I 1967, 1972) we have shown that the introduction of a particular heterochromatic

Transcription

1 FORMATION OF MEGACHROMOSOMES FROM HETEROCHROMATIC BLOCKS OF NZCOTZANA TOMENTOSZFORMIS1 J. A. BURNS AND D. U. GERSTEL Department of Crop Science, North Carolina State University at Raleigh, North Carolina Manuscript received July 12, 1973 ABSTRACT A heterochromatic block (HB) from N. otophora occasionally undergoes great enlargement to form a megachromosome in hybrids and hybrid derivatives with N. tabacum. This paper shows that the two large HB s of closely related N. tomentosiformis, and perhaps a smaller one, also have the same capability. The evidence that both large HB s form megachromosomes is twofold. In a segregating backcross population from a parent possessing the two large HB s, all segregants with one block produced megachromosomes at metaphase or large heterochromatic clumps at interphase. Second, those segregants which possessed both heterochromatic blocks produced megachromosomes of two visibly different types. Proliferation to make megachromosomes thus may not be the property of merely one particular segment but a more common property of heterochromatin in a hybrid or otherwise disturbed background. N previous papers (BURNS and GERSTEL 1969,1972; GERSTEL and BURNS 1966, I 1967, 1972) we have shown that the introduction of a particular heterochromatic block (HB) from Nicotiana otophora into N. tabacum leads to instabilities of two kinds, First, the alien HB undergoes breakage which causes chromatin loss; this can be observed cytologically and also phenotypically by the variegation it causes. Second, in some cells chromosomes with the HB from N. otophora proliferate enormously to many times their normal length, forming what we have called megachromosomes. In this paper we seek to determine whether heterochromatin from another species, N. tomentosiformis, can also enlarge. Furthermore, the question remains to be solved whether only one particular HB from a given species is involved in formation of megachromosomes, or whether this is a more general property of alien heterochromatin. HOEGERMAN (1969, 1972) demonstrated that at least three out of five HB s from N. otophora generate megachromosomes; however, his evidence was indirect and based on rates of transmission in interspecific hybrids. MATERIALS AND METHODS Three species were used in the investigation: N. tabacum L. (2n = e), N. tomentosiformis Goodsp. (2n = 24) and N. sylvestris Speg. et Com. (2n = 24). The coral line of Red Russian tobacco served as the N. tabacum parent. There are no large blocks of heterochromatin in N. Paper No of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, North Carolina. Genetics 75: November, 1973.

2 498 J. A. BURNS AND D. U. GERSTEL FIGURE 1.-Late prophase cell from root tip of N. iomeniosiformis showing heterochromatin. Below, the three pairs with the largest blocks. (The black spot in the middle of the rightmost chromosome is due to an overlap). FIGURE 2.- Extra-heterochromatin (left top cell) compared at interphase with normal HB s (below). FIGURE 3.--Somatic metaphase containing the two different HB-carrying chromosomes (arrows point at centromeres). FIGURE 4.4omatic metaphase with two megachromosomes having short arms of differing lengths. (FIGURES 1-2 ~1200; FIGURES 3-4 x1800).

