Molec. gem Genet. 170, 213-218 (1979) © by Springer-Verlag 1979

Induced Segregation in Interspecific Hybrids of Aspergillus nidulans and Aspergillus rugulosus Obtained by Protoplast Fusion Ferenc Kevei 1 and John F. Peberdy 2 Department of Microbiology, Attila Jozsef University, H-6701 Szeged, P O Box 428, Hungary 2 Department of Botany, University of Nottingham, University Park, Nottingham NG7 2RD, England

Summary. Interspecific hybrids produced by polyethylene glycol induced fusion of protoplasts from auxotrophic mutants of Aspergillus nidulans and Aspergillus rugulosus were grown in the presence of the recombinogens benomyl and chloral hydrate to stimulate segregation. The A. nidulans parental strains used had a known genetic marker in each linkage group. Hybrids grown on complete medium containing benomyl yielded more segregants. Analysis of the segregants showed that the distribution of A. nidulans linkage groups was random. No specific linkage group appeared in all the segregants. The two parents are closely related taxonomically and the findings from these experiments suggest that a high degree of chromosomal homology may exist between them.

to our understanding of the chromosomal and genetical relationships between different species of fungi. The limitation to this approach at present is the lack of fundamental genetical studies on all but a few fungal species thus restricting the availability of well marked strains. A virtue of A. nidulans for this work is the existence of master strains, developed for parasexual analysis (Clutterbuck, 1974), which have markers in each linkage group. As a first step experiments were carried out using certain of these strains for hybrid production in crosses with A. rugulosus. The induced segregation patterns of the latter, using benomyl and chloral hydrate as recombinogens, have been investigated and are described in this paper.

Materials and Methods Introduction

The development of interspecific hybrids in filamentous fungi following the fusion of isolated protoplasts has been rapidly established (Anne et al., 1976; Ferenczy, 1976; Ferenczy et al., 1977; Kevei and Peberdy, 1977; Peberdy et al., 1977). In a cross between two members of the A. nidulans group i.e. AspergiIlus nidulans and A. rugulosus diploid hybrids were obtained which gave rise to segregants either spontaneously or following induction (Kevei and Peberdy, 1977). In these experiments the parental strains used were genetically characterised by a few unmapped markers and as a consequence the behaviour of the individual linkage groups of the two parents during segregation cannot be established. The new combinations of linkage groups that may arise from these interspecific hybrids could provide useful information with regard For offprints contact: Dr. F. Kevei, Department of Microbiology, Attila Jozsef University, H-6701 Szeged, PO Box 428, Hungary

Fungal Strains' The genotypes and origins of the strains used in these crosses are shown in Table 1.

Protoplast Isolation and Fusion Methods The methods used for protoplast isolation, protoplast fusion and selection of fusion products ha;~e been described in previous publications (Ferenczy et aL, 1975a; Ferenczy et al., 1975b; Kevei and Peberdy, 1977).

Induced Segregation of Hybrids and Characterization of Segregants Subcultures of the hybrids were grown on complete medium (Ferenczy et ai., 1975a) containing either 0.75-1.0 gg ml-1 methyl-1(butylcarbamoyl)-2-benzimidazole carbomate (henomyl) (Hastie, 1970; Edwards et al., 1975) or 1.2 1.5 mg m1-1 chloral hydrate (Singh and Sinha, 1976). After purification master plates of the segregants were prepared and replicated, using a multipoint inocuIator, onto differential media for characterization.

