THE EFFECT OF CHROMOSOMES 5B, 5D, AND 5A ON CHROMOSOMALPAIRING IN TRITICUM AESTIVUM*

BY MOSHE FELDMAN

CURTIS HALL, UNIVERSITY OF MISSOURI, COLUMBIA

Communicated by E. R. Sears, April 14, 1966

The common wheat Triticum aestivum is an allohexaploid (2n = 6X = 42) con-sisting of three different genomes, A, B, and D. Genome A was derived from thediploid wheat, T'. monococcum," 2 genome B from T. speltoides (= Ae. speltoides),or from a close relative of this species,3' 4 and genome D from T. tauschii (= Ae.squarrosa).5-7 (The nomenclature used is after Morris and Sears.8) In hybridsbetween these diploid ancestors, considerable chromosome pairing (in some cellseven complete pairing) is observed regularly,9-"1 indicating that the homoeologouschromosomes of the diploids are still very closely related.In spite of this close relationship, T. aestivum behaves like a typical allopolyploid

and forms only bivalents at meiosis. No association takes place between the homoe-ologous chromosomes. Even in haploids there is very little pairing between homoe-ologues.'2The failure of the homoeologous chromosomes to pair is due to suppression of pair-

ing by chromosome 5B.12-14 In plants nullisomic for chromosome 5B, multivalentassociations are formed regularly which involve homoeologous chromosomes of allthe three genomes."5 Similarly, haploids deficient for chromosome 5B exhibit al-most the same amount of pairing as is expected from the pairing observed in hybridsbetween the three diploid ancestors, T. monococcum, T. speltoides, and T. tauschii."1Although genes for synapsis are known on other chromosomes, e.g., 3B (III) and

2A (IJ),16 none of these differentially affect pairing of homoeologous chromosomes.This activity is restricted to the long arm of chromosome 5B (5BL).17From the fact that no alteration in chiasma frequency was found in nullisomic 5B,

Riley'2 concluded that 5B interferes with the processes which lead to synapsis,decreasing the attraction to such a level that only pairing between fully homologouschromosomes can take place.The results of Okamoto'8 and Sears and Okamoto"3 indicate that under certain

circumstances chromosome 5B can also reduce pairing between homologous chromo-somes. The expected 14 bivalents (or their equivalent) were found in T. aestivum(AABBDD) X monococcum-tauschii (AADD) when 5B was absent, but only about10 bivalents when 5B was present.Study of chromosomal pairing in plants having different doses of the long arm of

chromosome 5B and of the homoeologous chromosomes 5A and 5D has shown thatthe gene or genes concerned are operative at a different time than previously sup-posed.

Materials and Methods.-Lines of T. aestivum var. Chinese Spring monosomic forisochromosomes of the long arm of chromosomes 5A, 5B, and 5D were kindly pro-vided by E. R. Sears. From these mono-isosomic lines, plants with four doses ofthe long arm of each chromosome (di-isosomics) were obtained through selfing,and these in turn gave rise to plants with six doses (tri-isosomics).The material was grown under normal greenhouse conditions except as otherwise

1447

1448 GENETICS: M. FELDMAN PROC. N. A. S.

specified. Spikes were fixed in Carnoy's solution and the anthers squashed in aceto-carmine.

In addition, cytological preparations were made available by E. R. Sears fromplants which were, respectively, nullisomic 5D, nullisomic 5D trisomic 5A, and nulli-somic 5D tetrasomic 5A.Results.-Chromosomal pairing at first meiotic metaphase in pollen-mother cells

was normal in all the mono- and di-isosomic combinations and in tri-isosomic 5ALand 5DL (Table 1). Twenty bivalents, mostly rings, were formed almost regu-larly, and the average number of chiasmata per cell was 40 or more except in di-iso5BL, where it was 37.7. The various arms of the isochromosomes paired to make abivalent or two univalents in the di-isosomics; and in tri-isosomic 5AL and 5DLthey appeared as a trivalent (sometimes ring), a bivalent, and a univalent, or threeunivalents. A univalent isochromosome usually appears as a small ring due topairing between its two arms.

