Field screening for red leaf spot
The 121 germplasm lines were evaluated for two years, where anthracnose is an endemic disease. Even though C. sublineolum is naturally present at each location, several plants per row were manually inoculated to ensure consistent symptoms in case of non-uniform distribution of inoculum in the field. We observed that of the 121 lines (Fig.1 and 2), 4 were consistently resistant, 27 were highly susceptible whereas 87 showed a variable resistance response during E1. Whereas in E2, 35 lines were highly susceptible and 80 lines had variable response.
Performance of diversity panel
For evaluation for red leaf spot disease score was taken at specific intervals and AUDPC was calculated and individual and pooled ANOVA was calculated (Table 2). ANOVA for both E1 and E2 showed significant genotypic variation for disease score. The pooled ANOVA across years revealed significant G x E interactions for disease score. Genetic parameters for disease score were presented in Table 3. Disease score range varied from 1-9 and genotypic mean values was 6.2 in E1 and 6.3 in E2. Disease score showed high narrow and broad sense heritability.
Table.2: Mean sum of squares for red leaf spot resistance traits under individual and pooled environment conditions
S.V
|
DF
|
DS
|
INDIVIDUAL ANOVA
|
GEN MSQ (E1)
|
120
|
7.4*
|
GEN MSQ (E2)
|
120
|
7.3*
|
POOLED ANOVA
|
ENV MSQ
|
2
|
36.4*
|
REP (ENV) MSQ
|
3
|
0.3
|
BLOCK (REP*ENV) MSQ
|
60
|
0.2
|
GEN MSQ
|
120
|
6.6*
|
GEN:ENV MSQ
|
240
|
6.9*
|
RESIDUALS MSQ
|
300
|
0.4
|
Table. 3: Genetic parameters for various disease score across years.
TRAIT
|
ENVIRONMENT
|
MEAN
|
RANGE
|
CV
|
GCV
|
PCV
|
HB
|
HN
|
Disease score
|
E1
|
6.2
|
0-9
|
10.5
|
3.5
|
3.7
|
0.94
|
0.99
|
E2
|
6.3
|
0-9
|
8.8
|
3.6
|
3.7
|
0.97
|
0.99
|
Table 4: Reaction of sorghum germplasm to red leaf spot disease
GENOTYPE
|
Disease score
|
Disease response
|
AUDPC
|
2021
|
2022
|
2021
|
2022
|
2021
|
2022
|
IS 7017
|
2
|
2
|
R
|
R
|
140
|
5.8
|
IS 7981
|
5
|
6
|
MR
|
S
|
600.8
|
1233
|
IS 8015
|
3
|
2
|
R
|
R
|
525.8
|
1173
|
IS 8256
|
2
|
3
|
R
|
R
|
178.3
|
163.3
|
IS 8364
|
5
|
4
|
MR
|
MR
|
817.5
|
625
|
SSV 9
|
5
|
3
|
MR
|
R
|
1010.0
|
296.7
|
SSV 15
|
5
|
5
|
MR
|
MR
|
855.8
|
767.5
|
SCL 2
|
5
|
4
|
MR
|
MR
|
804.2
|
471.7
|
SCL 3
|
6
|
4
|
S
|
MR
|
745.8
|
454.2
|
SCL 4
|
3
|
3
|
R
|
R
|
391.7
|
308.3
|
HC 308
|
4
|
4
|
MR
|
MR
|
396.7
|
445
|
SGL 87
|
5
|
5
|
MR
|
MR
|
641.7
|
1346
|
SSV 6
|
5
|
4
|
MR
|
MR
|
970
|
745.8
|
AUDPC: Area under disease progress curve
In this study, difference in response to disease was recorded among 121 sorghum genotypes. Four lines IS 7017, IS 8015, IS 8256 and SCL 4 were free from infection, indicating that these lines were resistant to red leaf spot (Table 4). Few lines showed moderate response to red leaf spot disease viz., IS 8364, SSV 15, SCL 2, HC 308, SGL 87 and SSV 6. Few lines like IS 7981, SSV 9 and SCL 3 showed variable response during E1 and E2.
AUDPC showed that resistant genotypes have lowest value for leaf infection i.e. 500. Genotype IS 7017 had AUDPC value 5.8 under environment E1 which depicts its resistance towards red leaf spot disease and also exhibited lower anthracnose infection than common susceptible line Swarna. Small changes in the environmental conditions in the field can favour a greater anthracnose disease pressure to which the plant can respond through a combination of pathogen recognition, activation of the plant immune system and activation of specific metabolic pathways that lead to the production of defence compounds.
