Associated chromosomal DNA changes in polyploids

Associated chromosomal DNA changes in polyploids S.N. RAINA, A. P A R I D A , ~ K.K. KOUL,S.S. SALIMATH,~M.S. B I S H T , ~AND V. RAJA Laboratory of Cellular and Molecular Cytogenetics, Department of Botany, University of Delhi, Delhi 110007, India AND

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T.N. KHOSHOO Tata Energy Research Institute, 9 Jor Bagh, New Delhi 110003, India Corresponding Editor: J.P. Gustafson Received August 23, 1993 Accepted February 3, 1994 RAINA, S.N., PARIDA, A., KOUL,K.K., SALIMATH, S.S., BISHT,M.S., RAJA,V., and KHOSHOO, T.N. 1994. Associated chromosomal DNA changes in polyploids. Genome, 37:560-564. The 2C and 4C nuclear DNA amounts were estimated in eight diploid species, belonging to three diverse genera (Vicia, Tephrosia, and Phlox) and their corresponding colchitetraploids. In P. drummondii, T. purpurea, and 7: oxygona tetraploids the deviation from the expectation was highly significant. The DNA in f? drummondii was further discarded in subsequent (C,, C,) generations, thus attaining an overall reduction of about 25%. The DNA content in the subsequent generations was the same as that of C,. It is concluded that rapid DNA loss in the first and subsequent generations was not only associated with the substantial increase (30-66%) in the seed set, but it also helped in the establishment and stabilization of the tetraploid. The possible relationship between such a nucleotypic change and success of polyploids is discussed. The DNA change from the expected value in the f? drummondii tetraploid was achieved by equal decrement to each chromosome independent of size, i.e., small chromosomes loose the same amount of DNA as the large chromosomes. Key words: colchjtetraploid, genome size, DNA loss, seed fertility, stability, DNA distribution. RAINA, S.N., PARIDA, A., KOUL,K.K., SALIMATH, S.S., BISHT,M.S., RAJA,V., et KHOSHOO, T.N. 1994. Associated chromosomal DNA changes in polyploids. Genome, 37 : 560-564. Les quantitCs d'ADN 2C et 4C ont CtC CvaluCes chez huit especes diploi'des appartenant a trois genres diffkrents : Vicia, Tephrosia et Phlox, ainsi que chez les plantes colchitCtraploi'des correspondantes. Chez les tCtraploi'des de f? drummondii, la teneur en ADN des gCnCrations C, et C2 subskquentes a fait l'objet de rejets plus prononcks, atteignant une rCduction d'ensemble d'environ 25%, et la teneur en ADN des gCnCrations subskquentes a CtC la m2me que celle de la C,. La conclusion dCgagCe est qu'une perte rapide d'ADN dans la premiere gCnCration et les subskquentes n'Ctait pas seulement associCe a un accroissement substantiel de la formation de graines (de 30 a 66%), mais elle a Cgalement favoris6 I'Ctablissement et la stabilisation de plantes tCtraploi'des. La relation possible entre une telle sorte de changement nuclCotypique et le succes des polyploi'des est discutCe. Le changement de la teneur en ADN de la valeur attendue chez le f? drummondii tktraploi'de a rCsultC d'une diminution Cgale chez chacun des chromosomes, indkpendamment de la dimension, c.-a.-d. que les petits chromosomes ont perdu la m2me quantitC d'ADN que les gros chromosomes. Mots cle's : colchitCtraploi'de, dimension des gCnomes, perte d'ADN, fertilitC des graines, stabilitC, distribution de I'ADN. [Traduit par la rCdaction]

Introduction The prevalence of polyploidy in angiosperms is estimated to be around 70% (Goldblatt 1980; Lewis 1980). In complete constrast with this situation, the performance of most of the numerous newly synthesized polyploids has consistently fallen short of expectations, and their contribution in plant improvement has made little impact (Dewey 1980). The multiplication of the same or different genomes in the formation of a polyploid is a major mutational event of far-reaching consequences on the nucleotype, physiology, chemistry, and genetics of the material. According to Stebbins (1980) the quantitative change in chromosome number and nuclear DNA is only one of a series of complex processes that must take place for polyploidy to be successful in nature. 'corresponding author. 'present address: M.S. Swaminathan Research Foundation, Taramani, Madras 600 113, India. 'present address: Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, U.S.A. 4 ~ r e s e naddress: t Biotechnology Centre, Indian Agricultural Research Institute, New Delhi 110012, India. Printed in Canada / lrnprirnk au Canada

