Marker-assisted pyramiding of two major, broad-spectrum bacterial blight resistance genes, Xa21 and Xa33 into an elite maintainer line of rice, DRR17B.

Bacterial blight (BB) disease reduces the yield of rice varieties and hybrids considerably in many tropical rice growing countries like India. The present study highlights the development of durable BB resistance into the background of an elite maintainer of rice, DRR17B, by incorporating two major dominant genes, Xa21 and Xa33 through marker-assisted backcross breeding (MABB). Through two sets of backcrosses, the two BB resistance genes were transferred separately to DRR17B. In this process, at each stage of backcrossing, foreground selection was carried out for the target resistance genes and for non-fertility restorer alleles concerning the major fertility restorer genes Rf3 and Rf4, using gene-specific PCR-based markers, while background selection was done using a set of 61 and 64 parental polymorphic SSR markers respectively. Backcross derived lines possessing either Xa21 or Xa33 along with maximum genome recovery of DRR17B were identified at BC3F1 generation and selfed to develop BC3F2s. Plants harboring Xa21 or Xa33 in homozygous condition were identified among BC3F2s and were intercrossed with each other to combine both the genes. The intercross F1 plants (ICF1) were selfed and the intercross F2(ICF2) plants possessing both Xa21 and Xa33 in homozygous condition were identified with the help of markers. They were then advanced further by selfing until ICF4 generation. Selected ICF4 lines were evaluated for their resistance against BB with eight virulent isolates and for key agro-morphological traits. Six promising two-gene pyramiding lines of DRR17B with high level of BB resistance and agro-morphological attributes similar or superior to DRR17B with complete maintenance ability have been identified. These lines with elevated level of durable resistance may be handy tool for BB resistance breeding.

Introduction

Rice production needed to be increased 42% by 2050to feed the demands of an ever-increasing human population globally [1, 2]. Exploitation of heterosis for grain yield through hybrid rice technology is one of the feasible options to enhance rice production and rice hybrids have15-20%yield advantage over inbreeds [3]. Even though rice hybrids were introduced in India in the early 1990s, their adoption has been slow and presently hybrid rice is cultivated in a limited area of 2.5 million ha. One of the principal reasons for slow adoption of rice hybrids in India is their susceptibility to major rice diseases like bacterial blight (BB) and blast [4]. Most of the commercial rice hybrids that have been released and cultivated in India do not possess broad spectrum resistance for BB disease [5].

BB disease is caused by a gram-negative bacterium called Xanthomonas oryzae pv. Oryzae (Xoo). It is one of the most devastating diseases in rice [6]. The bacterium infects rice at maximum tillering stage, leading to water soaking lesions (blighting) on the leaves, which gradually enlarge, wilts and causes yield losses ranging from 74 to 81% [7]. Application of chemicals or antibiotics against is very costly and is not very effective [8, 9]. Breeding BB resistant rice varieties and hybrids is the best strategy for managing the BB disease in rice [10]. To date, at least 41 BB resistance genes have been identified,and some of them viz., Xa4, xa5, xa13, Xa21 have been extensively used for development of BB resistant rice varieties [11, 12, 13, 14, 15] (). However, large scale and long-term cultivation of varieties and hybrids with a single gene results in the breakdown of resistance due to a high degree of pathogenic variation [12, 16, 17]. Pyramiding of two or three Xa genes can enhance the durability and spectrum of resistance against BB [18, 19].

The major BB resistance gene, ‘Xa21’ was identified from Oryza longistaminata. It is located on chromosome 11 and a tightly linked to gene-specific marker pTA248 [21]. Similarly, ‘Xa33’ was identified from Oryza nivara. It is located on chromosome 7 and tightly linked to a marker RMWR7.6 [22]. These markers can be used in marker-assisted breeding to introgress Xa21 and Xa33 genes into different rice varieties and hybrid parental lines. These two genes are found to be highly effective against several isolates of Xoo from India and hence, are ideal choices for pyramiding into popular rice varieties or hybrids through marker-assisted breeding.

DRR17B is a fine grain type and medium duration, stable promising maintainer line developed by ICAR-Indian Institute of Rice Research, Hyderabad, India [23]. It is however highly susceptible to BB of rice. In the present study, two major dominant BB resistance genes, Xa21 and Xa33 were introgressed into the genetic background of DRR17B through marker-assisted backcross breeding to develop improved DRR17B lines with broad spectrum resistance against BB.

Materials and methods

Plant materials

‘Improved Samba Mahsuri’ (ISM) is a recently released high-yielding and fine grain rice variety possessing BB genes, xa5, xa13, and Xa21 [18]. It was used as a donor for Xa21 [23]. A Near Isogenic Line (NIL) of ‘Samba Mahsuri’ (FBR1-15EM) served as the donor for Xa33 [22]. The popular but BB susceptible maintainer line DRR17B (APMS6B/BPT5204/IR69628B) was used as the recurrent parent. It was developed by ICAR-Indian Institute of Rice Research (IIRR), Hyderabad (17.3200° N, 78.3939° E), India.

