THE RELATIONSHIP BETWEEN CHROMOSOME VOLUME AND DNA CONTENT IN UNSQUASHED METAPHASE CELLS OF BARLEY, HORDEUM VULGARE CV. TULEEN 346

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1 J. Cell Sri. 56, IOI-III (198a) IOI Printed in Great Britain Company of Biologists Limited 1982 THE RELATIONSHIP BETWEEN CHROMOSOME VOLUME AND DNA CONTENT IN UNSQUASHED METAPHASE CELLS OF BARLEY, HORDEUM VULGARE CV. TULEEN 346 M. D. BENNETT, J. B. SMITH, J. P. WARD AND R. A. FINCH Plant Breeding Institute, Trumpington, Cambridge CB2 zlq, England SUMMARY The present work used haploid and diploid cells of barley, Hordeum vulgare L. cv. Tuleen 346 (an = 2x 14), which has three reciprocal translocations. All seven chromosomes of the haploid set are distinguishable using morphological criteria in Feulgen-stained root-tip squashes seen in the light microscope, as are five of the bivalents at diakinesis. The relative DNA content per bivalent was estimated in pollen mother cells at diakinesis. The results showed that all seven chromosomes or bivalents of Tuleen 346 can be identified using relative DNA content as sole criterion. The absolute and relative volumes of the seven chromosomes were estimated from electron micrographs of serial sections of unsquashed root-tip cells of a haploid. The results show that, using relative chromosome volume as sole criterion, it is highly probable that all seven chromosomes in single unsquashed cells of Tuleen 346 can be correctly identified. Consequently, teats for various non-random spatial arrangements of chromosomes in unsquashed cells of Tuleen 346 using this character to identify the chromosomes should be feasible. There was a very highly significant positive relationship (r>o<)o) between relative chromosome volume and mean relative DNA content per chromosome for each cell examined at metaphase of mitosis or meiosis. Thus, some mechanism ensures that the degree of condensation of all seven chromosomes within a cell is usually very similar in Tuleen 346, lespite its grossly abnormal karyotype. INTRODUCTION Using light microscopy, various authors have described close correlations between chromosome size and the DNA content of chromosomes at metaphase of mitosis in higher plants. For example, Barlow & Vosa (1969) showed a striking linear relationship (r = 0-99) between the DNA content and the volume of the mitotic metaphase chromosomes of Puschkinia Ubanotica (syn. Scilla libanotica). Similarly, the results of Heneen & Caspersson (1973) showed a correlation coefficient of 0-98 when the relative DNA contents and relative lengths of the seven root-tip metaphase chromosomes of Secale cereale were compared. However, Nishikawa (1970) reported a smaller correlation coefficient (r = 0-82) between the DNA contents and the lengths of the 21 chromosomes of hexaploid bread wheat (Triticum aestivum cv. Chinese Spring) measured as univalents at the first division of male meiosis. Examination of serial electron micrographs of sectioned cells has allowed the volumes of intranuclear structures to be determined more precisely than was possible

