phenotypic and chromosomal abnormalities …instability of the unsatellited abnormal chromosome than...

PHENOTYPIC AND CHROMOSOMAL ABNORMALITIES ASSOCIATED WITH THE INTRODUCTION OF HETEROCHROMATIN FROM

NICOTIANA OTQPHORA INTO N . TABACUMI

D. U. GERSTEL AND J. A. BURNS

Department of Crop Science, North Carolina State Uniuersiiy at Raleigh, North Carolina 27607

Received March 2, 1967

ENETIC instability frequently arises following interspecific hybridization Gand is of particularly common occurrence in hybrids of the genus Nicotiana (SMITH 1967). Several examples were observed by the authors among derivatives of crosses between Nicotiana tabucum (2n = 48) and the closely related species N . otophoru (2n = 24) from which two were chosen for detailed study. In one case the corollas were variegated with coral streaks on carmine background. This variegation was found to be caused by an instability of the carmine pigmentation controlled by the CO” allele from N . otophora (GERSTEL 1966; GERSTEL and BURNS 1966a). The other instability was cytological; one might call it also a variegation since only scattered cells were affected. In these cells one can see chromosomes of enormous length which we called “megachromosomes” ( GERSTEL and BURNS 1966b).

Previously corolla variegation and megachromosomes were investigated separately.* The study of the genetics of CO” was not accompanied by extensive cytological analysis. Megachromosomes, on the other hand, were first observed in cells of stocks which were not marked to reveal instability of CO”. Only at a later date were megachromosomes also discovered in a line which segregated for flower variegation. The family of plants studied here came from that line.

Besides determining the relation between variegation and megachromosomes we intended to study the inheritance of the latter. For these aims we needed a euploid plant which had variegated flowers and formed megachromosomes. Plant 1-638-9 (Table 1) combined these properties and since it also had 48 chromosomes like N . tabucum this plant appeared suitable for the analysis.

An additional relationship was observed in the course of the investigation. N . otophoru possesses large heterochromatic segments on five of the 12 chromosomes of the genome whereas N . tabucum has only scattered minute pieces of heterochromatin (BURNS 1966). The segments from N . otophora were incor- porated into a genome of N . tabucum by means of backcrosses. It was discovered that both carmine-coral variegation and megachromosome formation are associ-

’ Journal Series Paper No. 2352 from the North Carolina Agricultural Experiment Statim. Aided by Grant GB-2517 c l f the National Science Foundation.

2 \\‘e mentioned in the 1966b paper that megachromosomes were first found in a stock in which a chlorophyll factor froin A‘. otophora was unstable on a near-albino N . fahacum background. That line has not been investigated further and the relation between megachroniosr,mes and chlorophyll variegation is still unknown.

Genetics 56: 483-5M July 1967.

484 D. U. GERSTEL A N D J. A. B U R N S

I C C li

3 -

w L i t 2

c

(0 I

0 (0

(3 X -

X -

2

a M

Y

m 6

m 4

3

g 2

G" 2 4

a .+

m < + m

g

2 z 2

W .-.

E E

3 ffi

- 3 m

a &

r;l

il

D

g

- B 2 v

VI W .- < .- b 5 2

2

-c

r4

b 1

0

% 6 E

8 0

$'

E g p 2

2

3 m + m

rt

- 2

e:

MEGACHROMOSOMES A N D VARIEGATION 485

ated with one of these segments. The study led us therefore beyond a Mendelian analysis to an understanding of some causal aspects of megachromosome formation.

MATERIALS A N D M E T H O D S

Family J-500 represented the principal experimental material for the investigation. The pedigree of this family is given in Table 1 . The ancestral interspecific hybrid was an amphidiploid produced by crossing coral (CO) N . tabacum L. as female parent with N . otophora Griseb., race Cochabamba-I, and treating the resulting seedlings with colchicine. Four generations of backcrosses to coral N . tabacum served to reduce the chromosome number to that normal for N. tabacum. In each generation the parent derived from the hybrid had variegated flowers. Megachromosomes were first looked for and found in a plant (G-601-6) of the second backcross generation and they reoccurred in s3me plants of every subsequent generation regardless of the direction of the cross.

The cytological techniques employed were those described earlier (GERSTEL and BURNS

TABLE 2

Phenotypes and cytological characteristics of family J-500

Hetero- Abnormal chromosomes'

Plant Group number

I

I1

I11

IV

J-500-C -G -0 -P -T -Y

J-500-B -I -L -M -N -Q -U -V -W

1-638-9 J-500-A

-F -x

J-500-D -E -H -J -K -R -S

chromatic - Number of bodies Not Mega-

Flower color chromosomes i "HB") Satellited satellited chromosome&

Coral Coral Coral Coral Coral Coral High variegation High variegation High variegation High variegation High variegation High variegation Medium variegation High variegation High variegation High variegation High variegation High variegation High variegation Very high variegation Very high variegation Very high variegation Very high variegation Very high variegation Very high variegation Very high variegation

48 -t 48 48 48 4.8 48 48 + 48 + 48 + 48 + 48 + + 47 + 49 + 48 + 4.8 ++ 48 ++ 48 ++ 48 ++ 48 + 48 ++ 4.8 + 48 + 48 + 48 + 48 +

-

-

- -

-

- _ _ - _ - - _ - _ - - + - + - + - + - + - + - + - + - + + + + + + + + + + + + + + +

- - - - - - -

-

- -

- -

- + + + + + + ? + + + + + + + + + + + + +

* Shown in Figure 3. + Rare cells in all variegated plants, including U, showed the unusually large heterochromatic clumps mentioned in the

$ For - and + symbols see text. tcw and thought to be related to megachromosomes. These were not found in coral plants.

