.; .3. ”Si. .2 I? .. FRANK i an; LS EAR I ~ c _ ._ . ‘ ‘ . . . ‘ V . ‘ V 4a. 7 . . , . V ‘ ‘ . . . . ‘ . . HI . v . v . ,4 . _ H . , . . . | . , . . 7 A ‘ .3? I . . . v _ . .‘ :7 J .2 ....2 e. ....C.1_ U.Vfléii-raFcta . . . . . ‘ . . . .l .. 2 h . . . .. ‘. . ‘u? . “1.» Lu... .“ .51. J . . w _ t _ - . 2.. v, . z”! w. u. . ‘ . . ‘ ‘ y . . 31E.» 3. < . 5:...‘134t3 : 331.91....3?L1 3,131.3: “In”: ,.u.;.yn.r. .- H... a rzafimu. t , l q $.1Un . , . .. U #5015..4 < ' s» LIBRARy Michigan State University This is to certify that the thesis entitled The Role of Sexual Incompatibility ‘Factors in Somatic Recombination in'Schizophyllum commune presented by Carl Stephen Frankel has been accepted towards fulfillment of the requirements for Ph- D- degreein Botany and Plant Pathology Major professor Date August 6 1974 0-7639 "- 'IBRARY amoens 'IIIGPOIT. mama; m”. M‘ ' an: _ . ‘ "<1. ‘I‘Lm'— hu‘lzfi‘fi so Th1 the compati bination W‘r somatic Pat flmractepiz “1th meiot‘ ChaT'HCteri Dloidy and Th by mutager mutations from the : inder’eYXCle: know,“1 me: N mutaSEn & Of these ex1St1n? the othe, ABSTRACT THE ROLE or SEXUAL INCOMPATIBILITY FACTORS IN SOMATIC RECOMBINATION IN SCHIZOPHYLLUM COMMUNE By Carl Stephen Frankel This research was undertaken to determine whether the compatibility relationship of diploid cultures of Schizophyllum commune affects the type of somatic recom— bination which occurs in those diploids. Two types of somatic recombination were considered: ”meiotic-like”, characterized by direct breakdown from diploid to haploid with meiotic levels of crossing-over, and parasexual, characterized by breakdown to haploid via stages of aneu- ploidy and very little crossing-over. There were two additional objectives: (1) generation, by mutagenesis, of sufficient different biochemical ‘mutations to facilitate this study, and (2) determination, from the somatic haploidization data, of the number of independently segregating chromosomes represented in the known meiotic linkage groups of this organism. N—methyl-N'-nitro—N—nitrosoguanidine was used as a mutagen and seventy-four mutations were induced. Twenty-two of these were of usable ”new” genes and fourteen mapped to existing linkage groups. Twenty-five proved to be alleles of the other new or stock mutations. e. mi .14; v7 v. "n ‘: 3.1.0} ~ Ll)” 1"" .' 1 Th: comprised c but differs common-Ag d nutritional. tible diplo crosses of haploicis. (1 obtained.) Two and heteroz: Haploid and nletlium 88 Pt t1011 caused mPPPessor. % and a2 Haplom and non"diploid TWO to °°nta1n 4 cult“ 19. behaviollr 11 0th8r B rac‘ and. 3h0wed ‘ Hap all the (1 1p Q? Carl Stephen Frankel Three sets of diploids were obtained. Each set comprised cultures which were almost identical genetically but differed in their compatibility relationship. Four common~§§_diploids were isolated as spontaneous sectors from nutritionally forced common-A§_heterokaryons. Five compa- tible diploids were isolated from among the progeny of crosses of the common—Ag diploids with appropriately marked haploids. (No usable common-A or common-§,diploids were obtained.) Two of the sets of diploids were homozygous for Eggg and heterozygous for ggi, a recessive suppressor of 953g. Haploid and aneuploid products were selected on arginineless medium as rapidly growing sectors produced when haploidiza- tion caused loss of the chromosome bearing 235g without its suppressor. The third set of diploids was heterozygous for 993 and ad , linked ”pink“ adenine mutations in repulsion. Haploid and aneuploid products were selected as sectors of non-diploid morphology and/or pink colour. Two of the compatible "diploids' in one set proved to contain a high number of dikaryotic cells. One of these, Culture 19, possessed a g mating-type factor with anomalous behaviour in that it repressed the expression of certain other g factors, including the other g factor of Culture 19, and showed a marked tendency to non-disjoin. Haploid and/or aneuploid sectors were recovered from all the diploids, including those partially dikaryotic. Haploids 1| showed 31: the partie frequency somatic re mitotic (p dikarlotic their nucl like behav; meiotic-11] of the Mk: Am meiotic 11: indicated chromOSOme we. Cari Stephen Frankel Haploids from the common-fig and normal compatible diploids showed almost no evidence of crossing-over. Haploids from the partially dikaryotic cultures included a significant frequency of crossover genotypes. It appears that the somatic recombination process in diploids is classically mitotic (parasexual). The diploid cells of the partially dikaryotic cultures can undergo mitotic haploidization, but their nuclei give evidence of a previous history of meiotic- like behaviour involving crossing-over. The tendency toward meiotic-like somatic recombination is apparently a property of the dikaryotic state rather than of compatibility'pgg'gg. Analysis of segregation of markers on separate meiotic linkage groups during haploidization of the diploids indicated that each linkage group corresponds to a separate chromosome. There are at least seven chromosomes in §, commune. THE ROLE OF SEXUAL INCOMPATIBILITY FACTORS IN SOMATIC RECOMBINATION IN SCHIZOPHYLLUM COMMUNE By Carl Stephen Frankel A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 197h DEDICATION To the memory of Joe Treiger, a great and good friend 11 . ..,_. 21.“. I“. I . my major p deuce he e I Bromley, a manuscript I photograph W of th 'hlle in (1 My to Michael Damn an Th ACKNOWLEDGMENTS I am deeply grateful to Dr. Albert H. Ellingboe, my major professor, for his guidance and for the indepen- dence he encouraged in me in pursuing this research. I wish to thank Drs. H. G. Fields, H. Kende, S. C. Bromley, and J. Boezi for their help in preparing this manuscript. I would also like to thank Joe Martin for the photography and Bay Hancock for technical assistance and both of these honourable gentlemen for the good times, even while in despair at the recalcitrance of my fungus. My gratitude also to Le Club de Hockey Canadien and to Michael Kalin and Joe Vechter, as promised, for being partial answers to the existential dilemna. This research received support from the National Science Foundation for which I am indebted. 1H m! . LIST OF '. LIST OF ] HWBODUC! LITERATUI METHODS l Sch! Rd? Mute TABLE OF CONTENTS Page LIST OF TABLES. . . . . . . . . . . . . . . . . . . . . . vi LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . x INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . h METHODS AND MATERIALS . . . . . . . . . . . . . . . . . . 15 Schizoggzllum cultures . . . . . . . . . . . . . . .15 Me is. . 16 Hutagenesis with N-methyl-N'-Nitro-N-nitrosoguanidine NG O O O O O O O O ‘ O O O O O O I O O O O 1 Testing the requirement of new auxotrophs. . . . . . 1 Heterokaryotic allelic tests . . . . . . . . . . 1 Assigning mutations to their linkage groups and mapping... 0000000 000000019 Crossing, single spore isolation, percent gamination O O O O O O O O O O O O O O O 0 O I 19 Scoring for requirement. . . . . . . . . . . . . . . 20 Scoring for mating type. . . . . . . . . . . . . . 21 Synthesis of common-AB diploids. . . . . . . . . . . 21 Purification of cultures . . . . . . . . . . . . . . 22 Diploid x haploid matings. . . . . . . . . . . . . . 22 Mating type tests for progeny of diploid x haploid matings . . . . . . . . . . . . . . . 23 Recovery of progeny diploids from diploid x haploid matings O O O O O O O O O O O O I O O I 2 Verification and analysis of progeny diploids. . . . 2 Detection of somatic recombination . . . . . . . . . 25 Interpretation of recombinant sectors. . . . . . . . 26 Somatic sectoring in a dikaryon. . . . . . . . . . . 26 n1t°t1° mapping. 0 O O O O O O O O O I O O O O O O O 26 StBtiStical EMIYSIIB e o e o o e o o o o o e o o o e 27 iv Page RESULTS 0 O O O O I O O O O O O O O O O O 0 O O O O O O O 2 8 Mutagenesis with N—methyl-N'-nitro-N-nitrosoguanidine NG O O O I O O O O O O O O O O I O O O 28 Requirement of the mutations . . . . . . . . . . . . 28 Allelism among the mutations . . . . . . . . . . . . 33 Segregation of the mutations . . . . . . . . . . . . 3 Linkage of the mutations . . . . . . . . . . . . 3 Determination of the adenine synthetic pathway steps affected by the adenine mutations . . . . . . . 40 Synthesis of common—AB diploids. . . . . . . . #1 Verification of diploidy by mating to wild type haploid . . . . . . #6 Crossing-over in matings of .diploids 1 wild type hap101d8 O O O O. h 0 O O O O O O O O I O O O O O 51 Crosses to produce compatible, common-A, an common-B diploids . . . 52 Characterizafion of daughter diploids. Diploid C:2u. Diploid C h. . DiploidP 3. . . Culture P:21 . . Culture P-19 . . . . Somatic recombination: Type A Diploids Somatic recombination: Type C Diploids Somatic recombination: Type P Diploids Frequency of somatic crossing-over . . Mitotic mapping. . . . . . . . . . . . DISCUSSION 0 O O O O O O O O O O O O O O O O O O I O O C SUM! O O O O O O O O O O O O I O O O O O O O O O O O O 107 LIMWRE CITED 0 O O O O O O O O O O O I O O O O O O O O 1 10 e e e e e e e o o e e o e e o e e e o e e e o e o e e e e o o e o e o e o e o e e e e o e e e e e e o e e e e e e e o e e o o o e o e o o e e o e e e o e e e e e 0' o e o e e e e e o o o e e e e e e e O\ Ox 0 \O \J’i LIST OF TABLES Table Page 1. 2. 7. 9. 10. 11. Numbers of mutations of five general classes obtained by mutagenesis with NO in liquid culture flasks . . . 29 Symbol, linkage group, segregation ratio (mutant/ total) in a cross with wild type, requirement, and other characteristics of mutations obtained from mutagenesis with NG . . . . . . . . . . . . . . . . . 30 Distances between meiotically linked genes. . . . . . 35 Response of ad mutations to intermediates of the purine synthetic pathway. . . . . . . . . . . . . . . 42 Genotypes of forced common-AB heterokaryons and d1p101d8. O O O O O O O O I O O O O I O O O O O O O I “3 Recovery of markers in 216 progeny of a cross of Ahi Bhi s + ar 2 Diploid A-5 ARI BET + n12 ad2 su1 arg ) to Wild type Ah2 an haploid. . . . . #7 Recovery of markers in 17“ progeny of a cross of A41 B41 s ar 2 Diploid M (me sir rfir mgr)” t° "11“ type Ah2 Bh2 haploid. . . . . . Recovery of mzrkgzs in 125 progeny 0g a cross of , A 1 2 + + arg2 arg inol ni} Diploid 0' (AFT B52 ad2 sui erg? + + pan + ) to wild type Ahz Bhi haploid. . . . . . . . . . . . . #9 Recovery of markers in 79 progeny of a cross of Diploid 1L1: (“K-E} Bu?” + {b F—ib H 5-3739- 1'5) 0 . . . 5 to wild type Ahz 841 haploid. . . Expected and observed numbers of crossovers among 216 progeny of a cross of Diploid A—5 to wild type haploid O O O O O O O O O O O O O O O O O O O O O O O 53 Ex cted and observed numbers of crossovers among 17 progeny of a cross of Diploid A-8 to wild type haploj-deeeeeeeeeeeeoeeee00000053 vi in o C: ’1 O U) 15 o C) ’1 O m m 16 O '1 O on m 17 ExDect| 18 to (D O 0 4 (D 19 O ’1 O a: CO 0 20 £11 (D o O < .8 21 22. 23_ Cr 24, 25 o '1 o Table 12. 13. 1C. 15. 16. 17. 18. 19. 20. 21. 22. 23. 2h. 25. Expected and observed numbers of crossovers among 125 progeny of a cross of Diploid C to wild type hap1°1d O O O O O O O O O O O O O O C O O O O O O O 0 Expected and observed numbers of crossovers among 79 progeny of a cross of Diploid P—i to wild type hap101doe00.000000000000000... Cross of common-AB_Diploid A-5 x haploid, desired diploid progeny and their expected frequency, and expected frequency of progeny viable on selective medium 0 O O O O O O O O O O O O O O O O O O I O O O 0 Cross of common-AB Diploid C x haploid, desired diploid progeny and their expected frequency, and expected frequency of progeny viable on selective medium 0 O O O O O O O O O O O O O O O O O O O O O O 0 Cross of commongAB Diploid P—l x haploid, desired diploid progeny and their expected frequency, and expected frequency of progeny viable on selective medium 0 I O O O O O O O O O O O O O O O O O C O O O 0 Expected and observed frequencies of commongA, common-B and compatible diploids from crosses of comm-E d1p101d8 x haploids e e o o o e o o e e o 0 Recovery of segregating markers among 80 progeny from spontaneous fruiting of compatible Diploid 6-3” . . . Crossover frequency among 80 progeny from spontaneous fruiting of compatible Diploid C-BH . . . . . . . . . Recovery of segregating markers among 78 progeny from spontaneous fruiting of compatible Diploid C-Cfi . . . Crossover frequency among 78 progeny from spontaneous fmiting or compatible 131131015. C-llvl-l- e o o e e e o e 0 Recovery of segregating markers among 90 progeny from spontaneous fruiting of compatible Diploid P—3. . . . Crossover frequency among 90 progeny from spontaneous fruiting of compatible Diplbid P-3. . . . . . . . . . Recovery of segregating markers among 89 progeny from spontaneous fruiting of compatible Culture P—21 . . . Crossover frequency among 89 progeny from spontaneous fruiting Of compatible Culture P-21 0 e e e e e e e 0 vii Page 55 56 57 59 62 62 6h 65 65 67 67 28. 29. 30. 31. 36, 37. 38, 39. 40. Table 26. 27. 28. 29. 30. 31. 32. 33. 3h. 35. 36. 37. 33- 39. no. Recovery of segregating markers among 80 progeny from Page a mating of compatible Culture P—19 to wild type A“; ._B_1- haploid. O 0 O O O O O O 0 O O O O O O I O 0 O 69 Recovery of segregating markers among 122 progeny from a mating of compatible Culture P-19 to wild type AhlBliZhaplold.............o..... Recovery of segregating markers among 73 partially analysed progeny from a mating of P-19 to wild type Ahz B; haploid. Recombinant A—5 . Recombinant A-a o o e 0 Recombinant c O O O O O Recombinant C. Os. 0 o Recombinant 0-14“ 0 O O O Recombinant P-l O O O O sectors sectors sectors sectors sectors sectors isolated from O". O 0‘. O O isolated from isolated from isolated from isolated from isolated from Haploid families from recombinant common-Ag DiplOid P-1 o e e e e o e e e e e o e e e o Haploid sectors isolated from Raploid families from sectors 9-3.... Haploid sectors isolated from P-21. . . . Haploid families from sectors P-21. 0 O O Raploid sectors isolated from P-19. . . . viii compatible Culture commonpég'Diploid commongA§,Diploid commonaABIDiploid compatible Diploid compatible Diploid commongA§,Diploid sectors of compatible Diploid P-3. of compatible Diploid compatible Culture of compatible Culture compatible Culture 71 71 73 73 76 76 78 so 81 82 83 85 er? Table l #1. Hap’ P-l, #2. Pair stra 43. SortaI link are Table Page hi. Haploid families from sectors of compatible Culture 8 P-19. 0 o e e e e e e o o e o o e o e e e e o o e o e 9 #2. Pair-wise comparison of number of crossovers in strains P-1, P—3, P-21, and P-19. . . . . . . . . . . 92 #3. Somatic recombination frequencies between meiotic linkage groups in diploids P—1 and P—3. Sample totals are listed in parentheses . . . . . . . . . . . . . . 94 ix Figure 1. Linkag 2. Diploj 3- Compat Brgini 11. Sector dikary 50 secOnd Cultur- Figure LIST OF FIGURES 1. Linkage map of Schizophyllum commu g. . . . . . 2. 3. it. 5. Diploids of Schizophyllum commune . . . . . . . Compatible Diploid C-Bh on minimal medium with arginine. . Sectors arising from diploid and partially dikaryotic strains of Schizophyllum commune . . Secondar Culture i. sectoring of aneuploid isolates of 19. Page . 