DETECTION AND IDENTIFICATION OF TRANSLOCATIONS BY INCREASED SPECIFIC NONDISJUNCTION IN NIDULANS

ALAN UPs€3.ALL1p2AND ETTA Department of Biology, McGill University, Montreal, Canada Manuscript received June 28, 1973 Revised copy received September 24,1973

ABSTRACT A meiotic technique for visual detection of translocations has been applied to ten mitotically identified interchanges, and three new translocations were discovered using this method. Testcrosses between “standard” strains and potential translocation strains-e.g. strains with newly induced mutants or descendants from translocation crosse+are inspected for the frequency of abnormal-looking colonies. In all heterozygous translocation crosses “abnor- mals” are increased at least tenfold compared to the average control level of 0.15%. Most of these are rjisomics, and can be recognized by their character- istic phenotypes. Each translocation produces a few specific types, since nondisjunction is increased mainly in the linkage groups involved in the translocation (5GIOO-fold over control values). Therefore, translocations were not only detected but often tentatively assigned to linkage groups from the analysis of the disomic progeny in crosses. In addition, this technique allows reciprocal and nonreciprocal translocations to be distinguished, since only the latter produce one-third phenotypically abnormal duplication progeny. While results are clearcut in most cases, occasionally problems are encountered, e.g. when morphological mutants segregate in crosses, or when other genetic factors which increase or reduce the frequency of nondisjunction are present in certain strains.

IN many organisms chromosomal translocations produce specific patterns of genetic segregation, such as unusual linkages and reduced meiotic recombi- nation (e.g., in Drosophila, ROBERTS1970), reduced viability of a fraction of the offspring (e.g., in , BURNHAM1948), familial patterns of trisomy (in man, LEJEUNE,GAUTIER and TURPIN1959), etc. These can lead to the detection and mapping of translocations in genetically well-known organisms, but generally, in higher plants and animals, cytological investigations are used for identification or confirmation, when either characteristic meiotic metaphase configurations or rearranged banding patterns in mitotic can be observed. In fungi, cytological identification is not practicable as a routine procedure, although it has been successful in Neurospora (e.g., BARRY1967). A genetic method for detection is available in Neurospora where heterozygous translocations produce character- istic ascus-patterns, since inviable meiotic products form unpigmented ascospores

Supported by operating grant A2564 (to E.K.) from the National Researh Council of Canada. a On sabbatical leave from the Department of Biological Sciences, Lancaster University, Lancaster, England.

Genetics 76: 19-31 January, 1974. 20 A. UPSHALL AND E. GFER which can be observed in ordered or even unordered tetrads (PERKINS1967). In this , a large number of reciprocal and insertional translocations have been analyzed and genetically mapped in this way (PERKINS1974). In Aspergillus nidulans, techniques for the detection and identification of trans- locations are also of a genetic type. The system in general use relies upon the detection of complete mitotic linkage between all markers known to be located on two different chromosomes, when haploid segregants from translocation heter- ozygotes are tested (UFER1962)-analogous to the inversion technique used in Drosophila. This method does not differentiate between reciprocal and insertional translocations. It has been successfully employed in identifying and tracing the pedigrees of eight translocations, most of which were radiation-induced simul- taneously with the induction of new markers (=FER 1965). However, while such information is obtained automatically when new markers are mapped into their mitotic linkage groups (%FER 1958) this method is somewhat laborious for pedigree analysis; also it is not always easy to obtain translocation-free tester strains with suitable combinations of markers for every strain to be tested. A second method, which is the topic of this report, depends on the observation that in many organisms meiotic nondisjunction is significantly increased in trans- location heterozygotes, especially for the two homologs involved in the rearrange- ment (e.g., in Drosophila, GRELL1959). This was also found in Aspergillus nidulans, where crosses heterozygous for the reciprocal translocation TI (VI;VIZ) produced increased frequencies of nondisjunctionals, mainly disomic for VI or VII, and such increases could be detected visually in ascospore platings of moderate size (POLLARD,&FER and JOHNSTON1968). Assuming that other translocations would produce similar effects, we postulated that meiotic analysis could provide a convenient way (1) to detect such aberrations by observing an increased frequency of disomic, phenotypically abnormal progeny, and (2) in favorable cases, to identify the heterozygous translocations by characterizing the nondisjunctional progeny genetically and visually. The latter is possible in A. nidulans since disomics, as well as trisomics, for each of the eight linkage groups show specific phenotypes (KAFER 1961) and the last of the eight disomic types, n '+ 1 for group VI11 which has very poor viability, has recently been recovered and identified by segregation of genetic markers (UFERand UPSHALL 1973). Our investigations show that this meiotic method of detection and identification of translocations is applicable to all previously known cases as well as to those which were newly discovered in the course of this work. In heterozygous crosses all translocations yielded a significantly higher frequency of visually abnormal progeny than control crosses, and most of this increase was due to a very high specific increase of nondisjunctionals for one or both of the chromosomes involved in the rearrangement.

