Chromosome Differentiation and Pairing Behavior of ... fileinitiation points per chromosome and...

Copyright 0 1991 by the Genetics Society of America

Chromosome Differentiation and Pairing Behavior of Polyploids: An Assessment on Preferential Metaphase I Associations in Colchicine-Induced

Autotetraploid Hybrids Within the Genus Secale

Elena Benavente,*" and Juan Orellanat

*Departamento de Genitica, Facultad de Biologia, Universidad Complutense de Madrid, 28040-Madrid, Spain and ?Departamento de Genitica, E. T.S.Z. Agrdnomos, Universidad Politknica, 28040-Madrid, Spain

Manuscript received February 23, 1990 Accepted for publication February 22, 199 1

ABSTRACT Preferential chromosome association at metaphase I has been analyzed and compared in autotetra-

ploid cells obtained by colchicine treatment of hybrid diploid rye plants with different degrees of chromosomal divergence between homologs. The tendency to identical over homologous, but not identical, pairing preferences detected when homologous partners are contributed by less related parental lines indicates that chromosome differentiation may play an important role on preferential pairing behavior of polyploids. However, associations between more similar (identical) partners are not always favored, thus suggesting that additional factors must be considered. Other hypotheses for explaining pairing preferences in competitive situations are discussed. No clear relationship has been found between multivalent frequencies at metaphase I and chromosome differentiation between homologs or preferential pairing behavior. Therefore evolutionary divergences among related genomes should be carefully stated when evaluated from metaphase I configuration frequencies.

P AIRING competition, which may occur when more than two homologs are present, highly

influences meiotic behavior of polyploids. A choice between potential partners and preferences between specific chromosomes among all that compete may affect synapsis and recombination, and thus the meiotic stability of the plant.

Several theoretical models of meiotic behavior of polyploids have been developed to estimate the frequencies of the different kinds of meiotic configurations (multivalents, bivalents, univalents) with random pairing among all partners (JOHN and HENDERSON 1962; SVED 1966; SYBENCA 1975a; JACKSON and CASEY 1982;JAC~so~ and HAUBER 1982). With minor differences, all predict that if there are no pairing preferences in a tetraploid with synapsis initiation at both chromosome ends and pronounced distal chiasma location, the frequency of pachytene multivalents will be 2/3. If there are more than two synaptic initiation points per chromosome and chiasmata can occur at interstitial regions, then the probability of multivalent formation increases (SYBENGA 1975a; JACKSON and CASEY 1982).

In studies where all pairs of the complement must be considered as a whole because of the impossibility of distinguishing specific chromosomes, expected values are compared to the total numbers of multivalents and bivalents found (TIMMIS and REES 197 1 ; SYBENGA

' Present address: Departamento de Genitica, E.T.S.I. Agrbnomos, Uni- versidad PolitCcnica, 28040-Madrid, Spain.

Genetics 128: 433-442 (June, 1991)

1975b;JACKSON and HAUBER 1982; LENTZ et a/. 1983; WATANABE 1983; CALLOW, HAMEY and PATTRICK 1984; CHAPMAN 1984). Structural rearrangements or telocentric chromosomes have been employed in some cases as cytological markers to analyze pairing preferences of specific pairs of chromosomes (DOYLE 1963; SYBENCA 1972,1976; ELgand SYBENCA 1976; KIMBER and ALONSO 1984). Commonly, metaphase I multivalent frequencies lower than expected have been explained by the existence of a preferential bivalent association between specific partners. Pairing preferences have been inferred in a different manner from data on the segregation for genetic (GERSTEL and PHILLIPS 1958; REINBERCS et al. 1970; DOYLE 1979, 1982, 1986; HUTCHINSON et al., 1983; EVANS and DAVIES 1985; DITER, GUYOMARD and CHOUR- ROUT 1988) or chromosomal (STURTEVANT 1936; GRELL 1961 ; DE BOER and GROEN 1974) markers, or both (SUAREZ et al., 1988). In a few cases, preferences so detected have been found to be associated to some decrease in the frequency of multivalents at metaphase I (GERSTEL and PHILLIPS 1958; DOYLE 1982, 1986; EVANS and DAVIES 1985). It should be noted, however, that these results may have been influenced by gametic or zygotic selection.