38 ‘3" The firs accomt for s {1953). The nuclei via 51 over in the A in space and integral part EXperime: 592‘ rinus spp. hmokaryotic (‘ somatic recent INTRODUCTION The first description of a mechanism, other than heterokaryosis, to account for somatic recombination in fungi was presented by Pontecorvo (1953). The mechanism described, i.e. mitotic haploidization of diploid nuclei via stages of aneuploidy, was characterized by infrequent crossing- over in the Ascomycete Aspergillus nidulans. Haploidization was separated in space and time from crossing-over. This process was defined as an integral part of what was termed the "parasexual cycle." EXperiments with the Basidiomycetes Schizophyllum commune and Coprinus spp. utilized the illegitimate (or incompatible) dikaryotic - homokaryotic (di-mon) mating (e.g. l§l§l,+'ég§g7'x AlBZ) for detecting somatic recombination. Only a recombinational event which recombined mating types from the dikaryon nuclei gave a nucleus that was compatible with the nucleus of the homokaryon (e.g. ELLE; + gag] 19% 4 AZBl + Ail—.837). In one series of such experiments (Ellingboe and Raper 1962) two novel types of somatic recombination were described: a meiosis-like event in which there was a high frequency of crossing-over between linked genes, and Specific Factor Transfer in which only an incompatibility factor from each of the two nuclei of the dikaryon was transferred into the genetic background of the homokaryon nucleus. More recent work on §, commune was based on a selective system to detect haploidization and/or recombination within linkage groups in common- AB (fully incompatible) diploids (Mills and Ellingboe 1971). This is a system analogous to that used with Aspgrgillus. The recombinants obtained appeared to have originated via a mitotic parasexual process. These two systems used to detect somatic recombination differ in one fundamental respect. In the diploids, compatible mating type factors, which are ordinarily necessary for meiosis, are absent. In the di-mon matings they are present, Indeed, a recombinational event must produce a nucleus compatible with the nucleus of the homokaryon if it is to be detected. Thus the question can be posed: are the differences in somatic recombination mechanisms that were detected in these two systems due to differences in incompatibility factor relationships, or to some mechanistic or selective idiosyncracies? The former seems to be a reasonable alternative in light of the numerous published accounts of genes, in several organisms, affecting recombination in some way, of the known role of incompatibility factors in initiating meiosis (Raper 1966), and of the papular view of the incompatibility factors as "master switches" that control cascading arrays of genes and many developmental pathways (Raper 1966). Thus this research was initiated with the objective of (1) creating diploid strains of ‘g. commune identical in genetic background and marking but differing in the compatibility relationship of their constituent nuclei, and (2) to observe whether these incompatibility differences lead to different modes of somatic recombination detectable upon haploidization. A mutagenesis and mapping project was undertaken first to obtain new mutations marking the linkage groups of §, commune. This was done with two goals in mind: (1) generation of many different auxotrophio mutations so that extensive complementation tests were not necessary to analyse crosses between multiply marked strains, and (2) generation of sufficient markers in linkage groups to detect crossing-over with confidence. Finally, there was another goal of this research which would be obtainable as a by-product of analysis of haploidization. Meiotic map distances in §, commune can vary widely under a variety of circumstances and considerable doubt exists concerning the linkage relationships of known markers. Thus diploid strains were constructed of genotypes such that analysis of marker segregation during haploidization, together with meiotic linkage relationships, would give the necessary data to unambiguously assign the markers of g, commune to independent chromosomes. LITERATURE REVIEW Investigation of somatic recombination in fungi may be said to have begun with the study of dikaryotization of a homokaryon by a dikaryon in CoErinus lagopus by Buller (1931). This process was later termed the "Buller Phenomenon" and shown to comprise three different kinds of mating: (1) compatible, in which both nuclei of the dikaryon are compatible with the homokaryon: (g;§;,+'gg§§) x gzgz, (2) hemicompatible, in which only one nucleus of the dikaryon is compatible with the homokaryon: (gig; + aggg) x A121, and (3) incompatible, in which neither nucleus of the dikaryon is compatible with the homokaryon (§l§l_+'£§§§) x gig; (Quintanilha 1937). Dikaryotization of the homokaryon in the third type of mating is made possible only by a recombinational event. Such "illegitimate (incompatible) di-mon matings” in'Q. lagogus provided the first demonstration of genetic exchange between the nuclei of a dikaryon (£113; + 5%) -? (LIB-2 + $221). Such matings used the appearance of dikaryotic sectors in homokaryotic mycelium as the selective system for detecting recombination. A later study (Papazian 195#) used a variation of this basic technique. Two successive illegitimate diamon matings were made and hyphal tips that were isolated were found to contain nuclei of novel genotypes. No evidence of disomy was observed. Significant advance beyond this point was made possible by the deveIOpment of a technique for production of stable diploid strains of filamentous fungi (Reper 1952). Pontecorvo (1956) used diploids of 4 Aspgrgillus nidulans to characterize a system of somatic recombination he termed the parasexual cycle. The cycle consists of three basic phases: (1) fusion of unlike nuclei of a heterokaryon, (2) propagation of diploid cells during which mitotic crossing-over may occur, and (3) haploidization' of the diploid by successive random loss of chromosomes, i.e., via stages of aneuploidy. It became readily apparent that mitotic crossing-over was a rare event. Mitotic map distances were only a small fraction of meiotic distances and the two were not regularly proportional. The rarity of mitotic crossing-over means that genes on the same chromosome generally segregate together. Analysis of somatic recombination is thus a useful tool for assigning genes to their chromosomes. When a crossover: occurs on a chromosome marked by a series of heterozygous genes, all genes distal to the crossover can become homozygous. Thus genes on the same chromosome can be placed in linear order and the position of the centromere can be determined. This method was used together with a selective system based on recombination to homozygosity of heterozygous recessive suppressors of auxotrophic mutations. If the fungus was growing sparsely on a medium deficient in the requirement of the auxotrOphic gene (which was homozygous), recombination causing homozygosity of the suppressor would create a rapidly growing sector. These techniques made possible extensive mitotic mapping of Aspgrgillus nidulaps and extensive study of somatic recombination (Pontecorvo and Kafer 1958) (Kafer 1961). The discovery of parasexuality stimulated further work with diamon matings in basidiomycetes in an effort to understand the nature of the recombinational process in this system. Crowe (1960) studied compatible di-mon matings in §, commune and found dikaryotic sectors in the homokaryon which arose by migration of both nonrecombinant nuclei of the dikaryon through the homokaryon, and other sectors that resulted from association of the homokaryon nucleus with a nucleus from the dikaryon which may or may not have been recombined. The recombinant nuclei from the dikaryotic sector sometimes showed evidence of crossing-over, but the data were insufficient for Crowe to conclude whether she was observing parasexuality or a precocious form of meiosis. Swiezynski (1962, 1963) studied recombination in incompatible and hemicompatible di-mon matings in Q, lagogus. He experienced difficulty in analysing the genotype of the derived dikaryons due to poor germination of the basidiospores and low viability of those spores that germinated. Segregation ratios were mostly abnormal. The results obtained suggested a parasexual type of recombination with haploidization proceeding via stages of aneuploidy. A study of 178 recombinant dikaryons from 7,160 incompatible di-mon matings in §, commune (Ellingboe and Raper 1962) made use of several non-selective markers, one of which was linked to the é_incompatibility factor. With these it was possible to classify the recombinants obtained into two major groups: class I, in which non-selective markers represented various combinations of markers from the original dikaryon, and class II, in which non-selective markers were exclusively those of the homokaryon. There was no evidence of aneuploidy. Class I types most likely arose from familiar meiotic-like events in the original'dikaryon, but the authors had to suggest two successive recombinational events, segregation from a triploid nucleus, or "some heretofore unknown mechanism" to account for individuals of class II. Additional tests were performed with the use of two additional non- selective markers that were each less than one crossover unit from the Ag_ and Ae_loci, respectively (Ellingboe 1963). Twenty of eightyhsix derived dikaryotic cultures from several incompatible diamon matings were class II types in which an A_factor from the dikaryon was transferred to the background genome of the homokaryon without transferring the closely linked markers. Whatever process was Operative had a specificity of incorporation analogous to that of a temperate phage into a bacterium. It constituted a new, third mode of somatic recombination and was termed "Specific Factor Transfer." Sixty-one wellamarked recombinants from a single incompatible di-mon mating were shown in another study to be of forty-three different genotypes, none of which were class II types (Ellingboe 1964). This raised the possibility that Specific Factor Transfer may be a feature of only'certain matings and therefore under a specific form of control. A more recent study of §, commune yielded over three hundred derived dikaryotic sectors from incompatible diamon matings (Shalev et al 1972). Three different dikaryons were used in these matings but their constituent nuclei were from genotypically identical sibling strains and all three dikaryons were mated to the same homokaryon. The genotypes of the derived dikaryotic sectors were only partially analysed; auxotrophic markers shown to be heterozygous were not tested to show which nucleus of the derived dikaryon contained the wild-type or the mutant allele. However, the analysis was sufficient for the authors to conclude that the sectors had arisen in at least three ways: (1) migration of both nuclei of the original dikaryon, (2) production of a recombinant nucleus from fusion and and haploidization of the nuclei of the original dikaryon and mating of this nucleus with that of the homokaryon, and (3) production of a recombinant nucleus from fusion and haploidization of the homokaryon nucleus with a commonqg nucleus of the dikaryon and mating of this nucleus with the other of the dikaryon. This third process had not been previously reported, as such, in the literature. The recombinant genotypes revealed no evidence of Specific Factor Transfer, and, more interestingly, showed that although crossing-over was of essentially meiotic frequency where it occurred, it occurred only in the regions between mating-type factor subunits. The authors interpreted this as evidence for a mechanism controlling frequency of recombination in different parts of the genome. During this period data on somatic recombination in Basidiomycetes were also obtained from systems which did not have the selective bias of the incompatible di-mon mating system. Raper and Raper (l96h) obtained five recombinant prototrophic hyphal tips from an old §, commune heterokaryon which had been formed from homokaryons carrying a modifier mutation which eliminated fruiting. Three were homokaryotic, two were dikaryotic and there was no evidence of aneuploidy. A commonqA§.heterokaryon of’§, commune has been produced by’mating two homokaryons with complementing auxotrophic mutations on minimal medium (Middleton 1964). Fourteen hyphal tips isolated from the heterokaryon were prototrophic and stable, presumably diploids. ‘When crossed to wild- type homokaryons, ten gave progeny that were auxotrophic for one or more requirements. The combinations of auxotrophic mutations segregating in each cross were consistent with what would be expected if the hyphal tips had been diploid, the diploid underwent mitotic hyploidization (i.e. reassortment of whole chromosomes), and the resultant haploid nucleus participated in the mating with the wild-type homokaryon. An attempt was made to use recessive suppressors as a selective system to detect recombinational events in'Q. lagopus dikaryons (Cowan and Lewis 1966). The recombinational mechanism which produced rapidly growing sectors appeared to be parasexual. The total sample size was only six so it was impossible to draw any conclusions. A different selective system was used to detect recombination in a triheterokaryon of Caprinus radiatus (Prud'homme 1965). Two of the three nuclei were compatible with each other but possessed a morphological :mutation which prevented dikaryosis. The third nucleus did not have the morphological mutation but was incompatible with the other two. Thus a recombinational event was necessary to establish a dikaryon. The dikaryotic sectors which arose were genetically characterized by progeny analysis and dedikaryotization by maceration. Three conclusions were drawn: (1) haploidization proceeded via stages of aneuploidy, since numerous aneuploid and some diploid strains were recovered, (2) crossing- over is rare (none occurred in twentyaone recombinant nuclei), and (3) there was in many cases a parental association of independent non- selective markers. This last observation may be interpreted as evidence either for a mechanism similar to Specific Factor Transfer or for mutation to the wild type allele. Somatic recombinants have been obtained from Uredinales (Puccinia graminis tritici) using mixed inoculation of two dikaryotic urediospore isolates on wheat seedlings (watson 1957) (Ellingboe 1961). In both cases spore colour and virulence were used as markers. In neither case was it possible to determine the mechanism of recombination. Holliday (1961) induced mitotic recombination in Ustilago ggmgig with ultraviolet light (UV). Heterozygous prototrophic diploids 10 exhibited crossing-over by becoming auxotrOphic after irradiation. That these auxotrophs were still diploid was shown by a second UV treatment which initiated segregation of other auxotrophic markers. The results indicated mitotic crossing-over followed by'haploidization, similar to what would be expected with the parasexual cycle. It is apparent that the information about somatic recombination in basidiomycetes, which has come from a variety of techniques, is not consistent with any general theory. Recombination studies in basidiomycetes which used the techniques applied so successfully to Aspergillus had to await the development of stable diploid strains. The notion of transient diploidy in vegetative mycelium.gained acceptance with the demonstration of genetic exchange between the nuclei of a dikaryon (Quintanilha 1939). The first persistant diploids in basidiomycetes were produced from heterokaryons. They were stable as homokaryons but unstable when mated to a haploid (Casselton 1965) (Parag and Nachman 1966). In the former case Casselton made a commonqg heterokaryon in Q, lagopug with complementing auxotrophs. Oidia that could grow when plated on minimal medium were taken to be diploid since oidia are uninucleate. The instability in dikaryons was manifested as haploidization before fruiting. In the latter case, the authors used a commone§_heterokaryon of §, commune. The diploid, when mated with a compatible haploid, produced progeny which germinated poorly and had low viability. All mutations present in both parents were recovered, but genotypic frequencies were not in accord with triploid segregation. Diploids were also produced in Q, radiatus (Prud'homme 1965) as sectors from a triheterokaryon. 11 A stable diploid of’§, commune was picked up serendipitously as a prototrOphic product of the mating of two compatible, differently auxotrophic strains (Koltin and Raper 1968). The product had the morphology of a homokaryon instead of a dikaryon. The ability of the original homokaryons to produce a diploid when mated appeared to be controlled by a simple recessive gene. The authors named this gene ggg, and suggested that the gikf allele was necessary for the establishment and maintenance of a dikaryon. Finally, diploids of'§, commune were found to arise in a nutritionally forced commonqA§.heterokaryon (Mills and Ellingboe 1969b). These diploids were the first constructed expressly for the study of somatic v recombination. They were heterozygous for a recessive suppressor of the gaggDmutation and homozygous for agg§.(Mfills and Ellingboe 1969a). They thus comprised a system for examining somatic recombination exactly analogous to that used so successfully in A, nidulans (Pontecorvo and Kafer 1958). These diploids produced l5# sectors which grew rapidly on arginine deficient medium. Of these, 129 were apparently haploid, the remaining 25 aneuploid and diploid. Haploidization occurred via stages of aneuploidy. Since none of the recombinants showed evidence of crossing- over, the recombination mechanism appeared to be typically parasexual, i.e., mitotic. There seems no absolute pattern to the occurrence of the three modes of somatic recombination - parasexuality, meiosis-like, and Specific Factor Transfer. However, the most extensive studies (Pontecorvo 1956) (Ellingboe and Raper 1962) (Ellingboe 1963) (Ellingboe 1964) (Middleton 196#) (Mills and Ellingboe 1971) (Shalev et a1 1972) seem to suggest that 12 parasexuality might be expected in a homothallic fungus such as A, nidulans or a basidiomycete with common-A§,mating type factors, while meiosis-like recombination would occur in basidiomycetes with compatible mating type factors. Indeed, the incompatible diamon mating system selects for recombination to compatibility between;nuclei. (Specific Factor Transfer, of course, can only be detected in the presence of compatible mating type factors.) This pattern in turn suggests a role of the incompatibility factors in determining modes of recombination. The incompatibility factors are known to control specific sequences of events leading to the establishment of a dikaryon and thence to meiosis and fruiting (Raper 1966). Obviously such complex activities are unlikely to be the direct result of the products of only a few genes. Therefore, it is supposed that these factors are regulatory in nature, and that they control the activity of numerous other genes. It can be postulated that among the genes controlled by the incompatibility factors are ones which initiate the events of meiosis. There are precedents for gene effects specific to somatic or meiotic recombination, but not both. Jansen (1970) described a UVasensitive mutant in A, nidulans, Eygfilg, which when homozygous led to abnormally high frequency of mitotic recombination while meiotic recombination frequency remained unchanged. The author suggested that the high recombination activity of the ascus primordia may mask the effect of the gene, but that is only one possible explanation. Minute mutants in Drosthila melanogaster, known to have no effect on meiotic crossing-over, were shown to raise the frequency of mitotic crossing-over (Kaplanll953). The meiotic mutant £9.19 of Q. melanogaster virtually abolishes meiotic crossing-over but was shown to have no effect on somatic recombination 13 (Le Clerc 19%). Finally, a UV-sensitive mutant u_vs§_ in A. nidulans was shown to increase mitotic recombination more than ten-fold while not affecting meiotic recombination at all (Shanfield and Kafer 1969). Specific Factor Transfer is a more difficult problem to deal with. That its occurrence