486 D. U. GERSTEL A N D J. A. BURNS

1966b). One might repeat here that somatic tissues were generally pretreated before fixation with hydroxyquinoline to accumulate metaphases and contract chromosomes and that such pre- treatments als3 produced a visible differentiation between hetero- and euchromatin at prophase.

RES U LTS

Phenotypes: The plants of family 5-500 were arranged in four Groups, mainly according to corolla colors (Table 2). Five plants had self-colored coral flowers (Group I ) . Group I1 consisted of eight variegated plants which exhibited an amount of spotting comparable to that of the “high” class of a previous publication (GERSTEL and BURNS 1966a). A representative flawer is shown in Figure 1, left. Plant U with somewhat fewer coral spots, called “medium variegated” is included in Group I1 for reasons given below. The three members of the third Group were also highly variegated but were set apart from Group I1 on the basis of cytological findings. Parent plant 1-638-9 possessed the characteristics of the members of Group I11 and is therefore listed with them. Finally, seven plants had flowers with many more coral streaks and less carmine than did those of Groups I1 and 111; these plants with “very high variegation” constitute Group IV (Figure 1, middle).

Most flowers on a given plant exhibited a similar variegation pattern but exceptions occurred. Thus, on any variegated plant entirely coral flowers and sometimes even a coral branch could be found; changes toward a more stable carmine condition were also encountered (Figure 1, right). These last changes were rare in Group I1 but more frequent in plants of Groups I11 and IV.

Findings on heterochromatin and chromosomal breakage will be given next and observations on megachromosomes will follow.

Interphase nuclei: Interphase nuclei of the variegated plants contained con- spicuous heterochromatic bodies, chromocenters or prochromosomes as they are variously called. Figure 2 illustrates an interphase nucleus with such a heterochromatic body henceforth to be named “HB” for short. (The close resemblance to a mammalian “Barr body” [BARR and BERTRAM 19491 is deceiving, as will be shown). The term “heterochromatin” shall be used as defined by BURNS (1966), following HEITZ’S (1928) original description.

1

FIGURE 1.-Corollas showing different degrees of coral (light) streaking on carmine (dark) background. Left: Highly variegated. Middle: Very highly Variegated. Right: Very highly variegated with a more stable sector. (Natural size).

MEGACHROMOSOMES A N D VARIEGATION 48 7

FIGURE.^ 2 to 5: FIGURE 2.--Interphase nucleus with a heterochromatic body (“HB) from a heterochromatic segment of an “abnormal chromosome”. FIGURE 3.-Metaphase nucleus showing both abnormal chromosomes; the satellited one near upper right and the unsatellited one at lower left (from a plant of Group 111). FIGURE 4.-Metaphase plate with a dicentric chromosome in lower right. FIGURE 5.-Metaphase plate with a lengthened abnormal chromosome. (All approximately 1500 x ) .

Interphases of none of the coral plants of Group I contained HB’s. The plants of Group I1 had one HB in the majority of their nuclei, however there was variability. Some nuclei lacked HB’s, others had two and sizes were also not uniform. The very highly variegated plants of Group IV also had one HB in many cells, but there were more variant cells than in Group 11. Plant E was exceptional; it was placed in Group IV on the basis of phenotype, but two HB’s were frequent. Two HB’s were found in many cells of plants in Group 111.


These observations are summarized in the fifth column of Table 2. Minus signs signify that no HB’s were seen in any cells of a plant. One plus mark designates plants with one HB in a larger o r smaller proportion of cells and two plus signs those which frequently had two HB’s. Observations were made in part from root tips and in part from corollas; both kinds of tissue yielded similar results and the source is therefore not specified.

Metaphases: Metaphases will be considered before other mitotic stages. In the cells of coral sibs, no chromosomes were seen which differed visibly from those of the normal complement of N . tabacum. On the other hand, variegated plants unfailingly possessed one, or sometimes two, chromosomes which stood out by the unusual length of their long arms. There were two kinds of this type, differing mainly in their short arm. In plants of Group I1 the short arm was very small and possessed a satellite (Table 2). In corolla cells this satellite usually was close to the short arm; in root tip preparations it tended to be set off conspicuously by a long stalk. In plants of Group IV the second type of long-armed chromosome was found; the short arm was visibly longer than that of the type just described, but it had no satellite. These two chromosomes will be called the abnormal chromosomes. Both abnormal chromosomes occurred in plants of Group I11 and are illustrated in a cell of the latter Group (Figure 3 ) .

The abnormal chromosomes were not present in all cells. Thus, in plants of Group I1 the satellited abnormal chromosome with the unusually long arm was not observed in 14.5% of the cells (Table 3 ) ; similarly in cells of Group IV the other abnormal chromosome could not be seen in 70%. In Group I11 both abnormal chromosomes occurred in somewhat less than one half of the cells while the remainder had only one or the other or neither. The data indicate a greater instability of the unsatellited abnormal chromosome than of the satellited one. However, it is important to note that, with extremely rare exceptions, the chromosome number of.48 remained unchanged regardless as to whether a long-armed chromosome was present or not. In other words, the observed karyotypic changes were not losses of chromosomes but of segments and, as will be seen, of largely heterochromatic segments. It may be remembered that loss of heterochromatin was also reflected by the absence of HB’s from some interphase nuclei.

As further evidence for the occurrence of chromosome breakage in the varie-

TABLE 3

Frequencies of the two kinds of abnormal chromosomes at metaphase

Percentages of cells with*

Abnormal Abnormal Both Only normal satellited unsatellited abnormal Total

cells scored Group chromosomes chromosome chromosome chromosomes

I1 14.5 85.5 0 0 600 I11 10.0 25.0 20.0 45.0 40 I V 70.7 0 29.3 0 150

* Chromosomes were munted in several cells of each entry and found to be 48 in number in every case. Exceptional cells with dicentrics, fragments, or megachromosomes were omitted from the samples.

