The Relationship Between Chiasmata and Crossing Over in Triticum Aestivumi

THE RELATIONSHIP BETWEEN CHIASMATA AND CROSSING OVER IN TRITICUM AESTIVUMI

T. K. FU* AND E. R. SEARSS Department of Agronomy, University of Missouri, Columbia, Missouri 65201 Manuscript received March 19, 1973 Revised copy received June 8, 1973

ABSTRACT Telocentrics for the p arm of chromosome 4A and the long arm of 6B were used as cytological markers for the determination of chiasma frequency. In concomitant studies of recombination, terminal segments of rye and T. umbellulatum chromatin carrying Hp (Hairy peduncle) and Lr9 (Leaf-rust resistance), respectively, marked 4A and 6B. Two temperatures, 21" and 32", were used for bgth the 4A and 6B experiments.--Only one chiasma was observed in each heteromorphic bivalent. Because there was a substantial reduction in pairing between diakinesis and metaphase I, all determinations of chiasma frequency were made at diakinesis. In the 21" experiments, agreement was good between genetic recombination and cytological prediction on the basis of the partial chiasmatypy hypothesis that each chiasma represents a crossover. At 32" both chiasma frequency and crossing over, but particularly the latter, were strongly reduced. The fewer crossovers than expected are explained in part hy stickiness of chromosomes at the high temperature, sometimes resultkg in adjacent chromosomes being wrongly scored as having a chiasma, and in part by premetaphase disjunction of some recombined bivalents and subsequent independent behavior of the two resulting univalents.-Male transmission of the 4A telocentric from the heteromorphic bivalent was un- usually high: 51 % at 21" and 31% at 32".

0 explain chiasma formation and the pairing relations of the chromatids, two hypotheses have been advanced: the partial chiasmatypy hypothesis (JANS- SENS 1909,1924), which holds that each chiasma is the consequence of a previous crossover between nonsister chromatids; and the classical hypothesis (MCCLUNG 1927; SAX1930), which maintains that chiasmata are mere changes of partner resulting from alternate opening out of sister and nonsister chromatids from the pachytene bundle of four. With the partial chiasmatypy hypothesis, there is a one-to-one correspondence between chiasmata and crossovers; whereas with the classical hypothesis there is no such relation, for crossovers occur only when two of the four chromatids at a chiasma break and rejoin.

Cooperative investigations of the Department of Agronomy, Missouri Agriculmal Experiment Station, and the Agri- cultural Research Service, US. Department of Agriculture. Paper No. 6591 of the Journal Series of the Missouri Station. The material herein represents part of a dissertation presented by the senior author in partial fulfillment of the require- ments for the degree of Doctor of Philosophy at the University of Missouri, Columbia. * Presently Geneticist, VA Hospital, Wilshire and Sawtelle Blvds., Los Angeles, California 90073. Geneticist, Agricultural Research Service, U.S. Department of Agriculture.