or non-occurrence may be under genetic control is indirectly suggested by its absence in some studies (Ellingboe 1961+) (Shalev et a1 1972) which employed the same experimental methods and selective system as studies in which it was detected (Ellingboe 1963) (Ellingboe and Raper 1962). If the phenomenon is considered to be one of highly localized and regulated crossing-over there is ample precedent for its genetic control. Catcheside (1968) described genes which specifically control recombination frequencies in the am locus of Neurospora m. Simchen (1967) found that crossing-over between the A and .fi. subunits of the A incompatibility factor in §_. commune appeared to be under control of a major gene, linked to the A factor, and several minor genes. This conclusion was later modified to a more general form by Simchen and Stamberg (1969) who suggested that (l) recombination in 2 unlinked chromosome segments is regulated by different components of a control system, (2) the effects of certain loci on recombination seems localized to short segments, (3) probably at least some loci control recombination in more than one region, and (4) a specific relationship likely exists between controlling genes and their segments. A gene controlling recombination frequency between the .4 and ,fi subunits of the §,incompatibility factor in S, commune was mapped as being linked to the A factor itself, and described as’ having no effect on recombination in an unlinked region or a region contiguous with the _B_ factor (Koltin and Stamberg 1973)- 14 This information, in addition to the description of the specifically localized crossing-over between mating type factor subunits (Shalev et a1 1972), can be considered evidence for a process which, if not exactly Specific Factor Transfer, is mechanistically identical to it and under tight genetic control. METHODS AND MATERIAIS Schizopflllum cultures: All cultures used had a common background genome, strain 699 (Ellingboe and Raper 1962). Auxotrophic mutations and their linkage relationships were either previously known and published (Ellingboe and Raper 1962) or induced and characterized in this study. No markers were extensively used which affected morphology, were very leaky, slow growing, or which did not segregate normally. Various tester strains with single auxotrOphic mutations in the 699 background.were available, or, in the case of the new mutations, were prepared by backcrossing to 699. Multiply marked strains were synthesized by crossing single mutation stocks, their doubly'marked progeny, etc. Prototrophs representing most of the possible combinations of five A. and five I; incompatibility factors (g, Ag, A31, A12, Afl, and _B_}, B_2_, EQA, §4§,and 832) were maintained as mating type testers. Most auxotrophs were available in the nine possible combinations among AAA, 53g, A32, and AAA, gag, and.§flz. The subunit composition of the AIfactors is as follows: fl=Aslgl gauges A41= (3&5 Intran_factor recombinants among these A_factors were of three possible types: A42! 1, A41! 5, and 1N1! 4. Tester strains of each type were available with various _I; factors. No recombination between subunits of A factors was observed. 15 16 24.93%: All matings were made on migration complete medium (Snider and Raper 1958). Stock cultures were maintained on yeast medium in bottles at 4°. Yeast medium is made from minimal medium (Raper and Miles 1958) by substituting yeast extract and bacto-peptone at 2g/lit'er for asparagine. Growth on yeast medium was used as a criterion of viability in all tests for auxotrOphic mutations. The tests for auxotrophy were made on minimal medium and minimal supplemented with all possible nutrients necessitated by the mutations in the experiment except one, and called variously ”adenineless", ”sulphateless" etc. according to the missing supplement. Supplements were kept in stock solutions. All nucleic acid bases and amino acids were used at a final concentration of 10AM, except cysteine and methionine at 1.5 x 10AM and leucine, tyrosine, and phenylalanine at 2 x 10AM. Vitamins were used at the following concentrations: inositol .04%, pantothenate, ascorbate, and choline .02%, niacin .01%, pyridoxine and riboflavin .005%, para aminobenzoic acid .001%, and biotin .ooou%. Mutagenesis with N-methyl-N'-Nitro-N-nitrosog2nidine (NG): Wild type strain 699 was grown on three plates of minimal medium for five days at 32° and then macerated in sterile Waring Blenders for one minute with 25 m1 sterile distilled water. Five ml samples of the macerate were pipetted into 300 m1 Erlenmeyer flasks that each contained 20 ml liquid migration complete medium with 5 x 10'3 mg/ml NG. Fourteen flasks were prepared and placed on a shaker at 22° for three days, after which the contents of each flask were centrifuged in a clinical centrifuge at 6500 g for 3 minutes. Each pellet was resuspended in 25 ml sterile distilled water and macerated in a Waring Blender for 20 seconds. One ml l . L—www“'-—-‘m‘~w cf 17 of each suspension was pipetted onto each of four petri plates of yeast medium. The mycelial fragments were allowed to grow on these plates for 24 hours at 22°, after which they were examined under a dissecting microscope. Blocks of agar containing viable fragments were removed and placed on fresh plates of yeast medium, 16 to a plate. Twelve such plates were made from each of the fourteen original flasks. The plates were incubated at 220 for four days, by which time most of the fragments had produced macroscopically visible colonies. Small pieces of each colony were replicated onto yeast and minimal media and allowed to grow'fOr three days at 22°. AuxotrOphs were selected by their inability to grow on minimal medium. Several mutations affecting colony morphology were also isolated. Testing the rgguirement of new'auxotrths: Newly acquired auxotrophs were plated on three types of media: minimal to which had been added, respectively, a solution of the five nucleic acid bases, a solution of nine vitamins, and vitamin-free casein hydrolysate. Those that failed to grow only in the absence of nucleic acid bases were plated on minimal medium supplemented with either adenine, guanine, thymine, cytosine, or uracil. Requirements were, therefore, determined directly. Those that responded to the vitamins were placed on ”pool plates" - minimal with three supplements as follows: #; #2 #3________ j riboflavin pyridoxine inositol 15 niacin gpantothenate ascorbic acid ##6 para aminobenzoic acid choline biotin An auxotroph which grew, for example, only on pools #3 and #6 would appear to require biotin, the only supplement the two pools had in common. This would be verified by testing for growth on minimal plus biotin. and 1 guanf CODCG as ex tests was u and r Suppl occur infer StOQk test l8 Auxotrophs which responded to casein hydrolysate were tested on amino acid pool plates - minimal with either four or five supplements as follows: te utamic t An auxotrOph which grew, for example, only on pools #4 and #6 would appear to require serine, the only supplement the two pools had in common. This would be verified by testing for growth on minimal plus serine. Purine requirers were tested for growth response to inosine, xanthine, and 4-amino-5-imidazole-carboxamide, all intermediates of the adenine - guanine synthetic pathway. All these supplements were at a final concentration of 10-4 M. Heterokaryotic allelic tests: New mutants with the same requirements as existing stocks were tested for allelism. They were mated to the tester stocks and incubated at 32° for four or five days until a dikaryon was well established. Three small pieces of each dikaryon were removed and replicated onto minimal medium and minimal plus the appropriate supplement. Growth of the dikaryon on unsupplemented minimal medium would occur if the mutants were non-allelic and complementing. Homoallelism was inferred if no growth occurred on minimal medium. Different mutations implicated in this way to be allelic to the same stock strain were crossed with each other, and a heterokaryotic allelic test was performed with the two new mutations. Similarly, these mutations with the same requirement, but which were not allelic to any stock strain, were tested against each other. These tests were performed only after backcrossing the mutant strains to wild type to generate compatible mating types. 19 Heterokarytic allelic tests were also used to determine genotypes of progeny from crosses which involved markers with common requirements. For example, if a strain bearing p;§_were crossed with one bearing 2A2, niacin requiring progeny would be mated to ngg_and pgj.testers to determine whether their requirement for niacin were the result of carrying Egg, ni , or both Assigging mutations to their linkage groups apd mapping: New auxotrophs were mated with tester strains carrying, respectively,‘gg1, gagg, gggA, Aggé, and £23, each marking one of the known linkage groups of’§, commune. The necessity of heterokaryotic allelic tests was avoided when possible by the use of other genes in the linkage group, i.e. Ag; strains could be substituted by n_:'L_2_ strains, a_rg_2_ by a_d_2, and a_rgA by 9&4, Samples of 32 progeny spores were collected and tested for evidence of linkage. Each of these crosses also afforded an Opportunity to check for linkage to the A'or §,incompatibility factors. Auxotrophs which did not show linkage to any of the tester markers were tested against other markers of the same linkage groups and/or each other. When results of these tests were inconclusive larger samples of spores were collected and analysed. Once assigned, the new markers were placed in correct order with respect to the other markers of their linkage groups by 3-, 4-, or 5- point crosses. Each new marker was crossed in this way with all or most of its linked markers. Gene distances were averaged from all available data. Crossing, single spore isolation, pgrcent germination: Crosses were made by placing small mycelial plugs of compatible strains about 5 mm apart on migration complete medium. Each plate contained duplicate sever of ge field “28 C an .. 20 matings. They were incubated at 320 for four or five days or until fruit body initials formed. Chunks of agar bearing mcelium with the largest fruit body initials were out out and placed away from the mating on the plates. Plates were incubated upside down at 22°. When basidiospores began to be produced, the agar chunk was removed, placed on the cover of a yeast medium plate, and allowed to drOp spores onto the medium. After sufficient spores had collected, they were spread over the medium in several dr0ps of sterile distilled water with a bent glass rod. The spores were allowed to germinate for one day at 22°. The number of germlings vs number of ungerminated spores in three or more microscOpic fields (at least 50 total sample) was counted, and the percent germination was calculated. Small agar blocks with single germlings were removed with a small, flattened needle and transferred to yeast medium, sixteen to a plate. An effort was made to cut out every germling, regardless of size, in a microscopic field to assure a random sample. However, germlings growing too close together to separate with confidance were omitted. The germlings were incubated for four days at 22°. Scoring for rguirement: When isolated germlings had grown into macroscopically visible colonies they were replica plated individually with a dissecting needle onto minimal medium, minimal supplemented with all possible requirements for that cross, and the various test media in which all supplements except one were present. Yeast medium was frequently substituted for the completely supplemented control plates. In crosses involving only a single requirement, yeast and minimal media were used. Scoring for requirement was generally done after three days of incubation at 22°. 600 I' 21 Scoring for mating type: Four testers were generally used to determine the mating types of progeny from a cross. For example, the progeny of a cross of A4124; x AL£_B4_2_ would be tested with $111, 59313;: A2_B_lfl., and A2_B4_._2_. The first two testers were used to determine A mating types, the last two to determine 3 mating types. The reactions are as follows: Testers Genotype of Progeny A41Bl A4281 A2B41 A2B42 A41B41 _a + - + A41B42 _ + + __ ALPZBLP]. + _ _ + A42B42 b + _ + _ AxB41 + + - + AxBl+2 + + + - a + = compatible, - = incompatible b Ax = recombinant A factor Recombinant A factors were sometimes further typed to determine the subunit alleles. From the example above, since Ali]; is A4181 and AB is Aflfle 5, recombinant A factors would be tested with two strains such as A4 lBl and Adlg 5B1, only one of which should be compatible with a recombinant of crossing Afl by A4_2. Synthesis of common-4A2 diploids: Small mycelial plugs of selected strains with complementing auxotrophic mutations, but with the same mating type, were placed approximately 1 cm apart on migration complete medium, two pairs per plate, and incubated at 32° for five to seven days. A strip of agar about 2 mm wide was removed from the region of confluence and divided into thin slices. Each slice was placed in the center of a plate of minimal medium (or minimal plus arginine for common-fl heterokaryons homozygous for arg2 but heterozygous for the recessive suppressor s91). 22 Thus only nutritionally-forced commonqA§,heterokaryons could grow. The plates were kept at 22°. Rapidly growing sectors with good.morphology (presumptive diploids) were purified and analysed. Purification of cultures: Presumptive diploids, or any other culture in this study which arose as a sector in different genetic background, were purified by isolation of hyphal tips. Four samples were generally taken from a sector and placed on medium identical to that from which they had been removed. Wherever possible the samples were taken from the growing edge. Note_was made of the sector's mycelial morphology if it was visibly'different from.the background. The samples were incubated at 32°, usually for two days. Hyphal tips of two to four cells were then out out with a flattened isolating needle and transferred to fresh medium. Care was taken to select tips of the longest hyphae, or those with the correct morphology, whichever applied. The process was repeated after another two days of growth at 32°. The final tip cultures were kept at 320 until macrosc0pically visible colonies developed. Diploid x haploid matings: Presumptive diploids were crossed with wild type haploids. Spores were collected from fruiting bodies which develOped on the haploid side of the mating. All progeny'were tested for nutritional requirement. Recovery of all markers of the original common- A§_heterokaryon was taken as proof that the presumptive diploid was, in fact, diploid. Allele frequencies consistent with trisomic segregation were expected. The original inoculum of each germling onto the yeast control plates was transferred to an individual plate of’yeast medium. The subsequent growth was examined fer sectoring. Sectors were purified and tested for requirement as described previously. The process was continued until sectoring no longer occurred. 3L.-. tatil fact. mate reco prof dip] Hit 23 Mating tm tests for progeny of diploid x haploid matings: Mating type tests for diploid x haploid matings were carried out in a manner analogous to those for regular matings. Six testers were used, four for the parental A and .1; factors and two for the recombinant A factors. The latter two testers helped to distinguish between individuals disomic for the chromosome containing the A factor and those with a recombinant A factor. When a compatible diploid parent was used in diploid x haploid ‘ matings six tester strains were needed, three each for parental A and _8 factors. Progeny compatible with more than one A mating type tester were mated with two appropriate strains to determine whether they possessed a recombinant A factor . Recovery of progeny diploids from diploid x haploid matings: Experiments were initiated in an effort to obtain common-A, common-E, and compatible diploids marked identically to the parental cannon-Al: diploids by crossing the comon-A_B diploids to haploid strains containing all or most of the markers in the diploid. The matings were such that prototrOphic progeny (or progeny requiring only arginine in the case of diploids homozygous for a_rgg and heterozygous for §p_l_) had to be diploid with all markers heterozygous, aneuploid, or haploid and recombinant. The probability of obtaining progeny diploids with all markers heterozygous was calculated on the basis of four assumptions: (1) the pairing and distribution of homologous chromosomes during meiosis in a diploid x haploid cross is totally random, (2) the linkage relationships of the markers reflect real chromosomal constitution, (3) crossing-over is usually of no effect upon chances of acquiring the desired pregeny genotype and can be ignored, and (4) all progeny are, at least initially, equally viable . Che: TE '3 '1 .. 