2 M. D. Bennett, J. B. Smith, J. P. Ward and R. A. Finch 3 ~ f 2 V O

3 Chromosome volume and DNA content in barley cells 103 by light microscopy. Using this technique, it has proved possible to estimate the volumes of both whole chromosomes (Finch, Smith & Bennett, 1981) and small chromosome segments such as centromeres (Moens & Church, 1977; Bennett, Smith, Ward & Jenkins, 1981). It was decided, therefore, to compare the DNA content of individual chromosomes with their volumes estimated from serial electron micrographs of sectioned cells in a higher plant species. Such a study should show whether the relationship between DNA content and chromosome size is as precise as some previous light-microscopic studies have indicated. MATERIALS AND METHODS The chosen material was barley {Hordeum vulgare, in = zx = 14), stock 'Tuleen 346*. This stock, so named by us because it came from a plant numbered 346 by Professor N. A. Tuleen, is homozygous for the three reciprocal translocations (T), T1-5V, T2-6y and T3-7d (N. A. Tuleen, 1980, personal communication). Details of the origins and karyotype of this stock have been published recently (Finch & Bennett, 1982). Tuleen 346 is notable because all seven chromosomes can be identified in mitotic metaphase squashes seen in the light microscope using the morphological characters: relative length, arm ratio, and the presence or absence of a secondary constriction (Fig. 1). Moreover, compared with normal barley, the range of chromosome sizes is greatly increased. Thus, Tuleen 346 seemed a suitable material for the present study, especially as the increments between adjacent chromosomes, ranked by size order, was likely to be larger than in most materials including normal barley. Unequivocal identification of all the chromosomes in a cell using chromosome volume alone will depend on there being no overlap between any estimates of the relative volumes of any two chromosomes. It may be hard to identify homologous chromosomes in diploid material. Any misidentification would result in an error when calculating the mean relative volume and its variance for each chromosome. The need to identify homologues does not arise in a haploid. Consequently, estimates of relative chromosome volume were made using haploid Tuleen 346. Haploid plants of Tuleen 346 were obtained by pollinating emasculated spikes of diploid Tuleen 346 with pollen from diploid Hordeum bulbosum clones Ji or L6 (Simpson, Snape & Finch, 1980). The bulbosum chromosomes were eliminated during early seed development, as frequently occurs in hybrids between diploid H. vulgare and H. bulbosum (Subrahmanyam & Kasha, 1973; Bennett, Finch & Barclay, 1976). Haploid embryos were cultured until they formed plantlets large enough to survive transplantation to soil. When they had grown into large vegetative plants, one plant was divided into ramets and cultured in hydroponics (Finch et al. 1981) to produce clean new roots suitable for ultrastructural studies. An alternative way of avoiding the need to identify homologues with certainty is to study paired homologues during meiosis in diploid material. Such pairing is very regular in diploid Tuleen 346. For example, in a plant from a glasshouse a random sample of 20 pollen mother cells all contained seven bivalents and the mean chiasma frequency per cell was I4'8s. Thus, plants of diploid Tuleen 346 were grown in a glasshouse until they reached meiosis, when suitable tillers were sampled for DNA estimations or ultrastructural studies. A Vickers M86 integrating microdensitometer was used to estimate the relative DNA contents of the seven bivalents in suitable cells from Feulgen-stained anther squashes. Of many thousands of pollen mother cells examined, five at diakinesis were identified as most suitable for Fig. 1. The seven chromosomes of haploid Tuleen 346 at mitotic metaphase identified using morphological criteria in a Feulgen-stained root-tip squash preparation. Bar, 10 fim. Fig. 2. The seven bivalents of diploid Tuleen 346 at diakinesis of male meiosis identified using morphological criteria and relative DNA contents. Bar, 10 fim.

