Introduction

Rice (Oryza sativa L.) is one of the most important cereals, serving as a principal source of nutrition to half of the global population, and particularly in India, it accounts a major portion of total food grain production [1]. India must produce 120 million tonnes of rice by 2030 to maintain self-sufficiency and to fulfil future food demands, which must be accomplished with limited land, water, labour, and using minimum amount of chemicals (i.e., fertilizers and pesticides etc.), as well as a continuous fight against developing disease, pests and the possible harmful consequences of climate change [1, 2]. Biotic stresses like disease and insect pests cause major yield loss in rice varieties across the globe [3]. Among many biotic stresses that affect rice production, bacterial leaf blight (BLB) is one of the most dreadful disease, causing yield reduction upto 74–81% depending on weather, location, and rice cultivar used for cultivation [4]. In both tropical and temperate rice-growing zones, the gram-negative bacterium Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight (BLB) disease [5]. During the maximum tillering stage, the BLB infection occurs, resulting in water-soaked lesions on the leaves that steadily enlarge and finally cause the rice plant to wilt [5]. Chemical pesticides and antibiotic sprays do not work against BLB [6], however antibiotics are utilized to control bacterial disease such as Pseudomonas spp., and Xanthomonas campestris [7, 8]. Therefore, host plant resistance represents the most practical and cost-effective strategy for disease management [9,10,11,12,13]. Till date, 47 genes have been identified, that provide resistance from BLB, of which many of them have been incorporated into high-yielding, popular rice cultivars across the globe [14,15,16]. The deployment of resistant cultivars with a single major resistance gene has been proven significant due to the development of novel strain of Xoo due to mutation [17].

Hence, the development of varieties with several resistance genes offer a viable alternative for broad-spectrum, long-term resistance against BLB in rice [18]. To achieve the goal, multiple resistance genes can be stacked or pyramided into the elite genetic background as a breeding strategy [19]. Meanwhile, marker-assisted selection (MAS) is a more effective and simpler approach for gene introgression than conventional breeding [19, 20]. The utilisation of MAS holds significant potential in assisting plant breeders to accomplish their objectives. However, its influence on the development of plant varieties has been limited [20]. In order to fully exploit the potential of MAS, it is crucial to establish a higher level of integration between MAS and breeding programmes. Additionally, it is important to understand the existing barriers and develop suitable methods to overcome them. The utilisation of the benefits of MAS in comparison to traditional breeding methods has the potential to significantly influence the improvement of cereal crops [20, 21].

Therefore, the present experiment was conducted to transfer valuable genes in rice variety Pratikshya for BLB resistance using MAS with the objectives: validation of parental lines for BLB resistance genes by using molecular markers, development of F1 and the backcross populations and marker-assisted selection of lines possessing BLB resistance genes xa5, xa13, and Xa21 in the populations F1, BC1F1, BC2F1, BC2F3 (foreground selection), phenotypic screening of segregating population (BC2F2) for BLB resistance through artificial inoculation, marker-assisted selection of lines possessing background of recurrent parent Pratikshya in the population BC2F3 (background selection), and evaluation of pyramided lines for yield and agro-morphological characters in BC2F4 population.

Materials and methods

Plant materials

Pratikshya is a popular high-yielding rice variety of Odisha, India with good grain and cooking quality released by Odisha University of Agriculture and Technology (OUAT), Bhubaneswar, which is suitable for late sown rainfed medium land, moderately resistance to brown spot, sheath rot, sheath blight, leaf folder, white backed plant hopper, gall midge, stem borer, but highly susceptible to BLB. The donor parent for BLB resistance employed in the current crossing program was Swarna MAS (CR Dhan 800) released by ICAR-National Rice Research Institute (ICAR-NRRI), Cuttack, India that carries three BLB resistance genes, i.e., xa5, xa13, Xa21 in the genetic background of Swarna variety [22].

Hybridization and marker-assisted selection

A step-by-step marker-assisted backcross breeding approach was implemented as shown in Fig. 1 for the effective introgression of xa5, xa13, and Xa21 genes into Pratikshya. In Kharif season, 2017, F1 seeds were successfully generated by crossing recipient parent Pratikshya with donor parent Swarna MAS. In the shallow pots located within the net house, F1 seeds were planted in Rabi season 2017. True hybridity was checked in the F1 generation plants using the three SSR markers (Table 1) linked to the xa5, xa13, and Xa21 genes. The selected true F1 plants were hybridized with the recurrent parent, Pratikshya, during Rabi season 2017 to produce BC1F1 seeds. The BC1F1 plants were then subjected to both foreground selection in Kharif season 2018 using MAS and phenotypic selection for agronomic similarity with Pratikshya.

