Statistical analysis of agro-morphological trait performance of DHLs
The frequency distribution of the key agro-morphological traits indicates their normal distribution for three consecutive seasons (Figure 1). Significant phenotypic variation (Supplementary Table 1) was noted between the different traits in the DHL population. As shown in Supplementary Table 2, significant differences with respect to the traits, DFF, GP, TGW, PT, BM, FGP, PW, PL, FLL, FLW, PH and YLD, were observed among the individual entries of the DHL population through combined ANOVA. The statistical variation in agro-morphological data in the form of descriptive statistics table is presented in Supplementary Table 3. Genotypic correlation among the traits was observed to be very strong and positive than the phenotypic correlation. A significant and positive genotypic correlation among the traits was observed, particularly for the traits, YLD and GP (r = 0.02), TGW (r = 0.20), PW (r = 0.61), PT (r = 0.37), PL (r = 0.81), BM (r = 0.44) at 1% level of significance (Supplementary Table 4). At 5% level of significance, traits YLD and FGP were observed to be positively correlated (r = 0.04). A negative correlation was noted for the traits, YLD and DFF (r = -0.11), FLL (r = -0.06) at 5 % level of significance. Moreover, very strong and positive genotypic correlation was also observed between the traits PL and GP (r = 0.15), TGW (r = 0.24), PW (r = 0.59), PH (r = 0.22), FLW (r = 0.14), PT (r = 0.27) at 1% level of significance. Genetic variability estimates (Supplementary Table 5) demonstrated a very high level of genotypic variance (Vg) and phenotypic variance (Vp) for the traits viz., FGP (2750 and 3088, respectively), GP (2517 and 2950). Among all the traits, the lowest Vg and Vp values were observed for FLW trait (0.02 and 0.14), respectively. Cumulatively for all the agro-morphological traits, the phenotypic variance was observed to be higher than the genotypic variance. Higher genotypic coefficient of variance (GCV) and phenotypic coefficient of variance (PCV)(>20%) was observed for the traits PW, PL, BM and YLD among which the highest GCV-PCV value was observed for the trait BM (37.49 and 38.36, respectively). Moderate GCV-PCV values (between 10%-20%) was observed for the traits, GP, FGP, TGW, PH, FLL, FLW, PT. Low value (less than 10%) of the GCV-PCV was observed for the trait DFF. Broad sense heritability (H2) was noted to be in the range of 16.80% (FLW trait) to 95.52% (BM trait). The highest genetic advance (GA) value was observed for the trait FGP (101.94) whereas the lowest value of 0.13 was associated with FLW trait. The genetic advance per mean (GAM) was observed in the range of 75.49 (BM trait) and 6.41 (DFF trait). The DH population demonstrated significant statistical variability for the traits under study; hence, the population can be considered suitable to be subjected to QTL mapping analysis [50].
QTL identification with SSR markers
Using a set of 126 hyper-variable SSR and EST derived SSR markers (Supplementary Table 6), a total of 12 major effect-12 minor effect QTLs were detected among the 125 DHLs for all the traits except for the total number of grains per panicle (GP) and number of productive tillers (PT) (Additional file 1; Table 1 and Supplementary Table 7).
As per [54], a QTL with greater than 20 PVE % is considered to be having a major effect. Twelve major effect QTLs were detected for all the traits viz., days to fifty percent flowering (DFF), total grains per panicle (GP), test (1,000) grain weight (TGW), productive tillers (PT), biomass (BM), fertile grains per panicle (FGP), panicle weight (PW), panicle length (PL), flag leaf length (FLL), flag leaf width (FLW), plant height (PH) and total grain yield/plant (YLD) on chromosomes 3, 4, 6, 7, 9 and 12. The LOD scores of these QTLs were observed to be in the range of 2.70-16.51 with PVE% between 29.95%-56.75%. By using 126 hyper-variable SSRs, 15,750 data-points were noted to be amplified. Out of them, 652 data-points to the tune of 4.13% did not amplify and therefore considered to be missing data points.
