Development of Gene-Pyramid Lines of the Elite Restorer Line, RPHR-1005 Possessing Durable Bacterial Blight and Blast Resistance.

RPHR-1005, the stable restorer line of the popular medium slender (MS) grain type rice hybrid, DRRH-3 was improved in this study for resistance against bacterial blight (BB) and blast diseases through marker-assisted backcross breeding (MABB). In this study, four major resistance genes (i.e., Xa21 and Xa33 for BB resistance and Pi2 and Pi54 for blast resistance) have been transferred to RPHR-1005 using RPBio Patho-1 (possessing Xa21 + Pi2), RPBio Patho-2 (possessing Xa21 + Pi54) and FBR1-15EM (possessing Xa33) as the donors. Foreground selection was carried out using PCR-based molecular markers specific for the target resistance genes and the major fertility restorer genes, Rf3 and Rf4, while background selection was carried out using a set of parental polymorphic rice SSR markers and backcrossing was continued uptoBC2 generation. At BC2F2, plants possessing the gene combination- Xa21 + Pi2, Xa21 + Pi54 and Xa33 in homozygous condition and with >92% recovery of the recurrent parent genome (RPG) were identified and intercrossed to combine all the four resistance genes. Twenty-two homozygous, pyramid lines of RPHR-1005 comprising of three single-gene containing lines, six 2-gene containing lines, eight 3-gene containing lines, and five 4-gene containing lines were identified among the double intercross lines at F3 generation (DICF3). They were then evaluated for their resistance against BB and blast, fertility restoration ability and for key agro-morphological traits. While single gene containing lines were resistant to either BB or blast, the 2-gene, 3-gene, and 4-gene pyramid lines showed good level of resistance against both and/or either of the two diseases. Most of the 2-gene, 3-gene, and 4-gene containing pyramid lines showed yield levels and other key agro-morphological and grain quality traits comparable to the original recurrent parent and showed complete fertility restoration ability, with a few showing higher yield as compared to RPHR-1005. Further, the experimental hybrids derived by crossing the gene-pyramid lines of RPHR-1005 with APMS6A (the female parent of DRRH-3), showed heterosis levels equivalent to or higher than DRRH-3. The results of present study exemplify the utility of MABB for targeted improvement of multiple traits in hybrid rice.

Introduction

Hybrid rice is one of the proven technologies for increasing rice production and productivity. Through good management, a yield advantage of 1.0–1.5 t/ha can be obtained by cultivation of hybrids as compared to the high yielding varieties. India is the second country (after China) to adopt the hybrid rice technology and presently ~2.5 m. ha is under hybrid rice cultivation (Hari Prasad et al., 2014). One of the major problems encountered in hybrid rice cultivation is the susceptibility of many of the popular hybrids to various pests and diseases. For stable performance of hybrids across locations, it is necessary that they should possess resistance/tolerance to major biotic stresses like blast, bacterial blight (BB), stem borer, brown plant hopper, and white backed plant hopper, and gall midge. Among the rice diseases, BB and rice blast are the two major ones, which limit rice production significantly in India (Production Oriented Survey, 2008).

India has so far released 72 rice hybrids developed by both public and private sectors for commercial cultivation [Indian Council of Agricultural Report (ICAR), 2015]. Among the public bred rice hybrids, DRRH-3 is unique, as it is the first hybrid to be released with fine-grain type, preferred by many Indian farmers and rice consumers. The hybrid is similar in grain type and quality to the highly popular variety, Samba Mahsuri and possesses a 25–30% yield advantage over the variety and hence it is becoming increasingly popular across the country and has been licensed to several private seed companies for commercial seed production. Despite the popularity of DRRH-3, one of the major factors, which is limiting its spread, is its high susceptibility to several biotic stresses. Particularly, the hybrid and its parental lines (APMS 6A and RPHR-1005) are highly susceptible to BB and blast. It will be desirable to incorporate at least one or more dominant genes conferring resistance against the two diseases in the restorer parent (i.e., RPHR-1005) of DRRH-3, so that not only DRRH-3, but also any other hybrids developed using improved versions of RPHR-1005 will be resistant to both BB and blast. Marker-assisted backcross breeding (MABB), a time-tested and highly successful strategy was considered for deployment in this study for targeted improvement of BB and blast resistance of RPHR-1005 (and its hybrid DRRH-3) in the present study, as there several earlier reports where MABB has been successfully deployed for improvement of varieties and hybrids for a few target traits (Hittalmani et al., 2000; Singh et al., 2001; Chen et al., 2004; Joseph et al., 2004; Gopalakrishnan et al., 2008; Sundaram et al., 2008; Shanti et al., 2010; Zhan et al., 2012; Hari et al., 2013; Khanna et al., 2015).

The most effective approach to combat BB is the use of resistant varieties possessing different combination of resistance genes (Khush et al., 1989). To date, at least 40 BB resistance (Kim et al., 2015) genes conferring host resistance against various strains of Xanthomonas oryzae pv. oryzae (Xoo) have been identified. Among the resistance genes, many have been physically mapped and six have been cloned (Xa1, xa5, xa13, Xa21, Xa26, and Xa27; Suh et al., 2013; Sundaram et al., 2014). Xa21, a major resistance gene, originally introgressed from Oryza longistaminata (Ronald et al., 1992; Song et al., 1995) was observed to confer resistance to most Indian isolates of the bacterial pathogen and a simple, but highly efficient, PCR-based functional marker called pTA248, developed by Ronald et al. (1992) is available for marker-assisted selection of the gene. As far as choice of resistance genes for gene pyramiding along with Xa21 is concerned, one of the recently identified, wild-rice derived gene, Xa33 has attracted considerable attention as it has shown high level of resistance against multiple isolates of the BB pathogen (Gizachew and Kumaravadivel, 2015; Gizachew et al., 2015) and closely linked co-dominant molecular markers are available for marker-assisted selection of the gene (Natrajkumar et al., 2012). Therefore, considering these facts, in the present study, we selected Xa21 and Xa33 as the genes of choice for targeted improvement of RPHR-1005 for BB resistance.

Similar to BB disease, host-plant resistance is considered as the most effective strategy for management of blast disease and so far, at least 100 rice blast resistance genes (R-genes) have been identified (Sharma et al., 2012; Liu et al., 2013). Among the blast resistance genes widely deployed in breeding, Pi2 has been identified to be one of the most effective, broad-spectrum resistance genes in India. Pi2 was originally identified from a resistant indica rice genotype, 5173 and introgressed into the blast susceptible cultivar CO39. A near-isogenic line (NIL), named C101A51 possessing Pi2 in the genetic background of CO39 has been developed (MacKill and Bonman, 1992) and widely used in resistance breeding. Further, a gene-specific molecular marker, named AP5659-5 has been developed for MAS of Pi2 (Fjellstrom et al., 2006). Pi54 is another major blast resistant gene, which was originally derived from the Vietnamese cultivar, Tetep. It has been reported to be highly effective under Indian conditions (Sharma et al., 2010). Very robust, gene-specific markers are available for marker-assisted selection of Pi54 (Sharma et al., 2005; Ramkumar et al., 2011). Considering these points, Pi2 and Pi54 were selected as target blast resistance genes for pyramiding into RPHR-1005 in the present study.

Materials and methods

Plant materials

Two BB and blast resistant breeding lines in the genetic background of Samba Mahsuri viz., RPBio Patho-1 and RPBio Patho-2 (Prasad et al., 2011) possessing the resistance genes, Xa21 + Pi2 and Xa21 + Pi54, respectively were used as donors. Both the lines possessed medium slender (MS) grain type and yield levels similar to Samba Mahsuri and were derived from the crosses Improved Samba Mahsuri (ISM) X C101A51 and ISM X Tetep, respectively (Madhavi et al., 2011). Another breeding line, FBR1-15, possessing Xa33 gene in the genetic background of Samba Mahsuri (Natrajkumar et al., 2012) was also used as a donor. RPHR-1005 (the male parent of the elite, fine-grain type rice hybrid DRRH-3), derived from the cross BPT5204/SC5 126-3-2-4 (Ramesha et al., 2010) was used as the recurrent parent. In addition to the above mentioned rice lines, Taichung Native 1 (TN1) and HR12 rice varieties were used as susceptible checks for BB and blast, respectively. ISM (possessing Xa21, xa13, and xa5), FBR1-15 (possessing Xa33) were used as resistant checks for BB, while a NIL in the genetic background of Co39, C101A51 (possessing Pi2), and the Vietnamese landrace, Tetep (possessing Pi54) were used as resistant checks for blast, respectively.