In tri-isosomic 5BL, in contrast, the chromosomal pairing was much reduced(Table 1, Fig. 1). Asynapsis was found consistently in the four tri-isosomic 5BL

TABLE 1MEAN AND RANGE OF CHROMOSOMAL PAIRING AT FIRST METAPHASE OF T. aestivum

VAR. Chinese Spring WITH DIFFERENT DOSES OF THE LONG ARM OF CHROMOSOMES 5B AND 5DChromosomal No. Uni- - Bivalents Tri- No. Xtaconstitution 2n cells valents Rod Ring Total valents per cell

Disomic 42 30 0.06 2.10 18.87 20.97 0.00 43.40(0-2) (0-4) (17-21) (20-21) (40-47)

Tri-isosomic 5DL 43 30 1.67 1.94 18.23 20.17 0.33 42.20(0-3) (0-6) (14-21) (20-21) (0-1) (39-46)

Mono-isosomic 5BL 41 30 1.13 2.97 16.96 19.93 0.00 40.30(1-3) (0-7) (13-20) (19-20) (34-43)

Di-isOSOmic5BL 42 30 1.14 4.30 16.13 20.43 0.00 37.70(0-6) (1-8) (13-20) (18-21) (33-41)

Tri-isosomic 5BLPlant 1 43 50 14.20 7.34 6.34 13.68 0.48 21.36

(2-39) (1-13) (0-14) (2-19) (0-1) (2-34)Plant 2 43 50 16.74 7.74 4.88 12.62 0.34 18.80

(1-39) (2-12) (0-16) (2-21) (0-1) (2-36)

plants examined. (Detailed counts were made in only two plants.) The number ofunivalents varied greatly, from two or four in a very few cells to 20 or more in manycells. The bivalents were mostly rods with one, usually terminal, chiasma. Theaverage number of chiasmata per cell was 20 (from 2 to 36).

In many cells the three isochromosomes paired and formed a chain or ring tri-valent, or a bivalent and univalent (Table 1, Fig. 1). In a few cells they appeared asthree ring univalents showing interarm pairing. Because the isochromosomes wereindistinguishable from other univalents when they failed to pair intrachromosomally,it was impossible to determine their exact frequency of pairing with each other.However, there was no clear evidence that pairing of the 5B arms was affected anydifferently than the pairing of other chromosomes.Although it is difficult to analyze earlier stages of meiosis in T. aestivum, a few

observations were made at prophase. In tri-isosomic 5DL, only double strands wereseen at late pachytene and mostly bivalents at diplotene and diakinesis. (The iso-chromosomes appeared sometimes as univalents.) But in late pachytene of tri-

VOL. 55, 1966 GENETICS: M. FELDMAN 1449

_ .I~~~~'4o4

B

f

C DFIG. 1.-Meiotic first metaphase in Triticum aestivum var. Chinese Spring having six doses

of the long arm of chromosome 5B (tri-isosomic 5BL). (A) A cell showing much asynapsis.The three isochromosomes are paired as a chain trivalent at right. The central rod bivalent isheteromorphic, presumably involving homoeologous chromosomes. (B) A ring trivalent of thethree isochromosomes of 5BL. (C) A quadrivalent at right presumably representing pairingbetween homoeologous chromosomes. The three isochromosomes appear as ring univalents(indicated by arrows). (D) Interlocking of three ring bivalents (indicated by arrow).

isosomic 5BL single strands as well as double strands were seen, and at diplotene anddiakinesis many univalents were counted. Clearly, there was partial failure ofsynapsis in tri-iso 5BL.

In each of the three tri-isosomic 5BL plants examined, multivalent associationsindicating pairing between homoeologous chromosomes were observed at firstmetaphase (Fig. 1C). The multivalents, mostly tri- and tetra- and very rarelypentavalents, were formed in very low frequency, one per 100 or more cells. Hetero-morphic rod bivalents, were also observed (Fig. 1A).

1450 GENETICS: M. FELDMAN PROC. N. A. S.

Frequent interlocking of ring and rod bivalents, of two rings, and even of threerings was seen in tri-isosomic 5BL (Fig. iD). This was in spite of the large reductionin the number of bivalents (Table 1).

Multivalent associations and frequent interlocking, as well as asynapsis, were alsofound in di-isosomic 5BL plants which had been placed several days before meiosisat high (300C) and low (150C) temperatures. These extreme temperatures did notincrease the amount of asynapsis in tri-isosomic 5BL.The one available plant nullisomic for chromosome 5D exhibited a degree of asyn-

apsis. Two, four, and rarely more univalents were counted in many cells. Inaddition, rare multivalent associations (trivalents and quadrivalents) and frequentinterlocking bivalents were observed.