Pairing preferences in polyploids have been explained in a number of cases on the basis of the specificity of meiotic chromosome pairing itself (see RILEY and LAW 1965). DVORAK and MCGUIRE (1 98 1 ) have suggested that changes in chromosomes, even at

434 E. Benavente and J. Orellana

Line Y Line Z Parental Lines

P lln M n u 3 R, E.6Ri ANC m u IRA U D

Hybrids

FIGURE 1 .-C-banding pattern for chromosome IR in all parental lines and hybrids analyzed.

the level of nucleotide sequence, might be recogniz- able during the synaptic process. This level of chromosome differentiation might result in preferential pairing not only in allopolyploids, in which homologous and homoeologous partners compete for pairing, but also in those polyploids where homologs derived from a certain inbred line or cultivar (called “euhom- ologs”) vie with homologs from a different origin within the same taxon (“heterohomologs”) (REIN- BERGS et d. 1970). According to this supposition, any excess of bivalents at metaphase I in these polyploids could be ascribable to preferential association between those two specific partners which share higher homology. This assumption is commonly accepted in most cytogenetic studies on evolutionary divergence among related species where the patterns of genomic relationships are usually extrapolated from meiotic data of polyploid hybrids. However, methods of analysis traditionally employed prevent experimental verification of whether the more similar chromosomes are in fact those which are preferentially associated in bivalents. The development of differential staining techniques has provided a suitable tool to distinguish specific chromosomes in mitosis as well as in meiosis. Thus direct assessments of preferential MI association behavior can be made when C-banding procedure allows cytological identification of all potential distinct pairwise combinations in a given polyploid (KENTON and JONES 1985; further references in BENAVENTE and ORELLANA 1989b).

The primary aim of this study was to search for the influence of chromosome differentiation on pairing behavior in competitive situations within the genus Secale. For that purpose, association preferences at metaphase I between identical and homologous, but not identical, chromosomes have been analyzed and compared in autotetraploid meiocytes of hybrid plants

hybrid YZ (2x) SB

colchicine 1 tr88tment

1 eu-eu : 1 het-het : 4 eu-het u 11 2 H

FIGURE 2.-Chromosome constitution in colchicine-induced tetraploid cells heterozygous for C-bands.

of rye with different degrees of chromosomal divergence between homologs at the diploid level.

MATERIALS AND METHODS

The following parental lines were used to obtain the plants analyzed here:

Inbred lines P ( E O ) , M (I19), R (124) and E (123) of rye, Secale cereale ssp. cereale (noted as P, M, R and E, respectively).

Line 6Ri of S. cereale ssp. cereale (noted as 6Ri). S. cereale ssp. ancestrale (noted as Anc). S. vavilovii ssp. iranicum (noted as Ira). Crosses were designed to obtain hybrids heterozygous for

telomeric C-bands at chromosome 1R (Figure 1). The following hybrids were included in this study: (a) F1 plants of inbred lines of S. cereale ssp. cereale: PM, PR and PE, (b) intersubspecific hybrids within the species S. cereale: EAnc, PAnc and RAnc, and (c) interspecific hybrids within the genus Secale: EIra, 6RiIra and PIra. The offsprings examined were always obtained from a single cross. In all cases, the parental female line is noted first.

At the three-leaf stage, seedlings of the F, hybrids were treated with colchicine using a technique based on that of THOMAS and PICKERINC (1979). Some of the plantlets turned out to be diploid/tetraploid chimaeras. Anthers of the emerging spikes at meiosis were stained in 2% acetic orcein to determine their ploidy level and tetraploid anthers were then fixed in 1:3 acetic ethanol and stored at 4” for several months. The fixed material was squashed and stained according to the Giemsa C-banding procedure described by GIR~LDEZ, CERME~O and ORELLANA (1 979).

RESULTS

Following chromosome doubling by colchicine treatment of diploid plants, each chromosome is ac- companied by one identical and two homologous, but not identical, partners (Figure 2). Two types of two- by-two metaphase I associations are then possible for any chromosome arm: between identical chromo-

Pairing Preferences in 4X Hybrids 435

FIGURE 3.-Meiotic configurations for chromosome 1R in MI tetraploid cells. Arrows indicate associations in its short arm; arrowheads indicate associations in its long arm. Their type of pairing as noted in Table 1 is as follows: (a) IRS "21," JRL "21"; (b) IRS "lH," IRL "2H"; (c) IRS "2H," IRL "21"; (d) IRS "21," IRL "2H"; (e) IRS "2H," IRL "1H"; (0 IRS "U3," IRL "11" (het-het identical association); (g) IRS "0," I R L "U4." Larger than quadrivalent multivalent configurations never involving chromosome IR as in (a) are found in tetraploid M I cells of the interspecific hybrids examined.