Marker-assisted backcross breeding (MABB) strategy for targeted transfer of the selected BB and blast resistance genes into the genetic background of RPHR-1005

Three independent crosses, viz., RPBio Patho-1 X RPHR-1005 (Cross I), RPBio Patho-2 X RPHR-1005 (Cross II) and FBR1-15 X RPHR-1005 (Cross III) were made to transfer the genes Xa21 + Pi2, Xa21 + Pi54, and Xa33, respectively, into RPHR-1005 during dry season 2011. The methodology of MABB adopted in the study is depicted in Figure 1. The F1 plants were analyzed with the co-dominant PCR-based markers pTA248, RMWR7.6, AP5659-5, Pi54MAS specific for the resistance genes Xa21 (Ronald et al., 1992), Xa33 (Natrajkumar et al., 2012), Pi2 (Fjellstrom et al., 2006), and Pi54 (Ramkumar et al., 2011) to identify “true” F1s. They were then backcrossed with RPHR-1005 to generate BC1F1s, which were confirmed for the presence of resistance allele(s) of the genes, i.e., Xa21, Xa33, Pi2, and Pi54 in heterozygous condition, using the gene-specific markers. The resistance gene “positive” BC1F1 plants were then screened with the co-dominant markers DRRM-RF3-10 and DRCG-RF4-14, which are specific for the major fertility restorer genes, Rf3 and Rf4, respectively (Balaji Suresh et al., 2012) to identify those which are homozygous for both the genes. The restorer gene(s) “positive” BC1F1 plants were then screened with a set of polymorphic SSR markers to identify a solitary plant, possessing maximum recovery of the recurrent parent genome (RPG) through the procedure detailed in Sundaram et al. (2008). This plant was then backcrossed with RPHR-1005 to generate BC2F1s, which were then screened with markers specific for the target resistance genes to identify “positive” plants as described earlier. Among the resistance gene(s) “positive” BC2F1 plants, a solitary plant possessing maximum recovery of the RPG was identified through background selection and it was selfed to generate BC2F2s. Among them, plants homozygous for the respective target resistance genes, viz., Xa21 + Pi2 (i.e., from Cross I), Xa21 + Pi54 (i.e., from Cross II), and Xa33 (i.e., from Cross III) were identified with the help of gene-specific markers and the homozygous BC2F2 plants derived from each cross were then screened with the remaining parental polymorphic markers to identify a solitary BC2F2 plant from each cross (i.e., from Crosses I, II, and III) possessing maximum recovery of the RPG. One such BC2F2 plant from Cross I and Cross III were then crossed with another similar plant from Cross II, independently to generate intercross F1s (i.e., ICF1s) to combine the resistance genes Xa21 + Pi2 + Pi54 and Xa21 + Xa33 + Pi54 into the genetic background of the recurrent parent. The ICF1s were then selfed to generate ICF2 plants and plants homozygous for the three target resistance genes (i.e., Xa21 + Pi2 + Pi54 and Xa21 + Xa33 + Pi54) were identified through analysis with the gene-specific markers. A solitary ICF2 plant possessing the target resistance genes and closely resembling RPHR-1005 (based on visual traits) were identified from the two intercrosses and selfed. The three-gene pyramid lines (i.e., possessing the gene combination, Xa21 + Pi2 + Pi54 and Xa21 + Xa33 + Pi54) were advanced through pedigree method of breeding from ICF3 onwards for further evaluation of their resistance against BB, blast, yield, and to assess their key agro-morphological traits. Simultaneously, superior ICF2 plants from the first and second intercrosses were crossed with each other to generate double intercross F1s (DICF1s), in which all the four target resistance genes, viz., Xa21, Xa33, Pi2, and Pi54 are present in heterozygous condition. They were then selfed to generate DICF2 plants, among which, those possessing all the four target resistance genes in homozygous condition were identified through marker analysis. Homozygous DICF2 plants which were identical to or better than RPHR-1005 were identified through visual selection and then selfed to generate DICF3 lines. The improved versions of RPHR-1005 (single and two gene containing lines) viz., Xa21 + Pi2, Xa21 + Pi54 and Xa33 at BC2F7 generation, three-gene containing ICF5 lines, viz., Xa21 + Pi2 + Pi54 and Xa21 + Xa33 + Pi54 and four gene containing DICF3 lines, i.e., Xa21 + Xa33 + Pi2 + Pi54, which were subjected for evaluation of their resistance against BB, blast, yield and for key agro-morphological traits.

Figure 1

Schematic illustration of the crossing scheme for marker-assisted introgression of .

Mini-scale DNA isolation of parents, F1s and backcross derived lines was carried out from 25-day old seedlings following the procedure of Zheng et al. (1995). The PCR and gel electrophoresis protocols recommended by Sundaram et al. (2008) and Natrajkumar et al. (2012) were adopted for marker-assisted selection of Xa21 and Xa33, respectively, while the protocols recommended in Ramkumar et al. (2011) and Fjellstrom et al. (2006) were adopted for marker-assisted selection of Pi54 and Pi2, respectively. The protocol recommended by Balaji Suresh et al. (2012) was adopted for marker-assisted selection of Rf3 and Rf4. Background selection was done using polymorphic SSR markers as described in Sundaram et al. (2008). A total of 61 (RPBio Patho-1/RPHR-1005), 52 (RPBio Patho-2/RPHR-1005), and 59 (FBR1-15/RPHR-1005) parental polymorphic markers, which are reasonably well distributed throughout the 12 chromosomes of rice (i.e., ~ 3–5 polymorphic markers per chromosome) were identified and used for further analysis. More number of polymorphic markers were deployed on the carrier chromosomes (i.e., 18 polymorphic markers specific for Chr. 11, where Xa21 and Pi54 are located, 14 polymorphic markers specific for Chr. 6, where Pi2 is located, and 18 polymorphic markers for Chr. 7, where Xa33 is located) in order to minimize the linkage drag in the genomic region around the target resistance genes. Using the data from polymorphic SSR markers, a schematic map illustrating the genomic contribution of donor and recurrent parents was prepared using Graphical Genotype (GGT) Version2.0. (Van Berloo, 1999) to identify backcross derived lines possessing least introgression from donor genome in the vicinity of the target resistance genes.

Phenotypic screening for BB and blast resistance

BB resistance: The parents, 2-gene, 3-gene, and 4-gene pyramid lines RPHR-1005 along with TN1 (the susceptible check) and ISM (Resistant check) were screened when the plants were 50–55 day to assess their resistance against BB through artificial clip inoculation method (Kauffman et al., 1973) under glass house condition at ICAR-IIRR, Hyderabad, India. Two virulent isolates of X. oryzae pv. oryzae viz., DX-020 and DX-066 collected from Hyderabad, Telangana State, India and Raipur, Chattisgarh State, India, respectively, were cultured and maintained as explained in Laha et al. (2009). The inoculated plants were scored as per IRRI-standard evaluation system (IRRI-SES) scales (0–9), 1996 (IRRI, 1996) after 15 days of inoculation.

Blast resistance: All the promising gene-pyramid lines of RPHR-1005 (i.e., 2-gene, 3-gene, and 4-gene) along with RPHR-1005, HR12 (the susceptible check), C101A51, and Tetep (Resistant check) were screened for blast resistance under uniform blast nursery using SP-28, a local isolate of the blast pathogen, Magnaporthae oryzae (Madhan Mohan, 2011). These lines along with resistant and susceptible checks were scored for blast resistance as per IRRI-standard evaluation system (IRRI-SES) scales (0–9), 1996 (IRRI, 1996) after 15 days of inoculation.

Evaluation of agro-morphological characters of the backcross derived lines

Thirty-day-old seedlings of the selected 2-gene, 3-gene, 4-gene pyramid lines of RPHR-1005 were transplanted in the main the field and planted at a spacing of 15 × 20 cm with a fertilizer dosage of 220–70–80 (N: P: K) kg/ha during wet season (June–November) of 2015 along with the donor and recurrent parents. The experimental plots were arranged in a lattice design with six blocks and three replications maintained in each block. Standard agronomic practices were followed while growing the rice plants. Data were recorded for the agronomic traits, viz. days to 50% flowering DFF, mean days to maturity, mean plant height (cm), number of productive tillers per plant, panicle weight (gms), heterosis (%), panicle length (cm), grain yield per plant (gms), 1000-grain weight (gms), and grain type as explained in Hari et al. (2013). The data was tabulated and analyzed statistically for various agro-morphological traits with the help of standard techniques following Gomez and Gomez (1984). Coefficient of variation (CV), Least Significance Difference (LSD) values were calculated using standard errors of mean (S.E.M.±) at 5% level of significance using MS Excel package. Statistical analysis was performed with the software program, SAS Version 9.2 (SAS Institute Inc., Cary, NC, USA). The PROC GLM procedure of SAS was used to conduct analysis of variance (ANOVA) to determine the significant variation between the lines.