MEGACHROMOSOMES AND VARIEGATION

TABLE 4

Evidence for chromosome breakage in somatic tissuef

489

Anaphases

hletaphaaes Single bridges

Normal Frag- Noma1 No frag- 1 frag- 2 frag- Double Fragment Group complement Dicentrics ment separation ment ment meiits bridges: only

I 1200 0 0 195 2 0 0 2 1 I1 1320 5-f 2 44.0 50 0 3 3 4

I11 359 6t 4 316 43 0 0. 34 6 IV 915 9 5 492 71 4 0 13 3

'Part of the evidence came from root tips and part from corollas; since no relevant differences between the t w o tissues

f A satellite could be recognized on one free arm of several dicentrirs. t Derived either from dicentric chromosunies or from two isorhromatid breaks in the same cell. Both criss-cross and

were observed they are not listed separately.

interlocked double bridges were seen.

gated plants, dicentric chromosomes (Figure 4) and fragments were seen, and the frequencies are recorded in Table 4. The breakage products listed in this table were largely, but perhaps not entirely, due to independent events; e.g., the nine dicentrics listed for Group IV were found in five different plants. Many dicentrics in plants possessing the satellited abnormal chromosome had also a satellite on one free arm. Among the group of plants in which the abnormal chromosome was without a satellite none of the dicentrics were satellited, while in those plants which possessed both the satellited and the unsatellited abnormal chromosome some of the dicentrics were satellited. These observations suggest that the abnormal chromosomes were involved in the breakages and fusions which yielded dicentric chromosomes. This was corroborated by the fact that breakage-induced abnormalities were very rare among the coral plants in Group I which possessed neither of the abnormal chromosomes.

Breakages of bridges followed by healing of the breaks represents a way by which chromosomes may be either reduced in size or lengthened. Abnormal chromosomes of somewhat greater length than those pictured in Figure 3 were observed occasionally (Figure 5 ) . Of course, chromosome size can also change by processes other than bridge formation.

In this section too, data from root tip and corolla preparations were used inter- changeably since the only difference was the appearance of the satellites.

Prophases: Prophase chromosomes which are partially contracted by the action of hydroxyquinoline reveal locations of heterochromatin (TJIO and LEVAN 1950; BURNS 1966). At this stage, both types of abnormal chromosomes, the satellited and the unsatellited one, can be observed to possess a heavily stained segment occupying approximately one half of the long arms, depending on the degree of contraction. Figure 6 depicts the satellited abnormal chromosome; the unsatellited one looks rather similar since at this stage centromere position and satellite are ill defined in most cells. The heterochromatic segments are not quite terminal insofar as a small euchromatic piece is distal to the heterochromatin in both types. The hydroxyquinoline-prophases also serve to demonstrate occasional complete

490 -

D. U. GERSTEL A N D J. A. BURNS -

cr I

8 --

lo 9 1 FIGURFS 6 to 10: FIGURE 6.-The abnormal satellited chromosome in prophase. Note position

of heterochromatic segment and euchromatic distal end (Group 11). Arrow points to satellite. FIGURE 7.-Similar chromosome as in Figure 6, with distal euchromatin and some of the heterochromatin missing. FIGURE &-The heterochromatic segment approximately doubled in length. FIGURE 9.Xomparison of the satellited abnormal chromosome with a megachromosome from Group 11; both at metaphase. FIGURE 10.-Comparison of the unsatellited abnormal chromosome with a megachromosome in same Group IV; both at metaphase. Compare short arms in Figures 9 and 10. The long arm of the unsatellited megachromosome is highly variable (see text). (All approximately 3 2 0 0 ~ ) .

deletions or structural changes of the heterochromatin. In Figure 7 the distal euchromatic piece is lost and a shortened heterochromatic segment is now in the terminal position. Figure 8 represents a heterochromatic segment of approximately twice the usual size which could have originated by means of an isochro- matid break followed by sister reunion. The terminal position pictured in Figure 7 was seen several times in exceptional flowers or sectors from a number of plants; the one illustrated in Figure 8 was encountered only once.

Anaphases: Anaphases were obtained whenever pretreatment with hydroxy-


quinoline was omitted; the observations are summarized in Table 4. The coral plants of Group I produced very few abnormalities supporting the conclusion drawn from metaphases that plants without abnormal chromosomes were cytologically nearly stable. Plants in Groups I1 and IV which possessed one abnormal chromosome produced a number of single anaphase bridges and some fragments. Anaphases of Group IV showed only a small increase in instability over Group I1 (16.2% us. 12.0% of abnormal cells). Notable was the relatively high frequency of double bridges in plants of Group I11 which possessed both long-armed chromosomes. These observations largely reflect frequencies of separate breaks among the plants of three groups since the anaphase irregularities were scored in many separate preparations and from several plants. But again, some of the aberrations scored may have had a common origin.

Exceptional plants: J-500-E was exceptional. The variegation pattern placed this plant in the “very high variegation” Group IV as did the presence of one abnormal metaphase chromosome which had a very long arm and no satellite on the short arm. But interphase nuclei frequently possessed the two HB’s, one of which was smaller than the norm. Hydroxyquinoline treated prophases showed one chromosome in which a long heterochromatic segment was located in the usual subterminal position. In addition, there was a second slightly shortened and terminally located heterochromatic segment; no other plant in family 5-500 exhibited consistently such a terminal segment, though exceptional sectors with a terminal segment had been found on other plants, as mentioned (Figure 7). Presumably, the distal euchromatic segment had been lost together with some adjacent heterochromatin so shortening the chromosome that it could not be recognized as an abnormal one at metaphase.

