Verification of TH-625-491 progeny for disease resistance and confirmation of parental polymorphism of target genes between parents
The progeny of TH-625-491 (an improved Tellahamsa line possessing two BB (xa13 and Xa21) and two blast (Pi54 and Pi1) resistance genes with 95% Tellahamsa genome) consisting of 11 plants are verified for the presence of the target genes along with original donors B95-1 (xa13 and Xa21) and NLR145 (Pi54 and Pi1) and also GPP2 (xa13 and Xa21), Tetep (Pi54) and C Lac 101(Pi1) were included as positive checks for the resistance allele by using gene specific / tightly linked polymorphic markers viz., xa13-promo, pTA248, Pi54 MAS and RM224 for xa13, Xa21, Pi54 and Pi1 genes, respectively. The results revealed that all eleven plants possessed xa13, Xa21, Pi54 and Pi1 resistance alleles. These alleles were in homozygous condition as expected (supplementary table 2). These alleles were exactly identical to the alleles that were amplified in donor parents (B95-1 and NLR-145) and positive checks [GPP2 (xa13 and Xa21), Tetep (Pi54) and C Lac 101(Pi1)]. Apart from this distinctive polymorphism was observed between the donor and recipient parent that has greater value in selection work because this polymorphism can distinguish the two parental genotypes.
Parental polymorphism survey for assessing the background genome recovery using SSR markers
The previous study conducted by Jamaloddin (2016) (28) in whch565 SSR markers were tested and out of which 464 (82.12%) were found to be monomorphic, while 101 (17.88 %) (Supplementary table 3) commonly showed polymorphism between recurrent parent (Tellahamsa) and the two donor parents (B95-1 and NLR145) (Tellahamsa vs B95-1 with 78 and Tellahamsa vs NLR145 with 82) during the development of TH-625-491, an introgressed line of Tellahamsa carrying xa13, Xa21, Pi54 and Pi1 resistance genes. Among these 101 polymorphic markers, 5 markers were not recovered in TH-625-491, the plant with highest RPG 95%. In the present study, 394 SSR markers were tested, out of which 319 (80.96%) were found to be monomorphic, while 75 (19.03 %) showed polymorphism commonly between recurrent parent (Tellahamsa) and the two donor parents (B95-1 and NLR 145). The polymorphic markers between Tellahamsa and B95-1 were 66 and polymorphic markers between Tellahamsa and NLR 145 were 43. In addition to the above mentioned 75 polymorphic markers, 5 more polymorphic markers were included, which Jamaloddin, (2016) (28) could not recover in his study. Thus a total of 176 (96 markers from earlier study i.e., till TH-625-491 development and 80 from IBC1F1 to IBC1F2 of present study) polymorphic markers were used. The focus of background selection was mainly on chromosome 8 and Chromosome 11, as the carrier chromosomes (i.e. chromosome on which the target genes are located), justifies special consideration in backcross programs because of the selection for the donor alleles at the target loci in each generation. A total of 40 markers were observed on carrier chromosomes (19 markers from chromosome 8, a carrier of xa13 gene and 21 markers from chromosome 11 carrier for Xa21, Pi54 and Pi1 genes) were used for background selection as well as to determine the precise parental genomic contribution in carrier chromosomes.
Development of different combinations of back crossed introgression Lines (ILs) via MAS
One hundred and twenty IBC1F1 plants were obtained through crossing between recurrent parents (Tellahamsa) and donor (TH-625-491). However, due to the small population size, the backcross F1 progenies did not cover all re-combinations of the four genes. Results showed that only 17 plants out of 120 IBC1F1 were heterozygous after confirmation by all four genes specific/tightly linked to polymorphic markers viz., xa13-promo, pTA248, Pi54 MAS and RM224 for xa13, Xa21, Pi54 and Pi1 genes, respectively, which are proved polymorphic between the two parental lines. The selected IBC1F1 (IBC1F1 – 62) line was consequently selected for self-pollination based on the analysis of background genome recovery percentage (97.5 %) by using background polymorphic SSR markers. Nine hundred and twenty IBC1F2 plants were derived, in which Nineteen plants with four, three and two gene combinations were identified and proceeded for background analysis based on resistance to BB disease and agronomic characters like days to flowering, plant height and grain type. Four plants homozygous for four genes (xa13xa13Xa21Xa21Pi54Pi54Pi1Pi1), 9 plants carrying three homozygous genes in different combinations [2 plants carryingxa13xa13 Pi54Pi54Pi1Pi1, 1 plant carrying Xa21Xa21Pi54Pi54Pi1Pi1, 2 plants carryingxa13xa13 Xa21Xa21Pi1Pi1, 4 plants carryingxa13xa13 Xa21Xa21Pi54Pi54], and six plants carrying two genes in different combination of which, 2 plants carryingxa13xa13Xa21Xa21 and 4 plants carryingPi54Pi54Pi1Pi1 were taken into consideration for background genome recovery assessment. The selected nineteen IBC1F2 plants were selfed to produce IBC1F2:3 progenies and further selfing of nineteen IBC1F2:3 plants to produce IBC1F3:4 progenies for further blast resistance evaluation and agronomic performance.
