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Analysis of two additional loci in Neurospora crassa related to Spore killer-2
Fungal Genetics and Biology 39 (2003) 142–150 www.elsevier.com/locate/yfgbi
Analysis of two additional loci in Neurospora crassa related to Spore killer-2 Barbara C. Turner* Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020, USA Received 13 March 2002; accepted 18 February 2003
Abstract Two new loci found in one strain of Neurospora crassa (P2604) collected in Malaya are related to the meiotic drive system Spore killer Sk-2. Sk-2 was found in Neurospora intermedia and introgressed into N. crassa. P2604 showed high resistance to killing when crossed to Sk-2. This resistance was found to be linked to, but not allelic to, resistance locus r(Sk-2) on LGIIIL. Analysis showed that the high resistance phenotype of P2604 requires resistance alleles at two different loci on LGIIIR. Strains carrying a resistance allele at only the proximal or the distal locus, respectively, were obtained and intercrossed. Highly resistant strains were obtained by rejoining the two genes. The proximal locus alone confers a low level of resistance. This locus was named pr(Sk-2) for partial resistance to Sk-2. The distal locus was named mod(pr) because its only known phenotype is to modify pr(Sk-2). Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Meiotic drive; Spore killer; Neurospora
1. Introduction The terms ‘‘meiotic drive’’ and ‘‘segregation distortion’’ are used to describe certain matings in which an ordinarily viable chromosome, chromosome segment, or locus is recovered in less than half of the progeny, if recovered at all (see Lyttle, 1991 for a comprehensive presentation of the systems that have been studied extensively). An important question about meiotic drive is whether there are common evolutionary forces that can be discerned in these systems without regard to the species involved or the mechanism by which the drive operates (see Bengtsson and Uyenoyama, 1990). An example of a common outcome for three different systems is provided by crossover suppression. There are inversions that suppress recombination associated with the distorting chromosomes of Segregation distortion in Drosophila melanogaster (Temin et al., 1991) and the t haplotype in Mus spp. (Schimenti, 2000; Silver, 1989), while the Spore killer system of Neurospora includes a * Fax: 1-650-723-6132. E-mail address: [email protected].
virtually complete recombination block in an extensive region containing the Spore killer factor (Campbell and Turner, 1987). Mus and D. melanogaster have long been known to resemble each other in having many contributing loci, most of which are closely linked. The mouse t haplotype chromosomes (variants of chromosome 17) carry several trans-acting distorter loci and a responder locus. They also carry other mutations that enhance the segregation distortion. Silver, 1996, presented a map of the known loci of the distorter system and maps of partial haplotypes that have been used to dissect the system. In the Segregation Distorter (SD) system of D. melanogaster there are numerous loci on the SD chromosome, of which several have been studied in detail both for their effects and for their molecular basis (Temin et al., 1991). There is a main SD locus, a responder locus, an enhancer locus that strongly affects amount of distortion, and other loci that have smaller effects. It was speculated that there might be a similar situation of additional contributing loci in the meiotic drive systems of Neurospora. This report presents results of a search for additional loci related to one of those systems, Spore killer-2.
1087-1845/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S1087-1845(03)00018-5
B.C. Turner / Fungal Genetics and Biology 39 (2003) 142–150
Spore killers were reported by Turner and Perkins (1979), and cytological observations were reported by Raju (1979). (For summaries of subsequent findings see Raju, 1994; Turner, 2001; and Turner and Perkins, 1991). Spore killers Sk-2 and Sk-3 were found as rare variants in N. intermedia, a species that hybridizes readily with N. crassa. No Spore killer has been found in N. crassa, but for mapping and other genetic study, both Sk-2 and Sk-3 were introgressed from N. intermedia into N. crassa because of the far greater number of known genetic markers in N. crassa (Turner and Perkins, 1979). The effect of Spore killer is observed only in heterozygous crosses. In crosses of Spore killer sensitive, each ascus contains four viable black ascospores carrying the killer factor and four inviable, undersize, transparent (hyaline) ascospores (4:4 asci). The Spore killer element has no obvious effect on the vegetative growth of a strain carrying it nor on the survival of ascospores in homozygous Sk-2 Sk-2 or Sk-3 Sk-3 crosses. (In some previous publications, the killer elements were symbolized as Sk-2K and Sk-3K , but here are shortened to Sk-2 and Sk-3.) This study involved only Sk-2. Killing (which necessarily includes self-resistance) has been shown to segregate as a chromosomal locus or complex of loci in linkage group III. Recombination is almost completely blocked between Sk-2 and markers in a segment of linkage group III that is ordinarily at least 30 map units long (Campbell and Turner, 1987). Resistance to Spore killers was discovered in collected strains of N. intermedia very soon after the Spore killer elements themselves were discovered (Turner and Perkins, 1979). After the Spore killers were introgressed into N. crassa, a resistance locus, called r(Sk-2), was detected in a N. crassa strain collected in Louisiana. It was studied genetically by Turner and Perkins (1979) and by Campbell and Turner (1987). Resistance segregates as a single locus in the left arm of linkage group III in crosses of r(Sk-2) marked strains that are sensitive to Sk-2. In those crosses, the presence of the resistant allele has no effect on crossover frequencies in the linkage group. When strains carrying the resistant allele of r(Sk-2) and linked markers are crossed to N. crassa Sk-2, resistance does not alleviate the crossover suppression. The ongoing study of Spore killers overlapped with a project to set up a worldwide collection of Neurospora cultures from nature, now available as the Perkins collection from Fungal Genetics Stock Center (Perkins and Turner, 1988; Turner et al., 2001). One part of the program involved screening isolates of three Neurospora species from over 700 collection sites for further instances of Spore killer or genes modifying its effect (Turner, 1977 and Turner, 2001). In N. crassa, the only phenotype found that was related to Spore killers was resistance to Sk-2. This report describes a survey of resistant N. crassa strains from widespread locations and presents a detailed analysis of one of them.
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2. Materials and methods Procedures for culturing Neurospora were described by Davis and De Serres (1970), and laboratory procedures related to Spore killer were described by Turner (2001). 2.1. Identification of resistance to Spore killer The protocol initially adopted for this project was the one used for scoring resistance while mapping r(Sk-2) (Campbell and Turner, 1987). Each strain was crossed to an Sk-2 tester (FGSC 6647a or 6648A) that had been inoculated 4 or 5 days previously in a glass culture tube. After 9 days, ascospores shot to the walls of the culture tubes were examined to determine whether the asci were 8:0 (all ascospores black) or 4:4 (four full size black ascospores:four tiny transparent aborted ascospores). Experience showed that resistance could be scored reliably by examining the sides of the culture tubes to determine whether shot asci had 8 black ascospores or 4 black:4 aborted ascospores. If the scoring was uncertain because of too few spores, excessive spore abortion from other causes, or difficulty in identifying discrete asci among the shot spores, perithecia were removed from the tube and dissected. As this project progressed, an intermediate class of progeny was discovered. These progeny produced mostly 4:4 asci when crossed to Spore killer, but also a few 8:0 asci. This pattern had been overlooked when scoring was done by simply looking at the walls of the culture tubes. For all crosses involved in separating and recombining the two new loci described in the Section 3, scoring was done by removing at least six perithecia from the culture tube and opening them to observe intact asci. 2.2. Strains are listed in Table 1 The N. crassa species testers are referred to in the text simply as fl A and fl a where that is appropriate. The N. crassa Sk-2 testers are referred to as Sk-2 A or Sk-2 a. In the text, any strain that carries Sk-2 or resistance to Sk-2 is identified explicitly. Otherwise, the marked strains used for mapping were laboratory strains sensitive to Spore killers. (The marker r(Sk-3), which was introgressed from N. intermedia, does not interact with the Sk-2 system and was used simply because it marks the left end of the linkage group.) Both wild-collected strains from the Perkins collection (Turner et al., 2001) and laboratory strains with FGSC numbers can be obtained from the Fungal Genetics Stock Center, Department of Microbiology, University of Kansas Medical Center, Kansas City,
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KS 66160–7420. At the end of the study, a pair of strains from the final cross in the series of crosses to lab strains was deposited in FGSC. Their numbers are 8275A and 8276a. They are recommended for any work involving crosses of the new mutants to laboratory stocks.