J - 5 0 0 4 had fewer coral streaks than any other Variegated sib and was classi- fied as only “medium variegated”. Cytologically, this plant resembled those with high variegation in Group I1 but it had only 47 chromosomes. The missing chromosome ‘was not identified. J-500-V had 49 chromosomes, but it differed otherwise in no recognizable way from the sibs of Group 11.

Progenies: Progenies from members of the three variegated Groups 11,111 and IV were raised to see whether their characteristics, i.e. the extent of coral spotting and the type of abnormal chromosome were transmitted. Table 5 gives the data obtained from crosses with CO CO testers. The variegated offspring from plants of Group I1 were highly variegated like their parents. The very highly variegated Group IV produced very highly variegated progeny. There were exceptions in both tests, insofar as one and two plants, respectively, had less spotting than the typical class. Two plants belonging to Group I11 were subjected to testing. One was the parent plant 1-638-9 from which additional offspring were raised to enlarge upon family 5-500. J-500-X was also tested. According to expectation highly and very highly variegated plants resulted, in addition to coral flowered ones.

One and two-factor backcross ratios were expected among the progenies since the parents were heterozygotes having been obtained from backcrosses themselves. The expectations were fulfilled essentially (Table 5 ) , but an excess of

492 D. U. GERSTEL AND J. A. BURNS

TABLE 5

Progeny tests (X CO CO)

Offspring

Parents Variegated

Group Number Low High Very high Coral

I1 J-500-B P J-500-N 9 J-500-N 8

Total 111 1-638-9 0

1-638-9 8 J-500-X 0 J-500-X 8

Total IV J-500-D 9

J-500-H 8 J-500-H 9

0 18 0 1 20 0 0 22 0 1 60 0 0 25 9 0 12 18 0 19 9 0 16 12 0 72* 48 0 0 32 0 1 13 0 1 23

Total 0 2 68

Group I1 -Chi-square = 5.6; 0.02 > P > 0.01 (1 :1) Group 111-Chi-square = 13.0; P < 0.01 (2:l:l) Group IV-Chi-square = 14.1; P < 0.01 (1:l)

36 29 25 90 15 15 18 18 66 62 37 23

1 22

This class contained plants with characteristics of either Group I1 or Group 111.

coral off spring was observed among all three totals. This was hardly surprising from genetically unstable material. Also as expected, the less stable plants of Group IV gave a higher chi-square than those of Group 11. The results were in disagreement with the good fit to Mendelian ratios obtained earlier by GERSTEL and BURNS (Table 5 of 1966a) from some other lines of highly variegated plants.

A number of offspring from plants of Groups I1 and IV were checked cytologically for transmission of the abnormal chromosomes. The satellited abnormal chromosome with the long arm recurred in variegated offspring from Group I1 (four plants checked). The nonsatellited abnormal chromosome was present in offspring sampled from Group IV (seven plants). Two selected offspring were checked from crosses between plants of Groups I1 and IV and found to possess both abnormal chromosomes. Finally, homozygotes for each of the abnormal chromosomes were obtained by selfing plants of Groups I1 and IV. Heritability of the ahnormal chromosomes was thus ascertained.

Megachromosomes in metaphases: Several observations indicated a relation between megachromosomes and the two types of abnormal chromosomes with the long heterochromatic segments. First, no megachromosomes occurred in the six coral plants which had no abnormal chromosomes among a total of 1200 somatic metaphases. In all variegated plants megachromosomes were seen, with the exception of J-500-U. An appreciation of the frequency of megachromosomes, or rather of their rarity, was obtained by counting cells with and without megachromosomes from several plants in each group (Table 6). However, the data

MEGACHROMOSOMES AND VARIEGATION

TABLE 6

Occurrence of megachromosomes in uariegated plants (metaphases)