Days to fifty percent flowering (DFF):
A major effect QTL, qDFF12-1, was identified with a LOD score of 3.64 and PVE value of 48.60%. This QTL with size 5.01 cM was flanked by the markers RM27966 (46.68 cM) and RM235 (51.69 cM). Among them, RM27966 was the closest and with the additive effect value of 9.77 and it was inherited from IR58025A (Additional file 1; Table 1, Figure 2). The cumulative effect (RSq value) of the major and minor effect QTLs is shown in Additional file 1; Table 1 and Supplementary Table 7, respectively.
Total grain yield per plant (YLD):
Two major effect QTLs, namely, qYLD3-1 and qYLD6-1, were identified with LOD scores of 16.51 and 14.36, respectively and the PVE% of both these QTLs was 56.75% and 35.29%, respectively. The QTL, qYLD3-1, with a size of 19.71 cM was flanked by the markers, RM448 (93.41 cM) and RM15679 (113.12 cM) and it was closely associated with RM15679. The QTL, qYLD6-1 of 15.15 cM was observed to be flanked by RM7023 (53.95 cM) and RM586 (69.10) with RM7023 being the closely associated marker. The negative additive effect value, -7.81, of qYLD3-1 indicated that the favorable allele was inherited from KMR-3R (Table 1, Figure 2, Supplementary Figure 1a-I). An additive effect value of 7.43 for qYLD6-1, showed that it was inherited from IR58025A (Additional file 1; Table 1, Figure 2, Supplementary Figure 1b-I). The cumulative phenotypic variance (RSq) explained by both the QTLs accounted up to 97.26% indicating that both are major effect in nature.
Test (1,000) grain weight (TGW):
A total of two major QTLs namely, qTGW6-1 and qTGW7-1, were identified for this trait. The first major QTL, qTGW6-1 was observed to be flanked by RM19410 (25.11 cM) and RM19429 (27.50 cM) with RM19429 being the closest associated marker. The LOD score and PVE% of the 2.39 cM sized QTL, qTGW6-1, was observed to be 4.19 and 46.78%, respectively. The additive effect value of -2.11 for this QTL showed the inheritance of favorable allele from KMR-3R (Additional file 1; Table 1, Figure 2). qTGW7-1, was observed to be located in between the markers, RM20948 (167.52 cM) and RM21649 (172.53 cM) with RM21649 to be identified as closely associated marker. The LOD score and PVE% of this QTL was observed to be 3.06 and 29.95%, respectively. The size of this QTL being 5.01 cM, was identified to have a negative additive effect value of -1.24 demonstrating its inheritance from KMR-3R (Additional file 1; Table 1, Figure 2). The cumulative effect of both these QTLs on the phenotype accounted up to 72.94% (Additional file 1; Table 1).
Panicle weight (PW):
qPW9-1, a major effect QTL, was observed to be located between the flanking SSR markers namely, RM23959 (95.82 cM) and RM23958 (110.97 cM) with a LOD score of 3.44 and PVE% of 33.19%. Among the two markers, RM23958 was the closest associated with respect to this QTL. The additive effect of this 15.15 cM sized QTL was -6.46 indicating its inheritance from KMR-3R. The cumulative phenotypic variance (RSq value) of major and minor effect QTLs identified for this trait was observed to be 46.42% (Additional file 1; Table 1, Figure 2, Supplementary Figure 1b-II).
Plant height (PH):
A major effect QTL, qPH12-1, was identified between flanking markers, RM28275 (33.70 cM) and RM511 (41.66 cM) with LOD score of 2.72 and with PVE% of 43.88%. The additive effect value, -6.32, of this QTL demonstrated the inheritance of the favorable allele from KMR-3R. The cumulative phenotypic effect (RSq value) of this QTL on trait accounted up to 41.08%. (Additional file 1; Table 1, Figure 2).
