Crosses with Amut Bmut homokaryons of Coprinus cinereus

Yi Liu, José D. Granado*, Eline Polak, and Ursula Kües ­ Institut für Mikrobiologie, ETH Zürich, Schmelzbergstrasse 7, CH-8092 Zürich, Switzerland
Fungal Genetics Newsletter 46:16-20

*Present address: Institut für Pflanzenbiologie, University of Zürich, Zollikerstr. 107, CH-8008 Zürich, Switzerland

In this paper we describe the initial genetic analysis of some developmental REMI and UV mutants of the self-compatible homokaryon AmutBmut. We show that such homokaryons can mate with each other although in fruitbodies we often found spores of only one parent. Crosses with monokaryons of different mating types gave some indications about numbers of mutations and linkage of genetic markers. In most cases, however, we observed an uneven distribution of markers, most likely because of loss of certain progeny. Our results necessitate the construction of monokaryons as closely related to homokaryon AmutBmut as possible but with different mating type loci.

Monokaryon PG78 in analysis of defects in fruitbody development. A pab1 auxotrophic monokaryon was available to us with strain PG78 (A6, B42, pab1, trp1.1;1.6) (Granado et al. 1997, ibid). First, we performed a cross with REMI mutant B-1918 (A43mut, B43mut, pab1, ::pab1⁺), which has a single pab1⁺ plasmid integration (data not shown). Mutant B-1918 does not form mature fruitbodies. However, the stipes of fruitbody primordia elongate extensively under dark-light fruiting conditions without parallel development of the cap tissue (J. D. Granado, unpublished). Such a phenotype is known as "etiolated" or "dark stipe" phenotype (Elliott 1994 Reproduction in Fungi. Genetical and Physiological Aspects. Chapman & Hall: London). Progeny of PG78 x B-1918 were plated on YMG/T complete medium (Granado et al. 1997, ibid) and consecutively tested for auxotrophies. The distribution of the auxotrophic markers was unequal with 58% of prototrophic clones, 40% of pab1 + trp1 auxotrophic clones and 2% of pab1 auxotrophic clones (Table 1). This result suggested a close linkage between the pab1⁺ insertion and the natural trp1⁺ locus (P>0.99). We did not perform the tedious microscopical analysis with this F1 generation to identify clones with fused clamps, which indicates the presence of A43mut and B43mut. By simple statistics, 11 (25%) of the 43 analyzed clones should have had this genotype offering the possibility to fruit. However, only 2 out of all clones formed tiny fruitbody initials (~1 mm in size) and one, JG1-33 (A43mut, B43mut, pab1, ::pab1⁺), had the "etiolated stipe" phenotype. JG1-33 was backcrossed to the parent PG78. In the progeny, we found the same uneven distribution of trp1 and pab1 as before in the F1 generation (Table 1). Loss of trp1 progeny was also observed in the control cross PG78 x AmutBmut (Table 1), suggesting that this phenomenon is intrinsic to the PG78 and AmutBmut backgrounds. In the last two crosses, where we analyzed a high number of clones, trp1 : trp1⁺ clearly segregated in an 1:2 ratio (P>0.5 and P>0.25, respectively), indicating that 25% of the trp1 progeny was lost.

In the following, we examined clamp cell formation to monitor the inheritance of the A mating type locus amongst the PG78 x JG1-33 progeny. Only 39% of all germinated clones were without clamp cells (Table 2), indicating that half of the A6 progeny was missing (P>0.05). This was also the case in the progeny of a cross between monokaryon PG78 and the prototrophic REMI mutant E-1754 that has a defect in nuclear distribution (P>0.75; Table 2; E. Polak, unpublished) and in the progeny of a cross between monokaryon PG78 and homokaryon AmutBmut (P>0.25; Table 2). Interestingly, within the group of viable A6 clones trp1 : trp1⁺ distributed 1 : 1 unlike the A43mut clones (not shown). However, it is not clear from these data if there is a direct connection between the under representation of trp1 and that of the A6 mating type. Loss of certain phenotypes within progeny has been observed before in crosses with other strains. For example, a recessive gene slg is known to affect spore germination. Another recessive gene, bad blocks basidiospore development (Pukkila 1993 pp. 249-264 in Genetics and Breeding of Edible Mushrooms, Chang et al., Eds, Gordon and Breach: Y-Parc, Switzerland). Presence of such a mutation in one of the parental strains should lead to a 50% loss of all clones. The loss of 25% of certain phenotypes as found here and also in other studies (Moore 1981 Curr. Genet. 3:145-150) implies a negative interaction between two unlinked loci in a single haploid nucleus that came together from the different parental strains by karyogamy and meiosis. It is possible that the loss of 25% of progeny connects to translocations which commonly occur in C. cinereus, also with the trp1 chromosome (Pukkila and Casselton 1991 pp.126-150, in More Gene Manipulations in Fungi, Bennett and Lasure, Eds., Academic Press: San Diego).