somes (I) or between homologous nonidentical chromosomes (H). Colchicine-treated diploid plants heterozygous for a certain C-band provide suitable material for analyzing pairing preferences since such a cytological marker allows the identification of both types of associations in tetraploid meiocytes: identical associations will involve chromosomes with the same C-banding pattern, whereas homologous partners will show a different C-banding pattern in the marked arm (Figure 3, a-f). In those configurations where three or four chromosomes are associated within the marked arm, the type of associations at metaphase I cannot be actually determined (undetermined pairing,

U) (see chromosome arms 1RS and 1RL in Figure 3, f and g, respectively). Thus tetraploid cells can be classified according to their observed pattern of MI association for each arm of chromosome 1R (Table 1). It must be noted that the long arm of chromosome 1R in inbred line P and S. vavilovii iranicum have a similar sized block of C-heterochromatin (Figure l), making it impossible to distinguish identical from homologous nonidentical associations for this arm in tetraploid cells of hybrids PIra. In all cases 150 cells per plant were scored. The bound arm frequencies, estimated as the minimum number of chiasmata nec- essary to explain each observed meiotic configuration,


TABLE 1

Pattern of MI pairing for both arms of chromosome I R in tetraploid cells of all hybrids examined

Chromosome IR

Short arm Long arm

Plant 11 21

PM 1 PM2 PM3 PRl PR2 PE 1 PE2 PE3 PE4

EAnc 1 EAnc2 EAnc3 PAnc 1 PAnc2 PAnc3 RAnc 1 RAnc2 RAnc3

EIral EIra2 EIra3 6RiIral 6RiIra2 6RiIra3

PIral PIra2 PIra3

39 37 32 32 25 30 34 28 29

31 28 33 31 33 20 41 26 22

59 67 58 46 50 50

49 44 51

13 14 11 11 10 18 12 14 15

11 24 19 15 25 10 35 15 34

49 52 54 37 32 52

24 24 27

1H 2H U3 U4 0 ba/cell 11 21 1H 2H U3 U4 0 ba/cell

35 31 47 49 45 55 37 47 33

39 35 42 45 36 35 21 34 17

6 5 3

18 23 12

24 25 22

9 22 12 25 27 23 19 17 23

17 24 31 25 22 34 14 26 35

6 2 5 4

11 10

17 18 19

5 5 8 5 6

10 9 8 9

11 17 8

12 9

16 19 18 15

1 2

6 3 4

2 10 2

6 7 2 6 6 4 6 7 9

4 8 7 6

10 7 9

13 25

1 1 4 1 4 2

8 7 8

43 34 38 22 31 10 33 29 32

37 14 10 16 15 28 11 18 2

28 21 26 38 27 20

26 22 21

0.97 1.14 0.98 1.21 1.16 1.33 1.13 1.16 1.22

1.07 1.45 1.41 1.32 1.41 1.31 1.50 1.45 1.88

1.20 1.25 1.27 1.07 1.18 1.33

1.22 1.29 1.29

6 9 6

12 14 18 11 15 9

22 12 16

1

5 13 13 17

43 34 20 14 19 18

27 34 27 21 17 26 26 27 27

48 29 28 30 35 25 50 45 44

68 77 91 50 42 65

15 13 14 19 20 11 2 7 6

11 5 6 5 8 6

12 9

10

4 4 8

14 10 7

87 80 79 75 86 72 90 77 80

53 95 86

101 91

100 59 71 68

21 21 23 47 61 41

l I + l H 14 13 12

2I+2H 114 119 111

7 4 7 9 1 7 7 3 7

5 2 6 6 5 5 3 4 6

7 4

7 8 5

5 6 7

8 10 15 13 11 16 12 20 20

10 6 8 7

11 8

10 7 5

3 5 4

16 10 14

16 12 20

2 1 1

2 1 1

1 1

1 3 1

4 5 4 2

1

1.91 1.92 1.94 1.87 1.83 1.91 1.97 1.97 2.02

1.83 1.91 1.91 2.01 2.02 1.97 1.86 1.89 1.85

1.65 1.71 1.79 1.89 1.87 1.93

2.00 1.99 2.05

I, cells with one (11) or two (21) associations between identical partners; H, cells with one (1H) or two (2H) associations between homologous nonidentical partners; U, cells where three (U3) or four (U4) chromosomes were associated at the marked arm; 0, cells with the four partners unpaired; ba/cell, mean number of bound arms per tetraploid cell.

have also been included. It is worthy of mention that multivalent configurations larger than quadrivalents were found in some tetraploid cells of the interspecific hybrids between S. cereale and S. vavilovii (see Figure 3a), which are known to differ in two reciprocal translocations (KHUSH 1963); nevertheless, this does not affect our analysis because neither interchange involves chromosome 1R.