3. Results For those who are interested only in the location and characteristics of the two loci being reported, the section of these Results headed, ‘‘Mapping and characterization of the two new loci’’ will provide that information. Confirmation for the results in that section is provided in the final section, ‘‘Separation and resynthesis of the two components.’’ 3.1. Survey of strains of N. crassa resistant to Sk-2 This study was undertaken to find out whether more than one genotype can confer the phenotype of resistance to killing by Sk-2, a meiotic drive mutant in Neurospora. Strains resistant to Sk-2 occur in nature in both N. intermedia and N. crassa. N. crassa was
selected for study for two reasons: (1) unlike the case of N. intermedia, the N. crassa-resistant strains occur as isolated individuals in widely separated populations rather than as a significant segment of one widespread but contiguous region, and (2) genetic markers for mapping the site of a new locus are far more available in N. crassa. The traditional (non-molecular) method of determining whether a newly arisen mutation is allelic (or identical) to a previously identified gene with a similar phenotype was used for this survey. The ‘‘previously identified allele’’ was r(Sk-2), the Sk-2 resistance gene found in a wild-collected isolate from Louisiana (USA). A strain carrying r(Sk-2) was crossed to other wildcollected-resistant strains, and progeny were observed to determine whether or not recombinant (sensitive) progeny were produced. acr-2 r(Sk-2) A FGSC 7379 was crossed to Sk-2-resistant strains (see Table 1 and Turner, 2001) from Brazil, Haiti, India, Ivory Coast, Costa Rica, and Malaya. It was expected that an unlinked resistance locus should reassort with r(Sk-2), so that 25% of progeny would be sensitive to Sk-2 (scored by presence of asci with 4 large black:4 aborted ascospores) because of receiving the sensitive allele at both loci. A separate but linked locus would be expected to
Table 1 Strains used in crosses Description
Generation
FGSC number
A. Strains carrying pr(Sk-2) and mod(pr) P2604 cum pr(Sk-2) mod(pr) 1046-73 A pr(Sk-2) mod(pr) 1051-78 a pr(Sk-2) mod(pr) 1051-81 A pr(Sk-2) mod(pr) ser-1 1125-43 A pr(Sk-2) mod(pr) ser-1 1180-8 A mod(pr) ad-4 1186-17 A pr(Sk-2) ser-1 1186-147 a pr(Sk-2) mod(pr) ser-1 1187-1 a
original second third third fourth fifth sixth sixth progeny of 8272 8273
P2604 (Perkins)
B. Laboratory stocks used for mapping and analysis Description
Comments
FGSC Number
a
flA fla SK-2 a Sk-2 A acr-2 rðSk-2ÞA (3291) acr-2 ad-4 C39-20 a (3230) r(Sk-3) ser-1 902-98 a leu-1 thi-2 a ad-4 leu-1 a (3310) leu-1 a (3031) cum acr-7 A Sk-2 acr-2 leu-1 A Sk-2 acr-2 leu-1 a
Standard Standard Standard Standard
N. N. N. N.
crassa crassa crassa crassa
species tester species tester Sk-2 Sk-2
7398
8274 8272 8273
1838 1690 6647 6648
7162
7156 7375 7374
Note. For strains without FGSC numbers, other deposits with the same loci are available from FGSC. Numbers in parentheses are Perkins Lab silica gel numbers. fl (fluffy) is a mutation that results in absence of macroconidia, which makes shot ascospores much easier to observe. It also confers high fertility. a See Table 2 for the lineage of these strains.
B.C. Turner / Fungal Genetics and Biology 39 (2003) 142–150
yield some (fewer than 25%) sensitive progeny by crossing over. 3.1.1. Results of survey crosses Only strain P2604 from Malaya (cross 1005, Table 2) gave results suggesting a new resistance locus. When crossed to Sk-2, at least one, and probably three, of the 66 progeny from cross 1005 were sensitive to killing. For the strains from Brazil, Haiti, India, Ivory Coast, and Costa Rica, crosses to acr-2 r(Sk-2) A yielded only progeny resistant to Sk-2. For each of these five crosses, 50 to 80 progeny were fertile enough to be scored in crosses to the Sk-2 testers, and they were all resistant (not making 4:4 asci). Many more progeny tests would have been needed to rule out the presence of a second locus closely linked to r(Sk-2), but that was not the purpose of the survey. The aim was to look for loci that could be detected with progeny sets of this size. 3.1.2. Background and crossing characteristics of strain P2604 a from Penang, Malaya P2604 is a wild-collected N. crassa strain that does not carry a Spore killer itself but is resistant to killing by
Table 2 Crosses A. Crosses with P2604 from Penang, Malaya P2604 pr(Sk-2) mod(pr) a fl A FGSC 1838 P2604 pr(Sk-2) mod(pr) a Sk-2 A FGSC 6648 1005 P2604 pr(Sk-2) mod(pr) a r(Sk-2) acr-2 A 1046 P2604 pr(Sk-2) mod(pr) a cum acr-7 A 1156 P2604 pr(Sk-2) mod(pr) a acr-2 leu-1 his-7 A 1157 P2604 pr(Sk-2) mod(pr) cum acr-7 thi-4 348-22 A B. Initial 1051 1064 1065 1125 1126 1149 1165 1168 1170
mapping crosses with descendants of P2604 cum pr(Sk-2) mod(pr) 1046-73 A leu-1 thi-2 a pr(Sk-2) mod(pr) 1051-78a ad-4 leu-1 A pr(Sk-2) mod(pr) 1051-81A ad-4 acr-2 a pr(Sk-2) mod(pr) 1051-81A r(Sk-3) ser-1 a pr(Sk-2) mod(pr) 1051-81A ad-4 acr-2 a ser-1 pr(Sk-2) mod(pr) 1125-43 A Sk-2 acr-2 leu-1 a pr(Sk-2) mod(pr) 1157-15 a acr-2 leu-1 his-7 A pr(Sk-2) mod(pr) 1157-15 a pro-1 A (3135) ser-1 pr(Sk-2) mod(pr) 1168-7 A ad-4 acr-2 C39-21 a
C. Separation and resynthesis of the resistance genes of P2604 1180 pr(Sk-2) mod(pr) ser 1125-43 A leu-1 a 1186 pr(Sk-2) mod(pr) ser-1 1180-8 A ad-4 leu-1 a 1187 mod(pr) ad-4 1186-17 A pr(Sk-2) ser-1 1186-147 a 1188 mod(pr) ad-4 1186-17 A Sk-2 a (3213) 1189 pr(Sk-2) ser-1 1186-147 a Sk-2 A (3212) 1190 pr(Sk-2) mod(pr) ser-1 1187-1 a Sk-2 A Note. The lineage of all stocks carrying the new variants, pr(Sk-2) and mod(pr), can be derived by reference to their isolation numbers. The portion in bold face is the number of the cross from which the isolate was obtained, and this cross can be found higher up in the table. The cross numbers in bold italics in the first column show the lineage of pr(Sk-2) mod(pr) ser-1 1187-1 a, the strain in which pr(Sk-2) and mod(pr) were rejoined. A diagram of crosses 1186 and 1187 is presented in Fig. 2.
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Sk-2. It was collected at the easternmost border of the known range of N. crassa Penang, an island just west of the mainland of Malaya. Results for test crosses (to the species testers and Sk-2 testers listed in Table 1) of Perkins Collection strains from Malaya were reported previously in summary form (Turner et al., 2001; Turner, 2001), but this is the first report of specific crossing results for P2604. P2604 was not immediately diagnosed as N. crassa when crossed to the standard species tester of N. crassa (fl A FGSC 1838). Many of the cultures from Penang, including P2604, made mostly full-size, pigmented ascospores when crossed to the N. crassa testers, but more than 50% of those ascospores were inviable (tan and brown) (Turner et al., 2001). Some other Penang strains produced 90% or more black viable ascospores when crossed to the N. crassa species testers and thus were identified with certainty as N. crassa. P2614 A, one of the identified N. crassa strains from Penang, was selected as an auxiliary N. crassa species tester and was crossed to P2604 a. This cross produced at least 90% black ascospores, demonstrating that P2604 was also N. crassa. Because P2614 is sensitive to Sk-2, this cross also demonstrated (with more certainty than P2604 a flA) that P2604 does not carry Sk-2. Like all wild-collected N. crassa strains, P2604 was crossed to the Sk-2 tester of opposite mating type (Turner, 2001), and it was diagnosed as resistant. After cross 1005 showed that P2604 probably carried a new resistance factor, the cross to Sk-2 A was repeated and scrutinized more carefully. There were many full-size defective (brown, tan, and white) ascospores scattered randomly among asci. Some ascospores did have the characteristic (tiny, clear) appearance of ascospores killed by Spore killer, but they were usually found in completely aborted asci. Asci were not observed with a pattern of 4 viable:4 tiny ascospores. Most importantly, many asci had more than four large, pigmented ascospores. Therefore, P2604 was confirmed as resistant to Sk-2. The incidence and pattern of defective ascospores in crosses of P2604 to fl A and Sk-2 A was not remarkable. In all the known species of Neurospora, crosses of geographically distant strains may be poorly fertile or produce only a minority of viable ascospores (Turner et al., 2001). The pattern of defective ascospores for P2604 was random, not in pairs as it would be if the primary reason for defective ascospores were the presence of one or more rearrangements (Perkins and Barry, 1977). 3.1.3. Results for cross 1005:P2604 a acr-2 r(Sk-2) A Ascospore characteristics for the survey cross of P2604 were similar to the cross results just described for P2604 fl A and P2604 Sk-2A. Sixty-six progeny were obtained from cross 1005 and crossed to the Sk-2 testers. Many of these 66 crosses produced few viable
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ascospores, so they were difficult, and in some cases impossible, to score for resistance. However, the test crosses of three of the progeny gave no asci with more than four good ascospores (for two of these three progeny, the level of general spore abortion was high, making a diagnosis of sensitivity to Sk-2 only tentative). Based on this result of Sk-sensitive progeny segregating from P2604 acr-2 r(Sk-2) A (resistant resistant), further crosses were carried out to confirm and map the new resistance ‘‘locus.’’ 3.2. Development of stocks and initial mapping results Fig. 1 shows the markers from LGIII that were used in this study. In order to proceed with analysis of the new resistance ‘‘locus’’ it was desirable to develop stocks that would retain the resistance to Sk-2 but have normal fertility with laboratory stocks. After a second generation of back-crossing to laboratory stocks, ascospore viability improved greatly and scoring for Spore killing was much easier. Crosses are listed in Table 2. For progeny of the first generation of crosses ( P2604), resistance was not always scored with confidence. 3.2.1. Cross 1046 located the resistance ‘‘locus’’ far to the right of cum For cross 1046, among the cumþ progeny, 21 were resistant (parental coupling) and 24 were sensitive to Sk2 (recombinant). The map derived from the cross was cum—29—acr-7—24—(Sk-2 resistance). Since r(Sk-2) is only one map unit to the right of cum, the recombination results of cross 1046 were inconsistent with the survey cross result, (maximum of 9 map units between r-(Sk-2) and the new resistance ‘‘locus’’). These disparate results may be due to one or both of two phenomena previously observed in Neu-
Fig. 1. Map of LGIIIL and proximal portion of LGIIIR RB ¼ the region where recombination is blocked in crosses heterozygous for Sk2 (or Sk-3). The RB contains the locus or loci responsible for spore killing, but more precise mapping of Sk-2 is impossible because spore killing does not recombine with loci in the RB. O ¼ centromere The loci introduced in this report are in boldface type. Loci within square brackets have not been ordered. Analysis of r(Sk-2), locus that confers resistance to Sk-2, was reported previously (Turner and Perkins 1979; Campbell and Turner, 1987). r(Sk-3), which confers resistance to Sk-3, was introgressed into N. crassa from N. intermedia. It does not interact with the Sk-2 system. acr-2 and acr-7 confer resistance to acriflavin. They are completely silent under normal crossing and culture conditions. All loci are described in Perkins et al., 2000. Intervals in the diagram are not proportional to genetic map distance. Recombination rates varied greatly from cross to cross, as might be expected when working with loci obtained from a stock collected on the other side of the globe from the ancestors of laboratory stocks (see text).