Genetics 75 : 231-246 October, 1973 232 T. K. FU AND E. R. SEARS Although the partial chiasmatypy hypothesis has come to be widely accepted, the evidence favoring it over the classical hypothesis is not entirely conclusive. It is supported by studies on heteromorphic bivalents (DARLINGTON1936; BROWN and ZOHARY1955; DRISCOLLand SEARS1965), reciprocal translocations ( NODA 1960, 1967). and a dicentric chromosome (STEINITZ-SEARSand SEARS1953), but not by observations on male Drosophila (KAUFMAN1934; COOPER1949). Con- flicting evidence has been reported from double-interlocking bivalents (favoring partial chiasmatypy: MATHER1933; favoring the classical hypothesis: MAT- SUURA 1944), paracentric inversions (favoring partial chiamatypy : DARLINGTON 1936 and BROWNand ZOHARY1955; favoring classical: MATSUURA1950 and HAGA1953), and autoradiography (favoring partial chiasmatypy: CHURCHand WIMBER1969 and PEACOCK1970, 1971; favoring classical: TAYLOR1965 and MOENS1966). Almost all previous studies have been of two types, neither of which is capable of giving an unequivocal answer with the materials available. The more direct approach has been to compare the total amount of crossing over in all the chromosomes of an organism with the total number of chiasmata observed at meiosis. As HENDERSON(1970) points out, “this approach is unlikely to have the precision necessary to test unambiguously the equivalence of chiasmata and cross-overs, for a great deal depends upon the positions in which chiasmata form, in relation to the positions of the genes known within each linkage group.” The other approach has been the purely cytological one of trying to demonstrate a correspondence between chiasma formation and exchange of segments. As HENDERSON again says, this is not really a test for correspondence between chiasmata and genetic crossing over. for it is possible that genetic reconibination does not always lead to segmental exchange. A study by DRISCOLLand SEARS(1965) made use of a cytologically marked (heteromorphic) bivalent in which an alien segment in one chromosome consti- tuted a terminal marker. They were thus theoretically able to detect a direct relation between chiasmata and crossing over in this one chromosome arm. Unfortunately their results were inconclusive, perhaps because environmental conditions were not controlled and chiasmata were scored at a stage when some chiasmata had already disappeared. DRISCOLLand BIELIG’S(1968) similar study involving an alien segment very tightly linked to the centromere resulted in only a single crossover, and thus yielded a crossover value with too little reliability for critical comparison with chiasma frequency. In this report, evidence strongly supporting the partial chiasmatypy hypothesis is provided by the use of two genetically and cytologically marked lines of common wheat (Triticum aestiuum L.) tested at two temperatures. One of these lines was the same as that of DRISCOLLand SEARS.

MATERIALS AND METHODS

In experiments involving chromosome 4A, ‘Chinese Spring’ wheat and the following derivatives of Chinese Spring were used: 1. Homozygous Hp translocation line. Carries a gene Hairy peduncle (usually called Hairy neck) in a rye-chromosome segment that replaces the terminal portion of the so-called p arm CHIASMATA AND CROSSING OVER IN WHEAT 233 of chromosome 4A (DRISCOLLand SEARS1965). Hp behaves as a simple dominant to the absence of the locus. Because no crossing over occurs between the rye segment and the corresponding part of the unmodified 4A chromosome, Hp constitutes a terminal marker.

2. A line ditelocentric for the /3 arm of chromosome 4A and monotelocentric for the 01 arm of the same chromosome. In experiments involving chromosome 6B, the following derivatives of Chinese Spring were used: 1. 'Transfer.' Homozygous for a dominant gene Lr9 that conditions resistance to the leaf-rust fungus Puccinia recondita Rob. ex Desm. f. sp. tritici. Lr9 (henceforth shortened to Lr) lies on a Triticum umbellulatum (Zhuk.) Bowden (Aegilops umbellulata) chromosome segment that replaces the terminal region of the long arm of chromosome 6B (SEARS1956, 1966) and always remains unpaired in heterozygotes. On the same arm of the chromosome, located very close to the centromere, is another gene b2 (henceforth designated simply b) whose dominant allele suppresses awns. The short arm of the chromosome is marked by CO,whose recessive allele when homozygous or hemizygous causes necrotic patches in the leaves. 2. A line ditelocentric for the long arm of chromosome 6B carrying B and lacking the Lr segment.

3. A multiple-recessive line. Homozygous for b and CO, lacking the Lr segment. Crosses were made (Figures 1, 2) so as to give rise to plants heterozygous for both the terminal genetic marker and the cytological marker (absence of one arm). The plants were trans- ferred to a growth chamber at a constant 21" (optimum temperature) or 32" (high temperature) and 15-hour-per-day illumination about a week before they reached the meiotic stage, providing

NON CROSSOVER CROSSOVER FIGURE1.-Diagram sh3wing the crossing scheme and the progeny in the chromosome-4A exporiment. 234 T. K. FU AND E. R. SEARS