Table 1
Putative major effect QTLs for agronomic traits in 125 doubled haploid (DH) population derived from the cross of IR58025A × KMR-3R (KRH-2)
Trait Namea
|
QTLb
|
Flanking markers
|
Chr
|
Start position (cM)
|
End position (cM)
|
QTL Size (cM)
|
LOD peakc
|
PVE(%)d
|
Adde
|
RSqf
|
DFF
|
qDFF12-1
|
RM27966
|
RM235
|
12
|
46.68
|
51.69
|
5.01
|
3.64
|
48.60
|
9.77
|
51.01
|
YLD
|
qYLD3$-1
|
RM448
|
RM15679
|
3
|
93.41
|
113.12
|
19.71
|
16.51
|
56.75
|
-7.81
|
97.26
|
qYLD6#-1
|
RM7023
|
RM586
|
6
|
53.95
|
69.10
|
15.15
|
14.36
|
35.29
|
7.43
|
------
|
TGW
|
qTGW6-1
|
RM19410
|
RM19429
|
6
|
25.11
|
27.50
|
2.39
|
4.19
|
46.78
|
-2.11
|
72.94
|
qTGW7-1
|
RM20948
|
RM21649
|
7
|
167.52
|
172.53
|
5.01
|
3.06
|
29.95
|
-1.24
|
------
|
PW
|
qPW9-1
|
RM23959
|
RM23958
|
9
|
95.82
|
110.97
|
15.15
|
3.44
|
33.19
|
-6.46
|
46.42
|
PH
|
qPH12-1
|
RM28275
|
RM511
|
12
|
33.70
|
41.66
|
7.96
|
2.72
|
43.88
|
-6.32
|
41.08
|
FLL
|
qFLL6#-1
|
RM7023
|
RM586
|
6
|
53.95
|
69.10
|
15.15
|
3.09
|
30.67
|
4.06
|
42.89
|
FLW
|
qFLW4-1
|
RM252
|
RM3524
|
4
|
7.96
|
10.46
|
2.5
|
3.29
|
51.18
|
0.17
|
48.22
|
PL
|
qPL3$-1
|
RM448
|
RM15679
|
3
|
93.41
|
113.12
|
19.71
|
16.51
|
56.75
|
-7.80
|
97.26
|
qPL6#-1
|
RM7023
|
RM586
|
6
|
53.95
|
69.10
|
15.15
|
14.36
|
35.29
|
7.43
|
------
|
BM
|
qBM4-1
|
RM1388
|
RM17162
|
4
|
56.87
|
68.68
|
11.81
|
2.70
|
37.79
|
8.31
|
47.32
|
aDFF-Days to fifty percent flowering, YLD-Total grain yield per plant, TGW-1,000 grain weight, PW-Panicle weight, PH-Plant height, FLL-Flag leaf length, FLW-Flag leaf width, PL-Panicle length, BM-biomass
bDesignation of the QTLs is in accordance with the rules recommended by McCouch (2008)
cLogarithm of the odds ratio (LOD) score of ≥2.5 was set as threshold for this data
dTotal phenotypic variance (PVE%) in percentage explained by the QTL
eAdditive effect-Negative additive effect value indicates the direction of favorable allele from donor parent, KMR-3R, that increased the trait value
fPhenotypic variation explained by the final regression model
$QTL hotspot on chromosome 3 for YLD and PL, # QTL hotspot on chromosome 6 for YLD, FLL and PL
|
Flag leaf length (FLL):
A major effect QTL for this trait, qFLL6-1, was observed to be flanked by RM7023 (53.95 cM) and RM586 (69.10 cM) with RM7023 being the closely associated marker. The size of the QTL was 15.15 cM, with a LOD score of 3.09 and explaining 30.67% of PVE. The inheritance of this QTL was from IR58025A with an additive value of 4.06. As per the RSq value, this QTL accounted up to 42.89% of cumulative phenotypic variance (Additional file 1; Table 1, Figure 2).
Flag leaf width (FLW):
qFLW4-1, a novel major effect QTL with a LOD score of 3.29 with PVE% of 48.22% was identified in between the flanking markers, RM252 (7.96 cM) and RM3524 (10.46 cM) and the marker RM3524 to be closest to the QTL. The 2.5 cM size QTL was observed to be inherited from IR58025A with an additive effect value of 0.17 and accounting up to 48.22% of the cumulative phenotypic variance (RSq) (Additional file 1; Table 1, Figure 2).