4 104 M. D. Bennett, J. B. Smith, J. P. Ward and R. A. Finch Table i. Relative absorption per bivalent expressed as a percentage of the total absorption per cell in pollen mother cells of diploid Tuleen 346 at diakinesis Cell Bivalent Mean S.E. Variance T2-6 Ti-S T3-7 4 T7-3 T6-2 T i # * * * * * IO-I2 816 o-io O-II 0-16 o-34 O'I O'O2 Secondary constriction observed. microdensitometry because all seven bivalents were clearly separated from one another, and the five bivalents containing chromosomes T5-1, T6-2, T7-3, 4 and T3-7 were clearly identified using morphological criteria (e.g. Fig. 2). The other two chromosome pairs containing T1-5 and T2-6 could not be identified by this means at this stage of meiosis. Diakinesis rather than first metaphase was chosen as the optimal stage, because the degree of chromosome condensation at diakinesis allows cells with seven fully separated bivalents to be found, and both nucleolar organizers are sometimes clearly visible. In each cell the DNA content of each bivalent was estimated as the mean of three readings, and these means were expressed as the relative proportion of the total DNA content of the pollen mother cell. A root-tip about 1 cm in length was cut from a haploid Tuleen 346 plant and pretreated in ice-water at about o C for 24 h. This pretreatment increases the proportion of cells at metaphase (Subrahmanyam & Kasha, 1973). Root-tip and anther material were fixed, embedded and stained for ultrastructural studies as previously described (Bennett, Smith, Simpson & Wells, 1979). Sections o-i fim thick were cut using a Reichert Ultracut 4 microtome with a diamond knife, and collected on Formvarcoated 2x1 mm slot-grids, using the technique of Wells (1974). Chromosomes in selected cells were photographed at x 4500 using a Philips 201 electron microscope, and printed at a final magnification of x Four cells at metaphase (A, B, C and D, respectively) were identified in sections of a single haploid root-tip. The total number (n) of serial sections on which chromosomes of these nuclei appeared were 95, 96, 88 and 83, respectively. All the electron micrographs of chromosomes in a single cell were arranged in section number sequence in a binder and numbered from 1 to n. Photographs of all the sections of nuclei A, B, C and D were obtained except section 49 in nucleus A (which was obscured), and section 75 in B (which was lost). Two pollen mother cells at first metaphase (E and F, respectively) were identified in sections of a single anther of diploid material. In these cells bivalents appeared on 68 and 81 successive sections, respectively. Photographs, arranged as described above for root-tip nuclei, were available for all sections other than section 64 of nucleus F, which was lost. For each cell, each chromosome (in haploid material) or bivalent (in diploid material) was identified in the set of electron micrographs by its number (1-7) written in waterproof ink on each piece of chromatin belonging to that chromosome or bivalent. Different chromosomes or bivalents were numbered 1-7 according to the order in which each first appeared in the electron micrographs. Chromosome or bivalent volume was estimated using a Kontron Videoplan Digitizer, scaled to give areas in /tm 1. The outlines of a single chromosome or bivalent on each successive photograph where it appeared were traced on the digitizer tablet, and the resulting areas summed in the memory. The section thickness was o-i fim, so the volume of each chromosome or bivalent (in fim*) was obtained by multiplying the summed areas by a factor

5 Chromosome volume and DNA content in barley cells 105 Table 2. Absolute chromosome volume* and the relative chromosome volume expressed as a percentage of the total chromosome volume per cell in haploid Tuleen 346 root-tip cells at metaphase Cell Chromosome A B C D Mean S.E. Variance T2-6 Ti-S T3-7 4 T7-3 T6-2 Ts-i (11-07) (1022) 1806 (9-37) i6-57t (8-41) (7-24) (5-59) f (4-66) 8-23 (1092) 1934 (1025) 1896 (936) i7-32f (7-58) (6-88) (5-O9) 942t (4-44) 8-22 (1092) 1903 (1059) (io-oo) (8-53) 1469 (6-8 4 ) 1193 (5-77) (4-8o) 836 (1057) 1903 (10-27) (885) is-94 (810) I4-59 (7-48) 1346 (S-6i) io-iof (466) 8-39 Expressed as /*m' (in parenthesis). t Secondary constriction observed O'l8 o o-39 o-io o-oi of o-i. Where a section was lost or obscured (as noted above), the unknown area of a chromosome was estimated as the mean of the areas for the same chromosome on the two adjacent sections. RESULTS Relative DNA contents of the seven chromosomes Table 1 presents the relative absorption of the seven bivalents in five cells of diploid Tuleen 346 at male diakinesis expressed as a percentage of the total absorption for each cell. Examination of the estimates for bivalents containing the five chromosomes unequivocally identified using morphological criteria shows that they ranked in the same order in all five cells. Thus, estimates for T3-7, 4, T7-3, T6-2 and T5-1 ranked 3rd, 4th, 5th, 6th and 7th, respectively. Moreover, there was no overlap between any estimates of relative absorption for any two chromosomes with adjacent ranking. Thus, it is reasonable to assign estimates ranked first in each cell to the longest chromosome (T2-6) and those ranked second to the second longest chromosome (T1-5). It is then possible to estimate the mean relative DNA contents of the seven chromosomes in diploid Tuleen 346 (Table 1). These values show a range of about 2-4-fold from 8-16% in T5-1 to 1936% in T2-6. Microdensitometry of Feulgen-stained mitotic prophase nuclei of diploid Tuleen 346 and Sultan barley showed that their 4C DNA amounts were identical (22-2 pg)