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ample time for chromosome pairing to reach a stable level. From each plant, two spikes were collected for the determination of chiasma frequency in pollen-mother cells. At least three spikes from each were used for pollinations, as males in crosses to Chinese Spring in the 4A experiments and as females in crosses to the multiple-recessive line in the 6B experiments. Offspring of the 4A crosses were checked in root tips for chromosome number and as mature plants for liairi- ness of peduncle. The 6B offspring were inoculated with urediospores of Puccinia recondita tritici and scored at the appropriate times for leaf-rust reaction, awnedness, and corrodedness. For somatic chromosome counts, root tips from germinating seeds were treated with mono- Eromonaphthalene for five hours, fixed in glacial acetic acid, stained according to the Feulgen procedure, and squashed in aceto-carmine. For the meiotic studies, anthers were fixed in acetic- alcohol (1:3), stained in Feulgen, an2 squashed in aceto-carmine.

RESULTS

Comparisoln of metaphase I and diakinesis When the telocentric chromosome paired with its homolog and a chiasma was formed between them, the resulting heteromorphic bivalent could easily be distin- guished from the twenty complete bivalents (Figure 3). When the two chromosomes did not pair, they lay apart from the bivalents as univalents (Figure 4). CHIASMATA AND CROSSING OVER IN WHEAT 235

FIGURE3.-A telocentric chromosome 4A paired with its homolog to form a heteromorphic bivalent at metaphase I. FIGURE4.-A teleocentric chromosome not paired with its homolog. Both univalents lie apart from the bivalents at metaphase I. FIGURE5.--Hcteromorphic bivalent just started to separate. FIGURE6.-Heteromorphic bivalent separated completely. FIGURE7.-Two univalents that may have been paired at an earlier stage. FIGURE8.-Heteromorphic bivalent at diakinesis. 236 T. K. FU AND E. R. SEARS TABLE 1 Chiasm frequency in monotelodisoms at metaphase 1

Chromosome Chiasma muency (%) involved Temperature Range Average 4A Optimum 45.5-93.1 68.5 4A High 16.5-40.0 28.0 6B Optimum 40.0-92.7 72.1 6B High 15.9-52.7 37.1

Only one chiasma was seen in each heteromorphic bivalent. The frequency with which a chiasma was present at MI varied greatly from plant to plant and from anther to anther (Table 1). In the most extreme case, there was a range from 19.7% to 52.7% in chiasma frequency in different anthers of the same plant. On the other hand, chiasma frequency at diakinesis was very similar from anther to anther in the same plant and fairly constant in different plants grown at the same temperature. Also the frequency was substantially higher at diakinesis (Tables 3,5,7, and 9) than at metaphase (Table 1). Terminalization tended to be completed earlier in the heteromorphic bivalent than in the complete bivalents (Figures 5-77, and by full metaphase it was often not possible to tell that two desynapsed chromosomes had ever been paired. To some extent this lack of co-orientation can be attributed to shifts in position caused by flattening of the cells during preparation oi the slides, but early desynapsis and pre-metaphase disorientation cannot be ruled out as a major factor. Because crossing over is completed before diakinesis, the chiasma frequency at that stage is clearly more likely to be related to the frequency of crossing over than is the reduced frequency at metaphase I. Therefore, all comparisons were made using diakinesis data. A heteromorphic bivalent at diakinesis is shown in Figure 8.

Chiasma frequency and crossing over Chromosome 4A at optimum temperature: Data on chiasma frequency and crossing over at 21 O were obtained from nine 4A heterozygotes (Table 2, 3). Because a xz test revealed no significant differences among the nine plants in chiasma frequency (Table 3), the average frequency. 95.7%, was used to calcu- late the expected number of crossovers for each plant. It was assumed that each chiasma represents a crossover. As each crossover involves only two of the four chromatids, the expected crossover frequency equals one-half the chiasma frequency. None of the observed crossover values nor their total differed significantly from expectation. The distance between Hp and the centromere, determined from the pooled data, was 227/500 = 435.4 map units. This is actually the distance between the centromere and the beginning of the rye segment. CHIASMATA AND CROSSING OVER IN WHEAT 23 7