MATERIALS AND METHODS

Strains: All strains employed are descendants of the original wild-type strain of Aspergillus niduZans used by PONTECORVOand coworkers (1953). Details of the origin of the mutants used here are given by DORN(1967). The new symbols of the mutants used have been described DETECTION OF TRANSLOCATIONS 21 and current information on mapping and recent terminology changes have been summarized by CLUTTERBUCKand COVE(1974). Most of the translocations were found in strains with W- induced mutants and many of these are also in the Glasgow stock collection (several translocations detected by mitotic analysis have been reported previously-KXFm 1965). Most of the mitotic tester strains, i.e. translocation-free strains with markers in all or most linkage groups, are the same as used in the earlier work and many are available from the Fungal Stock Center (Humbolt College, Arcata, California; BAFIRAZT,JOHNSON and OGATA1965). “Meiotic tester” and control strains with the standard complement and a few suitable markers were isolated from crosses between the following strains: biA (1) (biotinless), induced by X-rays in the original wild type and used as “standard“ in Glasgow; the Montreal standard, which is an eighth-generation backcross strain containing the conidial color mutants yA(2) (=yellow), and WA(3) (=white), and the p-aminobenzoic-acid-requiringmutant pabd (1); two translocation-free strains with mutants induced by UV-treatment of biA, namely riboA (1) biA (1) and biA (1) ; pyroA (4) requiring riboflavine or pyridoxine respectively. The most frequently used meiotic testers were pdaA yA, riboA biA and pabaA yA; wA. One other meiotic tester AcrA; 1ysB; C~QAhad been obtained after five generations of backcrossing of the resistance mutant AcrA (1) (=acriflavine), the color mutant chaA (1) (=chartreuse) and the mutant lysB(5) requiring lysine (details of above backcrosses are given in BARRA~et al. 19%). All these strains are expected to be almost isogenic. Media and general techniques: Standard media and established methods were used with respect to incubating temperature, “perithecium analysis”, production of heterokaryons and diploids, etc. (PONTECORVOet al. 1953; for details of media see BARRATTet al. 1965). Experimental procedures a) Measurement of meiotic nondisjunction The frequency of meiotic nondisjunction is regarded as being represented by the frequency of disomic colonies recovered among the ascospore progeny. We observed no clones of premeiotically produced disomics, nor were any found previously among larger samples of disomics (then called “unstable mutants”; UPSHALL1966). This is, in fact, a minimum estimate, since even under optimal conditions aneuploid types are occasionally overgrown by normal colonies and in some translocation crosses certain expected disomic types have not been recovered and appear to be inviable. Controls: For measurements of the influence of structural nonhomology on meiotic nondis- junction, crosses between completely isogenic strains provide the theoretical control from which base-values of nondisjunction frequencies should be obtained. Aspergillus nidulans, being homothallic, provides such an isogenic system with the production of selfed cleistothecia. In our experiments (Table 1) such controls were obtained from platings of selfed ascospores from two prototrophic standard strains (descendants of the meiotic tester pubaA TA and the translocation- free biA; pyroA strain). Similarly, platings of selfed ascospores from a strain containing TI(V;VZ) are included which, as expected, also show control levels of meiotic nondisjunction. In addition, two crosses between the following closely related, practically isogenic, strains are considered valid controls: (1) between the wild type and an eighth-generation backcross strain to produce the ninth generation “backcross” and (2) between the Montreal and the Glasgow “standard” strains (described above). Testcrosses (Tables 1 and 2): These consist of two classes, namely, crosses of meiotic tester strains with (1) strains previously tested for translocations by mitotic analysis or with (2) previously untested strains. These latter are either UV-treated original mutants or descendants from heterozygous translocation crosses, some of which were simultaneously checked for trans- locations in test diploids. Ascospore samples were taken from a single cleistothecium (except where noted otherwise) and the data from comparable crosses have been pledon the basis of the close similarity of percent abnormals (e.g., for the translocation-free crosses, Table 1, statistical proof of homogeneity of individual crosses has not been attempted, partly because of the zero expectation in the smaller control samples; while the size of the samples ranged greatly, from 300 to 25,000, the frequency of abnormals