Plants nullisomic for chromosome 5D and trisomic for chromosome 5A showed asimilar picture in first metaphase; many cells exhibited interlocking bivalents and asmall amount of asynapsis. However, a plant nullisomic for 5D and tetrasomic for5A showed nearly regular pairing, with rare univalents and almost no interlockingbivalents. It seems that with respect to pairing, four doses of 5A compensate forthe nullisomic condition of 5D.

Discussion.-It is generally assumed that the chromosomes are distributed atrandom in the premeiotic nucleus. However, some doubt is cast on this assumptionby the demonstration by Kasha and Burnham"9 that pairing in barley is initiatedregularly in the terminal parts of the chromosomes. With pairing commencing atthe chromosome ends, bivalents formed from randomly distributed homologuesshould frequently be interlocked, but this is not the case.

Difficulty is also encountered in understanding the action of chromosome 5B inwheat if the premeiotic chromosomes are randomly distributed. If two doses of the5B gene simply reduce the attraction forces between homoeologues (and also homo-logues, to a lesser extent) as Riley"2 suggested, then by chance alone two homoe-ologues may be so much closer together than either is to its homologue that eventhe reduced attraction forces will be strong enough to cause them to pair. Suchhomoeologous pairing is almost never observed in disomic T. aestivum.Although extra dosage of the long arm of chromosome SB reduced the pairing of

fully homologous chromosomes as would be expected on the basis of Riley's hy-pothesis, it also induced pairing between homoeologous chromosomes and caused ahigh frequency of interlocking bivalents. Therefore, the action of the 5B gene isnot simply to reduce chromosomal attraction at meiotic prophase.The apparently paradoxical effect of extra dosage of 5BL can be explained in the

following way: in plants nullisomic for chromosome 5B the chromosomes are notrandomly scattered in the premeiotic nucleus, but homoeologues as well as homo-logues lie near each other and may perhaps even associate throughout most of theirlength. The meiotic pairing follows as a second step and brings the closely oriented(or loosely paired) homologous segments to a full and more intimate contact.

It may be assumed that the 5B gene suppresses the premeiotic association andthus tends to cause random distribution of chromosomes in the premeiotic nucleus.Two doses of this gene, while scarcely affecting the homologous chromosomes, keepthe homoeologues apart. This leads to exclusively homologous pairing. The homoe-ologues do show secondary association,20 indicating that two doses of 5B do notcompletely prevent them from approximating each other.

VOL. 55, 1966 GENETICS: M. FELDMAN 1451

Six doses of the 5B gene (in tri-isosomic 5BL) are assumed to suppress premeioticassociation completely. This leads to a random distribution of chromosomes inwhich the homologues may presumably be separated by a distance of as much asseveral microns. The meiotic attraction forces which usually cause pairing of veryclosely oriented homologues are not sufficient to bring about pairing of such distantlyseparated homologues, and as a result many chromosomes fail to pair. Since homoe-ologous chromosomes may by chance lie close to each other, homoeologous pairingcan take place. Although multivalent associations were rare in first metaphase oftri-isosomic 5BL because of the general reduction of pairing, heteromorphic rodbivalents occurred which were presumably also the result of homoeologous pairing.Finally, the wide occurrence of interlocking bivalents, in spite of the reduction in thetotal number of bivalents, shows clearly that some of the pairing here was betweenwidely separated chromosomes.Four doses of 5BL affected pairing only slightly in optimal temperature. This

effect was much increased in high and low temperatures, with the result that asyn-apsis, homoeologous pairing, and interlocking bivalents were quite common. Be-cause higher temperature did not increase the effect of six doses of 5BL, it is assumedthat the maximum possible effect, complete suppression of premeiotic association,was brought about by six doses at normal temperature.Although one or both of the homoeologues of chromosome 5B might also be ex-

pected to have a gene affecting synapsis, six doses of the long arms of 5A and 5D didnot provide evidence for the existence of such genes. However, from the fact thatnullisomic 5D is partially asynaptic and yet exhibits homoeologous pairing and in-terlocking bivalents, it seems clear that this chromosome does carry a gene condi-tioning premeiotic association. Its effect is inverse to that of the 5B gene.

