Copyright 0 1995 by the Genetics Society of America
Meiotic Chromosome Pairing in Triploid and Tetraploid Saccharomyces certwisiae
Josef h i d l
Znstitute of Botany, University of Vienna, A-1030 Vienna, Austria
Manuscript received September 26, 1994
Accepted for publication December 16, 1994
ABSTRACT
Meiotic chromosome pairing in isogenic triploid and tetraploid strains
of yeast and the consequences
of polyploidy on meiotic chromosome segregation are studied. Synaptonemal complex formation
at
pachytene was found to be different in the triploid and in the tetraploid. In the triploid, triple-synapsis,
that is, the connection of three homologues ata given site, is common. It can even extend all the way
along the chromosomes.In the tetraploid, homologous chromosomesmostly come in pairs of synapsed
bivalents. Multiple synapsis, that is, synapsisof more than two homologues in one and thesame region,
was virtually absent in the tetraploid.About five quadrivalents percell occurred dueto the switching of
pairing partners.From the frequencyof pairing partnerswitches it can be deduced thatin most chromosomes synapsis is initiated primarily at one end, occasionally at both ends and rarely at an additional
intercalary position. In contrastto a considerably reduced spore viability( -40% ) in the triploid, spore
viability is only mildly affected in the tetraploid. The good spore viability is presumably due to the low
frequency of quadrivalents and to the highly regular 2:2 segregation of the few quadrivalents that do
3:l nondisjunction that leads to
occur. Occasionally, however, quadrivalents appear to be subject to
spore death in the second generation.
M
EIOSIS is the division during which homologous
parental chromosomes are separated fromeach
other to reduce diploid sets to the haploid level. D i p
loidyis restored by the fertilization of haploid cells;
therefore meiosis is the necessary compensatory event
to sexual reproduction. Pairing of homologous chromosomes at meiotic prophase is a precondition of their
orderly segregation. Pairing requires recognitionof homology at theDNA level and is first seen as the parallel
alignment of homologous chromosomes at some distance(“presynaptic
alignment”) before they move
closer together and become intimately connected all
along their length by the synaptonemal complex (SC)
(for review see LOIDL1990; SCHERTHAN
et aZ. 1992).
The SC is a ladder-like, mainly proteinaceous structure
and is required, in a way not yet fully understood, for
chiasma formation and theregular disjunction of chromosomes. If homologous chromosomes are missing,
the SC can also be formed between nonhomologous
chromosomes ( LOIDLet al. 1991). Here an electron
microscopic analysis of SCs is performed to investigate
meiotic pairing when more than two homologous chromosome sets are present and chromosomes have to
compete for homologoussynapsis.
In polyploid or polysomic species synapsis occurs in
various ways. In triploids or trisomics, two possibilities
have been identified for how sets of three homologous
chromosomes can synapse. An SC may connect only
two chromosomes at any site, the third being excluded
Addressfur correspondence:Institute of Botany, University of’Vienna,
Rennweg 14, A-1030 Vienna, Austria.
Genetics 1 3 9 1511-1520 (April, 1995)
from synapsis ( 11+ I synapsis) or a triple SC can connect
all three chromosomes along some distance or even
along their whole length. In most organisms only 11+ I
synapsis of chromosome triplets has been observed,
whereas in a few only triple synapsis occurs (see SHERMAN et al. 1989). In the triploid basidiomycete Copn’nus
cinereus ( RASMUSSEN et al. 1981) and in triploid domeset aZ. 1991) both types of pairing have
tic fowl ( SObeen reported to occur in one and the same cell. In
tetraploids and tetrasomics, sets of four homologous
chromosomes usually come in pairs of bivalents or
quadrivalents with only two chromosomes synapsed at
any site; however, local quadruple synapsis in solanaet al. ( 1989)
ceous plants has been reported [SHERMAN
and literature cited therein]. In other plants where
higher degreesof ploidy wereinvestigated, higher multiple synapsis has not yet been found.
Synapsis in polyploids or polysomics is often accompanied by pairing partner switching,which occurs when
different regions of a chromosome synapse with different partners. Since pairing partner switches are generated by independently initiated stretches of SC, they
can be used to estimate the number of SC initiation
sites ( LOIDL1986; LOIDLand JONES 1986) . If crossovers
occur to both sides of a pairing partner switch, a pachytene multivalent is manifested as a metaphaseI multiva1975). Since multivalents are prone to
lent ( SYBENGA
nondisjunction at the first meiotic division, polyploidy
usually has an adverse effect on fertility. Triploid organisms segregate the chromosomes of trivalents randomly
in meiosis, which leads to the production of aneuploid
gametes. On fertilization these produce zygoteswith
1512
J. Loidl
TABLE 1
Yeast strains
Strain
Genotype
Source
~
~~~~~~~~
NKY857
MATa ho::LYS2 lys2 leu2::hisG his4X ura3
N.