varied only from 0 to 0.3%). No heterogeneity was 22 A. UPSHALL AND E. FER evident between the different groups of crosses listed in Table 1 (x2 for eight degrees of free- dom = 0.82, P > 0.95). b) Recovery and phenotypic classification of LLabmrmls’tfrom crosses In Aspergillus, aneuploid colonies have a much slower growth rate than euploid normal colonies and so are at a competitive disadvantage and can easily be lost when plated in crowded conditions. To counteract this, a low plating density of between 25 and 50 viable ascospores per plate was aimed for. Colonies of reduced size were initially marked after 24 hours’ incubation, and the plates reincubated for a further 24 or 36 hours. For some of the large platings of control crosses (Table 1, sections “a) 1” and “c) I”) the plating medium was supplemented with sodium deoxycholate (0.08%; MACKINTOSHand PRITCHARD1963). It was found that on this medium a density of up to 200 colonies per plate did not reduce the efficiency of recovery of abnormal, mainly disomic types. For final phenotypic classification the two- or three-day-old abnormal colonies were replated by the technique of needle plating (KAm 1961). Unstable abnormal colonies were classified as “typical disomics” for specific linkage groups by comparison with the eight standard phenotypes. Meiotic products with two or more extra chromosomes are generally not expected to survive, but a few are regularly found and probably represent the most viable types. Four types of disomics are expected to be found with increased frequency from heterozygous translocation crosses, namely in addition to the two typical disomics, two nondisjunctional types in which the extra chromosome is one of the two involved in the banslocation. These two new types will have the general characteristics of all disomics, i.e. “abnormal” center, instability, etc., but will be phenotypically dissimilar. Such colonies were phenotypically classified as “modified” disomics, provided they formed two phenotypic groups and/or clearly showed some of the characteristics of the phenotypes typical for the two relevant linkage groups. It is often not possible to be certain which of the two centromeres is actually disomic in each of the “modified” types without detailed genetic analysis (which will be described elsewhere). The frequency of nondisjunction for each of the two translocation linkage groups can therefore not be determined individually, and the sums of the values for both groups are used for all comparisons (e.g., in Tables 2 and 3). Occasionally, classification of a “modified” type may also become difficult, when it resembles a “typical” n+l for another linkage group (e.g. the modified nfl for VI from Tl(V;Vl) has large, slightly conidiating centers similar to the typical n+l for IV). Any additional colonies which did not have phenotypes of “typical” or “modified” disomics (or in very rare cases trisomics) are listed in all tables as “other abnormals”. These consist of genetically abnormal types showing various morphological deviations, some producing sectors with high frequency, others rarely, some not at all. To recognize with certainty stable abnormal types which show only slight morphological deviations, it is important that both parental strains show standard morphology. If morphological markers segregate or nutritional markers produce modified phenotypes, it is usually impossible to recognize the less extreme types of abnomals. In addition, in such crosses, some disomics may have modified phenotypes or reduced viability SO that the different types of abnormals become difficult to classify and their frequencies cannot be measured accurately. All such cases have been excluded from the analysis. c) Genetic analysis of disomics from crosses For standard aneuploids from heterozygous diploids, genetic proof of disomy can be obtained by observing segregation of the two alleles of any disomic marker in haploid sectors (KAFER 1961). This is seldom possible for disomics from translocation crosses, since meiotic tester strains carry few markers. Also, relevant markers may become homozygous due to a meiotic exchange, and haploid sectors of translocation disomics do not segregate for heterozygous markers (POLLAKD, KAFB and JOHNSTON 1968). Detailed genetic analysis was, therefore, not possible here (and will be described elsewhere for better marked crosses). However, for two translocations which were heterozygous in crosses containing suitable markers of the involved linkage groups, it was possible to confirm the phenotypic classification by genetic evidence. In the case of TI(1II;VII) this evidence was based on the segregation of ActA, an actidione-resistant mutant linked to the centromere of linkage group I11 (mapped by BAINBRIDGE1970). In the case of the translocation DETECTION OF TRANSLOCATIONS 23