6 io6 M. D. Bennett, jf. B. Smith, J. P. Ward and R. A. Finch Table 3. Absolute bivalent volume* and the relative bivalent volume expressed as a percentage of the total bivalent volume per cell in diploid Tuleen 346 cells at first metaphase of male meiosis Cell Bivalent E F Mean T2-6 Ti-S T3-7 4 T7-3 T6-2 Ts-i (2466) 1873 (2408) 1829 (2313) I7-S7 (2008) (1630) (1288) 979 (10-51) 799 Expressed (2913) 1959 (2865) 1927 (25-52) (21-15) (17-73) (14-52) 976 (1201) 808 as fim 3 (in parentheses) Mean relative chromosome DNA content Fig. 3. Relationship between mean relative chromosome volume and mean relative chromosome DNA content in haploid Tuleen 346 root-tip metaphase cells.

7 Chromosome volume and DNA content in barley cells Mean relative chromosome DNA content Fig. 4. Relationship between relative chromosome volume and mean relative chromosome DNA content in single cells of haploid Tuleen 346 cells A, B, C and D. (Finch & Bennett, 1982). Thus, in 4C cells of haploid Tuleen 346 the absolute DNA contents of the chromosomes were as follows: T2-6, 2-15 pg; T1-5, 2-06 pg; T3-7, 1-85 pg; 4, 162 pg; T7-3, 1-40 pg; T6-2, 1-12 pg; and, T5-1, 091 pg. A one-way analysis of variance showed very highly significant differences between relative absorption values for all chromosome pairs. Analysis also showed that the variance for estimates of the relative absorption for the same chromosome in different cells was small compared with the difference between pairs of chromosomes of most-similar DNA content. The results indicate that all seven bivalents in a cell would be identified correctly with a high probability using relative DNA content as sole criterion. A Bartlett test showed no significant differences between the variances for different chromosomes. Relative volumes of the seven chromosomes Table 2 presents the absolute volumes of the seven chromosomes in four cells of haploid Tuleen 346, together with the relative volumes expressed as a percentage within each cell. Estimates of the absolute volume of homologues in the different cells are remarkably similar. Examination of estimates of the relative volumes of the five unequivocally identified chromosomes (T3-7, 4, T7-3, T6-2 and T5-1) shows that they ranked in the same order and position in each cell. Moreover, there was no overlap between any estimates of relative volume for any two chromosomes adjacent in the ranking. It is reasonable, therefore, to identify estimates ranked first with the longest chromosome (T2-6) and those ranked second with the second longest chromosome (T1-5). It is, therefore, possible to calculate the mean relative volume for all seven chromosomes. These show a 2-3-fold range from 8-30% to 19-25%. These

8 io8 M. D. Bennett, jf. B. Smith, J. P. Ward and R. A. Finch 20 i E O > 15 ICD 10 i 5 Fig Mean relative chromosome DNA content Relationship between mean relative bivalent volume at diakinesis and the mean relative chromosome DNA content in diploid Tuleen 346. results are therefore in close agreement with estimates of relative chromosome length in light micrographs of haploid root-tip cells of Tuleen 346, which gave a 2-2i-fold range from 1868% for chromosome T2-6 to 8-44% for T5-1 (Finch & Bennett, 1982). A one-way analysis of variance showed a very highly significant difference (P<o-ooi) between estimates of relative chromosome volume for all pairs of chromosomes, with the exception of T2-6 and T1-5 where the difference was highly significant (P< o-oi). Analysis also showed that the between-cell variances for estimates of the relative volume of homologues were small compared with the differences between the mean relative volumes of any two chromosomes with adjacent ranking. A Bartlett test showed no significant difference among the variances for different chromosomes. These results indicate that all seven chromosomes in a cell would be identified correctly with a high probability using relative volume as sole criterion. However, chromosomes T2-6 and T1-5 would be confused more often than any other pair of chromosomes because the size difference between them is smaller than that between any other pair of chromosomes. Table 3 presents estimates of the absolute volumes of the seven bivalents in two pollen mother cells at first metaphase of meiosis from a single anther of diploid Tuleen 346. The relative bivalent volumes expressed as a percentage of the total volume of bivalents in each cell are also given, and showed ranges of 2'34-fold and 242-fold, respectively. Bivalents were not identified using morphological criteria at this stage since, for example, constrictions at nucleolar organizers are poorly expressed at metaphase. However, the mean relative volumes for bivalents (Table 3) are not significantly different from the mean relative volume of mitotic metaphase chromosomes ranked in corresponding positions. Moreover, the mean relative volume of each bivalent, except the two largest, is significantly different (P<o-O5) from the