TABLE 2

Chromosome constitution and phenotype of ofspring of chromome-4A heterozygotes grown at optimum temperature

Noncrossovers Crossovers Plant no. 42,Hp 41+t,hp 4l+t,Hp 42,hp Total 2.1-1 21 11 9 19 60 2.1-4 9 15 11 15 50 2.1-5 14 19 11 12 56 2.1-6 10 17 12 15 54 2.1-7 11 16 10 13 50 2.1-11 14 18 11 12 55 2.1-15 18 18 18 6 60 2.1-17 16 18 16 9 59 2.1-18 19 9 15 13 56 - - - - Total 132 141 113 114 500

TABLE 3

Relation between chiasma frequency in a chromosome-4A bivalent at optimum temperature and recombination in the resulting gametes

~~ ~ ~~ ~~ Cytological data Genetic data No. Chiasma Noncrossovers Crossovers Plant no. PMC's frequency (%) Obs. Exp. Obs. Exp. 2.1-1 142 95.8 32 31.3 28 28.7 2.1-4 154 96.8 21. 26.1 26 23.9 2.1-5 285 98.6 33 29.2 23 26.8 2.1-6 402 93.8 27 28.2 27 25.8 2.1-7 394 93.7 27 26.1 23 23.9 2.1-1 1 548 96.7 32 28.7 23 26.3 2.1-15 181 94.5 36 31.3 24 28.7 2.1-17 233 95.3 34 30.8 25 28.2 2.1-18 342 96.8 28 29.2 28 26.8 - __ - ~ _. __ Total 2681 95.7 273 260.7 227 239.3

Chromosome 4A at high temperature: High temperature (32") affected not only the pairing of the telocentric with its homolog, but also the pairing of the other chromosomes. The number of univalents per cell was greatly increased, and offspring aneuploid for chromosomes other than 4A were obtained. Because of this. not all plants could be identified with certainty as crossover or noncrossover. Hairy-necked plants with 39ft, 40ft, and 4lft (Table 4) were classified as crossovers (Table 5) because the telocentric could be assumed to be the only paternal 4A chromosome present and thus had to be the carrier of Hp. The possibility that some of the telocentrics were derived from other chromosomes, through asynapsis and misdivision, could be ignored, as no plant with two telocentrics was found. 238 r. K. FU AND E. R. SEARS

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Cytological data Genetic data No. Chiasma Noncrossovers Crossovers Plant no. PMC's frequency (%) Obs. Exp. Obs. Exp.

~ 2.1-23 102 52.9 27 23.7 5 8.3 2.1-24 91. 44.7 42 36.3 7 12.7 2.1-25 90 52.2 47 40.7 8 14.3 2.1-26 166 i .a 44 43.4 15 15.6 2.1-27 92 53.3 48 42.2 9 14.8 2.1-30 117 58.1 48 41.4 8 14.6 2.1-31 130 50.0 38 31.8 5 11.2 2.1-32 213 52.6 44 39.2 9 13.8 - __ - __. - __ Total 1QO+ 52.0 338 299.0 66 105.0