T2(Z;VZll) it depended on the sulphite-requiring mutant sD50 which is completely linked to the breakpoint on VI11 (MA and KAFER 1974). In each case several disomics of identical phenotype were analyzed by testing a few disomic centers and 12-25 of their haploid sectors for resistance or requirement. d) Mitotic identification of translocations The standard mitotic test for translocations in heterozygous diploids (=FER 1962, 1965) was carried out in parallel to the relevant testcrosses and some of the new translocations were detected, the others confirmed and identified in this way. Whenever possible, mitotic tester strains were used which permitted selection of haploids by the recessive adE-suppressor technique (PONTE- CORVO and K&ER 1958; KAFER 1958) in addition to the convenient method using complete medium supplemented with p-fluorophenylalanine (LHOAS1961 ; MCCULLYand FORBES1965), since the latter occasionally produces misleading selective effects (e.g. a TI(ZV;VZZZ) was prematurely postulated when from a translocation-free diploid, heterozygous for the two mutants in repulsion, all of fifty haploids tested were pyroA palE+).

RESULTS Frequency of nondisjunction in control and translocation-free crosses Table 1 shows that the same low frequency of “abnormals,” mainly of a non- disjunctional disomic type, was obtained not only in control crosses of varying isogenicity but also in many testcrosses. The latter group (Table IC) contains crosses of standard meiotic testers to ten strains which had been treated with mutagens to induce a biochemical mutant and to 20 descendants from crosses heterozygous for translocations. All these strains are, therefore, assumed to be free of reciprocal translocations. Of the 30 tested strains, nine were also checked by mitotic analysis and the above conclusions were confirmed in all cases. Relatiue frequency of disomics for the eight diflerent linkage groups: The inci- dence of disomics for any specific linkage group is very low (0.006-0.032% in the pooled total, Table 1). Impractically large samples would, therefore, be needed to demonstrate significant differences for each individual disomic type. However, the frequencies estimated from the pooled total suggest that, in trans- location-free crosses, the various disomic types may indeed be found with unequal frequencies (disomics for 111, VI and VI1 being the most common types). Frequencies and types of disomics in heterozygous translocation crosses Results in Table 2 show that all 13 translocations tested produced a high specific increase of nondisjunction. In all cases it was found that the frequency of “abnormals” of all types, which is easily determined visually in ascospore platings of moderate density, is considerably higher than in control crosses (10- 20-fold) when compared to the pooled control value of Table 1. This increase is seen to be almost entirely due to an increase of nondisjunctionals for the two link- age groups involved in the rearrangement (ranging from a 50- to over 150-fold increase over control levels; see Table 3). Table 3, in addition, lists a comparison between the observed and expected frequency of nondisjunction for the sum of the six chromosomes not involved in the translocation. In all cases these values are very similar and suggest that non- disjunction for these chromosomes was not affected by the translocations (in cases where translocation chromosomes segregated normally). 24 A. UPSHALL AND E. &FER TABLE 1 Ouerall frequency of “abnormls” and relative frequency of the eight disomic types from control and translocation-free crosses

Total Percent Number of dl’somicr Number of Type and number of colonlei abnormalr n + 1

c) Test crosses 1. Previously tested Translocation-free 5* 39441 0.15 7 7 7 2 5 13 10 51 9 Homozygous T1 (V1;VII) 1 1308 0.38 12 2 5 T2 ( I ;VI I I ) 1 593 0.17 1 2. Previously untested UV-treated original mutants 10 9341 0.19 2 1 2 2323 15 3 Descendants from T/+ crosses 29 39788 0.12 413 4 3 3 7 4 38 8

Total numbers 59 109778 [165] 12 15 27 8 10 35 24 7 138 27 Pooled frequencies (percent): 0.150 0.125 0.025 8 Disomic types (x~O-~): 11 13 24 7 9 32 22 6

* Pooled data from over 30 independent platings from different cldstothecia to reduce chances for recovery of clones of premeiotic origin.