rospora. (1) Because many loci affecting recombination occur in N. crassa (Perkins and Bojko, 1992; Yeadon and Catcheside, 1995), it is not unusual to see widely differing map distances in different crosses with the same markers, particularly when strains of different background are used. Variant recombination loci may explain not only the low recombination in cross 1005, but also variation from cross to cross for the map distances between any two markers in this study. (2) The apparent infrequency of recombinants in cross 1005 may have been attributable to unrelated loci that resulted in poor viability of crossover progeny (‘‘synthetic lethals’’) (see Turner, 1987). 3.2.2. Introgression of resistance In general, each generation of crossing to laboratory stocks halved the amount of the genome inherited from P2604 and thus decreased the potential for inviability of progeny ascospores in the following generation. Selecting for resistance to Sk-2, however, would tend to retain other segments of LGIII from P2604. Therefore, markers were deliberately used to increase the probability of obtaining stocks that would have full Sk-2 resistance from P2604 but eliminate material to the left of the region carrying it. 1046-2 A was selected for use in cross 1051 because it combined the left end marker cum (and presumably part of LGIII from the laboratory parent) with resistance to Sk-2. (Inexplicably, only about 20 of the 119 progeny obtained from cross 1051 carried cum, and only one of these had all the other parental markers.) Two strains carrying cumþ (and presumably eliminating even more of the LGIII material that was carried over from P2604) were selected from cross 1051 for use in further mapping crosses. Later, strain 1125-49 substituted ser-1 and any material to the left of ser-1 that remained from P2604. In cross 1125, 32 of 84 progeny (38%) were recombinant for r-(Sk-3) and resistance to Sk-2. 3.2.3. Mapping results for markers closely linked to the Sk-2 resistance from P2604 The crosses in Table 2, part B, were first analyzed assuming that resistance to Sk-2 was conferred by a single locus. Problems with this assumption were apparent, as illustrated by cross 1064 in Table 3. Looking at the data for cross 1064, two problems are seen: (1) Three of the classes do not have reciprocals, and (2) the double crossover class is unexpected considering the few progeny in the single crossover classes (:064 :046 ¼ :003 expected doubles). Pooled results for this stage of analysis are shown in Table 4. For all markers in IIIR, the Sk-2-sensitive/ markerþ recombinant classes were obtained. For markers between ser-1 and his-7, no Sk-2-resistant recombinant class was obtained. For example: pro-1 Sk-2-sensitive pro-1þ Sk-2-resistant produced pro-1þ
B.C. Turner / Fungal Genetics and Biology 39 (2003) 142–150
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Table 3 Example of presumptive progeny classes, assuming a single locus confers resistance to killing: Cross 1064 Sk-2 resistant 1051-78 a ad-4 leu-1 A CLASS
ad-4-region 1
Sk-2 resistance-region 2
leu-1
Parental
) +
S R
) +
45 49
) + ) + ) +
R S S R R S
+ ) + ) ) +
0 5 7 0 0 3
Crossover classes Single in region 1 Single in region 2 Double
Number
Total
109
Note. S, sensitive and R, resistant. Cross 1064 illustrates the anomalous results obtained when mapping crosses were analyzed under the initial assumption that there was only one resistance locus in strain P2604. There are three crossover classes without reciprocals, and the presumed double crossover class is unexpected considering the size of the presumed single crossover classes.
Table 4 Recombination of LG III markers with resistance to Sk-2 Cross
Marker
Recombinantsa Sk-2-sensitive/markerþ
Sk-2-resistant/marker
Total Progeny
1065 1126 1170 1156 Totals for acr-2
acr-2 acr-2 acr-2 acr-2
17 15 2 5 39
7 5 2 3 17
91 87 50 48
1125 Totals for ser-1
ser-1
5 5
1 1
84
1168 Totals for pro-1
pro-1
4 4
0 0
70
1064 1065 1126 1170 Totals for ad-4
ad-4 ad-4 ad-4 ad-4
8 1 10 2 21
0 0 0 0 0
109 91 87 50
1051 1064 1156 Totals for leu-1
leu-1 leu-1 leu-1
19 10 2 31
0 0 0 0
113 109 48
1156 1165 Totals for his-7
his-7 his-7
6 7 13
2 1 3
48 88
1051 Totals for thi-2
thi-2
27 27
12 12
113
Note. The loci are listed as they occur from left to right in the linkage group. This table illustrates a gradient of unequal recovery of reciprocal recombinants on either side of the zone (containing pro-1 ad-4 and leu-1) where no recombinants resistant to Sk-2 were recovered. a Recombinants between the marker in column 2 and resistance/sensitivity to Sk-2. For example, for cross 1065, the parents were Sk-2 sensitive acr-2 Sk-2-resistant acr-2þ . 17 progeny were Sk-2 sensitive acr-2þ , and 7 were Sk-2-resistant acr-2. Recombinants are tallied taking into account only one marker at a time (see text). Thus for cross 1064 (see Table 3) recombinants between Sk-2 resistance and leu-1 are listed without regard to ad4 genotype.
Sk-2-sensitive progeny but did not produce pro-1 Sk-2resistant progeny. Thus it appeared possible that in a stretch of the chromosome that included pro-1 ad-4 and leu-1, recombinants resistant to Sk-2 were not obtainable by a single crossover.
3.2.4. Two-gene hypothesis The mapping results shown in Tables 3 and 4 were similar to results for known two-locus systems (for example, the color loci reported in Turner, 1987). Thus it appeared possible that resistance required two interact-
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ing genes at two different loci, and that these loci were separated by crossovers between leu-1 and ad-4 in cross 1064. The consistent cumulative results summarized in Table 4 are more compelling evidence for a two-locus hypothesis than any single cross. If there were two loci, each one might independently confer some low level of resistance that had not been observed by the usual scoring method of observing ascospores shot to the walls of the crossing tubes. Among all the crosses used in Table 4, the test crosses to Sk-2 of a few of the progeny had been observed more closely. It appeared that the proximal element conferred some slight resistance on its own. For example, an acr-2þ ad þ progeny of cross 1170 was not resistant like the acr-2þ ad þ parent, but it had asci with more than four black ascospores mixed among the 4:4 asci. No evidence of partial resistance was found for the putative distal element. The two elements were provisionally named ‘‘partial resistance to Sk-2,’’ and ‘‘modifier of partial resistance,’’ symbolized as pr(Sk-2) and mod(pr). 3.3. Mapping and characterization of the two new loci—separating the two components of resistance Fig. 2 diagrams the genotypes of parents and critical progeny presented in this and the concluding section. 3.3.1. Cross 1186: pr(Sk-2) mod(pr) ser-1 1180-8 A ad-4 leu-1 a Cross 1186 was set up to obtain and characterize progeny that would have a mutant (wild variant) allele at one locus but not the other, and to continue genetic mapping. The 232 progeny are summarized in Table 5. All 232 were tested for the three biochemical markers, and all progeny recombinant for the biochemical markers were crossed to Sk-2. There were 102 progeny of the parental type, ser-1 ad-4þ leu-1þ . Seventy of these were crossed to Sk-2. Only a few of the 107 progeny with the three markers from the Sk-2-sensitive parent (ser-1þ ad-4 leu-1) were put through the time-consuming test for resistance. Even if a resistant progeny had been found among them, it would have arisen from a multiple crossover and would not have assisted in ordering the loci. 3.3.2. Phenotypes of the separated loci (1) pr(Sk-2) confers partial resistance in the absence of mod(pr). The crosses to Sk-2 testers were examined by opening perithecia and looking at intact asci. Among the 70 tested ser-1 ad-4þ leu-1þ progeny, nine showed the slight resistance tentatively attributed to pr(Sk-2) in the absence of mod(pr). Four of these nine test crosses were scored as ‘‘probable’’ because they had very few intact asci, but the other five had many individual perithecia with dozens of 4:4 asci and one or two 8:0 asci. One of these five progeny, pr(Sk-2) ser-1 1186-147
Fig. 2. Separation and rejoining of genes at the two loci that modify resistance to killing by Sk-2. Alleles from wild strain P2604 are in boldface type. (A) In cross 1186, single crossovers in two separate meioses produced the progeny shown. (B) In cross 1187, the two critical segments of 1180-8 that were separated by crossing over are reunited in isolate 1187-1. [Linkage Group IIIL is homozygous for the sensitive allele of r(Sk-2) in both crosses. The resistant allele of r(Sk2) would confer resistance regardless of the genotype at the pr(Sk-2) and mod(pr) loci].
a, was saved for stock and was used in the further analysis (cross 1187) reported in the final section and shown in Fig. 2, part B. Cross 1189: pr(Sk-2) ser-1 1186-147 a Sk-2 A was used to get an estimate of the level of resistance to killing of pr(Sk-2) in the absence of mod(pr). Fifty-four progeny were crossed to the fl testers to score for killing. Two of the 54 were non-killers. Incidence of non-killers
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Table 5 Recombination of LGIII markers with pr(Sk-2) and mod(pr) CLASS
ser-1 (1)
pr(Sk-2) (2)
Cross 1186 Sk-2-resistant ser-1 1180-8 A ad-4 leu-1 Parental ) R + S
ad-4 (3)
leu-1 (4)
mod(pr)
Number
+ )
+ )
R S
(89) (93)
Single in (1)
) +
S R
) +
) +
S? R
5 6
Single in (2)
) +
R S
) +
) +
S R?