KLECKNER
NKY860
MATa ho::LYS2 lys2 leu2::hisG his4B ura3
N.
KLECKNER
Strain 38
-MATa ho::LYS2 &
2
leu2::hisG his4X ura3
MATa ho::LYS2 lys2 h2::hisG his4B ura3
F. KLEIN
Strain 74
-~
MATa ho::LYS2 &
2 h2::hisG his4 ura3
F. KLEIN
MATa ho::LYS2 lys2 h2::hisG his4 ura3
Strain 75
-~
MATa ho::LYS2 &
2 h2::hisG his4 ura3
F.
KLEIN
F.
KLEIN
F.
KLEIN
F.
KLEIN
MATa ho::LYS2 lys2 leu2::hisG his4 ura3
Strain 76
_
MATa ho::LYS2
_ h 2 leu2::hisG
~ his4 ura3
-~
MATa ho::LYS2 k 2 leu2::hisG his4 ura3
-___
MATa ho::LYS2 &
2 h2::hisG his4 ura3
MATa ho::LYS2 lys2 leu2::hisG his4 ura3
Strain 77
-~
MATa ho::LYS2 &2 leu2::hisG his4 ura3
MATa ho::LYS2 &
2 leu2::hisG his4 ura3
MATa ho::LYS2 lys2 leu2::hisG his4 ura3
"
Strain 78
-~
MATa ho::LYS2 &
2 h2::hisG his4 ura3
_
MATa
__
ho::LYS2
_ _ h 2 h2::hisG his4 ura3
MATa ho::LYS2 lys2 leu2::hisG his4 ura3
multiple trisomy, which are frequently inviable. Also,
natural or artificial autotetraploids often suffer from
infertility due to the unequal distribution of homologous chromosomes (see GILLIES1989).
Here thepairing behavior and SC formation are studied in isogenic triploid and tetraploid strains of budding yeast. The occurrence of pairing partner switches
is used to estimate the numberof SC initiation sites per
chromosome. The effect of the triploid and tetraploid
conditions on fertility are quantitated by a spore viability test and the consequences of multivalent formation
on chromosome segregation are discussed.
MATERIALSANDMETHODS
Yeast s t r a i n s Haploid strains NKY857 and NKY860, which
are derived from SK1 (ME
and ROTH 1974),were obtained
from N. KLECKNER. They were mated and zygotes were isolated by micromanipulation to produce the diploid strain 38
(see Table 1 ) . To produce the isogenic MATa/a and MATa/a
strains 74 and 75 by recombination at the MAT locus, cells
of strain 38 were exposed to cobalt 60 irradiation (10 krad)
and plated for single colonies. Strains homozygous for MAT
were identified by their mating capability with appropriate
mating type tester strains. Strain 76 resulted from a cross of
strain 74 with strain 75. Tetraploidy of strain 76 was confirmed
by subsequent cytologicalanalysis. The triploid strain 77
( M A T a / a / a ) was obtained from a cross of strain 74 with
NKY860 and the triploid strain 78 ( M A T a / a / a )from a cross
of strain 75 with NKY857.
Growth and sporulation: Strains were grown in presporulation medium at 30" to a density of 2 X lo' cells/ml (ROTH
and HALVORSON
1969). Cells were then either left overnight
at 0" in the presporulation medium or immediately washed
and resuspended in 2% potassium acetate at a density of 4 X
l o 7 cells/ml. The sporulation time for maximum yieldof
pachytene nuclei was 5 hr for the polyploids, the same as for
the isogenic diploid.
SC spreading: We applied the protocol by LOIDL et al.
( 1991). Ten milliliters of cell suspension was harvested after
the appropriate period of sporulation, centrifuged and resuspended in 1 ml2% potassium acetate supplemented with 0.8
M sorbitol, 10 mM dithiothreitol and 10 p1of a 10 mg/ml
stock solution ofZymolyase20T
(Seikagaku Kogyo) . Cells
were left to spheroplast in this solution at 37". Spheroplasting
was optimal when -90% of cells lysed after mixing 10 pl of
suspension with 10 p1of 1% (w/v) Sarkosyl. Spheroplasting
was terminated by the addition of 10 ml icecold stopping
solution made of 1 M sorbitol, 0.1 M morpholino ethanesulfonic acid, 1 mM EDTA and 0.5 mM MgCIP. Spheroplasts
were centrifuged gently and resuspended in 1 ml stopping
solution; they were used for spreading immediately or stored
at 0" for several days.