In six cases (top parts of Tables 2 and 3) it was possible to distinguish four phenotypic classes of specific disomics, n + 1 for the structurally heterozygous linkage groups (two “typical” and two “modified” types) as expected in the case of reciprocal translocations. In two of these cases only a single colony of the second modified type was isolated (namely the modified n f I from TI (Z;ZV) and the modified n -I- I1 from TZ (ZZ;ZV), Table 2) ; however, their phenotypic classification was unambiguous and confirmed by genetic evidence in the second of the two cases (found to be heterozygous for wA, a marker in linkage group 11). Genetic analysis of such disomics from four crosses heterozygous for TI (1ZZ;VZZ) and the marker ActA of group I11 confirmed phenotypic classification into four types for this translocation: Only two of the four types showed heterozygosity for the centromere-linked marker ActA, namely one of the typical disomic types, (n + 111) , and one of the modified types. For the remaining six reciprocal translocations, only two or three types of DETECTION OF TRANSLOCATIONS 25 TABLE 2

Frequencies of various disomic types and L‘othrabnormals” from crosses heterozygous for different translocations

Frequency of abnomlr (XI Number and types of translocation group disomics T-linkage Number Total Diromics “Other” 9rO“pI of colonies All T-specific other abnor- a b (aib) cro~~ei examined types la;bl n + 1 mal$ typical modified typical modified Tl(1;IV) 1 1648 1.2 1.2 0.1 <0.1 4 1 a 6 Tl(I1;IV) 2 1623 3.0 3.0 <0.1 <0.1 3 1 26 19 Tl(II1;VII) 4 2944 4.2 3.8 0.1 0.2 31 47 9 26 Tl(1V;VIII) 1 690 2.7 2.5 <0.1 0.2 8 3 3 3 T1 (V;VI) 9 4557 2.7 2.3 0.2 0.3 49 12 15 27 Tl(V1;VII) 4 3643 4.4” 2.7 <0.1 1.5* 35 22 30 13

Tl(1;VII) 6 7858 2.8 2.4 0.1 0.3 a 0 184 0 TZ(1;VII) 1 644 5.0 3.6 <0.2 1.4 2 0 21 0 Tl(1;VIII) 3 3422 1.2 1.1 0.1 <0.1 11 0 0 22 TZ(1;VIII) 5 3885 2.0 1.5 0.1 0.4 14 0 43 0 Tl(I1;III) 2 1086 3.6 3.3 0.2 0.1 1 0 15 20 Tl(V;VIII) 2 2352 2.2 2.0

Tl(III+VIII) 1 1346 31.8t 3.9 0.3 27.5f 14 33 5 0

* Pooled data from four crosses homogeneous with respect to T-specific abnormals but not with respect to “other” abnormals. t ca. 30% duplication types, since unidirectional translocation. specific disomics were recovered. In some cases this may have been due to the relatively low incidence of nondisjunction in one linkage group [e.g. group I in TI(Z;VZZ) and T2(Z;VZZ),or group I1 in TI (ZZ;ZZZ)] ;or the typical and modified types may have been similar enough to produce an overlap of phenotypes. In other cases, however, clearly two-rather than four-classes seem to have been recovered. The latter was the case for T2(Z;VZZZ) since all recovered disomics were of “typical” types (see Table 2). Some of these were analyzed genetically and the phenotypic classification was confirmed. In the case of this (1;VIII) translocation, identification of typical disomics was facilitated by the completely linked mutant sD50. All n I+ 1 isolates phenotypically disomic for groups I or VI11 produced only sD- sectors. This is expected only if they are typical disomics, containing both translocation chromosomes in the haploid sector and an extra normal chromosome in the disomic center. The last case listed in Table 2 is the unidirectional translocation TI(ZZZ+VZZZ) which some time ago was traced to the original X-ray treated yA(2) strain (=FER 1965) and has recently been mapped in detail by BAINBRIDGE(1970). The meiotic results obtained here confirm the unidirectional nature of this trans- location (BAINBRIDGEand ROPER1966). In addition to one-third abnormal dupli- cation progeny (called “crinkled” and used for the meiotic mapping by BAIN- BRIDGE)three types of disomics were regularly found: two of these were “typical” 26 A. UPSHALL AND E. KAFER TABLE 3 Incidence of translocation-specific, and other, disom'cs in translocation crosses co"pared to controls