Multidisciplinary approach, therefore, might well explain why we have been unable to duplicate the successful polyploid evolution that is so ubiquitous in nature. Significant reduction in genome size in one (XTriticosecale) of the most promising man-made amphidiploids (Stebbins 1956), for example, gave a much better agronomic performance (Bennett 1985). Similar reports of concomitant DNA loss with increase in ploidy level might well be considered to be of primary importance in evolutionary and adaptational change in many successful polyploids, and this, according to Darlington (1958, 1963), is an adaptive adjustment of polyploids to resolve the nucleocytoplasm balance near the diploid possibly by the loss of bits of duplicated material. The amount of data available on genome size of polyploids vis-a-vis their corresponding putative diploids is limited and often contradictory. Such studies are, however, difficult to do in the light of more recent realization of enormous intraspecific DNA variation (4-288%) (Bennett 1985) and instances of multiple origin of polyploids (Soltis and Soltis 1990). There is, therefore, no certainity that the diploids used for comparison with tetraploid are similar, let alone identical, in DNA content to the true diploid ancestors of

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RAINA ET AL.

TABLE1. Nuclear DNA amounts in 2C nuclei Species

Material

2n

TABLE2. Mean nuclear DNA amounts in 2C and 4C P. drummondii nuclei

Mean DNA amount ( X 10-l2 g) DNA amounts ( ~ 1 0 - l g) 2

T. villosa

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Material

2n

2C

4C

Mean % deviation of the observed from expected

c 2

T. multiflora

2x

c0 c1 c 2

T. purpurea

2x

co c1 c2 2x co c1

T. polystachya

c 2

V monantha

2x

V atropurpurea

c0 2x c0

the polyploids. Moreover, without being certain of the pedigree of the large majority of natural polyploids, one cannot do such investigations. The other course, although with limitations, would be to monitor change(s), if any, in induced polyploids in primary (C,) and subsequent generations. We therefore decided to conduct experiments on eight diploid species, belonging to three diverse genera, whose DNA amounts (2.54-17.95 pg) differed markedly from one another. The present investigations have not only shown that direction of DNA change in polyploids is species specific, but it has also added new dimensions in polyploid evolution and improvement.

Materials and methods

Materials The list of the species utilized in the present investigation is given in Tables 1 and 2. The seeds of Phlox drummondii (2n = 2x = 14) cv. White Tall were obtained from Sluis and Groot, Eukhuizen, Holland. The seeds of Tephrosia (2n = 2x = 22) and Vicia (2n = 2x = 14) species were supplied by United States Department of Agriculture (USDA) and International Centre for Agricultural Research in Dry Areas (ICARDA), Aleppo, Syria, respectively.

Methods Induction of polyploidy For colchicine treatment two methods (cotton swab, cotyledonary leaf immersion) were employed. For Tephrosia species and P. drummondii, sterilized cotton swabs soaked either in 0.1, 0.15, or 0.2% aqueous colchicine (Sigma Chemical Co., St. Louis, Mo.) were placed on the emerging shoot tip between two cotyledonary leaves for 1-3 days with 4 or 6 h duration each day. Colchicine was added drop by drop at some intervals to the cotton swabs to avoid increase in the colchicine concentration and (or) to check air block(s). In Vicia species cotyledonary leaves along with the emerging shoot tips were immersed in three concentrations (0.1, 0.15, and 0.2%) of aqueous colchicine, and roots along with hypocotyl were placed between moist cotton

layers to avoid drying. The seedlings kept as such for 6 or 8 h for 1 day and 12 h, equally spread over 2 days, were washed in running tap water for 24 h and then planted in pots. Cytology Meiotic preparations were made from the anthers fixed in acetic alcohol (1:3) for at least 24 h and stained in the usual manner in Feulgen reagent. DNA estimation The method used for DNA measurement in 2C and 4C nuclei was carried out as described previously (Raina and Rees 1983a; Raina et al. 1986) using primary root tips fixed in ice-cold, phosphate-buffered 4% formaldehyde at pH 7.0 for 2 h, followed by washing in running tap water for 24 h. They were then fixed in acetic alcohol (1:3), hydrolyzed in 5M HCl for 1 h at room temperature, and stained in Feulgen (Sigma) solution (pH 2.2) for 1 h. The stained material was washed in three changes of SO2 water for 10 min each and squashed firmly in a drop of 50% glycerol. The batch of slides, constituting three replicates each of the standard, were converted from extinction (arbitrary) units into picograms (Allium cepa: 2C = 33.5 pg) (Bennett and Smith 1976), and the four samples were scanned using a Vickers M86 scanning microdensitometer in 4 days. Estimation of the DNA amounts of individual metaphase chromosomes in P drummondii complements was carried out as previously described (Raina and Rees 1983a; Raina and Bisht 1988) by taking photometric measurements of each of the Feulgen-stained chromosomes within the complement utilizing an adjustable rectangular mask. Knowing the total nuclear DNA amount in picograms from the scanning of nuclei, as described above, arbitrary values for individual chromosomes were readily converted into picograms.