Generation of experimental hybrids using improved versions of RPHR-1005 and their evaluation

The elite WA-CMS line, APMS6A, (the female parent of the hybrid DRRH-3) were crossed with selected 2-gene, 3-gene, and 4-gene containing gene-pyramid lines of RPHR-1005 possessing BB and blast resistance. Thirty-day-old seedlings of the experimental hybrids were transplanted to the field, planted at a spacing of 15 × 20 cm with three replications as explained in the earlier section. The plants were analyzed for their spikelet fertility, resistance against BB and blast and standard heterosis for grain yield during wet season of 2015 in the experimental farm of ICAR-IIRR at Rajendranagar, Hyderabad. The popular, high-yielding variety, BPT5204 (Samba Mahsuri) possessing medium-slender grain type served as a varietal check, while DRRH-3 served as the hybrid check. Appropriate statistical parameters were analyzed using MS Excel package as explained earlier.

Results

Marker-assisted introgression of Xa21 and Pi2 into RPHR-1005

A total of 44 F1s, which were produced by crossing RPBio Patho-1 × RPHR-1005 were screened for their heterozygosity with the help of gene specific markers i.e., pTA248 (Xa21) and AP5659-5 (Pi2) and 33 of these were identified to be “true” F1s and they were backcrossed with RPHR-1005 to generate 360 BC1F1s. Foreground analysis of these plants with the gene-specific markers revealed that 22 plants were heterozygous for both the target genes. They were then screened for identification of plants wherein the fertility restorer genes Rf3 and Rf4 are present in homozygous condition using trait-linked markers and a total of five “positive” BC1F1 plants were identified. Among these, one plant (# RPC-I-9-27), possessing maximum RPG recovery (77.4%) was identified through background selection using 61 parental polymorphic SSR markers. It was further backcrossed with RPHR-1005 to produce a total of 134 BC2F1 plants. Foreground selection among the BC2F1 plants revealed a total of eight plants possessing Xa21 and Pi2 in heterozygous condition, which were then subjected to background genome recovery analysis. A single BC2F1 plant (# RPC-I-9-27-79) with maximum RPG (86.9%) was identified and selfed to generate a total of 560 BC2F2s. Marker-assisted screening of these plants identified 35 plants possessing both Xa21 and Pi2 in homozygous condition. Among these, a single plant (# RPC-I-9-27-79-179) possessing maximum RPG recovery (91.8%) was identified through background selection. This plant was then analyzed to estimate the extent of “linkage drag” from the donor genome around the two target resistance genes, viz., Xa21 (chromosome 11) and Pi2 (chromosome 6). With respect to Xa21, a segment of 0.6 Mb was observed to be introgressed at the proximal end from the donor parent genome, while at the distal end, of the donor parent chromosome segment introgression was observed to be limited to 0.4 Mb. Thus, in total, a segment of 1.0 Mb was introgressed from the donor parent with respect to the genomic region in the vicinity of Xa21. With respect to Pi2, a segment of 0.2 and 0.3 Mb were introgressed from the donor parent genome in the proximal and distal sides of the gene, respectively (i.e., totaling to a segment of 0.5 Mb around Pi2) and the donor genome introgression is thus limited to ~1.5 Mb in the best BC2F2 plant (i.e., # RPC-I-9-27-79-179; Figures 2A,B) and this plant was forwarded for intercrossing.

Figure 2

Analysis of genome introgression associated with the blast resistance gene, Analysis of genome introgression associated with the both Xa21 and Pi54, a genomic region limited to ~1.3 Mb has been only introgressed from the donor parent (RPBio Patho-2) in the best backcross plant (i.e., plant # RPC-II–38-213-63-259; possessing Xa21 + Pi54). (D) Analysis of genome introgression associated with the Xa33, a genomic region limited to ~1.0 Mb has been only introgressed from the donor parent (FBR1-15) in the best backcross plant (i.e., plant # RPC-III-38-27-43-276; possessing Xa33). The position of the polymorphic SSR markers in Mb on Chr. 6, 11, and 7 is given in parenthesis adjacent to each marker, while each marker has also been positioned with respect to each other in terms of cM scale.

Marker-assisted introgression of Xa21 and Pi54 into RPHR-1005

A total of 53 F1s produced by crossing RPBio Patho-2 × RPHR-1005 were screened for their heterozygosity with the help of gene specific markers i.e., pTA248 (Xa21) and Pi54MAS (Pi54) and 38 of them were identified to be “true” F1s. They were then backcrossed with RPHR-1005 to generate 480 BC1F1s. Foreground selection of these plants with the gene-specific markers revealed that 29 were heterozygous for both the target resistance genes. Among these six BC1F1 plants were identified to possess both Rf3 and Rf4 in homozygous condition. After screening these plants through background selection using 52 parental polymorphic markers, a single plant (# RPC-II-38-213) possessing maximum RPG recovery (75%) was identified and was then backcrossed with RPHR-1005 to produce a total of 122 BC2F1 plants. Foreground selection among the BC2F1 plants, revealed a total of seven plants possessing Xa21 and Pi54 in heterozygous condition. A single BC2F1 plant (# RPC-II-38-213-63) with maximum RPG (86.5%) was identified through background selection and selfed to generate a total of 480 BC2F2s. Marker-assisted screening of these plants helped in identification of 22 plants which were homozygous for both Xa21 and Pi54. Among these, a single plant (# RPC-II-38-213-63-259) possessing maximum RPG recovery (92.3%; Figure 2C) was identified through background selection. This plant was then analyzed to estimate the extent of “linkage drag” from the donor genome around the two target resistance genes, viz., Xa21 and Pi54 (chromosome 11). With respect to Xa21, a segment of 0.6 Mb was observed to be introgressed at the proximal end from the donor parent genome, while at the distal end, a segment of 0.4 Mb was observed to be introgressed. Thus, in total, a segment of 1.0 Mb was introgressed from the donor parent with respect to the genomic region in the vicinity of Xa21. With respect to Pi54, a segment of 0.6 and 0.1 Mb were introgressed from the donor parent genome (totaling to 0.7 Mb) on the proximal and distal sides of the target gene, respectively and the donor genome introgression was observed to be limited to ~1.7 Mb in the best BC2F2 plant (i.e., # RPC-II-38-213-63-259; Figure 2C) and this plant was forwarded for intercrossing.

Marker-assisted introgression of Xa33 into RPHR-1005

Marker-assisted screening using RMWR7.6, resulted in the identification of 28 “true” F1s and they were backcrossed with RPHR-1005 to generate 175 BC1F1s. Eighty-seven of them were observed to be heterozygous for Xa33 and 43 among them were found to be homozygous for the fertility restorer genes Rf3 and Rf4. Among these 43 plants, a solitary plant, (i.e., # RPC-III-38-27) possessing maximum RPG recovery (78%) was identified through background selection using 59 parental polymorphic SSR markers. It was then backcrossed with RPHR-1005 to generate 121 BC2F1s. Among these, 60 BC2F1 plants were found to be heterozygous for Xa33 and a single BC2F1 plant (# RPC-III-38-27-43) with maximum RPG recovery (86.4%) was identified through background selection and selfed to generate 450 BC2F2 seeds. Marker-assisted screening of these plants identified 112 homozygous positive plants and among these, a single plant (# RPC-III-38-27-43-276) possessing maximum introgression of the RPG (93.2%), was identified through background selection. This plant was then analyzed to estimate the extent of “linkage drag” from the donor genome around the two target resistance genes, viz., Xa33 (chromosome 7). A segment of 0.1 Mb was observed to be introgressed at the proximal end from the donor parent genome, while at the distal end, a segment of 0.5 Mb was observed to be introgressed. Thus, in total, a segment limited to ~0.6 Mb was observed to be transferred from the donor parent in the best BC2F2 plant (i.e., # RPC-III-38-27-43-276; Figure 2D) and this plant was forwarded for intercrossing.