9 Chromosome volume and DNA content in barley cells 109 mean relative volume of any mitotic chromosome, except that ranked the same as the bivalent being compared. Thus, it is reasonable to assume that metaphase chromosomes of Tuleen 346 retain the same relative volumes at meiosis as at mitosis. If so, it should usually be possible to identify all seven metaphase bivalents in a single cell using relative volume as sole criterion. Relationship between chromosome volume and DNA content Fig. 3 illustrates the very highly significant (P<o-ooi) relationship between the mean relative chromosome volume (values from Table 2) and the mean relative chromosome DNA content (values from Table 1) for Tuleen 346. The regression coefficient and slope are i-ooo, and the intercept of the regression line with the abscissa ( 0-02) is not significantly different from the origin. Fig. 4A-D shows plots of the relative volume on relative DNA content for the seven chromosomes in each of the four haploid cells studied. In each case the relationship is very highly significant, the regression is not significantly different from unity, and the intercept is not significantly different from the origin. Fig. 5. illustrates the very highly significant relationship (P<o-ooi) between mean relative bivalent volume and mean relative chromosome DNA content (r = 0997; b = 1-009; a ~ O-I 3)- Similar separate plots for cells E and F also showed very highly significant relationships; P<o-ooi; r>0-099; b = 1-04 (E) and 1-08 (F); a = 0-13 (E) and 1-14 (F). DISCUSSION The precise relationship between chromosome volume and DNA content Estimates of chromosome volume in single cells at mitotic metaphase obtained by light microscopy are meaningful, but are subject to unavoidable errors; for example, the need to assume that chromatids are circular in cross-section and of constant diameter (Bennett & Rees, 1969). Photographs of the present serially sectioned cells show that chromatids are often not circular in cross-section (although in general they approximate to this configuration), and that the diameter of a chromatid varies considerably along its length. Nevertheless, striking correlations have been shown between chromosome DNA content and chromosome volume estimated as mean values for many cells using light microscopy (for references, see Introduction). Using serial sections of nuclei and electron microscopy, chromosome volume can be estimated more precisely in single cells than by light microscopy, since the abovementioned assumptions are unnecessary. The present results for Tuleen 346 barley, using chromosome volumes for four cells studied by electron microscopy, confirm the existence of a precise general relationship between chromosome volume and DNA content (Fig. 3) in a diploid angiosperm as described for Puschkinia Ubanotica (Barlow & Vosa, 1969). They also show that the precise relationship was displayed by each individual cell studied (Fig. 4). Thus, the present results indicate a precise intracellular control, which ensures that the relative condensation of all seven metaphase chromosomes in the haploid complement of Tuleen 346 is very similar. Tuleen 346