A 2Gp1 drop of cell suspension was pipetted onto a clean
microscopical slide and the following solutions were added
to the drop and mixed by tilting the slide: 40 pl fixative (4%
w/v paraformaldehyde, 3.4% w/v sucrose in water), 80 ~ 1 1 %
v/v Lipsol ( a mixture of anionic and nonionic detergents;
L.I.P. Ltd., Shipley, England) in waterand finally 80 p1 fixative
(as above) . The solutions were mixed and spread evenly over
the slide with a glass rod. (Lipsol acts as spreading agent by
solubilizing cell and nuclear membranes and the application
of some fixative before the addition of the detergent appears
to prevent overspreading. Lysis of nuclei can be monitored
by phase contrast microscopy.)
Staining and microscopy: After spreading, the slides were
left to dry, rinsed with 0.2%v/v Photo-Flo (Kodak) and dried
again. For the staining of the SCs a few drops of 50% silver
nitrate solution were placed on aslide and covered with nylon
mesh (Nybolt PA-100/31, Swiss Silk Bolting Cloth Mfg., Zurich) trimmed to the size of the slide. Preparations were incubated in a moist chamber at 60" for 40 min, rinsed with distilled water and dried.
Meiosis in Polyploid Yeast
FIGURE1.-SCs in silver-stained spread preparations dip
of
loidwildtype.Sixteen pachytene bivalents are present; the
large patchof mediumdensity material to the left
is the nucleolus that is organized by bivalent XII. Arrows point to the
sites where the arms of bivalent XI1 project from the nucleolar
mass. The doublespindle pole can be seen in the upper part
of the panel. Scale bar, 1 pm.
For the transfer of nuclei to electron microscopic grids,
slides with spread nuclei were coated with Formvar ( 1 % w/
w in chloroform). Good areas on the slides were preselected
in the light microscope at low magnification and markedwith
a waterproof pen. The
plastic coating aroundthe area bearing
the marks was scratched.A few drops of 1% hydrofluoric acid
(caution: toxic and corrosive!) applied to the scores detached
the plastic film together with the probe from the glass. Upon
addition of water the plastic film floats freely. Electron microscopic grids were placed on the marks, and the film together
with the grids were picked up from the water surface with a
pieceof Benchkote (Whatman) plasticcoated paper. After
drying, the grids with the attached film were picked off the
paper and were ready for examination in the electron microscope.
RESULTS
SC morphology: Similar to most eukaryotes, diploids
of the yeast Saccharomyces cerevisiae show a tripartite SC.
It consists oftwo parallel longitudinal axial elements
with acentralelement
in between. The SC is surrounded by a haloof chromatin from the homologously
synapsed chromosomes. Sixteen linear SCs indicate that
each pachytene bivalent is synapsed all the way from
end to endwith the exception of the chromosome X I I
bivalent, whose SC is interrupted by a large median
nucleolus (DRESSERand GIROUX1988; LOIDLet al.
1991 ) (Figure 1 ) . Ultrastructural details like the parallel arrangement of axial and central elements remain
unchanged in the polyploids. Likethe diploid, they also
form a single nucleolus and a normal double spindle
pole body at pachytene. Multiple homologous pairing
1513
in isogenic triploid and tetraploid strains is, however,
accompanied by complicated synaptic patterns. The formation of a joint SC by more than two chromosomes
and the switching of synapsed partners was observed
(Figures 2and 3 ) . SC polycomplexes, which are aggregates of SC components devoid of chromatin, were
found as a frequent feature of the triploid but not of
the tetraploid (Figure 2 ) .
Synapsis inthe triploid: (Near-) complete SCs were
most frequent after 5 hr in sporulation, which agrees
with the isogenic diploidstrain (Table 2 shows the
result of a typical experiment).Inboth
triploid
strains, different formsof SCs were found (Figure 2 ) .
In some nuclei triple synapsis extended all over the
complement (Figure 2 b ) . Five longitudinal elements,
three axial and two central, can be seen in favorably
flattened trivalents (Figure 2b). Thisindicatesthat
triple-synapsed trivalents are flat and ribbon-shaped in
cross section rather than triangular (see
also SHERMAN
et al. 1989). In most nuclei, however, trivalents were
not or only partially triple synapsed. In these trivalents
the axial element formed by the third chromosome
ran parallel at some distance to the SC formed by its
two partners (II+ I synapsis) (Figure 2c). The third
chromosome was frequently associated at its ends and
occasionally at one or several interstitial sites with its
synapsed partners.
The proportion of nuclei with (near-) complete triple synapsis generally extended from < 1 % to 5%. In
two experiments, however, it was as high as 38% ( n =
125) in the MATa/a/a strain and 23% ( n = 113) in
the MATa/a/a strain after 5 hr sporulation. This suggests that triple synapsis may be physically unstable ( i.e.,
sensitive to the spreading procedure) or vary between
cultures due to the variable physiologyof the strain
and/or be a short transientevent. To detect a possible
correlation of triple us. II+I synapsis with pachytene
substages, relative amounts of predominantly tripleand
II+I synapsed nuclei were scored in cultures from 4.5
to 6.0 hr (Table 3 ) . At all time points triple-synapsed
nuclei were rare and their frequencies did not differ
significantly. Therefore variability isgreater between experiments than within different time points in one and
the same experiment and it seems that the proportion
of triple synapsis is not stage dependent.