The non-hairy-necked plants with 42, 43, 44, and 58 complete chromosomes could be assumed to have from the pollen a chromosome 4A which had lost the Hp segment by chromatid exchange; thus these plants also belonged to the crossover class. The hairy-necked plants with 39 to 44 complete chromosomes must have had a complete chromosome with Hp, and those non-hairy-necked plants with 39+t to 43+t and with 48-l-t presumably had a 4A telocentric lacking Hp. These were therefore noncrossovers. The remaining plants were classified as noncrossovers. Those that were hairy- necked and had 42ft or 43+t were believed to have received both the telocentric and the complete 4A chromosomes through the pollen; while the non-hairy- necked plants with 39 to 41 complete chromosomes were considered to have received neither parental 4A. It was assumed that only after failure of the 4A chromosomes to pair could both be distributed to the same pole or both be lost. As will be argued later, this assumption may not be valid; paired chromosomes that disjoin before metaphase could conceivably behave independently of each other at anaphase, particularly under the stress of unfavorably high temperature. Also, some of the plants may have been aneuploid for chromosomes other than 4A. A hairy-necked plant with 42+t, for example, may have had an exchanged telocentric carrying Hp and, instead of a complete paternal 4A, have had an extra dose of some other chromosome. Similarly, a non-hairy-necked plant with 41 may have had a paternal, exchanged, complete 4A and hence may have been monosomic for a different chromosome. When chiasma frequency was compared with amount of recombination (Table 5),all eight testcross progenies showed fewer crossovers than expected, but none was significantly deficient at the 1% point. When the data for the eight test- crosses were pooled, however, the x2 value proved to be highly significant. Chromosome 6B at optimum temperature: There were mainly four classes in the testcross progeny (Figure 2, Table 6): two noncrossover types and two types 240 T. K. FU AND E. R. SEARS TABLE 6

Gametes produced by chromosone-6B heterozygotes at optimum temperature

Single crossovers Apparent double crossovers Noncrossovers LX-B Ecentromere Lr-¢romere Plant no. LrbCo lrBco LrBco lrbCo Lxbco IrBCo LrBCo lrbco 12.61 19 14 13 13 1 0 0 0 12.62 11 14 19 16 0 0 0 0 12.63 12 14 10 11 1 1 1 3 12.64 15 13 13 12 0 0 0 3 12.65 18 15 7 15 0 1 1 0 12.66 13 12 14 20 0 0 0 0 12.610 15 12 15 12 0 a 1 4 12.6-1 1 11 15 9 14 1 1 2 2 12.6-15 16 14 14 9 1 0 a 3 12.6-17 16 14 12 17 0 0 0 0 12.620 13 20 15 11 0 0 0 1 12.623 12 23 13 9 0 0 0 3 12,624 13 15 20 6 1 0 0 2 12.628 8 19 9 13 0 0 0 1 12.637 17 10 13 17 0 0 0 3 12.6-46 11 17 14 11 0 0 1 2 ------Total 220 241 210 206 5 3 6 27

TABLE 7

Relation between chiasma frequency in a chromosome-6B bivalent at optimum temperature and recombination in the resulting gametes

Cytological data Genetic data No. Chiasma Noncrossovers Crossovers Plant no PMMc's frequency (%) Obs. Exp. Obs. Exp. 12.61 217 97.7 33 30.8 27 29.2 12.62 126 94.4 25 30.8 35 29.2 12.63 136 97.1 30 27.2 23 25.8 12.64 752 95.9 31 28.8 25 27.2 12.65 4 65 98.1 34 29.3 23 27.7 12.66 1,053 95.6 25 30.3 34 28.7 12.610 418 99.1 32 30.3 27 28.7 12.611 505 97.4 30 28.3 25 26.7 12.615 176 lm.o 33 29.3 24 27.7 12.617 5 08 97.4 30 30.3 29 28.7 12.620 158 98.7 34 30.8 26 29.2 12.623 340 97.4 38 30.8 22 29.2 12.624 547 98.7 30 29.3 27 27.7 12.628 323 96.0 28 25.7 22 24.3 12.637 534 98.9 30 30.8 30 29.2 12.646 327 95.7 31 28.8 25 27.2 ___ __ - - - __ Total 6,615 97.2 494 471.7 424 446.1 CHIASMATA AND CROSSING OVER IN WHEAT 241 with exchange between Lr and B. Crossovers also occurred between B and the centromere but were rare because B is very close to the centromere. Double crossovers should have been even more rare but in fact appeared to exceed the number of B-centromere singles. However, because asynapsis of the telocentric and its homolog is known to occur (Tables 1 and 7), we may assume that all or nearly all of these apparent double crossovers were actually plants that had received either both or neither of the maternal 6B chromosomes after asynapsis. Therefore they were added to the noncrossovers. When the data were corrected and analyzed (Table 7), neither the individual progenies nor the total of the entire experiment differed significantly from what was expected if each chiasma represents a crossover. Map distances were: Lr-B (actually B to beginning of umbellulatum segment), 416/918 = 45.3; B-centromere, 8/918 = 0.87. Chromosome 6B at high temperature: The classification of noncrossover and crossover types (Table 8) was made in the same way as in the optimum-temperature experiment, with the apparent double crossovers being pooled with the noncrossovers (Table 9). None of the testcross progenies was significantly different (at the 1% level) from expectation on the basis of chiasma frequency. When the data for the ten progenies were combined, however, the x2 value for the pooled data reached a highly significant level, with many fewer crossovers observed than expected. As in the high-temperature experiment involving chromosome 4A, a substantial fraction (33%) of the population were classified as having received either both paternal chromosomes or neither. These were again all assumed to have been produced following asynapsis and hence not to have crossed over. But again this assumption may not be valid. Possibly pairing and crossing over were sometimes followed by early disjunction and subsequent univalent behavior.

