2.1. Morphological results
Morphological evaluation of selected Germplasm revealed the highest 100-grain weight of (26.74 g) for PS2, while the lowest (20.06 g) were recorded for Rachna Basmati. Similarly, the highest grain length (12.18 mm) was recorded for PS2. Although JP-5 showed the shortest grain of (7.27 mm) but had the widest grain (3.51 mm) among the Germplasm. Narrowest grains of (2.21 mm) were recorded for Rachna Basmati. We also measured the grain thickness, and the JP-5 (2.19 mm) showed the highest value, while Rachna Basmati showed the lowest (1.90 mm) measure for grain thickness. Grand mean of 22.87g, 9.59mm, 2.47mm, and 1.94mm were recorded for grain weight, respectively. The highest standard deviation was recorded for grain weight while the lowest was recorded for grain thickness. However, the highest coefficient of variation was recorded for grain width, while the lowest was recorded for grain thickness (Table 1).
Table 1
Grain evaluation for Grain weight, grain length, grain width, and grain thickness
S.No
|
Landraces
|
Grain weight (g)
|
Grain Length (mm)
|
Grain Width (mm)
|
Grain Thickness (mm)
|
1
|
JP-5
|
24.78
|
7.27
|
3.51
|
2.19
|
2
|
Fakhr-e-malakand
|
25.69
|
8.06
|
3.10
|
2.01
|
3
|
GA-5015
|
25.44
|
11.15
|
2.30
|
1.91
|
4
|
PS-2
|
26.74
|
12.18
|
2.44
|
1.85
|
5
|
Swat-1
|
18.35
|
7.99
|
2.81
|
1.93
|
6
|
Swat-2
|
25.42
|
9.83
|
2.45
|
1.99
|
7
|
DR-2
|
24.67
|
9.45
|
2.49
|
1.88
|
8
|
Dilrosh
|
24.91
|
10.55
|
2.45
|
1.96
|
9
|
Malhar-346
|
19.75
|
8.80
|
2.22
|
1.9
|
10
|
Kashmir Basmati
|
20.13
|
9.023
|
2.21
|
1.92
|
11
|
Rachna Basmati
|
20.06
|
9.11
|
2.21
|
1.90
|
12
|
KS-282
|
26.00
|
10.43
|
2.44
|
2.02
|
13
|
Basmati-370
|
19.02
|
9.33
|
2.17
|
1.84
|
14
|
KSK-133
|
26.91
|
9.82
|
2.64
|
2.09
|
15
|
KSK-434
|
23.70
|
10.09
|
2.37
|
1.98
|
16
|
MG-Basmati
|
20.38
|
10.78
|
2.14
|
1.79
|
17
|
Lateefy
|
16.86
|
9.13
|
2.01
|
1.9
|
|
Grand mean with standard deviation
|
22.87+3.32
|
9.59+1.23
|
2.47+0.37
|
1.94+0.09
|
|
CV
|
14.52
|
12.85
|
15.22
|
5.02
|
|
MAX
|
26.91
|
12.18
|
3.51
|
2.19
|
|
MIN
|
16.86
|
7.27
|
2.01
|
1.79
|
Information based on an association of various traits is essential to gather them for yield and quality improvement in rice. To understand the nature and magnitude of the association between the four-grain traits, i.e., 1000-grain weight, grain length, grain width, and grain thickness were calculated in Table 2.
Pearson correlation revealed the highest positive association between grain width and grain thickness, and after that thousand-grain weight and grain thickness, both correlations were highly significant and significant, respectively. However, the highest negative correlation was observed between grain length and width, which were statistically significant, followed by between grain length and grain thickness, which were not statistically significant. The correlation between thousand-grain weight and grain length were not statistically significant. Further, the correlation revealed that the contribution of grain traits in the selected Germplasm to 1000-grain weight one of the main constituents of yield, were in order TGW=GT>GW>GL (Table 2).
Table 2
Pearson correlation among the grain phenotype
|
TGW
|
GL
|
GW
|
GL
|
0.3818
P=0.13
|
|
|
GW
|
0.4545
P=0.06
|
-0.532
P=0.02
|
GT
|
0.4908
P=0.04
|
-0.4581
P=0.06
|
0.7748
P=0.00
|
TGW= Thousand Grain Weight, GL= Grain Length, GW= Grain Width, GT= Grain thickness. LSD test revealed that the three traits mean compared were statistically different from one another except grain width and grain thickness at P=0.05 (Table 3).
