Inheritance of false smut resistance in RIL population
The RYT2668 (resistant) and PR116 (susceptible) parents showed contrasting responses to false smut disease for all the three seasons (Fig. 1, Supplementary table 5). The mean data of the crop season 2013, 2015, and 2016 was used for QTL analysis (Table 1). The mean of the IP/plant observed for the parental line RYT2668 and PR116 was 2.84 and 32.17, respectively. The mean IP/plant on the RIL population was ranged from 0-36.54. The mean of the TSB/panicles was 1.3 and 9.9 for the RYT2668 and PR116, respectively. The mean TSB/panicle on the RIL population was ranged from 0-26.44. Similarly, the mean score observed for the RYT2668 and PR116 was 1.6 and 7.0, respectively. The mean score on RIL population ranged from 0-5.44.
Table 1
Mean phenotypic data of the RIL population for the false smut disease components
S. No. | RILs | IP/planta | TSB/panicleb | Score | S. No. | RILs | IP/plant | TSB/panicle | Score |
1 | RIL-1 | 7.4 ± 7.2 | 4.3 ± 5.1 | 1.8 ± 2 | 49 | RIL-107 | 13 ± 9.1 | 5.4 ± 5.7 | 2 ± 1.6 |
2 | RIL-2 | 8.7 ± 7.8 | 6.7 ± 7.6 | 2.1 ± 1.8 | 50 | RIL-109 | 11 ± 6.8 | 5.7 ± 3.3 | 3.2 ± 1.8 |
3 | RIL-6 | 3.3 ± 4.1 | 1.2 ± 1.5 | 1.2 ± 1.5 | 51 | RIL-111 | 21.7 ± 13.5 | 10.2 ± 7.1 | 3.6 ± 2.3 |
4 | RIL-7 | 11.1 ± 12.3 | 2.7 ± 3.1 | 1.6 ± 1.3 | 52 | RIL-115 | 9 ± 4.9 | 2.9 ± 2.5 | 1.4 ± 1.2 |
5 | RIL-9 | 5.2 ± 6.4 | 3.3 ± 4.1 | 1.2 ± 1.5 | 53 | RIL-119 | 21.6 ± 9.4 | 8 ± 3.9 | 3.2 ± 1.2 |
6 | RIL-10 | 2.8 ± 3.4 | 1.6 ± 1.9 | 0.9 ± 1.1 | 54 | RIL-120 | 13 ± 5.2 | 5.8 ± 3.2 | 3 ± 1.4 |
7 | RIL-13 | 5 ± 6.1 | 1.4 ± 1.8 | 1 ± 1.2 | 55 | RIL-127 | 4.1 ± 5 | 1.6 ± 1.9 | 1 ± 1.2 |
8 | RIL-14 | 30.7 ± 8.6 | 23.8 ± 6.9 | 5.4 ± 1.5 | 56 | RIL-130 | 6.9 ± 8.5 | 2.2 ± 2.7 | 1.2 ± 1.5 |
9 | RIL-17 | 17 ± 9.5 | 7.8 ± 4 | 3.2 ± 1.9 | 57 | RIL-136 | 7.9 ± 9.7 | 5.7 ± 6.9 | 1.2 ± 1.5 |
10 | RIL-18 | 27.9 ± 11.7 | 19.8 ± 10 | 4.2 ± 2.3 | 58 | RIL-140 | 22.1 ± 10.3 | 16.6 ± 5.4 | 4.6 ± 1.7 |
11 | RIL-19 | 19.4 ± 12.3 | 8 ± 6 | 3.6 ± 2.2 | 59 | RIL-142 | 16.1 ± 7.3 | 4.3 ± 2.5 | 2.8 ± 1.7 |
12 | RIL-20 | 22.7 ± 12.5 | 7 ± 5.3 | 3.3 ± 2.5 | 60 | RIL-144 | 1.9 ± 2.3 | 0.7 ± 0.8 | 0.3 ± 0.4 |
13 | RIL-21 | 17.5 ± 10.9 | 7.9 ± 5.3 | 2.9 ± 2 | 61 | RIL-154 | 3.2 ± 3.2 | 1 ± 1 | 0.8 ± 0.8 |