Combining multiple BB and blast resistant genes into RPHR-1005

At BC2F2, a single homozygous plant, possessing maximum RPG was identified from each cross and intercrossed as follows: Cross-I plant # RPC-I-9-27-79-179 X Cross-II plant # RPC-II-38-213-63-259 (to combine Xa21, Pi2, and Pi54, i.e., Intercross- IC1) and Cross-II plant # RPC-II-38-213-63-259 X Cross-III plant # RPC-III-38-27-43-276 (to combine Xa21, Xa33, and Pi54, i.e., Intercross 2; IC2). A total of 61 and 51 plants were confirmed to be heterozygous in IC1F1 and IC2F1, respectively, with respect to the target genes (i.e., Xa21 + Pi2 + Pi54 and Xa21 + Xa33 + Pi54). The “true” intercross F1s were selfed to obtain 900 IC1F2 and 1050 IC2F2 plants, respectively. Among these, a total of 14 and 16 homozygous triple positive plants were identified in IC1F2s (i.e., Xa21 + Pi2 + Pi54) and IC2F2s (Xa21 + Xa33 + Pi54), respectively. A single plant was identified among the homozygous IC1F2s and IC2F2s (i.e., plant # RPIC1F2-12-196 and RPIC2F2-53-640, respectively) to be closer to RPHR-1005 in terms of agromophological traits and they were then intercrossed to obtain double intercross F1s (i.e., DICF1s), in which all the four target resistance genes (viz., Xa21, Xa33, Pi2, and Pi54) were combined in the genetic background of RPHR-1005. In addition, the selected three gene-pyramid plants from IC1F2 and IC2F2 were also further advanced by selfing up to ICF5 generation through morphology based pedigree selection for further evaluation. A total of 49 DICF1 plants were identified to be “true” F1s and they were selfed to obtain 5650 DICF2 plants. Screening of these plants with the gene-specific markers resulted in identification of 350 plants which were homozygous for all the four target resistance genes (Figure 3). Among these, a single plant (i.e., RPDICF2-9-786) was identified to be similar to RPHR-1005 through phenotype-based morphological selection. This plant was advanced by selfing through pedigree method up to DICF3 generation. Five promising DICF3 lines (Tables 1, 2) were identified for further evaluation.

Figure 3

The gene linked markers pTA248, RMWR7.6, AP5659-5, and Pi54MAS were used for screening of . The numbers shown on the top of the each gel represents the double intercross F2 plant numbers. Gel (i), (ii), (iii), and (iv) represents the screening of the double intercross F2 plants derived from the cross IC1F2 X IC2F2. Lanes M: 100 bp molecular weight ladder; R-Recurrent parent (RPHR-1005); D- Donor parent (i & iii- RPBio Patho-1 and ii-FBR1-15 & iv- RPBio Patho-2), 1-25- DICF2 plants; Arrow indicates “tetra homozygous positive plants.”

Table 1

Screening of selected backcross, intercross and double intercross plants for their resistance against BB and blast.

S. No.	Plant identity	Allelic status of Xa21, Xa33, Pi2 and Pi54	*Disease scoring scale for rice blast (0–9 scale)	#Disease scoring scale/Average lesion length for BB (cm)
				DX-020	DX-066
KHARIF 2015
1	RPHR-1005	xa21xa21, pi2pi2, pi54pi54	9	21.6 ± 0.7	20.2 ± 0.4
2	RPBio Patho-1/C101A51	Xa21Xa21, Pi2Pi2	3	3.0 ± 0.6	–
3	RPBio Patho-2/Tetep	Xa21Xa21, Pi54Pi54	0	1.0 ± 0.2	–
4	FBR1-15	Xa33Xa33	–	–	0.7 ± 0.3
5	HR12 (Susceptible check for blast)	–	9	–	–
6	TN1 (Susceptible check for BB)	–	–	20.2 ± 0.4	20.7 ± 0.7
CROSS (C-I) — (Xa21 + Pi2)
7	RPC-I-9-27-79-179-7	Xa21Xa21, Pi2Pi2	2	3.0 ± 0.6	–
8	RPC-I-9-27-79-179-29	Xa21Xa21, Pi2Pi2	1	2.3 ± 0.3	–
9	RPC-I-9-27-79-179-120	Xa21Xa21, Pi2Pi2	0	1.3 ± 0.3	–
CROSS (C-II) — (Xa21 + Pi54)
10	RPC-II-38-213-63-259-3	Xa21Xa21, Pi54Pi54	3	3.0 ± 0.6	–
11	RPC-II-38-213-63-259-9	Xa21Xa21, Pi54Pi54	2	2.0 ± 0.0	–
12	RPC-II-38-213-63-259-62	Xa21Xa21, Pi54Pi54	0	1.7 ± 0.3	–
CROSS (C-III) — (Xa33)
13	RPC-III-38-27-43-276-6	Xa33Xa33	–	–	2.0 ± 0.2
14	RPC-III-38-27-43-276-17	Xa33Xa33	–	–	1.3 ± 0.3
15	RPC-III-38-27-43-276-24	Xa33Xa33	–	–	1.0 ± 0.3
INTERCROSS (IC1) — Xa21 + Pi2 + Pi54
16	RPIC1-12-196-12	Xa21Xa21, Pi2Pi2, Pi54Pi54	0	1.7 ± 0.3	–
17	RPIC1-12-196-15	Xa21Xa21, Pi2Pi2, Pi54Pi54	1	2.3 ± 0.3	–
18	RPIC1-12-196-36	Xa21Xa21, Pi2Pi2, Pi54Pi54	1	2.7 ± 0.3	–
19	RPIC1-12-196-45	Xa21Xa21, Pi2Pi2, Pi54Pi54	0	3.0 ± 0.6	–
INTERCROSS (IC2)—Xa21 + Xa33 + Pi54
20	RPIC2-53-640-6	Xa21Xa21, Xa33Xa33, Pi54Pi54	0	1.7 ± 0.3	1.3 ± 0.3
21	RPIC2-53-640-18	Xa21Xa21, Xa33Xa33, Pi54Pi54	1	1.0 ± 0.0	1.7 ± 0.3
22	RPIC2-53-640-35	Xa21Xa21, Xa33Xa33, Pi54Pi54	1	0.7 ± 0.3	0.7 ± 0.3
23	RPIC2-53-640-43	Xa21Xa21, Xa33Xa33, Pi54Pi54	1	0.0 ± 0.0	0.0 ± 0.0
DOUBLE INTERCROSS (DIC)—Xa21 + Xa33 + Pi2 + Pi54
24	RPDIC-9-786-1	Xa21Xa21, Xa33Xa33, Pi2Pi2, Pi54Pi54	0	1.0 ± 0.0	0.7 ± 0.3
25	RPDIC-9-786-19	Xa21Xa21, Xa33Xa33, Pi2Pi2, Pi54Pi54	0	1.0 ± 0.0	0.0 ± 0.0
26	RPDIC-9-786-25	Xa21Xa21, Xa33Xa33, Pi2Pi2, Pi54Pi54	0	0.0 ± 0.0	0.0 ± 0.0
27	RPDIC-9-786-46	Xa21Xa21, Xa33Xa33, Pi2Pi2, Pi54Pi54	1	0.0 ± 0.0	0.0 ± 0.0
28	RPDIC-9-786-78	Xa21Xa21, Xa33Xa33, Pi2Pi2, Pi54Pi54	0	1.0 ± 0.0	0.0 ± 0.0

The backcross derived lines at BC.

The backcross derived lines at BC.

Table 2

Analysis of agro-morphological characters of improved RPHR-1005 lines along with parents under field conditions.

