Population genetic diversity and variation
In the current study, 127 alleles were amplified by 39 SSR loci examined in the 177 S. album accessions and the average number of alleles (Na) was detected 9.39 followed by the highest number of expected alleles (9.12), and expected heterozygosity (0.87) was harbored in Telangana S. album SuryaL2TS plantations amongst the selected fourteen numbers of populations. However, the Chennarayapatna natural population of S. album from Karnataka state showed lower than average Ne (7.90) and He (0.83) (Table 2).
Table 2
Number of alleles (Na), effective number of alleles (Ne), observed heterozygosity (Ho), Expected heterozygosity (He), fixation index (F), Polymorphic Information Content (PIC) of fourteen populations of S. album by 39 genic and genomic SSR markers
State
|
Population
|
Na
|
Ne
|
Ho
|
He
|
F
|
I
|
PIC
|
Karnataka
|
IWC L 1 KA
|
11.38
|
8.90
|
0.18
|
0.87
|
0.86
|
2.24
|
0.96
|
Nel L2 KA
|
8.51
|
7.36
|
0.19
|
0.85
|
0.79
|
2.02
|
0.83
|
KFD HaL5 KA
|
8.00
|
6.83
|
0.15
|
0.83
|
0.89
|
1.95
|
0.80
|
Che L4 KA
|
7.90
|
6.75
|
0.15
|
0.83
|
0.84
|
1.94
|
0.79
|
KFD DhL7 KA
|
8.38
|
6.62
|
0.18
|
0.84
|
0.80
|
1.91
|
0.83
|
Shim L6 KA
|
8.23
|
7.01
|
0.18
|
0.84
|
0.79
|
1.98
|
0.88
|
GoL3 KA
|
8.45
|
7.24
|
0.19
|
0.84
|
0.76
|
1.99
|
0.87
|
Kerala
|
Mara L1 KL
|
9.23
|
7.65
|
0.18
|
0.65
|
0.79
|
2.07
|
0.74
|
Mara L2 KL
|
7.84
|
6.60
|
0.13
|
0.63
|
0.88
|
1.93
|
0.74
|
Mara L3 KL
|
8.27
|
7.18
|
0.15
|
0.70
|
0.84
|
1.99
|
0.72
|
Telangana
|
SuryaL1 TS
|
12.13
|
9.12
|
0.27
|
0.80
|
0.77
|
2.21
|
0.78
|
Surya L2 TS
|
11.40
|
8.58
|
0.19
|
0.87
|
0.79
|
2.22
|
0.88
|
Kham L3 TS
|
8.27
|
7.17
|
0.19
|
0.84
|
0.78
|
1.97
|
0.88
|
Hyd L4 TS
|
8.50
|
7.42
|
0.22
|
0.85
|
0.74
|
2.02
|
0.84
|
Overall pop
|
9.39
|
7.50
|
0.18
|
0.85
|
0.80
|
2.04
|
0.85
|
As per the obtained results of S. album genetic diversity analysis results, the highest polymorphic information content (PIC) was detected in natural population IWCL1KA (0.96) followed by ShimL6 KA (0.88) (Table 2) in Karnataka followed by the lowest value in natural population of MaraL3KL in Kerala state (Table 2). In terms of Polymorphic information, out of fourteen populations, six populations of Karnataka state (IWC L1KA (NP) NelL2KA (P), KFDHaL5KA (NP), KFDDhL7 KA (NP), ShimL6KA (NP) and GoLKA (P) and three plantations populations of S. album (SuryaL2TS (P), KhamL3TS (P) and HydL4TS) (P) found to be highly informative alleles with higher \(\ge\)0.85 PIC value (Table 2) whereas, one natural population (ChenL4KA) (NP) from Karnataka state and three natural populations of Kerala state (MaraL1 KA, MaraL2 KL, Mara L3 KL) were moderately informative polymorphic (Table 2).
In the genetic diversuty analysis by GenAlex 6.20, the Shannon information index (I) was reported highest in IWST (2.24) among Karnataka state followed by Suryapet population 2 (2.22) and Suryapet population 2 (2.21). However the lowest I was observed in Kerala population Mara2KL (1.93) followed by Dharwad sandalwood population (1.91) of Karnataka (Table 2).
The Highest Fixation index (F) was observed in KFDHaL5KA (P) (0.89) and MarayurL2KL (0.88) (NP) and IWST (0.86) (NP). The lowest F was obtained in Hyd4TS (0.74) (P) with the average value of entire population was \(\ge\)8.0 (Table 2). Population wise FST was described in (Table 3). The obtained results showed the range of FST 0.03 − 0.02. The value 0.03 was obtained in IWST (P), Nelamangala (P) and Khammam (P) (0.03). Where as the lowest value 0.02 was shown in other eleven sandalwood populations with the mean value of 0.02 respectively (Table 3).