T w o types of pairing involving either euchromatic (lacking C-bands) or heterochromatic (bearing C- bands) chromosome arms can be distinguished in cells with identically paired partners (see chromosome arm 1RL in Figure 3c). In addition, there are four cytolog- ically indistinguishable pairwise combinations between homologous partners, involving one euchromatic and one heterochromatic chromosome arm. Therefore, if initial pairing and chiasma formation take place at random among the four partners, then homologous associations will be twice as common as

identical ones (Figure 2). Identical and homologous pairing frequencies could be calculated either from the total number of associations observed in MI configurations (in which case cells with two bonds involving a certain chromosome arm are considered twice) or from the number of cells with identical or homologous nonidentical pairing pattern irrespective of the number of bonds present for the marked arm. In the former, preferences might be overestimated because of the lack of independence between the pattern of pairing when two associations occur within a cell ( i e . , if one association involves identical chromosomes, a second potential bond between the remaining two partners necessarily will show the same type of pairing). This is why we used the numbers of cells with at least one identical or homologous association to test randomness (Table 2). Table 3 summarizes the preferences in all hybrid plants analyzed. Frequencies of identical pairing (Fip) were calculated as the probabil-


TABLE 2

Comparison between the numbers of cells with at least one identical or homolopun asmciation at metaphase I and the

expected valuer calculated under a ratio of 12, I:H

Chromosome 1R

Short arm Long arm

Plant I H x* (d.f. = 1) I H x' (d.f. = 1)

PM1 52(+) 44 18.75** 33 102 (+) 4.8* PM2 51 (+) 53 11.54** 43 93 0.18 PM3 43 59 3.57 33 93 2.89 PRl 43 74 0.62 33 94 3.09 PR2 35 72 0.02 31 106(+) 7.07** PE 1 48 78 1.29 44 83 0.10 PE2 46 (+) 56 6.35* 37 92 1.26 PES 42 64 1.89 42 84 0.0 PE4 44 (+) 56 5.12* 36 86 0.80

EAncl 42 (+) 56 4.0* 70(+) 64 21.55** EAnc2 52(+) 59 9.12** 41 100 1.15 EAnc3 52 (+) 73 3.844* 44 92 0.06 PAncl 46 70 2.09 31 106(+) 7.07** PAnc2 58 (+) 58 14.5** 35 99 3.14 PAnc3 30 69 0.41 30 106(+) 7.78** RAncl 76(+) 35 61.66** 63 (+) 71 11.29** RAnc2 41 60 2.40 58 (+) 80 4.70* RAnc3 56 (+) 52 16.67** 61 (+) 78 6.96**

EIral 108(+) 12 173.4** 111 (+) 25 142.68** EIra2 119 (+) 7 21 1.75** 111 (+) 25 142.68** EIra3 112 (+) 8 194.4** 111 (+) 31 128.45** 6Rilral 83 (+) 22 98.74** 64 (+) 61 17.96** 6RiIra2 82 (+) 34 72.84** 61 (+) 71 9.85** 6RiIra3 102 (+) 22 133.56** 83 (+) 48 53.15** PIral 73 (+) 41 48.36** PIra2 68 (+) 43 38.96** PIra3 78 (+) 41 55.57**

I, cells with identical MI association; H, cells with homologous non-identical MI association; (+), type of association significantly in excess under the random ratio; *, significant at the 5% level; **, significant at the 1 % level.

ity of identical association in the total number of distinguishable (identical plus homologous) associations. Values of Fip for both arms of chromosome 1R are also included in Table 3.

Contingency chi-square tests made to compare the numbers of identical and homologous associations in plants belonging to the same cross (Table 4) indicate that the distributions of both types of MI associations significantly differ only in those offsprings where open-pollinated materials were used as parental plants (6Ri or Anc). By contrast, no differences were found with inbred (P, R, M or E) and/or autogamous (Ira) parental lines.