Observations Efficiency of colchicine Induction of tetraploidy in I? drummondii and Tephrosia and Vicia species was achieved using 0.15% colchicine for 12, 18, and 12 h, respectively. Meiosis a n d seed set In the control (diploid taxa) meiosis was perfectly normal. The tetraploids (C,) (barring 30-60% unequal distribution at A1 in T. villosa, T. polystachya, and T. purpurea) of the eight species did not show much variation in the meiotic details. They showed a much lower frequency of quadrivalents per cell, ranging from 2.76 in I? drummondii (2n = 4x = 28) to 5.72 in T. oxygona (2n = 4x = 44), than many autotetraploids (see Morrison and Rajhathy 1960; Gottschalk 1978). Most of the quadrivalents being of ring or chain configuration would, because of greater flexibility (Jones 1964; Darlington 1958), disjunct normally at MI. The seed set, however, showed considerable variation, ranging from about 30% in P. drummondii to complete seed sterility in the

GENOME, VOL. 37, 1994

TABLE3. The mean DNA amounts (pg) of individual metaphase chromosomes (in decreasing order of size) in P. drummondii

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Chromosomes Material

I

II

111

IV

V

VI

VII

Diploid Diploid (polysomatic cell) Tetraploid (C,)

2.95

2.89

2.78

2.62

2.45

2.22

2.04

2.95 2.29

2.85 2.22

2.80 2.12

2.61 2.01

2.51 1.85

2.20 1.70

2.02 1.54

2 Mean D N A

3 (X~O-~'~)

FIG. 1. The DNA amounts in chromosomes of the haploid and tetraploid (C,; x) f? drummondii complements of diploid (0) plotted against mean DNA. tetraploids of two Vicia species. The colchitetraploids of P. drummondii showed a significant increase in seed set in the second (43%) and third (66%) generations. No such trend was observed in the tetraploids of Tephrosia species. Genome size Tables 1 and 2 present estimates of nuclear DNA content in induced tetraploids. In three species the absorption estimates obtained were significantly (P 5 0.001) higher (T oxygona and T purpurea) or lower (P. drummondii) than expected for cells at the tetraploid (C,) levels. The DNA loss in P. drummondii was consistent in that the average 2C and 4C values in different C, plants was lower by 16.70% than expected. The DNA was further discarded in two subsequent (C C,) generations, thus attaining an overall reduction of about 25%. The drop in DNA content from C, to C , was 6.6%, while it was only 2.75% from C , to C, (Table 2). The DNA content in C, and C, plants corresponded to the values estimated for C, plants. The DNA amounts determined for subsequent generations (C,, C,) in Tephrosia species did not show marked deviation from the C, plants (Table I). The tetraploid-derived callus cells in I? drummondii, exhibiting complete uniformity in the chromosome number, also had DNA amounts corresponding to that of the tetraploid parent plants (Raja et al. 1992).

,,

The distribution of DNA change The important question that arises is the way in which nuclear DNA change in the colchitetraploids of P. drummondii is achieved within the chromosome complement. To determine the nature of such change, we decided to estimate relative absorption values of the individual chromosomes in the diploid and tetraploid (C,) complements and a rare polysomatic complement in the diploid root tip. This led to

the finding that there was a substantial fall in the absorbance values in all the 28 chromosomes (constituting four homologous chromosomes in each of the seven groups) in the tetraploid complement (Table 3), ranging from 23.05 to 24.49% with an overall mean (23.53%) corresponding to the total percentage DNA loss in C, plants. On the other hand, the DNA difference between the corresponding chromosomes in the diploid and polysomatic complements was negligible. Figure 1 shows the distribution of DNA amounts in the haploid set of chromosomes within diploid and tetraploid complements. The slopes are virtually parallel with no significant heterogeneity of regression between the slopes. This is indicative of the fact that the DNA loss of about 25% was achieved by an equal decrement in all chromosomes within the complement.