Table 3
LSD test for the grain phenotypes
|
TGW
|
GL
|
GW
|
GL
|
13.28*
|
|
|
GW
|
20.40*
|
7.12*
|
GT
|
20.92*
|
7.64*
|
0.52
|
TGW= Thousand Grain Weight, GL= Grain Length, GW= Grain Width, GT= Grain thickness |
The LSD test which basically uses a t-test to compare the means revealed that the traits 1000-grain weight, grain length, and width were statistically different from one another. Pearson correlation showed a negative correlation of grain length with grain width, and from the picture of accessions (Figure 1), it is evident that profoundly meaningful Germplasm was selected to distinguish the individual effect of GS3 and GW2 on their respective traits.
2.2. Molecular genotyping
2.2.1. GS3 C-A allele genotyping
The extracted DNA was amplified for SF28 respective for GS3 amplified ~140bp fragment size. After the amplification, the product size was digested with a PST1 restriction enzyme. Those accessions which carry wild type allele C nucleotide carries a splicing site CTGCAG for PstI which cleaved the wild type (JP-5 as positive control) PCR product into(~110bp) and (~30bp) fragment size while did not digest the domesticated allele carry C-A substitute i.e., CTGAAG (Figure 2).
The accessions with a non-digested fragment of (~140bp) (Bas-370 as negative control) fragments were represented with (+) for presence and those with digested fragments of (~110bp) and (~30bp) were represented with (-) for absence. Out of 17 accessions, Germplasm the two accessions JP-5 and Fakhr-e-Malakand carried dominant allele for while the remaining fifteen accessions i.e., GA-5015, PS-2, PS-2, Swat-1, Swat-2, DR-2, Dilrosh, Malhar-346, Kashmir Basmati, Rachna Basmati, KS-282, Basmati-370, KSK-133, KSK-434, MG-Basmati, and Lateef carried the domesticated allele (Table 4).
Table 4
List of accessions carrying GS3
S.No
|
Landraces
|
GS3
|
S.No
|
Landraces
|
GS3
|
1
|
JP-5
|
+
|
10
|
Kashmir Basmati
|
-
|
2
|
Fakhr-e-malakand
|
+
|
11
|
Rachna Basmati
|
-
|
3
|
GA-5015
|
-
|
12
|
KS-282
|
-
|
4
|
PS-2
|
-
|
13
|
Basmati-370
|
-
|
5
|
Swat-1
|
-
|
14
|
KSK-133
|
-
|
6
|
Swat-2
|
-
|
15
|
KSK-434
|
-
|
7
|
DR-2
|
-
|
16
|
MG-Basmati
|
-
|
8
|
Dilrosh
|
-
|
17
|
Lateefy
|
-
|
9
|
Malhar-346
|
-
|
|
(+) for the presence of dominant GS3 gene (-) for the absence of GS3 |
3.2.2. GW2 allele genotyping
The GW2 marker, when amplified, also revealed a monomeric fragment size of 51bp for each accession. The amplified product digest with APO1 exhibited two different banding patterns, i.e., one a 51bp, which did not carry the restriction site for APO1, and the other cleaved of 30bp and 21bp [18] (Figure 3).
The two accessions JP5 and Fakhr-e-Malakand carries the domesticated allele while Bas-370, GA-5015, PS-2, PS-2, Swat-1, Swat-2, DR-2, Dilrosh, Malhar-346, Kashmir Basmati, Rachna Basmati, KS-282, KSK-133, KSK-434, MG-Basmati, and Lateefy carried the dominant allele for GW2 (Table 5).
Table 5
List of accessions carrying GW2
S.No
|
Landraces
|
GW2
|
|
S.No
|
Landraces
|
GW2
|
1
|
JP-5
|
-
|
|
10
|
Kashmir Basmati
|
+
|
2
|
Fakhr-e-malakand
|
-
|
|
11
|
Rachna Basmati
|
+
|
3
|
GA-5015
|
+
|
|
12
|
KS-282
|
+
|
4
|
PS-2
|
+
|
|
13
|
Basmati-370
|
+
|
5
|
Swat-1
|
+
|
|
14
|
KSK-133
|
+
|
6
|
Swat-2
|
+
|
|
15
|
KSK-434
|
+
|
7
|
DR-2
|
+
|
|
16
|
MG-Basmati
|
+
|
8
|
Dilrosh
|
+
|
|
17
|
Lateefy
|
+
|
9
|
Malhar-346
|
+
|
|
|
(+) for the presence of dominant GW2 gene (-) for the absence of GW2
|