In II+I synapsed trivalents the observed intercalary
associations may be caused by pairing partnerswitching.
Associationsmay,however, also occur through short
triple SCs or homologous interlocks (see Figure 4a for
possible interpretations). Thelimited resolution of the
finestructureprecludesthe
assessment of the frequency of pairing partner switches in the triploid.
In a small subset of pachytene nuclei, no individual
trivalents could be discerned. The SCs and axial elements were interconnected atmany sites and generated
a network of partially paired regions (Figure 2 d ) . This
resembles the situation in late pachytene of triploid
1514
J. Loidl
<. ..
. ..
,. . .
.
I
f
'
.
'
.
, .
.
.
.
.
.
.
FIGURE 2.--Aspects of synapsis in silverstained spreadsof triploid yeast. (a) Several single axialelements plus afew synapsed stretches
are present at presumptive zygotene. The absence of triplesynapsed regions at the earliest pairing stage suggeststhat there is no initial
simultaneous synapsis of all three homologues at one site. ( b ) Triple synapsis. Five longitudinal structures (three axial elements and
two central elements) can be discerned in some regionsof trivalents. (c) 11+ I synapsis. Arrows point to end-toend associations between
homologues (see Figure 4a). The arrowhead denotes a possible pairing partner switch (for interpretation see Figure4a). (d)Network
of interconnected SO. ( e ) Putative diplotene. Most axial elements are unpaired. This aspect was only observed in preparations from
time points later than 4.5 hr. In b and d SC polycomplexes can be seen as dense striated bars. Scale bar, 1 pm.
Allium sphderocephalon, where chromosomes thatare
prevented from homologous synapsis become engaged
in random SC formation ( LOIDLand JONES 1986).
Apart from pachytene nuclei showing different
modes of pairing, the spread preparations contained
meiotic nuclei where synapsis was incomplete. At early
Polyploid
Meiosis in
Yeast
1515
FIGURE
3.-Synapsis in tetraploid yeast. Homologues are present as pairs of bivalentsor quadrivalents with one or two pairing
partner switches (arrows). ( a and b ) Total views. ( a ) Single axial elements are rarely present (asterisks) ; one is associated to
whatmay be the homologous bivalent. ( b ) One quadrivalent shows two pairing partner switches; two aligned presumptive
homologous bivalents are denoted by an arrowhead. (c-f) Individual quadrivalents from different nuclei shown at higher
magnification. Even relatively smallchromosomes can have two pairing partner switches ( e ) . Incompletely synapsed quadrivalent;
an axial element (open arrowheads) is split off for about half its length. For interpretation see Figure 4c. Scale bar in a, 1 pm
in a and b; scale bar in c, 1 pm in c-f.
time points (3.5-4.0 hr) , nuclei withSC fragments
predominated over pachytene nuclei (Table 2 ) . These
putative zygotene nuclei comprised axial elements together with short synapsed stretches that involved only
two axial elements (Figure 2a). This observation suggests that synapsis firstoccurs between two homologues
only and that in trivalents with triple synapsis the third
chromosome joinsin later. Nuclei with incomplete SCs
were less frequent than pachytene nuclei from 4.5 to
5.5 hr but increased again at later time points (Table
2 ) . Close examination revealed that most of these late
nuclei hadtheir axial elementsseparated and they
looked somewhat fuzzy (Figure 2e). This phenotype
has been observed in several diploid yeast strains other
than SK1 before (J. LOIDL,unpublished observations)
and may be interpreted as diplotene considering its
resemblance to diplotene SCs in animals.
Synapsis in the tetraploid In the tetraploid, homologues are mostly present as pairs of bivalentsat pachytene (Figures 3 and 4b). Sometimes, they form quadrivalents by switching pairing partners. A total of 218
quadrivalents was found in 41 nuclei, that is, 5.3 per
nucleus. Thus, on average one outof three homologous
chromosome sets formed a quadrivalent. Thirty-six
J. Loidl
1516
TABLE 2
Relative frequencies (%) of nuclei at different meiotic prophase stages throughout sporulation of a triploid
SCs
“diplotene”
Incomplete
Hours
“zygotene”,
sporulation
142
3.5
4.0
4.5
5.0
5.5
6.0
6.5
(Near-)complete SCs
“pachytene”
3
7
27
14
8
39
13
75
206
0
<1
51
55
48
26
12
Empty nuclei“
n
97
93
50
31
44
55
199
152
223
206
192
“Nuclei without silver-stained SC structures. This class includes both premeiotic/early meiotic and late
meiotic (postdiplotene) stages.
quadrivalents ( i.e., less than one per nucleus) showed
more than one pairing partner switch.