Fig. 1
figure 1

Steps in pyramiding bacterial leaf blight resistance genes into the recipient variety, Pratikshya via Marker-assisted backcross breeding

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Backcrossed seeds from selected BC1F1 plants were grown in the next generation as BC2F1 plants in Rabi season 2018. Selected plants from BC2F1 carrying the xa5, xa13, and Xa21 genes were allowed to self-pollinate and then carried forward to BC2F2 generation during Kharif season, 2019. Resistant plants from BC2F2 generation were selected by phenotypic screening using clip inoculation technique of Xoo inoculum (Xanthomonas oryzae pv. oryzae brought from Crop Protection Division, National Rice Research Institute, Cuttack, India) by following a standard method [23], and by assessing the similarity of agronomic characteristics to Pratikshya. During Rabi season, 2019, plants from the BC2F3 generation were used for foreground selection and selected plants bearing all three resistance genes were subjected to background selection with markers showing parental polymorphism, and phenotypic selection for agronomic similarity with Pratikshya. Plants having all three resistance genes and good recipient parent genome recovery percentage were carried forward to BC2F4 generation during Kharif season, 2020 for agronomic trials.

Molecular characterization and SSR markers analysis

Total genomic DNA was isolated by the modified CTAB method of DNA extraction for rice [24], and quantified on Nanodrop - Spectrophotometer (NANODROP 2000c), by diluting in 1X TE buffer to a final concentration of around 50 ng/µl. The PCR reaction mixture for foreground selection of xa5, xa13, and Xa21 consisted of 50 ng of genomic DNA, 10 mM of each primer, 10 mM of each dNTP, 10x PCR buffer, 3U Taq polymerase in a volume of 10 µl. The amplified products were subjected to electrophoresis on a 1.5% agarose gel for xa13 prom primer (for xa13 gene), pTA248 primer (for Xa21 gene), 3% for RM122 primer (for xa5 gene) (Table 1), and visualized on a gel documentation system (Bio-Rad Laboratories Inc., USA). To confirm the presence of the target genes, foreground selection was conducted till BC2F3 generation. However, 45 polymorphic SSR markers after the parental survey (Table 2) were employed for background selection to detect parental genome recovery in the pyramided lines [25].

Table 1 Markers used for foreground selection of bacterial leaf blight resistant line in rice variety Pratikshya
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Table 2 List of background primers used in the present study
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The SSR bands from the gel photograph of BC2F3 generation were manually scored as 1 (if band present) and 0 (if band absent) to obtain the binary data for molecular analysis. Missing data were verified twice by repeating the genotyping process. The genetic distance between pyramided lines was calculated through the construction of the distance matrix using Jaccard’s similarity coefficient from the binary data to deduce the genetic relationships between the pyramided lines and two parents, by constructing a dendrogram by following sequential UPGMA (unweighted pair group method with arithmetic mean) using software package TASSEL 5.0 [26].

Screening for bacterial blight resistance and morphological characterization

During Kharif season, 2017, field screening for BLB resistance was conducted by utilizing the inoculum of Xanthomonas oryzae pv. oryzae. Each Plant artificially inoculated by the leaf clip inoculation method, in which, the top leaves of each plant were clipped at the maximum tillering stage and then inoculated [23]. The symptoms started to develop five to six days after the inoculation, and the observation period was between 14 and 21 days after inoculation. The plants with a score of 1 were regarded as resistant, those with a score of 3 as moderately resistants, those with a score of 7 as susceptible, and a score of 9 as highly susceptible [27]. The observations were recorded for percentage of diseased leaf area (DLA) followed by a standard evaluation system [27]. However, a multi-location trail was conducted at Rice Research Station, OUAT, Bhubaneswar, India (GPS co-ordinates: latitude NL 20° 15′ 55″, longitude of EL 85° 48′ 33″), and at agricultural farm, OUAT, Bhubaneswar, India (GPS co-ordinates: latitude of 20° 16’ 09.3’’ N, longitude of 85° 47’ 29.0’’ E), for proper validation of the screening.