Genetic diversity analysis of Sorghum germplasm set
The SNP density plot (Fig.3) showed distribution of SNPs across all 10 chromosomes. The horizontal axis represented the chromosome length in Mb and vertical line showed number of chromosomes. Different colours respond to density of SNPs. Density was calculated from number of SNPs per Mb. SNP density plot revealed that there were some regions in all chromosomes where there is no SNP has been called and some regions have more than 473 SNPs per Mb. Population structure (Fig.4) was analysed based on origin of different germplasm lines. Based on diversity analysis, the germplasm lines were grouped into 4 major groups and 3 minor groups based on colour difference.
Genotyping-by-sequencing (GBS)
In present study 86 germplasm lines were using restriction endonuclease based approach and the Illumina sequencing technology, which generated 42678 SNPs markers distributed across the entire genome covering 10 chromosomes on the basis of minor allele frequency (MAF) 0.05. Each chromosome had varying number of SNPs. Chromosome wise SNP distribution (Table 5) showed that the number of SNPs on the chromosome was variable. Chromosome 2 (4899 SNPs) and 5 (4605 SNPs) had maximum number of SNPs while the chromosome 10 (3656 SNPs) had the minimum number.
Table 5: Genome-wide distribution of markers on all chromosomes of S. bicolor
Chromosome
|
01
|
02
|
03
|
04
|
05
|
06
|
07
|
08
|
09
|
10
|
No. of SNPs
|
4483
|
4899
|
4600
|
4337
|
4605
|
3796
|
4168
|
4141
|
3992
|
3656
|
Table 6: SNP associated with AUDPC of red leaf spot disease in E1
SNP ID
|
Chromosome
|
Method
|
LOD score
|
'-log10(P)'
|
MAF
|
Genotype
|
47095273
|
1
|
1
|
3.8
|
4.5
|
0.2
|
CC
|
71693738
|
2
|
1,2,4,6
|
7.4
|
3.8
|
0.2
|
CC
|
30008363
|
2
|
1
|
3.7
|
4.4
|
0.1
|
TT
|
58636620
|
6
|
1
|
3.4
|
4.1
|
0.1
|
GG
|
39566904
|
6
|
1
|
5.0
|
5.8
|
0.1
|
CC
|
32445546
|
5
|
2,4
|
4.1
|
4.9
|
0.5
|
AA
|
5833783
|
8
|
2
|
5.7
|
6.5
|
0.2
|
AA
|
35313655
|
10
|
2,6
|
3.0
|
3.7
|
0.2
|
CC
|
56241495
|
7
|
2
|
3.0
|
3.7
|
0.4
|
GG
|
41592784
|
4
|
4
|
3.5
|
4.2
|
0.1
|
CC
|
37943897
|
6
|
4
|
4.0
|
4.8
|
0.4
|
CT
|
56493379
|
10
|
4
|
4.8
|
5.5
|
0.1
|
AA
|
5832672
|
8
|
6
|
7.6
|
8.5
|
0.2
|
GA
|
(1)mrMLM, (2) FASTmrMLM, (4) pLARmEB, (6) ISIS EM BlASSO
Table 7: SNP associated with AUDPC of red leaf spot disease in E2
SNP ID
|
Chromosome
|
Method
|
LOD score
|
'-log10(P)'
|
MAF
|
Genotype for code 1
|
17346634
|
1
|
1
|
4.9
|
5.7
|
0.3
|
AA
|
71693738
|
2
|
1,2,4,6
|
3.2
|
3.9
|
0.2
|
CC
|
73157939
|
3
|
1
|
5.2
|
6.0
|
0.1
|
CC
|
12630178
|
4
|
1
|
3.3
|
4.0
|
0.1
|
GG
|
48679317
|
4
|
1
|
3.1
|
3.8
|
0.1
|
GG
|
70158260
|
2
|
2
|
3.3
|
4.0
|
0.4
|
CT
|
32445546
|
5
|
2
|
7.0
|
7.9
|
0.5
|
AA
|
56938505
|
6
|
2,6
|
4.5
|
5.3
|
0.3
|
TT
|
51200419
|
9
|
2,6
|
5.6
|
6.5
|
0.5
|
AG
|
77795390
|
1
|
4,6
|
3.8
|
4.5
|
0.1
|
TT
|
61276027
|
2
|
4
|
3.9
|
4.6
|
0.2
|
AA
|
32445546
|
5
|
4
|
5.9
|
6.7
|
0.5
|
AA
|
779383
|
6
|
4
|
5.0
|
5.8
|
0.4
|
TC
|
11223299
|
6
|
4
|
3.9
|
4.6
|
0.5
|
AG
|
(1)mrMLM, (2) FASTmrMLM, (4) pLARmEB, (6) ISIS EM BlASSO
Total of 27 SNPs were identified for AUDPC of red leaf spot disease across both environments (Table 6 and 7). In E1, 13 SNPs found significantly associated with trait as they were above Bonferroni significance threshold. These SNPs were present on chromosome 1, 2, 4, 5, 6, 7, 8 and10 and their p values ranged from 3.9-8.5. For E2, 15 SNPs were found associated with trait and present on chromosome number 1, 2, 3, 4, 5, 6 and 9 and p values ranged from 3.7-7.9.