Panicle length (PL):
Two major effect QTLs, qPL3-1 and qPL6-1, were identified with a LOD score of 16.51 and 14.36, respectively and the PVE% for was observed to be 56.75% and 35.29%, respectively. The 19.71 cM sized QTL, qPL3-1, was flanked by RM448 (93.41 cM) and RM15679 (113.12 cM), with RM15679 being the closely associated marker. A negative additive effect value of -7.80 indicated that the inheritance of the favorable allele of this QTL was from KMR-3R. qPL6-1, (15.15 cM sized) was observed to be flanked by RM7023 (53.95 cM) and RM586 (69.10 cM) and RM7023 being the closest marker. The additive effect value of 7.43 indicated that the inheritance of this QTL was from IR580525A. The cumulative phenotypic variance (RSq) of both these QTLs was observed to 97.26% indicating that they are major QTLs (Additional file 1; Table 1, Figure 2, Supplementary Figure 1a-(I)-1b (I).
Biomass (BM):
A novel biomass QTL, qBM4-1, (11.81 cM sized QTL) between RM1388 (56.87 cM) and RM17162 (68.68 cM) was identified with a LOD score of 2.70 and PVE% of 37.79%, with closest associated marker being, RM1388. The additive effect value of this QTL, 8.31, demonstrated its inheritance from IR58025A with an RSq value of 47.32 (Additional file 1; Table 1, Figure 2, Supplementary Figure 3a-II). The details of co-localization of major effect QTLs with earlier studies is presented in Additional file 1; Table 2.
Table 2
Co-validation of putative major effect QTLs for various agronomic traits in 125 doubled haploid (DH) population derived from the cross of IR58025A × KMR-3R (KRH-2) with previous studies
Trait Namea
|
QTLb
|
Flanking markers
|
Chr.
|
QTL position(s) in this study
(cM/Mb)
|
Published QTLs names in previous study
|
Flanking marker(s) in previous study
|
Published QTL position(s) in previous study
(cM/Mb)
|
Previous study
|
DFF
|
qDFF12-1
|
RM27966
|
RM235
|
12
|
12.16-26.17 Mb
|
qtl12.1
|
RM3103
|
RM511
|
7.46-17.44 Mb
|
Bernier et al. (2007)72
|
12
|
12.16-26.17 Mb
|
qDF12.1
|
id12006721
|
12884952
|
20 Mb
|
Descalsota-Empleo et al. (2019)73
|
YLD
|
qYLD3-1
|
RM448
|
RM15679
|
3
|
34.49-26.87 Mb
|
GY QTL
|
RM416
|
31.24 Mb
|
Agrama et al. (2007)74
|
qYLD6-1
|
RM7023
|
RM586
|
6
|
6.98-1.47 Mb
|
qGY6-1
|
RM217
|
4.05 Mb
|
Dejun et al. (2002)75
|
TGW
|
qTGW6-1
|
RM19410
|
RM19429
|
6
|
2.91 Mb-3.41 Mb
|
qTGW6-1
|
RM6273
|
RM204
|
0.13-3.16 Mb
|
Marathi et al. (2012)50
|
qTGW7-1
|
RM20948
|
RM21649
|
7
|
2.5-18.8 Mb
|
gw7.1
|
RM125
|
5.46 Mb
|
Septiningsih et al. (2003)76
|
PW
|
qPW9-1
|
RM23959
|
RM23958
|
9
|
95.82-110.97 cM
|
--------
|
--------
|
--------
|
--------
|
Novel QTL
|
PH
|
qPH12-1
|
RM28275
|
RM511
|
12
|
14.1-17.6 Mb
|
--------
|
--------
|
--------
|
--------
|
Novel QTL
|
FLL
|
qFLL6-1
|
RM7023
|
RM586
|
6
|
6.9-1.4 Mb
|
QFll6
|
RM588
|
RM589
|
1.61-1.38 Mb
|
Bing et al. (2006)77
|
FLW
|
qFLW4-1
|
RM252
|
RM3524
|
4
|
24-22.8 Mb
|
--------
|
--------
|
--------
|
--------
|
Novel QTL
|
PL
|
qPL3-1
|
RM448
|
RM15679
|
3
|
93.41-113.12 cM
|
--------
|
--------
|
--------
|
Novel QTL
|
qPL6-1
|
RM7023
|
RM586
|
6
|
6.9-1.4 Mb
|
qPL6.1
|
YJInDel-207
|
YJInDel-208
|
5.2–7.9 Mb
|
Kinoshita et al. (2017)78
|
BM
|
qBM4-1
|
RM1388
|
RM17162
|
4
|
56.87-68.68 cM
|