0 LP 9 D '1 fro: shc fat 00!: 24 While these assumptions taken together are almost certainly false, they had some heuristic value. The calculations provided a rough estimate of the number of spores to be collected in order for there to be a realistic chance of recovering the desired diploids. Spores were spread at a density of about 100 per plate on minimal medium (or minimal plus arginine where appropriate). Each plate was checked daily, under a dissecting microscope, for six to eight days for thriving germlings. Spores germinating close to each other were separated in an attempt to avoid dikaryotization among progeny. The germlings which grew best under these selective conditions were isolated. Those which sectored spontaneously (presumptive aneuploids) were eliminated. Those kept were stored in bottles of yeast medium. Verification and angysis of progepy diploids: Presumptive diploids from the diploid x haploid matings were checked for mating type. Strains showing compatibility to both parental A factors and to recombinant A factors, those compatible with both parental E factors, and those compatible with all the testers were assumed to be diploid, or stable aneuploid, and were saved for further analysis. They were then crossed to wild type homokaryons for a determination of markers present. There was a possibility that those scoring as compatible diploids were actually dikaryons, having arisen undetected on the original germling plates. (It was highly unlikely that diploids scoring as comon-}_3_ were actually heterokaryotic since they were maintained on unselective medium which would allow them to dedikaryotize rapidly.) Each strain was stained with a simplified modification of the Feulgen technique and the number of nuclei per cell was observed. The staining procedure was as follows: Pieces of wcelia were fixed in 3:1 ethanol - acetic acid for twenty dens WCG CI‘OS PGCO' rem Step . 25 minutes, hydrolysed in IN NCl at 60° for ten minutes, transferred to Schiff Reagent and stored in the dark for forty minutes, at. 22°, then removed and teased apart in a drop of 10% acetic acid on a slide, pressed firmly under a cover slip, and mounted with Permount. With this technique nuclei were visible in only a fraction of the cells. Appearance of two nuclei in 10% or more of those with visible nuclei was arbitrarily taken as sufficient indication that the ”diploids” were actually dikaryotic. Detection of somatic recombinatiop: Diploids homozygous for _a_1_'g2_ but heterozygous for £11.33 a recessive suppressor of a_rgg, were plated on minimal medium supplemented with all the requirements of the constituent haploid strains except arginine. These diploids could grow slowly and thinly without arginine. Somatic crossing-over could lead to homozygosity for _s_ul_. Haploidization could produce a cell line carrying both Egg and Asu_l_. In both cases the arginine requirement is eliminated and rapid, dense sectors would be expected. Occasionally, slow growing sectors - even slower than the background mycelium - were detected. These were predicted as the product of somatic crossing-over that led to elimination of s_uA (the reciprocal product of the crossover which led to homozygosity of 59;) or haploidization and recovery of pig; without .s_uA. Some of the diploids constructed for this study contained, in repulsion, 29.9. and $429 the two loci for the enzyme controlling the last step in adenine synthesis. Adenine requirers containing _a_._dA or §._d2 grown on supplemented or complete medium turn pink due to accumulation of an adenine precursor. Thus these diploids grown on medium supplemented with all possible requirements of their constituent haploids produced pink sects some: rapid color stat A! 26 sectors as a result of either haploidization or recombination to homozygosity of either marker. These diploids also produced sectors of variant morpholog - slow or rapid growth, thinner or irregular hyphae - with or without pink colouration. Thus recombinants from diploids heterozygous for Ag.- and _a_d2 were not selected only on the basis of colour. Integretation of recombinant sectors: All sectors were purified ' and tested for nutritional requirement (and mating type when from other than common-A}; diploids). Auxotrophy for non-selective markers was taken as one indication of haploidization. Recombinant sectors were isolated from completely supplemented minimal medium, but transferred to both completely supplemented minimal and yeast media. They were grown on these media and allowed to either produce further sectors or demonstrate stability (presumably due to complete haploidization) by lack of sectoring. The genotypes of the haploid products revealed the occurrence of crossing-over in their parent diploids. The various diploids were compared, in this way, for frequency of somatic crossing-over. Somatic sectoring ip a dikapyon: Dikaryons bearing all or most of the desired heterozygous markers were accidentally formed in a cross of haploid x cannon-A122, diploid heterozygous for Adi-l; and £12 in repulsion. They were plated and examined for somatic sectoring in exactly the same way as were the diploids. Mitotic mapping: The mutations used in this study marked all seven known linkage groups. The compatible diploids and dikaryons heterozygous for all and Egg in repulsion each had all seven linkage groups marked. This provided an opportunity to determine whether the meiotic linkage groups correspond to actual chromosomes. Mutations on the same chromosome would be predicted to segregate togetherduring haploidization. 27 The cannon-fl and compatible diploids proved to have very low levels of somatic crossing-over and were thus ideal for this purpose. The comonqfl diploids had no nutritional markers in the A factor or Q factor linkage groups, but in compatible diploids the incompatibility factors themselves could be used as markers for their linkage groups. Thus a mitotic map of all seven meiotic linkage groups was obtained. No attempt was made to determine mitotic gene distances. Statistical analfiis: Variability in gene distances, when apparent, was calculated as standard deviation among samples. The comparisons among diploids and dikaryons for frequency of somatic crossing-over was done by Contingency Chi Square with a 5% level of significance. RESULTS Mutagenesis with N-methyl-N'-nitro-N-nitrosoganidine (NG): NG proved to be a very effective mutagen. A total of 2,688 colonies were isolated, 192 from each incubation flask. It is likely that many of the colonies were originally plated as mycelial fragments of more than one viable nucleus, in which case mutant cells would have been hidden by growth of prototrophs. Thus the apparent rate of mutation, excluding lethals, (74 colonies of 2,688, or 2.8%) is a conservative estimate. Reguirement of the mutations: The initial characterization of the mutations into five classes is summarized in Table l. Twenty-five mutations were originally of undefined requirement, but eight of these were classified after being backcrossed to wild type. The requirement of each mutation, in so far as it was determined, is shown in Table 2. All but two of the sixty nutritional mutations were assigned specific biochemical requirements, although six of them had to be first backcrossed and/ or tested on pool plates with double the normal concentration of supplement. Several nucleic acid base requirers are peculiar in responding equally (and poorly) to all five bases. Strains bearing t_hy grow perhaps half as well given cytosine as they do with thymine, while strains bearing 253.]; respond somewhat to almost anything, but best to pyrimidines. Growth of pp; strains is promoted almost as well by guanine as by adenine, which, in toms of the direct purine synthetic pathway, should 28 29 Table 1. Numbers of mutations of five general classes obtained by mutagenesis with NC in liquid culture flasks. Flask Mutation Class ’ nucleic amino acid morpho- acid vitamin base unknown logical total 1 0 1 0 0 1 2 2 4 1 1 0 7 3 1 0 1 2 1 5 4 1 1 1 1 0 4 5 1 0 O 0 2 3 6 o 1 1 u 2 8 7 0 1 1 1 1 4 8 0 1 2 O 3 6 9 0 0 4 O 2 6 10 1 2 2 2 1 8 11 2 1 0 1 1 5 12 0 2 3 1 0 6 13 0 2 2 2 1 7 14 0 1 1 0 1 3 g__ Total 7 19 17 17 1a 74 30 Table 2. Symbol, linkage group, segregation ratio (mutant/ total) in a cross with wild type, requirement, and other characteristics of mutations obtained from mutagenesis with NG. Linkage Segre- Name Group gation Requirement Description ad2-b IV 33/63 adenine ad2-c IV 32/63 adenine ad3-b III 28/60 adenine ad3-c III 29/62 adenine ad4-b V 29/58 adenine pink ad5-b I 31/60 adenine tighter than ad5 adS-c I 28/59 adenine tighter than ad5 ad6 ? 34/64 adenine ad?‘ 7 29/63 adenine poor response ad7-b 7 21/61 adenine poor response ad8 7 32/62 adenine ad9 V 32/63 adenine pink ad10 ? 35/64 adenine adll I 29/62 adenine ad12 IV 34/63 adenine arg1-b* V 29/59 arginine argl-c V 30/64 arginine arg2-b IV 28/63 arginine are VII 26/57 aromatic amino poor response to acids tryptophan cho-b IV 33/63 choline leaky cho-c IV 34/63 choline leaky cho-d IV 30/61 choline leaky ino1 VI 31/62 inositol Table 2 continued Linkage Segre- 31 Name Group gation Requirement Description v ino1-b VI 30/59 inositol incl-c VI 32/61 inositol in02 ? 29/62 inositol very leaky leu III 30/62 leucine responds less well to isoleucine . lys 7 6/123 lysine low germling viability ni1-b IV 32/63 niacin niZ-b III 35/64 niacin niZ-c III 34/63 niacin niZ-d III 34/64 niacin n15 III 29/61 niacin adaptive n15-b III 30/60 niacin adaptive pan VI 32/63 pantothenate pdx III 30/59 pyridoxine pdx-b III 31/62 pyridoxine pur ? 28/61 purine responds best to adenine rib IV 29/63 riboflavin 8 III 31/64 cysteine or methionine thy III 32/60 thymine responds less well to cytosine, slow growth ul-b III 30/57 uracil leaky u3 __ 29/59 uracil poor response com VII 32/62 none compact, highly branched com-b VII 31/64 none compact, highly branched min, V 32/62 none very compact, highly branchedg(dome?) 89 81 n1 13 x3 x3: 13.‘ X3 :3 X3 X3 X3 xh x14 xh xhg 32 Table 2 continued Linkage Segre- Name Group gation Requirement Description minpb V 28/61 none very compact, highly branched (dome?) O. spi V 22/h5 none viewed from top forms cw spiral a1 - 29/62 7 responds to casein hydrolysate n1 - 35/63 7 responds slightly to any nucleic acid base 130 - 31/59 7 x31 - 32/63 7 responds somewhat to pyrimidines x32 - 28/6h 7 :33 - 3h/63 ? 13h - 31/6h 7 x35 - 27/59 7 :36 - 31/63 ? x37 - 32/60 ? x38 - 30/60 ? x39 - 31/63 7 x40 - 34/63 ? xhi - 33/6h 7 x42 - 30/63 7 xh3 — 32/61 7 responds slightly to casein hydrolysate 14h - 28/62 7 xhj - 24/63 7 leaky Egg - 2/2h 7 low germling viability * mutations induced previously by others ** spontaneous mutation found in stock 33 be impossible. Strains carrying Eflfl_will grow somewhat without vitamins and bases, but not at all without amino acids. None of the amino acid pool plates, however, increased its growth significantly. Virtually every mutant grows as well on migration complete as on yeast medium. Mutations for the same requirement at the same locus were considered alleles if they came from different incubation flasks but separate isolates of the very same mutation if they came from the same flask. The latter situation did not arise in this study. Allelism among the mutatiog_; For most of the mutations listed in Table 2, complementation in heterokaryotic allelic tests was satisfactory as a demonstration of allelism. However, in the cases of‘ggz,and ni , the former slow growing and the latter leaky, growth response was somewhat ambiguous. The dikaryons were allowed to fruit and the progeny'were tested before any conclusions were reached. The two isolates of the morphological mutations gig.and ggm.would not fruit when mated. Allelism was assumed on the basis of colony morphology and mapping to the same position. figggggation of the mutatioggg The segregation frequencies presented in Table 2 represent, in all but one case, the sums of two separate first backcrosses to wild type. The exception is that of lyg, in which the total of 123 represents the sum of the two original and three additional generations of backcrosses to wild type. The segretation ratios approximate 1:1 for all but two mutations, ly§,and.§él. Germling viability is only about 50%'with both of these and the survivors are almost all wild type. Chromosomal abnormalities may be associated with these mutations. 31+ Linkage of the mutations: Linkage tests were done only with those mutations listed in Table 2 deemed useful for this study. Of these, all but four mapped to six of the seven linkage groups previously described (Ellingboe and Raper 1962) (Middleton 1964-). The four mutations (_a_d_6_, g2, gig and $13) which showed no linkage to any gene also showed no linkage with each other. Meiotic distances between loci, separated into values from Z-point and 3- or multi-point crosses, are presented in Table 3. ‘hm-point data are not available for most of the gene pairs, but were included in an effort to find some order in the erratic behaviour of a few mutations - specifically pg, u_l_, and M. The first two both show different cross- over frequencies with the same marker in different crosses, with u_l. (and 31:12) always appearing in excess of their wild type allele in multi-point crosses (261+ progeny among the 359 from such crosses were uracil auxotrOphs). Egg appeared to be linked to nil (67 of 207 = 32 units) but not to ar_q, which is linked to n_’j_. Nor did a_dfi or ad , flanking markers of pig, appear to be linked to _n_i_3_. The order of the markers shown in Figure l is based entirely on 3- point analysis of 3- or multi-point crosses. These analyses indicated unambiguous orders for almost all sets of three markers. One exception occurred in a cross of 311.; to 3d; a_rgg in which _r_1_i_l_ appeared to be to the left of fig. In a sample of 100 progeny, if p_i;l._ were to the left of 533;, there were 39 crossovers between 93; and El, 2 between a_rg_Z and _n_i_;L_, and no double crossovers. If 93; were to the right of 3353, there would have been 2 double crossovers and there would have been no single crossovers between pi; and Egg. The other exception was in a cross of m ul-b with Elli 9g agl in which progeny genotypes could not be 35 Table 3. Distances between meiotically linked genes. 2-point crosses 3- & multi-point crosses genes Bngie (cegtimggggns) simple (cegtggggggns) ad11, pab 79 23 141 3112 ni5, ui-b 77 39 412 #3111 niS-b, ul-b 111 hi n15, s 210 #715 n15, ad3 39 31 131 aniz niS-b, ad3 an 13 222 3811 n15, thy 196 6612 ni5, pdx-b 609 “8:11 ni5-b, pdx-b 197 u7iu ui-b, s 210 22 ui-b, ad3 216 20t5 ui-b, ad3-c n7 10 ui-b, n12 78 #6 9a 3111 ui-b, pdx-b 252 27 s, ad3 135 6 97 12 s, ad3-b 111 12 s, n12 #10 2316 s, thy 209 2017 s, leu 202 31:8 5, pdx-b 38h 25th ad3, ni2 281 19th ad3, niZ-c #8 13 ad3-b, n12 158 2513 Table 3 continued 36 2-point crosses 3- & multi-point crosses sample distance sample distance ,genes size (centimorgans) size (centimoggans) ad3, thy 2k“ 26th ad3, leu #6 26 111 an ad3, pdx-b 22 27 511 uiiu n12, thy 369 712 n12, leu 105 16 n12, pdx-b 308 26th thy, leu 105 17 thy, pdx-b 7a 29 321 1915 leu, pdx-b 303 3817 rib, ad2 58 in rib, arg2 91h 22:5 rib, sui 96 23 rib, adi 125 “5:5 ad2, arg2 97 21 ad2, sui 59 29 ad2, nil 97 20 arg2, n11 197 2t1 arg2, adi 225 36th arg2, ad12 83 hi ni1, adi 106 37 100 u3 nii, ad12 185 #2111 cho, ad12 #5 #1 185 h9i9 37 Table 3 continued 2-point crosses 3- & multi—point crosses sample distance sample distance genes size (centimorgans) size (centimorgans)A argi, spi 17# 12i7 argi, ad# 316 1118 argi, adh-b 58 32 78 33 argi, min 82 22 217 9:2 argi, ad9 108 22 spi, ad# 157 19:3 spi, ad#-b 78 37 ad#, min 317 121# ad#, ad9 109 37 min, ad9 108 16 arg6, inoi 120 18113 arg6, pan #12 21:15 inoi, pan 160 511 are, ni3 #5 32 16# 2#i10 are, com 10# 3# n13, com 92 8 38 Figure 1. Linkage map of Schizgphyllum commune. The seven meiotic linkage groups correspond to seven independent mitotically segregating chromosomes. Solid arrows indicate genes discovered in this study, while outlined arrows represent previously studied genes. Spacing between adjacent markers is approximately to scale and based on meiotic linkage data from this study (Table 3) and previous publications (Ellingboe and Raper 1962), (Middleton 196#). 39 J1 4 Soc 2: one lllqllf 4 _> can 3.: amen. J1 4 < 4‘ < > mom EE :6 Em _wam «:3 an 0&0 Rx Lu: «Who awn a... >— . 2.5 Jam-alt 1 1111 1 1 1 4 1|. 