S. No.	Plant identity	Days to 50% flowering (DFF)	Mean plant height (cm)	No. of productive panicles/plant (NPP)	Panicle weight (gm)	No. of grains/panicle	Panicle length (cm)	Grain yield per plant (gm)	1000 seed weight (gm)	Grain type
1	RPHR-1005	105.3 ± 0.3	90.3 ± 0.7	16.7 ± 0.3	2.53 ± 0.04	187.3 ± 2.73	15.5 ± 0.3	26.3 ± 0.1	16.3 ± 0.1	MS
2	RPBio Patho-1	96.0 ± 1.0	68.1 ± 0.2	15.0 ± 0.6	2.06 ± 0.07	163.7 ± 0.88	18.8 ± 0.6	25.2 ± 0.3	17.0 ± 0.1	MS
3	RPBio Patho-2	103.7 ± 0.9	70.1 ± 0.5	16.0 ± 0.6	2.20 ± 0.04	167.3 ± 1.20	19.2 ± 0.4	26.4 ± 0.1	17.9 ± 0.4	MS
4	FBR1-15	101.7 ± 0.7	76.9 ± 0.5	13.0 ± 0.6	1.99 ± 0.13	180.0 ± 1.00	15.6 ± 0.3	19.4 ± 0.2	16.3 ± 0.3	MS
CROSS (C–I) POSSESSING Xa21 + Pi2
5	RPC-I-9-27-79-179-7	100.3 ± 0.3	89.9 ± 0.1	15.7 ± 0.3	2.24 ± 0.07	183.0 ± 1.15	16.8 ± 0.6#	26.0 ± 0.1	16.8 ± 0.2#	MS
6	RPC-I-9-27-79-179-29	102.7 ± 0.7	88.8 ± 0.5	17.7 ± 0.9#	2.45 ± 0.03	190.3 ± 1.45#	19.2 ± 0.4#	26.0 ± 0.1	16.9 ± 0.4#	MS
7	RPC-I-9-27-79-179-120	101.0 ± 0.6	91.5 ± 0.6#	21.0 ± 0.6#	2.88 ± 0.02#	205.7 ± 1.76#	20.3 ± 0.3#	27.3 ± 0.3#	17.7 ± 0.2#	MS
CROSS (C–II) POSSESSING Xa21 + Pi54
8	RPC-II-38-213-63-259-3	104.3 ± 0.3	91.8 ± 1.1#	17.3 ± 0.9#	2.48 ± 0.04	198.0 ± 1.53#	16.1 ± 0.1#	27.1 ± 0.6#	16.7 ± 0.4	MS
9	RPC-II-38-213-63-259-9	99.7 ± 0.7	93.1 ± 0.6#	18.0 ± 0.6#	2.59 ± 0.02#	188.0 ± 1.53	17.1 ± 0.2#	27.8 ± 0.2#	17.1 ± 0.1#	MS
10	RPC-II-38-213-63-259-62	102.7 ± 0.7	95.3 ± 0.6#	19.0 ± 0.6#	2.66 ± 0.02#	191.0 ± 1.53#	18.3 ± 0.7#	28.0 ± 0.6#	17.5 ± 0.2#	MS
CROSS (C–III) POSSESSING Xa33
11	RPC-III-38-27-43-276-6	97 ± 1.5	88.8 ± 0.5	15.7 ± 0.3	2.24 ± 0.07	165.0 ± 1.73	15.3 ± 0.1	23.3 ± 0.4	16.8 ± 0.2#	MS
12	RPC-III-38-27-43-276-17	101.3 ± 1.3	91.0 ± 0.5#	16.7 ± 0.3	2.12 ± 0.02	177.7 ± 2.40	15.6 ± 0.3	24.0 ± 0.2	17.3 ± 0.2#	MS
13	RPC-III-38-27-43-276-24	105.0 ± 1.0	91.5 ± 0.6#	17.7 ± 0.9#	2.59 ± 0.00#	191.3 ± 1.20#	16.8 ± 0.6#	26.0 ± 0.1	17.4 ± 0.2#	MS
INTERCROSS (IC1) POSSESSING Xa21 + Pi2 + Pi54
14	RPIC1-12-196-12	100.7 ± 0.3	91.2 ± 0.8	16.3 ± 0.3	2.15 ± 0.03	177.7 ± 1.45	16.1 ± 0.1#	24.7 ± 0.3	16.8 ± 0.2#	MS
15	RPIC1-12-196-15	102.3 ± 0.9	93.9 ± 0.9#	17.7 ± 0.7#	2.52 ± 0.04	181.7 ± 1.45	17.1 ± 0.2#	25.3 ± 0.2	16.9 ± 0.2#	MS
16	RPIC1-12-196-36	99.7 ± 0.7	95.3 ± 0.6#	18.3 ± 0.9#	2.63 ± 0.01#	198.3 ± 2.60#	18.6 ± 0.3#	26.6 ± 0.7#	17.0 ± 0.1#	MS
17	RPIC1-12-196-45	104.7 ± 0.7	101.9 ± 0.5#	19.7 ± 0.3#	2.67 ± 0.03#	201.7 ± 2.40#	20.5 ± 0.3#	27.5 ± 0.3#	17.6 ± 0.1#	MS
INTERCROSS (IC2) POSSESSING Xa21 + Xa33 + Pi54
18	RPIC2-53-640-6	97.0 ± 1.5	91.0 ± 0.5#	17.7 ± 0.9#	2.58 ± 0.04#	194.3 ± 2.40#	18.6 ± 0.3#	26.6 ± 0.7#	16.7 ± 0.3#	MS
19	RPIC2-53-640-18	104.3 ± 0.3	89.9 ± 0.1	19.0 ± 0.6#	2.63 ± 0.01#	186.0 ± 2.65	20.5 ± 0.3#	26.9 ± 0.1#	17.3 ± 0.3#	MS
20	RPIC2-53-640-35	94.7 ± 0.7#	88.8 ± 0.5	20.0 ± 0.0#	2.79 ± 0.03#	205.7 ± 2.40#	20.8 ± 0.3#	27.1 ± 0.6#	17.4 ± 0.0#	MS
21	RPIC2-53-640-43	102.7 ± 0.3	102.5 ± 0.6#	22.7 ± 0.3#	3.08 ± 0.08#	201.3 ± 1.45#	22.7 ± 0.3#	27.3 ± 0.3#	17.3 ± 0.2#	MS
DOUBLE INTERCROSS (DIC) POSSESSING Xa21 + Xa33 + Pi2 + Pi54
22	RPDIC-9-786-1	99.7 ± 0.7	93.1 ± 0.6#	17.7 ± 0.7#	2.63 ± 0.06#	198.3 ± 2.52#	19.5 ± 0.5#	26.6 ± 0.7#	17.9 ± 0.2#	MS
23	RPDIC-9-786-19	102.3 ± 0.9	95.3 ± 0.6#	19.7 ± 0.3#	2.73 ± 0.07#	201.1 ± 1.73#	20.8 ± 0.3#	27.7 ± 0.3#	17.9 ± 0.2#	MS
24	RPDIC-9-786-25	93.7 ± 0.3#	101.0 ± 0.6#	20.0 ± 0.0#	2.79 ± 0.02#	203.0 ± 2.00#	20.7 ± 0.3#	27.8 ± 0.1#	18.1 ± 0.1#	MS
25	RPDIC-9-786-46	106.3 ± 0.7	102.5 ± 0.6#	22.3 ± 0.3#	3.03 ± 0.08#	245.3 ± 2.73#	22.8 ± 0.1#	28.4 ± 0.1#	18.4 ± 0.2#	MS
26	RPDIC-9-786-78	104.7 ± 0.7	102.3 ± 0.6#	22.7 ± 0.3#	3.01 ± 0.06#	249.0 ± 1.73#	22.4 ± 0.3#	29.2 ± 0.4#	19.0 ± 0.0#	MS
	F	17.53	229.08	16.22	42.38	127.07	42.84	25.86	9.23
	p-value	< 0.0001	< 0.0001	< 0.0001	< 0.0001	< 0.0001	< 0.0001	< 0.0001	< 0.0001
	CV (%)	1.33	1.05	5.43	3.13	1.52	3.18	2.43	2.04	–
	LSD (p = 0.05)	2.21	1.57	1.62	0.15	4.83	0.98	1.05	0.58	–

RPHR-1005-Reccurent parent; RPBio Patho-1, RPBio Patho-2 and FBR1-15-Donar parents; MS-Medium slender; CV, Coefficient variance; LSD-Least significance difference at 5% probability level; ± -Standard error and values given are mean of three replications.

Better than the recurrent parent in terms of DFF, Plant height, NPP, Panicle weight, No. of grains per panicle, Panicle length, Grain yield per plant, and 1000 seed weight.