Because of the partial loss of certain progeny, care has to be taken in mapping the locations of genes within chromosomes. Nevertheless, we concluded from the 12% recombinant phenotypes (pab1⁺ trp1 and pab1 trp1⁺) within the PG78 x JG1-33 progeny (Table 1) that the chromosomal distance between the ectopic pab1⁺ insertion in JG1-33 and trp1⁺ is about 0.12 map units. In order to determine whether this pab1⁺ insertion links to the "etiolated stipe" defect, we submitted selected groups of the PG78 x JG1-33 progeny (Table 3) to standard fruiting conditions (Granado et al. 1997). Within these groups we did not discriminate between the presence of B43mut or B6. Since B43mut and B6 distribute equally within the progeny (data not shown), half of the analyzed clones (B43mut clones) would be expected to initiate fruiting. However, none of the cases came even close to the expectations (Table 3). Thus, we analyzed fruiting within the A43mut (B43mut + B6) progeny of a control cross PG78 x AmutBmut. Twenty-one percent of the clones were able to initiate fruiting (Table 3), corresponding to half of the B43mut clones (P>0.25). Twenty nine out of the 31 fruiting clones were trp1⁺, which suggests either a negative effect of the trp1 auxotrophy on fruiting and/or, on the PG78 chromosome, a linkage between trp1 and a non-functional gene that is necessary for fruitbody initiation. From other strains, we recently learned that a trp1 auxotrophy interferes with fruitbody initiation (U. Kües and M.J. Klaus, unpublished observations). Co-transformation of a compatible A gene with a trp1⁺ selection marker did not induce the fruiting pathway in strain PG78 unlike in other trp1 monokaryons (Kües et al. 1998 Mol. Gen. Genet. 260:81-91). Together, these observations lead to the conclusion that monokaryon PG78 is not well suited in the genetic analysis of fruiting mutants of homokaryon AmutBmut. Nevertheless, we still might conclude from data obtained from the PG78 x JG1-33 progeny that the locus responsible for the "etiolated stipe" phenotype in mutant JG1-33 links to the pab1⁺ insertion. Clones forming etiolated stipes predominate amongst the few clones that were able to induce fruiting (Table 3). Initials of the 3% exceptional cases (Table 3) did not develop far enough to determine whether primordia are of wild-type or of the "etiolated stipe" phenotype. 80 out of the 81 fruiting inducing clones were pab prototrophs. Sometimes, we specifically selected for trp1⁺ progeny and thus preferentially for a pab prototrophic progeny as well because of the close linkage of trp1⁺ to the pab1⁺ insertion (Table 3). With the distance of 0.12 map units, we would expect at least 9-10 pab1 clones amongst those initiating fruitbody development, if the "etiolated stipe" mutation is unlinked to the pab1⁺ insertion.