0 11
Single in (3)
) +
R S
+ )
) +
S Ra
0 1
Single in (4)
) +
R S
+ )
+ )
S R
(13) (14)
Note. For each of the resistance loci, ‘‘S’’ stands for the allele that does not contribute to resistance (and hence is sensitive), and ‘‘R’’ stands for the allele that does contribute to resistance. The allocation of progeny between the classes ‘‘Parental’’ and ‘‘Single crossover in region 4’’ is only an estimate based on crossing 70 of the 102 ser leuþ ad þ progeny to Sk-2. 61 were pr(Sk-2) mod(pr) (fully resistant), and 9 were pr(Sk-2) modðprÞþ (partially resistant). 107 progeny were serþ leu ad. a mod(pr) cannot be scored directly when the sensitive allele of pr(Sk-2) is present. This isolate, ad-4 mod(pr)1186-17, was scored for mod(pr) by progeny analysis (see text and Fig. 2).
from a cross of sensitive Sk-2, is usually less than 103 (Campbell and Turner, 1987), so the partial resistance effect of pr(Sk-2) was verified. (2) mod(pr) has no effect in the absence of pr(Sk-2). It was expected from previous crosses that likely recombinants between pr(Sk-2) and mod(pr) could be selected from reciprocal recombinants between ad-4 and leu-1. Contrary to expectations, no ad-4þ leu-1 isolate was obtained. Fortunately, one isolate, 1186-17A (see Fig. 2), was ser-1þ ad-4 leu-1þ . In its cross Sk-2, no ascus had more than four viable ascospores, indicating, as expected from previous crosses, that it had the sensitive allele of pr(Sk-2). For confirmation, 65 progeny were obtained from this test cross, and all were Sk-2. The confirmation that it carried the resistant allele of mod(pr) was obtained by cross 1187, presented in the final section. 3.3.3. Mapping of the two loci The results summarized in Table 4 had already indicated that the two loci were between acr-2 and his-7. Six progeny of cross 1186 were serþ ad þ leuþ (resulting from a crossover between ser-1 and all the other loci) and were fully resistant. This result located both of the resistance loci to the right of ser-1. These six progeny can be paired with a presumptive reciprocal class of five ser-1 ad-4 leu-1 Sk-2-sensitive (fully sensitive). Eleven progeny were serþ ad þ leuþ and fully sensitive. This result located pr(Sk-2) left of ad-4, with the 11 progeny resulting from a single crossover between pr(Sk-2) and ad-4. There were no progeny reciprocal to these eleven, and contrary to expectations from previous crosses, only one isolate, 1186-17A, resulted from a crossover between ad-4 and leu-1 (see Section 4: Prospects for future studies).
The proximal locus, pr(Sk-2), is within the ‘‘recombination block’’ (Campbell and Turner, 1987), but mod(pr) appears to be outside it. The right end of the recombination block was found by Campbell and Turner to be essentially inseparable from leu-1. In the results presented here for cross 1186, mod(pr) appeared to be about 13 map units distal to leu-1 (9/70 ser-1 ad-4þ leu-1þ progeny were partially resistant). 3.4. Reuniting the two components of resistance 3.4.1. Cross 1187: mod(pr) ad-4 1186-17 A pr(Sk-2) ser-1 1186-147 a The parents in cross 1187 were described in the previous section and are shown in Fig. 2. Thirty-seven progeny were obtained. Fifteen were ad-4 ser-1þ , and 22 were ad-4þ ser-1. The 22 ad-4þ ser-1 progeny were crossed to Sk-2. Three of them produced abundant 8:0 asci in their test crosses to Sk-2 and thus must have had the genotype pr(Sk-2) mod(pr) ser-1, resulting from crossing over between ad-4 and mod(pr). Therefore, the mod(pr) genotype of their parent, 1186-17 was confirmed. One of these three test crosses, ser-1 1187-1 a Sk-2 A, was designated cross 1190 and was further analyzed. 3.4.2. Cross 1190: Further confirmation of the reconstitution of high resistance The usual criterion for scoring full resistance is the observation of mostly 8:0 asci. As an additional check on the pr(Sk-2) mod(pr) genotype of 1187-1 a, one hundred progeny were obtained from cross 1190 [1187-1 ser-1 pr(Sk-2) mod(pr) Sk-2] to see whether the level of survival (resistance) of the non-Sk-2 ascospores would be comparable to that of the pr(Sk-2) mod(pr)
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strains in their ancestry. Forty of the 100 progeny were ser-1, and all of these 40 were non-killers, meaning that they were pr(Sk-2) mod(pr). Recovery of non-killers from crosses of the original Malaya stock ranged from 30% to 50%, so this result is within the range expected of a reconstituted pr(Sk-2) mod(pr) resistant genotype.
to the two anonymous reviewers who worked hard to understand the complexities of the results and to suggest more understandable ways to present them. This work was carried out as part of a comprehensive program for Neurospora genetics in the Perkins laboratory, supported by Research Grants AI-01462 from the National Institutes of Health and MCB-9728675 from the National Science Foundation.
4. Discussion For the Sk-2 system in Neurospora, only the killer chromosome segment and one resistance locus, r(Sk-2), had been identified. The two new loci described in this report have significantly altered our concept of the complexity of the system. While r(Sk-2) is toward the left end of LGIII, very close to the left end of the Recombination Block associated with Sk-2, the two new loci are in the right arm of LGIII. pr(Sk-2) is within the Recombination Block, but mod(pr) is distal to it. By showing further complexity in the Sk-2 system, the results of this study enhance the previous findings that meiotic drive in Neurospora resembles meiotic drive in Mus and Drosophila. Prospects for future studies. No further work involving these new loci is contemplated. The recombination anomalies observed in many of the crosses may signal previously unknown recombination loci. There may be undiscovered factors (including rearrangements too small to produce a recognizable pattern of aborted ascospores) affecting the recombination patterns of the region between pr(Sk-2) and leu-1. As shown by this study alone, such tangential problems arise constantly when working with strains from nature, and it is not possible to resolve each one. Strains that carry one or both variant alleles and close markers have been deposited in FGSC (see Table 1). Obviously the present study has not exhausted the possibilities for finding variation in the resistance to spore killing of N. crassa, nor has it addressed the distribution of pr(Sk-2) and mod(pr) among collected strains. Sk-2 is far more abundant in N. intermedia than in N. crassa, and Sk-3 is known only in N. intermedia. For those working with either population biology or meiotic drive, there are hundreds of N. intermedia strains available for continuing analysis of resistance to Spore killers.
Acknowledgments I am grateful to David Perkins, Edward Barry, N.B. Raju, and David Jacobson for advice and comment on the research project, Dorothy Newmeyer and Edward Barry for reviewing the first drafts of the report, and David Jacobson for preparing the figures. I am indebted
References Bengtsson, B.O., Uyenoyama, M.K., 1990. Evolution of the segregation ratio: Modification of gene conversion and meiotic drive. Theoretical Population Biology 38, 192–218. Campbell, J.L., Turner, B.C., 1987. Recombination block in the Spore killer region of Neurospora. Genome 29, 129–135. Davis, R.H., De Serres, F.J., 1970. Genetic and microbiological research techniques for Neurospora crassa. In: Tabor, H., Tabor, C.W. (Eds.), Methods in Enzymology, Vol. 17. Academic Press, New York, pp. 79–143. Lyttle, T.W., 1991. Segregation distorters. Annu. Rev. Genet. 25, 511– 557. Perkins, D.D., Barry, E.G., 1977. The cytogenetics of Neurospora. Adv. Genet. 19, 133–285. Perkins, D.D., Bojko, M., 1992. The basis of decreased recombination in certain outcrosses of Neurospora crassa. Genome 35, 503–509. Perkins, D.D., Turner, B.C., 1988. Neurospora from natural populations: Toward the population biology of a haploid eukaryote. Exp. Mycol. 12, 91–131. Raju, N.B., 1979. Cytogenetic behavior of Spore killer genes in Neurospora. Genetics 93, 607–623. Raju, N.B., 1994. Ascomycete Spore killers: Chromosomal elements that distort genetic ratios among the products of meiosis. Mycologia 86, 461–473. Schimenti, J., 2000. Segregation distortion of mouse t haplotypes: The molecular basis emerges. Trends Genet. 16, 240–243. Silver, L.M., 1989. Gene dosage effects on transmission ratio distortion and fertility in mice that carry t haplotypes. Genet. Res. 54, 221– 226. Silver, L.M., 1996. Maps of the mouse t complex, third ed.. In: Lyon, M.F., Rastan, S., Brown, S.D.M. (Eds.),. Genetic Variants and Strains of the Laboratory Mouse, Vol. 2. Oxford University Press, London, pp. 925–928. Temin, R.G., Ganetzky, B., Powers, P.A., Lyttle, T.W., Pimpinelli, S., Dimitri, P., 1991. Segregation distortion in Drosophila melanogaster: Genetic and molecular analyses. Am. Nat. 137, 287–331. Turner, B.C., 1977. Resistance to Spore killer genes in Neurospora strains from nature. Genetics 86, s65–s66 (Abstr.). Turner, B.C., 1987. Two ecotypes of Neurospora intermedia. Mycologia 79, 425–432. Turner, B.C., 2001. Geographic Distribution of Neurospora Spore killer strains and strains resistant to killing. Fung. Genet. Biol. 32, 93–104. Turner, B.C., Perkins, D.D., Fairfield, A., 2001. Neurospora from Natural Populations: A Global Study. Fung. Genet. Biol. 32, 67– 92. Turner, B.C., Perkins, D.D., 1979. Spore killer, a chromosomal factor in Neurospora that kills meiotic products not containing it. Genetics 93, 587–606. Turner, B.C., Perkins, D.D., 1991. Meiotic drive in Neurospora and other fungi. Am. Naturalist 137, 416–429. Yeadon, P.J., Catcheside, D.E.A., 1995. The chromosomal region which includes the recombinator cog in Neurospora crassa is highly polymorphic. Curr. Genet. 28, 155–163.