493

~~~~~~~

Number Cells with Total G1 oup Tissue plants rnegaLhromosomes cells

11* Root tips Corollas

I11 Root tips Corollas

IV Root tips Corollas

Total+

6 4 700 1 0 100 2 5 199 1 3 100 3 3 4.00 4 5 4.00

20 1899

* 'Ibc relatively stable nionosoniic plant J-50GU is not included; no megachromosomes were found in 700 metaphases. t For statistical bios (overestimate) see text.

were biased in two ways. Counts were sometimes discontinued when a cell with a megachromosome was found in a given plant. Also, if a megachromosome could be distinguished the cell was included regardless as to whether the chromosomes were well spread or not, whereas negative entries came only from cells in which the size of each chromosome could clearly be delimited. The actual frequency of megachromosomes was probably lower than the 1/95 ratio indicated by the totals in Table 6. (By the way, this ratio is commensurable with the 1/85 ratio obtained earlier which was combined from several independent lines; see Table 1 in GERSTEL and BURNS 1964). The frequency appeared to be greater in plants of Group I11 with two abnormal chromosomes than in the other two groups.

A second reason for associating megachromosomes with the abnormal partially heterochromatic ones is based on their morphology. Megachromosomes in Group I1 characteristically had satellites on their short arms just like the abnormal chromosomes of that group (Figures 9, 16). The satellite could not be seen in all cells, but in general, satellites are not recognizable in all preparations. It is not unlikely that all of the megachromosomes in Group I1 were satellited. Signifi- cantly, the s%ort arms of the satellited abnormal chromosomes and of the megachromosomes were of similar proportions. They are compared in Figure 9. On the other hand, none of the megachromosomes in Group IV were satellited and in absolute length the short arms matched those of the abnormal chromosomes in that Group (Figure 10). Though the nonsatellited short arm is less distinctive, by analogy one may presume that also in Group IV the short arms of the megachromosomes came from the unaltered short arms of the abnormal chromosomes.

Counts of all the chromosomes in cells containing megachromosomes should indicate whether one chromosome, and only one, contributes to a megachromosome. Chromosomes could be counted with confidence in only nine cells possessing megachromosomes (Table 7 ) . Seven of these cells had the same total number of chromosomes as normal cells, indicating that a single chromosome can become a megachromosome with 2n-1 chromosomes remaining unaltered. However devi- ant cells were also observed. In one cell from Group I11 the megachromosome was dicentric but the total number of centromeres here too equalled 2n. One cell had

494 D. U . GERSTEL A N D J. A. B U R N S

FIGURFS 1 1 to 15: FIGURE 1 1.-Megachromosomr fraginents ((;roup 11). FIGURE 12.-Twn very large mrgarhromosomes (Fragments? Crntromrrrs not seen: Group 111). FIGURE 1 %.-Mrg;i- chromosomrs in neighhxing cells (rxtrrmely rarr). In cell on right an acentric ring nwgarhro- mosome; thr visihlr centromere Iwlongs to a normal chromosome in a slightly highrr planr. FIGURE 14.-Another ring mrgachromosonir. FIGURE 15.-Megachroinnsomr with onr chroma- t id hrokrn. (All approxiniatrly l i W X ).

MEGACHROMOSOMES AND VARIEGATION 495

TABLE 7

Cells with megachromosomes ( M ) in which all chromosomes could be counted

Trisomic Disomics (J-500-V) Group I11 Group IV

Cells with 2n centromeres 4 7 + M 2 2 1 4 7 + M + fragment 46 f dicentric M f fragment M - - 4.8+M

4 5 + M - - -

-

- - - 1 - 1

- - - 1 Cell not with 2n centromeres

1

a diminished number of centromeres. These counts suggest a greater regularity than was reported previously ( GERSTEL and BURNS 1966b) ; this discrepancy seems significant and will be discussed further on.

In addition to cells in which all chromosomes could be counted, megachromosomes were studied in many other cells and the following conclusions could be drawn: In plants from Group I1 nearly all megachromosomes were of similar morphology and dimensions. Occasional deviations occurred. A second dicentric megachromosome was found; one free arm was satellited. One may note the parallel observations made on dicentric chromosomes of .normal proportions (Table 4, footnote 2). Fragments of large proportions were seen in several cells (Figure 11 ) . More irregularity occurred in Group IV and even more in Group 111. Figures 12 to 15 illustrate some of the observations which indicate occurrence of breakages and fusions. Direct evidence came from two sources. Occasionally, circular megachromosomes were seen (Figures 13, 14) which could have been formed only by means of breakage and end-to-end fusion. One megachromosome in Figure 15 had one fractured chromatid and the chromosome was held together by the other intact one.

Nondisjunction was rare in cells without as well as in cells with megachromosomes; in one cell from Group I1 two megachromosomes of identical proportions and both satellited could be seen (Figure 16) which may have had such an origin.

Extra-heterochromatin in interphases: One or several unusually large heterochromatic clumps were seen in some cells of all plants possessing megachromosomes. Such clumps were described previously by GERSTEL and BURNS (1966b) as “intensely stained bodies of different sizes and irregular outlines visible in some resting nuclei.” They varied widely in proportion and in number. The largest ones covered as much as one half of a nucleus (Figure 17), but ranged down in size to some that were indistinguishable from HB’s. Sometimes they occurred in tiers of neighboring cells (Figure 18).

One apparent exception to the rule that plants with extra-heterochromatin also had cells with megachromosomes was plant J-500-U. As noted, this monosomic plant differed from other variegated plants also in having fewer coral streaks. Extra-heterochromatin occurred in this plant but extremely rarely as compared

4IXi D. U. GERSTI'.L A S D J. A. BURNS

I;IGUIIL~ I O to 19: FIGURE 16.-Two satrllited nirgachromosomrs in Same cell; from nnw disjunction (Group 11). FIGURE 1 i.-Intrrphnw with w r y lnrgr clump of "rxtra-hrtrrortirn111;1- tin". I ~ I G U H E 18.-Tirr of intrrphasr crlls with "rxtra-hrtrroctiromatin". FIGURE 19.-Hrtc.ro- pyrnotic mrg;cclironiosoni~~(s) at rarly prophase. l'nusually large. (All approniniatrly I i0Ox ).

