Variation in yield contributing traits and biotic stress
A total of 10,500 mutants (M1) were generated upon treatment of Samba Mahsuri with EMS mutagen in two different concentrations. Phenotypic observations at M2 generation for various morphological, physiological and yield parameters identified 1231 mutants (~ 12% of population) showing promising variation for the traits of interest (Table 1; Table S2 to S7). These mutants were categorized for different traits, among those, the mutants showing variation in the yield contributing traits occupied the major proportion (43%), followed by physiological and panicle emergence mutants (15% each) and the panicle type and morphological trait mutants (Fig. 1). The selected mutants were subsequently advanced to M3 and M4 generations wherein 418 and 276 mutants were showing stable inheritance of variations respectively (Table S2 to S7). Considering the traits of interest, 180 mutants were selected and advanced up to M5 generation for further studies (Table S2 to S7).
Upon screening of mutagenic population of M2 for three biotic stresses (YSB, ShB and BLB), 1453 promising mutant lines were identified, of which ShB mutants occupied the major proportion (55%). These mutants were advanced to the subsequent generations and a total of 35 stable mutants were recovered in M5 generation for the three biotic stresses (Table S8 to S10).
Variations in the morphological traits
Around (0.79%, 83 mutants) of the total population showed variation in various morphological traits (viz., tall, dwarf, grassy bonsai and pink apiculus) (Fig. 2A & 2B). Among these, mutants having tallness (130-155cm) were the highest proportion (51%, 43 mutants) of the total morphological traits, followed by dwarf mutants (43.3%; 36 mutants) which have a length <110cm. We also identified one extreme dwarf mutant (bonsai type having a shoot length of 30-45cm). This mutant had less number of internodes (3) and very narrow internodal length with grassy appearance. The details of the traits and the number of mutants observed in each generation are given in Table 1 and Table S2.
Variations in the physiological traits
The M2 mutants were characterized for various physiological traits viz., flag leaf variation, leaf variations, albino, xantha, coloration, strong culm, stay green, early maturity, shattering and sterility. Of the total population, 1.8% mutants (191) showed variations in the physiological traits. Among these, Strong culm that imparts lodging resistance is an important trait and we identified 26 mutants (0.24%) with culm strength ranging between 26-32 N/m2, while the wild type showed culm strength of 25N/m2. (Fig. 2C). Early maturing (108-136 days) mutants were observed at a frequency of 0.69% (73 mutants) (Fig. 2D). Stay-green in the post-anthesis period is found to be an efficient drought tolerance trait and we obtained 4 stay green mutants (0.03%) in the M2 generation. We also obtained a good frequency (0.26%) of sterile plants; however these could not be maintained further due to non-uniform segregation (Table S3).
Variations in panicle types
Panicle types (sparse, long and dense) were studied and a proportion of 0.8% (85) of the total M2 population was observed. Among these, long panicle (30-32cms) type mutants were the highest with 0.47% (50 mutants) (Fig. 2E). The dense panicle mutants recorded 300-350 seeds per plant, while the wild type had only 180-200 seeds (Fig. 2F; Table S4).
Variations in panicle emergence
Complete panicle emergence (CPE) and Elongated Upper Internode (EUI) are two major traits that were studied under the category of panicle emergence. In this category we identified 182 mutants (1.72% of the population) of which CPE mutants were more numerous (1.47% of the population) and the EUI mutants were less frequent (0.25% of the population). The CPE mutants exhibited 2 to 2.5cms of panicle exsertion and thus no panicle choking (Fig. 2G) whereas 10-20% panicle chocking was observed in wild type. The EUI mutants exhibited a length of 7-26cm for the upper most internode (Fig. 2H) (Table S5).
Variations in yield and yield related traits
The yield-related traits included more number of tillers, number of productive tillers and better grain filling, grain number and phenotypic acceptability (PA). In the M2 generation, the mutants in yield traits were the most frequent; i.e. a total of 532 mutants representing 5.06% of the total mutant population (Fig. 2I). Among these traits, number of productive tillers and better grain filling occupied the major proportion with 4.06% (484 mutants), while more number of tillers (15-26 tillers) occupied the least with a proportion of 0.12%. Overall acceptability of the mutant lines was also recorded and we obtained a frequency of 0.19% (20 mutants) in the M2 generation (Table S6).
Variations in the Grain types:
The wild type has medium slender grain type, however upon treatment with EMS we observed a wide range of grain types (Table 1). The grain type mutants occupied 1.5% (158) of the total M2 population, of which medium bold grain without awns were the highest frequency (0.36%), followed by long slender grain without awns (0.34%). Interestingly, we also observed seven (0.06%) sickle shaped seed mutants. The types of variants obtained are indicated in Fig. 2J. All these mutants were forwarded to M5 generation, however in subsequent generations we focused on medium slender and long slender grain types, as these are the commercially preferred grain types (Table S7).
Biotic stress variations among the mutants
The mutant population that had been generated was large and screening such a large population at one time for various biotic stresses in the same season was not practical. For ease of operations, 2500 M2 families each were screened in two seasons for all three biotic stresses. While going for the second season screening, the selected tolerant mutants from the first season were carried forwarded for rescreening in the next generation along with the base mutant population. A high frequency (13.83%) of the total population showed tolerance for at least one of the three biotic stresses at M2 generation. But rescreening was continued up to M5 generation, the frequency of tolerance was reduced from 13.83% to 0.33%. Among those, Sheath blight tolerant mutants were the most frequent (7.62%) followed by the bacterial leaf blight tolerant (4.59%) while the YSB tolerant mutants were the least (1.61%) at the M2 generation (Table S8 to S10). The reduction in frequency of tolerant mutants following rescreening indicates that there were a large number of false positives possibly due to lines that escaped infection. The details of the tolerant mutants obtained in each generation for various biotic stresses are given in Table 2 and the visual symptoms are given in fig. 2K, 2L and 2M.
Mutants with multiple trait variations
Most of the mutants showed trait-specific variations in comparison with Samba Mashuri, but a few mutant lines (8) showed variation in multiple traits. For instance, the mutant line TI -24 showed different trait combinations like strong culm, early flowering, medium bold, pink apiculus, complete panicle emergence, broad flag leaf and dense panicle and stay green. Similarly, another mutant TI-21 also exhibited variations in multiple traits viz., Strong culm, broad flag leaf and early flowering yet showed medium slender grain like wild type. The details of the mutants which showed multiple trait variations are given in the Table S11.
Genotyping of mutant lines:
To know the genomic similarity between mutants and wild type, a set of 60 SSR markers distributed uniformly across the genome were used. A total of 49 mutants were selected randomly for the estimation of genomic similarity, which revealed a similarity range of 85.0-98.3% in comparison to the wild type (Table S12).
We further performed whole genome resequencing of the wildtype (three genetic stocks) and fifteen mutant lines showing different traits to study the extent of mutagenesis. We obtained high quality sequences with an average depth of 41X (Table 3). Greater than 97% of the reads from the mutant lines mapped to the Samba Mahsuri reference genome (Table 3). Analyses of the EMS-induced SNPs revealed that the dissimilarity between Samba Mahsuri and the mutant lines ranges from 0.008% to 0.089% (Table 4). The number of EMS-induced homozygous SNPs in the mutant lines was between 29850 and 348872. Annotation of the SNPs showed that among the total number of SNPs, 16 to 20% of the SNPs were in the coding region of the mutant lines (Table S13) and relatedness analysis revealed clustering of the mutant lines (Figure S1).