Strategy for marker-assisted introgression of Xa21 and Xa33 into DRR17B

Marker-assisted backcross breeding strategy was adapted for targeted introgression of Xa21 and Xa33 genes into the genetic background of the elite maintainer line of rice, DRR17B. Each of these genes was separately introgressed into DRR17B through two sets of crosses, i.e., Cross I, viz., DRR17B/ISM and Cross II, viz., DRR17B/FBR1-15 (). The F1s obtained from the two crosses were analysed by extracting DNA through the method described by [24] and using that DNA by keeping Polymerase Chain reaction with gene-specific markers pTA248 (specific for Xa21; [21]) and RMWR7.6 (specific for Xa33; [22]) to identify ‘true’ heterozygotes. The ‘true’ F1s were backcrossed with the recurrent parent DRR17B to generate BC1F1s, which were then screened for the presence of the target resistance genes using the gene-specific markers. The positive plants for Xa21 and Xa33 were selected and further screened for the non-presence of major fertility restorer genes, Rf4 and Rf3 using tightly linked markers, viz., DRCG-RF4-14 and DRRM-RF3-10, respectively [25]. BC1F1 plants possessing BB genes and a non-restoring allele concerning Rf4 and Rf3 in homozygous condition were selected following the procedure described by [23]. These plants were later screened with a set of parental polymorphic SSR markers (61 markers specific to the cross DRR17B/ISM and 64 markers specific for the cross DRR17B/FBR1-15EM) through background selection to identify a single BC1F1 plant from each cross possessing maximum recovery of the recurrent parent genome. The selected plant was backcrossed once again with DRR17B.

The process of marker-assisted backcrossing was repeated until BC3 generation, and BC3F1 plants of DRR17B possessing either Xa21 or Xa33 and maximum recovery of recurrent parent genome were then selfed to obtain BC3F2s. Plants homozygous for either Xa21 or Xa33 were identified among the BC3F2 plants and the best plants from the two crosses were intercrossed to obtain intercross F1s (i.e., ICF1s). ‘True’ ICF1 plants were identified by screening with molecular markers specific for Xa21 and Xa33 and were then selfed to generate intercross F2s (i.e., ICF2s). Plants homozygous for both Xa21 and Xa33 were identified among the ICF2 plants using the gene-specific markers. The identified plants were advanced through the pedigree method of selection (involving selfing followed by morphological trait-based visual selection) up to ICF4generation. Marker-assisted selection procedures were followed as recommended by [21] and [22] for detection of Xa21 and Xa33 genes, while the protocol described by [23] was adopted for background selection and detection of non-restoring alleles of Rf4 and Rf3.

Screening for BB resistance

Eight virulent isolates of the BB pathogen, Xanthomonas oryzae pv. oryzae (Xoo) collected from BB disease endemic across major rice cultivation agro climatic locations in India, viz., IX-020 (Hyderabad, Telangana), IX-133 (Raipur, Chhattisgarh), IX-027 (Chinsurah, West Bengal), IX-200 (Pantnagar, Uttarakhand), IX-002 (Faizabad, Uttar Pradesh), IX-403 (Thanjavur, Tamil Nadu), IX-090 (Ludhiana, Punjab) and IX-281 (Tanuku, Andhra Pradesh) were used to screen the ICF4 lines of DRR17B (possessing the gene combinations Xa21+Xa33, Xa21 alone or Xa33 alone) along with the donor parents/resistant check, ‘ISM’ (possessing xa5+xa13+Xa21), FBR1-15 (possessing Xa33) and BB recurrent parent and susceptible check (DRR17B and TN1) were screened under glasshouse conditions for their resistance/susceptibility against BB. The Xoo strains were cultured and stored as described by [16]. The rice plants were clip-inoculated with a bacterial suspension of 108−9 CFU/ml at maximum tillering stage (45 to 55 days after transplanting) through the methodology of [26]. Approximately, 5 to 10 leaves were inoculated per plant, and the disease reaction was scored 14 days after inoculation [27]. In addition to measurement of BB lesion length, the disease score was calculated as per IRRI Standard Evaluation System (SES) scale, which is based on percent diseased leaf area [28].

Screening for agro-morphological traits

Improved lines (ILs) of DRR17B (ICF4) were field evaluated in randomized complete block design in Kharif 2014 (i.e. July-October/Wet season 2014) for the following agro-morphological traits involving days to 50% flowering (days), plant height (cm), number of productive tillers (No.), panicle length (cm), grains per panicle (No.) and spikelet fertility. Each entry was planted in 20 rows of 4m length with a spacing of 15 x 20 cm between rows and within rows. Days to 50 percent flowering was recorded based on number of days from sowing to 50% population flowering on a whole plot basis. Plant height (cm), number of productive tillers (No.) and panicle length (cm) were recorded from 5 competitive plants from each plot chosen at random and the mean values computed for different lines. Five individual panicles harvested separately from five plants were collected to compute for the average grain number per panicle (No.). The ILs were crossed with IR58025A line and evaluated for spikelet fertility based on seed setting of each cross. The percentage was calculated based on seed setting per panicle as described in [23].

Statistical analysis

Agro-morphological and phenotypic BB screening data were analysed using standard procedures by calculating Mean, significant standard error of Mean (S.E.M ±), Analysis of variance (ANOVA) and Least Significance Difference (LSD) [29]. Analysis of variance (ANOVA) and Duncan’s multiple range test (DMRT) and Least Significance Difference (LSD) at 5% level of significance, significant standard error of Mean (S.E.M ±) were calculated by using MS Excel and Statistical computer software Statistix8.1 [30] software to analyze the variation between ILs and parents.