5 XUQ :2 >5“ 2: mum m —: 2: mm = m 14 < < 4 4 ‘1 +|II| - 2x mom a and!” Zoe :3 _ #0 interpreted to indicate any single gene order, but rather several different inconsistent orders, depending upon which three markers were‘ being considered. The problem in this case seems to have been due to the erratic behaviour of’glgb; results from a cross identical but for the omission of‘gl:b_indicated a consistent gene order. Meiotic crossing-over frequencies in §, commune are highly variable and it is not surprising that, even with standard deviations taken into account, distances are rarely additive. There are several instances of markers recombining more frequently with adjacent than with more distal markers. Thus it is cautioned that while the gene order of Figure l is very likely correct, the distances listed in Table 3 are only approximations. Determination of the adenine sypthetic pgthway steps affected g! the adenine mutations: There are nine steps leading from ribose-S-phosphate to 5'-phosphoribosyl-5-aminoimidazole-#-carboxamide, two more to inosine- 5'-phosphate, which marks the branch point for synthesis of adenosine-Sl- phosphate and guanosine-5'-phosphate, and two more to adenosine-5'-phosphate. Xanthosine-5'-phosphate is the intermediate between inosine-5'-phosphate and guanosine-5'-phosphate. The adenine mutations were tested for growth response on adenine, inosine, xanthine, and 5-aminoimidazole-#-carboxamide (AIC). It was assumed that the mutants were enzymatically capable of adding the phosphate and ribose moieties to these intermediates. Xanthine was included as a check for the leakiness of the pathway - i.e. the organisms' ability to convert a similar substance to adenine indirectly by other biochemical :means. The mutants did not respond well to.AIC, even applied at 10'3M. But the data do provide a basis for the classification of some of the #1 mutations with a high degree of certainty and an indication for some of the others as to which part of the pathway they operate upon. None of the mutants responded positively to xanthine. Both gg#,and §g2_responded only to adenine (Table #) and are "pink" mutations, therefore known to code for the two polypeptides of adenylosuccinase, the enzyme for both the last synthetic step and the conversion of 5'-phosphoribosyl-5- aminoimidazole-#—(N-succino) carboxamide to 5'-phosphoribosyl—AIC. This dual role explains the partial restoration of growth achieved,by'agflland ad2 strains with inosine. The only other mutation which did not respond fully to inosine was aglg, aglg.must code for adenylosuccinate synthetase, the enzyme mediating the penultimate step. ggl, Egg, 225: and gde definitely responded to AIC and can be assigned to the first eight steps of the pathway. ag1,showed some response to AIC and may possibly also be in this group. Growth of'ggll,and EQZDstrains was apparently supported only by inosine and adenine, so these can be each assigned one of the two steps between 5'-phosphoribosyl_AIC and inosine-5'-phosphate. The responses of‘§g§.and ag§_to AIC were uncertain. §Dthesis of common-Jig diploids: Nineteen different common-fl heterokaryons were set up in an attempt to obtain variously marked diploids (Table 5). Only four produced rapidly growing sectors (Figure 2), and these four did so with different facility. Each was later found to be diploid. A diploid of heterokaryon A was isolated nine times from eighty plates. One diploid was isolated from three hundred plates of heterokaryon C, and two diploids were isolated from 160 plates of heterokaryon K. Heterokaryon P produced many sectors (about thirty on eighty plates) but only two were analysed. Both proved to be diploid. Those heterokaryons from which no diploids arose were grown on 150 to 800 #2 Table #. Response of fig mutations to intermediates of the purine synthetic pathway. Growth response to intermediates mutation 3:22:23;26232018- inosine xanthine adenine adi +* + - + ad2 + + - + ad3 i + - + ad# - i - + adS-b + + - + ad6 7 + - + ad? - i - i ad8 7 + - + ad9 - i - + adiO + + - + ad11 - + _ + ad12 - - _ + a 43 Table 5. Genotypes of forced common-Ag heterokaryons and diploids. ‘ -_’ Name Genotype of heterokaryon Diploid obtained A A#1 B#1 s + + + a 2 es 353' EFT + ni2 ad2 sui arg y B A#2 B#2 s + + + arg2 no IE2 3E2 + n12 ad2 su1 arg2 A#1 B#2 + + arg6 inol + hi} 0 I171 55—2 57123111 arg2 + + pan 4- yes b A#1 + B#1 + arg2 + D + 151 ad5-b EET sui arg2 ni3 no b A#1 + B#2 + ar 2 + E + IE? adS-b B#2 su1 arg2 n13 no F A#1 B#2 n 2 + + ad2 sui ar 2 no IE? ‘852 + leu pdx-b + + arg2 G A#1 ad -b B#2 1, sui arg2 + n13 + + 8E2 s + arg2 pan + ad6 no A#1 ad -b B#2 .i sui ar 2 + ni3 + H + B#2 s + arg2 pan + ‘538 no A#1 ad -b B#2 + ar 2 + + 11 + WWW “0 A#1 ad -b B#1 + ar 2 + + 12 1 + 53? sul arg2 n15 aro no b A#1 B#1 + + + ar 2 J + Ifii 'EET n12 rib sui arg no A#1 B#1 ad# + ar 6 + n “WWW—$7.3? M- L A# B#1 g;g_ + ar 2 Egg + + no EET + sui arg + n53 aro M ad11 A#2 B#1 + + + ar 2 no + IE2 B#1 s n52 sui arg b A#1 B#2 + ar 2 + + 1 2 sui arg2 n13 aro no i“ a Table 5 continued Name Genotype of heterokaryon Diploid obtained A#1 adfi-b B#2 1 sui ar2 n1} 4- O + 8752 s + arg2 + ad1U no A#1 B# -b + + ad + + P m an; t—%—- m ass—=2 sac-2??” m V” A#1 B#2 g + arg2 pan + + ad6 Q m 8172 + sui arg2 + n13 are + no A#1 B#2 g + arg2 an + + ad8 B m 5732 + sui arg2 + n13 are + no #5 Figure 2. Diploids of Schizophyllum commune. A. Sector arising on twelve-day-old plate of common-Ag heterokaryon A on minimal medium plus arginine. B. Sectors arising on fourteen-day-old plate of common-Ag heterokaryon P on minimal medium. #6 plates each. The proclivity to diploidize is apparently a highly variable property among strains. verification of diploidy py mating to wild type haploid: Tables 6 thru 9 show the frequency of recovery of auxotrophic markers and of mating type factors in crosses of the four commonqg§,diploids with wild type haploids. Two different expected marker frequencies are listed in these tables. The second columns list the marker frequencies predicted on the basis of trisomic segregation of chromosomes and no subsequent loss of any disomic chromosomes from the progeny. The third columns list predicted frequencies based in trisomic segregation followed by total haploidization of all progeny. The feurth and fifth columns contain marker frequencies observed originally upon colony maturation, and subsequently after total haploidization of the aneuploid progeny, respectively. Only those progeny'with irregular growth pattern were considered probable aneuploids. These progeny were isolated and allowed to sector. Hyphal tips of a single sector were isolated and allowed to sector again. The process was continued until stable growth was obtained, at which point the progeny were considered haploid. The data from Tables 6 thru 9 show that the original observed frequencies of each marker were almost always between the values expected from no loss of chromosomes and those expected from total haploidization-- usually closer to the latter. This indicates either poor germination of aneuploids or instability and extensive loss of disomic chromosomes among the aneuploid progeny; The former is suggested by spore germination j percentages of about 75% from diploid.x haploid matings. The latter is suggested by the tremendous initial variation in growth rate of the germlings, a difference which tended to disappear with time. #7 Table 6. Recovery of markezs £3 216 progeny of a cross A 1 s + + + prgz Of Diploid A'5 (A31 EH? + n12 ad2 sui arg2 to wild type A#2 B#2 haploid. Marker Frequency expected with tri- somic segg-egation and obperveL no hap- total hap— after hap- Marker loidization loidization originally loidization A#1 99 132 109 1#0 11111 “2' 66 - 113 A#2 33 66 #1 57 Ax 18 19 B#1 108 1## 123 1#1 B#1 B#2 72 - 50 B#2 36 72 #3 75 ad2 36 72 #1 69 arg2" 72 72 38 56 1112 36 72 54 98 s 36 72 3# 67 * either A#1 or A#2 may occasionally be replaced by g; *1! those progeny bearing arg2 coupled with sui are here scored as arg2 #8 Table 7. Recovery of markers in i7# progeny of a cross of Diploid A-8 (“1-55— EL” 1% W) to wild type A#2 B#2? haploid. Marker Frequency expected with tri— gomic segregation and observed no hap— total hap- after hap- Marker loidization loidization originally loidization A#i 77.5 103.3 90 102 A#1 192' 51.7 - 32 auz 25.8 51.7 33 53 Ax 19 19 B#1 87 116 95 121 B#1 B#2 58 - #3 B#2 29 58 36 53 ad2 29. 58 #1 55 arg2" 58 58 #2 #8 ni2 29 58 #8 67 s 29 58 35 51 * either A#1 or A#2 may occasionally be replaced by_ those progeny bearing arg2 coupled with sui are here scored as p_g2+ 49 Table 8. Recovery of markers in 125 progeny of a cross of Diploid C: A#1 B#2 + + apgz ang6 inoi + ni (IET'EE2 ad2 sui arg2 + + pan'—:1) to wild type 52;,p3; haploid. Marker Frequency expected with tri- somic segregation and observed no hap- total hap- after hap- Marker loidization loidization originally loidization A#1 56 7#.7 67 72 A#1 Anz' 37.3 — 18 A#2 18.7 37.3 27 38 AI 13 15 B#2 62.5 83.3 71 87 B#2 B#1 #1.? - 3o B#1 20.8 #1.? 2# 38 ad2 20.8 #1.? 32 38 sui'” #1.7 36 arg2** #1.? 32 ., 55.5 69.# 3# arg6 #1.? 22 inoi 20.8 #1.? 16 21 n13 20.8 #1.? #2 51 pan 20.8 #1.? 33 ## either A#1 or 53;,may occasionally be replac by g; as these markers cannot be counted unless the progeny are totally haploidized. Thus values offset between lines in the second, third, and fourth columns represent total number of arginine auxotrophs 50 Table 9. Recovery of markers in 79 progeny of a cross of Diploid 1°...1:b # (KAT-18b, 7+ + Mribfizfififi) to wild type A#2 B#1 haploid. Marker Frequency czpected with tri- somic segregation and observed no hap— total hep. after hap— Marker loidization loidization originally loidization A#1 3# #5.3 #0 #6 11111 2112' 22.7 - 10 A#2 11.3 22.7 18 22 Ax 11 11 an? 39.5 52.7 #3 55 B47 B#1 26.3 - 17 B#1 13.2 26.3 19 2# adh" 26.3 28 ad9" 26.3 526?3 45 25 arg6 13.2 26:3 9 13 n13 13.2 26.3 22 36 pan 13.2 26.3 25 nu Pal-b 13.2 26.3 18 27 rib 13.2 26.3 20 29 s 13.2 26.3 19 23 either §#1_or A#2 may occasionally be replaced by __, we 1 these markers cannot be counted unless the progeny are totally haploidized. Thus values offset between lines in the second, third, and fourth columns represent total number of adenine auxotrophs 51 The observed marker frequencies upon total haploidization usually matched the predicted values well, although there appears to be selection against progeny carrying arg6 or incl (Tables 8 and 9). It was not possible to predict the frequency of recombinant g mating type factors. Thus their numbers were merely subtracted from the total and the predicted numbers of the parental A factors were based upon this reduced total. Progeny which were compatible with both parental and recombinant A factors were scored as disomic and containing the two parental A factors. However, such progeny, having completely haploidized, occasionally proved to have a recombinant A factor (Tables 6. and 8). 3 They were thus originally disomic with one of the A factors being recombinant. It was considered undesirable to perform heterokaryotic allelic tests for aneuploid, and therefore unstable, progeny. It should therefore be noted that Eng, arg2, and arg6 in Table 8 and Lag and a__d2 in Table 9 were not distinguished and scored except in haploidized progeny. erg and arg6 tester strains were used in heterokaryotic allelic tests on the progeny of Diploid C x haploid (Table 8). Arginine prototrophic progeny were tested ' at the same time with the arg2 tester to distinguish those truly wild type from those bearing E with arg2. Crossing-over in matings of diploids x wild typg haploids: The flli'equency of crossing-over in suitably marked regions in the diploid x 1'1apll.oid crosses provided support for two assumptions made about these maftings, namely, that the three homologous chromosomes participate 991.1211le, and that intimate pairing over any chromosome region involves any two of the three at random. Expected numbers of crossover genotypes “Ere generated when these assumptions were considered along with normal 52 meiotic distance between two genes and the fraction of crossovers between these genes which would actually be detectable in fully haploidized progeny (Tables 10 thru 13). Only two-thirds of crossovers between:g factors and between markers in coupling were detectable since one-third of the crossovers would be between chromatids with identical A_factors or identical wild type alleles of the coupled.markers. Only one-sixth of crossovers between markers in repulsion were detectable since only the doubly auxotrOphic crossover product is distinguishable from.the three parental genotypes. Crossing-over between pg2.and gpl,could only be detected one-sixth of the time in Diploids A—5 and A-8, but two-thirds of the time in Diploid C in which splgwith ppg§_was distinguished from wild type. There was a fairly good match between eXpected and observed frequencies in all but one instance, and the assumptions therefore seem reasonable. Crosses to produce compatible, common-A, and cannon-2 diploids: Crosses were made between each of the commonqg§_diploids and apprOpriate haploids with most of the same markers in an attempt to synthesize a series of commonqé, commonfifi, and compatible diploids with the same, or similar, arrangements of the mutations as were present in the commonqgfi diploids. The strains which were crossed to make the haploids fer these crosses were either the very same ones that were incorporated into the common5§§_diploids or else siblings marked identically but for mating type. The series of diploids should then provide a basis for determining the effect of the incompatibility factors in the diploid on the mechanism(s) of somatic recombination. The crosses and the expected frequency of the desired diploids among the progeny are given in Tables l#, 15 and 16. The expected frequency of 53 Table 10. Expected and observed numbers of crossovers among 216 progeny of a cross of Diploid A-5 to wild type_haploid. Fraction of Number of crossovers crossover! Marked Meiotic detectable region length in haploids expected observed 1.1 .. a, 16* 2/3 23 19 ad2 .. sui 21 1/6 8 10 n12 - s 2316 1/6 812 1# Table 11. Expected and observed numbers of crossovers among 17# progeny of a cross of Diploid A—8 to wild type haploid. Fraction of Number of crossovers crossovers Marked Meiotic detectable region length in haploids expected observed Aw - As 16* 2/3 19 19 ad2 - su1 21 1/6 6 7 ni2 - s 2316 1/6 712 11 ‘ highly variable 5# Table 12. Expected and observed numbers of crossovers among 125 progeny of a cross of Diploid C to wild type haploid. Fraction of Number of crossovers crossovers Marked ' Meiotic detectable region length in haploids expected observed Ax - A. 16* 2/3 13 15 ad2 - sui 21 2/3 17 13 arg6 - inoi 18113 2/3 15111 1? incl - pan 511 1/6 1 # arg6 - pan 21115 1/6 #13 Table 13. Expected and observed numbers of crossovers among 79 progeny of a cross of Diploid P-i to wild type haploid. ‘J‘ I {I Fraction of Number of crossovers crossovers Marked Meiotic detectable region length in haploids expected observed Act - As 16' 2/3 8 11 s — pdx-b 251# 2/3 1312 28 aufl# - ad9 37 1/6 5 6 55rg6 — pan 21115 1/6 312 3 . highly variable 55 .mw .w _m osHusao + HeadoHs no scream H H H H H H H H weHeeHanoe neeHeeeemHeeee osouosoaso no noupoohm Hmhm w m m w. m. m. m. 3033 masons—co e .Hou H canopHsu mooHpeastuooo cacaosoaSo ho noauomhm w 1. 1%.. HI. HI. i f WWW fl god—«o muses—Hoe 695qu Em m m a m w m w eHeHeHe «.858 e to H H H H H H H H baneeHsu neeHeesewHaeee osoeoEOASo no noHpomam + + + who + + NH: + H .I H .+. ... $3 4.1.. mm 1% eHeHeHH. 18.80 85.8 memm m m w m m m m eHeHmHe 623886 e .Hec H H H H H H H H oHofiHse 36388886 osouosowno no :oHuooam m m. m m We; mwelm mm mm 3638 33838 8.225 M H H mmee Ham «88 NH: e N N: x mmoao H .H .H mam + + was + Hum «mm + + + N no Ham Noe + m Hem «:4 anemone HH> H> > >H HHH HH H Hence no aoauomhm nacho owexsaq .asHeoa osHueoHou no canoHp anemone no hoaosvoau eouooawo use «honoswmam copoemmo .523 as Remote 23.5 8.338 .3393 a n4 eHeHHHHa 3.88.8 co 896 .1: e33 56 H ooHsHmao + HoaHsHs no sproam ml. m _w H m. H H H wsprHahoo mnoHpoaouHuooo oaomosohno no :oHpooam BR m m m w e m m 332. .9888 e 8. H H H H H H H H capopHsa esoHaoasuHcaoo oeomoeoaso mo :oHuooam w m .1 mmm :MmHmswm+ne m. WMMMIwmmlmwm _m _mmm._mw« oHoHaHe mucosaoo ooaHmoa E m. m m m w m m 3.3:. «.88.. e .8. H H H H H H H H eHoeeHse eeoHpesemHeeoe osomoeoaso no :oHuooam s on who mu :m .I I.” a. w . .1 m T .M m .w. a. PM.“ PM.“ 2...... 1...... .28.. meow m m m. .m _m .m _m eHeHaHe eHnHeeaaee u see H H H H H H H H ereeHze eeoHeessmHeeee osomosoaso mo soHuomam m H m“: own HmoH 0+.He m. wwmw1memwo m. Mam mw« oHOHoHo oHpHumaeoo coaHmoa mHo + HooH page H. + New H. H Nc< macho mm: + HooH owed H. “Hm. MHK + coax + + + mam Hod anemone HH> H> > >H HHH HH H Home» mo ooHpoeam ozone omoonq .ssHooa opHpooHoe so oHner anemone no honosooau cocooowo one .mwsosooau eouooawo aHosn use haemoao oHoHoHe oohHmoe .oHoHaos x o eHoaaHn mdlsoaaoo co macho .nH oHpoa 57 Bum. w. M m. N .m- H H aHHHooa HmaHHHHs so 539% H H H H H H wsHHpHshoo usoHueasmHuaoo oaomoaoaso mo :oHuoeam E m m m. m n m m. eHeHeHe .9858 e .Hoe H H .d H H oHnepHsa nooHpeasmHusoo . o ., . oaoeosoaso no soHpoeam a who a .I WWI mWIwHII mwfluwmm .orwl a...“ M whom mm 3033 muwoaaoo 3.239 E m m. m. .n. m... M m 33.33 fllsoseoo -e pom H H H H H H H H manmpHam mnovahouHunoo . oaoeoaoaso mo :oHooohm o e a .I mml. we + mml. 91+ m mmm .wmm oHoHoHe duoossoo eoaHmon mane .n. m. m. .m .m .m .n. eHeHeHe 83858. e .Hee H H H H H H H H oHnmqum mmovahsuHmcoo oeomosoaso no soHpomam : mam o a PHI mmmlwfll i wwl o-.. M WWW. mm. eHeHeHe 023858 82.8 one . e 8 13.1.18 .8 e. . HI. 8.... x euopo + e oHa + + a Hu< em + + on a an H34 anemone HH> Hs s 5 HHH HH H Hope» we soHuooam ozone oweonH [1" l ' .ssHoos osHpooHeu no oHnoH> hsowoaa Ho hososdoau oouooawo one .hooosdoam oopmmnwo aHonp one anemone eHoHoHo oosHmoe .eHOHoen x Hum eHoHoHo_Mflrooeaoo no mocha .oH oHnea 58 of the desired diploid was calculated as the product of the expected frequency of appropriate marker configurations for each linkage group without selection. These figures were then used to determine the number of spores to be plated to have a realistic chance of isolating the desired diploid progeny. The expected frequency of progeny able to grow on a particular selective medium was calculated in the same way. The predicted and observed numbers of both progeny able to grow on their selective medium.and those among them which were the desired diploids are summarized in Table 17. The lower than "expected" numbers of progeny able to grow on their selective medium.is likely due in large part to a lower than ”expected" incidence of disomy, as suggested by the crosses of these diploids to wild type haploids. Progeny of.A-5 and those of C that grew on minimal medium plus arginine were screened for inability to grow on minimal medium. .All the progeny were observed for stable growth, acceptable morphology, and were tested for mating type. This lowered the number of potential desired diploids to 6 from.A-5, # from C, and 7 from P—l. All these were mated to wild type haploids or, if compatible, allowed to fruit. These tests provided a basis for elimination of most of the presumptive diploids. The recovery of’desired strains is listed in Table 17. The one possible commonqé,progeny'diploid from.A-5 would not cross with haploids and exhibited deteriorating morphology. Characterization of dagghter diploids: Spontaneous fruiting of the diploids was encouraged or matings were made to wild type haploids, and the progeny were screened for markers for which the diploid should have been heterozygous. Nuclei were stained and counted to distinguish compatible diploids from "accidental" dikaryons. The results of the 59 m N Em N n u m ummw o m u m mmmm ooo.oH Hum coon o coca mu< eonasn hopes: :oHuomhm .mno doueeoxo mcHOHQHU OHDHuanaoo o H mwmm. we as nmm o H mmmm. 33H ooa .mw «H H emwm :OH ooe .mw hopes: hopes: :oHpoehh popes: hopes: :oHuoehm .mp0 oopoonxo .uno dopoooxo uoHOHqHH. Wflnoaeeo 53.008 33033 no no diocesan seem on OHDe hsOMOLm oopeHo copes mononu eHOHnHo Hence .ndHOHnun N ueHOHoHu_Mflrooeaoo no gouache seem uoHoHoHe oHnHaeneoo one mrooeaoo qflrnoaaoo no moHooosooAu dophomno one cocoonuu .nH oHpoa 6O latter test correlated with the morphology of the strain. Compatible diploids have a rather flat, uneven appearance on migration complete or completely supplemented minimal media, with fruiting bodies scattered singly or in small groups (Figure 3), whereas dikaryons have a characteristic knotty appearance and large groups of fruiting bodies. 