Chromosome pairing occurs at pachytene, when any direct analysis of pairing behavior should be made. However, as in most other meiotic studies of polyploids, our data were obtained from MI observations after synapsis and chiasma formation. Nonrandom association at metaphase I may arise either from pref-

TABLE 3

Summary of the preferential MI pairing behavior detected in all hybrids examined

Chromosome 1R

Short arm Long arm

Plant MI pairinE Fip MI pairing Fip

PM 1 PM2 PM3 PR 1 PR2 PE 1 PE2 PE3 PE4

EAncl EAnc2 EAnc3 PAncl PAnc2 PAnc3 RAnc 1 RAnc2 RAnc3

Elra 1 EIra2 EIra3 6Rilra1 6RiIra2 6RiIra3 PIral PIra2

Identical Identical Random Random Random Random Identical Random Identical

Identical Identical Identical Random Identical Random Identical Random Identical

Identical Identical Identical Identical Identical Identical Identical Identical

PIra3 Identical

0.551 Homologous 0.464 Random 0.432 Random 0.353 Random 0.3 13 Homologous 0.395 Random 0.436 Random 0.409 Random 0.428 Random

0.421 Identical 0.478 Random 0.406 Random 0.391 Homologous 0.509 Random 0.280 Homologous 0.694 Identical 0.394 Identical 0.508 Identical

0.897 Identical 0.950 Identical 0.927 Identical 0.822 Identical 0.7 17 Identical 0.828 Identical 0.626 0.601 0.636

0.241

0.259 0.242 0.200 0.31 1 0.257 0.300 0.275

0.502 0.246

0.228 0.269 0.21 1 0.465 0.406

0.308

0.288

0.418

0.796

0.789 0.514 0.438 0.624

0.803

Fip, frequency of identical pairing = number of identical associations ["lI" + 2x"2IW]/total number of distinguishable associations ["lI" + "1H" + 2X("2I" + "2H")J.

erentiai pairing with random chiasma formation, or from random pairing with preferential chiasma formation (or failure) depending on the pattern of pairing (identical or homologous nonidentical) previously established, or from nonrandom pairing and chiasma formation. Because we cannot determine the origin of the preferences found here, they should be more appropriately considered as preferences for the type of pairing more effective in crossing over, ie., preferential effective pairing.

DISCUSSION

Chromosome differentiation, preferential association behavior and multivalent frequencies at metaphase I: Identical partners obtained by chromosome doubling both have exactly the same structure and nucleotide sequence when analyzed, as here, in the same generation of colchicine treatment. (Following recombination, differences between two originally identical copies may arise in the offspring of a colchi-


TABLE 4

Comparison between the numbers of identical and homologous MI associations among plants belonging to the same offspring

Contingency x'

Cross d.f. 1 RS 1 RL

PM 2 2.89 1.90 PR 1 0.40 0.40 PE 3 1.56 1.48 EAnc 2 0.70 18.18** PAnc 2 8.65* 0.72 RAnc 2 16.86** 0.70 EIra 2 1.91 0.71 6RiIra 2 4.82 8.20* PIra 2 0.47

*, significant at the 5% level; **, significant at the 1% level.

cine-induced autotetraploid.) However, some differences can potentially exist between homologous nonidentical partners contributed by parents from distinct lines. Moreover, the level of chromosome differentiation between homologs may be expected to be related to the degree of evolutionary divergence between the corresponding parental lines. Accordingly, homologous partners of hybrids obtained from parental lines within a certain subspecies will share higher homology than those of intersubspecific hybrids which, in their turn, will be less different than partially homoeologous homologs present in interspecific hybrids.

Results in Tables 2 and 3 indicate that F1 plants of inbred lines within the subspecies S. cereale ssp. cereale show a wide range of preferential behavior; a clearer tendency for preferences between identical partners is detected in hybrids between subspecies of S. cereale, whereas this is the sole type of preferential pairing observed in interspecific hybrids. As also expected, higher values of Fip (frequency of identical pairing) are obtained for both arms of chromosome 1R when homology between nonidentical partners decreases (Table 3). This likely reflects some role of chromosome differentiation on pairing preferences in polyploids.