14 | RIL-23 | 14.3 ± 11.1 | 9.2 ± 11.1 | 2 ± 1.9 | 62 | RIL-155 | 8.9 ± 8.6 | 4.9 ± 5.6 | 2.1 ± 2.2 |
15 | RIL-24 | 25.6 ± 12.8 | 19.4 ± 13.8 | 3.8 ± 2.3 | 63 | RIL-158 | 1.2 ± 0.8 | 0.4 ± 0.4 | 0.4 ± 0.4 |
16 | RIL-25 | 6.9 ± 8.4 | 0.8 ± 1 | 0.8 ± 1 | 64 | RIL-160 | 6.2 ± 6.5 | 2.8 ± 3.2 | 1.3 ± 1.1 |
17 | RIL-26 | 14.3 ± 8.7 | 20.6 ± 9.7 | 3.2 ± 1.4 | 65 | RIL-162 | 14.8 ± 7.6 | 7.2 ± 4.5 | 3.2 ± 2 |
18 | RIL-27 | 14.4 ± 7.5 | 7.8 ± 4.6 | 3 ± 1.7 | 66 | RIL-164 | 9.3 ± 7.4 | 2.6 ± 2.7 | 1.7 ± 0.9 |
19 | RIL-29 | 18 ± 10 | 6.2 ± 3.9 | 3 ± 2.2 | 67 | RIL-170 | 3.6 ± 2.3 | 0.9 ± 0.9 | 1.2 ± 0.8 |
20 | RIL-30 | 22.4 ± 12.7 | 10.7 ± 7.1 | 3.1 ± 2 | 68 | RIL-174 | 23.8 ± 15.5 | 12 ± 8.6 | 3.3 ± 2.2 |
21 | RIL-31 | 16.3 ± 11 | 7.3 ± 6.5 | 2.4 ± 2.4 | 69 | RIL-182 | 23.8 ± 8.2 | 17 ± 8.8 | 4 ± 1 |
22 | RIL-35 | 14.7 ± 11.5 | 4.3 ± 3.2 | 2.9 ± 2 | 70 | RIL-185 | 14.7 ± 1.5 | 4.6 ± 1.6 | 3.4 ± 0.7 |
23 | RIL-36 | 8.9 ± 9.7 | 3.8 ± 4 | 1.8 ± 2 | 71 | RIL-186 | 21.7 ± 7.1 | 7.1 ± 2.9 | 3.8 ± 1.1 |
24 | RIL-37 | 26.8 ± 13 | 16.8 ± 7.4 | 4.4 ± 1.7 | 72 | RIL-187 | 20.7 ± 4 | 7.8 ± 1.9 | 3.4 ± 1 |
25 | RIL-44 | 10 ± 4.1 | 4.3 ± 3.2 | 1.7 ± 0.6 | 73 | RIL-188 | 36.5 ± 10.1 | 24.2 ± 11.8 | 5.2 ± 1.4 |
26 | RIL-47 | 2.7 ± 2.3 | 0.4 ± 0.4 | 0.6 ± 0.4 | 74 | RIL-190 | 14.3 ± 8.9 | 4.2 ± 3.1 | 2.7 ± 1.8 |
27 | RIL-48 | 6.4 ± 7.8 | 2.3 ± 2.9 | 0.8 ± 1 | 75 | RIL-192 | 19.4 ± 8.9 | 8.7 ± 4 | 3.6 ± 1.7 |
28 | RIL-50 | 14.7 ± 10.3 | 5 ± 5.5 | 1.3 ± 1.4 | 76 | RIL-193 | 3.7 ± 3.1 | 0.9 ± 0.9 | 1.3 ± 0.8 |
29 | RIL-53 | 3 ± 2.3 | 0.3 ± 0.2 | 0.6 ± 0.4 | 77 | RIL-195 | 32.8 ± 11.8 | 19.8 ± 11.5 | 4.9 ± 2.6 |
30 | RIL-55 | 1.3 ± 1.3 | 0.2 ± 0.2 | 0.5 ± 0.5 | 78 | RIL-205 | 2.2 ± 1.4 | 0.6 ± 0.5 | 0.6 ± 0.5 |
31 | RIL-59 | 20.1 ± 11.8 | 14 ± 11.4 | 3.8 ± 1.1 | 79 | RIL-206 | 21.8 ± 2.4 | 10.7 ± 2.9 | 3.7 ± 0.9 |
32 | RIL-61 | 29.8 ± 10.5 | 14.7 ± 12.5 | 3.9 ± 2.2 | 80 | RIL-212 | 4.1 ± 5 | 0.9 ± 1.1 | 0.4 ± 0.5 |
33 | RIL-66 | 20.4 ± 11.1 | 9.4 ± 5.6 | 3.2 ± 2.1 | 81 | RIL-216 | 12.1 ± 4.4 | 4.1 ± 2.5 | 2.3 ± 0.9 |
34 | RIL-67 | 0 ± 0 | 0 ± 0 | 0 ± 0 | 82 | RIL-219 | 4.9 ± 4.8 | 1 ± 1 | 1.1 ± 0.8 |
35 | RIL-68 | 16.9 ± 5.4 | 5.9 ± 2.4 | 2.7 ± 1.3 | 83 | RIL-221 | 14.2 ± 7.7 | 7.7 ± 5.7 | 2.9 ± 1.2 |