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F. Kevei and J.F. Peberdy : Segregation in Aspergillus Hybrids

214 Table 1. Strains used

Species

Strain

Marker genotype a

Origin

Aspergillus nidulans

M.S.E. FGSC 407

s u A a d E 2 0 , y, adE20 ; wA ; gal A ; p y r o A 4 ; f a e A 3 0 3 ; S B ; nicB ; riboB. Professor J.A. Roper a d E 2 O b i A l , 4 c r A 1 ; p h e n A 2 ; p y r o A 4 ; lysB5; laeA 1; choA1; riboB2chaA1; Dr. A. Upshall

Aspergillus rugulosus

RI R2

prol metl

)mutants induced. (uv) ~in A. rugulosus J I M I 19120a

Mutant symbols: A c r (acriflavine resistance) ad (adenine), bi (biotin), cha (chartreuse spores), cho (choline), f a c (acetate non-utilisation), gal (galactose non-utilisation), lae (lactose non-utilisation), lys (lysine), m e t (methionine), nie (nicotinic acid) phen (phenylalanine), pro (proline), p y r o (pyrodoxine), ribo (riboflavin), s (sulphate non-utilising), suad (suppressor of adenine requirement), w (white spores), y (yellow spores)

Table 2. Parentage of hybrids used in induced segregation experi-

Results

ments

Hybrid Development. Following exposure to the fu-

Hybrid

Parents

HI H2 H3

A. nidulans M S E + A . rugulosus R2 A. nidulans FGSC 407 + A. rugulosus R1 A. nidulans FGSC 407 + A. rugulosus R2

Table 3. Frequency of segregants induced by benomyl or chloral

hydrate in A. nidulans x A. rugulosus hybrids Hybrid

H1

H2 H3

Recombinogen Benomyl

Chloral hydrate

52 34 133

48 32 70

sion treatment heterokaryotic colonies grew up from protoplasts plated on the selective minimal medium in a similar manner to that previously described (Kevei and Peberdy, 1977). From subcultures of these heterokaryons sectors of the hybrid appeared which after purification were used in the segregation experiments. Three hybrids were produced and their parentage is shown in Table 2. The frequency of segregation obtained for these hybrids grown in the presence of benomyl and chloral hydrate is shown in Table 3. The sectors developing in 10 colonies of each hybrid, grown in the presence of the two recombinogens, were taken and single spore isolates obtained. These segregants included stable types, which proved to be haploid, and unstable colonies, probably aneuploids, which gave stable haploids after subsequent purification. All segregants studied in detail were deemed to be haploid on the basis of spore size, DNA content

Table 4. Benomyl-induced segregation in hybrid H1

Strain phenotypes a' b Number of segregants

fac fac fac fac fac fac fac nic nic

nic ribo met nic m e t ribo m e t nic ribo m e t

Frequency of parental colony types" Table 5. Chloral hydrate induced segregation in hybrid H1 A. nidulans

A. rugulosus

18

3

15

6 1 2 2 1 1

6 2 1 1

1 2 -

8

8(6+1 +1) d

-

ribo m e t

1

1

-

prototrophs

12

1

11(9 + 2) d

a The suad, a d and s markers were not scored in the segregants b All isolates were gal + , p y r o + ° Colony type denotes form and not spore or colony pigmentation d Two or three morphological variations

Strain phenotypes a, b fac fac met f a c nie ribo f a c nic m e t nic met nic m e t ribo m e t

prototrophs " b c

see Table 4 see Table 4 see Table 4

Number of Frequency of parental colony types ~ segregants A. nidulans A. rugulosus Mixed 2 6 1 2

1 1 2

1 6 -

-

10

10

12 8 1

3 8

6 -

3 1

6

4

2

-

F. Kevei and J.F. Peberdy : Segregation in A s p e r g i l l u s Hybrids

215

Fig. l a and b. Hybrid 1 grown on CM containing a benomyi and b chloral hydrate. Upper: controls growing on CM

Table 6. Benomyl induced segregation in hybrid H2

Table 7. Chloral hydrate induced segregation in hybrid H2

Strain phenotype"' b

Strain phenotype"

Number of segregants

Frequency of parental colony types b A.