Background genome recovery analysis
Background analysis was carried out in 17 IBC1F1 plants along with parents. These plants were tested with 80 (75 from present study and 5 from previous study which were found to be still in heterozygous condition) polymorphic SSR markers. The results revealed that the genome recovery percentages ranged from 95.5% to 97.5% (table 4) with an average of 96.35%. The IBC1F1 – 62 (97.5%) plant scored the highest genome recovery among seventeen heterozygous plants, while IBC1F1 – 4 plant scored the lowest genome recovery (95.5%). Out of 80 polymorphic markers, 75 markers were recovered in IBC1F1-62 plant where remaining five markers were found to be in heterozygous condition. Five markers were used in selected 19 IBC1F2 plants and the recurrent parent genome recovery percentage ranged between 97.5 and 98.5% (Table 5). IBC1F2 - 62 - 515 (98.5%) plant scored the highest genome recovery, while IBC1F2 - 62 - 18, IBC1F2 - 62 - 127, IBC1F2 - 62 - 187, IBC1F2 - 62 - 423, IBC1F2 - 62 - 499 and IBC1F2 - 62 - 611 scored (97.8%) the lowest genome recovery among four gene, three gene and two gene homozygous plants in IBC1F2 generation with an average of 97.8%. At IBC1F2 generation all 19 plants exhibited increased RPG compared to theoretically expected value of 93.75%.
Biotic stress resistance validation
Bacterial leaf blight resistance validation
Artificial field screening for rice bacterial blight disease was carried out in IBC1F2 population during the kharif season, 2016 along with resistant donor parent, B95-1 and susceptible check, Tellahamsa using DX-020, a virulent isolate of Xoo collected from Indian Institute of Rice Research (IIRR). The donor genotype, B95-1 showed an average <3% leaf area diseased with disease score “1” while the recurrent parent, Tellahamsa has an average >90% leaf area diseased with a disease score of “9”. All the four gene (xa13xa13Xa21Xa21Pi54Pi54 Pi1Pi1) combination plants showed an average range of 1-2.8% diseased leaf area with very high level of resistance to bacterial blight with disease score of “1” and three gene combination BLB transferred plants showed disease score values of “3 and 5” (table 6). It is significant to note that the three gene combination plants carrying xa13 alone exhibited moderate susceptibility with a score of 5 (14.5 % leaf area diseased), while the three gene combination plants carrying Xa21 alone also exhibited moderate level of resistance with a score of 3 (7.2 and 8.0% leaf area diseased).
Blast resistance validation
Nineteen IBC1F2:3 lines which are of different gene combinations with bacterial leaf blight resistance were screened for Blast resistance at ICAR-Indian Institute of Rice Research (ICAR-IIRR), Hyderabad, Agricultural Research Station (ARS), Nellore and Andhra Pradesh Rice Research Institute (APRRI), Maruteru, Acharya NG Ranga Agricultural University (ANGRAU), during November and December months of 2016. Local isolates of Magnaporthe oryzae from their respective stations viz., SPI-40 (ICAR-IIRR), ID-14 (ARS, Nellore) and IB-45 (APRRI, Maruteru) were used to screen the donor, recurrent parents along with IBC1F2:3 lines under uniform blast nursery (UBN) method. Blast disease reaction scores of IBC1F2:3 lines along with their parents were mentioned in Table 7.
The results revealed that all the 19 IBC1F2:3 lines exhibited moderate resistance to susceptible reaction with scores ranging from “5 to 9” scores in all three locations even though the major blast resistance genes are present in those plants. The plants with two gene combination i.e., Pi54+Pi1 showed moderate resistance at Hyderabad, Maruteru whereas moderate susceptibility at Nellore. The three gene as well as four gene combination plants showed susceptible reaction in all three locations with the score of “7-9”. To confirm above results nineteen IBC1F2:3:4 lines (Table 8) were once again screened for blast resistance at ICAR-Indian Institute of Rice Research, Hyderabad during November and December, 2017. The results revealed that differences in level of resistance were observed with disease scores between 3 and 9 means highly resistance to highly susceptible reaction. The lines with Pi54 and Pi1 genes alone showed high resistance to blast during 2017. While remaining entries with both bacterial leaf blight and blast resistance genes showed moderate resistance to highly susceptibility.
Agronomic and yield performance
Agro-morphological evaluation was carried out in 19 IBC1F2:3 lines. Majority of the gene pyramided lines showed non-significance (similarity with Tellahamsa) especially for days to 50% flowering, plant height, grain type in addition to yield and yield parameters. (Table 9).