Analysis of two additional loci in Neurospora crassa related to Spore killer-2 Barbara C. Turner* Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020, USA Received 13 March 2002; accepted 18 February 2003
Abstract Two new loci found in one strain of Neurospora crassa (P2604) collected in Malaya are related to the meiotic drive system Spore killer Sk-2. Sk-2 was found in Neurospora intermedia and introgressed into N. crassa. P2604 showed high resistance to killing when crossed to Sk-2. This resistance was found to be linked to, but not allelic to, resistance locus r(Sk-2) on LGIIIL. Analysis showed that the high resistance phenotype of P2604 requires resistance alleles at two different loci on LGIIIR. Strains carrying a resistance allele at only the proximal or the distal locus, respectively, were obtained and intercrossed. Highly resistant strains were obtained by rejoining the two genes. The proximal locus alone confers a low level of resistance. This locus was named pr(Sk-2) for partial resistance to Sk-2. The distal locus was named mod(pr) because its only known phenotype is to modify pr(Sk-2). Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Meiotic drive; Spore killer; Neurospora
1. Introduction The terms ‘‘meiotic drive’’ and ‘‘segregation distortion’’ are used to describe certain matings in which an ordinarily viable chromosome, chromosome segment, or locus is recovered in less than half of the progeny, if recovered at all (see Lyttle, 1991 for a comprehensive presentation of the systems that have been studied extensively). An important question about meiotic drive is whether there are common evolutionary forces that can be discerned in these systems without regard to the species involved or the mechanism by which the drive operates (see Bengtsson and Uyenoyama, 1990). An example of a common outcome for three different systems is provided by crossover suppression. There are inversions that suppress recombination associated with the distorting chromosomes of Segregation distortion in Drosophila melanogaster (Temin et al., 1991) and the t haplotype in Mus spp. (Schimenti, 2000; Silver, 1989), while the Spore killer system of Neurospora includes a * Fax: 1-650-723-6132. E-mail address: [email protected].
virtually complete recombination block in an extensive region containing the Spore killer factor (Campbell and Turner, 1987). Mus and D. melanogaster have long been known to resemble each other in having many contributing loci, most of which are closely linked. The mouse t haplotype chromosomes (variants of chromosome 17) carry several trans-acting distorter loci and a responder locus. They also carry other mutations that enhance the segregation distortion. Silver, 1996, presented a map of the known loci of the distorter system and maps of partial haplotypes that have been used to dissect the system. In the Segregation Distorter (SD) system of D. melanogaster there are numerous loci on the SD chromosome, of which several have been studied in detail both for their effects and for their molecular basis (Temin et al., 1991). There is a main SD locus, a responder locus, an enhancer locus that strongly affects amount of distortion, and other loci that have smaller effects. It was speculated that there might be a similar situation of additional contributing loci in the meiotic drive systems of Neurospora. This report presents results of a search for additional loci related to one of those systems, Spore killer-2.
1087-1845/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S1087-1845(03)00018-5
B.C. Turner / Fungal Genetics and Biology 39 (2003) 142–150
Spore killers were reported by Turner and Perkins (1979), and cytological observations were reported by Raju (1979). (For summaries of subsequent findings see Raju, 1994; Turner, 2001; and Turner and Perkins, 1991). Spore killers Sk-2 and Sk-3 were found as rare variants in N. intermedia, a species that hybridizes readily with N. crassa. No Spore killer has been found in N. crassa, but for mapping and other genetic study, both Sk-2 and Sk-3 were introgressed from N. intermedia into N. crassa because of the far greater number of known genetic markers in N. crassa (Turner and Perkins, 1979). The effect of Spore killer is observed only in heterozygous crosses. In crosses of Spore killer sensitive, each ascus contains four viable black ascospores carrying the killer factor and four inviable, undersize, transparent (hyaline) ascospores (4:4 asci). The Spore killer element has no obvious effect on the vegetative growth of a strain carrying it nor on the survival of ascospores in homozygous Sk-2 Sk-2 or Sk-3 Sk-3 crosses. (In some previous publications, the killer elements were symbolized as Sk-2K and Sk-3K , but here are shortened to Sk-2 and Sk-3.) This study involved only Sk-2. Killing (which necessarily includes self-resistance) has been shown to segregate as a chromosomal locus or complex of loci in linkage group III. Recombination is almost completely blocked between Sk-2 and markers in a segment of linkage group III that is ordinarily at least 30 map units long (Campbell and Turner, 1987). Resistance to Spore killers was discovered in collected strains of N. intermedia very soon after the Spore killer elements themselves were discovered (Turner and Perkins, 1979). After the Spore killers were introgressed into N. crassa, a resistance locus, called r(Sk-2), was detected in a N. crassa strain collected in Louisiana. It was studied genetically by Turner and Perkins (1979) and by Campbell and Turner (1987). Resistance segregates as a single locus in the left arm of linkage group III in crosses of r(Sk-2) marked strains that are sensitive to Sk-2. In those crosses, the presence of the resistant allele has no effect on crossover frequencies in the linkage group. When strains carrying the resistant allele of r(Sk-2) and linked markers are crossed to N. crassa Sk-2, resistance does not alleviate the crossover suppression. The ongoing study of Spore killers overlapped with a project to set up a worldwide collection of Neurospora cultures from nature, now available as the Perkins collection from Fungal Genetics Stock Center (Perkins and Turner, 1988; Turner et al., 2001). One part of the program involved screening isolates of three Neurospora species from over 700 collection sites for further instances of Spore killer or genes modifying its effect (Turner, 1977 and Turner, 2001). In N. crassa, the only phenotype found that was related to Spore killers was resistance to Sk-2. This report describes a survey of resistant N. crassa strains from widespread locations and presents a detailed analysis of one of them.
143
2. Materials and methods Procedures for culturing Neurospora were described by Davis and De Serres (1970), and laboratory procedures related to Spore killer were described by Turner (2001). 2.1. Identification of resistance to Spore killer The protocol initially adopted for this project was the one used for scoring resistance while mapping r(Sk-2) (Campbell and Turner, 1987). Each strain was crossed to an Sk-2 tester (FGSC 6647a or 6648A) that had been inoculated 4 or 5 days previously in a glass culture tube. After 9 days, ascospores shot to the walls of the culture tubes were examined to determine whether the asci were 8:0 (all ascospores black) or 4:4 (four full size black ascospores:four tiny transparent aborted ascospores). Experience showed that resistance could be scored reliably by examining the sides of the culture tubes to determine whether shot asci had 8 black ascospores or 4 black:4 aborted ascospores. If the scoring was uncertain because of too few spores, excessive spore abortion from other causes, or difficulty in identifying discrete asci among the shot spores, perithecia were removed from the tube and dissected. As this project progressed, an intermediate class of progeny was discovered. These progeny produced mostly 4:4 asci when crossed to Spore killer, but also a few 8:0 asci. This pattern had been overlooked when scoring was done by simply looking at the walls of the culture tubes. For all crosses involved in separating and recombining the two new loci described in the Section 3, scoring was done by removing at least six perithecia from the culture tube and opening them to observe intact asci. 2.2. Strains are listed in Table 1 The N. crassa species testers are referred to in the text simply as fl A and fl a where that is appropriate. The N. crassa Sk-2 testers are referred to as Sk-2 A or Sk-2 a. In the text, any strain that carries Sk-2 or resistance to Sk-2 is identified explicitly. Otherwise, the marked strains used for mapping were laboratory strains sensitive to Spore killers. (The marker r(Sk-3), which was introgressed from N. intermedia, does not interact with the Sk-2 system and was used simply because it marks the left end of the linkage group.) Both wild-collected strains from the Perkins collection (Turner et al., 2001) and laboratory strains with FGSC numbers can be obtained from the Fungal Genetics Stock Center, Department of Microbiology, University of Kansas Medical Center, Kansas City,
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KS 66160–7420. At the end of the study, a pair of strains from the final cross in the series of crosses to lab strains was deposited in FGSC. Their numbers are 8275A and 8276a. They are recommended for any work involving crosses of the new mutants to laboratory stocks.