Results

Marker-assisted transfer of Xa21 and Xa33 into DRR17B

The true F1s derived by crossing DRR17B with ‘ISM’ (i.e., Cross I) and FBR1-15 (i.e., Cross II) were backcrossed with DRR17B to obtain BC1F1s, which were then screened with the gene-specific markers. A total of 61 and 65 BC1F1 plants were observed to be positive for the target genes in Cross I and Cross II, respectively. The positive plants were screened with markers specific for Rf3 and Rf4,and a total of 15 and 11 plants were identified to be devoid of both the fertility restorer genes concerning Cross I and Cross II, respectively. These plants were then subjected to background selection using a set of polymorphic SSR markers (61 markers for Cross I and 64 for Cross II). Plant # IIRRGP3 from Cross I, with a recurrent parent genome (RPG) recovery of 73.7% and Plant # IIRRGP22 from Cross II, with a RPG recovery of 75% were identified to be the best ones (i.e. having a maximum recovery of DRR17B genome) and were used for further backcrossing. The process of marker-assisted backcrossing was carried out until BC3F1 generation (details given in ). At BC3F1, plant # IIRRGP3-87-64 from Cross I with RPG recovery of 93.4% and plant # IIRGP22-73-10 with RPG recovery of 93.7% were identified to be superior and were selfed to generate BC3F2s. With regards to the BC3F2s produced from Cross I and Cross II, 39 and 52 plants were identified to be homozygous for Xa21 and Xa33, respectively. Among these, a solitary plant, which was morphologically similar to DRR17B, was identified from Cross I (i.e., plant # IIRRGP 3-87-64-22 and Cross II (i.e., plant # IIRRGP 22-73-10-15) and intercrossed with each other to generate intercross F1s (i.e., ICF1s). Out of 68 ICF1s, 63 were identified to be heterozygous for both Xa21 and Xa33 (i.e. true intercross F1s), and they were selfed to obtain ICF2 generation. At ICF2, a total of 309 plants were screened with markers specific for Xa21 and Xa33 and 18 were identified to be double homozygotes (; ). A total of nine plants out of the 18, which were identified to be phenotypically similar to DRR17B, were further advanced until ICF4 generation through phenotype-based pedigree selection. At ICF4 generation, six promising lines which were similar to the recurrent parent were identified (line #IIRRIC 10-8-94, IIRRIC 10-19-138, IIRRIC 102-26-7, IIRRIC 123-34-84, IIRRIC 123-58-3 and IIRRIC 172-77-12) and analysed for their resistance to BB, sterility maintenance ability and also characterized for important agro-morphological traits. Among the six ILs, line # IIRRIC102-26-7 exhibited the highest recurrent parent genome recovery with more than 95% along with minimal linkage drag on carrier chromosomes ().

Screening of ICF2 population for identification of double homozygotes for the target resistancegenes, viz., Xa21 and Xa33.

The ICF2 plants were screened through PCR to analyze the allelic status of Xa21 (A) and Xa33 (B) using the gene-specific markers. M–Marker, R–Recurrent parent (i.e. DRR17B) and D–donor parent [i.e., ‘ISM’ (A) and FBR1-15EM (B)]. Arrows indicate plants which possess target genes Xa21 and Xa33 in homozygous condition.

Graphical genotyping representation for the best improved line (IIRRIC102-26-7) of DRR17B.

Graphical genotyping representing that the best line of improved two-gene-containing DRR17B line (Xa21+ Xa33), IIRRIC102-26-7 exhibiting the highest genome recovery of the recurrent parent with more than 95%, along with minimum linkage drag on carrier chromosomes 7 and 11, with less than 2 Mb donor parent chromosome (1. DRR17B, 2. ‘ISM’, 3. FBR1-15, 4. IIRRIC102-26-7).

Phenotypic evaluation of ILs for BB resistance

The recurrent parent, DRR17B (11) (with lesion lengths ranging from 18.8 to 33.1 cm) and susceptible check TN1 (12) (with lesion lengths ranging from 20.9 to 33.8 cm) showed a disease score of 9 against all the eight isolates of the Xoo (; depicted as graph). The resistant check and the donor for Xa21 gene ‘ISM’ (9) (possessing Xa21, xa13, and xa5) showed a score of 3 against all the isolates (with an average lesion length ranging from 1.6 to 3.6 cm). FBR1-15 (10), the donor for Xa33 gene and improved DRR17B lines possessing Xa33 (# IIRRGP22-73-10-15-13-2 (2)) showed a resistance score of 3 with most of the isolates (with lesion lengths ranging from 1.7 to 4.8 cm), with two isolates, IX-002 and IX-281 recorded moderate resistance reaction with a score of 5 (average lesion lengths ranging from 7.3 to 9.7 cm and 7.5 to 9.2 cm).The ILs of DRR17B containing only Xa21 (# IIRRGP3-87-64-22-4-50 (1)) showed a resistance reaction against two isolates viz., IX-002 and IX-090 with a score of 3 (with lesion lengths of 2.8 to 4.3 cm and 2.0 to 2.8 cm, respectively), while with three isolates,viz., IX-020, IX-027 and IX-281, the line with only Xa21 exhibited moderately susceptibility with a score of 7 (with lesion lengths of 12.5 to 14.7 cm, 13.1 to 14.5 cm and 13.0 to 14.6 cm, respectively). Further, the line showed highly susceptible reaction with a score of 9 (with lesion lengths of 20.1 to 23.5cm, 22.2 to 25.6 cm and 21.9 to 24.4cm, respectively)with three other three isolates viz., IX-133, IX-200 and IX-409, respectively. The ILs of DRR17B containing both Xa21 + Xa33 (3–8) exhibited a significantly higher level of resistance, showing a score of 1 against all eight isolates with lesion lengths ranging from 0.1 to 1 cm (; ).

Screening of the single-gene and two-gene pyramid lines of DRR17B against different virulent isolates of the bacterial blight pathogen.