0n old plates the difference disappears. The parental configurations of linked genes in the cultures derived from the diploid x haploid crosses were determined from the segregation patterns in selfing or crosses of the cultures with wild type haploids. From the data presented in Tables 19, 21, 23, and 25, it can be argued that there is approximately the same frequency of recombination in fruiting of compatible diploids as in compatible dikaryons. Diploid 0-21}: 0-31} was derived from a cross of Diploid C with a homokaryon (Table 15). No cells with two nuclei were visible under the microscOpe. The colony morphology was typical of a compatible diploid. It fruited spontaneously. Eighty progeny were obtained and the recovery of markers was determined (Table 18). One of the 5 factors was recombinant and thus had one subunit in common with £31.A11 auxotrophic mutations present in Diploid C were recovered. Three - £12, sui, and ni - were recovered in approximately 1:1 segregation with the wild type alleles. The arrangements of linked markers and frequency of crossing-over between them in 0-314, are given in Table 19. There appeared to be extreme selection against a_rgé and in_ol_, two genes that were present in coupling. Selection against these markers - a_rgé in particular - was observed repeatedly in this study and is the probable cause of the great excess of pan, a gene linked to arg6 and incl but in repulsion to them. 61 Figure 3. Compatible diploid 0-31} on minimal medium plus arginine. Arrows indicate fruiting bodies. 62 Table 18. Recovery of segregating markers among 80 progeny from spontaneous fruiting of compatible Diploid C—3h, Marker Number of progeny Ah1 37 Aoup 5 “3 B41 41 B42 39 ad2 #8 sul #5 arg6 u incl 1 pan 76 n13 35 Table 19. Crossover frequency among 80 progeny from spontaneous fruiting of compatible Diploid C-3n. Parental Marked Meiotic arrangement‘ Crossovers region length cis trans expected observed ad2,sui 21 X 17 19 arg6,inol 18113 X intio 3 arg6,pan 21i15 X 17i12 6 inoi,pan 5&1 X til 3 63 Diploid 0.144: 0-141» was derived from a cross of Diploid C with a homokaryon (Table 15). The colony morphology was typical of a compatible diploid. Only about 2% of the cells with a visible nucleus had two nuclei. Seventy-eight progeny were obtained from spontaneous fruiting of C-M». The marker frequency among the progeny indicated a 1:1 segregation with the wild type allele in each case (Table 20). Interestingly, however, twenty-eight of the progeny were initially slow growing, requiring two extra days for the germlings to become large enough to transfer. All twenty-eight bore arg6 and in_o_I_L_. This showed that selection was operating against these alleles in 0-144 as well as in 0-31}. The arrangements and recombination frequencies of linked markers in CM are presented in Table 21. Diploid P-3: P—3 was derived from a cross of Diploid P-l with a homokaryon (Table 16). The colony morphology was typical of a compatible diploid and only a few percent of the cells appeared to have two nuclei. It fruited spontaneously. Ninety progeny were obtained and tested for marker frequency (Table 22). One of the _A; factors was recombinant, having a subunit in common with {$1. All markers present in Diploid P—l were recovered and segregated approximately 1:1 with their wild type alleles, except nil for which there was a marked excess of Qi. The arrangements and recombination frequencies of linked markers in P—3 are presented in Table 23. Culture P-21: P-Zl was derived from a cross of Diploid P-l with a homokaryon (Table 16). The colony morphologt had the knotty texture typical of a dikaryon. It had two nuclei in about no% of those cells in which nuclei were stained. Only a small percent of the cells took up stain, indicating that the 40% figure might be due to unstained second 6h Table 20. Recovery of segregating markers among 78 progeny from spontaneous fruiting of compatible Diploid c-u4. Marker Number of progeny M1 3‘1 A42 38 Ax 6 B#1 39 B#2 39 ad2 36 sui 35 arg6 38 incl 3“ pan #2 n13 “5 Table 21. Crossover frequency among 78 progeny from spontaneous fruiting of compatible Diploid C-flh. Parental Marked Meiotic arrangement Crossovers region length cis trans expected observed Act,Ap 16* 12 6 ad2,su1 21 X 16 19 arg6, 11101 18.113 x 111110 12 arg6,pan 21115 X 16111 12 in01,pan 5:1 X #11 2 * highly variable 65 Table 22. Recovery of segregating markers among 90 progeny from spontaneous fruiting of compatible Diploid P-3. Marker Number of prageny Ahi 45 Aolgs “5 B#1 #8 Ba? #2 s 49 pdx-b #3 rib 35 ad“ 42 ad9 39 arg6 #3 pan 52 mi} 15 Table 23. Crossover frequency among 90 progeny from spontaneous fruiting of compatible Diploid P—B. Parental Marked Meiotic arrangement Crossovers region length cis trans expected observed s,pdx-b 2511 x 231“ 30 M“.8119 37 X 33 25 arg6,pan 21115 X 19t1h 11 66 nuclei. The 40% is still much' higher than the percentages obtained for C-34, C-44, and P—3, and too high to be due to cells in the process of division or random cellular mistakes. The culture fruited and progeny were tested for segregation of markers (Table 24). All markers present in P-l were recovered and all segregated approximately 1:1 with their wild type alleles. The arrangements and recombination frequencies of linked markers in P-Zl are presented in Table 25. Culture P-l9: P-l9 was derived from a cross of Diploid P-l with a homokaryon (Table 16). It was originally tested against stocks of mating types A_41 2142, _Al %, A_41_ 132;, and _A_4_2_ EL. It dikaryotized all but the first and was thus scored as common-E (B_4_Z). However, haploid sectors from P-19 all proved to contain E41, although this factor had not been thought to be present in P—l9. Several tests were therefore initiated to determine the nature and mating behaviour of the P-19 culture. P—l9 had highly irregular morphology and was never observed to fruit spontaneously. MicroscOpic investigation revealed many clamp connections and at least equal numbers of pseudo-clamps, usually septate, and many with nuclei trapped within the pseudo-clamps. Many cells contained two nuclei. This description is similar to that of a common-_B_ heterokaryon (Raper 1966). The observed inability of P-l9 to dikaryotize an A_4_Z B4_Z haploid was reconfirmed in matings with. another strain of the same mating type. P-19 also proved unable to dikarytize an fl B_41 strain. It did, however, successfully dikaryotize M .B_l_ and A_4}_ §4_2 haploids, the former with no difficulty, the latter rather more slowly (it required six weeks to fruit). From all these observations it was concluded that P-l9 was partially dikaryotic but had some irregularity in the function’iof its E mating type ,.....' 3‘43139 17.15.; .IIIIMl 67 Table 24. Recovery of segregating markers among 89 progeny from spontaneous fruiting of compatible Culture P-21. 4 Marker Number of progeny A41 38 A42 40 Ax 11 B47 46 1341 43 s 42 pdx-b 48 rib 45 ad4 46 ad9 “3 arg6 “3 pan 44 n13 47 Table 25. Crossover frequency among 89 progeny from spontaneous fruiting of compatible Culture P—21. Parental Marked Meiotic arrangement Crossovers region length cis trans expected observed Ad,Ap 16' 14 11 s,pdx-b 2514 X 22:4 21 adhad9 37 x 33 22 arg6,pan 21115 X 15111 17 . highly variable 68 factors. The E factor was apparently unable to participate in matings when in the presence of the Q41 factor, but it fimctioned normally when alone in the haploid sectors from P-l9. The 3 factor was never found in the haploid sectors. Mating type tests of the progeny of P-19 crossed with either an M _B_]_._ or an A_41_ B_4_2_ haploid showed that those progeny, and only those progeny, which had inherited the B_4_Z factor of P-l9 had also inherited anomalous mating behaviour. Specifically, they could dikaryotize Al §4_l_._, A_2 132, and (for those progeny £41) £142 El; and one £41 .84]; testers. With another particular M ill-L1 strain, it gave the "barrage” reaction typical of common-J} interactions. This latter A42 RE. strain was a great-great- grandparent of P-19, but had contributed none of P-l9's mating type factors. The £12 at haploid parent of P-19, which had contributed its _B_4_;l._ factor to P-l9, was mated to both $1 841 haploids which had gone into P-l, the diploid parent of P-l9. One of these matings was normal. The other was neither normal nor ”barrage", but produced instead a weak, slow growing dikaryon. Substitution of control strains with the same mating type for either ancestor of P-l9 in this mating restored normal dikaryotization. The other data relevant to understanding of P-l9 are the marker frequencies among the progeny from crosses of P-19 with $42 13;, fl B_4§_, and £42 £1. The last of these was a mating type test cross originally done on P-19 which produced a fruiting body. In the cross with £41 I}; (Table 26) the All; factor from P-19 and the £41 factor from the haploid segregated approximately 1:1, but the £42 factor of P-l9 was absent from the progeny. Recombinant 5 factors, which would have been predicted as a 69 Table 26. Recovery of segregating markers among 80 progeny from a mating of compatible Culture P-19 to wild type 541 B; haploid. Marker Number of progeny A41 35 A47 45 A42 0 Ax 0 B47 60* BI 37* B41 0 s 8 pdx-b 6 rib 41 ad4 2 ad9 43 pan 41 ni3 O 1!» 17 progeny were disomic for the B chromosome and possessed both Eiz.and.§1- i h I‘vu. ".l "- 70 result of crossing-over between & and A_4_Z or A_42_ were also absent. There was an excess of‘§41_(from P-l9) relative to §1’(from the haploid). Three markers - gig, Egg, and pg_.- were recovered at 1:1 ratios with their wild type alleles while the other markers - ggfl (in repulsion with a_d2), _s_ and Eggs; (in coupling) and 931 - were absent or rare. In the cross of P-l9 with 541_§42 (Table 27) most biochemical markers were recovered at near 1:1 ratios with their wild type alleles. The sole exception was git which was totally absent from the progeny. The A_4_2_ factor from P-19 segregated 1:1 with.§41'even though there were supposedly two doses of £1; entering the cross. Progeny of the cross of P-l9 with A42 8; were not fully analysed (no mating types, no distinction between ad4,and‘§g2). Nineteen of the progeny'scored as auxotrophic for all markers, even for‘ggg§_which,'by its absence from the progeny of the other P-19 crosses and haploid sectors, was thought not to be present in P419. Probably'these progeny were just not viable on sub-Optimal media of any kind. They'have been excluded from the data of Table 28. Two markers - pij_and.g - were rare in this sample. The latter marker is particularly interesting as §_is linked to $112 which was not rare. This would indicate that _s_ and 31222 were present in repulsion. Data from the other P-19 crosses and the haploid sectors indicated that §_and_pg§:b_were in coupling. None of the progeny from these three crosses involving P—l9 ever sectored, except seventeen progeny of the cross to £42.81, This group proved to be disomic for only the chromosome containing the'§_factor. The varying frequencies of the various markers in the progeny of the three crosses of P-l9 make determination of crossover frequenoy difficult. Therefore the arrangements of linked markers in P-19 have been determined by analysis of its haploid sectors. 71 Table 27. Recovery of segregating markers among 122 progeny from a mating of compatible Culture P—19 to wild type A41 B42 haploid. Marker Number of progeny A41 58 A42 52 Ax 12 B47 51 B42 71 B41 0 8 55 PdX-b 59 rib 69 ad4 O ad9 62 pan 53 n13 53 Table 28. Recovery of segregating markers among 73 partially analysed progeny from a mating of compatible Culture P-19 to wild type A42‘§;_haploid. Marker Number of progeny s 1 pdx-b 16 rib 16 ad4 and/or ad9 37 pan 8 n13 1 72 Somatic recombination: 3222 A Diploids: Arginine prototrophs were isolated from common-Ag diploids A-5 and A-8 as rapidly growing sectors. (See Table 5 for genotype of Diploid A.) Haploid sectors which arise by mitotic haploidization should show a requirement for either gig or 3 since these heterozygous linked markers were present in repulsion. Sectors which arose as a result of mitotic crossing-over between £31 and its centromere, followed by reassortment to give homozygosity for s_u1_, should be prototrophic for _n_i_2_ and g. Crossovers between 9% and the centromere would lead to homozygosity for gd_2_ also. Crossovers between gig and s_u_.1_ would not lead to homozygosity for ad2. About one plate in four eventually produced a sector. Tables 29 and 30 show that, of 123 sectors purified from these two diploids, only 4 from A-5 had p_i_2_ or g and were therefore considered to be haploid. These data also show that the frequency of crossovers in the region centromere .- fi relative to the frequency in region ggg - §_11.l_ was greater in A-5 than in A-8. No sectors showed any instability which would have suggested aneuploidy, and none of the four haploids were both _rg._2_ and. g. Somatic recombination: Type C Diploids: Common—Ag Diploid C (see Table 5 for genotype) produced arginine prototrophic sectors more rarely than did the type A diploids - about one sector per 25 plates (Figure 4A,B). There was, as in A-5 and A-8, a high ratio of diploid to haploid recombinants (51:14, see Table 31), as judged by the recovery of non- selective markers heterozygous and in repulsion. Haploids bearing Egg and m were recovered only once. The one that was isolated was recovered from a sector of. very weak growth in the less sparse background. No crossovers between 5:36 and 313; or 213% and m were recovered, nor was there any instability that could have been associated with aneuploidy. 73 Table 29. Recombinant sectors isolated from commonggg Diploid A-5. GenOtype Number ad2 arg2 n12 s 30 + + + + 36 ad2 + + + 1 ad2 + n12 + 3 ad2 + + s Table 30. Recombinant sectors isolated from commongég Diploid A-8. Genotype Number ad2 arg2 n12 s 7 + + + + 46 ad2 + + + 74 Figure 4. Sectors arising from diploid and partially dikaryotic strains of Schizophyllum commune. A. Rapid-growing sector from nine-day-old growth of Diploid C on arginineless medium. B. Rapid-growing sector from twenty-day-old growth of Diploid C on arginineless medium. C-D. Thin morphological sectors from twenty- two—day-old growth of Diploid P-1 on completely supplemented minimal medium. E-F. Thick morphological sectors from fourteen-day-old growth of Culture P—19 on completely supplemented minimal medium. 75 11. 1t:- mu )ld Table 31. Recombinant sectors isolated from commonggg 76 Diploid C. Genotype Number ad2 arg2 arg6 inoi pan n13 8 + + + + + + 43 ad2 + + + + + 9 ad2 + + + pan + ad2 + + + pan n13 1 + arg2 arg6 incl + + Table 32. Recombinant sectors isolated from compatible Diploid 0-34. Genotype v Number A B ad2 arg2 arg6 incl pan n13 7 1g; 3% ad2 + + + + + 5 If; 42 + + + + + + 1 1-5 41 ad2 + + + pan + 2 1-5 41 ad2 + + + pan n13 1 41 41 ad2 + + + pan n13 2 41 42 ad2 + + + pan + 77 The compatible diploids C-34 and C-44 had an uneven type of morphology (Figure 3). Detection of rapidly growing sectors was very difficult on any but young plates. The twenty-four isolated (Tables 32 and 33) included only six haploids, (a percentage similar to that in Diploid C), all from C-34. Two of these haploids were secondary sectors from sectors that were presumably aneuploid. No crossovers in the Lrgé m; m linkage group were in evidence. _S_omatic recombination: Type P Diploids: It was originally supposed that haploid sectors from these diploids (see Table 5 for genotype) should be detectable as pink sectors due to presence of id}; or idfi (present in repulsion). However no such sectors ever arose from P-l when plated on yeast or migration complete medium. When grown on completely supplemented minimal medium, the diploid had denser morphologr with a more clearly defined growing edge. A few pink sectors were found on supplemented minimal medium. More importantly, many sectors appeared which were of different morphology i.e. thinner, uneven, and, if not actually growing more rapidly, at least expanding laterally at a faster rate (Figure 4C,D). When isolated, these usually proved to be aneuploid or haploid. Almost all the unstable and, therefore, presumably aneuploid sectors eventually produced stable colonies which were considered haploid. In some cases two or more different haploids from the same original aneuploid were isolated and these "haploid families", sometimes of three sector generations, were numbered to indicate the order in which they arose. It was found that all adj and a_d2 strains eventually turned pink on any medium, but at highly different rates, which accounted for the early difficulty in identifying pink sectors. Every allele in every type P Kid—7T. {E Table 33. Recombinant sectors isolated from compatible Diploid 0-44. 