Monokaryon PG78 in analysis of defects in hyphal development. Despite the limited use of monokaryon PG78 in analyzing fruiting defects, the strain still has some value for analyzing defects in vegetative development. This is indicated from studies on the prototrophic REMI mutants E-1281 and E-2095, both of which lost the ability to form clamp cells at the hyphal septa (E. Polak, unpublished data). As expected from a single mutation unlinked to A43mut, the relative numbers of clones with and without clamp cells reversed in the progeny of cross PG78 x E-1281 with 24% : 75% (Table 4), compared to the 70% : 30% in cross PG78 x AmutBmut (Table 2). pab1⁺ : pab1 distributes 1 : 2 (P>0.25) within progeny of PG78 x E-1281 (Table 4), indicating 25% loss of pab1⁺ clones, but we currently do not know whether this relates to the 25% loss of A6 mating type. In the progeny of cross PG78 x E-2095, only 8% of all clones formed clamp cells (Table 4). Therefore, the loss of clamp cell formation in E-2095 could be caused by a mutation linked to A43mut. 8 of the 20 identified A43mut clones were pab prototrophs suggesting that the insertion of pab1⁺ segregates independently of the mutation (P>0.5; Table 4). In accordance, 10 out of 22 randomly chosen clampless pab1^- strains formed clamp cells in backcrosses with PG78 and thus should have the A43mut genotype. However, only 19% of all clones of the PG78 x E-2095 progeny were pab prototrophs (Table 4). One possible explanation for this observation is a loss of the ectopic pab1⁺ insertion during meiosis (Kües and Stahl 1990 Prog. Bot. 52: 201-225).

Mating between strains of related mating type specificities. The complications in the crosses with PG78 described above demand another strategy for analyzing defects in fruiting. The mutations in the mating type loci in homokaryon AmutBmut overcome the natural incompatibility between the endogenous mating type products (Hiscock and Kües 1999 Int. Rev. Cytol. in press). Therefore, one might expect that Amut Bmut homokaryons are also compatible with monokaryons carrying the wild-type mating type loci. We mated homokaryon AmutBmut on several YMG/T plates (~20) with its progenitor strain 5026 (A43, B43; kindly provided by T. Kamada) and with the distantly related monokaryon AT8 (A43, B43, trp3, ade8; Kües et al. 1992 Genes Dev. 4:568-577). We readily observed fruitbody formation on the AmutBmut side of the crosses. In contrast, we never observed any fruitbodies on the outer edge of an AT8 colony and only once a fruitbody on the outer edge of a 5026 colony. Basidiospores of this exceptional fruitbody and of fruitbodies from the AmutBmut sides of the crosses were always pab1 which suggests that there was no interaction between the nuclei of the two different mating partners at least during later stages of basidiome formation. To test the situation between A43mut B43mut homokaryons we first crossed the clamp cell defective REMI mutants E-1281 x E-2095 and found clamp cells at hyphae grown in the intermediary growth zone of the two strains (not shown). Therefore, the two different mycelia must have fused and their nuclei complement each other in their defects in clamp cell formation. To study complementation of defects in fruitbody formation, we crossed mutants that are defective in initiation of fruiting, taking advantage of the pab auxotrophy of an UV mutant (6-031) and the pab prototrophy of REMI mutants (B-436, B-2798 and E-1593). Fruitbodies were formed in the intermediary zones of the crosses suggesting that the defects in fruitbody initiation are recessive. However, when basidiospores of six independent fruitbodies of 6-031 x B-2798 were analyzed, all were pab1⁺. This result suggests that only one type of nucleus migrated into cells of the fruitbodies, although the defects in fruitbody initiation could be complemented either by the presence of the two different nuclei within the same hyphae or by feeding effects of diffusible substances. In contrast, pab1 : pab1⁺ distributed 1 : 1 in the progeny of the one tested fruitbody from cross 6-031 x E-1593 (P>0.75) and 1 : 3 in one out of three tested fruitbodies from cross 6-031 x B-436 (P>0.5); basidiospores of the other two fruitbodies were 100% pab1⁺. Obviously, it depends very much on the crossing partners whether both or only one type of nuclei migrate into the basidia of the fruitbodies.