Table 3
Analysis of Molecular Variance (AMOVA) of 177 S. album accesssions based on 39 genic and genomic SSR markers
Source of Variation
|
Df
|
SS
|
Variance component
|
Estimated variation
|
% of variation
|
FST
|
P value
|
Overall
|
Among population
|
13
|
95.808
|
7.370
|
0.000
|
3%
|
0.08
|
0.001
|
Among individuls
|
162
|
1646.883
|
10.166
|
0.000
|
5%
|
Within individulas
|
176
|
1930.000
|
10.966
|
10.966
|
92%
|
Karnataka
|
Among population
|
6
|
45.288
|
7.548
|
0.000
|
2%
|
-0.011
|
0.001
|
Among individuls
|
73
|
740.675
|
10.146
|
0.000
|
7%
|
Within individulas
|
80
|
876.000
|
10.950
|
10.950
|
91%
|
Kerala
|
Among population
|
2
|
15.350
|
7.675
|
0.000
|
4%
|
-0.012
|
0.001
|
Among individuls
|
27
|
273.867
|
10.143
|
0.000
|
6%
|
Within individulas
|
30
|
328.500
|
10.950
|
10.950
|
90%
|
Telangana
|
Among population
|
3
|
22.475
|
7.492
|
0.000
|
2%
|
-0.008
|
0.001
|
Among individuls
|
63
|
605.791
|
10.097
|
0.000
|
6%
|
Within individulas
|
67
|
703.500
|
10.992
|
10.992
|
92%
|
The Analysis of Molecular Variance (AMOVA) showed the highest gene flow (Nm) was detected in the Sandalwood plantation populations of Shimogga with the value of 12.95 followed by Telangana state sandalwood plantations SuryaL2TS with the value of 12.44 (Table 3). The lowest gene flow was shown in Kerala state natural populations Marayur L1/L2 (8.66) followed by Nelamangala plantations (8.18) in Karnataka with the mean value of 10.55 in this study (Table 3).
Population genetic structure and genetic differentiation
The AMOVA analysis was conducted to investigate the genetic variation among fourteen sandalwood populations. Hierarchical AMOVA in the current study revealed that 3.0% of the total genetic variation occurred among selected fourteen populations and 92% of the total variation was distributed within the individuals. Population genetic structure of S. album revealed strong population genetic structure at the species level (P < 0.001) (Table 3). AMOVA indicated that a small fraction of the observed genetic diversity was attributable to differences among the populations by 5% (Table 3).
Cluster analysis based on the neighbour-joining (NJ) dendrogram method was established to demonstrate the relationship between 177 accessions of S. album from fourteen populations was clearly clustered into four clusters. Cluster I comprised three natural populations from Karnataka and Kerala viz, MaraL3KL (NP), ShimL6KA (NP), and CheL4KA (NP). Cluster II consisted of two natural populations and nine plantations admixtures viz, SuryaL1TS (P), SuryaL2TS (P), MaraL1KL (NP), MaraL2KL (NP), HydL4TS (P), KFDDhL7KA (P), IWCL1KA (P), NelL2KA (P), KhamL3TS (P), KFDHaL5KA (P), and GoL3KA (P). Overall in cluster II natural populations and plantations both were grouped together whereas, cluster I only plantations were segregated. Notably many neighboring populations cluster together specifically all four populations of Telangana state cluster II (Fig. 1).
The pairwise population Matrix of (Nie’s unbiased genetic distance) between any two provenances, the index values varied from 0.04–0.28. The smallest value 0.04 was found in between Karnataka populations IWCL1KA and ChenL4KA along with the highest distant differentiation (0.286) was observed in Kerala (MaraL2KL) and Karnataka KFDDhL7KA, which originated from two geographical distant states in southern India (Table 5).