Eight selected ICF4of DRR17B possessing either Xa21 (IIRRGP3-87-64-22-4-50) or Xa33 (IIRRGP22-73-10-15-13-2) or Xa21 + Xa33 (# IIRRIC10-8-94, IIRRIC10-19-138, IIRRIC102-26-7, IIRRIC123-34-84, IIRRIC123-58-3, and IIRRIC172-77-12) were screened for their BB resistance with eight virulent isolates of Xanthomonas oryzae pv. Oryzae (Xoo) along with the recurrent parent (DRR17B) and donor parents (‘ISM’ and FBR1-15). While all the lines showed excellent resistance against the multiple isolates of Xoo screened, the two-gene pyramid lines (i.e., Xa21 + Xa33) were observed to show a higher level of resistance to the different isolates of the pathogen.

Characterization of ILs for maintenance ability and agro-morphological traits

The current study screened the six ILs for their maintenance ability. Out of six, three lines showed partial spikelet fertility, while the remaining three lines (viz., line # IIRRIC102-26-7, IIRRIC123-34-84, and IIRRIC172-77-12) showed complete spikelet sterility when crossed with the WA-CMS line, IR58025A (). Comparison of five agro-morphological parameters (days to 50% flowering, plant height, number of productive tillers, panicle length and number of grains per panicle) revealed thatall the six ILs are isophenic in their panicle length and number of productive tillers to DRR17B, while significant differences were observed with respect to the number of grains per panicle. The ILs viz., IIRRIC10-8-94, IIRRIC102-26-7, IIRRIC123-58-3 and IIRRIC172-77-12 possessed more number of grains per panicle with respective to DRR17B viz., 301.6, 360.4, 308 and 317 respectively (). However, all selected six lines showed comparatively shorter plant height than recurrent parent. While panicle length of, line # IIRRIC102-26-7 was observed longest among all six panicle (24.16 cm), the remaining five ILs exhibited equal or less than the recurrent parent DRR17B (average length of 23.24 cm: ). Line # IIRRIC102-26-7 exhibit highest numbers of productive tillers per plant (average of 12), all remaining five ILs were similar to thir recurrent parent (10–11.2). The to 50% flowering, of all the six ILs flowered earlier (92–102 days), as compared to DRR17B (105 days).

Discussion

Several studies indicate that global rice production needs to be doubled by 2050 to meet the demands of ever growing population [2]. However, rice grain yield is badly affected by biotic and abiotic stresses [31]. The present study was taken up to improve, an elite maintainer of rice, DRR17B, for its resistance against BB resistance. DRR17B is a fine grain type and medium duration maintainer line of rice, possessing stable maintenance ability was developed by ICAR-Indian Institute of Rice Research, Hyderabad, India [23]. As DRR17A and its maintainer parent- DRR17B are highly susceptible to BB disease, considering this deficiency in the elite maintainer line, the current study was carried out with an objective to introgress two major dominant BB resistant genes, viz., Xa21 and Xa33 through MABB in order the make the maintainer line durably resistant to BB. These two selected genes are known to confer resistance against multiple isolates of the BB pathogens for large rice cultivated area; hence, the hybrids developed from ILs of DRR17A will also be sustainable resistant against this disease.

Introgression of BB resistance genes through conventional breeding involving patho-phenotypic selection which is very laborious, time and resource consuming process and its success significantly depends on accurate disease scoring, the existence of environmental conditions which favour disease development and the availability of appropriate virulent strains of the pathogen causing the disease [11]. As compared to conventional breeding, marker-assisted selection (MAS) breeding strategy is more useful for targeted introgression of resistance genesas it does not depend on the availability of virulent strains or existence of ideal environmental conditions, since the selections are indirect, and are based on the presence or absence of specific alleles of molecular markers linked to the resistance genes. Previous studies, [23, 32, 33] reported on successfully developed bacterial blight resistant versions of hybrid rice parental lines PRR78 and IR58025B, through marker-assisted selection for target traits in the initial stages and phenotype-based selection at later stages and hence at the same methodology was adopted in the current study.

So far, at least 41 genes conferring resistance against BB have been identified in rice [11, 12, 13]. Among them, the wild rice derived gene, Xa21 encoding a receptor kinase-like protein has been successfully deployed by many research groups across the world, as it has been documented to confer broad-spectrum resistance against the BB disease [17, 18, 23, 32, 34, 35, 36, 37, 38]. The commonly used BB resistance gene Xa21 has been tagged and mapped on chromosome 11 with a tightly-linked PCR-based marker pTA248 [21].Xa33, the wild rice derived BB resistance gene has been reported to confer broad spectrum resistance [22] and the gene has been deployed by the research group at Tamil Nadu Agricultural University, Coimbatore, India and the breeding lines possessing Xa33 were observed to be very effective in terms of their BB resistance [39, 40]. Hence, these two broad spectrum resistance genes were selected for introgression into the DRR17B.