TABLE 8 Gametes produced by chromosome-6B heterozygotes at high temperature

Single crossovers Apparent double crossoyers

Noncroscovers Lr-B B-centromere Lr-B-centromere

Plant no. LrbCo lrBco LrBco GbCo Lrbco lrBCo LrBCo lrbco 12.6-52 10 5 3 7 2 2 3 16 12.6-53 8 8 2 2 1 1 6 IO 12.6-57 15 10 10 2 0 0 4 7 12.6-58 6 9 2 2 0 1 7 7 12.6-60 10 10 5 5 1 3 6 8 12.6-62 11 7 3 2 1 0 2 9 12.6-68 8 6 3 5 0 1 4 7 12.669 15 15 3 6 0 0 7 8 12.6-70 11 11 2 8 1 0 3 14 12.682 17 13 3 5 2 0 9 10 ------Total 111 94 36 44 8 8 51 96 242 T. K. FU AND E. R. SEARS TABLE 9 Relation beteen chiasma frequency in a chromosome-6B bivalent at high temperature and recombination in the resulting gametes

Cytological data Genetic data No. Chiasma Noncrossover Crossovers Plant no. PMC’s frequency (%) Ohs. EXp. Obs. Exp. 12.652 406 63.8 34 33.2 14 14.8 12.653 99 53.5 32 26.3 6 11.7 12.657 102 62.7 36 33.2 12 14.8 12.658 80 57.5 29 23.5 5 10.5 12.&60 92 62.0 34 33.2 14 14.8 12.662 93 58.1 29 24.2 6 10.8 12.6-68 61 63.8 25 23.5 9 10.5 12.669 148 62.3 45 37.3 9 16.7 12.670 155 65.8 39 34.6 11 15.4 12.682 136 59.9 49 40.8 10 18.2 ~ - ~ - Total 1,372 61.7 352 309.6 96 138.3

The number of plants that evidently received both paternal chromosomes (51 LrBCo) relative to the number that received neither (96 Zrbco) indicates that the transmission rate of univalent 6B was much closer to the 39% observed by TSUNEWAKI(1964) for 6B than to the 25% commonly assumed for wheat univalents (SEARS1954). In fact a transmission rate of 42% fits the data best. The two classes listed as having crossovers between B and the centromere should probably not be accepted as crossovers, because the 16 of them would make the crossover value for the B-centromere interval 3.57% when it was only 0.87% in the optimum-temperature experiment. For the eight ZrBCo plants, two explanations other than B-centromere crossovers are possible: (1) Those plants (or some of them) were actually ZrBco individuals that failed to develop the corroded phenotype. (2) They (or some of them) arose from gametes with both a recombined lrbCo chromosome and an ZrB telocentric. Such gametes would occur after inclusion of the two members of a desynapsed bivalent in the same TI nucleus; at the next division a recombined ZrbCo chromatid would have a 50% chance of being included in the same megaspore as an ZrB chromatid. The other class, Lrbco. can be accounted for as the result of misdivision of an unpaired LrbCo chromosome, followed by transmission of a resulting Lrb telo- czntric or isochromosome only. Misdivision of univalents is known to occur fre- quently enough in this variety of wheat (SEARS1952) that all eight Lrbco plants could have arisen in this way. A complementary class, which received only a CO telocentric or isochromosome, would be expected, and probably a fraction of the 44 lrbCo individuals were of this complementary type rather than Lr-B crossovers.