Since four doses of 5A compensate for the absence of 5D, it seems likely thatchromosome 5A also has a gene which promotes premeiotic association. However,since three doses of 5A do not compensate for nullisomic 5D, the 5A gene musthave a weaker effect. If it were as strong as the 5D gene, nulli-5D tri-5A would beequivalent to mono-5D and would therefore have normal synapsis.

Plants mono-isosomic for the long arms of chromosomes 5D and 5A have regularpairing. Thus, the synapsis genes of these chromosomes are located on their longarms, like that of 5B.17 Since the long arms of 5A, 5B, and 5D are homoeologous,'all three genes are probably derived from one ancestral locus.Every diploid species in the wheat group may be assumed to have a gene similar

to the one on chromosome 5D which promotes premeiotic association. The genesin the different species evidently differ in potency, for it was founid2l that the genomesof T. speltoides and T. tripsacoides (Ae. mutica) are much more effective than thoseof other diploids in reducing the effect of chromosome 5B. The first tetraploidwheat, believed to have been a doubled hybrid of T. monococcum and T. speltoides,presumably had homoeologous pairing and resultant meiotic irregularity until amutation occurred on chromosome 5B to suppress the homoeologous pairing.4 Thesimplest assumption is that the gene on 5B which promoted premeiotic associationmutated to a form with the opposite effect.The conclusion that premeiotic association serves as an essential step in regulating

meiotic pairing invites reconsideration of certain assumptions concerning chromo-some behavior. Oite of these is the existence of long-range forces. Since it has

1452 GENETICS: M. FELDMAN PROC. N. A. S.

been assumed that homologues are randomly distributed throughout the nucleus,the existence of a mechanism capable of bringing homologous segments togetherover long distances has been widely accepted. However, the fact that in T. aestivumwith six doses of 5BL the presumably randomly distributed chromosomes fail to pairsuggests that the meiotic forces do not operate effectively at long range.

Smith22 thought that synapsis begins at the preceding anaphase and terminatesat late pachytene. However, premeiotic association and meiotic pairing should beconsidered as two distinct phenomena which are conditioned by different factors.This is clearly apparent from the fact that the 5B gene suppresses only the pre-meiotic association.

It may be that association of homologues (and homoeologues in nulli-5B) takesplace in all the somatic cells throughout the life of the plant. However, this associ-ation may be difficult to observe, for it may be more pronounced at interphase thanduring division stages. Close association during interphase would bring homologousgenes into proximity at the time when they are most active, and this might be ad-vantageous. During mitosis the chromosomes move and divide, and as a con-sequence may become more scattered. Yet somatic association was found individing cells in tissue culture of Haplopappus gracilis,23 and it has been shown thathomologous human chromosomes tend to lie closer to each other on the metaphaseplate than expected by chance.24

It has been suggested25 that the chromosomes occupy definite positions in theinterphase nucleus. It is possible that they are attached by their centromeres ortelomeres in a specific order to the nuclear membrane26' 27 with homologues lyingclose to each other. Support for this assumption comes from reports28-30 that thechromosomes take an active part in the formation of the nuclear membrane inmitotic telophase.Summary.-In normal dosage, the long arm of chromosome 5B (5BL) prevents the

pairing of homoeologues. In four doses (di-isosomic 5BL) it slightly reduces chiasmafrequency between homologues. In six doses (tri-isosomic 5BL) it causes consider-able asynapsis of homologues but permits some pairing between homoeologues andinduces a high frequency of interlocking bivalents. The explanation is advancedthat 5BL regulates the premeiotic association of homologous and homoeologouschromosomes. Two doses of 5BL suppress partially this premeiotic association,especially that of the homoeologues. Six doses of 5BL suppress all the premeioticassociation, causing random distribution of the chromosomes in the premeioticnucleus. The meiotic attraction forces are not strong enough to cause pairing of thedistantly separated chromosomes, and thus partial asynapsis results. Under theseconditions, homoeologous pairing takes place when two homoeologues happen to becloser to each other than to their respective homologues. Pairing between sepa-rated chromosomes results in a high frequency of interlocking bivalents.Chromosomes 5D and 5A have an effect on premeiotic association opposite to that

of their homoeologue 5B. It is assumed that the 5B gene was derived as an anti-morphic mutation from an association-promoting gene like those carried by 5D and5A.