Discussion The present manuscript reports rapid DNA loss to the adaptive advantage in the establishment of colchitetraploids in P. drummondii. Earlier studies have shown that the rapid DNA deviation from the expected values is not uncommon (see Walbot and Cullis 1985). In particular, the results of Evans (1968) and Cullis (1983) in flax show that heritable changes of about 8% (gain or loss in DNA content) could be induced, by specific environmental conditions, in the ongoing generation itself, as well as in first 5 weeks of the growth period. Similarly, in Microseris, the interspecific (F,) hybrid plants did not show identical DNA content (Price et al. 1983). The seedlings obtained from seeds collected off of different portions of single heads from plants belonging to a selfed line of Helianthus annuus had different DNA contents (Cavallini et al. 1986). The instances of rapid DNA change have also been observed to occur in in vitro conditions (Larkin and Scowcroft 1983). Apart from such a rapid change, there are reports of DNA loss in long-established polyploids as well (Christensen 1966; Becak et al. 1967; Southern 1967; Grant 1969; Dowrick and El Bayoumi 1969; Yamaguchi and Tsunoda 1969; Pai and Swaminathan 1960; B haskaran and Swaminathan 1960; Upadhya and Swaminathan 1963). Further, there is overwhelming evidence to suggest that many polyploids have attained, during evolution, a smaller chromosome size of adaptive value (Darlington 1958; Stebbins 1950) by loosing DNA within chromosomes (Darlington 1963; Bennett 1985; Webster and Buckner 197 1; McWilliam 1974; Cauderon 1977). The polyploidy success comes more readily in plants that benefit most or suffer least from the two universal effects of induced polyploidy, increased cell size, and reduced fertility (Dewey 1980). The present study is in agreement with the conclusions reached by Morrison and Rajhathy (1960), Gottschalk (1978), and Evans and Davies (1983) that there is no evidence to indicate that chromosomal nondisjunction

R A I N A ET AL.

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is the only source for infertility in tetraploids. The reason(s) may, therefore, lie elsewhere. The nature of .the DNA gained through polyploidy is quite clear. The genetic and nucleotypic consequences of this DNA gain are also quite well understood. On one hand the multiplication of gene sequences is reflected directly by multiplication of gene products (Ohno et al. 1968; Becak 1969) and on the other hand the increase in DNA content with corresponding increases in the cell size might influence the rate and duration of various physiological and developmental processes; such alterations of profound nature might have an adverse effect (including seed set) on the neopolyploid. The present results suggest that such alterations are partly neutralized by the remarkably inherent capacity in the I? drummondii genome to bring about rapid adaptational change in nuclear DNA content and thereby leading among other things to good seed set. This is further supported by the fact that given the same set of conditions with no appreciable change in parameters other than DNA values, .the seed fertility increases substantially in subsequent generations by further loss in the DNA amount. Such kind of nucleotype change, including (possibly) triggering the expression of advantageous physiological processes and (or) functional derepression of previously silent structural genes in the neopolyploids (Tal and Gardi 1976; Tal 1977; Levy 1976), might lead to increased genetic and physiological adaptability for better performance, and consequently upon establishment of I? drummondii tetraploids. While details about the tetraploid level being the optimum ploidy level in I? drummondii and that the tetraploid is a better ornamental than the diploid cytotype is not described here, it is worthwhile mentioning that the tetraploids now in the sixth generation are very well established and stable. Further, a sudden change from the profound chromosomal instability in the diploid derived calli to a conservative mode of cell multiplication in the tetraploid (Raja et al. 1992) assumes significance in the sense that the changed nucleotypic and (possibly) genetic conditions might have enabled the neopolyploid to withstand in vitro stress and thereby lead to a favourable set of environmental and (or) nutritional conditions for a more stable mitotic condition. Another remarkable feature about the tetraploid cytotype of I? drummondii is that the 25% DNA loss is achieved by equal DNA decrement in all the chromosomes, and the balance, therefore, is achieved by a change in all, not some, of the chromosome complement. This is in agreement with the conclusions reached by Seal and Rees (1982), Narayan and Durrant (1983), Raina and Rees (1983a, 1983b), Kenton et al. (1990), and Parida et al. (1990). This is probably the first report giving direct evidence about such a well-defined and rigid pattern of change in the relative size of chromosomes within the neocomplement, which is in some way of adaptive significance (Mizuno and Macgregor 1974; Bennett 1982; Seal and Rees 1982).

Acknowledgements The financial support provided by CSIR and DBT, New Delhi, is gratefully acknowledged. Thanks go to Sluis and Groot, USDA, and ICARDA for providing seed samples. Becak, W. 1969. Gene action and polymorphism in polyploid species of amphibians. Genetics, 61: 183-190. Becak, W., Becak, M.L., Laville, D., and Schrieber, G. 1967.

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