Identification of BB resistant accessions of O. glaberrima
O. glaberrima accessions were screened repeatedly for four seasons to assess and reconfirm their level of resistance against Xoo (Supplementary Table 1). Plants were inoculated with bacterial blight pathogen culture (strain IX-020) and were scored at 14 days after inoculation (DAI) Fig 1(A). In the first experiment, the reactions of the genotypes were scored based on SES scale and in the subsequent experiments, the reactions were recorded by measuring the lesion length. Based on observations of multiple screenings, out of 31 accessions, 28 accessions showed high level of resistance with lesion length less than 3 cm. The average lesion length among the resistant entries ranged from 0.3 cm (EC # 861795, 861810) to 1.8 cm (EC861811). Two accessions, viz., EC861819 and EC861820 showed a moderate resistance reaction with average lesion length ranging from 3.9-4 cm while EC861804 showed extreme susceptibility with an average lesion length > 30 cm. The cultivar, IR64, exhibited a score of 7 with a lesion length ranging from 7.3-8.2 over the seasons.
Molecular characterization using reported genes BB resistance
Thirty accessions of O. glaberrima which showed moderate to high level of BB resistance in repeated phenotyping were genotyped for the presence/absence of major BB resistance genes namely xa5, xa13, Xa21, Xa38 and xa41(t) using gene-specific PCR based markers xa5FM, xa13prom, pTA248, Oso4g53050-1 and OsSWEET14 promoter markers, respectively. BB resistant rice variety, Improved Samba Mahsuri possessing three BB resistance genes, xa5, xa13 and Xa21 (Sundaram et al., 2008) and breeding line PR114-Xa38 possessing BB resistance gene, Xa38 (Bhasin et al., 2012) were used as positive checks while the BB susceptible rice variety IR64 was used as negative check. Presence of resistance allele with respect to different BB resistance genes in the positive checks was revealed through amplification of fragment sizes of 134bp (xa5), 450bp (xa13), 950bp (Xa21), 269bp (Xa38) and 489bp (xa41), respectively. None of the resistant accessions of O. glaberrima showed the presence of the resistant allele for the above mentioned known BB resistance genes (Table 1; Figure 2).
Development and identification of BB resistant introgression lines
Among the 31 resistant accessions of O. glaberrima, EC861812 consistently showed high level of BB resistance with lesion length of 0.4-1.0 cm across different seasons; hence it was selected as the donor parent (Table 2). The bacterial blight susceptible cultivar, IR64 (used as the female parent and recurrent parent) was crossed to O. glaberrima accession (EC861812) during wet season, 2016 to produce inter-specific F1 seeds. The seed set was very low (less than 20%), as the F1s were products of inter-specific hybridization. The F1 plants (nine F1 plants) were grown and inoculated with the Xoo strain IX-020 during wet season of 2017. The mean lesion length of the F1 plants was below 4 cm (Table 3 and Figure 1B). The F1s plants were found to be partially/completely pollen sterile and there was very low seed set on selfing. Hence, the F1 plants were backcrossed with recurrent parent, IR64 to generate BC1F1 seeds. A total of 10 BC1F1 seeds were obtained as the seed set percentage was very low. The BC1F1 seeds were grown during dry season of 2017-18 and selfed to produce 206 BC1F2 seeds. High level of spikelet sterility was also observed among plants of BC1F1 population.
During wet season of 2018, BC1F2 population was grown under field conditions and the plants were inoculated with Xoo strain, IX-020 for their reaction to BB. The resistant plants identified in BC1F2 population were further backcrossed to recurrent parent IR64 and 16 BC2F1 seeds were obtained. The seeds of BC2F1 were sown during dry season 2018-19 and inoculated with Xoo isolate (IX-020). Out of 16 BC2F1 plants grown and evaluated for BB resistance, two plants were found to be highly resistant (lesion length ranging from 2.5-3cm), five were moderately resistant (lesion length ranging from 3.3- 5.5 cm) and nine plants were moderately susceptible (lesion length ranging from 6.2-8.8cm) (Figure 1D, and Supplementary Table 4). Three resistant/moderately resistant plants of BC2F1 (58*-4-14*-3; 58*-5-6*-1 and 58*-5-6*-3) were further selfed to generate 26 BC2F2 seeds.
During wet season 2019, 26 BC2F2 plants were grown and screened for BB reaction, of which, 17 were resistant (0.1-2.4cm,), and six moderately resistant (3.2-5.4cm), two moderately susceptible (7.1cm and 8.7cm), while one was susceptible (9.7cm) (Figure 1E & F, Table 4 and Supplementary Table 5). The resistant BC2F2 plants were further selfed to generate BC2F3 population.
Evaluation of BC2F2 population for yield attributing traits
The mean data of different yield attributing traits (days to 50% flowering, plant height, number of tillers per plant, number of productive tillers per plant, panicle length, number of spikelets per plant and spikelet fertility) of BC2F2 plants are presented in Table 4. Details of plant wise data of different morphological characters are presented in Supplementary Table 6. The mean days to 50% flowering varied from minimum of 75 days (Plant # 58*-5-6*-1-3) to maximum of 88 days (Plant # 58*-4-14*3-6) with mean of 83.61 days. The mean plant height was 83.38 cm and ranged from 70 cm (Plant # 58*-5-6*-1-5) to 101 cm (Plant # 58*-5-6*-1-7). Interestingly, in terms of total tillers per plant, all the BC2F2 plants recorded higher values than both parents IR64 and EC861812. Mean total tillers per plant was 15.96 with ranging from 8 (Plant # 58*-5-6*-1-2, 58*-5-6*-3-8) to 26 tillers (Plant # 58*-4-14*3-7). In case of productive tillers per plant, all the plants recorded higher values than the parents, IR64 and EC861812. The mean productive tillers per plants was 11 and ranged from 4 (Plant # 58*-5-6*-1-3) to 22 (Plant # 58*-5-6*-3-4). The plants showed mean panicle length of 22.48 cm and the values ranged from 19.3 cm (Plant # 58*-5-6*-1-4) to 25cm (Plant # 58*-4-14*3-7, 58*-4-14*3-8). In case of spikelets per panicle, the mean was 101.79 ranging from 56 spikelets (Plant # 58*-5-6*-3-6) to 175 (Plant # 58*-4-14*3-1), while mean spikelet fertility was 32.71% ranging from 14.81% (Plant # 58*-4-14*3-4) to 88.9% (Plant # 58*-4-14*3-8).
Estimation of recovery of RP genome in BB resistant introgression lines
Six introgression lines (at BC2F2 generation) showing high level of BB resistance (with lesion length 0.1-0.8 cm) were selected for estimation of recovery of recurrent parent genome. A total of 70 parental polymorphic SSR markers identified (out of 428 screened) across all the 12 chromosomes were used for genotyping of BB resistant introgression lines. The total number of markers per chromosome ranged from four (Chromosome 9 and 10) to seven (Chromosome 1, 6 and 12) with average of six markers per chromosome (Table 5). The recurrent parent genome recovery percentage among the six selected plants was observed to range from 52.4 (Chromosome 2) to 86.8 (Chromosome 1) with an average of 72.8% (Table 5). The heterozygous component of the genome ranged from 0% (chromosome 4 and 10) to 25.3% (chromosome 6) with an average of 7.98% (Table 5; Plate 4.7). Among the six introgression lines, the recurrent genome per individual line ranged from 65.1% (Plant # 58*-4-14*-3-6) to 84.5% (Plant # 58*-4-14*-3-5) with an average of 67.5% (Table 6; Fig 4 (A)).