36 | RIL-70 | 18.9 ± 5.8 | 9.1 ± 3.8 | 3.6 ± 1.3 | 84 | RIL-222 | 24 ± 13.4 | 8.7 ± 4.3 | 4.3 ± 2 |
37 | RIL-71 | 8.5 ± 9.3 | 3.4 ± 4 | 1.1 ± 0.8 | 85 | RIL-223 | 19.4 ± 11.4 | 7.8 ± 4.9 | 3.2 ± 1.5 |
38 | RIL-73 | 17.4 ± 11.6 | 5.6 ± 3.2 | 2.6 ± 1.4 | 86 | RIL-224 | 32.9 ± 10.1 | 26.4 ± 11.6 | 4.8 ± 2.6 |
39 | RIL-75 | 33.5 ± 7.9 | 13.1 ± 5 | 5 ± 1.2 | 87 | RIL-229 | 16.3 ± 3.8 | 6.9 ± 1.9 | 3.2 ± 0.7 |
40 | RIL-79 | 23 ± 12.7 | 12.1 ± 8.3 | 3.5 ± 2 | 88 | RIL-230 | 9.9 ± 9.5 | 2.8 ± 2.8 | 1.4 ± 1 |
41 | RIL-80 | 5 ± 4.8 | 1.6 ± 1.7 | 0.8 ± 0.5 | 89 | RIL-234 | 11.8 ± 7.8 | 4.6 ± 3.6 | 2.7 ± 1.5 |
42 | RIL-85 | 10.7 ± 13.1 | 7 ± 8.6 | 1.4 ± 1.8 | 90 | RIL-236 | 4.3 ± 4.3 | 0.9 ± 0.9 | 1.1 ± 0.8 |
43 | RIL-87 | 17.1 ± 6.7 | 6.3 ± 3.7 | 3 ± 1.6 | 91 | RIL-238 | 17.5 ± 9.6 | 7 ± 5.1 | 2.2 ± 1.4 |
44 | RIL-89 | 7.2 ± 7.3 | 1.6 ± 1.7 | 1.3 ± 1.1 | 92 | RIL-240 | 17.4 ± 7.7 | 7.8 ± 2 | 4 ± 1.5 |
45 | RIL-92 | 3.7 ± 4.5 | 0.4 ± 0.5 | 0.4 ± 0.5 | 93 | RIL-248 | 25.8 ± 10.9 | 13.8 ± 6.2 | 4.6 ± 1.5 |
46 | RIL-93 | 4.1 ± 5 | 2.4 ± 3 | 0.9 ± 1.1 | 94 | RIL-249 | 20.1 ± 8.4 | 9.2 ± 4.2 | 1.9 ± 0.6 |
47 | RIL-97 | 24.6 ± 7.6 | 20.4 ± 9.8 | 4.8 ± 1.5 | 95 | RYT 2668 | 2.8 ± 0.7 | 1.3 ± 1.3 | 1.6 ± 0.9 |
48 | RIL-101 | 7.4 ± 7.5 | 3.2 ± 3.7 | 1.6 ± 1.3 | 96 | PR116 | 32.2 ± 13 | 9.9 ± 5.0 | 7.0 ± 1.2 |
a Infected panicle/plant |
b Total smut balls/panicle |
High-density linkage mapping
A total of 3348 polymorphic SNP markers were obtained between the two parents (Table 2). After removal of distorted markers, a total of 2983 SNP markers were used for the construction of the linkage map. We kept only one representative marker from the set of co-segregating markers showing identical segregation patterns in the linkage analysis. Finally, a high-density linkage map was prepared from a set of 2326 high-quality singleton SNPs markers and assigned them on all 12 linkage groups (LGs) of rice (Supplementary table 1). The total genetic length of all the linkage groups was 2081.5 cM with an average of 1.11 SNPs per cM across the genome (Supplementary Fig. 1, Supplementary table 2). The genetic length of smallest LG was 46.871 cM for Chromosome 12 (Chr12) whereas the largest linkage group was chromosome 2 (Chr02) with 388.266 cM (Table 2).