There is some remnant alignment between putative
homologous pairs of synapsed bivalents (Figure 3 b ) .
This suggests that initial homologue recognition and
presynaptic alignment has involvedall four chromosomes, even in cases where only pachytene bivalents
ensue. Although all four chromosomes of a homologous set are genetically identical, no quadruplesynapsis
was found; synapsis occurs between pairs of axial elements only. Very rarely (two examples) trivalents with
partial triple synapsis occurred with the fourth homologue remaining single. Occasionally, incomplete synapsis resulted in the formation of univalents (Figure
3a) or quadrivalents with unsynapsed arms (Figures 3f
and 4c).
Also in the tetraploid a change in the
relative frequencies of configurations with sporulation time could
not be established. Zygotene is not readily analyzable
in yeast SC spreads because the axial elements are fragmentary. Therefore it was not possibleto
decide
whether a reduction of pairing partner switches from
zygotene to pachytene occurs through the dissolution
and reassembly of the SC. The mean frequency of quadrivalents at 4.5 hr sporulation was 4.4 per pachytene
nucleus ( 14 nuclei) ; at 5 hr it was 5.5 ( 10 nuclei) and
at 6 hr 5.9 (17 nuclei). Thus,although the number of
examined nuclei islow,it seems that the number of
switches per nucleus remains fairly constant throughout
pachytene.
TABLE 3
Relative frequencies (%) of pachytene nuclei
with triple and I1 I synapsis
+
Hours
sporulation
Triple
synapsis
4.5
5.0
5.5
6.0
3
5
2
I1
+ I synapsis
97
95
98
96
n
233
226
125
287
The segregation of trivalent and quadrivalent chromosomes: The effect of meiotic malsegregation of
multivalents on spore viability was tested in the polyploids by dissecting asci on W D plates. Spore viability
of the two triploid strains was 37% (224 spores dissected) and 41% (of 128 spores) (difference not significant, x 2 ;P 0.5). This is higher than the 15-25%
germination reported by PARRY
and COX ( 1970; E. M.
PARRY,
personal communication) and the 16-20% via-
b
FIGURE4.-Aspectsofsynapsis
in triploid ( a ) and tetraploid ( b and c ) yeast. ( a ) Interpretation of possible interactions of chromosomes in a pachytene trivalent. Upper part:
II+I synapsis. Two chromosomes are connected by SC, the
third is aligned in parallel with them at some distance. II+I
synapsed homologues are often attached end-to-end ( A ) .
Partner switches in 11+ I synapsed trivalents ( B ) may be easily
confused with interlocks ( C ) or local triple SC ( D ) . Lower
part: Triple synapsis. ( b ) The numberof partner switches can
be used to calculate the minimal number of independent
synaptic initiation sites on a chromosome, which is the number of switches + 1. In a quadrivalentwith one switch, as shown
here, each chromosome is involved in two SC initiation events.
Two neighboring SC initiation sites between the same two
chromosomes would not be detectable as a switch, but the
chance of this event occurring is negligible in yeast. ( c ) Interpretation of the quadrivalent depicted in Figure 3f with two
arms lacking SC initiation.
1517
Meiosis in Polyploid Yeast
a
b
@
Parental cells
(tetraploid)
69
Sporulation
(1 st round)
F1 sporedcolonies
(mostlydiploid)
Sporulation
(2nd round)
I I I I
I I I I I I
F2 spores
(mostlyhaploid)
FIGURE5.-Experimental approach to study nondisjunction in tetraploid meiosis
by viability tests of spores of the first
(F1)
and second ( F2) daughter generations. ( a ) Tetraploid meiosis will produce four viable diploid F1 spores if only bivalents are
formed or if quadrivalents segregate 2:2. Colonies grown from these spores will produce asci with four haploid F2 spores. ( b )
4:O segregation of a quadrivalent at tetraploid meiosiswill produce two nullisomic, deadF1 spores and four tetrasomicF1 spores
that give rise to viable disomic F2 spores. ( c ) 3:l segregation of a quadrivalent will produce four viable F1 spores (two trisomic
and two monosomic) and thereforego unnoticed at thefirst generation. The resulting trisomic colonies will produceasci with
four viable F2 spores (two disomic and two normal haploid) whereas the monosomic colonies will produce asci with only two
viable haploid F2 spores. The remainingtwo F2 spores are nullisomic and dead. More complicated situations, such as spores that
are trisomic or tetrasomic for more than one chromosome
may be rare, given the rarityof nondisjunction events as documented
of F1and F2 spores. Bars within the circles correspond
to the numberof homologous chromosomes
by the fairly good viability rates
per cell.
bility reported by CAMPBELL et al. (1981)for triploid
strains. Nevertheless, it means a highly significant reduction ( x 2 ;
P < 0.001) compared with spore viability
in the isogenic diploid SKI of 98% ( n = 128) . Probably
as the consequence of theiraneuploidconstitution,
most of the resulting colonies were very small and it
took 4 days until all of them became visible.