--------
|
--------
|
--------
|
--------
|
Novel QTL
|
aDFF-Days to fifty percent flowering, YLD-Total grain yield per plant, TGW-test (1,000) grain weight, PW-Panicle weight, PH-Plant height, FLL-Flag leaf length, FLW-Flag leaf width, PL-Panicle length, BM-biomass
bDesignation of the QTLs is in accordance with the rules recommended by McCouch (2008)
|
A total of 12 minor effect QTLs were identified for the following traits: fertile grains per panicle (FGP), panicle weight (PW) and panicle length (PL). For the trait FGP, a total of nine QTLs namely, qFGP4-1, qFGP5-1, qFGP6-1, qFGP6-2, qFGP8-1, qFGP8-2, qFGP8-3, qFGP9-1, qFGP12-1, whose LOD scores were in the range of 2.66-4.25 with PVE% in the range of 9.06%-9.78% were identified. The cumulative phenotypic effect (RSq) of all these QTLs on the trait accounted up to 45.89%. Two minor effect QTLs for trait panicle weight (PW), qPW3-1 and qPW8-1, were identified with LOD scores, 2.60 and 3.44, respectively with PVE% of 17.90% (qPW3-1) and 18.56% (qPW8-1). The RSq value of these two QTLs accounted up to 46.42. qPL8-1, a minor effect QTL for panicle length trait was identified with a LOD score of 7.56 and PVE% of 7.08%. The negative additive effect value of -3.74 demonstrated the inheritance of favorable allele from KMR-3R. The cumulative phenotypic variance (RSq) value of this QTL with two major effect QTLs accounted up to 97.26%. The details of these minor effect QTLs is shown in Supplementary Table 7. The details of co-localization of minor effect QTLs from earlier studies are presented in Supplementary Table 8.
Epistatic interactions of QTLs with LOD peak > 2.5 and PVE% > 20% can be considered as significant and major effect in nature. Significant epistatic interactions were observed between the QTLs located on chromosomes 2, 3, 4, 6 and 11 and controlling three traits namely, days to fifty percent flowering (DFF), panicle weight (PW) and flag leaf length (FLL) (Additional file 1; Table 3, Supplementary Figure 2). For the trait DFF, epistatic interaction was observed between the QTLs located on chromosomes 11 and 12 with LOD score of 6.63 and PVE% accounting up to 79.58%. The RSq value of this significant epistatic interaction was observed up to 51.02%. Similarly, for the trait panicle weight (PW), complex epistatic interactions were observed as shown in Supplementary Figure 2. Among these, three significant QTL epistatic interactions were observed between the QTLs located on chr 2 – 6, chr 6 – 7 and chr 3 – 9, with total phenotypic variance (RSq) accounting for 46.4%. Epistatic interaction was also observed between QTLs located on chr 4 and 12 for the trait flag leaf length (FLL) with a LOD score of 5.25 and phenotypic variance (PVE%) value of 78.26%. As shown in Supplementary Figure 3, among the DHL population, two high yielding DHLs namely, DHL-1 (RP6301-189-17-2) and DHL-2 (RP6301-188-15-47) were observed to be best performing with 32.13 g and 31.28 g of total grain yield per plant (YLD), respectively. DHL-1 was observed to have longer panicles, more number of productive tillers and medium bold (MB) grain type whereas DHL-2 had longer-dense panicles with highly desirable medium slender (MS) grain type.
