.

196

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