Table 4
F statistics and Nm (Gene flow) of 14 populations of S. album by 39 genic and genomic SSR markers; Nm = [(1/Fst)- 1]/4
States
|
Populations
|
|
Nm
|
Karnataka
|
IWC L1 KA
|
0.03
|
9.50
|
Nel L2 KA
|
0.03
|
8.18
|
KFD DhL7 KA
|
0.02
|
12.95
|
KFD HaL5 KA
|
0.020
|
10.75
|
Che L4 KA
|
0.02
|
8.75
|
GoL3 KA
|
0.02
|
9.57
|
Shim L6 KA
|
0.02
|
8.06
|
Kerala
|
Mara L1 KL
|
0.02
|
13.47
|
Mara L2 KL
|
0.02
|
8.66
|
Mara L3 KL
|
0.02
|
8.66
|
Telangana
|
SuryaL1 TS
|
0.02
|
14.57
|
Surya L2 TS
|
0.02
|
12.44
|
Kham L3 TS
|
0.03
|
10.51
|
Hyd L4 TS
|
0.02
|
11.62
|
Average
|
|
0.02
|
10.55
|
Table 5
Pairwise Population Matrix of Nie’s (1973) unbiased genetic distance by using 39 genic and genomic SSR markers
SuryaL1 TS
|
SuryaL2 TS
|
Kham L3 TS
|
Hyd L4 TS
|
Mara L1 KL
|
Mara L2 KL
|
Mara L3 KL
|
IWC L1 KA
|
Nel L2 KA
|
KFD HaL5 KA
|
Che L4 KA
|
KFD DhL7 KA
|
Shim L6 KA
|
GoL3 KA
|
|
***
|
|
|
|
|
|
|
|
|
|
|
|
|
|
SuryaL1 TS
|
0.08
|
***
|
|
|
|
|
|
|
|
|
|
|
|
|
SuryaL2 TS
|
0.09
|
0.01
|
***
|
|
|
|
|
|
|
|
|
|
|
|
Kham L3 TS
|
0.08
|
0.06
|
0.04
|
***
|
|
|
|
|
|
|
|
|
|
|
Hyd L4 TS
|
0.04
|
0.00
|
0.00
|
0.06
|
***
|
|
|
|
|
|
|
|
|
|
Mara L1 KL
|
0.14
|
0.12
|
0.08
|
0.00
|
0.14
|
***
|
|
|
|
|
|
|
|
|
Mara L2 KL
|
0.19
|
0.09
|
0.11
|
0.05
|
0.09
|
0.193
|
***
|
|
|
|
|
|
|
|
Mara L3 KL
|
0.09
|
0.05
|
0.01
|
0.00
|
0.00
|
0.098
|
0.11
|
***
|
|
|
|
|
|
|
IWC L1 KA
|
0.18
|
0.10
|
0.05
|
0.00
|
0.11
|
0.078
|
0.09
|
0.07
|
***
|
|
|
|
|
|
Nel L2 KA
|
0.12
|
0.01
|
0.00
|
0.03
|
0.07
|
0.123
|
0.16
|
0.00
|
0.19
|
***
|
|
|
|
|
KFD HaL5 KA
|
0.07
|
0.09
|
0.03
|
0.00
|
0.05
|
0.064
|
0.16
|
0.04
|
0.13
|
0.05
|
***
|
|
|
|
Che L4 KA
|
0.22
|
0.18
|
0.07
|
0.12
|
0.17
|
0.286
|
0.17
|
0.10
|
0.17
|
0.05
|
0.14
|
***
|
|
|
KFD DhL7 KA
|
0.14
|
0.05
|
0.09
|
0.03
|
0.03
|
0.153
|
0.13
|
0.02
|
0.04
|
0.09
|
0.15
|
0.22
|
***
|
|
Shim L6 KA
|
0.14
|
0.10
|
0.12
|
0.10
|
0.13
|
0.171
|
0.08
|
0.04
|
0.16
|
0.08
|
0.08
|
0.13
|
0.13
|
***
|
GoL3 KA
|
The structure analysis with the proportion of cluster members of each accession from 14 populations was shown in (Fig. 1). The graph showed that the geographical distance was relatively not clear. The first cluster (red lines) had a lower proportion than second cluster (green lines) (Fig. 1). Structure analysis was conducted for highest value K = 4 which means that all populations were divided into four genetic structure groups with admixtures. At K = 4 the estimated Ln probability of all data was < 0.75, inferred cluster value 28.85%; red lines) 30.45%; green lines) 20.34%; blue and 22.45%; yellow lines) revealed that genetic structure of all the selected populations showed admixtures of mostly in Telangana and Karnataka state populations whereas, Kerala populations were found pure which were showed in yellow lines (Fig. 1).
State wise, structure analysis and cluster analysis of seven populations with 80 accessions of sandalwood from Karnataka state divided into four clusters (K = 4) with admixtures. The mean assignment probabilities were observed < 0.7 in Karnataka. In group I (red colour) with the 18 sandalwood accessions divided into three sub clusters i, ii and iii were shown in UPGMA dendrogram, whereas, in group II (green color) 31 sandalwood accessions were grouped into two sub-clusters i & ii. The III group (blue colour) with 16 accessions of sandalwood were divided into three sub-clusters i, ii & iii. However group IV (yellow colour) were divided into five sub-clusters i, ii, iii, iv & v respectively with 12 accessions of sandalwood (Fig. 2 (i & ii), Fig. 5A).