with other sibs in Group 11. The fact that no megachromosomes could be found among 700 metaphases might thus be ascribed to a relative stability of this plant and a consequent rarity of megachromosomes.

Megochromosomes at prophase: The pycnotic conformation of megachromosomes in somatic prophases was mentioned already (GERSTEL and BURNS 1966b). At this stage highly condensed chromosomes of great length were visible in some cells; since the ends of these convoluted structures were usually not recognizable one could not be certain whether the entire length of the chromosome was hetero- pycnotic. An example of extreme size is shown in Figure 19.

DISCUSSION

Origin of the satellited abnormal chromosome: The heterochromatic segment of the satellited abnormal chromosome resembles the long segments of hetero-

MEGACHROMOSOMES A N D VARIEGATION 49 7

chromatin found in N . otophora but not in N . tabacum (BURNS 1966); a detailed comparison of pachynemata to determine accurately the homology still needs to be made. The chromosomes of the two species synapse quite readily during meiosis in hybrids ( GOODSPEED 1954) and the heterochromatic segment from N . otophora presumably was substituted for an N . tabacum segment by crossing over in an ancestor of the 5-500 family. Selection for CO" in each backcross generation kept the associated heterochromatic piece in the line. Also the morphology of the satellited abnormal chromosome bespeaks a dual origin since the satellite does not resemble the large N . otophora satellites borne on chromosomes with long heterochromatic segments but looks like those of N . tabacum (BURNS 1966). Earlier work had located the introgressed CO" in a homologue of the satellited F-chromosome of N . tabacum (GERSTEL and BURNS 1966a; that work was done with a different variegated line, however). Presumably, the F-chromosome of N . tabacum and the CO" bearing chromosome of N . otophora are at least partially homologous. This is further supported by the fact that the alleles CO (coral) and CO (carmine) are located in the F-chromosome (CLAUSEN and CAMERON 1944; an analysis of the biochemical effects of CO, CO and CO" was made by KITTILSON 1966).

The parent plant and the origin of the unsatellited abnormal chromosome: Plant 1-638-9 was chosen because of a numerically balanced complement of 48 chromosomes. As it turned out, the plant was not genetically balanced since it carried duplicate CO" alleles with adjoining heterochromatin in nonhomologous chromosomes. A translocation must have occurred in an earlier generation where- upon the two segments were introduced into the same gamete.

(The amphidiploid ancestor, necessarily, had two CO" segments. Most likely the backcross plant F-14-35-see Table l-did also. F-14-35 had at the time been singled out for further study because it had fewer coral spots than any other sib. Subsequently, this lower spot frequency could be attributed to a double dose of CO". The translocation, followed by nondisjunction of the CO" segments, presumably occurred in F-14-35).

The two distinctive abnormal chromosomes (Figure 3) were recognized in 1-638-9. They were transmitted to members of the J-500 family for which there is both genetical and cytological evidence. The observed segregation of 19 variegated and 6 coral plants fits far better a 3: 1 (P > .9) than a 1: 1 (p < .Ol) ratio. Occurrence of plants having both abnormal chromosomes as well as plants with either one during two successive generations served as a confirmation.

Variability of the abnormal chromosomes: 1-638-9 was cytologically unstable as were all variegated plants among its progeny 5-500. This instability was revealed by the occurrence of breakage products such as dicentric chromosomes, ring chromosomes, fragments and anaphase bridges. Since the coral sibs were nearly stable, instability may be ascribed to the presence of the heterochromatic segments associated with CO". The variability of HB's-i.e. presence or absence, variation in size-indicates such an instability of the heterochromatic segments themselves. As will be shown elsewhere (BURNS and GERSTEL 1967), the phenotypic variability, i.e. variegation, is the result of deletions of CO". Breakage ap-


parently is not limited to the abnormal chromosomes alone but may also affect other chromosomes if the heterochromatic blocks are present. This is shown by the occurrence of dicentric chromosomes with distinctly different free arms. Thus, dicentric chromosomes in Group I1 may possess a satellite at one end, whereas the other free arm is of different dimensions and unsatellited. Such dicentrics must have arisen from fusions between nonhomologues.

Further evidence for breakage comes from metaphases which have the full complement of 48 chromosomes but lack chromosomes of abnormal proportions (Table 3, column 1 ) . Part of the long arm of the abnormal chromosomes must have become deleted, such that they can no longer be recognized. Hydroxyquino- line treated prophases in which the heterochromatic segment can be seen in a terminal instead of the usual subterminal position and/or is changed in length, provide the most direct proof (Figures 7,8).

Origin of mgachromosomes: Origin and time of formation of the megachromosomes are fascinating but as yet elusive problems. Apparently megachromosomes are formed from the two abnormal chromosomes which may be assumed for the following reasons. First, plants of Group I which lacked the abnormal chromosomes failed to form megachromosomes. Second, the megachromosomes of plants in Groups I1 and 1V possessed short arms matching the short arms of the abnormals found in these groups (Figures 9, 10). This is particularly strik- ing in the case of the satellited abnormal chromosome. Third, plants of Group I11 with two abnormal chromosomes produced megachromosomes with an increased frequency.