Table 2
Distribution of SNP markers on 12 chromosomes of rice
Linkage groups (LG) | Total No. of polymorphic SNP markers | Polymorphic SNP markers used for linkage mapping | Singleton SNP | Length of genetic map (cM) | Average markers/ cM |
Chr01 | 441 | 372 | 274 | 191.378 | 1.43 |
Chr02 | 380 | 348 | 291 | 328.266 | 0.88 |
Chr03 | 521 | 479 | 427 | 272.009 | 1.56 |
Chr04 | 189 | 169 | 142 | 128.373 | 1.10 |
Chr05 | 163 | 158 | 112 | 143.479 | 0.78 |
Chr06 | 409 | 369 | 295 | 218.815 | 1.34 |
Chr07 | 182 | 159 | 131 | 162.380 | 0.80 |
Chr08 | 155 | 145 | 107 | 108.950 | 0.98 |
Chr09 | 263 | 233 | 103 | 120.550 | 0.85 |
Chr10 | 128 | 105 | 85 | 69.594 | 1.22 |
Chr11 | 411 | 355 | 293 | 291.294 | 1.00 |
Chr12 | 106 | 91 | 66 | 46.871 | 1.40 |
Total | 3348 | 2983 | 2326 | 2081.959 | 1.11 (mean) |
QTL identification
QTL analysis with IP/plant, TSB/panicle, and score detected a total of seven QTLs on chromosomes 2, 4, 5, 7, and 9 controlling rice false smut disease (Table 3). Two QTLs (tentatively designated as qRFSr5.3 and qRFSr7.1) were detected for the IP/plant on chromosome 5 and 7. The qRFSr5.3 was located on chromosome 5 between SNP markers C5.4852660 and C5.4888145 at LOD score 3.3 explaining 9.3% phenotypic variance. Whereas, qRFSr7.1 was mapped between SNP markers C7.13373853 and C7.13369043 at 3.2 LOD score with 8.9% phenotypic variance. Genomic region of the locus qRFSr7.1 was fairly close to another QTL qRFSr7.2 detected on same chromosome for score. A single QTL qRFSr9.1 was detected for TSB/plant on chromosome 9 between SNP markers C9.8029326 and C9.9023440 at LOD score 6.2 exhibiting 20.6% phenotypic variance. Four QTLs (designated as qRFSr2.2, qRFSr4.3, qRFSr5.4, and qRFSr7.2) were detected for score on chromosome 2, 4, 5, and 7. The QTL qRFSr2.2 was detected on chromosome 2 between two SNP markers C2.33882274 and C2.33796392 at 3.9 LOD score explaining 11.0% phenotypic variance. The qRFSr4.3 was mapped on chromosome 4 between C4.31529767 and C4.31393008 at LOD scores 3.5 displaying 8.9% phenotypic variance. The QTL qRFSr5.4 was detected between two SNP markers C5.1021716 and C5.2489245 on chromosome 5 at 3.0 LOD score explaining 8.0% phenotypic variance. The qRFSr7.2 was detected on chromosome 7 between two SNP markers C7.13373853 and C5.13369043 at LOD scores 5.1 accounting for 14.2% phenotypic variance. The phenotypic variance conferred by qRFSr2.2, qRFSr5.3, and qRFSr5.4 had a negative additive effect indicating that the resistance conditioned in progenies was contributed by the alleles from susceptible parent PR116. Three QTLs qRFSr2.2, qRFSr7.2, and qRFSr9.1 were identified as major QTLs exhibiting larger effect on the traits whereas the remaining four QTLs were accounting minor effect.