A.

nidulans

rugulosus 1

Mixed

a d p h e n lys lac a d p h e n lys lac ribo

1 1

1

a d p h e n p y r o lys lac cho ribo A c t p h e n p y r o lys cho Act" p h e n p y r o lys cho ribo Act" lys cho p r o (Act') ( A c r ) p h e n lys ribo ( A c r ) p h e n lys cho ( A c r ) p h e n lys lac cho ( A c t ' ) p h e n p y r o lys cho ( A c t ) p y r o lys ( A c r ) p y r o [ys cho ( A c t ' ) p y r o lys ribo ( A c r ) p y r o lys cho ribo ( A c r ) lys cho ( A c r ) lys cho ribo ( A c t ) cho p h e n p y r o lys lac cho p y r o lys ribo p y r o lys p r o lys lys lac cho ribo p r o lys cho lys cho ribo lys ribo

1 1 1 1

1 1

-

1

-

3

2(i + 1) ° 1

-

1 1 1 3 1 1 2 2 1 1 1 1 1 1 1 1 1 1 1

1 1 2 1 2 1 1 1 1 1 1 1 1 -

1 -

-

prototrophs

2

-

2

1

1 -

1 2 -

biAcr bi ( A c r ) cho p r o bi lys p r o bi lys lac p r o bi lys cho p r o bi p r o bi cho p r o bi cho ribo p r o (Acr) lys p r o lac p r o pro ribo p r o

Frequency of parental colony types b A . nidulans

A . rugulosus

Mixed

1 1

1

1 -

-

4

-

4(3 + 1) ~

1 1 8 4 1 3 1 1 4 2

1 1 4 1 -

"

Act" and ( A c r ) segregants - see Table 6

b °

see Table 4 Two morphological variations

-

8

3 1 1 4 -

2

-

-

1 -

1

-

1

A c r segregants grow in the presence of acriflavine at 100 gg ml- i ( A r c ) segregants are intermediate growing in the presence of acriflavine at 50 pg ml- 1 b see Table 4 c Two morphological variations a

Number of segregants

of conidia in some cases and stability when cultivated in the presence of haploidizing agents.

Segregation of Hybrid l. The segregants obtained from this hybrid were scored for all the auxotrophic except adenine requirement and sulphate non-utilization. Up to nine classes of segregants were obtained (Tables 4 and 5) but the A. nidulans parent was not included in these (Fig. 1), Of the met segregants only half were like A. rugulosus in colony morphology, the remainder were like A. nidulans or had a morphology intermediate between the two parental types. Distri-

216

F. Kevei and J.F. Peberdy : Segregationin AspergillusHybrids

Fig. 2a and b. Hybrid 2 grown on CM containing a benomyl and b chloral hydrate. Upper left and upper: controls growing on CM

bution of thefae and met markers amongst the segregants was very different for the two recombinogens; with benomyl 51.6% of the segregants were fac dropping to 18.2% of segregants isolated in the presence of chloral hydrate. In contrast the values for the met marker were 11.7% and 45.3% for the two respective recombinogens. The frequencies for nic and ribo were very similar for hybrids grown on benomyl and chloral hydrate and segregation of the gal and pyro markers was not observed. A characteristic of the segregants, also found in earlier studies (Kevei and Peberdy, 1977) was the variety in colony morphology, pigmentation and capacity for cleistothecia production. Segregants obtained from hybrids grown in the presence of benomyl were more of the A. ruguIosus type but this was reversed for hybrids grown in the presence of chloral hydrate when A. nidulans types predominated. Colony pigmentation included both parental types but several novel forms were also obtained.

Segregation of Hybrid2. This hybrid yielded more segregant classes, but this was partly due to the increase in number of markers being screened. The number of segregants obtained was similar for the two recombinogens (Tables 6 and 7) but as with hybrid 1 differences in the patterns of segregation were apparent (Fig. 2). An interesting observation related to the segregation of the ad and bi markers in the A. nidulans linkage group I. The ad marker was isolated at a very low

frequency from hybrids grown on medium containing benomyl but these segregants were not bi. Growth of the hybrid in the presence of chloral hydrate yielded 28.7% of the segregants carrying bi but none of these were ad. The frequency distribution of the other markers again differed for the two recombinogens. Three types of response were found with respect of the Acr marker; as well as isolates sensitive to the inhibitor and resistant to the tolerance of the A. nidulans parent, a third group showing resistance to an intermediate level was also found. In comparison to hybrid 1 the benomyl-induced segregants were more of the A. nidulans colony form and chloral hydrate yielded more A. rugulosus types.

