3. MATERIALS AND METHODS
3.1.1 Cultures used in the study
The Neurospora crassa cultures (wild-type and mutant cultures having genetic markers) used during the study were obtained from Fungal Genetic Stock Center, Kansas City, USA. The following cultures were obtained during the study:
S. No. FGSC# Genotype Mating Type
1 FGSC #2489 74-OR23-IVA A
2 FGSC #4200 ORS6a a
3 FGSC #3661 alcoy csp-2 A
4 FGSC #3434 alcoy csp-2 a
5 FGSC #1206 al-1;arg-5 A
6 FGSC #4347 fl a
7 FGSC #4317 fl A
8 FGSC #2997 pyr-4 arg-12 A
9 FGSC #2998 pyr-4 arg-12 a
10 FGSC #7192 thr-2 arg-5 A
11 FGSC #7193 thr-2 arg-5 a
12 FGSC #8596 eas trp-3 A
13 FGSC #1290 cys-3 arg-5 A
3.1.2 Growth and maintenance of cultures
Vogel’s minimal medium having 1.5% dextrose and 1.5% agar was used for growth and maintenance of Neurospora cultures (Vogel 1956, 1964). The Vogel’s medium was prepared as follows:
Vogel’s minimal medium
Dextrose1.5 g
Agar1.5 g
Vogel’s stock solution (50X)2 ml
Distilled water100 ml
The method of preparation of stock solution is described below. While preparing stock solutions, salts were added in serial order and each salt was dissolved by continuous stirring before adding the next salt. CaCl2 was predissolved in 2 ml distilled water and added drop wise.

Composition of (50 X) Vogel’s stock solution
dH2O 75 ml
Na3citrate.2H2O12.672 g
KH2PO4 (anhydrous)25.00 g
NH4NO310.00g
MgSO4.7H2O1.0g
CaCl2 (fused)0.378g
Biotin stock (5 mg biotin in 0.5 ml
100 ml of 50% ethanol)
Trace element stock0.5 ml
The final volume of the stock solution was made 100 ml using dH2O and 0.5 ml chloroform was added for preservative. The stock solution was kept in brown bottle at room temperature. The composition of Trace element stock was as follows
Composition of Trace element Stock Solution
dH2O47.5 ml
Citric acid. 1H2O2.50g
ZnSO4. 6H2O2.34g
Fe(NH4)2(SO4)2. 6H2O 0.662 g
CuSO4. 5H2O 0.125g
MnSO4. 1H2O 0.025 g
H3BO3 0.025 g
Na2MoO4. 2H2O0.025 g
Chloroform (0.5 ml) was added for preservation and final volume was made 50 ml by adding distilled water. The solution was stored in at 4-8oC in refrigerator. The cultures were grown at 34±2oC unless otherwise mentioned and stored at 4 oC for short term preservation and -20 o C for long term.

3.1.3 Mutagenesis
The macroconidia of seven day old wild-type Neurospora culture (FGSC #2489 ‘A’) were mutagenised using Ethyl Methane Sulphonate (0.1 M). The mutagenesis was done using the modified method described by Mukati et al., 2015. The suspension was prepared in 0.05 M phosphate buffer and contained 42.5 x 106 conidia/ ml. Conidial count was done in Neubauer counting chamber. Conidia were treated with EMS for different time intervals (0 h, 0.30 h, 1 h, 1.30 h, 2 h and 2.30 h) after which centrifugation was done. Conidial suspension was washed twice with phosphate buffer and stored in refrigerator at 4±2°C. Mutagenised conidia were enriched by cold treating at 7-8°C for two days. The filtrate containing enriched mutagenised conidia was plated on 1% sorbose containing Vogel’s agar media and incubated at 34±2°C for 48 h. Ninety well isolated colonies were picked and transferred to Vogel’s minimal agar medium slants after 48 hours at 34±2oC. The slants were kept 34±2oC for 4-5 days and well growing Neurospora cultures were obtained. The subculturing was done in same medium for purifying the cultures.
3.1.4 Determination of mating type
Mating type of mutant cultures were determined in Synthetic crossing medium by Spot inoculation method. The fluffy culture (FGSC #4347 ‘a’) of known mating type was allowed to grow on Synthetic Crossing Medium for 5 days at 25oC. After 5 days, mutant cultures whose mating was to be determined was rubbed in circular manner on the grown fluffy plate. The plate was observed after two days of incubation at 25oC. The opposite mating type cultures (‘A’) had reacted with the fluffy culture (mating type ‘a’) and developed black colored ascospores while the same mating type did not show any change. Hence, the mating type of unknown mutant cultures were determined. The mating type reaction is shown in Figure . The composition of Synthetic Crossing medium is described below.