Phenotypic screening for BB resistance was carried out in this study among selected single gene containing BC3F6 lines possessing either Xa21 or Xa33 and two-gene containing intercross derived lines at ICF4 generation possessing Xa21+Xa33 along with the donor and recurrent parents (‘ISM’, FBR1-15, and DRR17B, respectively) using eight virulent isolates of Xoo. All the ILs possessing Xa21+Xa33 were observed to show significantly higher level of resistance against BB when compared to the donor parents, ‘ISM’ and FBR1-15. Single gene containing lines of DRR17B (i.e. possessing either Xa21 or Xa33), the recurrent parent DRR17B and the BB susceptible check TN1 ().It is earlier known that Xa21 confers broad spectrum resistance against many of the virulent pathotypes of Xoo in India [17,18] and several studies have indicated the suitability of Xa21 in BB resistance gene pyramiding programmes [10, 18, 34, 41, 42]. However, in this study, a few isolates of the pathogen were observed to be compatible with Xa21 containing lines of DRR17B indicating that Xoo isolates, which are capable of overcoming Xa21 conferred resistance are fast-developing [17, 43, 44]. Interestingly, the ILs of DRR17B possessing Xa33were observed to show a better level of resistance as compared to the lines having Xa21. Furthermore, DRR17B lines possessing both Xa21 and Xa33 were observed to be highly resistant against all the eight virulent isolates of Xoo, thus, indicating the suitability of deployment of Xa33 either singly or in combination with Xa21. Earlier, two elite restorer lines, KMR3R, and RPHR1005 were improved for BB resistance by introducing Xa21 [23, 33, 36, 38]. Similarly, Xa33 has been deployed in different genetic backgrounds by different research groups [19, 22, 39, 40]. However, this is the first report wherein Xa21 has been combined with Xa33 in the genetic background of an elite maintainer line, i.e., DRR17B and the gene-pyramid lines demonstrated a higher level of resistance as compared to lines possessing a single resistance gene ().

It is a known fact that long term cultivation of rice varieties possessing single resistance gene can result in the breakdown of resistance by faster development of virulent pathogens [43, 44, 45].Hence, pyramiding of multiple resistance genes has been advocated to be one of the best strategies to achieve durable dual-resistance [18, 46, 47]. In our present study, the genotype ‘ISM’ (with Xa21 + xa13 + xa5) has displayed satisfactory level of resistance with a score of 3 against all eight isolates. Interestingly, when Xa21 gene was combined with another major dominant gene- Xa33, such breeding lines exhibited the highest level of resistance with a score of 1. This indicates that the gene combination Xa21 + Xa33 displayed a broad spectrum of resistance and hence can be recommended for deployment in hybrid rice improvement programs as both Xa21 and Xa33 are both dominant and will express in the F1 hybrid.

Similar to the approach adopted in the current study, several earlier studies also resorted to phenotype-based selection for key agro-morphological traits along with marker-assisted selection while improving elite varieties and parental lines for one or more target traits through MABB [18, 23, 33, 35, 36, 37, 38, 48].The approach of deployment of MABB strategy for the target resistance genes along with negative selection for major fertility restorer genes, Rf3 and Rf4, coupled with phenotype-based selection for certain key agronomic characters helped in near-complete recovery of good features of DRR17B along with identification of few ILs with complete maintenance ability (). In addition to improving BB resistance of DRR17B, The current study also focused on the identification of ILs of DRR17B possessing plant height which is significantly shorter than DRR17B, as shorter plant is preferred for deployment as good maintainers [23]. Significant differences in plant height were observed in many improved DRR17B lines viz., RMSIC 10-8-94, RMSIC 10-19-138, RMSIC 102-26-7, RMSIC 123-34-84, RMSIC 123-58-3 and RMSIC 172-77-12 and these lines could serve as better maintainers as compared to DRR17B. Interestingly, when compared to DRR17B, some of the ILs exhibited advantage concerning grain number per panicle. These lines include RMSIC 10-8-94, RMSIC 102-26-7, RMSIC 123-58-3 and RMSIC 172-77-12 ().For the panicle length, all the ILs showed values equivalent to DRR17B, except one line viz., RMSIC 102-26-7, a wherein slight improvement over the recurrent parent was noticed. Significant differences (i.e., shorter duration) were observed concerning number of days to 50% flowering in some of the backcross derived plants (). No significant differences were observed between improved versions of DRR17B and recurrent parent DRR17B concerning other agro-morphological characters or grain type and the lines mostly resembled the original recurrent parent. The approach of coupling of MABB with phenotypic selection adopted in this study helped to regain most of the key agro-morphological traits of DRR17B, while simultaneously helping in the selection of some superior backcross derived segregants of DRR17B possessing BB resistance.

Fig 5

IL of DRR17B displaying high grain number under field conditions at IIRR, Panicles of ILs along with donor and recurrent parents.

A. IL # IRIC102-26-27 displaying high grain number in field conditions, B. The ILs of DRR17B possessing Xa21 + Xa33 genes(# IIRRIC10-8-94, IIRRIC10-19-138, IIRRIC102-26-7, IIRRIC123-34-84, IIRRIC123-58-3, and IIRRIC172-77-12)were displayed more number of grains per panicles and similar panicle length (except number 6) when compared with DRR17B.

The ILs of DRR17B were test crossed with IR58025A (WA-CMS line) to check their maintainer ability. Three lines (viz., IIRRIC102-26-7, IIRRIC123-34-84, and IIRRIC172-77-12) displayed complete maintainer ability highlighting the necessity of phenotypic confirmation for maintenance ability, despite a rigorous marker-assisted selection for rf3 and rf4 alleles in this study. This could be attributed to the existence of minor fertility restorer genes/QTLs as reported earlier [49].The three ILs of DRR17B, possessing Xa21 + Xa33 are being converted as CMS lines by crossing with DRR17A through MABB.

The six ILs of DRR17B exhibited high level of BB resistance against the BB isolates, when compared with the recurrent parent DRR17B. Whereas in agro-morphological characters like plant height, day to 50% flowering and number of grains per panicle etc, variations were observed. All the improved lines were shorter than the recurrent parent. With regards to Days to 50% flowering all the improved lines were little early (92–103 days) than DRR17B (105 days). Some the improved lines Viz., IIRRIC10-8-94, IIRRIC102-26-7 IIRRIC123-58-3 and IIRRIC172-77-12 were exhibited significantly more number of grains per panicle then DRR17B (280 per panicle). The ILs Viz., IIRRIC102-26-7, IIRRIC123-34-84, and IIRRIC172-77-12 were exhibited complete maintainer ability as like DRR17B and remaining lines were partial maintainers.