One may assume that megachromosomes are produced de nouo in each plant in which they are found and not passed on as such by the gametes. GERSTEL and BURNS (1966b) did not find megachromosomes in metaphases of pollen grains from a plant which produced them in somatic tissues. Further search among plants of Group I1 brought no positive evidence, nor was extra heterochromatin found in pollen; there were only a few grains with structures suggestive of megachromosomes, but they were rare. Since half of the backcross progeny of Group I1 plants have cells with megachromosomes, the latter are generated within the plant and not introduced intact by the gametes. Furthermore, most megachromosomes are likely to originate during the interphases preceding the mitoses in which they were observed, as suggested by the usual failure of megachromosomes to appear in clusters of cells. It is unlikely that megachromosomes are gradually built up during several cell cycles, since they are of uniform appearance and size (at least in Group 11) and no intermediate sizes occurred. This was less clearly expressed in Groups 111 and IV, presumably because of the greater amount of breakage going on in those plants. As yet it is completely unknown what stimulus may induce formation of megachromosomes in only an occasional interphase. One might suspect specific breakage events to be required. Whether the distal ends of the partially heterochromatic arms are perhaps broken off is still unknown. Even though in prophases distal ends of the abnormal chromosomes are distinctly marked by a short segment of euchromatin (Figure 6), towards metaphase the marking is obscured by the condensation. But metaphase


is the only stage at which the dimensions of the megachromosomes are exactly revealed. Also the question whether only the heterochromatin of the abnormal chromosomes assumes gigantic proportions or whether the euchromatic sectors are also affected remains unsolved since the distinctions are obscured in metaphases. The unchanged appearance of the short arms suggests that either euchromatin remains unaffected or only one arm is transformed.

(The previous paper, GERSTEL and BURNS 1966b, reports variability of the total number of chromosomes in cells with megachromosomes and also a variable number of megachromosomes. Now it appears that the report was based on an erroneous interpretation. We analysed again the old drawings and photographs and found that only part of the megachromosomes pictured there possessed centromeres and that it is more likely that those cells contained only one or sometimes two megachromosomes which had fragmented. In several instances it was possible by counting centromeres rather than chromatin units to reconcile the counts with the somatic chromosome number characteristic of the normal cells of the particular plant. The relatively high breakage rate may have been due to the fact that the plants belonged to early generations with several heterochromatic segments from N . otophora. The former report thus fails to contradict the surmise that a particular member of the complement can become transformed into a megachromosome).

Since the blocks of “extra-heterochromatin” seen in interphases varied in size they may represent stepping stones in the construction of megachromosomes within an interphase. In that case the amount of material included in the blocks should fall into a geometric series which could be detected by photometric means-a suggestion for which we are grateful to DR. G. T. RUDKIN. The blocks could also represent breakage products fragmented during divisions of cells with megachromosomes. This is supported by frequent occurrence of several pieces of heterochromatin in the same and sometimes in adjacent cells.

Structure of megachromosomes: The nature of megachromosomes is still an enigma. For the reasons given, megachromosomes are not likely to be built up gradually by the addition of segments to their length during successive breakage- fusion-bridge cycles. The main argument is failure of large chromosomes to pass through anaphase, and additional points ‘were discussed by GERSTEL and BURNS ( 196613). Alternatively, one may compare megachromosomes with polytene chromosomes found not only in Diptera but also in higher plants, though in the latter only in cells of the embryo-sac (TSCHERMAK-WOESS 1963) or in suspensor cells (NAGL 1962, 1965). Discovery in parasite infested cells of Rhynchosciara angelae of polytene chromosomes which were even larger than those normally occurring (DIAZ and PAVAN 1965) is proof that cells can extend tremendously the synthesis of chromosomal material, given the proper stimulus. A somewhat different example testifying to the synthetic potential during an individual interphase was found in cases of repeated endoreduplication in mouse tissue cultures (LEVAN and Hsu 1961).

The appearance of megachromosomes differs from that of polytene chromosomes and casts doubt upon the interpretation. There is no trace of the banding

5 00 D. U. GERSTEL AND J. A. BURNS

characteristic of the euchromatic parts of polytenes; megachromosomes, however, are largely heterochromatic, as described. Megachromosomes often show a dis- tinct split between chromatids whereas published pictures of polytene chromosomes do not demonstrate separate chromatids, though in the latter the many single strands are not always fused (TSCHERMAK-WOESS 1963). (The split often noticeable in salivary chromosomes of Diptera separates somatically paired chromosomes, not chromatids). Furthermore, megachromosomes are not, or only a little, wider than normal chromosomes in the same cell; lengthening or unspiral- ling would have to be rather precisely adjusted to compensate for the increased number of strands. In polytene chromosomes of plants, as well as of Diptera, increase in both length and width are the rule (NAGL 1962). Lastly, all chromosomes in a cell are affected in such cases.

The conventional morphology of the megachromosomes resembles more that of the polynemic chromosomes named by DARLINGTON (1955) than that of polytene structures. Manifold differences in chromosome size within one taxonomic group (GREGORY 1941; HUGHES-SCHRADER and SCHRADER 1956; and many others) or even in somatic differentiation of the same organism (tabulation in DARLINGTON 1965; NAGL 1962) are not rare and have been ascribed, by some, to a lateral multiplicity of basic units ( DARLINGTON 1955). (If adaptive increases and decreases of chromosome size during evolution (STEBBINS 1966) should be due to changes in the number of parallel arranged DNA units, an impact on the current controversy on whether the eukaryotic chromosome is unineme or mul- tineme should be obvious; see LEWIS and JOHN 1963).

Again, the polynemic chromosomes illustrated in the literature are not only larger but also thicker than those for which a lesser number of strands may be argued. However, recently reports are accumulating of cases where only certain parts of the genome are undergoing extra replications. This happens, e.g., in oogonial cells of the fly Tipula oleracea where LIMA-DE-FARIA and MOSES (1966) observed very large “DNA-bodies” which they interpreted as highly replicated nucleolar organizer regions or adjacent segments of the chromosome. If the comparison between such a phenomenon and megachromosomes is at all valid, one must consider the fact that in Tipula the extreme enlargement of a part of the complement is part of normal developmental processes while in Nicotiana it occurs only casually in a synthetic genome. Also mentioned might be the view of BEERMANN (1966) that the chromomere is the unit of replication and that the different chromomeres of the same complement can replicate with different frequencies in the same interphase. Furthermore, all chromosomes in a complement are again of comparable dimensions.