Translocation Disomic$ f01 T-linkage 91OUPS Frequency of other and linkage Wined frequencies six disomic types gmupr Jnvalued Ti* crosleP* controlt Factor Of .TI+ crorser* CO" tro1 s t percent percent increase percent Pelcent

Tl(1;IV) 1.21 0.018 67 0.06 0.106 Tl(I1;IV) 3.02 0.02u 151 <0.1 0.104 Tl(II1;VII) 3.84 0.046 77 <0.1 0.078 TI( IV;vIII) 2.46 0.013 189 <0.15 0.111 T1 (V;VI) 2.26 0.041 55 0.14 0.083 Tl(V1;VII) 2.74 0.054 51 0.05 0.070

Tl(1;VII) 2.44 0.033 74 0.04 0.091 T2( I ;VI I) 3.57 0.033 108 <0.15 0.091 T1 (I;VI1 I ) 1.05 0.017 62 0.12 0.107 T2 ( I ;VI I I) 1.71 0.017 101 0.10 0.107 T1 (II ;I I1 ) 3.31 0.037 89 0.20 0.087 Tl(V;VIII) 1.96 0.015 130 0.04 0.109

T1 (I114111) 3.86 0.030 129 0.30 0.094

* Values from Table 2. t Sum of pooled values for individual linkage groups from Table 1. disomics, with either a normal I11 or a normal VI11 extra, and the third showed a phenotype like n 4- I11 but less extreme, and is most likely the more viable of the two "modified" types, namely the disomic with the non-translocated part of I11 extra. Since n 4-VI11 disomics grow extremely slowly it is quite likely that the fourth type, with VI11 as well as part of I11 extra, would be too inviable to be recovered. One case which has been omitted from Tables 2 and 3 involves crosses with original strain containing adE (20), a UV-induced, adenine-requiring mutant. Crosses to meiotic testers or translocation-free strains gave rise to 2% abnormal progeny (over 1000 colonies inspected). Many of these were phenotypically similar to n '4-VI1 disomics, suggesting an aberration involving linkage group VII. Mitotic analysis of this strain with two different translocation-free strains, however, did not confirm the presence of any translocation involving group VII, but showed various unusual segregation ratios among the haploids selected on complete medium containing p-fluorophenylalanine. On the other hand, consist- ent results were obtained from the analysis of diploids formed with two descend- ants of the adE biA strain, one of which showed the presence of a 1I;VII trans- location, while the other was translocation-free. Further mitotic analysis of the TI (ZZ;VZZ) descendant confirmed that linkage group I1 is involved in an aberra- tion in this F, strain; however, when it was crossed with a translocation-free strain the expected pattern of specific disomics was not observed. Further analysis DETECTION OF TRANSLOCATIONS 27 TABLE 4 TI (1;VIII) strains showing different levels of nondisjunction in Crosses with meiotic testers or a translocation-free non-standard strain

Crosses with meiotic teste?’ strains Crosses with T-free non-standard rtrafn Total Frequency of abnomalr Total Frequency of abnormals Tl(1;VlII) examtned All types T-rpeci fic diroml cs examined All types T-specific disomic5 strain “&er PePcent Number Percent Number percent Number Pelcent F1 1120 0.7 (5) 0.4 1712 0.3 (2) 0.12 F2 857 0.8 (7) 0.8 3096 0.1 (1 1 0.03 F5 1445 1.7 (24) 1.6 21 39 0.7 (12) 0.56

will be needed to determine the nature of the original UV-induced aberration in the adE biA strain.