The author is grateful to Professor E. R. Sears for his encouragement and advice during thecourse of t.his work and for his invaluable contribution in the interpretation of the data and the

VOL. 55, 1966 GENETICS: JOHNSON AND BACH 1453

preparation of the manuscript. Thanks are also due to Drs. L. M. Steinitz-Sears and T.Mello-Sampayo for their critical reading of the manuscript.

* This work was supported by a grant from the National Science Foundation to Dr. E. R.Sears. Journal series no. 3085 of the Missouri Agricultural Experiment Station. Approved bythe Director.

1 Kihara, H., Botan. Mag. (Tokyo), 32, 17 (1919).2 Sax, K., Genetics, 7, 513 (1922).3 Sarkar, P., and G. L. Stebbins, Am. J. Botany, 43, 297 (1956).4 Riley, R., J. Unrau, and V. Chapman, J. Heredity, 49, 91 (1958).5 McFadden, E. S., and E. R. Sears, Rec. Genet. Soc. Am., 13, 26 (1944).6 McFadden, E. S., and E. R. Sears, J. Heredity, 37, 81 (1946).7 Kihara, H., Agqri. Hort. (Tokyo), 19, 889 (1944).8 Morris, R., and E. R. Sears, in Wheat and Wheat Improvement, ed. K. S. Quisenberry and

L. P. Reitz, in press.I Sears, E. R., Res. Bull. Missouri Agri. Exptl. Sta., 337 (1941).10 Mochizuki, A., and M. Okamoto, Chromosome Inform. Serv., 2, 12 (1961).11 Kimber, G., and R. Riley, Can. J. Genet. Cytol., 5, 83 (1963).12 Riley, R., Heredity, 15, 407 (1960).13 Sears, E. R., and M. Okamoto, Proc. Intern. Congr. Genet., 10th, 2, 258 (1958).14 Riley, R., and V. Chapman, Nature, 182, 713 (1958).15 Riley, R., and C. Kempanna, Heredity, 18, 287 (1963).16 Sears, E. R., Res. Bull. Missouri Agri. Exptl. Sta., 572 (1954).17 Riley, R., V. Chapman, and G. Kimber, Nature, 186, 259 (1960).18 Okamoto, M., Wheat Inform. Serv., 5, 6 (1957).19Kasha, K. J., and C. R. Burnham, Can. J. Genet. Cytol., 7, 620 (1965).20 Kempanna, C., and R. Riley, Heredity, 19, 289 (1964).21 Riley, R., and V. Chapman, Nature, 203, 156 (1964).22 Smith, S. G., Can. J. Res., 20, 221 (1942).23 Mitra, J., and F. C. Steward, Am. J. Botany, 48, 358 (1961).24 Schneiderman, L. J., and C. A. B. Smith, Nature, 195, 1229 (1962).21Mirsky, A. E., and S. Osawa, in The Cell, ed. J. Brachet and A. E. Mirsky (New York:

Academic Press, 1961), vol. 2, p. 695.26 Sved, J. A., Genetics, 53, 747 (1966).27Vanderlyn, L., Botan. Rev., 14, 270 (1948).28 Barer, R., S. Joseph, and G. A. Meek, Exptl. Cell Res., 18, 179 (1959).29 Porter, K. R., and R. D. Machado, J. Biophys. Biochem. Cytol., 7, 167 (1960).30 Ito, S., J. Biophys. Biochem. Cytol., 6, 433 (1960).

THE ANTIMUTAGENIC ACTION OF POLYAMINES: SUPPRESSIONOF THE MUTAGENIC ACTION OF AN E. COLI MUTATOR GENE

AND OF 2-AMINOPURINE

BY HERBERT G. JOHNSON AND MICHAEL K. BACH

DEPARTMENT OF BIOCHEMISTRY, THE UPJOHN COMPANY, KALAMAZOO, MICHIGAN

Communicated by C. B. van Niel, April 21, 1966

In a recent paper' we reported that the presence of nontoxic concentrations ofspermine during growth of cultures of Escherichia coli markedly reduced the spon-taneous mutation rate to streptomycin resistance and decreased the number ofrevertants in a tryptophan-requiring mutant. Similar conditions also markedlyreduced the mutation rate when caffeine or ultraviolet light were used as mutagenic