10 no M.D. Bennett, J. B. Smith, J. P. Ward and R. A. Finch is abnormal in having three translocations, which involve six of the seven chromosomes and increase the range of chromosome sizes to from i to 2-4 compared with the range from 1 to 1-3 in normal barley. It is interesting to note that the precise control of chromosome condensation is unaffected by the large rearrangement of segments in the complement of Tuleen 346 due to the three reciprocal translocations. The advantages of highly synchronized chromatin condensation among chromosomes within a cell in facilitating balanced and efficient mitosis are obvious. Thus, it seems reasonable to expect that precisely synchronized condensation among chromosomes, as seen in Tuleen 346, is normal and that examples of gross allocycly among chromosomes within a genome are exceptions. Use of chromosome volume to identify chromosomes Experiments to test for various types of non-random spatial distribution of chromosomes have mainly been done with squashes, although it has been suspected that considerable rearrangement of relative chromosome disposition may be unavoidable in their preparation (Feldman, Mello-Sampayo & Sears, 1966; Stack & Brown, 1969; Wagenaar, 1969). It is obviously preferable to make such tests using undistorted cells, but it has rarely been possible to identify all the chromosomes in unsquashed cells of organisms with more than a few (i.e. zn > 6) chromosomes, using either light or electron microscopy. The present results show that using serial sections of nuclei of suitable materials, it is possible to identify most or all of the chromosomes at both mitotic and meiotic metaphase in single cells undistorted by squashing, using relative chromosome volume alone as a criterion. Moreover, by comparing the dispositions of chromosomes identified in this way it should be possible to test for various non-random chromosome arrangements in unsquashed dividing nuclei, including somatic association (see Avivi & Feldman, 1980), secondary association (Kempanna & Riley, 1964), affinity (Wallace & Gunn, 1965) and specific end-to-end arrangements (Ashley, 1979). Preliminary studies indicate that such tests are quite feasible using Tuleen 346, and show that useful results should be obtainable from small numbers of replicate cells. REFERENCES ASHLEY, T. (1979). Specific end-to-end attachment of chromosomes in Ornithogalum virens J. Cell Sd. 38, AVIVI, L. & FELDMAN, M. (1980). Arrangement of chromosomes in the interphase nucleus of plants. Hum. Genet. 55, BARLOW, P. W. & VOSA, C. G. (1969). The chromosomes of Puschkinia libanotica during mitosis. Ckromosoma 37, BENNETT, M. D., FINCH, R. A. & BARCLAY, I. R. (1976). The time, rate and mechanism of chromosome elimination in Hordeum hybrids. Chromosoma 54, BENNETT, M. D. & REES, H. (1969). Induced and developmental variation in chromosomes of meristematic cells. Chromosoma 27, BENNETT, M. D., SMITH, J. B., SIMPSON, S. & WELLS, B. (1979). Intranuclear fibrillar material in cereal pollen mother cells. Chromosoma 71, BENNETT, M. D., SMITH, J. B., WARD, J. & JENKINS, G. (1981). The relationship between nuclear DNA content and centromere volume in higher plants. J. Cell Sd. 47,

11 Chromosome volume and DNA content in barley cells 111 FELDMAN, M., MELLO-SAMPAYO, T. & SEARS, E. R. (1966). Somatic association in Triticum aestrvitm. Proc. natn. Acad. Set. U.S.A. 56, FINCH, R. A. & BENNETT, M. D. (1982). The karyotype of Tuleen 346 barley. Theor. appl. Genet. (In Press.) FINCH, R. A., SMITH, J. B. & BENNETT, M. D. (1981). Hordeum and Secale mitotic genomes lie apart in a hybrid. J. Cell Set. 53, HENEEN, W. K., & CASPERSSON, T. (1973). Identification of the chromosomes of rye by distribution patterns of DNA. Hereditat 74, KEMPANNA, C. & RILEY, R. (1964). Secondary association between genetically equivalent bivalents. Heredity, Lond. 19, MOENS, P. B. & CHURCH, K. (1977). Centromere sizes, positions, and movements in the interphase nucleus. Ckromosoma 61, NISHIKAWA, K. (1970). DNA content of the individual chromosomes and genomes in wheat and its relatives. Rep. Kihara Inst. biol. Res. 23, SIMPSON, E., SNAPE, J. W. & FINCH, R. A. (1980). Variation between Hordeum bulbostm genotypes in their ability to produce haploids of barley, Hordeum vulgare. Z. PflZCcht. 85, STACK, S. M. & BROWN, W. V. (1969). Somatic and premeiotic pairing of homologues in Plantago ovata. Bull. Torrey bot. Club 96, SUBRAHMANYAM, N. C. & KASHA, K. J. (1973). Selective chromosomal elimination during haploid formation in barley following interspecific hybridization. Chromosonia 4a, m-125. WAGENAAR, E. B. (1969). End-to-end chromosome attachments in mitotic interphase and their possible significance to meiotic chromosome pairing. Ckromosoma a6, WALLACE, M. E. & GUNN, R. E. (1965). Affinity in cotton. Heredity, Lond. 20, WELLS, B. (1974). A convenient technique for the collection of ultra-thin sections. Micron 5, (Received 27 January 1982)