DISC US S ION Diakinesis was clearly a better stage for determining chiasma frequency than metaphase I. Even at optimum temperature, many diakinesis chiasmata had dis- CHIASMATA AND CROSSING OVER IN WHEAT 243 appeared by metaphase I. This disappearance meant that chromosomes which had paired and had a chance to cross over would have been scored at metaphase as though they had never been synapsed. Obviously this wrong scoring would have tended to obscure any correspondence that existed between chiasmata and crossing over. Reduction in chiasma frequency between diakinesis and metaphase I was also found by TSUCHIYA(1970) in hexaploid Triticale. LIN and Ross (1969) observed in triploid sorghum that the number of univalents increased as metaphase I proceeded. The pre-metaphase desynapsis observed here was much more frequent in the 4A and 6B heteromorphic bivalents than in other bivalents in the same cells. Presumably synapsis in the heteromorphics could involve only a part of one arm and seldom, if ever, led to the formation of more than one chiasma. Terminaliza- tion was evidently able to proceed more rapidly in these heteromorphic bivalents. There is some evidence that the terminalization usually was completed at early metaphase I after the two chromosomes had become co-oriented on the metaphase plate: (1) In some cells at full metaphase I, such as shown in Figure 6, the unpaired chromosomes were oriented as though they had been paired and lying on the plate; (2) at optimum temperature, if the desynapsed chromosomes behaved as ordinary univalents, there should have been offspring in the 4A experiment with neither (41) or both (42l-t) chromosomes, and many more such plants (Zrbco and LrBCo) in the 6B experiment than were observed; ar?d (3)the high chiasma frequency at metaphase I in some anthers (Table 1) is best explained by assuming that these observations were made at early metaphase before separation of loose pairs began. Some of the genetic data, on the other hand, favor the idea that much desynapsis occurred before metaphase, particularly at high temperature, leaving the desynapsed chromosomes to behave as ordinary univalents or possibly to tend to go to the same pole, because they presumably lay near each other. High temperature could conceivably cause terminalization of chiasmata to proceed more rapidly and result in disjunction before metaphase. Early work with an appar- ently desynaptic line of maize (RHOADES1946) suggested that crossover bivalents could desynapse and the resulting univalents reach the same pole, but MILLER (1963) could detect no desynapsis of crossover bivalents in this line. MOENS (1969) found in some tomato mutants that desynapsis occurred between pachytene and diakinesis, but he could not determine whether crossover bivalents ever desynapsed. At optimum temperature and with diakinesis data, an excellent fit was obtained for both chromosomes 4A and 6B to the hypothesis that each chiasma represents a crossover. High temperature, which greatly reduced the chiasma frequency and crossing over, caused an apparent poor fit to the hypothesis. In the chromosome-4A experiment, the fit can be improved somewhat by recognizing that a few crossover plants were probably included in the noncrossover class because of a missing or extra chromosome that was considered to have been 4A but was not necessarily so. Even after this correction, however, a large deficiency of crossovers 244 T. K. FU AND E. R. SEARS still exists. In the 6B experiment, various adjustments can be made, but most of these decrease, rather than increase, the number of crossovers. Assuming that each chiasma does represent a crossover, there are two possi- bilities for explaining the deficiency of crossovers in the high-temperature experiments: either (1) too many chiasmata were recorded, or (2) some of the crossovers escaped observation. Possibility ( 1) is particularly plausible because the chiasma frequencies were determined at diakinesis, which in wheat is a less favorable stage for observation than metaphase I. With some stickiness of chromosomes perhaps being caused by the unfavorably high temperature, some chromosomes that were paired at pachytene but did not form chiasmata may have remained associated until after diakinesis and have