Monokaryon 5401 in analysis of defects in fruitbody development. Since crosses between strains of related mating type specificities not always lead to karyogamy of different parental nuclei, we performed crosses with a more closely related monokaryon of a different mating type, in the hope not to introduce any unwanted and unknown genetical traits into the progeny. Prof. T. Kamada kindly supplied wild-type monokaryon 5401 [A1(m), B1(m); note that the nomenclature of these mating types relates to strains originally described as C. macrorhizus, Maida et al. 1998 Curr. Genet. 32:231-236]. This strain was derived from the same genetic background as monokaryon 5026 and homokaryon AmutBmut. Strain 5401 had been used before to identify gene loci in primordiumless and sporeless AmutBmut mutants (Kanda et al. 1989 Bot. Mag. Tokyo 102:561-564 and Mol. Gen. Genet. 216:526-529). We crossed monokaryon 5401 with UV mutant 6-031 and with homokaryon AmutBmut and found in both progeny an equal distribution of the pab1 and pab1⁺ marker (P>0.75 and P>0.05, respectively; Table 5). In contrast, the A43mut mating type was slightly over represented (Table 5). The A locus and the pab1 gene are closely linked on chromosome I (0.5 map units; Lukens et al. 1996 Genetics 144:1471-1477). Thus, for unknown reason, the 3-7% recombination frequency between pab1 and the A locus (Table 5) was somewhat higher than expected from the literature.

Analyzing the fruiting behavior of the A43mut [B43mut + B1(m)] progeny, we found 50% of all clones (66 out of 129 clones) from the cross 5401 x AmutBmut and 27% of all clones (34 out of 124 clones) from the cross 5401 x 6-031 to initiate fruitbody formation. This reduction by half correlates well with a defect in fruitbody initiation in a single gene (fbi1 for fruitbody initiation gene 1) that is unlinked to A43mut (P>0.5). However, another 16% of the clones (20 out of 124 clones) formed fruitbodies in crosses with both parental strains, indicating that the fbi1⁺ wild-type gene and B43mut was present within these clones. Furthermore, 17% of the A43mut progeny (21 out of 124) developed fruitbody initials only when crossed to 6-031 suggesting that these contained the active gene fbi1⁺ and probably the B1(m) mating type genes. 22% of the A43mut progeny (27 out of 124) initiated fruitbody development only in crosses with monokaryon 5401 showing that they contain B43mut and likely the mutated gene for fruitbody initiation. The remaining 18% of the clones (22 out of 124) did not form fruiting bodies with either parental strain. Adding up these data, 60% of the clones of the A43mut progeny (34 + 20 + 21 clones) should carry the wild-type gene and a maximum of 40% the mutated gene. Moreover, at least 65% of the A43mut clones (34 + 20 + 27 clones) should contain B43mut. This over representation of B43mut within the A43mut progeny agrees with values we obtained within the limitations of certainty of the microscopical test with which we analyzed all the A43mut mycelia for the presence of fused clamp cells. According to this visual test, 78% of all A43mut clones (97 out of 124) of cross 5401 x 6-031 and 70% of all A43mut clones (90 out of 129) of cross 5401 x AmutBmut had fused clamp cells. These results show that markers also did not segregate perfectly in the progeny of crosses with monokaryon 5401 although the higher frequency of fruitbody formation within progeny of crosses makes it a better strain for such analysis than monokaryon PG78.

General conclusions. We discovered a number of possible problems (loss of progeny, instabilities of ectopic DNA insertions, migration of only one nucleus into fruitbody cells) to follow genetical traits within progeny of crosses involving REMI and UV mutants of homokaryon AmutBmut. In consequence, it can be difficult to decide whether a mutant contains one or more mutations and whether a pab1⁺ insertion in REMI mutants links to the mutation of interest. It appears that it is better to maintain the same genetic background. Truly isogenic strains distinguished only in their mating type specificity so far do not exist in C. cinereus. We will thus have to develop an optimal partner for crosses, e.g. by repetitive backcrossing to the AmutBmut background (see Pukkila 1993 for further discussion). For analyzing REMI mutants we will have to introduce a pab1 auxotrophy into a generated suitable tester strain by mutagenesis.

Acknowledgments. We thank Rob Boulianne for critical reading of the manuscript and language corrections. This work was supported and by the Swiss National Science Foundation (Grant 31-46'940.96) and the ETH Zürich.