3 MEGACHROMOSOMES IN NICOTIANA 499 tubacum, but only scattered small knobs which can be seen clearly only at pachytene. The U.S.D.A. line of N. tomentosiformis which was used possesses a haploid complement with two large HB s at the distal ends of their chromosomes and one medium-sized intercalary block. In addition, there are several very small HB s or knobs. Figure 1 shows a late somatic prophase. At interphase the large HB s are large enough to be visible as darkly stained bodies while the medium and smaller ones cannot be identified. The Catamarca race of N. syluestris which was used has no HB s but only small knobs like N. tabacum. Thus, all the large HB s encountered in the study were derived from N. tomentosiformis. The aim was to analyze the behavior of HB s from N. tomentosiformis on a background with N. tubacum. Since N. tubacum is an allopolyploid and N. tomentosiformis a diploid, we circumvented the complications of aneuploidy by first synthesizing an amphidiploid from the ancestors of N. tabacun, viz. N. tomentosiformis and N. syluestris. The amphidiploid was crossed to N. tabucum to give a triple hybrid, N. tabacum-sylvestris-tomentosiformis. This triple hybrid is quite fertile, since the chromosomes of N. syluestris pair with those of one genome of N. tubacum and the N. tomentosiformis chromosomes pair with those of the other N. tubacum genome (GOODSPEED 1954). The triple hybrid was backcrossed to N. tabucum to derive the plants used in the investigation; in this generation the HB s from N. tomentosiformis were separated by assortment. The details of the cytological methods used were described earlier (BURNS 1964.; BURNS and GFZSTEL 1969). All fixations were made of young petal tissue or root tips in a Carnoy mixture. Prophase and metaphase preparations were pretreated in 8-hydroxyquinoline, fixed, hydrolyzed in 10% concentrated HCl and stained with acetocarmine. This treatment made the heterochromatin visible at late prophase. Preparations for the study of interphase were neither pretreated nor hydrolyzed; nearly enhre, young and unfixed corollas were placed on slides in acetocarmine solution, heated, covered and then scanned under the mlcroscope. This permitted observation of very large numbers of cells. It was essential to find out which plants were capable of forming megachromosomes. At interphase these are visible as very large blocks of heterochromatin ( extra heterochromatin = XHC; Figure 2). A piece of heterochromatin was scored as XHC if it was at least four times the size of a normal HB. As pointed out previously (GERSTEL and BURNS 1966) megachromosomes are not transmitted intact through cell division and are found only in widely scattered cells or groups of cells. In some plants it was sufficient to score a single corolla or part of a corolla to find five or more separate cells or small groups of cells with XHC, in which case the plant was diagnosed as XHC positive. In other plants more than one corolla had to be scanned before five separate cases of XHC were found. Negative diagnoses were based on at least three coroilasi.e. if three corollas showed no XHC the plant was judged unable to make megachromosomes. In two doubtful plants (see below) interphases of five corollas were scanned. RESULTS Interphases: Interphases were useful for two purposes. They permitted counting of the number of HB s in a plant; there were either 0, 1 or 2 large HB s in a segregant. As mentioned, the smaller pieces of heterochromatin could not be recognized at interphase. Second, from interphases we could determine whether or not a plant could make megachromosomes, which at this stage form extremely large heterochromatic clumps in single scattered cells (Figure 2) or in small groups of cells. All plants without HB s failed to show these large blocks (Table 1 ), with two exceptions to be discussed later. All plants, with one exception, which possessed one or two HB s had cells with XHC. There was, therefore, a nearly complete correspondence between the presence of HB s and that of XHC. The frequency of cells with XHC varied widely, which is not surprising in such heterozygous material. Since all 21 plants with one HB made XHC, one must con-

4 500 J. A. BURNS AND D. U. GERSTEL TABLE 1 Distribution of XHC in interphases in backcross from triple hybrid No. of HB s per plant No. of plants No. of plants with XHC * XHC = extra-heterochromatin, i.e. megachromosomes in interphase. HB = heterochromatic block. = minute amount of XHC-see text. t clude that both large HB s from N. tomentosiformis could make megachromosomes. Since, on the whole, plants without visible HB s failed to make XHC, it appeared that the ability to make heterochromatic megachromosomes was limited to plants with large HB s. But there were two exceptional plants without visible HB s which made XHC extremely rarely; one plant had, in five corollas, three groups of cells with XHC and the other had one cell with XHC among five corollas. Prophase preparations of these plants showed that they possessed the medium-sized HB, which could be a possible source of the rare XHC. Metaphases: We studied somatic metaphases of 12 of the 21 plants with one HB. The HB-carrying chromosomes could be recognized at this stage because their long arms were longer than those of any other chromosome, as observed at late prophase. There were two types (Figure 3) : one with a subterminal centromere (HB1) and the other with a more submedian centromere (HB2). Seven plants had an HB1 type chromosome and five had HBz. This made it appear, initially, that the two different HB-carrying chromosomes could be distinguished by the position of the centromere. However, observation of somatic metaphases in eleven plants with two HB s revealed that eight had two chromosomes of the HB type and three had one of each type. Since all eleven were backcross plants and heterozygotes, they all should carry one of each long homolog. This meant that one chromosome in each plant had to be a homolog of HB even though, in the majority of plants, both HB-carrying chromosomes had the HB1 morphology. This led to the conclusion that one of the HB chromosomes appeared in two forms, one of which was indistinguishable from HB1 while the other was HB2. Another possibility is that HB1 undergoes frequent nondisjunction, but this is less likely since no plants with three large HB s were recovered. How the two forms could have arisen is not clear; one possibility is that the more submedian form arose by crossing over with an N. tabacum chromosome (all large N. tomentosiformis chromosomes are subterminal). For the metaphase analyses described below, made to determine if both large HB s from N. tomentosiformis could form megachromosomes, backcross plants were used which had both HB1 and HBz, and which were presumably nonhomologs. In megachromosomes derived from N. otophora, only the long arm is enlarged, whereas the short arm retains its short proportions (GERSTEL and BURNS 1967). If the same is true in megachromosomes from N. tomentosiformis, one has the 2t 21 10