Conclusion

The present study has resulted in development of improved versions of an elite maintainer of rice, DRR17B possessing durable resistance against BB through incorporation of two major dominant genes conferring broad-spectrum resistance, Xa21 and Xa33 by marker-assisted backcross breeding (MABB) strategy. The double gene pyramided lines of DRR17B expressed high level of resistance against eight different virulent isolates of Xoo and their resistance levels was comparable with triple resistance gene pyramided rice variety, ‘ISM’ (possessing Xa21 + xa13 + xa5) and were also significantly better than the single gene containing lines (possessing Xa21 or Xa33). Three promising double-gene pyramided lines of DRR17B with high level of BB resistance, agro-morphological attributes similar to or superior to the DRR17B with complete maintainer ability would be helpful in development of superior rice hybrids with durable, broad-spectrum resistance.

Table 1

Agroclimatic zones according ICAR- IIRR [4] and ICAR-NARP [20], existing pathotypes and recommended genes.

S.No.	Agro climatic Rice growing zones (AZ) ICAR-IIRR (ACRIP)	Agro climatic zone (AZ) ICAR-NARP	State	Number of Xoo Pathotypes	Recommended Resistance genes and gene combinations
1	Zone I	AZ6-AZ9	Himachal Pradesh	2 (1 & 6)	xa13, Xa21, xa5+xa13, xa13+Xa21, Xa4+xa5+xa13, Xa4+xa5+Xa21, xa5+xa13+Xa21 & Xa4+xa5+xa13+Xa21
2	Zone II	AZ10-AZ14	Punjab	15 (1,2,5,6,7 8,9, 11, 12,14,17,19 20,21 & 22)	xa5+xa13+Xa21 & Xa4+xa5+xa13+Xa21
3	Zone II	AZ15-AZ16	Haryana	9 (1,2,5,14,17,18,19,21 &22)	xa5+xa13+Xa21 & Xa4+xa5+xa13+Xa21
4	Zone I, Zone II, Zone III	AZ26-AZ35	Uttaranchal & Uttar Pradesh	12(2,3,6,7,10,11,12,14,17,19,20 &22)	xa5+xa13+Xa21 & Xa4+xa5+xa13+Xa21
5	Zone I, Zone III	AZ36-AZ41	West Bengal	8 (1,4,6,7,12,17,19 &22)	xa5+xa13+Xa21 & Xa4+xa5+xa13+Xa21
6	Zone IV	AZ42-AZ47	Assam	8 (4,7,11,14,17,19,21 & 22)	xa5+xa13+Xa21 & Xa4+xa5+xa13+Xa21
7	Zone IV	AZ53	Tripura	11 (1,2,7,9,11,13,14,17,19,21 & 22)	xa5+xa13+Xa21 & Xa4+xa5+xa13+Xa21
8	Zone III	AZ54-AZ59	Bihar & Jharkhand	2 (19 & 22)	xa5+xa13+Xa21 & Xa4+xa5+xa13+Xa21
9	Zone III	AZ60-AZ69	Odisha	8 (1,2,4,7,11,16,17 & 19)	Xa4+xa5+xa13, Xa4+xa5+Xa21, xa5+xa13+Xa21 & Xa4+xa5+xa13+Xa21
10	Zone V	AZ 70-AZ81	Madhaya Pradesh & Chattisgarh	7 (1,2,6,14,17,19 & 22)	xa5+xa13+Xa21 & Xa4+xa5+xa13+Xa21
11	Zone VI	AZ82-AZ89	Gujarat	6 (2,3,6,7,17 & 19)	Xa4+xa5+xa13, Xa4+xa5+Xa21, xa5+xa13+Xa21 & Xa4+xa5+xa13+Xa21
12	Zone V and VI	AZ90-AZ98	Maharashtra	5 (6,14,17,19 & 22)	xa5+xa13+Xa21 & Xa4+xa5+xa13+Xa21
13	Zone I, Zone VII	AZ99-AZ108	Karnataka	4 (2,5,19 & 22)	xa5+xa13+Xa21 & Xa4+xa5+xa13+Xa21
14	Zone VII	AZ109-AZ113	Kerala	9 (1,2,6,7,12,14,17,19 & 22)	xa5+xa13+Xa21 & Xa4+xa5+xa13+Xa21
15	Zone I, Zone VII	AZ114-AZ120	Andhra Pradesh & Telangana	12 (1,5,6,7,9,11,12,14,17,19,21 &22)	xa5+xa13+Xa21 & Xa4+xa5+xa13+Xa21
16	Zone I, Zone VII	AZ121-AZ127	Tamil Nadu	11 (1,5,6,7,9,11,12,14,17,19 & 21)	xa5+xa13+Xa21 & Xa4+xa5+xa13+Xa21

Zone I: Hilly Areas, Zone II: Northern, Zone III: Eastern, Zone IV: North Eastern, Zone V: Central, Zone VI: Western, Zone VII: Southern

Table 2

Details of plants generated and analyzed with markers in each generation of backcrossing/intercrossing.