Fig. 1 : Mating type determination by Spot inoculation
Synthetic Crossing Medium stock solution composition (Westergaard and Mitchell, 1947)
dH2O950 ml
KNO31.0 g
K2HPO40.7 g
KH2PO40.5g
MgSO4. 7H2O0.5g
NaCl0.1g
CaCl2 (Predissolved in 20 ml dH2O)0.1 g
Trace element stock (same as used in 0.1 ml
Vogel’s minimal medium)
Biotin stock (5 mg biotin in 100 ml0.1 ml
of 50% ethanol)
The final volume was made 1000 ml with distilled water.

3.1.5 Characterization of mutants
The morphological studies of mutant cultures were done by:
1. Observing colony characteristics- The mutant cultures were grown on Vogel’s minimal agar medium and incubated at 25oC and 34oC. After 24 h of incubation the colony morphology was observed and noted.

2. Microscopic observation- The mutant cultures were grown on Vogel’s minimal agar medium, incubated at 25oC and 34oC at different magnification (10X, 40X and 100 X). The defects in tip growth, hyphal morphology, branch initiation and branch formation were observed and recorded.

3. Growth rate- The growth rates of wild-type and mutant cultures were determined in race tubes (Ryan et al., 1943).

3.1.6 Inheritance of defects
The mutants were reciprocally crossed with the wild-type (FGSC #4200) culture. A month after crossing, random ascospores were individually picked, heat shocked at 60oC for 30 minutes and incubated at 34oC for 24 h so that a single ascospore grows into a mature culture. Twenty viable progeny were studied under the microscope to see the inheritance of defects in mutant cultures. The ratio of parental and mutant progeny was calculated. The mutant progeny was again reciprocally crossed with wild-type (FGSC #4200) to confirm the results. Twenty viable progeny were again studied to see the inheritance of defects in mutant progeny. The cross tube is shown in Figure 1.

Figure 1: Wild-type (FGSC #4200), cross tube (centre) and mutant (VU13-16)
3.1.7 Linkage group analysis
The tester strain, alcoy was used for determining the position of an unmapped gene on chromosome by a single cross. This strain contains four phenotypic markers which can be clearly. For assisgning the gene on the two chromosomes, it has three translocations, each translocation tagged with a phenotypic marker. The right arm of chromosome I and II was tagged by the marker albino-1 (al-1), right arm of chromosome IV and V was tagged by the marker colonial temperature sensitive-1 (cot-1), right arm of chromosome III and VI was tagged by the marker yellow-1 (ylo-1) and the linkage group VII was tagged by the marker conidial separation-2 (csp-2).

The cross PS #57 was set up in which mutant, PS-4-11 (progeny of VU13-16) was used as a male parent and the alcoy tester strain (FGSC #3434) was used as a female parent. After a month of crossing, 132 random ascospores were isolated and studied to determine the linkage group on which the mutant gene was present.

3.1.8 Follow up cross
After determining one of the four linkage group, next step was to determine the chromosome number on linkage group on which the mutant gene was present. For this, the cross #PS-98 was set up in which the mutant strain (PS-4-11) was used as a female parent and the tester strain (FGSC #1206) was used as a male parent. After a month of crossing, random ascospore analysis was done.
3.1.9 Fine mapping
3.1.10 Amylase, Protease and Lipase assay
The wild-type and mutant cultures were grown in starch agar, milk agar and Tween 80 agar plates for 24 hours to determine the protease, amylase and lipase activity in mutant cultures. Compositions of starch agar, milk agar and tween 80 agar medium is described below:
Composition of Starch agar medium (100 ml)
dH2O100 ml
Starch0.5 g
Agar1.5 g
Vogel’s salt2 ml
Composition of Milk agar medium (100 ml)
dH2O100 ml
Sterilized milk5 ml
Agar1.5 g
Vogel’s salt2 ml
Composition Tween 80 agar plates
dH2O100 ml
Tween 801 ml
Agar1.5 g
Vogel’s salt2 ml
3.1.11 HPLC analysis
Well grown wild-type (FGSC #2489 and FGSC #4200) and mutant cultures (VU13-16, PS-4-9, PS-4-11, VU13-37, PS-142-1, VU13-12, AM-232-10 and VU-82) were inoculated and allowed to grow in Vogel’s liquid medium (50 ml) in conical flasks for 5 days in static condition in sets of duplicates. After 5 days of incubation at 25oC the mycelium and culture filtrate were harvested and sent for HPLC analysis in sterilized screw capped tubes to PG Tech Laboratory, Indore.