78 Genotype Number A B ad2 arg2 arg6 incl pan n13 41 42 2 “2 “1 ad2 + + + + + 41 42 4 “2 41 + + + + + + 79 diploid or culture was recovered (though often at frequencies far different from 50%), except the gulrector from P—l9. Forty sectors isolated from comm-Ag diploid P-l were haploid or ultimately produced haploids or haploid families. These are listed in Tables 34 and 35. Thirty-two of the sectors produced only one stable haploid (Table 34). Eight eventually produced families of stable subcultures including two or more genotypes (Table 35). Crossing-over had occurred in only one of the forty, and that crossover was between arg6 and 33911. (There is the possibility that the sector is stably disomic for the chromosome on which Egg and M are located.) Compatible Diploid P-3 sectored less often than its parent P—l. Twenty-six sectors were isolated which, upon subculturing, eventually produced stable haploid colonies (Tables 36 and 37). Some aneuploids were fairly stable. Total haploidization often took five or six weeks of continuous growth. Twenty—two of the sectors produced only one stable genotype (Table 36). Four sectors eventually produced stable subcultures of two genotypes (Table 37). There were no crossover types. Culture P-Zl sectored rarely compared to P-1 and P-3. Nineteen sectors eventually yielded haploids (Tables. 38 and 39). Fifteen of the sectors produced only one stable genotype (Table 38). Four sectors eventually produced stable subcultures of two genotypes (Table 39) . Four were the result of crossing-over. Tm crossovers were between pig; and a_rg_é (also possibly stably disomic for this chromosome), one was between §_ and m, and one was between $4 and a_dfi. Culture P-l9 grew very irregularly. Many thick clumps or well defined feathery arms were common. Eyen small morphological or pink sectors were easily detectable at the edge of such growth (Figure 4E,F). 80 Table 34. Recombinant sectors isolated from commonggg Diploid P-1. Genotype Number 8 pdx-b rib ad4 ad9 arg6 pan n13 5 + + + + ad9 + pan n13 4 + + rib + ad9 + pan n13 3 + + + ad4 + + pan n13 2 + + rib + ad9 + pan + 2 + + + ad4 + arg6 + n13 1 + + + + ad9 arg6 + n13 u s pdx-b + ad4 + + pan n13 2 s pdx-b rib + ad9 + pan n13 2 + + + + ad9 + pan + *1 + + + + ad9 + + n13 2 s pdx-b + + ad9 + pan n13 1 + + rib ad4 + arg6 + n13 1 s pdx-b + + ad9 arg6 + + 2 s pdx-b + + ad9 + pan + 32 Total mutant at each locus (of 32) 11 11 9 10 22 5 26 25 * crossover genotype 81 Table 35. Haploid families from recombinant sectors of common-AB Diploid P-l. Genotype Order 8 pdx-b rib ad4 ad9 arg6 pan n13 1 s pdx—b rib + ad9 + pan + 2 + + + + ad9 + pan + 1 + + rib ad4 + + pan ni3 2 + + + + ad9 + pan n13 is + + rib ad4 + + pan n13 1b + + + ad4 + + pan n13 2a + + rib + ad9 + pan n13 2b + + + + ad9 + pan, n13 1 + + rib ad4 + arg6 + n13 2 + + rib ad4 + + pan n13 1 s pdx-b + + ad9 + pan n13 2a 3 pdx—b + ad4 + + pan n13 2b + + rib ad4 + + pan n13 2c + + 4 ad4 + + pan n13 1 s pdx-b + + ad9 + pan n13 2 s pdx-b rib + ad9 + pan n13 1 + + rib + ad9 + pan ni3 2 + + + + ad9 + pan n13 3 s pdx—b rib + ad9 + pan n13 1 + + + ad4 + + pan n13 2 s pdx-b + + ad9 + pan n13 Total mutant at each locus 5 5 9 5 7 1 8 7 Of: 12 12 14 12 12 9 9 8 82 Table 36. Haploid sectors isolated from compatible Diploid P-3. Genotype Number A B s pdx-b rib ad4 ad9 arg6 pan n13 2 1—5 47 s + rib ad4 + + pan n13 1 42 41 s + + ad4 + + pan + 1 1-5 41 + pdx-b rib + ad9 + pan + 1 1-5 47 + pdx—b + ad4 + arg6 + + 1 42 41 s + + + ad9 arg6 + + 1 1-5 41 s + rib + ad9 + pan + 1 42 47 + pdx—b + ad4 + + pan + 1 1-5 47 s + + + ad9 arg6 + + 1 1-5 47 + pdx-b + + ad9 arg6 + + 1 1-5 41 + pdx-b + ad4 + arg6 + n13 1 1-5 41 s + + + ad9 arg6 + + 1 42 47 + pdx—b + ad4 + + pan n13 1 1-5 47 + pdx-b rib + ad9 + pan + 1 1-5 41 s + + ad4 + arg6 + + 1 42 47 + pdx—b rib + ad9 + pan n13 1 42 41 s + + ad4 + arg6 + + 1 1-5 41 + pdx-b rib ad4 + + pan + 1 1-5 47 s + + + ad9 + pan + 1 42 41 + pdx-b rib + ad9 arg6 + + 1 42 41 + pdx-b + + ad9 + pan + 1 1-5 47 s + + ad4 + + pan + 22 Total mutant at each locus (of 22) lggi§h fiéii: 11 11 8 11 11 9 13 5 83 Table 37. Haploid families from sectors of compatible Diploid P-3. -; E Genotype Order A B s pdx-b rib ad4 ad9 arg6 pan n13 1 42 41 s + rib ad4 + + pan + 2 42 41 s + + ad4 + + pan ni 3 1 42 47 s + + + ad9 + pan n13 2 42 47 + pdx-b + + ad9 + pan n13 1 1-5 41 8 + + + ad9 + pan + 2 1-5 47 + pdx—b + + ad9 + pan + 1 1-5 41 + pdx—b + ad4 + + pan n13 2 1-5 41 s + + ad4 + + pan n13 Total mutant at each locus 1'5‘2 “1‘3 4 3 1 2 2 o 4 3 42:2 47:2 0f: 4 5 7 7 5 4 4 4 4 5 84 Table 38. Haploid sectors isolated from compatible Culture P-21. Genotype Number A B s pdx-b rib ad4 ad9 arg6 pan n13 2 41 47 s pdx-b + ad4 + + pan + 1 41 47 s pdx-b + + ad9 arg6 + + *1 41 47 + + + + ad9 + + + 1 41 41 + + rib ad4 + arg6 + + 2 42 47 s de-b + + ad9 + Pan + 1 42 47 + + + ad4 + + pan + 1 42 41 + + + ad4 + arg6 + n13 1 41 41 + + rib ad4 + + pan n13 1 41 47 s pdx—b rib ad4 + arg6 + + 1 42 47 s pdx—b rib + ad9 + pan + 1 41 41 + + + ad4 + + pan n13 1 42 47 + + rib ad4 + + pan n13 1 42 41 + + rib ad4 + + ‘pan + 15 41:8 “1:5 Total mutant at each locus (of 15) 42:7 47:10 7 7 6 1° 5 4 11 4 * crossover genotype 85 Table 39. Haploid families from sectors of compatible Culture P-21. Genotype Order A B s pdx-b rib ad4 ad9 arg6 pan n13 1 42 47 s pdx-b rib + ad9 + pan + *2 42 47 + pdx-b rib + ad9 + pan + 1 41 47 + + + ad4 + arg6 + n13 2 42 47 s pdx-b rib ad4 + arg6 + + **1 42 41 s pdx-b rib ad4 ad9 + + n13 *2 42 41 s pdx-b rib ad4 ad9 + pan + 1 41 41 + + + ad4 + arg6 + n13 2 41 47 + + + ad4 + arg6 + n13 4 4 Total mutant at each locus 17%;; “$3 3 3 3 3 2 2 2 Of: 5 6 6 5 4 4 5 5 * crossover genotype ** two-crossover genotype 86 Forty-six sectors were recovered," thirty of which produced one stable haploid genotype (Table 40) and sixteen of which produced two or more stable haploid genotypes (Table 41 and Figure 5). Fifteen crossovers were evident - thirteen between _s_ and my and two between §_d_4 and £2. Frequency of somatic crossing-over: The C diploids produced few haploids, none the result of crossing-over. This made comparisons of crossover frequency impossible. The P diploids and cultures are compared in Table 42. There were four regions, .A_Il_ - A_l_, g - E22: adj-l - a_d2, and a_rg_é - p_a_n, in which crossovers were detectable, but each diploid or culture did not have all of these regions marked. Crossover frequencies were compared assuming that each suitably marked region common to the two strains compared represented an independent Opportunity for crossover. Numbers of crossovers per common Opportunity were then compared for each strain in every possible pair-wise combination. The significance of their crossover frequency differences were calculated by Contingency Ghiz. Significant differences are apparent between all combinations except P-1 and P-3, and also P-21 and P-l9. Mitotic mapping: Since it is apparent that crossovers are very rare in the cannon-AB and compatible diploids, these strains were used to identify independently assorting chromosomes. The compatible diploid was particularly valuable as all seven meiotic linkage groups were marked. Linked genes were used in calculations as single units, with the one crossover type from P-l omitted. Each gene or unit was compared with every other gene or unit, (in pairs, for frequency of recombination. The pairs were arranged in their two possible reciprocal allele combinations and the number of each combination was counted. The minority combinations were considered recombinant types. Recombination frequencies 87 Table 40. Haploid sectors isolated from compatible Culture P-19. Genotype Number A B pdx-b rib ad4 ad9 pan n13 1 41 41 pdx—b rib + ad9 + + 42 41 pdx-b rib ad4 + + + 1 41 41 + rib ad4 + + + 1 41 41 pdx-b rib ad4 + pan n13 2 41 41 + rib ad4 + pan n13 *1 42 41 pdx-b + + ad9 + + *1 41 41 + rib ad4 ad9 + n13 1 42 41 + + ad4 + pan + 1 42 41 + rib + ad9 pan + *1 42 41 + + ad4 ad9 + + *1 41 41 de-b rib ad4 + + n13 *1 42 41 pdx-b rib ad4 + + + 1 42 41 pdx-b rib + ad9 + + 1 42 41 pdx-b rib ad4 + + n13 2 41 41 + rib ad4 + pan + 2 42 41 + rib ad4 + + + 5 41 41 + rib ad4 + + n13 *1 42 41 pdx—b rib + ad9 pan + *1 41 41 pdx-b rib ad4 + + + 1 41 41 de-b rib ad4 + + + 88 Table 40 continued Genotype Number A B s pdx-b rib ad4 ad9 pan n13 1 42 41 s pdx—b + ad4 + + n13 i 42 41 + + rib ad4 + pan n13 1 42 41 + + + ad4 + + + Eu 0 Total mutant at each locus (of 30) 41:19 41:30 5 42:11 47:0 7 12 25 25 7 9 13 5 * crossover genotype 4 89 Table 41. Haploid families from sectors of compatible Culture P-19. Genotype Order A B s pdx-b rib ad4 ad9 pan n13 1 42 41 + + rib ad4 + + n13 Za 41 41 + + + ad4 + + n13 2b 41 41 + + rib ad4 + + n13 1 41 41 + + rib ad4 + + + *2 41 41 + pdx-b rib ad4 + + mi} 3 42 41 + + rib ad4 + + n13 1 42 41 + + rib ad4 + + + *2 42 41 + pdx-b rib ad4 + + + is 42 41 + + + ad4 + + n13 1b 42 41 + + rib + ad9 pan + 2a 42 41 + + rib + ad9 + + *2b 42 41 + pdx-b rib + ad9 + + *1 42 41 + pdx-b + + ad9 pan n13 *2 41 41 + pdx-b + + ad9 pan + *3 42 41 + pdx-b rib + ad9 pan n13 1a 42 41 s pdx-b rib ad4 + -+ + 1b 42 41 8 de-b rib ad4 + + n13 2 42 41 + + rib ad4 + + + 1 42 41 + + rib ad4 + + + 2 41 41 + + rib ad4 + + + 1 41 41 + + rib ad4 + + + *2 41 41 + pdx—b rib ad4 + + + *1 41 41 + pdx-b rib ad4 + pan + *2 41 41 + pdx—b rib ad4 + pan n13 1 41 41 s pdx-b rib ad4 + + n13 2 41 41 + + rib ad4 + + + 1 41 41 + + rib ad4 + + n13 2 41 41 + + rib ad4 + pan + 1 41 41 s pdx-b rib ad4 + + n13 2 42 41 s pdx-b rib ad4 + + n13 Table 41 continued 90 Genotype Order A B s pdx-b rib ad4 ad9 pan n13 1 42 41 + + rib ad4 + pan n13 2 42 41 + + rib ad4 + + n13 1 41 41 s pdx-b rib ad4 + + n13 2 41 41 + + rib ad4 + pan + *1a 41 41 + pdx-b rib ad4 + + + 1b 41 41 s pdx-b rib ad4 + + + 2 41 41 s pdx-b rib ad4 + pan + *1 42 41 + pdx-b + + ad9 pan n13 2 42 41 + + + + ad9 pan n13 4 4 6 Total mutant at each locus 1:11 1:1 42:10 47:0 5 13 15 14 3 8 12 0f: 21 16 24 24 19 17 17 21 24 * crossover genotype 91 Figure 5. Secondary sectoring of aneuploid isolates of culture P-19. A. Two sectors from an aneuploid plug on yeast medium. B. Irregular growth and sectors from hyphal tip isolates of an aneuploid sector on completely supplemented minimal medium. Sectors are indicated by arrows. 92 Table 42. Pair-wise comparison of number of crossovers in strains P-1LP-31 P-21L and P-19. Crossovers Strains Total Common in common 2 compared haploids regions regions Chi Significance 5:3 32 3 3 0.67 .3 - .5 5:21 13 3 i 5.44 .02 52:9 12 2 1; 11.37 .001 5:21 is 3 3 5.05 .02 5:29 ES 2 12 9.42 .01 - .001 £213 12 3 1% 2.79 .10 93 are listed in Table 43 together with their sample numbers. In haploid families where both "parental" and "recombinant" types were present, both were counted. For any pair of genes or gene units, a difference between ”parental" and ”recombinant" frequencies significantly different from that predicted by 1:1 segregation would.have been considered evidence that those genes or units were actually oyntenic and that recombination between them could only'have occurred by crossing-over. The recombination frequencies within P-1 and P-3 were considered together, and.among them the lowest value was that between (aggérpgn) and pi}, and even this value showed no significant deviation from the 1:1 ratio that was predicted by independent assortment of whole chromosomes (Chi2 = .10 - .05). Thus it appears that the meiotic linkage groups correspond to independent chromosomes, and that there are, therefore, at least seven chromosomes in Schizopgyllum.commune. Figure 1 illustrates the seven chromosomes with.markers separated approximately to scale according to their meiotic distances from.their nearest neighbours. 94 nlm UHOHQHQ named noted lone no. load «a. “and He. lama or. “and on. land as. one Acne as. land me. name me. lend me. Adm. no. oiuoe.o land as. Aamv an. “mud ca. name He. and Acme on. lame an. none on. soo.oanc Aamv we. lose on. mee.eoo .oea me. «one no. load on. lore He. lend no. load on. Away on. Away on. “one we. Arne mm. 1 1 1 1 1 Humv an. m 1 1 1 1 1 1 4 an n1xeo.u an noo.mwne moo.:ee m < unoxnea ozone ownquq Hun oHoHeHe "ostn nodes .eouosanonoo nH eoauHH one eHopop oHoemm .n1m coo H1m oeHoHoHe oH oesonw omoerH oHpoHos monsoon eoHonosoonn eoHuoeraoeen oHpesom .n: oHnoB DISCUSSION The mutations induced by NC were numerous and proved to be very useful. The exact mode of action of the mutagen is unknown, but it is capable of acting as an alkylating agent that can induce chromosome breaks and aberrations, and also as a very potent point mutagen (Hollaender 1971). The former occurs at alkaline pH, the latter optimally at about pH 6. LNG was used at pH 7, so most of the mutations are likely point mutations. Chromosomal aberrations of some kind would probably'account for the two mutations that have a high degree of lethality, and perhaps deletions over at least two cistrons could explain some of the auxotrophs with unknown requirement. The total linkage data - mitotic and meiotic - are very satisfactory in that all linkage groups except II have at least three usable markers, and one in particular - group III - has a series of eight auxotrophic markers for seven different substances, only two of which present difficulties when used in multi-point crosses. Despite statistical analysis of the mitotic data which indicate that all seven linkage group are on different chromosomes, it may be argued that some of the groups are syntenic but separated by distances long enough to make even mitotic crossing-over likely. It is mmmh.mere probable, however, that meiotic data indicating such synteny are spurious. Spurious indications of loose linkage were found commonly during this study. 95 96 In largely restricting the mutagenesis project to a search for simple auxotrophs, rather than fer genes that suppress or modify other genes, affect colony morphology, or confer resistance to drugs, in verifying the existence of seven chromosomes and indicating the direction of the centromere on chromosome IV, a map has been produced which should make §, commune more amenable to easy} standard genetic analysis. The spontaneous production of diploids by commonqA§,heterokaryons proved to be a highly strain-specific phenomenon. While it is impossible to compare the total number of cells containing both nuclei of the heterokaryon among plates of different heterokaryons, it is likely safe to say that differences in diploidization frequencies such as those between heterokaryon P (30 apparent diploids from.80 plates) and heterokaryons G and H (no sectors from 800 plates) are real differences. This large variation was also noted by Casselton (1965) working with oidia from commonHA,heterokaryons of‘g. lagopgg. It is unfortunate that among those heterokaryons which failed to yield diploids were those which contained the adenine mutations for which no linkage was apparent, and those with mutations bracketing the A_mating-type factor which were necessary for studying Specific Factor Transfer. The purpose of crossing the commonqA§_diploids to wild type haploids was to ascertain that they were, in fact, diploids containing all the markers originally put into the heterokaryons from which they arose. It was not expected that the alleles be recovered in frequencies predicted by simple trisomic segregation. Aneuploids are unstable and it is reasonable that many of the disomic chromosomes in progeny of these crosses will have been lost by the time the progeny'have grown to testable size. This process would bring allele frequencies closer to 97 those expected from total haploidization. Indeed, a majority of the progeny did not sector at all after their initial five days of growth and were presumed haploid. Most of the allele frequencies fell between those predicted by trisomic segregation with no subsequent loss of chromosomes and those predicted by trisomic segregation followed by total haploidization. When allowed to haploidize, the progeny showed allele frequencies close to expected values. Those that deviated usually'did so 1. because of selection effects noted throughout this study - for niacin requirement or against aggé. Trisomic segregation assumes equal participation of the three homologous sets of chromosomes and close pairing of any two of these three, randomly, in any chromosome region. This mechanism generates a prediction of the percent of detectable cross- over genotypes, compared to standard meiotic values, which should be found in the progeny. The four commongA§_diploids, upon crossing, produced crossover-type progeny in close to the expected numbers in most cases. A glaring exception were crossovers in the §.pgx:b’region from the cross of P—l with haploid. Whether this reflects selection peculiarities or preferential pairing or crossover ”hot spots” cannot be determined from the data. Initial efforts to detect daughter diploids with desired genotypes by testing entire samples of progeny'from the appr0priate diploid x haploid crosses were totally'unsuccessful. Thus it became necessary to test much larger samples and also to use a selective system to pick out likely candidates. In calculating the number of spores from each cross to plate on selective medium.it was assumed that trisomic pairing is random, that crossing-over could be ignored and that all progeny'were equally viable and stable. The first assumption has been borne out by 98 crossover data. The second is only partially true; crossovers would not alter the number of progeny able to grow on selective medium but would lower the number marked in the desired way. The last assumption is certainly false, as aneuploids are unstable and tend toward.haploidy. Haploidy would lower the number of viable progeny on the selective plates, as fewer auxotrOphic markers would be ”covered" by a wild type allele. The results showed that numbers of both selected progeny and desired diploids were lower than "predicted". The absence of commong§,and, with one possible exception, commonHA, diploids from the selected progeny'was puzzling. There is no‘gupgiggi reason to suspect that they might be unstable or inviable. Diploids have been obtained from both commonHA,heterokaryons of Q, lagopgg (Casselton 1965) and common-§,heterokaryons of‘§, commune (Parag and Nachman 1966). The compatible diploids and partially'dikaryotic cultures that were recovered by this procedure present several interesting questions. Although there is no necessity to suppose that compatible diploids would be unstable, they do, by virtue of being compatible, contain the genetic machinery necessary'fOr the maintenance of a dikaryon - of coordinated but separate nuclei. For a diploid to persist we must assume that the machinery to keep nuclei apart either is not operating or will not separate them if they have originally been "tricked” together. It is established, since recombination is known to occur between the nuclei of a dikaryon (Crowe 1960) (Parag 1962), that spontaneous diploid nuclei must arise in dikaryons. But they are transient. Thus the mere occurrence of diploidy does not guarantee its persistence, and the question remains as to what allows the diploid state to persist in the compatible diploids. 