Evaluation of BB and blast resistance of the improved RPHR-1005 lines

The resistance checks RPBio Patho-1/C101A51 and RPBio Patho-2/Tetep harboring the blast genes Pi2 and Pi54, respectively, showed a blast disease score of 0–3, while the susceptible checks, RPHR-1005 and HR12 showed a score of 9. All the IC1F5,IC2F5,and DICF3 lines of RPHR-1005 (possessing single or two blast resistance genes) displayed high level of resistance with scores in the range of 0–1 (Table 1; Figure 4). With respect to screening for BB, the donor RPBio Patho-1, RPBio Patho-2, and FBR1-15 displayed average lesion length for BB 1.0 ± 0.3 to 3.0 ± 0.6 cm (i.e., high level of resistance), while the recurrent parent, i.e., RPHR-1005 showed average lesion length >20 cm (i.e., highly susceptible). The improved (at IC1F5) single-BB resistance gene containing RPHR-1005 lines (Figure 5) displayed resistance equivalent to the donor parent with score from 1.7 ± 0.3 to 3.0 ± 0.6 cm, whereas lines containing two BB resistance genes, (at IC2F5 and DICF3;Table 1; Figure 5) showed a higher level resistance reaction (i.e., immune level of resistance).

Figure 4

Phenotypic screening of the selected improved and double intercrossed lines against rice blast disease. (A) Screening of the selected IC1F5 lines (Xa21 + Pi2 + Pi54) against blast disease following UBN Method. All the intercross derived lines IL-1 to IL-4 (RPIC1-12-196-12, RPIC1-12-196-15, RPIC1-12-196-36, and RPIC1-12-196-45) were highly resistant. (B) Screening of selected IC2F5 lines (Xa21 + Xa33 + Pi54) against blast disease following UBN Method. All the intercross derived lines IL-1 to IL-4 (RPIC2-53-640-6, RPIC2-53-640-18, RPIC2-53-640-35, and RPIC2-53-640-43) were highly resistant. (C) Screening of selected DICF3 lines (Xa21 + Xa33 + Pi2 + Pi54) against blast disease following UBN Method. (A51) C101A51- Resistant check; (T) Tetep- Resistant check; (RP) RPHR-1005- Recurrent parent (susceptible), (H) HR-12- Susceptible check and all the double intercross derived lines IL-1 to IL-5 (RPDIC-9-786-1, RPDIC-9-786-19, RPDIC-9-786-25, RPDIC-9-786-46, and RPDIC-9-786-78) were highly resistant.

Figure 5

Phenotypic screening of the selected intercrossed and double intercrossed lines against rice blast disease. (A) Phenotypic screening of IC1F5 lines (Xa21 + Pi2 + Pi54) against rice BB resistance with DX-020 isolate. (B) Phenotypic screening of IC2F5 lines (Xa21 + Xa33 + Pi54) against rice BB resistance with DX-066 isolate. (C) Selected DICF3 lines against rice BB resistance with DX-020 isolate. (D) Selected DICF3 lines against rice BB resistance with DX-066 isolate. Along with (RP) Recurrent parent (RPHR-1005), (D) donor parents [RP Bio patho-1 for (A,C) and FBR1-15 for (B,D)], (1) to (6) were selected lines. Forty-five days old seedlings were inoculated by clip inoculation method (Kauffman et al., 1973). The selected lines have shown high level of resistance equal to the donor parents. The lesion length and disease score were calculated based on IRRI- SES (IRRI- standard evaluation system; IRRI, 1996).

Screening of the improved RPHR-1005 lines for agro-morphological traits

The days to 50% flowering (DFF) of the recurrent parent i.e., RPHR-1005 and the donors (i.e., RPBio Patho-1, RPBio Patho-2, and FBR1-15) ranged from 93 to 106 days respectively. The DFF of selected backcross derived lines at BC2F7 of RPHR-1005 (possessing Xa21 + Pi2, Xa21 + Pi54, and Xa33) ranged from 97 to 105 days (Table 2). With respect to the selected intercross F5 lines of RPHR-1005 (possessing Xa21 + Pi2 + Pi54 and Xa21 + Xa33 + Pi54), the DFF ranged from 97 to 104 days respectively (Table 2). The DFF of selected double intercross F3 lines of RPHR-1005 possessing Xa21 + Xa33 + Pi2 + Pi54 ranged from 93 to 106 days (Table 2). While most of the improved breeding lines of RPHR-1005 were observed to flower similar to the original recurrent parent (i.e., RPHR-1005), two lines, viz., IC2F5 line # RPIC2-53-640-35 and DICF3 line # RPDIC-9-786-25 flowered significantly earlier (i.e., 11–12 days) as compared to the recurrent parent, RPHR-1005.

Significant differences were noticed in plant height among some of the improved lines, which were taller than the parent RPHR-1005 (Table 2). Some such lines include the BC2F7 lines- RPC-I-9-27-79-179-120, RPC-II-38-213-63-259-3, RPC-II-38-213-63-259-9, RPC-II-38-213-63-259-62, RPC-III-38-27-43-276-17, and RPC-III-38-27-43-276-24, ICF5 lines- RPIC1-12-196-12, RPIC1-12-196-15, RPIC1-12-196-36, RPIC1-12-196-45, RPIC2-53-640-6, RPIC2-53-640-43 and all the DICF3 lines.

The mean values for number of grains per panicle of selected backcross derived lines at BC2F7 ranged from 165.0 ± 1.73 to 205.7 ± 1.76 (Table 2). With respect to the selected ICF5 lines the values ranged from 177.7 ± 1.45 to 205.7 ± 2.40, while the values for DICF3 lines ranged from ranged from 198.3 ± 2.52 to 249.0 ± 1.73. Many improved lines, viz., BC2F7 lines-RPC-I-9-27-79-179-29, RPC-I-9-27-79-179-120, RPC-II-38-213-63-259-3, RPC-II-38-213-63-259-9, RPC-II-38-213-63-259-62, RPC-III-38-27-43-276-24, ICF5 lines-RPIC1-12-196-36, RPIC1-12-196-45, RPIC2-53-640-6, RPIC2-53-640-35, RPIC2-53-640-43 and all the DICF3 lines showed significantly higher grain number per panicle as compared to RPHR-1005.

The mean values for grain yield per plant of selected backcross derived lines at BC2F7 ranged from 26.0 ± 0.1 to 28.0 ± 0.6 (Table 2). With respect to the selected ICF5 lines the values ranged from 24.7 ± 0.3 to 27.5 ± 0.3, while the values for DICF3 lines ranged from ranged from 26.6 ± 0.7 to 29.2 ± 0.4. BC2F7 lines viz., # RPC-I-9-27-79-179-120, RPC-II-38-213-63-259-3, RPC-II-38-213-63-259-9, RPC-II-38-213-63-259-62, and ICF5 lines # RPIC1-12-196-36, RPIC1-12-196-45, RPIC2-53-640-6, RPIC2-53-640-18, RPIC2-53-640-35, RPIC2-53-640-43, and DICF3 lines # RPDIC-9-786-1, RPDIC-9-786-19, RPDIC-9-786-25, RPDIC-9-786-46, and RPDIC-9-786-78 had significantly higher grain yield per plant as compared to the recurrent parent, RPHR-1005.

Evaluation of the newly derived experimental hybrids for fertility restoration ability and heterosis

The mean value of spikelet fertility recorded for the three experimental hybrids derived from the crosses between selected BC2F7 lines, RPC-I-9-27-79-179-120 (possessing Xa21 + Pi2) and RPC-II-38-213-63-259-62 (possessing Xa21 + Pi54) with APMS 6A (i.e., Hybrids H-1 to H-6) was 86.0 ± 1.7 and 91.7 ± 2.0, respectively (Table 3). With respect to intercross F5 lines, the mean spikelet fertility values of the experimental hybrids lines ranged from 81.7 ± 1.8 to 92.0 ± 1.2%, while the values for hybrids derived from selected DICF3 ranged from 88.0 ± 1.2 to 93.3 ± 0.9%.

Table 3

Details of spikelet fertility (%) and grain yield of the parents, improved lines of RPHR-1005 and their derived experimental hybrids and standard heterosis of the hybrids during wet season 2015.