Similarly in three populations of Kerala state with (n = 30) revealed four major groups I, II, III and IV with low admixtures by using structure software analysis (K = 4) with the mean assignment probabilities < 0.8. In group I (red colour) with the eleven sandalwood accessions divided into three sub clusters i, ii and iii were shown in UPGMA dendrogram, whereas, in group II (green color) nine sandalwood accessions were grouped into two sub-clusters i & ii. The III group (blue colour) with four number of sandalwood accessions were divided into three sub-clusters i, ii & iii. However group IV (yellow colour) were divided into five sub-clusters i, ii, iii, iv & v respectively with eight number of sandalwood accessions (Fig. 3 (i & ii), Fig. 5B). Structure analysis of four populations of Telangana with (n = 67) accessions divided into three major groups with some admixtures at (K = 3) with the mean assignment probabilities at < 0.9 inferred cluster value. In group I (red colour) with the eighteen sandalwood accessions were divided into three sub clusters i, ii and iii which were shown in UPGMA dendrogram, whereas, in group II (green color) 23 sandalwood accessions were grouped into five sub-clusters i, ii, iii, iv and v. However group III (blue colour) with 24 numbers of sandalwood accessions were divided into three sub-clusters i, ii & iii. Around sixteen sandalwood accessions amongst four populations of Telangana were found admixtures (Fig. 4 (i & ii), Fig. 5C).
Similarly the three clusters with admixtures were distinguished by PCA analysis (Fig. 6); the populations from Karnataka, Kerala and Telangana were mixed and scattered in all three clusters (Fig. 6). The localization of individuals was defined by the first principal component (PC1) and second principal component (PC2). The results revealed that PC1 reflected the highest contribution in genetic variation (6%) followed by PC2 (5.5%). Eigen values of 25 PC components were found higher than 1 and 15 PC components were found to be lower than 1 revealed high variability in selected S. album accessions. The result showed the combined variation 87.5% and cumulative variation 98.6% (Table 6). The obtained result showed that there was no significant correlation was found between genetic distance and geographic distance among the investigated populations.
Table 6
Principal Component Analysis (PCA) of selected S. album accessions by using 39 genic SSR and genomic SSR markers
PC
|
Eigen value
|
Variation (%)
|
Cumulative (%)
|
PC1
|
3.81
|
6.02
|
6.03
|
PC2
|
3.52
|
5.53
|
11.67
|
PC3
|
3.36
|
5.36
|
16.83
|
PC4
|
3.23
|
5.18
|
22.07
|
PC5
|
2.94
|
4.63
|
26.62
|
PC6
|
2.73
|
4.41
|
30.99
|
PC7
|
2.75
|
4.38
|
35.22
|
PC8
|
2.56
|
4.02
|
39.29
|
PC9
|
2.43
|
3.88
|
43.11
|
PC10
|
2.31
|
3.71
|
46.97
|
PC11
|
2.12
|
3.45
|
50.23
|
PC12
|
2.17
|
3.37
|
53.57
|
PC13
|
2.05
|
3.22
|
56.83
|
PC14
|
2.01
|
3.25
|
59.98
|
PC15
|
1.95
|
3.01
|
63.03
|
PC16
|
1.79
|
2.77
|
65.79
|
PC17
|
1.69
|
2.64
|
68.32
|
PC18
|
1.63
|
2.59
|
70.98
|
PC19
|
1.53
|
2.41
|
73.32
|
PC20
|
1.45
|
2.35
|
75.68
|
PC21
|
1.41
|
2.27
|
77.82
|
PC22
|
1.34
|
2.13
|
79.96
|
PC23
|
1.27
|
2.02
|
81.93
|
PC24
|
1.15
|
1.81
|
83.75
|
PC25
|
1.08
|
1.74
|
85.48
|
PC26
|
0.93
|
1.55
|
86.86
|
PC27
|
0.91
|
1.47
|
88.31
|
PC28
|
0.88
|
1.48
|
89.62
|
PC29
|
0.83
|
1.33
|
90.93
|
PC30
|
0.77
|
1.21
|
92.24
|
PC31
|
0.76
|
1.24
|
93.37
|
PC32
|
0.70
|
1.15
|
94.49
|
PC33
|
0.62
|
1.07
|
95.53
|
PC34
|
0.57
|
0.95
|
96.32
|
PC35
|
0.57
|
0.93
|
97.23
|
PC36
|
0.50
|
0.87
|
98.06
|
PC37
|
0.45
|
0.72
|
98.71
|
PC38
|
0.34
|
0.57
|
99.35
|
PC39
|
0.28
|
0.48
|
99.73
|