To assess the frequency of nondisjunction at tetraploid
meiosis, the tetraploid parental strain was sporulated and
the viability of the diploid spores of the first daughter
generation ( F1) and the haploid spores of the second
daughter generation ( F2) was checked (Figure 5 ) . One
hundred eight diploid F1asci resulting from the first
sporulation were dissected. Spore viability was 94%. This
is slightly lessthan in the isogenic diploid. Six asci ( 5%)
contained only two viable spores. A possible explanation
for their occurrenceis the 4:Onondisjunction of a quadrivalent, which produces two tetrasomic and two nullisomic diploid spores (Figure 5b) . Some asci produced
two apparently normal and two small colonies. The reduced vigor of two spores could be due to monosomies
caused by 3:l nondisjunctions.
To quantitate the incidence of 3:l nondisjunction at
tetraploid meiosis, cells were allowed to undergo two
rounds of sporulation. A 3:l nondisjunction will produce viable trisomic and monosomic F, offspring.
Monosomic F1 cells willyield 50% viable monosomic
and 50% nonviable nullisomic haploid F2 spores after
another round of sporulation (Figure 5 c ) .
Fifty-four randomly selected F1 tetrads were dissected
(Table 4). Twenty-three of these contained spores that
were all viable and grew to colonies that were able to
TABLE 4
Sporulation competence and viabilityof the F, and F2progeny of 54 tetraploid parental cells
Number
17
of tetrads
FI spores/colonies
(diploid)
F2(haploid)
spores
S,
5
s
s
s
s
s
s
14
1
s
s
s
s
s
1 1 1 1
1 1 1 1
1 1 1 1
v
v
v
v
v
v
v
v
v
v
v
v
v
v
v
v
v
v
v
v
v
v
v
v
v
v
v
v
v
v
15
(
s
v
v
v v d dd d d d
v v d dd d d d
sporulating colony;n, nonsporulating colony;v, viable spore; d, dead spore.
s
s
n
n
n
1
n
n
n
2
)
s s s d
1
determined
not
J. Loidl
1518
undergo a second round
of sporulation. The remaining
tetrads contained two or fourspores thatwere homozygous at the MATlocus (as tested by their ability tomate
with h4ATa or MATa tester strains) or they contained
less than four viable spores. Of the 23 F, tetrads that
produced colonies that were sporulating for another
round, 17 produced only F2asci withfour viable spores,
5 producedF2 asci of which halfhad fourviable spores
and half two viable and two dead spores and one produced only FP asci with two viable spores (Table 4 ) .
The best explanation for F1 tetrads with two spores producing all viable and two spores producing 50% viable
F2 offspring is that 2 F1 spores were monosomic for
at least one chromosome as the consequence of a 3:l
nondisjunction in the tetraploid meiosis ofthe parental
cell (Figure 5c) . The single F1 tetrad that gave rise to
F2 tetrads withonly two viable second generation F2
spores (Table 4) is possibly the product of a meiosis
with two 3:l nondisjunction events, which produced 4
monosomic F1 spores.
a
d
DISCUSSION
Homologous alignment and synapsis in polyploids:
In many organisms, homologous chromosomes have
been observed to be arranged in parallel at some distance before their intimate pairing by the SC. It is believed that this presynaptic alignment reflects primary
homologous contacts at multiple sites along chromosome pairs (see VON WETISTEINet al. 1984). In yeast,
presynaptic alignment was detected as the parallel arrangement of chromosomes delineated by in situ hybridization, at a distance greater than the width of the
SC ( SCHERTHAN
et al. 1992). In polyploids showing
II+ I synapsis, alignment between nonsynapsed homologues is often maintained into pachytene ( RASMUSSEN
1977; LOIDLand JONES 1986). To a lesser extent this
can be seen also in triploid yeast where often terminal
associations of nonsynapsed homologues seem to contribute to their alignment (Figures 2c and 4a).
In tetraploid yeast there is some alignment at pachytene between homologous pairs of bivalents (Figure
3b) . Also the pictures by BYERSand GOETSCH( 1975)
of sectioned tetraploid yeast SCs suggest that all four
chromosomes contact each other in some regions, but
in the sections no discrimination was possibleas to
whether quadruple synapsis, nonsynaptic alignment or
convergence of SCs at sites of partner switches had occurred.The
nonsynaptic alignmentfoundhere
in
spreads is by far not as close and extensive as in polyploid plants ( LOIDL1986;LOIDLandJONES 1986;LOIDL
et al. 1990) ; therefore it seems that in yeast either the
chromosomal sites by which it is mediated are fewer or
that alignment of unsynapsed homologues is abolished
by pachytene.