S. No.	Cross/Genotype	Genes are present	Spikelet fertility (%)	Grain yield per plant (gm)	Grain yield heterosis over Standard check BPT 5204 (%)	Grain yield heterosis over hybrid check DRRH3 (%)
1	RPHR-1005	–	93.0 ± 1.5	26.3 ± 0.1	–	–
2	RPBio Patho-1	Xa21 + Pi2	84.0 ± 1.5	25.2 ± 0.3	–	–
3	RPBio Patho-2	Xa21 + Pi54	84.7 ± 3.2	26.4 ± 0.1	–	–
4	FBR1-15	Xa33	78.3 ± 1.2	19.4 ± 0.2	-	–
5	Samba Mahsuri	–	94.3 ± 1.5	15.9 ± 0.8	–	–
6	DRRH3 (RPHR-1005 X APMS6A)	–	87.0 ± 2.0	18.5 ± 0.9	16.3*	–
7	H-1 (RPC-I-9-27-79-179-7 X APMS6A)	Xa21 + Pi2	84.7 ± 2.0	19.2 ± 0.1	20.7*	3.7
8	H-2 (RPC-I-9-27-79-179-29 X APMS6A)	Xa21 + Pi2	84.0 ± 1.5	19.2 ±0.2	20.7*	3.7
9	H-3 (RPC-I-9-27-79-179-120 X APMS6A)	Xa21 + Pi2	86.0 ± 1.7	19.7 ±0.1	23.8*	6.4#
10	H-4 (RPC-II-38-213-63-259-3 X APMS6A)	Xa21 + Pi54	87.0 ± 1.5	19.5 ±0.2	22.6*	5.4#
11	H-5 (RPC-II-38-213-63-259-9 X APMS6A)	Xa21 + Pi54	90.0 ± 1.0	20.2 ± 0.1	27.0*	9.1#
12	H-6 (RPC-II-38-213-63-259-62 X APMS6A)	Xa21 + Pi54	91.7 ± 2.0	20.5 ±0.1	31.4*	10.8#
13	H-7 (RPIC1-12-196-12 X APMS6A)	Xa21 + Pi2 + Pi54	81.7 ± 1.8	19.9 ± 0.6	25.1*	7.5#
14	H-8 (RPIC1-12-196-15 X APMS6A)	Xa21 + Pi2 + Pi54	82.0 ± 1.5	19.5 ± 1.5	22.6*	5.4#
15	H-9 (RPIC1-12-196-36 X APMS6A)	Xa21 + Pi2 + Pi54	87.7 ± 2.0	19.9 ± 1.4	25.1*	7.5#
16	H-10 (RPIC1-12-196-45 X APMS6A)	Xa21 + Pi2 + Pi54	92.0 ± 1.2	20.9 ± 1.0	26.4*	12.9#
17	H-11 (RPIC2-53-640-6 X APMS6A)	Xa21 + Xa33 + Pi54	87.7 ± 1.9	19.4 ± 0.8	22.0*	4.8
18	H-12 (RPIC2-53-640-18 X APMS6A)	Xa21 + Xa33 + Pi54	88.0 ± 1.5	19.5 ± 0.5	22.6*	5.4#
19	H-13 (RPIC2-53-640-35 X APMS6A)	Xa21 + Xa33 + Pi54	88.0 ± 3.8	19.5 ± 0.9	22.6*	5.4#
20	H-14 (RPIC2-53-640-43 X APMS6A)	Xa21 + Xa33 + Pi54	90.0 ± 2.3	20.7 ± 0.7	25.7*	11.8#
21	H-15 (RPDIC-9-786-1 X APMS6A)	Xa21 + Xa33 + Pi2 + Pi54	88.0 ± 1.2	19.7 ± 1.2	23.8*	6.4#
22	H-16 (RPDIC-9-786-19 X APMS6A)	Xa21 + Xa33 + Pi2 + Pi54	92.3 ± 1.2	20.1 ± 0.7	26.4*	8.6#
23	H-17 (RPDIC-9-786-25 X APMS6A)	Xa21 + Xa33 + Pi2 + Pi54	92.7 ± 1.9	20.1 ± 0.4	26.4*	8.6#
24	H-18 (RPDIC-9-786-46 X APMS6A)	Xa21 + Xa33 + Pi2 + Pi54	93.0 ± 2.0	21.1 ± 0.4	32.7*	14.0#
25	H-19 (RPDIC-9-786-78 X APMS6A)	Xa21 + Xa33 + Pi2 + Pi54	93.3 ± 0.9	22.0 ± 0.7	38.3*	18.9#

Significantly higher heterosis (i.e., by >10%) as compared to the check variety (i.e., BPT5204).

Significantly higher heterosis (i.e., by >5%) as compared to the check hybrid (i.e., DRRH-3).

All the F1s derived from BC2F7 lines, i.e., H-1 to H-6, from the respective crosses (i.e., C-I, C-II, and C-III X APMS 6A) were fertile, indicating that all the improved lines of RPHR-1005 analyzed, are indeed complete restorers. The standard heterosis values of selected backcross derived lines at BC2F7 ranged from 3.7 to 10.8% (Table 3). Some (i.e., H-3, H-4, H-5, and H-6) of the newly derived hybrids (Table 3) displayed higher level of standard heterosis (when compared to DRRH-3). With respect to ICF5 lines, the standard heterosis values of the experimental hybrids lines ranged from 4.8 to 12.9% (Table 3), while the values for hybrids derived from selected DICF3 ranged from 6.4 to 18.9%.

Discussion

The hybrid, DRRH-3 besides having the highly desirable MS grain type and other superior grain quality features, similar to the elite rice variety, Samba Mahsuri (also known as BPT5204), matures earlier (by about 10 days) with a yield advantage of 20–25% over the elite variety. Despite its superior grain and yield qualities, DRRH-3 and its parents RPHR-1005 and APMS6A are highly susceptible to two major rice diseases, viz., BB and blast. The present study was therefore carried out with an objective to improve RPHR-1005 and DRRH-3 for durable resistance against BB and blast by targeted introgression of two major genes, each conferring resistance against the two diseases through MABB coupled with phenotype-based selection, while retaining the premium grain quality and high yield potential of the parental line and the hybrid.

Among the different strategies available for improvement of BB and blast resistance in hybrids, marker-assisted introgression of the resistance genes into hybrid rice parental lines, particularly the restorer line is considered as the ideal choice (Hari et al., 2011, 2013; Balachiranjeevi et al., 2015). Among the blast resistance genes available for deployment in gene-pyramiding programs, Pi2 and Pi54 are major dominant genes, which have been reported to be highly effective against the pathogen populations in India (Sharma et al., 2002). The durable resistance gene combination of Pi2 + Pi54 was not only effective in northern and eastern parts of India, but also in the Southern parts of the country such as Pattambi, Kerala, and Gudalur, Tamil Nadu (Ellur et al., 2016). Similarly several earlier studies have established that the BB resistance gene, Xa21 to be highly effective under Indian conditions (Gopalakrishnan et al., 2008; Sundaram et al., 2008) and the newly identified wild rice derived BB resistance gene, Xa33 has also been found to be effective against several Indian isolates of the pathogen (Natrajkumar et al., 2012). Considering these points, in the present study, two major BB resistance genes, viz., Xa21 and Xa33 and two major blast resistance genes, Pi2 and Pi54 were selected for improvement of RPHR-1005 (the restorer parent of DRRH-3) for durable resistance against BB and blast through MABB strategy coupled with phenotype-based selection (mainly in the later generations of backcross breeding).

Earlier, Sundaram et al. (2008, 2009) and Hari et al. (2011, 2013) developed BB resistant versions of the varieties, Samba Mahsuri and Triguna, the restorer line, KMR-3R and the maintainer line IR58025B, respectively. Similarly, PRR78, an elite restorer line possessing Basmati type grain quality was improved for resistance against BB and blast by Basavaraj et al. (2010) and Singh et al. (2013), respectively. Recently, Balachiranjeevi et al. (2015) and Abhilash Kumar et al. (2016) improved the maintainer line DRR17B and restorer line RPHR-1005, respectively, against BB and blast (Xa21 + Pi54) by transferring a major resistance gene each for BB and blast resistance, implementing the approach similar to that used in the present study. As done in some of our earlier studies (Hari et al., 2011, 2013; Balachiranjeevi et al., 2015; Abhilash Kumar et al., 2016), in the present study also, molecular markers specific for two major fertility restorer genes (viz., Rf3 and Rf4) were deployed in the initial stages of backcrossing in order to obtain complete fertility restorer lines after backcrossing. Thus, through the present study, improved breeding lines RPHR-1005 possessing tall plant type, excellent resistance against BB and blast, medium-slender grain type along with stable fertility restoration and suitable for use as a restorer parent in three line breeding system have been developed. Even though there are some reports wherein breeders have improved hybrid rice parental lines for either resistance against BB (Chen et al., 2001; Liyong et al., 2003; Basavaraj et al., 2010; Shanti et al., 2010; Hari et al., 2011; Zhou et al., 2011) or blast (Amante-Bordeos et al., 1992; Hittalmani et al., 2000; Arunakanthi et al., 2008; Singh et al., 2013), reports on combining resistance against both the biotic stresses are very limited (Singh et al., 2011; Fu et al., 2012; Hari et al., 2013; Balachiranjeevi et al., 2015). The present study, wherein two genes each have been introgressed into RPHR-1005 for resistance against BB and blast is a positive step in this direction, making the improved RPHR-1005 lines and hybrids developed from it, highly resistant against two deadly diseases of rice. Significantly, all the improved lines of RPHR-1005 were observed to display complete fertility restoration, when test crossed with CMS lines of APMS 6A (Table 3). This indicates that the molecular markers specific for Rf3 and Rf4 developed by Balaji Suresh et al. (2012) are highly suitable for targeted improvement of hybrid rice parental lines. Based on the results of this study, the use of these markers is advocated in studies attempting improvement of hybrid rice parental lines through MAS.