3. Results For those who are interested only in the location and characteristics of the two loci being reported, the section of these Results headed, ‘‘Mapping and characterization of the two new loci’’ will provide that information. Confirmation for the results in that section is provided in the final section, ‘‘Separation and resynthesis of the two components.’’ 3.1. Survey of strains of N. crassa resistant to Sk-2 This study was undertaken to find out whether more than one genotype can confer the phenotype of resistance to killing by Sk-2, a meiotic drive mutant in Neurospora. Strains resistant to Sk-2 occur in nature in both N. intermedia and N. crassa. N. crassa was
selected for study for two reasons: (1) unlike the case of N. intermedia, the N. crassa-resistant strains occur as isolated individuals in widely separated populations rather than as a significant segment of one widespread but contiguous region, and (2) genetic markers for mapping the site of a new locus are far more available in N. crassa. The traditional (non-molecular) method of determining whether a newly arisen mutation is allelic (or identical) to a previously identified gene with a similar phenotype was used for this survey. The ‘‘previously identified allele’’ was r(Sk-2), the Sk-2 resistance gene found in a wild-collected isolate from Louisiana (USA). A strain carrying r(Sk-2) was crossed to other wildcollected-resistant strains, and progeny were observed to determine whether or not recombinant (sensitive) progeny were produced. acr-2 r(Sk-2) A FGSC 7379 was crossed to Sk-2-resistant strains (see Table 1 and Turner, 2001) from Brazil, Haiti, India, Ivory Coast, Costa Rica, and Malaya. It was expected that an unlinked resistance locus should reassort with r(Sk-2), so that 25% of progeny would be sensitive to Sk-2 (scored by presence of asci with 4 large black:4 aborted ascospores) because of receiving the sensitive allele at both loci. A separate but linked locus would be expected to
Table 1 Strains used in crosses Description
Generation
FGSC number
A. Strains carrying pr(Sk-2) and mod(pr) P2604 cum pr(Sk-2) mod(pr) 1046-73 A pr(Sk-2) mod(pr) 1051-78 a pr(Sk-2) mod(pr) 1051-81 A pr(Sk-2) mod(pr) ser-1 1125-43 A pr(Sk-2) mod(pr) ser-1 1180-8 A mod(pr) ad-4 1186-17 A pr(Sk-2) ser-1 1186-147 a pr(Sk-2) mod(pr) ser-1 1187-1 a
original second third third fourth fifth sixth sixth progeny of 8272 8273
P2604 (Perkins)
B. Laboratory stocks used for mapping and analysis Description
Comments
FGSC Number
a
flA fla SK-2 a Sk-2 A acr-2 rðSk-2ÞA (3291) acr-2 ad-4 C39-20 a (3230) r(Sk-3) ser-1 902-98 a leu-1 thi-2 a ad-4 leu-1 a (3310) leu-1 a (3031) cum acr-7 A Sk-2 acr-2 leu-1 A Sk-2 acr-2 leu-1 a
Standard Standard Standard Standard
N. N. N. N.
crassa crassa crassa crassa
species tester species tester Sk-2 Sk-2
7398
8274 8272 8273
1838 1690 6647 6648
7162
7156 7375 7374
Note. For strains without FGSC numbers, other deposits with the same loci are available from FGSC. Numbers in parentheses are Perkins Lab silica gel numbers. fl (fluffy) is a mutation that results in absence of macroconidia, which makes shot ascospores much easier to observe. It also confers high fertility. a See Table 2 for the lineage of these strains.
B.C. Turner / Fungal Genetics and Biology 39 (2003) 142–150
yield some (fewer than 25%) sensitive progeny by crossing over. 3.1.1. Results of survey crosses Only strain P2604 from Malaya (cross 1005, Table 2) gave results suggesting a new resistance locus. When crossed to Sk-2, at least one, and probably three, of the 66 progeny from cross 1005 were sensitive to killing. For the strains from Brazil, Haiti, India, Ivory Coast, and Costa Rica, crosses to acr-2 r(Sk-2) A yielded only progeny resistant to Sk-2. For each of these five crosses, 50 to 80 progeny were fertile enough to be scored in crosses to the Sk-2 testers, and they were all resistant (not making 4:4 asci). Many more progeny tests would have been needed to rule out the presence of a second locus closely linked to r(Sk-2), but that was not the purpose of the survey. The aim was to look for loci that could be detected with progeny sets of this size. 3.1.2. Background and crossing characteristics of strain P2604 a from Penang, Malaya P2604 is a wild-collected N. crassa strain that does not carry a Spore killer itself but is resistant to killing by
Table 2 Crosses A. Crosses with P2604 from Penang, Malaya P2604 pr(Sk-2) mod(pr) a fl A FGSC 1838 P2604 pr(Sk-2) mod(pr) a Sk-2 A FGSC 6648 1005 P2604 pr(Sk-2) mod(pr) a r(Sk-2) acr-2 A 1046 P2604 pr(Sk-2) mod(pr) a cum acr-7 A 1156 P2604 pr(Sk-2) mod(pr) a acr-2 leu-1 his-7 A 1157 P2604 pr(Sk-2) mod(pr) cum acr-7 thi-4 348-22 A B. Initial 1051 1064 1065 1125 1126 1149 1165 1168 1170
mapping crosses with descendants of P2604 cum pr(Sk-2) mod(pr) 1046-73 A leu-1 thi-2 a pr(Sk-2) mod(pr) 1051-78a ad-4 leu-1 A pr(Sk-2) mod(pr) 1051-81A ad-4 acr-2 a pr(Sk-2) mod(pr) 1051-81A r(Sk-3) ser-1 a pr(Sk-2) mod(pr) 1051-81A ad-4 acr-2 a ser-1 pr(Sk-2) mod(pr) 1125-43 A Sk-2 acr-2 leu-1 a pr(Sk-2) mod(pr) 1157-15 a acr-2 leu-1 his-7 A pr(Sk-2) mod(pr) 1157-15 a pro-1 A (3135) ser-1 pr(Sk-2) mod(pr) 1168-7 A ad-4 acr-2 C39-21 a
C. Separation and resynthesis of the resistance genes of P2604 1180 pr(Sk-2) mod(pr) ser 1125-43 A leu-1 a 1186 pr(Sk-2) mod(pr) ser-1 1180-8 A ad-4 leu-1 a 1187 mod(pr) ad-4 1186-17 A pr(Sk-2) ser-1 1186-147 a 1188 mod(pr) ad-4 1186-17 A Sk-2 a (3213) 1189 pr(Sk-2) ser-1 1186-147 a Sk-2 A (3212) 1190 pr(Sk-2) mod(pr) ser-1 1187-1 a Sk-2 A Note. The lineage of all stocks carrying the new variants, pr(Sk-2) and mod(pr), can be derived by reference to their isolation numbers. The portion in bold face is the number of the cross from which the isolate was obtained, and this cross can be found higher up in the table. The cross numbers in bold italics in the first column show the lineage of pr(Sk-2) mod(pr) ser-1 1187-1 a, the strain in which pr(Sk-2) and mod(pr) were rejoined. A diagram of crosses 1186 and 1187 is presented in Fig. 2.