Preferential association between less similar ( i e . , homologous nonidentical) partners has been found for chromosome arm 1RL in some hybrids (Table 3). Obviously, if pairing preferences exclusively de- pended on chromosome similarities, then identical associations should always be favored over homologous ones. However, our results indicate that pairing involving partners with a higher level of differentiation, at least greater than that which exists between homologs from two distinct subspecies, can be more effective for crossing over than identical pairing (see plants PAncl and PAnc3 in Table 3). Therefore chromosome differentiation may not be the major cause of preferential pairing in competitive situations.

Many evolutionary analyses of genomic affinities

TABLE 5

Multivalent frequencies for chromosome 1R in tetraploid cells of all hybrids examined

Preferential behavior a b C

1 RS 1RL Plant FIV Plant FIV Plant FIV

Random Homologous PR2 0.49 PAncl 0.49 PAnc3 0.48

Random Random PM3 0.46 PRl 0.61 PE1 0.67 PE3 0.55

Random Identical RAnc2 0.53 Identical Homologous PM 1 0.5 1 Identical Random PM2 0.48 EAnc2 0.51

PE2 0.51 EAnc3 0.57 PE4 0.55 PAncP 0.52

Identical Identical EAncl 0.39 EIral 0.21 RAncl 0.55 EIra2 0.22 RAnc3 0.62 EIra3 0.22

6RiIra1 0.49 6RiIra2 0.51 6RiIra3 0.37

Identical ? PIral 0.63 PIra2 0.60 PIra3 0.65

FIV, frequency of multivalents (quadrivalents plus trivalents) at metaphase I; a, F, plants between inbred lines of S. cereale ssp. cereale; b, intersubspecific hybrids within the species S. cereale; c, interspecific hybrids within the genus Secale.

assume that chromosome differentiation between related genomes is the origin of pairing failure in interspecific hybrids and the main cause of the excess of bivalents either in interspecific polyploid hybrids or in allopolyploids (e .g . , ARMSTRONG 1984; LEGGETT 1984; SMITH 1984; VON BOTHMER, FLINK and LAND- STROM 1988; LUGAS andJAHIER 1988). It is commonly accepted that bivalent over multivalent formation is there favored because of the preferential association between more similar, closely related partners. With some exceptions where the existence of Ph-like genes has been demonstrated, the lack of markers providing cytological discrimination of different pairwise combinations prevents further verification on that supposition. Only in EIra plants we find a significant decrease in MI multivalent frequencies (FIV) associated with preferences between identical over partially homoeologous partners (Table 5). However, this can hardly be attributed to the exclusive effect of chromosome differentiation since interspecific hybrids PIra (where nonidentical partners are supposed to share a similar level of homology than those of EIra) have chromosome 1R FIV values even higher than most F1 plants between inbred lines of S. cereale ssp. cereale and intersubspecific S. cereale hybrids.

The total numbers of bound arms per cell (ba/cell) for chromosome 1R in plants EIra are not very different than those of the rest of the plants analyzed (Table 1). However, their extremely high Fip values (Table


3) indicate that most of the MI associations involve identical partners, thus reflecting a very low effectivity of nonidentical pairing for chiasma formation at both sides of the centromere. Since no multivalent can be formed with identical associations at both IRS and IRL chromosome arms, such a pronounced restric- tiveness of identical pairing for crossing over may be the main cause for the low frequency of 1R multivalent formation in interspecific EIra plants. Very likely, higher FIV values are found in most of the remaining “identical IRS-identical 1RL” plants because of a sof- ter requirement of identical pairing for chiasma formation, as deduced from their corresponding Fip values given in Table 3. Preferential behavior of chromosome arm IRL could not be analyzed in hybrids PIra because of the lack of any cytological marker to distinguish identical from homologous, but not identical, associations (Figure 1). Nevertheless, low Fip values can be expected for such an arm, even if it showed identical over homologous MI preferential pairing, thus explaining multivalent frequencies close to 0.67 in all PIra plants (Table 5).

So, with the above exception where a strong preferential MI association behavior seems to notoriously influence multivalent formation, FIV values lower than 0.67 and close to 0.5 have been found in most plants under analysis, not only irrespective of their type of preferential behavior but also at any of the three levels of homolog divergence considered. These results indicate that multivalent frequencies in polyploids may not be mainly determined by the degree of chromosome differentiation between homologs. This should be taken into account in those studies where evolutionary divergences are stated from analysis of genomic affinities based on MI configuration frequencies as well as in theoretical approaches of meiotic pairing in polyploid hybrids (e.g., KIMBER and ALONSO 198 1 ; CRANE and SLEPER 1989), at least when closely related genomes are concerned.