Table 1. Inheritance of auxotrophic markers in progeny of crosses between monokaryon PG78 and Amut Bmut homokaryons

Cross

Phenotype of progeny, number and percentage of clones*

Total isolates

	trp pab	trp pab+	trp+ pab	trp+ pab+
PG78 x B-1918	17 (40%)	-	1 (2%)	25 (58%)	43 (100%)
PG78 x JG1-33	38 (30%)	8 (6%)	7 (6%)	74 (58%)	127 (100%)
PG78 x AmutBmut	76 (36%)	n. a.	134 (64%)	n. a.	210 (100%)

*Basidiospores from each one fruitbody were plated on YMG/T and auxotrophies determined on minimal medium MM (Granado et al. 1997) supplemented with 100 mg/L trp and 5 mg/L pab where appropriate; n.a.: not applicable.

Table 2. Inheritance of the A mating type locus in progeny of crosses between monokaryon PG78 and Amut Bmut homokaryons

Cross

Phenotype of progeny, number and percentage of clones*

Total isolates

	Clamps (A43mut)	No clamps (A6)
PG78 x JG1-33	145 (61%)	91 (39%)	236 (100%)
PG78 x E-1754	66 (65%)	36 (35%)	102 (100%)
PG78 x AmutBmut	147	63	210

* Basidiospores from each one fruitbody were plated on YMG/T. Presence of the respective mating type in germinated clones was microscopically determined by presence (A43mut) or absence of clamp cells (A6) at the hyphal septa.

Table 3. Fruiting behaviour in progeny of crosses between monokaryon PG78 and Amut Bmut homokaryons

Cross

Analyzed Genotypes

Phenotype of progeny, number and percentages of clones*

Total isolates

		Wild-type primordia	"Etiolated stipes"	Tiny initials
PG78 x JG1-33a,c	A43mut trp1+ + A43mut trp1	-	10d (7%)	2e (1%)	145 (100%)
PG78 x JG1-33b,c	A43mut trp1+	-	45e (8%)	1e (0.2%)	534 (100%)
PG78 x JG1-33b	A43mut trp1+ + A6 trp1+	-	23e (8%)	-	300 (100%)
PG78xAmutBmuta,c	A43mut trp1+ + A43mut trp1	31 (21%)	-	-	147 (100%)

* Fruiting was tested as described by Granado et al. (1997).
^a Basidiospores of each one fruitbody were germinated on YMG/T and consequently clones were either trp or trp⁺ and either pab or pab⁺.
^b Basidiospores of each one fruitbody were germinated on MM/pab and consequently clones were either pab or pab⁺ but always trp⁺.
^c The A6 progeny was eliminated by microscopy for clamp cells at hyphal septa.
^d One clone was trp⁺ pab, all others trp⁺ pab⁺.
^e All clones were trp⁺ pab⁺.

Table 4. Clamp cell production in the progeny of crosses between monokaryon PG78 and clampless mutants of homokaryon AmutBmut

Cross

Phenotype of progeny, number and percentages of clones*

Total isolates

Clamps

No clamps

	pab+	pab	pab+	pab
PG78 x E-1281	-	42 (24%)	53 (30%)	78 (45%)	173 (100%)
PG78 x E-2095	8 (3%)	12 (5%)	41 (16%)	188 (75%)	249 (100%)

* Basidiospores of each one fruitbody were plated on YMG/T and auxotrophies determined on minimal medium MM supplemented with 100 mg/L trp and 5 mg/L pab where appropriate. Presence of clamps at hyphal septa was determined by microscopical inspection.

Table 5. Inheritance of the A mating type locus in progeny of crosses between monokaryon 5401 and Amut Bmut homokaryons

Cross

Phenotype of progeny, number and percentages of clones*

Total isolates

pab-

pab+

	Clamps (A43mut)	No clamps [A1(m)]	Clamps (A43mut)	No clamps [A1(m)]
5401 x 6-031	108 (49%)	-	16 (7%)	95 (43%)	219 (100%)
5401 x AmutBmut	123 (56%)	-	6 (3%)	90 (41%)	219 (100%)

*Basidiospores of each one fruitbody were plated on YMG/T and auxotrophies determined on minimal medium MM (Granado et al. 1997) supplemented with 5 mg/L pab where appropriate. Presence of the respective mating type in germinated clones was microscopically determined by presence (A43mut) or absence of clamp cells [A1(m)] at the hyphal septa.

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