Table 3
QTLs associated with rice false smut (RFS) disease detected by composite interval mapping (CIM)
Traits | QTLs | Chromo-some | Left marker | Right marker | Physical distance (Kb)a | Genetic distance | Peak marker (cM)b | LOD | Additive | R2 | Favourable alleles c |
IP/plant | qRFSr5.3 | 5 | C5.4852660 | C5.4888145 | 35.485 | 102.6-106.9 | 104.8 | 3.3 | -2.6 | 9.3 | PR116 |
qRFSr7.1 | 7 | C7.13373853 | C7.13369043 | 4.811 | 106.1-113.2 | 111.6 | 3.2 | 2.8 | 8.9 | RYT2668 |
TSB/panicle | qRFSr9.1 | 9 | C9.8029326 | C9.9023440 | 994.114 | 48.9–51.2 | 50 | 6.2 | 3.0 | 20.6 | RYT2668 |
Score | qRFSr2.2 | 2 | C2.33882274 | C2.33796392 | 85.880 | 0.9–2.5 | 1.2 | 3.9 | -0.4 | 11.0 | PR116 |
qRFSr4.3 | 4 | C4.31529767 | C4.31393008 | 136.759 | 101.4-102.2 | 102.1 | 3.5 | 0.4 | 8.9 | RYT2668 |
qRFSr5.4 | 5 | C5.1021716 | C5.2489245 | 1467.529 | 132.9–138.0 | 135.8 | 3.0 | -0.4 | 8.0 | PR116 |
qRFSr7.2 | 7 | C7.13373853 | C7.13369043 | 4.811 | 106.1-113.2 | 111.6 | 5.1 | 0.5 | 14.2 | RYT2668 |
a Physical position was determined on the basis of Nipponbare-reference-IRGSP-1.0 |
b Value showed the position of peak marker |
c Alleles are inherited from the mentioned parents |
Stable QTLs
QTL analysis was also extended for each trait observed in 2013, 2015 and 2016 separately to determine the stability of QTLs across the season. A total of twenty-four QTLs were mapped on chromosome 1, 2, 3, 4, 6, 7, 9, 11, and 12 (Supplementary table 3). However, QTLs on chromosome 1, 3, 6, 11, and 12 were mapped for the first time when QTL analysis was performed separately but not detected in the analysis of mean data of all seasons. The QTLs on chromosome 2, 4, 7, 9, 11 and 12 were highly consistent and detected for multiple traits across the seasons. Among them, QTLs on chromosome 2, 4, 7, and 9 were detected in analysis of mean data as well. QTLs on chromosome 1, 3 and 6 were highly unstable which were detected only once for at least one trait.
Analysis of ORFs underlying the QTLs
Based on the physical position of linked SNP markers, the physical map of the Nipponbare reference-IRGSP-1.0 pseudomolecule was searched for the ORFs. The physical intervals of all the QTLs were ranged from 4.811 Kb to 1467.529 Kb (Table 3). The detailed list of ORF predicted in the QTL intervals was presented in supplementary table 4. The ORFs were analyzed for all the QTLs except qRFSr5.4 due to larger chromosomal region (1.467 Mb). The qRFSr5.3 affecting IP/plant was spanned in 35.485 Kb regions containing six genes. Of which, three genes LOC_05g08810, LOC_05g08840, and LOC_05g08850 encode phosphotidylinositol-3-kinase, DnaK protein, and cytochrome P450, respectively and rest three genes encode expressed proteins. Another QTL qRFSr7.1 affecting IP/plant harbours one ORF (LOC_Os07g23690) encoding a root cap protein spanned in the 4.811Kb regions of the interval. The QTL qRFSr7.1 was found highly stable across the crop seasons that share the genomic location with qRFSr7.2 responsible for score. The qRFSr9.1 affecting TSB/panicle spanned in 994.114 Kb regions on chromosome 9 harbouring a total of 116 ORFs. Of which, three genes (LOC_Os09g14010, LOC_Os09g14060, and LOC_Os09g14100) encode RPS2-type disease resistance proteins and one gene (LOC_Os09g14490) encodes a toll interleukin-1 receptor (TIR)-nucleotide binding sites (NBS) domain-containing disease resistance protein (Table 4). Additionally, on chromosome 9 QTLs for IP/plant and TSB/panicle were also observed on the short arm during the crop season 2016. This region spans a 102.7 Kb interval harbouring 11 ORFs including one functional protein-coding ORF, LOC_Os09g12150 encoding an F-Box domain-containing protein. Similarly, the qRFSr2.2 locus affecting score spanned in 85.880 Kb regions on the chromosome 2 harboring 16 ORFs that belong to diverse biological functions. The genomic region of the locus qRFSr4.3 contains 16 ORFs spanned in 136.759 Kb regions including one disease resistance gene (LOC_Os04g52780) encodes a leucine-rich repeats (LRR) receptor protein kinase.