In our study, background selection strategy was also adopted for accelerated recovery of RPHR-1005 genome and backcross derived, disease resistant plants, which were equivalent to the original parent in terms of most agromorphological traits and grain type were identified after just two rounds of backcrossing. A few intercross and double intercross derived lines which have shown significantly higher yields as compared to the recurrent parent (Table 2), fully exserted panicles and better plant height as compared to RPHR-1005 (which is ideal for a good restorer line; Table 2) were also developed in this study. This was possible only because, morphology based phenotypic selection coupled with stringent MAS involving both foreground and background selection was adopted in this study, resulting in the development of lines which are equivalent or superior to RPHR-1005 and possessing high level of BB and blast resistance (Table 1; Figures 4, 5). Background strategy used in this study involves additional markers on carrier chromosome which limit the number of backcrosses to just two generations.

At BC2F2 generation, the recovery of RPG was observed to be nearly equivalent to the theoretically expected value of 93%, viz., 91.8% (for the cross RPBio Patho-1//RPHR-1005, Plant # RPC-I-9-27-79-179), 92.3% (RPBio Patho-2//RPHR-1005, plant # RPC-II-38-213-63-259) and 93.2% (FBR1-15//RPHR-1005, plant # RPC-III-38-27-43-276). Further, the introgression of donor chromosomal segment was limited on either side of the target genes to a small region of ~1.4 Mb (Chr.6; near Pi2; Figure 2A and Chr.11; near Xa21; Figure 2B), ~1.3Mb (Chr.11; near Xa21 + Pi54; Figure 2C), and ~1.0 Mb (Chr.7; near Xa33; Figure 2D) and the non-carrier chromosomes were observed to carry only small segments of donor parent genome. The level of BB and/blast resistance in the improved versions of RPHR-1005 in all the desired gene combinations viz., Xa21 + Pi2, Xa21 + Pi54, and Xa33 (Table 1; Figures 4, 5) was observed to be significantly higher than the original parent, RPHR-1005 (21.6 ± 0.7 cm) and equivalent to that of the donor parents i.e., RPBio Patho-1, RPBio Patho-2, and FBR1-15, respectively at BC2F7 generation. Thus, one of the key objectives of the present study, i.e., near-complete recovery of all the good features of RPHR-1005, while introgressing multiple BB and blast resistance genes, was achieved combining MAS in the early and mid-stages of backcrossing and phenotype-based selection in the later stages of backcrossing. Two-gene pyramids possessing one BB resistance gene (Xa21) and blast resistance genes (i.e., Pi2 or Pi54) and single gene containing lines possessing BB resistance gene, Xa33 were intercrossed and selfed to generate ICF2 lines (adopting pedigree-based, morphological selection for key agronomic traits) in order to combine either a single BB resistance gene and two blast resistance genes (i.e., Xa21 + Pi2 + Pi54) and two BB and a single blast resistance gene (i.e., Xa21 + Xa33 + Pi54) in the genetic background of RPHR-1005 in homozygous condition. Four-gene pyramids were developed by deploying double intercross program involving stacking the two major resistance genes, two of each conferring resistance against BB and blast (Xa21 + Xa33 + Pi2 + Pi54). The level of BB resistance in the improved versions of intercross (at ICF5) and double intercross (at DICF3) lines of RPHR-1005 was observed to be significantly higher than the original parent, RPHR-1005 and equivalent to that of donor parents, RPBio Patho-1, RPBio Patho-2, and FBR1-15 (Table 1; Figure 5). Similarly, the level of blast resistance in the improved versions of RPHR-1005 at BC2F7, ICF5, and DICF3 (Table 1; Figure 4) was also observed to be significantly higher than the original parent, RPHR-1005 and equivalent to the donor parents.

Even though the donor parents, RPBio Patho-1, RPBio Patho-2, and FBR1-15 possessed desirable features like BB and blast resistance and good grain type, their semi dwarf plant type nature however is not desirable for ideal restorer lines. Hence, selection for taller plant stature was carried out from BC2F1 generation and three lines at BC2F7 generation possessing taller plant type along with target resistant genes were identified. In addition to BC2F7 plant, some lines at IC1F5 (possessing Xa21 + Pi2 + Pi54), IC2F5 (possessing Xa21 + Xa33 + Pi54), and DICF3 (possessing Xa21 + Xa33 + Pi2 + Pi54) possessed desirable features like BB and blast resistance and good grain type in addition to taller plant stature. Significantly, five lines at DICF3 generation (Table 2), which were taller than RPHR-1005 were identified. Recently, a similar strategy of phenotype based stringent background selection during MABB followed by Ellur et al. (2016), while improving Pusa Basmati 1121and Pusa Basmati 6 for BB and blast resistance.

All the improved lines of RPHR-1005 were observed to display complete fertility restoration when test crossed with CMS lines of APMS 6A (Table 3). The newly developed improved versions of RPHR-1005 (possessing Xa21 + Pi2 + Pi54, Xa21 + Xa33 + Pi54, and Xa21 + Xa33 + Pi2 + Pi54) lines can be used as a replacement of RPHR-1005 for developing disease resistant hybrids i.e., DRRH-3 with superior grain quality characters. Among the improved lines of RPHR-1005, two lines (viz., RPIC1-12-196-36 and RPIC1-12-196-45) possessing Xa21 + Pi2 + Pi54, single line (i.e., RPIC2-53-640-43) possessing Xa21 + Xa33 + Pi54 and five lines (viz., RPDIC-9-786-1, RPDIC-9-786-19, RPDIC-9-786-25, RPDIC-9-786-46, and RPDIC-9-786-78) possessing Xa21 + Xa33 + Pi2 + Pi54 have been identified as best restorer lines as they possesses all the good phenotypic traits of RPHR-1005, better plant height, complete panicle exsertion, and superior plant yield. These lines have been nominated for All India Coordinated Rice Improvement Project trials under National Screening Nursery 1 in the year 2015 to validate their biotic stress resistance and for development of experimental hybrids for station trials. Further, in the improved lines of RPHR-1005 mentioned above, no apparent yield penalty associated with the presence of the resistance genes, IC1F5 (Xa21 + Pi2 + Pi54), IC2F5 (Xa21 + Xa33 + Pi54), and DICF3 (Xa21 + Xa33 + Pi2 + Pi54) was noticed.

Most of the experimental hybrids from the cross between backcross derived improved lines of RPHR-1005 (possessing resistance against BB and blast, developed through this study) and APMS 6A (the female parent of DRRH-3) were observed to be superior to DRRH–3 in terms of heterosis (Table 3). The best hybrids developed from selected improved versions of RPHR-1005 (mentioned earlier) will be again validated in the forthcoming season and nominated for multi-location trails under All India Coordinated Rice Improvement Project (AICRIP) for their evaluation and possible release for the benefit of rice farmers. Cultivation of such hybrids possessing highly desirable medium-slender grain type along with durable BB and blast resistance would be of great advantage in BB and blast endemic areas and the improved versions of hybrids developed using improved RPHR-1005 lines could replace popular hybrids possessing desirable grain type like DRRH-3 and others.

In conclusion, through the present study, we have developed improved versions of the elite restorer line, RPHR-1005 and possessing resistance against BB, blast, better panicle exsertion along with complete fertility restoration, MS grain type and demonstrated the heterotic potential of the experimental hybrids (i.e., improved versions of DRRH-3) derived from crosses between improved lines of RPHR-1005 and APMS 6A.

Author contributions

Designed experiment RS and VA, Research work performed by VA, Phenotypic screening of bacterial blight facilities given by GL and AY, Phenotypic screening of blast facilities given by MP, RR, KV, and JA, Heterosis experimental facilities given by AH, Supported by CB, SB, GR, GH, SH, KP, MA, MHK, MK, MM, SB, Facilities provided in Indian Institute of Rice research by VR.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.