Figure 1.
Phenotypic variation in pigmentation, period and phase in N. crassa conidiation revealed by inverted race tube assay.
Representative accessions are shown to highlight variation in pigmentation (A), period and phase (B). The accessions in A are, from top to bottom, FGSC#4833, FGSC#852, FGSC#4829, FGSC#5914, FGSC#4817 and FGSC#4723. Conidial density in B was estimated using the Chrono program and plotted as a line graph for visual effect. The accessions in B are FGSC#6634 (black) and FGSC#3968 (grey). Accessions were grown at 25°C and represent one replicate to highlight circadian regulated conidiation (banding).
Figure 2.
Significant phenotypic variation in period, phase and temperature compensation across 143 N. crassa accessions.
Circadian period (A, B) and phase (D, E) were estimated using the Chrono program for 118 and 117 accessions at 20°C (grey) and 25°C (black) respectively that had an average rhythmicity index (RI) ≥ 3 (Materials and Methods). Period (B), temperature compensation (C) and phase (E) were analyzed for the 106 accessions with a RI≥3 at both temperatures. Temperature compensation (C) was measured as Q10 (Materials and Methods). (F) Summary of period, phase and RI across the rhythmic accessions at 20°C and 25°C. 87-3 is the N. crassa reference accession that harbours the bd mutation and is the primary lab strain. All values presented are an average of four biological replicates (Table S1).
Figure 3.
Extensive genotypic variation in WC-1 SSR.
(A) The wc-1 gene contains a 5′UTR, an intron, a 3′UTR (solid line) and two exons (boxes). WC-1 protein structure: AD, activation domain (striped boxes); LOV, light, oxygen and voltage domain (horizontal line boxes); PAS, PER-ARNT-SIM domain (horizontal line boxes); ZnFn, GATA-type zinc transcription factor DNA binding domain (grey dotted boxes). The number WC-1 5′AG/GA (B), NpolyQ (C) and CpolyQH (D) repeats were estimated across 143 N. crassa accessions (Materials and Methods). Repeat number was confirmed by sequencing the repeats in multiple accessions (Figure S1 and Table S1).
Figure 4.
Genotypic variation WC-1, WC-2, FRQ and VVD.
The number of 5′frq (A) and WC-2 CpolyN (B) repeats were estimated in the N. crassa accessions. (C) wc-1, frq and vvd SNPs were scored across 143 accessions. The allele frequency of the reference strain (black) is shown in comparison to the other allele (grey) across the accessions. (D–E) Associations between repeat length of NpolyQ and CpolyQH (D), and WC-2 CpolyN and NpolyQ (E). (F) The average length of both the NpolyQ and CpolyQH were significantly different by a student's t-test when grouped for the presence of the FRQ: R753 or FRQ: Q753 allele (p<0.05). All data is available in Table S1.
Table 1.
Associations between genotype, phenotype and circadian parameters across Neurospora crassa accessions at 25°C
Table 2.
Parameter estimates and tests from a multiple linear regression of period length on latitude, wc-1 and frq genotypes; NpolyQ length and FRQ: R753Q, respectively.
Figure 5.
WC-1 genotype segregates with period length in a cross between two N. crassa accessions.
(A) Period, phase, genotype and latitude of collection information for the parental strains FGSC#3223 and FGSC#4724 at 25°C. Period (B) and phase (C) were measured for both parental strains (FGSC#3223 and FGSC#4274) as well as for 69 progeny from the cross (Table S2). (D) Association between period and phase of the progeny. (E) Period length was significantly associated with WC-1 genotype, regardless of FRQ genotype (P<0.001). Since FRQ and WC-1 are on the same chromosome (linkage group), we scored FRQ: Q753R for crossover events between the two loci. Eleven crossover events were identified in the 69 progeny, FRQ: Q753 (black) and FRQ: R753 (grey). Average period and phase of two biological replicates are presented, and data available in Table S2.