Thirty-days-old seedlings of BC2F4 generation carrying BLB resistance genes were transplanted along with the donor and recipient parents in the main field of Rice research station, OUAT, Bhubaneswar with 20 cm × 15 cm spacing. The crop was successfully grown using conventional agronomic practices in Kharif season, 2020. The phenotypic trait observations were recorded for 10 plants in three replications and the replicated data were used to calculate mean, coefficient of variation (CV), and critical difference (CD). The observations were recorded for yield component characters viz., days to 50% flowering, plant height (cm), total number of tillers per plant, number of productive tillers per plant, panicle length (cm), filled grains/panicle, 1000-grain weight (g), and seed yield per plant (g). Data analyses were carried out with the use of the software, Grapes version 1.0.0 [28].

Results

Molecular validation of parental lines

The resistant parent (Swarna MAS) and susceptible parent (Pratikshya) were validated for the presence of BLB resistance genes xa5, xa13, and Xa21 with help of gene-linked markers RM122, xa13 prom, and pTA248, respectively. Genomic DNA from both parents was amplified using the above-mentioned three SSR markers and then parental polymorphism was revealed from gel electrophoresis. The resistant allele of xa5 showed a band at 200 bp using the RM122 marker. The resistant allele of the xa13 gene generated a fragment at 500 bp, whereas the resistant allele of the Xa21 gene was found in the resistant parent Swarna MAS at 1000 bp (Fig. 2).

Fig. 2
figure 2

Gel photographs of parents indicate (A) presence of expected base pair specific band for xa5 (200 bp), (B) specific band for xa13 (500 bp), (C) specific band for Xa21 (1000 bp). Lane 1 represents DNA ladders (100 bp); Lane 2: P1 - Swarna MAS; Lane 3: P2 – Pratikshya, and L represents 100 bp ladder

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Pyramiding of bacterial leaf blight resistance genes

During each generation from F1 to BC2F3 (except for BC2F2, due to more number of population and use of clip inoculation technique), foreground selection was conducted to select plants having all three resistance genes (positive plants) and only those plants were advanced to the next generation. The hybridity of F1 plants was assessed using molecular markers, and it was determined that out of a total of 26 F1 plants, 18 exhibited characteristics consistent with being true F1 plants. True F1 plants were backcrossed with the recipient parent Pratikshya to generate BC1F1 seeds. In the BC1F1 generation, 117 plants were grown and out of these, 58 plants were identified to possess all three resistance genes. These 58 BC1F1 plants positive for resistance genes were backcrossed with recurrent parent Pratikshya. Of the 170 BC2F1 plants grown, 40 were detected to possess three-resistance genes. Thus only these 40 BC2F1 plants were allowed to self-pollinate and advanced to BC2F2 generation (Fig. 3).

Fig. 3
figure 3

Foreground selection of plants from BC2F3 generation, (A) for xa5 gene with 100 bp ladder, (B) for xa13 gene with 100 bp ladder, (C) for Xa21 gene with 1 kb ladder

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In BC2F2 generation phenotypic screening procedures were followed instead of the use of molecular screening procedures to identify resistant plants. Selected 35 BC2F2 plants were grown as a total of 35 lines in BC2F3 generation and plants from those lines homozygous for three resistance genes combinations were identified (Fig. 2). Out of the 35 lines only thirty-one plants were found to possess all the three genes and hence were subjected to background selection. The background selection of these 31 BC2F3 plants with forty-five polymorphic SSR markers showed genome recovery of Pratikshya in the range of 64.44–93.33%. Out of 31 plants, 5 were showed genome recovery from 91.11 to 93.33% (Table 3; Fig. 4). Thereafter, thirty-one BC2F3 plants were allowed to self-pollinate to obtain plants for BC2F4 population.