Whatever structure may be proposed for megachromosomes, whether polyteny, polynemy or any other arrangement of nucleic acids and proteins that might be conceived, the nature of the specific stimulus exerted upon only one element of the complement and the manner of response remains to be discovered. A further complication, both from the structural aspect as well as from the point of view of regulation, resides in the fact that only one arm of the megachromosome is magnified. Lastly, it remains uncertain whether indeed megachromo-

MEGACHROMOSOMES AND VARIEGATION 501

somes have an increased content of DNA; this probably is the case but has not yet been proven in a critical manner.

SUMMARY

Carmine-coral flower variegation occurs in plants of N . tabacum which carry alien heterochromatic segments introduced from N . otophora. The carmine (CO") allele is associated with, though not necessarily located in, the heterochromatic block; coral ( C O ) comes from N . tabacum. In the presence of the heterochromatin, chromosomes break; this occurs largely in the heterochromatic segments themselves but to some extent also in other chromosomes. The same block of heterochromatin has been found either in a satellited chromosome or in another one; when in the latter the instability is enhanced.-Instability was revealed at various stages: in interphasic nuclei by presence, absence or variations in size of the heterochromatic body, at metaphase by the variation in length of the chromosomes in question, and in the phenotype by variegation.-"Megachromosomes" of several times the normal length occurred in a few and widely scattered cells. The evidence indicated that they were formed from chromosomes with the alien heterochromatic segments. The total number of chromosomes in a cell usually remained unaltered by the presence of a megachromosome. Plants carrying the heterochromatic block in a satellited chromosome formed satellited megachromosomes and unsatellited megachromosomes were constituted when the heterochromatic block was present in a chromosome without satellite.-The mode of origin of megachromosomes and the nature of the stimulus leading to the astound- ing increase of a particular element in the cell are as yet obscure, as is the internal structure of megachromosomes. Various hypothetical interpretations are put forward; e.g., megachromosomes are compared with polytenic or polynemic chromosomes and weaknesses of each proposal are discussed.

LITERATURE CITED

BARR, M. L.. and E. G. BERTRAM, 1W9 A morpholagical distinction between neurones of the male and female, and the behaviour of the nucleolar satellite during accelerated nucleo- protein synthesis. Nature 163: 676-677.

Differentiation at the level of the chromosomes. pp. 24-54. Cell Differ- entiation and Morphogenesis. Edited by W. BEFAMANN, R. J. GAUTHEXET, P. D. NIEUWKOOP, C. W. WAADSL~W, V. B. WIGGLESWORTH, E. WOLFF, and J. A. D. ZEEVAART. North-Holland Publishing Co., Amsterdam.

The heterochromatin of two species of Nicotiana: Cytological observations. J. Heredity 57: 43-47.

heterochromatin in Nicotiana. Genetics 57: (in press).

mmic analysis. Genetics 29: 447-477.

1 1 4 4 . -- 1965 Cytology. Churchill, London.

BEERMANN, W., 1966

BURNS, J. A., 1966

BURNS, J. A., and D. U. GERSTEL, 1967

CLAUSEN, R. E., and D. R. CAMERON, 1944

DARLINGTON, C. D., 1955

Flower color variegation and instability of a block of

Inheritance in Nicotiana tabacum. X V I I I . Mono-

The chromosome as a physico-chemical entity. Nature 176: 1139-

5 02

DIAZ, M., and C. PAVAN, 1965 Changes in chromosomes induced by microorganism infection. Proc. Natl. Acad. Sci. US. 54: 1321-1327.

GERSTEL, D. U., 1966 Effect of monosomy on variegation in hybrids between two species of Nicotiana. J. Heredity 57: 126-128.

GERSTEL, D. U., and J. A. BURNS, 1966a Flower variegation in hybrids between Nicotiana tabacum and N . otophora. Genetics 53: 551-567. - 1966b Chromosomes of unusual length in hybrids between two species of Nicotiana. Proc. 1st Oxford Chromosome Conf., pp. 41-56. (Chromosomes Today Vol. 1; Suppl. Heredity 19: 1 W ) .

D. U. GERSTEL AND J. A. BURNS

GOODSPEED, T. H., 1954 GREGORY, W. C., 1941

HEITZ, E., 1928 HUGHES-SCHRADER, S., and F. SCHRADER, 1956 Polyteny as a factor in the chromosomal evolu-

tion of the Pentatomini (Hemiptera). Chromosoma 8: 135-151. KITTILSON, H. F., 1966 A biochemical and genetical study of the anthocyanins and some related

polyphenols present in various strains of Nicotiana tabacum and related species. Ph.D. thesis, North Carolina State Univ. Library.

Repeated endoreduplication in a mouse cell. Hereditas 47:

The Genus Nicotiana. Chronica Botanica, Waltham, Mass. Phylogenetic and cytological studies in the Ranunculaceae Juss. Trans.

Amer. Phil. Soc. n.s. 31: 443-521. Das Heterochromatin der Moose. I. Jahrb. Wiss. Bot. 69: 762-818.

LEVAN, A., and T. C. Hsu, 1961

LEWIS, K. R., and B. JOHN, 1963 LIMA-DE-FARIA, A., and M. J. MOSES, 1966

NAGL, W., 1962

69-71. Chromosome Marker. Little, Brown, Boston.

Ultrastructure and cytochemistry of metabolic DNA

Uber Endopolyploidie, Restitutionskernbildung und Kernstrukturen im SUS- -

Die SAT-Riesenchromosomen der Kerne des Suspensors von Phaseolus coccineus und

Recent cytogenetic studies in the genus Nicotiana. Advan. Genet. (In Press).

in Tipula. J. Cell Biol. 30: 177-192.

pensor von Angiospermen und einer Gymnosperme. Gster. Bot. Ztschr. 109: 431-494. 1965 ihr Verhalten wahrend der Endomitose. Chromosoma 16: 511-520.

SMITH, H. H., 1967 STEBBINS, G. L., 1966 TJIO, J. H., and A. LEVAN, 1950

Exp. Aula Dei 2: 21-64. TSCHERMAK-WOESS, E., 1963

plasmologia (Handbuch der Pr3toplasmaforschung) Vol. V. 1. 158 pp.

Chromosomal variation and evolution. Science 152 : 1463-1469. The use of oxyquinoline in chromosome analysis. Ann. Est.

Proto- Strukturtypen der Ruhekerne von Pflanzen und Tieren.

phenotypic and chromosomal abnormalities …instability of the unsatellited abnormal chromosome than...

Documents