Table 8: SNP associated with red leaf spot disease score in E1
SNP ID
|
Chromosome
|
Method
|
LOD score
|
'-log10(P)'
|
MAF
|
Genotype
|
63370375
|
2
|
1
|
8.4
|
9.3
|
0.4
|
CC
|
14648159
|
4
|
2
|
5.1
|
5.9
|
0.2
|
AC
|
3196136
|
6
|
2
|
3.5
|
4.2
|
0.2
|
GG
|
32470060
|
9
|
2,4,6
|
6.1
|
7.0
|
0.5
|
GA
|
28540216
|
10
|
2,6
|
4.2
|
4.9
|
0.1
|
TT
|
72875406
|
2
|
4
|
4.4
|
5.2
|
0.5
|
GG
|
5058055
|
4
|
4
|
5.6
|
6.4
|
0.1
|
GG
|
56706374
|
6
|
4
|
6.6
|
7.5
|
0.3
|
AG
|
43651047
|
10
|
4
|
7.1
|
8.0
|
0.2
|
AA
|
72830398
|
2
|
6
|
4.9
|
5.7
|
0.4
|
GT
|
(1)mrMLM, (2) FASTmrMLM, (4) pLARmEB, (6) ISIS EM BlASSO
Table 9: SNP associated with red leaf spot disease score in E2
SNP ID
|
Chromosome
|
Method
|
LOD score
|
'-log10(P)'
|
MAF
|
Genotype
|
42048994
|
9
|
1,2
|
4.5-5.1
|
5.2-5.9
|
0.4
|
CC
|
57034183
|
4
|
2
|
4
|
4.8
|
0.4
|
TC
|
56855881
|
5
|
2
|
5.2
|
6
|
0.4
|
TA
|
14438260
|
3
|
4
|
4
|
4.7
|
0.1
|
CC
|
58965172
|
3
|
4
|
3.5
|
4.3
|
0.1
|
GG
|
50256818
|
6
|
4
|
3.8
|
4.6
|
0.1
|
CA
|
42266729
|
7
|
4
|
3.5
|
4.2
|
0.1
|
GG
|
58028298
|
5
|
6
|
3.2
|
3.9
|
0.1
|
TT
|
18419367
|
6
|
6
|
4.4
|
5.2
|
0.4
|
AT
|
51309845
|
7
|
6
|
4.1
|
4.9
|
0.1
|
CC
|
(1)mrMLM, (2) FASTmrMLM, (4) pLARmEB, (6) ISIS EM BlASSO
Total of 20 SNPs were identified for red leaf spot disease across both environments (Table 8 and 9). In E1, 10 SNPs found significantly associated with trait as they were above Bonferroni significance threshold. These SNPs were present on chromosome 2, 4, 6, 9 and10 and their p values ranged from 4.2-8. For E2, 10 SNPs were found associated with trait and present on chromosome number 3, 4, 5, 6, 7 and 9 and p values ranged from 3.9-5.9.
Linkage disequilibrium (LD)
Linkage disequilibrium decay was computed using the PopLDdecay package in Linux, resulting in an LD decay of 50 kb for an r2 value of 0.2 (Fig. 7). To identify potential candidate genes, DNA sequences from a 50 kb genomic region on both sides of the significant SNPs were selected as the confidence interval. Information regarding the genes within the confidence interval of the SNPs was obtained from NCBI. The functions of the predicted candidate genes were reviewed to assess their significance in enhancing resistance against shoot fly and red leaf spot in S. bicolor.