S. No.	Generation	No. of plants screened		No. of positive plants for target genes and negative Rf3 and Rf4		Recurrent parent genome recovery (%) of the selected backcross plant
		Xa21	Xa33	Xa21	Xa33	Xa21	Xa33
1	BC1F1	187	134	15	11	73.7	75.0
2	BC2F1	112	157	42	59	85.2	85.9
3	BC3F1	144	142	47	48	93.4	93.75
4	BC3F2	178	213	39	52	-	-
		No. of intercross plants screened		No. of homozygous double positive plants for Xa21 and Xa33
5	ICF1	68		63		—
6	ICF2	309		18		—

Table 3

Reaction of the breeding lines of DRR17B possessing Xa21 and Xa33, singly or in combination against eight virulent isolates of the bacterial blight pathogen, Xanthomonas oryzae pv. oryzae (Xoo).

Xoo Isolates		Breeding Lines								Parents/Resistant/Susceptible Check				CV	LSD	H2	F Value
		1	2	3	4	5	6	7	8	9	10	11	12
IX-020	Mean ± SE	13.66 ± 0.42 b	3.8 ± 0.28c	0.68 ± 0.07e	0.5 ± 0.06e	0.58 ± 0.06e	0.66 ± 0.07e	0.54 ± 0.05e	0.56 ± 0.05e	2.66 ± 0.19d	3.68 ± 0.27cd	31.02 ± 0.87a	30.12 ± 0.95a	11.5	1.08	0.99	909.86
	Range	12.5–14.7	3.2–4.7	0.5–0.9	0.3–0.7	0.4–0.7	0.5–0.7	0.4–0.7	0.5–0.7	2.1–3.1	3.0–4.4	28.6–33.1	27.6–32.8
	Score	7	3	1	1	1	1	1	1	3	3	9	9
IX-027	Mean ± SE	13.88 ± 0.24c	4.08 ± 0.24d	0.28 ± 0.10e	0.44 ± 0.07e	0.42 ± 0.07e	0.34 ± 0.06e	0.30± 0.09e	0.36 ± 0.09e	2.90 ± 0.23d	3.94 ± 0.30d	21.60 ± 0.90b	25.20 ± 1.30a	17.8	1.39	0.99	335.00
	Range	13.1–14.5	3.5–4.9	0.1–0.6	0.2–0.6	0.2–0.6	0.2–0.5	0.1–0.6	0.1–0.6	2.1–3.5	3.1–4.8	18.8–23.5	20.9–28.5
	Score	7	3	1	1	1	1	1	1	3	3	9	9
IX-133	Mean ± SE	22.04 ± 0.79c	3.56 ± 0.25d	0.2 ± 0.00f	0.24 ± 0.02f	0.26 ± 0.04f	0.26± 0.04f	0.28 ± 0.04f	0.22 ± 0.02f	2.2 ± 0.19e	3.4 ± 0.21d	24.14 ± 0.75b	26.06 ± 0.57a	11.91	1.04	0.99	809.23
	Range	20.1–23.5	2.7–4.1	0.2	0.2–0.3	0.2–0.4	0.2–0.4	0.2–0.4	0.2–0.3	1.7–2.7	2.8–3.9	22.9–26.5	24.8–27.5
	Score	9	3	1	1	1	1	1	1	3	3	9	9
IX-200	Mean ± SE	23.42 ±0.70b	3.52 ± 0.21c	0.82 ± 0.08d	0.78 ± 0.04d	0.7± 0.07d	0.76 ±0.08d	0.68 ± 0.08d	0.64 ±0.07d	2.74 ± 0.18c	3.68 ± 0.17c	30.56 ± 0.76a	31.62 ± 0.79a	10.49	1.11	0.99	1006.00
	Range	22.2–25.6	3.0–4.2	0.6–1	0.7–0.9	0.5–0.9	0.5–1	0.4–0.8	0.4–0.8	2.3–3.1	3.3–4.2	28.2–32.8	29.3–33.8
	Score	9	3	1	1	1	1	1	1	3	3	9	9
IX-002	Mean ± SE	3.72 ± 0.26c	8.58 ± 0.49b	0.86 ± 0.05e	0.74 ± 0.07e	0.8 ±0.04e	0.72 ± 0.06e	0.84 ± 0.05e	0.8 ± 0.04e	2.34 ± 0.22d	8.72 ± 0.43b	24.88 ± 0.90a	24.76 ± 0.65a	13.22	1.09	0.99	558.46
	Range	2.8–4.3	7.2–9.8	0.7–1	0.5–0.9	0.7–0.9	0.6–0.9	0.7–1	0.7–0.9	1.8–2.7	7.3–9.7	22.6–27.7	22.5–26.2
	Score	3	5	1	1	1	1	1	1	3	5	9	3
IX-403	Mean ± SE	23.34 ± 0.53b	2.52 ±0.15c	0.48 ± 0.06d	0.52 ±0.07d	0.48 ± 0.06d	0.5 ± 0.07d	0.48 ±0.06d	0.52 ± 0.02d	2.28 ± 0.19c	2.84 ± 0.17c	21.34 ± 0.83a	25.92± 0.98a	13.21	1.19	0.99	658.00
	Range	21.9–24.4	2–2.7	0.3–0.6	0.3–0.7	0.4–0.7	0.3–0.7	0.3–0.6	0.5–0.6	1.6–2.7	2.4–3.3	23.9–28.1	23.4–28.8
	Score	9	3	1	1	1	1	1	1	3	3	9	9
IX-090	Mean ± SE	2.46 ± 0.13cd	2.88 ±0.17c	0.56 ± 0.04e	0.56 ± 0.06e	0.54 ± 0.07e	0.58 ± 0.06e	0.54 ±0.04e	0.6 ± 0.04e	1.96 ± 0.26d	3.22 ± 0.19c	25.78 ± 0.74b	29.42 ± 0.66a	12.03	0.88	0.99	1101.20
	Range	2–2.8	2.4–3.5	0.5–0.7	0.4–0.7	0.3–0.7	0.4–0.7	0.5–0.7	0.5–0.7	1.7–2.8	1.7–3.7	20.9–28.5	23.2–31.6
	Score	3	3	1	1	1	1	1	1	3	3	9	9
IX-281	Mean ± SE	13.68 ± 0.40c	8.3 ± 0.45d	0.54 ± 0.09f	0.56 ± 0.04f	0.62 ± 0.08f	0.66 ± 0.05f	0.46 ± 0.05f	0.6 ± 0.04f	2.92 ± 0.22e	8.24 ±0.30d	24.1 ± 0.56b	28.68 ± 0.55a	9.43	1.53	0.99	987.94
	Range	13.0–14.6	7.1–9.4	0.3–0.8	0.5–0.7	0.4–0.8	0.5–0.8	0.3–0.6	0.5–0.7	2.4–3.6	7.5–9.2	23.5–25.7	27.9–30.3
	Score	7	5	1	1	1	1	1	1	3	5	9	9