It is worthy of noting that correlation coefficients significantly different from 0 were obtained when the total frequency of bound arms per cell for chromosome 1R ( i .e . , ba/cell in IRS chromosome arm + ba/ cell for IRL chromosome arm in Table 1) were cor- related with the IR multivalent frequency (Table 5) , either when all plants used were tested for correlation ( r = 0.5401, d.f. = 25), or in the three groups of hybrids analyzed (F, hybrids: r = 0.6850, d.f. = 7; intersubspecific hybrids: r = 0.9039, d.f. = 7; interspecific hybrids: r = 0.7097, d.f. = 7). These results only refer to one out of the seven sets of rye chromosomes. However, if extended to the whole chromosome complement, they could be reflecting how MI multivalent frequency in polyploids is certainly affected by the actual level of pairing and chiasma formation.

TABLE 6

Comparison between the frequencies of both types of identical associations and the expected values assuming no differential pairing ability for both homologs (ratio of 1:1, eu-eu:het-het)

Chromosome I R

Short arm Long arm

Plant eu-eu het-het Y’ (d.f. = 1) eu-eu het-het Y* (d.f. = 1)

PM1 29 36 0.75 PM2 33 32 0.02 PM3 31 23 1.19 PRl 31 23 1.19 PR2 30 (+) 15 5.0* PEl 36 PE2 26 PE3 24 PE4 30

EAncl 24 EAnc2 39 EAnc3 28 PAncl 19 PAnc2 31 PAnc3 14 RAncl 56 RAnc2 31 RAnc3 41

EIral 90 EIra2 87 EIra3 83 6RiIra1 64 6RiIra2 66 6RiIra3 88 PIral 56 PIra2 49

30 0.55 32 0.62 32 1.14 29 0.02

29 0.47 37 0.05 43 3.17 42 (+) 8.67** 52 (+) 5.31* 26 3.6 55 0.01 25 0.64 49 0.71

67 3.37 84 0.05 83 0.0 56 0.53 48 2.84 66 3.14 41 2.32 43 0.39

PIra3 64 (+) 41 5.04*

32 28 0.27 40 37 0.12 28 32 0.27 29 25 0.30 22 26 0.33 36 34 0.06 33 30 0.14 32 37 0.36 32 31 0.02

53 65 1.22 33 37 0.2 34 38 0.22 30 31 0.02 35 35 0.0 30 25 0.45 56 57 0.01 52 51 0.01 53 52 0.01

88 91 0.05 92 96 0.09

103 99 0.08 63 51 1.26 52 51 0.01 77 71 0.24

eu-eu, identical associations between the two partners lacking C- bands; het-het, identical associations between the two partners bearing C-bands; (+), type of identical association significantly in excess. *, significant at the 5% level; **, significant at the 1% level.

Differential pairing ability and MI association preferences: The random ratio employed (1 :2 identical us. homologous nonidentical pairing) assumes that all four chromosomes have an equal probability of pairing. Where this is not so, an excess of associations between more active partners may lead to deviations from the expected random ratio. As pointed out previously (BENAVENTE and ORELLANA 1989a), identical chromosomes necessarily will have equal pairing abilities, but differences may exist between homologous nonidentical chromosomes. If one of them (that which bears (het) or lacks (eu) the C-band) shows a higher pairing ability than the other, then deviations from the ratio 1: 1, het-het:eu-eu, will be observed, identical associations for the more active partner (het- het or eu-eu) being more frequent. When MI associations are used for this analysis, differences in chiasma- forming activity between the chromosomes involved would have a similar effect. Results given in Table 6


do not show differences in the frequencies of both types of identical associations (eu-eu and het-het) for any of the two arms of chromosome 1R in most plants analyzed, no matter what their preferential pairing behavior (Table 3). Similar results were obtained in a previous report on pairing preferences at metaphase I in autotetraploid rye heterozygous for interstitial C- bands (BENAVENTE and ORELLANA 1989b). Further- more, differences in pairing ability between homologous partners in rye involving metacentric and telocentric chromosomes did not explain the preferences detected there (BENAVENTE and ORELLANA 1989a). Therefore, the existence of MI association preferences in our materials cannot be ascribed to differential pairing (or chiasma formation) abilities between homologs.