Table 3 Recurrent parent genome recovery in thirty-one pyramided lines for BLB resistance
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Fig. 4
figure 4

Background selection of plants from BC2F3generationwith different polymorphic SSR markers. The amplified fragments with respect to marker RM26885 is present in Pratikshya, plant no. 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 17, 18, 19, 20, 21, 22, 23, 24, 28, 29, 30, 31. The amplified fragments with respect to marker RM27180 is present in Pratikshya, plant no, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 17, 18, 19, 21, 22, 23, 27, 28, 29, 31.The amplified fragments with respect to marker RM27446 is present in Pratikshya, plant no 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31. Lane 1- 100 bp ladder, Lane 2- Pratikshya (P). However, the best improved line no. 19 - OR 2772-19 gave a seed yield/plant of 65.5 g

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Genetic similarity of the pyramided lines with the recipient parent using SSR markers

The dendrogram was constructed from SSR data grouped the 31 three-gene pyramided lines along with both parents into two major clusters with Swarna MAS in cluster I and the remaining 31 pyramided lines, including Pratikshya, in cluster II along with the similarity matrix of the pyramided lines (Fig. 5; Table 4). Cluster II was further divided into two sub-groups, cluster II-A and cluster II-B (Table 5). Cluster II-A was further subcategorized into two sub-groups, cluster II-Aa, and cluster II-Ab. Cluster II-Aa consists of pyramided lines OR 2772-4, OR 2772-28, OR 2772-73, OR 2772-31, OR 2772-2, OR 2772-20, OR 2772-15, OR 2772-74, OR 2772-77, OR 2772-92, OR 2772-1, OR 2772-17, OR 2772-32, OR 2772-90, OR 2772-16, OR 2772-3, OR 2772-19, OR 2772-21, OR 2772-27 and OR 2772-30, OR 2772-33, OR 2772-54, OR 2772-64, OR 2772-51, OR 2772-65, OR 2772-62, OR 2772-10, and OR 2772-93, while Cluster II-Ab contains OR 2772-13 and OR 2772-18. Cluster II-B consists of OR 2772-76.

Fig. 5
figure 5

Dendrogram representing the genetic relationship between pyramided lines of the BC2F3 generation. The donor parent Swarna MAS (P1) is in cluster I, whereas the recipient parent (Pratikshya (P2) and the other 31 pyramided lines are clustered into a separate cluster II.

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Table 4 Similarity matrix of the pyramided lines used in the present study
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Table 5 Clustering of the pyramided lines into different major clusters and sub-clusters
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Phenotypic screening of the BC2F2 against bacterial leaf blight

The recipient parent Pratikshya showed a disease score of 7 indicating a greater level of susceptibility to BLB. Swarna MAS displayed a high level of resistance to the infection with a disease score of 0. The disease score of BC2F2 plants ranged from 0 to 1 showing resistance to the disease. Out of all the plants of the BC2F2 population (1598 plants), 1412 were observed to be resistant and 186 were susceptible, across the multi-location trail.

Evaluation of pyramided lines for agro-morphological traits in BC2F4 generation

The mean values of eight agronomic traits viz., days to 50% flowering, plant height (cm), number of tillers per plant, number of productive tillers per plant, panicle length (cm), number of filled grains per panicle, seed yield per plant (g) and 1000 grain weight (g) are presented in Table 6. From the statistical analysis, it was noticed that a significant difference was observed between the pyramided lines and both of the parents for all the characters except for total tillers, though, the difference was very less. The pyramided line OR 2772-19 displayed the highest seed yield per plant of 65.5 g. The average seed yield per plant of pyramided lines in the field was found to be 42.37 g, and the yield is varying due to the change in their genotyping composition after introgression.

Table 6 Agro-morphological and yield characters of thirty-one bacterial leaf blight resistance genes pyramided lines
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The recurrent parent, Pratikshya, recorded a mean seed yield of 44 g/plant, while the donor parent (Swarna MAS) recorded 49.4 g/plant. Among pyramided lines, ten lines attained more seed yield per plant than Pratikshya. For panicle length, among the pyramided lines, OR 2772-21 had the longest panicle length (34.7 cm) followed by OR 2772-20 (28.9 cm). The parent Pratikshya displayed a panicle length of 24.5 cm. Out of thirty-one pyramided lines, nineteen lines produced longer panicles, while ten lines produced smaller panicles than Pratikshya. The highest plant height of 109.6 cm was observed in OR 2772-76 followed by 107 cm in OR 2772-27, while the shortest plant height of 88.3 cm was observed in OR 2772-13. Majority of the pyramided lines attained more grain weight than the Pratikshya, while OR 2772-92 showed a similar grain weight with the recurrent parent. From these observations, it was concluded that agro-morphological features play a significant part in the yield of the plant.