Segregation of Hybrid 3. This hybrid gave the highest frequency of segregants; the 10 colonies gave 133 and 70 when grown in the presence of benomyl and chloral hydrate respectively. The benomyl-induced segregants fell into 47 phenotype classes and those produced in the presence of chloral hydrate were in 15 classes; however, many of these classes were represented by only one isolate (Tables 8 and 9, Fig. 3). The behaviour of the ad and bi markers in these segregants differed from that found for hybrid 2 segregants. A proportion of the ad segregants were also bi but ad or bi types separately were also isolated. Variation in frequency of segregation of the other markers was again noted with the exception of Aer and cho which were similar for both recombinogens. The lac marker was recovered only in segregants iso-

F. Kevei a n d J . F . P e b e r d y : S e g r e g a t i o n in Aspergillus H y b r i d s

217

T a b l e 8. B e n o m y l i n d u c e d s e g r e g a t i o n in h y b r i d H 3

T a b l e 9. C h l o r a l h y d r a t e i n d u c e d s e g r e g a t i o n in h y b r i d H 3

Strain phenotype a

Strain phenotype"

Number Frequency of parental of c o l o n y types b segregants A. A. Mixed

nidulans ad bi Acr phen lys cho 2 ad bi pyro lys cho 1 ad bi pyro lys cho met 1 ad bi lys cho met 1 ad A crphen pyro lys ribo 1 ad Act phen lys cho 2 ad Acr lys cho 1 ad (Acr) lys eho 1 ad (Acr) lys 1 ad lys lac cho met 1 ad lys lac met 1 bi Acr phen lys cho 1 bi Acr lys cho ribo 1 bi (Act) lys cho ribo 1 bi phen lys cho i bi phen lys 1 bi pyro ribo met 1 bi pyro 2 bi met i Acr phen cho 3 Aer phen 1 A c t lys cho 1 Acr cho 25

1 --

1

1

1

-

1

1

--

--

-

1

--

1

--

-

1

1

1 23

(Act) phen pyro lys cho (Act) phen lys cho ribo (Acr) phen lys (Act) phen (Acr) lys cho ribo (Acr) lys ribo (Acr) cho ribo (Acr) cho (Acr) ribo (Act) met (Acr) phen lys cho

1 1 2 4 1 2 1 2 2 1 6 2

phen cho pyro ribo lys cho lys lac cho met cho met cho ribo met

3 1 2 1 1 1 6 1 2

prototrophs

14

ad (Act) lys cho Acr cho Acr (Acr) pyro cho (Act) lys cho (Acr) cho (Acr) phen cho pyro met lys cho ribo cho met cho met prototrophs

1

--

1

-

1

1

1

-

5

5 ( 4 + I) c

-

5 1 2

5 1 2

-

19

Mixed

2 1 1 1 1 7 5 12 7

19(17+2) ~

1 1 1 1 5 5 ( 4 + 1) c -

--

2

3(2+1) ~

-

--

"

Acr a n d (Acr) - see T a b I e 6

-

b °

see T a b l e 4 Two morphological variations

1 1 -

2

12 7

1

1 1

4 (2+2 c

21(13+4

1

8(4+2 +1+1) ~ i

15(13 +1+I) -

-

-

2

-

-

4

-

-

2

1

-

-

-

1

1

1

-

-

2

5(2 + 1

1

1 2(1+1)

~

3(2+1) ° 1 2(1

+ 1) ° 1

1 1

--

--

6

-

-

1 1

1

1

13

A 'r a n d (Acr) s e g r e g a n t s - see T a b l e 6 see T a b l e 4 T w o to five m o r p h o l o g i c a l v a r i a t i o n s

lated in the presence of benomyl and the A. rugulosus marker, met, again segregated at high frequency from hybrids grown in the presence of chloral hydrate.