In the triploid it was found that both triple synapsis
and 11+ I synapsis occur in different nuclei or at differ-
FIGURE6.-Interpretation of the interrelationship of the
different synaptic configurations in triploid yeast. There is a
strong bias of axial elements toward synapsis. This may be
achieved by triple synapsis if two independently initiated SC
stretches ( a ) extend without interfering so that they finally
will overlap ( b ) . Sometimes (as is common in other organisms) synapsis comes to a halt where the two synapsing regions
meet at the site of apairing partner switch ( c ) . If SC is
initiated between two chromosomes only ( d ) , I I + I synapsis
will ensue ( e ) . However, the third chromosome tends to synapse heterologously either with itself by forming a hairpinloopor with otherhitherto unsynapsed chromosomes ( f ,
compare with Figure 2 d ) . In some other organisms pairing
partner switches are eliminated by resolution and reformation
of the SC (see JENKINS and REES1991) . It is unknown if this
“pairing correction” ( g ) occurs in yeast and if I1+ I synapsis
and triple synapsis are convertible ( h ) .
ent proportions in one and the same nucleus and that
the ratio of triple-synapsed us. 11+ I-synapsed trivalents
does not notably change during pachytene (Table 3 ) .
Also in the triploid basidiomycete Coprinus cinereus no
significant tendency from triple synapsis at early pachytene toward I1 I synapsis at mid to late pachytene has
been observed ( RASMUSSEN et al. 1981) . The observation in yeast, however, that the earliest stretches of SC
that appear atzygotene are made up of only two chromosomes (Figure 2a) suggests that triple synapsis does
not occur by simultaneous synapsis of allthree chromosomes at zygotene but might take place slightly later.
From the presence of different synapsed configurations the following picture of the synaptic processin the
triploid emerges (Figure 6 ) : the occurrence of both
+
Polyploid
in
Meiosis
(partial) triple synapsis and II+I synapsismay be explained on the basis of different starting conditions. If
SC is initiated between different chromosomes of a homologous set, then a11+ I synapsed trivalent witha pairing partner switch willbe formed first. As synapsis continues, a triple-synapsed region will appear. Ifsynapsisis
initiated at one or several sites between only two out of
three homologues, then II+I synapsis will ensue. At a
later stage, the hitherto unsynapsed chromosome may
engage in nonhomologous synapsis. Bothtriple synapsis
and heterologous synapsis might be explained by a
strong bias of axial elements toward synapsis (see LOIDL
and JONES 1986). In the tetraploid these phenomena
do not occur because all axialelements are saturated by
involvement in homologous bivalent synapsis.
Pairing partnerswitches: Inthe triploid, pairing
partner switches between 11+ I synapsed axescan easily
be confused with local triple synapsis or homologous
interlocks (see Figure 4a). The occurrence of pairing
partner switches in trivalents of a trisomic strain has
been reported by MOENS and ASHTON ( 1985). However, theinterpretation of these configuations from
electron microscope thin sections is also ambiguous.
In the tetraploid, pairing partner switches were much
better visible than in the triploid and it was found that
28% of chromosomes were paired in quadrivalent configurations with one switch (Figure 4b) and 5.5% of
chromosomes were part of a quadrivalent withtwo
switches.
From the numberof pairing partner switches in trivalents and quadrivalents the number of independent SC
initiation sites in bivalents of an isogenic diploid can
be estimated under theassumption that all homologues
participate equally in SC initiation ( LOIDL1986; LOIDL
and JONES 1986). A switch indicates that SC initiation
has occurred on both of its sides (Figure 4a). Thus
there is at least one moreSC initiation site than switches
observed.' In the tetraploid there mostly occur additional SC initiations that, however, can be regarded as
dependent events as they simply maximize synapsis
after
the switches have been formed (Figure 4 b ) . If additional SC initiations fail to occur, either
imperfect quadrivalents (Figures 3f and 4c) or trivalents plus univalents are formed. The presence of quadrivalents with
two switches means that interstitial initiation of synapsis
does occur. This is noteworthy because yeast chromosomes are the smallest among eukaryotes and pairing
initiation in regions in addition to those near the telomeres was assumed to be a property of large chromosomes (see VON WETTSTEINet al. 1984).
'
It should be noted that the formulas for the relationship between
switches and initiation sites given by LOIDL ( 1986) for a tetraploid
and by LOIDLandJONEs ( 1986) for a triploid plant generate higher
values for the likeliest number of independent SC initiation sites that
accompany a given number ofswitches. They apply to situations
where SC initiations and switches are frequent and the chance for
two neighboring SC initiations involving the same two chromosomes
is high.