Other genetic factors influencing frequency of CLubnormuls’’ During the course of our work we experienced three independent genetic situ- ations which either altered the expected pattern of nondisjunction or gave rise to other abnormal phenotypes with relatively high frequency. The first of these was found in crosses heterozygous for TI (VZ;VZZ).Of the four crosses analyzed (Table 2) three gave, in addition to the expected increase of disomics for linkage groups VI and VII, a relatively high (1.5%) frequency of other abnormal pheno- types, mainly of a class of colonies with well conidating centers which were of reduced size and showed an instability pattern typical of duplication strains. The remaining strain yielded only typical disomics. Further analysis will be needed to explain the differences in these crosses which do not appear to be dependent on the TI (‘VZ;VZZ) strain alone. The second case involves three strains carrying the TI (Z;VZZZ)translocation, two of which are closely related in the pedigree (being the F, and F, strains) and the third of which is quite distant (F,-progeny from a sister F,-strain; for details see Figure 6, =FER 1965). The results are shown in Table 4. When crossed to meiotic testers, the F, strain produces the typical pattern of disomic types, while the F, and F, strains yield a consistently lower frequency of T-specific disomics. In addition, when crossed to a specific translocation-free strain carrying markers of group VIII-called “T-free non-standard” strain in Table &practically no abnormal progeny were recovered from the F, and F2 strains while a reduced frequency was recovered from the F, strain. The third situation is of the opposite type and arose in testcrosses involving a previously untested strain and several F, descendants. Among the progeny of testcrosses with these strains a high frequency (2% among ca. 1500 progeny) of abnormals was observed. These were mainly disomic types, but there was no specificity for any one or any pair of linkage groups. In fact, there appeared to be a proportional increase for most disomic types. Mitotic analysis of the relevant strains has ruled out the presence of translocations and the cause for this unusu- ally high level of general nondisjunction remains unknown. 28 A. UPSHALL AND E. FER

