1. Isolation and Purification of Neurospora species from soil
Soil sample were collected from the field of P.G. Dept. of Biotechnology of T. M. Bhagalpur University. Three flasks (250 ml) were taken, transfer 90 ml distilled water in each flask and plugged them properly, then labeled 1-3 and autoclave at 15 psi for 30 minutes. Small amount of soil was collected from 3 different places of a desire field and mixed to make one lot. 1 g soil was weighed and 1 ml of homogenized sample was transferred into tube 1 containing 9 ml sterilized water. Flask was shaken gently for 10 minutes by using electric shaker. 1 ml suspension transferred from dilution in to test tube containing 9 ml Distil water similarly, dilution of the sample was achieved upto 10-7. Then the tubes of higher dilution factors were used for inoculation in PDA and Vogel’s N- Media [7].
Table1.0: Composition of Vogel’s N- Media
Compound
|
Amount
|
Glucose
|
10 g
|
Na3 Citrate
|
2.5 g
|
KH2PO4 (anhydrous)
|
5.0 g
|
NH4NO3 (anhydrous)
|
2.0 g
|
MgSO4.7H2O
|
0.02 g
|
CaCl2.2H2O
|
0.1 g
|
Citric Acid (anhydrous)
|
5.26 mg
|
ZnSO4.7H2O
|
5.26 mg
|
Fe(NH4)2(SO4)2.6H2O
|
1.05 mg
|
CuSO4.5H2O
|
0.26 mg
|
MnSO4.4H2O
|
0.05 mg
|
H3BO3
|
0.05 mg
|
Na2Mo4.2H2O
|
0.05 mg
|
Biotin
|
0.05 mg
|
2. Preparation of pure culture
To grew the isolates fungal media PDA and Vogel’s N- media [7] plate was prepare by doing streak plate method. Then after 72 hours fungal colonies was grown then the individual colony was purified in slants.
3. Morphological characterization of isolated Neurospora
The suspected Neurospora identified by staining and biochemical detection methods. For staining, the isolates were stained with Lactophenol Cotton Blue (LPCB) to observed the morphological characteristics such as hyphae, conidial headed and arrangements under microscope. Gilman’s and Barnet manual[5] used for the characterization and thus identification of Neurospora.
Table 1.0: Identification of different types of morphological characters
Test Tube
|
Colony color
|
Size of Sporangium (mm)
|
Size of spore (mm)
|
Septa
|
Length of sporangium (mm)
|
Color of spore
|
Shape of sporangium
|
T1
|
Light grey
|
37.73
|
6.3
|
+
|
168.96
|
Grey
|
Globular/ Spherical
|
T2
|
Whitish
|
19.04
|
3.32
|
+
|
374.4
|
Grey
|
Dumble
|
T3
|
Whitish
|
35.9
|
95.9
|
+
|
93.61
|
White
|
Spherical
|
T4
|
Whitish
|
23.76
|
6.61
|
+
|
105.6
|
White
|
Spherical
|
T5
|
Whitish
|
19.8
|
5.3
|
-
|
132
|
Grey
|
Spherical
|
T6
|
Grey
|
26.45
|
5.29
|
-
|
88.88
|
White
|
Spherical
|
T7
|
Grey
|
21.16
|
7.14
|
+
|
192.72
|
Grey
|
Spherical
|
T8
|
Whitish
|
17.68
|
6.3
|
-
|
150.06
|
Grey
|
Spherical
|
T9
|
Whitish
|
36.96
|
9.24
|
-
|
475.2
|
Grey
|
Spherical
|
T10
|
Whitish
|
20.21
|
8.76
|
+
|
390.72
|
White
|
Spherical
|
T11
|
Grey
|
29.04
|
9.5
|
+
|
114.84
|
White
|
Spherical
|
T12
|
White
|
52.8
|
7.9
|
+
|
25.8
|
White
|
Spherical
|
T13
|
Grey
|
35.3
|
8.9
|
-
|
467.2
|
White
|
Spherical
|
T14
|
White
|
27.3
|
5.9
|
+
|
94.6
|
White
|
Spherical
|
T15
|
White
|
20.1
|
7.4
|
-
|
190.3
|
Grey
|
Spherical
|
T16
|
Grey
|
18.23
|
6.8
|
-
|
155.2
|
White
|
Spherical
|
T17
|
White
|
22.6
|
5.9
|
+
|
104.3
|
Grey
|
Spherical
|
T18
|
Yellowish grey
|
36.7
|
8.9
|
-
|
97.4
|
White
|
Spherical
|
T19
|
Yellowish grey
|
27.9
|
6.8
|
+
|
90.5
|
Grey
|
Spherical
|
T20
|
White
|
21.2
|
17.02
|
-
|
149.3
|
White
|
Spherical
|
4. Assessment of Morphological character
Microscopic examination of culture
a) Color of colony
b) Septa
c) Shape of spore
5. Preparation of linkage map
Genome mapping was an important tool for locating a specific gene to a particular region of a chromosome and to determine its relative distances between genes and molecular markers on the chromosome.
Study of frequency of characters-
a) Color of colony
b) Size of sporangium
c) Size of spore
d) Septa
e) Length of sporangium
f) Color of spore
g) Shape of sporangium
Table2.0: Study of different characters of Neurospora
Study of frequency of character
|
Color of Colony (Neurospora)
|
Light Grey
|
1
|
Whitish
|
5
|
Grey
|
5
|
Yellowish Grey
|
2
|
White
|
7
|
Size of Sporangium
|
Group 1 (11-20mm)
|
6
|
Group 2 (21-30mm)
|
8
|
Group 3 (31-40mm)
|
5
|
Group 4 (41-50mm)
|
1
|
Size of Spore
|
Group 1 (3-5mm)
|
1
|
Group 2 (6-8mm)
|
12
|
Group 3 (9-11mm)
|
6
|
Group 4 (11-13mm)
|
1
|
Septa
|
Positive
|
11
|
Negative
|
9
|
Length of Sporangium
|
Group 1 (80-180mm)
|
13
|
Group 2 (181-280mm)
|
5
|
Group 3 (281-380mm)
|
1
|
Group 4 (381-480mm)
|
2
|
Color of Spore
|
Grey
|
10
|
White
|
10
|
Shape of Sporangium
|
Spherical
|
18
|
Globular
|
1
|
Dumble
|
1
|
The data generated in the manuscript is the original experimental data, compiled in tabular and graphical forms. The data is not used for any other publication, dissertation and thesis work.