99 The gik,gene (Koltin and Raper 1968) and perhaps others like it seem to be agents for maintaining a dikaryon. The diploids produced in these experiments cannot have been due to gik;mutations since progeny and sectors from the diploids mate normally. But the ability of compatible diploids to persist may well be due to failure, fer some reason, of gikf to act. Perhaps the product of gikf, or other genes that it may control, require some time to build up to effective concentration in the cytoplasm. .A germinating compatible diploid spore might establish a diploid mycelium before any of its cells become dikaryotic. This would predict a degree of instability in compatible diploids, especially in older cells. The compatible diploid samples examined microscopically in this study came from young regions close to the growing edge, yet still contained a small number of two-nucleus cells. Perhaps these were true dikaryotic cells and not just cells seen in the act of division. The notion of cyt0p1asmic accumulation of substances which would cause a diploid nucleus to form two haploid nuclei is consistent with the observation that diploid nuclei of'g. lagopgs, stable when vegetative, become highly unstable in a dikaryon (Casselton 1965). The nature and behaviour of the partially dikaryotic cultures used in this study can also be viewed in this perspective. Microscopic observations left no doubt that many cells of P-19 and P-21 were dikaryotic. While it was possible that the cells which appeared monokaryotic did so only because the second nucleus failed to pick up stain, it is also possible that there was a real and significant population of'monokaryotic diploid cells. Transient diploidy in dikaryons is a widely accepted reality, but the fact that the "dikaryons” haploidized via stages of aneuploidy 100 indicates that diploidy, in these "dikaryons" at least, is quite persistant. Haploidization is understood to be a phenomenon of diploid nuclei in which a non-disjunctional event creates a 2n+l and 2n-l pair of daughter cells. The 2n+l cell loses one of its trisomic chromosomes at random and the 2n-l cell becomes unstable and proceeds toward haploidy. Any analogous process in a dikaryon would necessitate either a control mechanism.to keep the two nuclei from losing homologous chromosomes, and t“ then have them unite to become a haploid, or else loss of one of the two fi nuclei. The former is highly improbable and the latter would create 3 basically two classes of haploid products, each reflecting the constitution of one of the two nuclei. The data of Tables 38 thru.4l, by if} 1.- contrast, show almost as many classes of haploid products as there are haploid products. It is more likely that haploidization in ”dikaryons” actually occurs in some diploid cells. The frequency of haploidization and the persistence of the aneuploid stages indicate that these diploid cells are neither especially rare nor short-lived. The origin of these dikaryons also merits consideration. It was realized that the selective plating technique allowed the possibility of occasional undetected mating of young germlings. The probability that any two adjacent germlings would be of proper genotype to form these dikaryons is about half the probability of properly marked compatible diploids being formed at meiosis. In addition there is the necessity of the adjacent haploids being close enough to mate undetected, and.being able to grow together sufficiently to mate despite bearing several auxotrophic mutations which cause them to be selected against on the medium. ll I'll I III. llllllllll l IIIIIlIlIrI I Jin. III.|| “a! I ‘6‘ 101 In view of these considerations and the likelihood of considerable diploidy in the dikaryons and of some instability. in compatible diploids, the possibility exists that the dikaryons are partial breakdown products of compatible diploids. The only strain which exhibited behavioural peculiarities was P-19. Its morphology was irregular and the behaviour of its 11 factors highly anomalous. Although it contains compatible A and g factors it never fruited. Only fl participated in matings and this cannot be attributed to nuclear selection since the selection was absolute; crosses of P-l9 to B_l+_Z strains would not dikaryotize. Only _Bil; was found in haploid sectors. Progeny of P-l9 bearing its B_1+_Z factor were unable to dikaryotize an A_’+_Z Eli strain (the comm-_B_ "barrage" reaction occurred instead) which, although a great—great—grandparent of P-l9, had no mating-type factors in common with it. These same progeny successfully dikaryotized other .Aiz _Bfl strains. The haploid parent of P-l9 could mate only with difficulty with a compatible strain that constituted one genome of the diploid parent of P-l9. Progeny from the one partially analysed and two completely analysed crosses of P-l9 to wild type haploids were almost never aneuploid. This indicates that only one nucleus from P-l9 participated in what were therefore essentially haploid x haploid crosses. The marker frequency data from these crosses can best be explained by assuming that the participant nucleus from P-l9 was different in each case. (No matter how the mating-type factors are arranged into nuclei, and in order for all three of these crosses to have occurred, one of the three haploids would have had to have mated with a recombinant nucleus from P-l9.) This anomalous mating behaviour seems to be due to P-l9's 2+1 factor or at least the chromosome with B_’+_Z. Since mutations of _B_ regularly 102 abolish I_3_'s ability to confer incompatibility, the mutation, if such it is, would more likely be of a modifier gene than B_’+Z itself. A cluster of modifiers has been mapped close to B (Raper 1973). w seems able to suppress certain E's, both the ELI of P-l9, which it rendered incapable of participating in mating, and that of its great-great-grandparent, with which it reacted as if the interaction were cannon-Q. The g4; factor of P-l9, on the other hand, behaves normally once isolated from P-l9. The exclusive appearance of E in haploid products is best explained by a high tendency of the M chromosome to non-disjoin. If this were so, gill would almost always be included in the 2n+l mitotic non-disjunctional product rather than the Zn-l product which ultimately becomes the haploid. Beyond this, an anomaly which can act as a dominant allele to certain Pig's - perhaps, specifically, ones in its lineage - and also cause its chromosome to non-disjoin defies easy definition. Recombination in cannon—A}; diploids appears, as expected, to be mitotic as in the parasexual cycle. Only one of forty haploids from P-l was a crossover type, and stages of aneuploidy were easily isolated; only one of the forty was originally isolated as a complete; haploid. Data from A-5, A-8 and C are scant, but none of the 18 haploids they produced was a crossover type. The large percentage of sectors that were apparently still diploid was unexpected. A preponderance of haploids among sectors collected in this way had been reported earlier (Mills and Ellingboe 1971). These recombinant sectors cannot be used in calculating frequency of crossing-over in the diploids because they are selected for. It should be noted that some of the diploid prototrophs which were homozygous for id; in addition to gu_l_, and therefore scored as products of crossing-over between ;ad_2 and the centromere, may have been 103 "non-disjunction" diploids - 2n+l products of non-disjunction of chromosome IV from which the Egg? grg§.homelogue was subsequently lost. None of the haploids isolated were originally collected as aneuploids. Recombination in compatible diploids 0-314, C-M, and P—j also appeared to be parasexual. 0f the 32 haploids they gave rise to, 26 were originally isolated as aneuploids and none was of crossover genotype. Again the input from the C diploids was disappointing as sectors were difficult to detect and most of those isolated were recombinant diploids. The mode of recombination in the "dikaryons” is not as clear. Sixty-five haploid sectors were collected. All but nine of them went through stages of aneuploidy during isolation, which is predicted by a parasexual.mechanism. Yet nineteen had crossover genotypes. That is a very high figure for parasexuality and.mere in accord.with a meiotic type of mechanism. Both cultures had significantly higher crossing-over frequency'than did compatible or commenqA§_diploids. ‘And perhaps surprisingly, despite the peculiarities of P-l9, no significant difference in crossover frequency was indicated between Pal9 and P-Zl. The cross- over type haploid products of P—l9 are, however, peculiar in that thirteen of the fifteen represent crossing-over between §.andlpg§:b_and that all of these are gfpg§:_. In an attempt to explain these results it is useful to return to the idea of a tension between diploid and dikaryotic states. Diploidy'is only maintained in vegetative cells whereas the dikaryotic state marks a step in a cell's committment to meiosis and reproduction. Therefbre, in a dikaryon, given a tendenoy fer nuclei to occasionally unite, a high degree of’meiotic competence could be achieved in other than basidial cells and "precocious meiosis" occur. Similarly, particularly low 104 levels of whatever gene products confer meiotic competence in other cells might lead to diploidization. The fission yeast Schizosaccharmces m, while not dikaryotic, is a haplontic organism in which the diploid is normally confined to a single cell, briefly, prior to meiosis. Several meiotic mutants have been identified in this organism (reviewed by Breach et a1, 1968). Four of these genes which are necessary for successfiil meiosis have been found to cause, when mutant, persistence of the diploid state (Egel 1973). Thus a tension between meiotic competence and diploidy is demonstrated. For the purposes of this study, the terms diploid and dikaryon applied to whole organisms become relative terms, referring to a variable majority but probably not all of their cells. It is possible that the "dikaryons” used are less dikaryotic than most, if they formed originally by partial breakdown of diploid germlings. A compatible diploid is then, of necessity, an organism in which, for whatever reason, the vegetative state is maintained in most cells. Somatic recombination in such cells would be parasexual. An organism very predominantly dikaryotic would probably undergo somatic recmbination in the form of precocious meiosis. Techniques such as incompatible di-mon matings can be used to pick out recombinant nuclei. Organisms in an intermediate state such as that argued for P-19 and P-21 should be able to undergo parasexual reduction by stages of aneuploidy, but the genotype of some of the haploid nuclei produced might reveal an earlier dikaryotic history in which meiotic-like recombination occurred. The fact that thirteen of the fifteen crossover type haploids from P-l9 were of genotype §+ m could be due to selection. However, no selection has been previously observed for these markers. A more likely 105 explanation would be that a gf' -b recombinant nucleus arose early in P-19, proliferated, and eventually'mnde up a substantial fraction of the mosaic P-l9 genome. Accepting this idea would.mean considering all thirteen sf pgx:b'recombinants as results of one event. This is an inherent difficulty in studying somatic recombination via analysis of mitotic haploidization. Any crossover types which are identical or reciprocals of each other may have arisen earlier from the same event, but they are scored as independent events. Pal9 is only'the most glaring case. Despite this difficulty, the Observations have been made that (1) cultures which are largely or completely'diploid, whether compatible or commonqgfi, haploidize via stages of aneuploidy and show almost no evidence of somatic crossing-over, and (2) cultures which are compatible and substantially dikaryotic also haploidize via stages of aneuploidy and show definite evidence of somatic crossing-over. The discussion above is presented as a reasonable way of viewing these two sets of Observations which, on the surface, appear irreconcilable. A final assumption which must be made is that whatever gene or genes may be defective in P-l9 are not those which confer ability to perfOrm crossing-over. The role of'incompatibility factors in determining modes of somatic recombination is then an indirect one. Compatibility'itself will not cause a switch from a vegetative state, in which parasexual phenomena occur, to a premeiotic state. It will, however, activate many genes of the type of <_i_ik_, or the meiotic genes of S. mbg, which can effect the change. In such an indirect system slippage can occur. The genes fer dikaryosis and.meiosis will not immediately and totally'be derepressed 106 by compatible mating-type factors, and under extreme conditions they might become derepressed'without compatibility'(haploid fruiting). Specific Factor Transfer, if it is a phenomenon of highly specific and frequent crossing-over, would only occur in a meiotically'competent cell, not in compatible diploids or probably even metastable diploid- dikaryons. CommengA,and commenq§,diploids would be expected to behave like any other diploid and show parasexual somatic recombination. There were no data from this study bearing on these predictions. SUMMARY Seventyzfour mutant strains of Schizopgyllum.commune were induced by mutagenesis with Nmefilyl-N'-h'itro-N-nitrosoguanidine. Twenty-two of these, two morphological and twenty auxotrophic, were ultimately characterized as "new" genes and fourteen mapped to existing linkage groups. Another twentyafive mutations proved to be alleles of the newly isolated or stock strains. Tests of growth response to intermediates of purine synthesis made it possible to assign most of the twelve different adenine genes to regions of the purine pathway. Three of these genes could be assigned specific enzymes. CommonqA§,diploids were isolated as spontaneous sectors from nutritionally forced commonqA§,heterokaryons. Their genotypes were determined by analysis of progeny from crosses to wild-type haploid strains. Two of these diploids were homozygous for gagg,and heterozygous for .gul, and produced rapidly growing sectors on selective media via mitotic crossover leading to homozygosity of‘gul, or via haploidization with loss of one 5352, A majority of the sectors were still diploid, as determined by prototrophy for non-selective heterozygous markers. No aneuploid sectors were fermed, but those that were haploid showed no evidence of crossing-over and thus likely arose via a mitotic mechanisms Another diploid was heterozygous for gg&,and.ad , linked "pink" adenine mutations in repulsion. This diploid produced sectors of non- 107 108 diploid morphology and/ or pink colour, almost all of which were aneuploid. When finally haploidized, these sectors showed only a single incidence of crossing-over and thus also appeared to have arisen by mitotic haploidization. The cannon-Ag diploids were crossed to haploids optimally marked for selection of progeny diploids heterozygous for all markers (save those homozygous for aggg and heterozygous for §u_l) . Five progeny proved, via analysis of progeny from crosses to wild type haploid, or from spontaneous fruiting, to be 2n and heterozygous for the desired markers. Staining and counting of nuclei was done to distinguish diploids from dikaryons. Two of the daughters were compatible diploids of the §.u_l5 - ar 2 selective type. Three daughters were of the Ldfil; - a_d2 selective type, one diploid and two largely dikaryotic. One of the dikaryons possessed a 1_3_ mating type factor with anomalous behaviour. A series of tests indicated that the anomalous .3 factor repressed the expression of certain other Q factors, including that of the other dikaryon nucleus, making the dikaryon pseudo-coman-E. It also indicated a marked tendency to non- disjoin. No usable cannon-A or common-§ progeny diploids were obtained. Recombinant sectors were collected fran the progeny diploids and dikaryons in the same ways they were collected from the diploid parents. Analysis of the sectors showed a common incidence of aneuploidy in all cases. Haploids from the compatible diploids showed no evidence of crossing-over, while those from the dikaryons showed a significant frequency of crossing-over. It appears that the somatic recombination process in diploids, comon-Aj and compatible, is classically mitotic. The dikaryons apparently contain a percentage of diploid cells which can 109 undergo mitotic haploidization but whose nuclei give evidence of a previous history of meiotic-like behaviour involving crossing-over. The tendency toward "precocious meiosis" is apparently a property of the dikaryotic state rather than of compatibility Ell s_e_. 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