DISCUSSION The meiotic method for detection of translocations To detect translocations by this method ascospores are plated at a moderate density (to give 20-30 colonies per plate) and a fair-sized sample (500-1000 colonies) is checked for the presence of abnormal-looking, especially sectoring, colonies. The results presented here show that, under these conditions, one can detect the presence of a translocation in heterozygous crosses against standard strains by observing an increased frequency of morphologically abnormal, mainly disomic, progeny compared to the control value of 04.3%.Similar to methods commonly used in other organisms, e.g. Neurospora or Drosophila, this is basic- ally a genetic test which permits identification of the presence of the aberration by inspection of meiotic segregants, eliminating the need for cytological analysis. The meiotic technique developed here is regarded as complementary to the previously available mitotic technique. Both have specific features and, depend- ing on the nature of the investigation, either one may have certain advantages 01 both may regularly be used. The latter is the case, for example, for newly induced which are routinely located to the appropriate linkage group by mitotic analysis and also invariably crossed into a desired genotype. In both tests the additional work to detect any translocations is negligible. On the other hand, it is in the analysis of progeny from crosses found heterozygous for a translocation that the meiotic system is advantageous. To eliminate translocations at the F1 stage, one need only cross a few segregants of the required genotype and then use those which give a low frequency of disomics. Or, should one be interested in translocations per se, those isolates which yield abnormal progeny in crosses can be retained for further analysis. If the abnormal colonies are needle-plated and the various disomics classified, one or both of the linkage groups affected by the rearrangement may be identified. However, to recognize the involved chromo- somes with certainty, subsequent mitotic analysis is usually needed. On the other hand, when several descendants from heterozygous translocation crosses need to be analyzed, e.g. for the tracing of translocations in stock strains, the meiotic tech- nique is the more suitable one (especially if strains with interacting transloca- tions are used as testers). In addition, the meiotic system has the following three advantages. First, it can differentiate between reciprocal and nonreciprocal translocations, since only the latter produce one third duplication progeny with characteristic phenotypes. Second, only a few meiotic tester strains varying in color and a limited number of requiring markers are needed for crosses with all potential translocation strains in general use, in contrast to the relatively large number of testers with many different markers needed for mitotic analysis (e.g. over 20 used here in the diploid testing strains for translocations). It is important, however, that the meiotic testers should not only be translocation-free, but preferably be proven standard strains, since in some strains we have encountered genetic factors other than translocations which affect nondisjunction frequencies. Finally, the meiotic method can be used even for other wild isolates of Aspergillus nidulans which are DETECTION OF TRANSLOCATIONS 29 heterokaryon-incompatible with Glasgow strains ( GRINDLE1963). These strains have less advanced genetic maps but their disomics show the same phenotypic specificity ( UPSHALL19 71 ) . Frequency of nondisjunction for different chromosome pairs in control and translocation crosses The control crosses show that in A. nidutans meiotic nondisjunction is low (0-0.3% ) in homozygous normal, or homozygous translocated . While it is possible that each chromosome has a specific nondisjunction frequency, our data only show that three are found disomic more frequently than the remaining five. This could be a reflection of various known phenomena, e.g. chromosome size or, at least partly, relative viability and ease of detection (disomics for IV and VI11 may well have been missed in some of the controls, the former because they grow and conidiate almost normally, the latter because they grow extremely slowly). Translocations, when heterozygous in crosses, lead to increased missegregation of centromeres and therefore increased frequencies of disomics for the two groups involved in the rearrangement. Such effects are already well known, especially in Drosophila where they have been the subject of extensive study (GRELL1959; ROBERTS1970). In addition, however, GRELLnoted that other chromosomes of the Drosophila complement not involved in the translocation also underwent more frequent nondisjunction. This gave rise to the theory of “distributive pair- ing” (GRELL1962). Preliminary results from a translocation cross in Aspergillus were compatible with such a hypothesis (POLLARD,UFER and JOHNSTON1968). The following was found in the much more extensive data with heterozygous translocation crosses described here: when only the progeny which segregated normally for the translocation chromosomes are considered, the frequency of nondisjunction for the other six chromosome pairs is no higher than in the con- trols. On the other hand, among the cases which showed missegregation for one of the chromosome pairs involved in the translocation, simultaneous nondisjunction for a normal pair would produce aneuploids with two extra chromosomes (n 4- 2) as the only viable product. Such types might be recovered in some cases while others are expected to be too inviable to form colonies. The frequency of double nondisjunction can, therefore, not be measured in any reliable way. It was observed, however, that certain n 4- 2 types occurred with increased frequencies in crosses with specific translocations; for example several crosses with TI (V;VZ) produced an increased number of n 4- 2 types, disomic for V or VI as well as disomic for 111. One further observation invites speculation: The four expected types of disomics are not recovered with equal frequency for over half of the transloca- tions analyzed. In some cases disomics for mainly one chromosome pair are found which might be a reflection of differences in sizes between the translocated seg- ments. In other cases, only one rather than two types of abnormals, disomic for a specific linkage group, are recovered, even though they are expected to be pro- duced with equal frequency. This could be due to large differences in viability, 30 A. UPSHALL AND E. KAFER when one abnormal chromosome is much longer and the other much shorter than the normal one, as for example in the case of the non-reciprocal translocation. Or, if translocated segments are small, the phenotypes might be overlapping, pre- venting classification into two groups. Other genetic effects As is to be expected from most experimental material, exceptions to the basic pattern occurred. We have observed four such situations, three of them involving various translocation strains. The first appears to be a case of interaction in crosses with the translocation TI (VZ;VZZ) which results in an increase in the frequency of nondisomic, abnormal colonies resembling duplication types. The factor responsible can segregate out of association with the translocation since trans- location progeny are recovered which no longer show the increase, but transloca- tion-free progeny have not been recovered which only show the “other” abnor- mals. A second interactive effect is of the opposite type, apparently resulting in suppression or reduction of nondisjunction in crosses between the TI (Z;VZZZ) translocation complex and a translocation-free, but non-standard, strain carrying various mutants on VIII. Such a mechanism of suppression is difficult to visualize since other aberrations would more likely enhance the frequency of abnormal progeny (as in the case reported by POLLARD,KAFER and JOHNSTON 1968). The third exception is the most interesting one at this stage, since it does not involve gross chromosomal rearrangements and manifests itself in a general (ca. tenfold) increase of nondisjunction for most linkage groups. The fourth case is that of a 1I;VII translocation which does not give a consistent pattern of increased non- disjunction in crosses; it originated from an irradiated strain which appears to contain other aberrations (similar to some UV-treated strains of Neurospora analyzed by PERKINS1967).

The expert technical assistance of MRS.P. MARSHALLis gratefully acknowledged. We greatly appreciate the criticisms and helpful comments of DR.D. D. PERKINSin the preparation of this publication. DRS.E. R. BOOTHROYDand D. J. COVEvery kindly read the manuscript and made helpful comments.

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