Table 3
Major effect epistatic interactions between QTLs in 125 doubled haploid (DH) population derived from the cross of IR58025A × KMR-3R (KRH-2)
Trait Namea
|
Chr 1
|
QTL nameb
|
Left Marker 1
|
Right Marker 1
|
Chr 2
|
QTL nameb
|
Left Marker 2
|
Right Marker 2
|
LOD peakc
|
PVE(%)d
|
Add by Adde
|
RSqf
|
DFF
|
11
|
qDFF11-1
|
Chr 11-28.1
|
RM286
|
12
|
qDFF12-1
|
RM511
|
RM27966
|
6.63
|
79.58
|
-7.10
|
51.02
|
PW
|
2
|
qPW2-1
|
RM12434
|
RM13709
|
6
|
qPW6-1
|
RM19429
|
Chr 6-21.4
|
5.11
|
24.49
|
5.66
|
46.43
|
PW
|
6
|
qPW6-2
|
RM7023
|
RM586
|
7
|
qPW7-1
|
RM21941
|
RM20775
|
6.55
|
20.29
|
-1.15
|
0.00
|
PW
|
3
|
qPW3-1
|
RM422
|
RM16238
|
9
|
qPW9-1
|
RM23959
|
RM23958
|
5.90
|
49.14
|
4.05
|
0.00
|
FLL
|
4
|
qFLL4-1
|
RM1388
|
RM17162
|
12
|
qFLL12-1
|
RM519
|
RM27920
|
5.25
|
78.26
|
5.52
|
78.26
|
a DFF-Days to Fifty percent Flowering; PW-Panicle weight; FLL-Flag leaf length
bDesignation of the QTLs is in accordance with the rules recommended by McCouch (2008)
cLogarithm of the odds ratio (LOD) score of ≥2.5 was set as threshold for this data
dTotal phenotypic variance in percentage explained by the QTL
eAdditive by additive effect
fPhenotypic variation explained by the final regression model.
|
QTL × environment
QTL (genotype) × environment interaction of both major and minor effect QTLs in three consecutive seasons namely wet season (2014-2015), dry season (2015) and wet season (2015-2016) is presented in Supplementary Table 9. Some of the crucial parameters namely the LOD score for additive by environment effects (LOD(AbyE)), Phenotypic variation explained by additive by environment effect at the current scanning position (PVE(AbyE)), Additive by environment effect which determine the genotype × environment interactions (AbyE) were observed to zero in both major and minor effect QTLs. Cumulative phenotypic variance (RSq values) were also observed with least significant variation in all the identified QTLs.
In-silico analysis of the major effect QTLs
Supplementary Table 10 enlists the details of the putative candidate genes located in the vicinity of the physical positions of novel major effect QTLs identified through in-silico analysis. Pertaining to the physical positions of the plant height QTL, qPH12-1, out of 136 annotated genes in QTARO’s database, three putative candidate genes with possible biological function related to trait phenotype were identified in the region around 17.57 Mb. Out of 1,344 annotated genes within the physical positions of panicle length, qPL3-1, only one putative candidate gene located at 30.48 Mb, with possible biological function related to trait phenotype was identified. Similarly, out of 137 annotated genes identified within the physical positions of biomass QTL, qBM4-1, only one putative candidate gene located at 25.10 Mb position in the genome was observed to have a close biological function with respect to the trait, biomass. Further, no candidate gene, having a possible biological role in regulating the QTLs viz., qPW9-1 and qFLW4-1 could be identified in the physical positions of these QTLs.
Molecular assessment of the DHL population with functional markers specific for Rf3 and Rf4 revealed the existence of four groups of DHLs (Supplementary Table 1). The first group comprised of 104 individuals was observed to have both fertility restoration loci. The second group consisting of nine individuals had Rf3 locus only. The third group consisted of 11 individuals of Rf4 locus only and one DHL was negative for both the loci.