been wrongly scored as chiasmata. However, it seems unlikely that the entire decrease in chiasmata from diakinesis to MI at high temperature was due to the early separation of the members of such spurious bivalents. It is reasonable to assume that at optimum temperature few or no spurious bivalents occurred, and that the disappearance of chiasmata between diakinesis and MI (30% for 4A and 26% for 6B) was due to premature disjunction of truly chiasmate bivalents. A similar percentage disappearance of true chiasmata in the high-temperature experiments would mean that there were 20 or 30% more true chiasmata at diakinesis than expected from the crossover data. Possibility (2), that some crossovers escaped observation, arises from the fact that gametes containing both homologs or neither of them were assumed to have come from sporocytes in which the two chromosomes had not paired and therefore could not have crossed over. It appears, however, that some of the metaphase univalents resulted from premetaphase desynapsis of bivalents which had been held together by true chiasmata, and such univalents were presumably crossover chromosomes. Most of these particular univalents must have behaved like those that had never been paired (i.e., they did not go preferentially to opposite poles) ; otherwise, the number of gametes with neither chromosome or both in the high- temperature experiments could not have been so high. For example, in the high- temperature 4A experiment, the two chromosomes concerned were unpaired at diakinesis in 48% of the cells (Table 5). Since each of the two univalents would have been transmitted to only about 114 of the gametes, approximately 9/16 of the gametes from sporocytes with non-synapsis would have received neither chromosome-i.e., about 27% of all the gametes. In fact 39% of the gametes transmitted lacked both chromosomes. Like possibility (l),possibility (2) plausibly accounts for much of the discrepancy between chiasmata and crossing over in the high-temperature experiments but has difficulty in explaining the entire discrepancy. Probably both phenomena were involved, some of the apparent chiasmata at diakinesis being due to mere stickiness, and some of the univalents distributed randomly at TI being crossover chromosomes. DRISCOLLand SEARS(1965) using the same 4A material as in the present experiments, obtained more crossing over than was expected from the 40% CHIASMATA AND CROSSING OVER IN WHEAT 245 metaphase pairing observed. The discrepancy may presumably be attributed to the premetaphase completion of terminalization of chiasmata. If a reduction in chiasmata from diakinesis to metaphase similar to what we observed is assumed, a diakinesis value of 60 to 65% is obtained, in good agreement with DRISCOLL and SEARS’crossover value of about 30%. Male transmission of the 4Ap telocentric chromosome was strikingly more frequent in the present experiments than in the DRISCOLLand SEARSstudy. Whereas they observed only about 10 % transmission of the telocentric, we found 51 % at optimum temperature and 31 % at high temperature. Because the stocks used were the same, it appears that constant temperature, particularly if optimum, favors male transmission of this particular telocentric chromosome. For chromosome 6B, the ¢romere value of 0.87 should supercede the 0.44 calculated by SEARS(1 966) on the basis of a single crossover. The present Lr-B value of 45.3 is much higher than that SEARSobtained for Lr-centromere (25.0 or lower) j it is nearer the 45.1 he found for Sr-centromere. The Sr-centromere distance is substantially the same as that for Lr-B, as Sr maps only a fraction of a unit distal to Lr; but in the 1966 experiment the presence of the alien Lr segment evidently reduced crossing over greatly in the interval concerned. Presum- ably it did not do so in the present experiment.

The authors express their thanks to C. J. DRISCOLLand L. M. S. SEARSfor their valuable comments and suggestions during the experimental work and for critical reading of the manuscript. Their special thanks are due to W. Q. LJEGERINGfor providing leaf-rust spores, assisting in making the inoculations and reading the reactions, and permitting the use of his growth chambers.

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