°

+1+1) °

a b

A. nidulans A. rugulosus

1

+2+1 +1) c

Acr cho met Acr

Frequency of parental c o l o n y types

rugulosus

2

--

Number of segregants

-

Discussion

Evidence was presented in our previous experiments (Kevei and Peberdy, 1977) to demonstrate the formation of hybrids, from interspecific heterokaryons, following fusion of complementing nuclei. The observations described in this paper substantiate our earlier findings but also allow us to understand further the segregation behaviour of the hybrids. The occurrence of a diverse spectrum of segregants in which one or more A. nidulans markers (and presumably therefore chromosomes) were obtained leads to the conclusion that the exclusion of chromosomes during haploidization is as much a random process in these interspecific hybrids as in an intraspecific diploid. One obvious criterion in the segregation of interspecific haploid nuclei is the complementation of gene activities, donated by the two parents, so producing viable progeny. The degree of homology in gene activities between these two species is unknown, but our results suggest that it is quite substantial. The failure to isolate certain markers in the segre-

218

F. Kevei and J.F. Peberdy : Segregation in Aspergillus Hybrids

Fig. 3a and b. Hybrid 3 grown on CM containing a benomyl and b chloral hydrate. Upper left: controls growing on CM

gants is unknown but could be due to numbers of segregants tested relative to the number of markers in the crosses. Also of interest is the behaviour of the ad and bi, which are linked on chromosome I in A. nidulans, in H2. Mitotic recombination between chromosome I and an A. rugulosus chromosome is an obvious possibility but were this so, the isolation of the two markers might be expected. The failure to do so might again be due to the relatively small number of segregants that were tested. Support for this idea can be seen in the data for H3 segregants where considerably more were examined and ad bi, ad and bi segregants were obtained.

References Ann6, J., Eyssen, H., Somer, P. De: Somatic hybridization of Penicilliurn chrysogenum with P. roquefortii after protoplast fusion. Nature 262, 719-721 (1976) Clutterbuck, A.J. : Aspergillus nidulans. In: Handbook of genetics, Vol. 1 (R.C. King, ed.), pp. 447 510. New York: Plenum Press 1974 Edwards, G.F.St.U, Normansell, I.D., Holt, G. : Benlate induced haploidisation in diploid strains of Aspergillus nidulans and Penicillium chrysogenum. Aspergillus Newsl. 12, 12 (1975)

Ferenczy, L. : Some characteristics of intra- and interspecific protoplast fusion products of Aspergillus nidulans and Aspergillus fumigatus. In: Cell genetics in higher plants (D. Dudits, G.L. Farkas and P. Maliga, eds.), pp. 171 182. Budapest: Akademiai Kiado 1976 Ferenczy, L., Kevei, F., Szegedi, M. : High frequency fusion of fungal protoplasts. Experientia 31, 1028 1029 (1975a) Ferenczy, L., Kevei, F., Szegedi, M.: Increased fusion frequency of fungal protoplasts. Experientia 31, 50 52 (1975b) Ferenczy, L., Szegedi, M., Kevei, F. : Interspecific protoplast fusion and complementation in Aspergilli. Experientia 33, 184-186 (1977) Kevei, F., Peberdy, J.F. : Interspecific hybridzation between Aspergillus nidulans and Aspergillus rugulosus by fusion of somatic protoplasts. J. Gen. Microbiol. 102, 255 262 (1977) Hastie, A.C. : Benlate induced instability of Aspergillus diploids. Nature 226, 771 (1970) Peberdy, J.F., Eyssen, H., Ann6, J.: Interspecific hybridization between Penicillium chrysogenum and Penicillium cyaneo-fulvum following protoplast fusion. Mol. Gen. Genet. 157, 281-284 (1977) Singh, M., Sinhar, U.: Chloral hydrate induced haploidization in Aspergillus nidulans. Experientia 32, 1144-1145 (1976)

Communicated by G. Melchers Received November 6, 1978