145
Sk-2. It was collected at the easternmost border of the known range of N. crassa Penang, an island just west of the mainland of Malaya. Results for test crosses (to the species testers and Sk-2 testers listed in Table 1) of Perkins Collection strains from Malaya were reported previously in summary form (Turner et al., 2001; Turner, 2001), but this is the first report of specific crossing results for P2604. P2604 was not immediately diagnosed as N. crassa when crossed to the standard species tester of N. crassa (fl A FGSC 1838). Many of the cultures from Penang, including P2604, made mostly full-size, pigmented ascospores when crossed to the N. crassa testers, but more than 50% of those ascospores were inviable (tan and brown) (Turner et al., 2001). Some other Penang strains produced 90% or more black viable ascospores when crossed to the N. crassa species testers and thus were identified with certainty as N. crassa. P2614 A, one of the identified N. crassa strains from Penang, was selected as an auxiliary N. crassa species tester and was crossed to P2604 a. This cross produced at least 90% black ascospores, demonstrating that P2604 was also N. crassa. Because P2614 is sensitive to Sk-2, this cross also demonstrated (with more certainty than P2604 a flA) that P2604 does not carry Sk-2. Like all wild-collected N. crassa strains, P2604 was crossed to the Sk-2 tester of opposite mating type (Turner, 2001), and it was diagnosed as resistant. After cross 1005 showed that P2604 probably carried a new resistance factor, the cross to Sk-2 A was repeated and scrutinized more carefully. There were many full-size defective (brown, tan, and white) ascospores scattered randomly among asci. Some ascospores did have the characteristic (tiny, clear) appearance of ascospores killed by Spore killer, but they were usually found in completely aborted asci. Asci were not observed with a pattern of 4 viable:4 tiny ascospores. Most importantly, many asci had more than four large, pigmented ascospores. Therefore, P2604 was confirmed as resistant to Sk-2. The incidence and pattern of defective ascospores in crosses of P2604 to fl A and Sk-2 A was not remarkable. In all the known species of Neurospora, crosses of geographically distant strains may be poorly fertile or produce only a minority of viable ascospores (Turner et al., 2001). The pattern of defective ascospores for P2604 was random, not in pairs as it would be if the primary reason for defective ascospores were the presence of one or more rearrangements (Perkins and Barry, 1977). 3.1.3. Results for cross 1005:P2604 a acr-2 r(Sk-2) A Ascospore characteristics for the survey cross of P2604 were similar to the cross results just described for P2604 fl A and P2604 Sk-2A. Sixty-six progeny were obtained from cross 1005 and crossed to the Sk-2 testers. Many of these 66 crosses produced few viable
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ascospores, so they were difficult, and in some cases impossible, to score for resistance. However, the test crosses of three of the progeny gave no asci with more than four good ascospores (for two of these three progeny, the level of general spore abortion was high, making a diagnosis of sensitivity to Sk-2 only tentative). Based on this result of Sk-sensitive progeny segregating from P2604 acr-2 r(Sk-2) A (resistant resistant), further crosses were carried out to confirm and map the new resistance ‘‘locus.’’ 3.2. Development of stocks and initial mapping results Fig. 1 shows the markers from LGIII that were used in this study. In order to proceed with analysis of the new resistance ‘‘locus’’ it was desirable to develop stocks that would retain the resistance to Sk-2 but have normal fertility with laboratory stocks. After a second generation of back-crossing to laboratory stocks, ascospore viability improved greatly and scoring for Spore killing was much easier. Crosses are listed in Table 2. For progeny of the first generation of crosses ( P2604), resistance was not always scored with confidence. 3.2.1. Cross 1046 located the resistance ‘‘locus’’ far to the right of cum For cross 1046, among the cumþ progeny, 21 were resistant (parental coupling) and 24 were sensitive to Sk2 (recombinant). The map derived from the cross was cum—29—acr-7—24—(Sk-2 resistance). Since r(Sk-2) is only one map unit to the right of cum, the recombination results of cross 1046 were inconsistent with the survey cross result, (maximum of 9 map units between r-(Sk-2) and the new resistance ‘‘locus’’). These disparate results may be due to one or both of two phenomena previously observed in Neu-
Fig. 1. Map of LGIIIL and proximal portion of LGIIIR RB ¼ the region where recombination is blocked in crosses heterozygous for Sk2 (or Sk-3). The RB contains the locus or loci responsible for spore killing, but more precise mapping of Sk-2 is impossible because spore killing does not recombine with loci in the RB. O ¼ centromere The loci introduced in this report are in boldface type. Loci within square brackets have not been ordered. Analysis of r(Sk-2), locus that confers resistance to Sk-2, was reported previously (Turner and Perkins 1979; Campbell and Turner, 1987). r(Sk-3), which confers resistance to Sk-3, was introgressed into N. crassa from N. intermedia. It does not interact with the Sk-2 system. acr-2 and acr-7 confer resistance to acriflavin. They are completely silent under normal crossing and culture conditions. All loci are described in Perkins et al., 2000. Intervals in the diagram are not proportional to genetic map distance. Recombination rates varied greatly from cross to cross, as might be expected when working with loci obtained from a stock collected on the other side of the globe from the ancestors of laboratory stocks (see text).
rospora. (1) Because many loci affecting recombination occur in N. crassa (Perkins and Bojko, 1992; Yeadon and Catcheside, 1995), it is not unusual to see widely differing map distances in different crosses with the same markers, particularly when strains of different background are used. Variant recombination loci may explain not only the low recombination in cross 1005, but also variation from cross to cross for the map distances between any two markers in this study. (2) The apparent infrequency of recombinants in cross 1005 may have been attributable to unrelated loci that resulted in poor viability of crossover progeny (‘‘synthetic lethals’’) (see Turner, 1987). 3.2.2. Introgression of resistance In general, each generation of crossing to laboratory stocks halved the amount of the genome inherited from P2604 and thus decreased the potential for inviability of progeny ascospores in the following generation. Selecting for resistance to Sk-2, however, would tend to retain other segments of LGIII from P2604. Therefore, markers were deliberately used to increase the probability of obtaining stocks that would have full Sk-2 resistance from P2604 but eliminate material to the left of the region carrying it. 1046-2 A was selected for use in cross 1051 because it combined the left end marker cum (and presumably part of LGIII from the laboratory parent) with resistance to Sk-2. (Inexplicably, only about 20 of the 119 progeny obtained from cross 1051 carried cum, and only one of these had all the other parental markers.) Two strains carrying cumþ (and presumably eliminating even more of the LGIII material that was carried over from P2604) were selected from cross 1051 for use in further mapping crosses. Later, strain 1125-49 substituted ser-1 and any material to the left of ser-1 that remained from P2604. In cross 1125, 32 of 84 progeny (38%) were recombinant for r-(Sk-3) and resistance to Sk-2. 3.2.3. Mapping results for markers closely linked to the Sk-2 resistance from P2604 The crosses in Table 2, part B, were first analyzed assuming that resistance to Sk-2 was conferred by a single locus. Problems with this assumption were apparent, as illustrated by cross 1064 in Table 3. Looking at the data for cross 1064, two problems are seen: (1) Three of the classes do not have reciprocals, and (2) the double crossover class is unexpected considering the few progeny in the single crossover classes (:064 :046 ¼ :003 expected doubles). Pooled results for this stage of analysis are shown in Table 4. For all markers in IIIR, the Sk-2-sensitive/ markerþ recombinant classes were obtained. For markers between ser-1 and his-7, no Sk-2-resistant recombinant class was obtained. For example: pro-1 Sk-2-sensitive pro-1þ Sk-2-resistant produced pro-1þ
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147
Table 3 Example of presumptive progeny classes, assuming a single locus confers resistance to killing: Cross 1064 Sk-2 resistant 1051-78 a ad-4 leu-1 A CLASS
ad-4-region 1
Sk-2 resistance-region 2
leu-1
Parental
) +
S R
) +
45 49
) + ) + ) +
R S S R R S
+ ) + ) ) +
0 5 7 0 0 3
Crossover classes Single in region 1 Single in region 2 Double
Number
Total
109
Note. S, sensitive and R, resistant. Cross 1064 illustrates the anomalous results obtained when mapping crosses were analyzed under the initial assumption that there was only one resistance locus in strain P2604. There are three crossover classes without reciprocals, and the presumed double crossover class is unexpected considering the size of the presumed single crossover classes.
Table 4 Recombination of LG III markers with resistance to Sk-2 Cross
Marker
Recombinantsa Sk-2-sensitive/markerþ
Sk-2-resistant/marker
Total Progeny
1065 1126 1170 1156 Totals for acr-2
acr-2 acr-2 acr-2 acr-2
17 15 2 5 39
7 5 2 3 17
91 87 50 48
1125 Totals for ser-1
ser-1
5 5
1 1
84
1168 Totals for pro-1
pro-1
4 4
0 0
70
1064 1065 1126 1170 Totals for ad-4
ad-4 ad-4 ad-4 ad-4
8 1 10 2 21
0 0 0 0 0
109 91 87 50
1051 1064 1156 Totals for leu-1
leu-1 leu-1 leu-1
19 10 2 31
0 0 0 0
113 109 48
1156 1165 Totals for his-7
his-7 his-7
6 7 13
2 1 3
48 88
1051 Totals for thi-2
thi-2
27 27
12 12
113
Note. The loci are listed as they occur from left to right in the linkage group. This table illustrates a gradient of unequal recovery of reciprocal recombinants on either side of the zone (containing pro-1 ad-4 and leu-1) where no recombinants resistant to Sk-2 were recovered. a Recombinants between the marker in column 2 and resistance/sensitivity to Sk-2. For example, for cross 1065, the parents were Sk-2 sensitive acr-2 Sk-2-resistant acr-2þ . 17 progeny were Sk-2 sensitive acr-2þ , and 7 were Sk-2-resistant acr-2. Recombinants are tallied taking into account only one marker at a time (see text). Thus for cross 1064 (see Table 3) recombinants between Sk-2 resistance and leu-1 are listed without regard to ad4 genotype.
Sk-2-sensitive progeny but did not produce pro-1 Sk-2resistant progeny. Thus it appeared possible that in a stretch of the chromosome that included pro-1 ad-4 and leu-1, recombinants resistant to Sk-2 were not obtainable by a single crossover.