Table 4
Putative disease resistance genes identified within the QTL intervals conferring rice false smut resistance
QTLs | Locus Id | Encoded protein |
qRFSr4.3 | LOC_04g52780 | Leucine-rich repeat receptor protein kinase |
qRFSr9.1 | LOC_Os09g14010 | Disease resistance protein- RPS2 |
LOC_Os09g14060 | Disease resistance protein- RPS2 |
LOC_Os09g14100 | Disease resistance protein- RPS2 |
LOC_Os09g14490 | TIR-NBS type disease resistance protein |
Some QTLs were observed only in either of the Kharif crop season and analysed for the putative ORFs (Supplementary table 3). Locus 11 was mapped in the year 2015 at 94.5 cM and 266.4 cM for TSB/plant and score, respectively. This region (94.5 cM) spanned in 377.7 Kb intervals containing 65 ORFs including four disease resistance genes. Among them, four genes, LOC_Os11g17014 encodes a NB-ARC domain containing protein, LOC_Os11g17110 and LOC_Os11g17120 encode a RPM1-type disease resistance protein and LOC_Os11g17110 encodes a stripe rust resistance protein-Yr10. Two loci were mapped on chromosome 12 for TSB/panicle and score. Both the loci were flanked by same markers spanned in the 655.8 Kb regions. Therefore, QTLs mapped on chromosome 12 was considered as single locus that contains 91 ORFs including LOC_Os12g17480 encodes a MLA12, LOC_Os12g17490 encodes a stripe resistance protein-Yr10 and LOC_Os12g18360 and LOC_Os12g18374 encode NB-ARC domain containing proteins.
Analysis of structural variations in the QTL regions
Structural variations (SV) and presence and absence variations (PAV) in the QTL regions were analyzed among RILs including parents. A total of 2362 genome-wide insertion and deletion markers (InDels) were found with reference genome. Of which, 1410 InDels were polymorphic between two parents (Supplementary table 5). Among them, 34 InDels were identified within seven QTLs. After careful examination, we found that 2 InDels C9.8620154 (TA/TAA allele) and C9.8760422 (ATTTT/ATTT allele) within qRFSr9.1 and one InDel C5.1169072 (TTG/T allele) within qRFSr5.4 were co-segregate with the trait TSB/panicle and score, respectively (Supplementary table 5). The physical location of the InDel marker C9.8620154 was analyzed in rice genome annotation project. This marker was located at 44.9 Kb downstream from a TIR-NBS domain containing disease resistance protein (LOC_Os09g14540). Similarly, SNP C9.8541647 was located at just 22.6 Kb upstream of the LOC_Os09g14540. Additionally, 138 SNPs were polymorphic between two parents within the QTL regions. Among them, 32 SNPs were co-segregated with different component traits of false smut disease (Supplementary table 5). Based on the physical location, C9.8149063 and C9.8518466 was flank the cluster of three disease resistance genes (LOC_Os09g14010, LOC_Os09g14060, and LOC_Os09g14100) within the qRFSr9.1 locus. These two SNP markers C9.8149063 and C9.8518466 cover 369.4 Kb in the rice genome. These InDels and SNPs can be converted into PCR-based markers for efficient transfer of the genes using marker-assisted breeding (MAB). The sequence information of these InDels and SNPs was given in supplementary table 6.