Breeding line 1 represents DRR17B line containing Xa21 gene that was screened with eight different isolates under glass house conditions at IIRR. With two isolates (viz., FZB and Lud-05-1 Xa21) introgressed lines that showed resistance reaction having a score of 3. With remaining isolates, Xa21 introgressed lines showed moderate susceptibility with ascore of 7 and high susceptibility having a score of 9 (Lore et al., 2011). Breeding line 2 represents DRR17B line containing Xa33 gene, except for two isolates (viz., FZB and TNK12-3 moderate resistance with a score of 5) while the remaining isolates showed a resistance reaction score of 3. Breeding lines 3–8 represent ILs of DRR17B containing Xa21 + Xa33 genes that exhibited a high level of resistance with a score of 1 against all eight isolates. Resistant checks 9 and 10 represent ‘ISM’, which showed resistance against all eight isolates with a score of 3 and another resistant check, FBR1-15, which showed resistance reaction score of 3 except the two isolates that showed moderate resistance with a score of 5. Recurrent parents 11 and 12 represent DRR17B and susceptible check TN1 that exhibited highly susceptible reactions against all eight isolates with a score of 9. (1-IIRRGP3-87-64-22-4-50 (Xa21), 2-IIRRGP22-73-10-15-13-2 (Xa33), 3-IIRRIC10-8-94, 4-IIRRIC10-19-138, 5-IIRRIC102-26-7, 6-IIRRIC123-34-84, 7-IIRRIC123-58-3,8-IIRRIC172-77-12, 9-‘ISM’, 10-FBR1-15, 11-DRR17B and 12-TN1).

Table 4

Agro-morphological features of selected backcross derived lines of DRR17B possessing Xa21 + Xa33.

Plant identity	Days to 50% flowering	Plant height in cm		No. of productive tillers		Panicle length in cm		No. of grains per panicle		Spikelet fertility (%) (IR58025A X selected ICF4 Plant)
		Mean ± SE	Range	Mean ± SE	Range	Mean ± SE	Range	Mean ± SE	Range
IIRRIC10-8-94	97	81.84 ± 1.51de	76.8–86.8	10.6 ± 1.03a	8–14	23.64 ± 0.60a	21.9–25.5	301.6 ± 7.14bc	284–322	2
IIRRIC10-19-138	92	78.36 ± 1.19ef	74.1–82.4	10 ± 0.71a	8–12	21.52 ± 1.05b	18.7–23.8	263.8 ± 9.49de	235–294	5
IIRRIC102-26-7	101	89.86 ± 1.18b	85.1–93.2	12 ± 1.10a	10–16	24.16 ± 0.63a	21.9–25.5	360.4 ± 9.98 a	331–390	0
IIRRIC123-34-84	95	78.62 ± 1.10ef	74.7–82.4	10.8 ± 1.16a	8–14	23.28 ± 0.45ab	21.7–24	279.8 ± 8.86cd	265–311	0
IIRRIC123-58-3	95	84.92 ± 1.21cd	80.6–89.4	11.2 ± 1.11a	7–13	22.52 ± 0.65ab	20–23.1	308.0 ± 9.48b	278–328	18
IIRRIC172-77-12	97	77.06 ± 1.13f	81.1–73.2	11.2 ± 1.20a	9–15	21.62 ± 0.72b	19.2–23.4	317.0 ± 7.00b	298–339	0
DRR17B	105	96.08 ± 1.86a	90.6–102.1	10.8 ± 0.97a	9–14	23.24 ± 0.64ab	21.7–25.6	280.0 ± 11.60cd	284–307	-
ISM	108	86.78 ± 1.04bc	83.1–90.3	11.6 ± 1.21a	9–15	19.58 ± 0.72c	17.7–21.9	252.0 ± 7.98e	224–270	-
FBR1-15	103	77.02 ± 1.34f	73–82.1	9.8 ± 0.66a	8–12	18.76 ± 0.72c	16.0–19.9	200.2 ± 11.10f	169–228	-
CV	-	4.22	-	22.11	-	6.6	-	7.02	-	-
LSD (p = 0.05)	-	4.5287	-	3.1018	-	1.8724	-	25.734	-	-
H2	-	0.88	-	0.25	-	0.68	-	0.9	-	-
F	-	17.6	-	0.43	-	8.14	-	25.52	-	-

Values in a column followed by common letters do not differ significantly by Duncan’s Multiple Range Test (DMRT). DMRT (P = 0.05)