3.2.4. Two-gene hypothesis The mapping results shown in Tables 3 and 4 were similar to results for known two-locus systems (for example, the color loci reported in Turner, 1987). Thus it appeared possible that resistance required two interact-
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ing genes at two different loci, and that these loci were separated by crossovers between leu-1 and ad-4 in cross 1064. The consistent cumulative results summarized in Table 4 are more compelling evidence for a two-locus hypothesis than any single cross. If there were two loci, each one might independently confer some low level of resistance that had not been observed by the usual scoring method of observing ascospores shot to the walls of the crossing tubes. Among all the crosses used in Table 4, the test crosses to Sk-2 of a few of the progeny had been observed more closely. It appeared that the proximal element conferred some slight resistance on its own. For example, an acr-2þ ad þ progeny of cross 1170 was not resistant like the acr-2þ ad þ parent, but it had asci with more than four black ascospores mixed among the 4:4 asci. No evidence of partial resistance was found for the putative distal element. The two elements were provisionally named ‘‘partial resistance to Sk-2,’’ and ‘‘modifier of partial resistance,’’ symbolized as pr(Sk-2) and mod(pr). 3.3. Mapping and characterization of the two new loci—separating the two components of resistance Fig. 2 diagrams the genotypes of parents and critical progeny presented in this and the concluding section. 3.3.1. Cross 1186: pr(Sk-2) mod(pr) ser-1 1180-8 A ad-4 leu-1 a Cross 1186 was set up to obtain and characterize progeny that would have a mutant (wild variant) allele at one locus but not the other, and to continue genetic mapping. The 232 progeny are summarized in Table 5. All 232 were tested for the three biochemical markers, and all progeny recombinant for the biochemical markers were crossed to Sk-2. There were 102 progeny of the parental type, ser-1 ad-4þ leu-1þ . Seventy of these were crossed to Sk-2. Only a few of the 107 progeny with the three markers from the Sk-2-sensitive parent (ser-1þ ad-4 leu-1) were put through the time-consuming test for resistance. Even if a resistant progeny had been found among them, it would have arisen from a multiple crossover and would not have assisted in ordering the loci. 3.3.2. Phenotypes of the separated loci (1) pr(Sk-2) confers partial resistance in the absence of mod(pr). The crosses to Sk-2 testers were examined by opening perithecia and looking at intact asci. Among the 70 tested ser-1 ad-4þ leu-1þ progeny, nine showed the slight resistance tentatively attributed to pr(Sk-2) in the absence of mod(pr). Four of these nine test crosses were scored as ‘‘probable’’ because they had very few intact asci, but the other five had many individual perithecia with dozens of 4:4 asci and one or two 8:0 asci. One of these five progeny, pr(Sk-2) ser-1 1186-147
Fig. 2. Separation and rejoining of genes at the two loci that modify resistance to killing by Sk-2. Alleles from wild strain P2604 are in boldface type. (A) In cross 1186, single crossovers in two separate meioses produced the progeny shown. (B) In cross 1187, the two critical segments of 1180-8 that were separated by crossing over are reunited in isolate 1187-1. [Linkage Group IIIL is homozygous for the sensitive allele of r(Sk-2) in both crosses. The resistant allele of r(Sk2) would confer resistance regardless of the genotype at the pr(Sk-2) and mod(pr) loci].
a, was saved for stock and was used in the further analysis (cross 1187) reported in the final section and shown in Fig. 2, part B. Cross 1189: pr(Sk-2) ser-1 1186-147 a Sk-2 A was used to get an estimate of the level of resistance to killing of pr(Sk-2) in the absence of mod(pr). Fifty-four progeny were crossed to the fl testers to score for killing. Two of the 54 were non-killers. Incidence of non-killers
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149
Table 5 Recombination of LGIII markers with pr(Sk-2) and mod(pr) CLASS
ser-1 (1)
pr(Sk-2) (2)
Cross 1186 Sk-2-resistant ser-1 1180-8 A ad-4 leu-1 Parental ) R + S
ad-4 (3)
leu-1 (4)
mod(pr)
Number
+ )
+ )
R S
(89) (93)
Single in (1)
) +
S R
) +
) +
S? R
5 6
Single in (2)
) +
R S
) +
) +
S R?
0 11
Single in (3)
) +
R S
+ )
) +
S Ra
0 1
Single in (4)
) +
R S
+ )
+ )
S R
(13) (14)
Note. For each of the resistance loci, ‘‘S’’ stands for the allele that does not contribute to resistance (and hence is sensitive), and ‘‘R’’ stands for the allele that does contribute to resistance. The allocation of progeny between the classes ‘‘Parental’’ and ‘‘Single crossover in region 4’’ is only an estimate based on crossing 70 of the 102 ser leuþ ad þ progeny to Sk-2. 61 were pr(Sk-2) mod(pr) (fully resistant), and 9 were pr(Sk-2) modðprÞþ (partially resistant). 107 progeny were serþ leu ad. a mod(pr) cannot be scored directly when the sensitive allele of pr(Sk-2) is present. This isolate, ad-4 mod(pr)1186-17, was scored for mod(pr) by progeny analysis (see text and Fig. 2).
from a cross of sensitive Sk-2, is usually less than 103 (Campbell and Turner, 1987), so the partial resistance effect of pr(Sk-2) was verified. (2) mod(pr) has no effect in the absence of pr(Sk-2). It was expected from previous crosses that likely recombinants between pr(Sk-2) and mod(pr) could be selected from reciprocal recombinants between ad-4 and leu-1. Contrary to expectations, no ad-4þ leu-1 isolate was obtained. Fortunately, one isolate, 1186-17A (see Fig. 2), was ser-1þ ad-4 leu-1þ . In its cross Sk-2, no ascus had more than four viable ascospores, indicating, as expected from previous crosses, that it had the sensitive allele of pr(Sk-2). For confirmation, 65 progeny were obtained from this test cross, and all were Sk-2. The confirmation that it carried the resistant allele of mod(pr) was obtained by cross 1187, presented in the final section. 3.3.3. Mapping of the two loci The results summarized in Table 4 had already indicated that the two loci were between acr-2 and his-7. Six progeny of cross 1186 were serþ ad þ leuþ (resulting from a crossover between ser-1 and all the other loci) and were fully resistant. This result located both of the resistance loci to the right of ser-1. These six progeny can be paired with a presumptive reciprocal class of five ser-1 ad-4 leu-1 Sk-2-sensitive (fully sensitive). Eleven progeny were serþ ad þ leuþ and fully sensitive. This result located pr(Sk-2) left of ad-4, with the 11 progeny resulting from a single crossover between pr(Sk-2) and ad-4. There were no progeny reciprocal to these eleven, and contrary to expectations from previous crosses, only one isolate, 1186-17A, resulted from a crossover between ad-4 and leu-1 (see Section 4: Prospects for future studies).
The proximal locus, pr(Sk-2), is within the ‘‘recombination block’’ (Campbell and Turner, 1987), but mod(pr) appears to be outside it. The right end of the recombination block was found by Campbell and Turner to be essentially inseparable from leu-1. In the results presented here for cross 1186, mod(pr) appeared to be about 13 map units distal to leu-1 (9/70 ser-1 ad-4þ leu-1þ progeny were partially resistant). 3.4. Reuniting the two components of resistance 3.4.1. Cross 1187: mod(pr) ad-4 1186-17 A pr(Sk-2) ser-1 1186-147 a The parents in cross 1187 were described in the previous section and are shown in Fig. 2. Thirty-seven progeny were obtained. Fifteen were ad-4 ser-1þ , and 22 were ad-4þ ser-1. The 22 ad-4þ ser-1 progeny were crossed to Sk-2. Three of them produced abundant 8:0 asci in their test crosses to Sk-2 and thus must have had the genotype pr(Sk-2) mod(pr) ser-1, resulting from crossing over between ad-4 and mod(pr). Therefore, the mod(pr) genotype of their parent, 1186-17 was confirmed. One of these three test crosses, ser-1 1187-1 a Sk-2 A, was designated cross 1190 and was further analyzed. 3.4.2. Cross 1190: Further confirmation of the reconstitution of high resistance The usual criterion for scoring full resistance is the observation of mostly 8:0 asci. As an additional check on the pr(Sk-2) mod(pr) genotype of 1187-1 a, one hundred progeny were obtained from cross 1190 [1187-1 ser-1 pr(Sk-2) mod(pr) Sk-2] to see whether the level of survival (resistance) of the non-Sk-2 ascospores would be comparable to that of the pr(Sk-2) mod(pr)
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strains in their ancestry. Forty of the 100 progeny were ser-1, and all of these 40 were non-killers, meaning that they were pr(Sk-2) mod(pr). Recovery of non-killers from crosses of the original Malaya stock ranged from 30% to 50%, so this result is within the range expected of a reconstituted pr(Sk-2) mod(pr) resistant genotype.
to the two anonymous reviewers who worked hard to understand the complexities of the results and to suggest more understandable ways to present them. This work was carried out as part of a comprehensive program for Neurospora genetics in the Perkins laboratory, supported by Research Grants AI-01462 from the National Institutes of Health and MCB-9728675 from the National Science Foundation.
4. Discussion For the Sk-2 system in Neurospora, only the killer chromosome segment and one resistance locus, r(Sk-2), had been identified. The two new loci described in this report have significantly altered our concept of the complexity of the system. While r(Sk-2) is toward the left end of LGIII, very close to the left end of the Recombination Block associated with Sk-2, the two new loci are in the right arm of LGIII. pr(Sk-2) is within the Recombination Block, but mod(pr) is distal to it. By showing further complexity in the Sk-2 system, the results of this study enhance the previous findings that meiotic drive in Neurospora resembles meiotic drive in Mus and Drosophila. Prospects for future studies. No further work involving these new loci is contemplated. The recombination anomalies observed in many of the crosses may signal previously unknown recombination loci. There may be undiscovered factors (including rearrangements too small to produce a recognizable pattern of aborted ascospores) affecting the recombination patterns of the region between pr(Sk-2) and leu-1. As shown by this study alone, such tangential problems arise constantly when working with strains from nature, and it is not possible to resolve each one. Strains that carry one or both variant alleles and close markers have been deposited in FGSC (see Table 1). Obviously the present study has not exhausted the possibilities for finding variation in the resistance to spore killing of N. crassa, nor has it addressed the distribution of pr(Sk-2) and mod(pr) among collected strains. Sk-2 is far more abundant in N. intermedia than in N. crassa, and Sk-3 is known only in N. intermedia. For those working with either population biology or meiotic drive, there are hundreds of N. intermedia strains available for continuing analysis of resistance to Spore killers.
Acknowledgments I am grateful to David Perkins, Edward Barry, N.B. Raju, and David Jacobson for advice and comment on the research project, Dorothy Newmeyer and Edward Barry for reviewing the first drafts of the report, and David Jacobson for preparing the figures. I am indebted
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