5 MEGACHROMOSOMES IN NICOTIANA 501 opportunity to see whether both HB1 and HBZ can make megachromosomes. Plant P-604-A-1 had both HB' and HB2 (Figure 3) and we looked at 1300 metaphases in which 23 cells with megachromosomes were found. Fifteen of these were useless because the position of the centromere was not clear. Two cells each had two megachromosomes, one with a short arm like HB' (subterminal) and one with a longer short arm (submedian) of the HB2 type (Figure 4). One cell had a dicentric megachromosome with distal arms of the two lengths. One cell had a megachromosome with an HB1 short arm, and in four others the short arm was of the longer HBz type. Smaller samples were taken of two more plants with two HB's. One-hundred cells were scored of plant , among which four had single megachromosomes. Two of these had the short short arm of HB' and two were of the submedian HBz type. Among 100 cells of plant , six had megachromosomes. One of these had two megachromosomes, one of each type. In addition, there was one cell with a megachromosome of type HB' and four cells had a megachromosome with the longer HBZ short arm. DISCUSSION The observations show that both large heterochromatic blocks of N. tomentosiformis, and perhaps, rarely, the medium-sized block also, can produce megachromosomes as can heterochromatin of N. otophora. The ability to produce megachromosomes, in a background of N. tabacum, is thus not a property of a specific piece of heterochromatin but apparently a more general characteristic of block heterochromatin. However, it should be noted that N. tomentosiformis and N. otophora are closely related members of the section Tomentosae (GOODSPEED 1954), and some blocks in the two species may well have a common origin. In an earlier paper (BURNS and GERSTEL 1969) we discussed the possible involvement of controlling elements in chromosome breakage and formation of megachromosomes. If, as now appears likely, all or most large HB's of N. otophora and N. tomentosiformis and perhaps also HB's from other species may undergo these aberrations, the existence of separate controls for each HB is not plausible. Perhaps, there is in each species, or in N. tabacum, a common control for the induction of the aberrations in several blocks of heterochromatin. As an alternative, the aberrations may be due to properties of heterochromatin itself-e.g., a physical proneness to break and a tendency of broken heterochromatin to continue replication beyond one cycle. We had found previously (BURNS and GERSTEL 1972) that intact genomes of N. tomentosiformis suppress chromosome breakage and the generation of megachromosomes and concluded that this species contains one or more suppressors. The observation that nearly all plants in the first segregating generation produced megachromosomes makes it likely that more than one element is needed for suppression. A possible alternative which may also explain the high frequency of progeny with megachromosomes is that the N. syluestris parent contributed element (s) which counteracted the suppressor element (s) from N. tomentosiformis.

6 5 02 J. A. BURNS AND D. U. GERSTEL Megachromosomes in Nicotiana do not seem to be unique. BURNS and GERSTEL (1969) and HOEGERMAN (1972) have reviewed a number of cases in the literature where single extremely long chromosomes occurred in scattered cells following hybridization, exposure to radiation and in tumors in a variety of species. The reader is referred to those publications. The mechanism of origin of megachromosomes is completely unknown; of importance seems the fact that chromosome breakage occurs in the same plants which form megachromosomes, as shown in all our previous publications on the subject; yet it seems unlikely that megachromosomes arise from breakage-fusionbridge cycles (GERSTEL and BURNS 1966). They are too large to go through anaphase but zigzag from pole to pole or lag between poles and are apparently fragmented by the forming cell plate. Also, there is a general absence of chromosomes of intermediate sizes. As an alternative, one might mention that heterochromatin seems capable of ~altational ~ increases (BRITTEN and KOHNE 1968; YUNIS and YASMINEH 1971). It appears that some heterochromatin in the background of foreign species, or under otherwise abnormal conditions, is capable of sudden amplification. LITERATURE CITED BRITTEN, R. J. and D. E. KOHNE, 1968 Repeated sequences in DNA. Science 161: BURNS, J. A., 1964 A technique for making preparations of mitotic chromosomes from Nicotiana flowers. Tobacco Science 8 : 1-2. BURNS, J. A. and D. U. GERSTEL, 1969 Consequences of spontaneous breakage of heterochromatic chromosome segments in Nictotiana hybrids. Genetics 63 : Inhibition of chromosome breakage and of megachromosomes by intact genomes of Nicotiana. Genetics 69 : GERSTEL, D. U. and J. A. BURNS, 1966 Chromosomes of unusual length in hybrids between two species of Nicotiana. Chromosomes Today 1: , 1967 Phenotypic and chromosoma1 abnormalities associated with the introduction of heterochromatin from N~co~~Q~u otophora into N. tabacum. Genetics 56: , 1972 On the absence of cytoplasmic determination of the formation of megachromosomes in Nicotiana. J. Heredity 63 : GOODSPEED, T. H., 1954 The Gems Nicotiana. Chronica Botanica, Waltham, Mass. HOEGERMAN, S. F., 1969 Megachromosomes in the progeny of Nicotiana tabacum x N. otophora backcrossed to N. tabacum. Genetics 61: S27-S28. -, 1972 The enlargement of heterochromatin from Nicotiana otophora Griseb. in hybrids with N. tabacum L. Ph.D. thesis, North Carolina State University Library. YUNIS, J. J. and W. G. YASMINEH, 1971 Heterochromatin, satellite DNA and cell function. Science 174: Corresponding editor: 0. E. NFLSON