Further comments on chromosome pairing ability can be made from these results. T w o partners bearing conspicuous C-bands pair with each other as fre- quently as those which lack them, thus suggesting that C-heterochromatin likely has no relevant effect on pairing (or chiasma-forming) ability. Moreover, no correspondence seems to exist between pairing ability and chromosome differentiation since both types of identical pairing almost never differ significantly for the two arms of chromosome IR, at any level of homology between the homologs. From our point of view, this indicates that pairing ability is an inherent property of each chromosome and does not depend on its potential pairing partners.

Differential pairing affinity and MI association preferences: SANTOS, ORELLANA and GIR~LDEZ (1983) pointed out that preferential pairing occurs in a polyploid when the distinct chromosome combinations show a differential pairing affinity. Here the term “affinity” refers to the capacity of two specific partners for pairing with each other. The resulting preferential behavior shown by every single competitive situation will depend on the relative pairing affinities of all potential (either identical and homologous nonidentical) pairing combinations. As far as homologous pairwise combinations are concerned, pairing affinity will be determined by the two specific chromosomes involved, so that it could differ for a given chromosome from one hybrid to another depending on its actual partner in each case. This assumption may be indirectly confirmed from the comparison between the values of Fip in those hybrids where inbred lines E and P were used either in intersubspecific and interspecific crosses (Table 3). Plants EAnc and EIra always have higher Fip values than those of PAnc and PIra, respectively, even when the same preferential MI association behavior has been found, as occurs for chromosome arm 1RS in the interspecific hybrids EIra and Plra. This seems to indicate that homologous nonidentical combinations involving

chromosome 1R from inbred line E show lower pairing affinities than those involving chromosome 1R from inbred line P, at any level of chromosome differentiation between homologs.

RILEY and LAW ( 1 965) stated that the pairing sys- tem of normal outbreeding materials such as S. cereale operates most efficiently when heterozygous combinations of genes are frequent. In a previous report (BENAVENTE and ORELLANA 1989b), structural chromosomal homozygosity derived from inbreeding was proposed to play a role in the desynaptic MI behavior detected in diploid inbred lines of rye (ORELLANA, ALAMO and GIRALDEZ 1984) and other allogamous species (KARP and JONES 1982). Then complete homozygosity may be the source of lower pairing affinities for identical combinations, thus explaining the homologous nonidentical over identical preferential MI pairing reported here (Table 3), and in previous anaiyses of autotetraploids of rye (SANTOS, ORELLANA and GIR~LDEZ 1983; ORELLANA and SANTOS 1985; BENAVENTE and ORELLANA 1989b).

Recombination processes could provide slight genotypic or structural differences (not necessarily cyto- logically detectable), for a given chromosome in those plants obtained from a single cross where a certain level of heterozygosity exists in any of the corresponding parental lines. Consequently, a specific homologous pairing combination could show distinct affinities within the same offspring. This would explain significantly different frequencies of identical and homologous nonidentical associations among hybrids belonging to the same offspring when open-pollinated materials (6Ri or Anc) are used as parental lines (Table 4), even when all of them show the same preferential MI association behavior, as occurs for chromosome arm IRL in hybrids 6RiIra (Table 3). Accordingly, no differences should be found in the offspring when inbred lines (P, M, R or E) or autogamous materials (Ira) are crossed since any homologous combination will show the same pairing affinity in all hybrid plants. As expected, chi-square contingency tests do not de- tect differences in the distribution of identical and homologous nonidentical associations in the latter case (Table 4). Thus our results provide indirect evidence of the proposed origin of differential pairing affinities based on genotypic or structural differences among partners in competition (SANTOS, ORELLANA and GIR~LDEZ 1983).

Concluding remarks: From this analysis we suggest that preferential MI association behavior in polyploids is mainly determined by the relative pairing affinities of all potential pairwise combinations, which seem not to be strictly dependent on the level of homology between chromosomes involved. Nevertheless, a clear tendency to identical over homologous nonidentical preferential association has been detected when hom-

Pairing Preferences in 4X Hybrids 44 1

ologs are contributed by less related parental lines. Thus chromosome differentiation might result in lower pairing affinities as homology between pairing partners decreases.

This work has been supported by grant 2062/83 from the Comisibn Asesora de Investigacibn Cientifica y Ticnica of Spain. The authors wish to thank R. JACKSON for his critical comments and helpful suggestions on a previous version of this paper.

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