Yeast
1519
From the frequency of pairing partner switches in
the tetraploid, one can estimate that stable synapsis is
initiated at -22 sites per diploid nucleus. This is less
than the mean number of crossovers ( 75) per nucleus
(JACOBSON et al. 1975). This finding is at variance with
observations in Sordaria macrospora, where a correspondence between pairing initiation sites and crossovers
was found ( ZICKLER et al. 1992) and in maize where
there is a 1:l relationship between the occurrence of
crossing over and SC formation in an inversion loop
( MACUIREand &ESS 1994) . The discrepancy of SC initiation sites and crossovers in yeast argues against models
where SC is initiated at sites of reciprocal recombination.
Evidence from a large number of organisms suggests
that synapsis starts near the ends of chromosomes. If
synapsis intetraploids starts at both ends of all chromosomes, then 67% of configurations should be quadrivalents, assuming that any combinations of four homologous chromosomes are equally involved inpairing (see
SYBENCA
1975). Since in the tetraploid used here the
four genomes are virtually identical, preferential pairing between any two homologues is not likely and an
average of 10.7 quadrivalents per cell should occur.
However, onlya mean of 5.3 quadrivalents per nucleus
was found, that is, 33% of chromosomes were involved
in quadrivalents. The presence of 33% quadrivalents
indicates that only in -50% of homologous chromosome sets ( i e . , 1.5 times the number of quadrivalents;
see aboveand SYBENCA
1975) pairing is initiated at both
ends (and sometimes at an additional intercalary site).
In the remainder
synapsis must be assumed to start
from one end only, resulting in exclusive bivalent formation. One possible explanation for this is that SC is
initiated at one site between any two chromosomes of
a homologous set of four and proceeds very fast. Because of the smallness of the yeast chromosomes it will
extend all along the bivalent before another initiation
event takes place. Subsequent initiation events would
therefore be restricted to the remaining homologues
and produce another bivalent.
Consequences of polyploidy on nondisjunction and
spore viability: Pachytene multivalents become metaphase I multivalents if crossovers occur in the synapsed
portions and the resulting chiasmata stabilize the bonds
between chromosomes after the SC has disappeared.
In yeast, where there is an average of three crossovers
for the smallest chromosome to nine for the largest
one ( MORTIMERet al. 1989), it seems likely that most
pachytene multivalents become metaphase multivalents
and are thussubject to potential nondisjunction. In the
triploid, this means that trivalents segregate randomly
as 2:l chromosomes, producing highly genetically imbalanced monosomic/disomic spores (see CAMPBELLet
al. 1981) . Therefore, spore viability in the triploids is
reduced to 40% and the resulting colonies are very sick.
In tetraploid yeast, high spore viability is maintained
1520
J. Loidl
compared with the considerably reduced fertility found
in other autotetraploid organisms. This is partly due to
the preferential formation of bivalents that segregate
regularly as 2:2 chromosomes and hence mimic diploid
behavior (see above) . Chromosomes paired in quadrivalents, ontheotherhand,
mightbe
subject to
malsegregation. In fact, it was already noted by ROMAN
et al. ( 1955) that tetraploid meiosis may produce aneuploid spores in yeast. The slightly reduced sporeviability
combined with the occurrence ofasciwithonly
two
viable spores observed here could be interpreted as the
result of rare 4:0 nondisjunction (Figure 5). Moreover,
by determining inviable spores after two rounds of sporulation (Figure 5), it is calculated here that in -25%
of meiotic divisions at least one chromosome is nondisjoined in a 3:l manner. Since on average 5.3 quadrivalents are formed atpachytene (and practically the same
frequency can be inferred for metaphases), one can
estimate that 1 of -22 quadrivalents is subject to 3:l
nondisjunction.
Nondisjunction in yeast is likely to be caused by a 3:l
centromere orientation of metaphase quadrivalents (3
centromeres pointing to one pole and 1 centromere
pointing to the opposite pole) at the meiotic spindle.
Assuming arandomorientation
and segregation of
chromosomes in a quadrivalent, one would expect relative frequencies of 3:4:1 for 22,3:l and 4:0 disjunction,
respectively (see SYBENGA
1975,p. 223).The observed
underrepresentation of malsegregating quadrivalents
suggests that some correction mechanism promotes 2:2
disjunction. This might be constituted by the strong
bias of the spindle apparatus toward separating equal
masses of chromosomes (see, e.g., ARANA and NICKLAS
1992;RIEDER and SALMON
1994). Therefore, preferential bivalent formation together with preferential 2:2
segregation of the few quadrivalents that do form ensures the high spore viability in tetraploid yeast.
I am indebtedto FRANZK L E I N for constructing the polyploid strains
KLEIN
and tn NANCXKLECKNER for the haploid parent strains. FRANZ
and RNUD
NAIRZgave me a lot of advice and support with the genetical part of the work. I am also extremely grateful to DAVID KARACK
for reading the manuscript and making many useful comments. This
project was supported by grant S5807 from the Austrian Fund for
the Advancement of Scientific Research (F.W.F.) .
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Communicating editor: S. JINKS-RORERTSOK