Discussion

Pratikshya (Parentage: Swarna × IR64) is late-maturing, popular high-yielding rice variety in Eastern India [25]. It can be harvested in 146 days, and has a high degree of adaptability, and it creates opportunities for cropping system intensification in the coastal zone of India by earlier establishment of Rabi season crops [29]. However, the main limitation of Pratikshya is its susceptibility to BLB, which significantly reduces yield, despite the fact that it has potential for high yield with a desirable plant type. BLB acts as a biotic constraint in the decline of productivity in South-East Asian countries. The extent of severity of 10-20% annual reduction in rice production worldwide caused by BLB in rice demands the development of effective management strategies [30]. However, in order to prevent the spread of BLB, it has been found that host plant resistance is the most efficient environmental friendly method [31]. The incorporation of resistance genes in combination remains as an effective strategy for managing BLB and providing durable resistance [32]. In contrast to conventional phenotypic evaluation, marker-assisted backcross breeding permits for the selection of recessive alleles, selection of desirable plants at the seedling stage before the formation of a visible phenotype, and the pyramiding of important traits into a single genetic background. The application of molecular markers allow for the genetic categorization of progeny at each generation and speeds up the selection process [33].

Pyramiding multiple resistance genes with potential characteristics into a single genotype through MAS can improve the efficiency of generating new crop varieties exhibiting disease resistance, as well as other desirable traits, however, it is crucial for the maintenance of yield stability in the rice variety Pratikshya [34]. Meanwhile, in several studies, this approach has been successfully employed to introduce resistance genes into the elite genetic background [11, 30, 31, 34]. The BLB resistance genes xa5, xa13, and Xa21 have been successfully incorporated into different genetic backgrounds of rice [11, 35].

Hence, these three resistance alleles covering three chromosome regions were selected to transfer into the popular variety Pratikshya in the present investigation. Out of 46 identified BLB resistance genes, three major R genes xa5, xa13, and Xa21 have been used in the current study. However, previous research has successfully cloned and described all three of these resistances, i.e., Xa21 resistance gene is dominant and has been cloned with a high level of resistance from many Xoo strains [36]. The gene xa5 is in the recessive gene category and codes an alternative form of transcription factor cIIa [37]. The xa13 is a recessive gene that originated because of a mutation in the promoter region of a gene which is homolog to nodulin MtN3 [38].

The pyramided lines carrying these three genes in the present study exhibited higher degrees of resistance than both of the parents. This aligns with the study of some scientists elucidating the contribution of synergistic action of resistance genes in achieving increased levels of resistance [39, 40]. These three R genes are resistant to all the prevalent races of BLB pathogen in the coastal region of East India. However, previously many workers have used these three genes for the transfer of BLB resistance to many traditional varieties of Odisha, India, as well as in other parts of the country [22, 41].

Meanwhile, due to the masking effect of dominant genes over recessive genes, combining them at the same time might be a challenge via phenotypic selection. In such cases, molecular markers for both recessive and dominant resistance genes can assist in the identification and selection of desirable plants with multiple resistance genes [41, 42]. However, pyramided lines with the highest recovery of the recipient parent genome were found with the help of marker-assisted background selection, in the present study. In the BC2F3 generation, the highest genome recovery rate was 91.11–93.33% in five pyramided lines. The low recovery of the background of recurrent parent observed in a few lines can be attributed to linkage drag, referring to the reduction in fitness in cultivars due to deleterious genes introduced along with the beneficial gene during backcrossing [42]. It is possible that the functional portion of the genome is not recovered since SSR markers are often used to target non-coding and heterochromatic areas of chromosomes [43].

The phenotypic selection was also used in each generation to identify plants that are similar to Pratikshya in terms of agronomic characteristics, but also better from it. However, the functionally expressed area of the genome is an indirect target of phenotypic selection and helps to accelerate the recovery of the recurrent parent phenotype [41, 42]. Meanwhile, the linkage drag is kept to a minimum in this study by using genetically related parents in the crossing program. Swarna MAS (CR Dhan 800) is a derivative of the variety Swarna, which is a highly adaptable variety. Previous researchers concluded that using a highly adaptable variety as a donor parent results in better performance and less linkage drag than using a wild species or landrace as a donor parent [42]. However, the parentage of the recipient parent is Swarna and IR64, and the shared ancestry of Pratikshya with donor parent Swarna MAS speeds up the recovery of the recipient parent genome [42, 43].

Yield and agro-morphological parameters from thirty-one pyramided lines, Pratikshya and Swarna MAS showed that the pyramided lines have the outstanding yielding ability of recipient parent and resistance to BLB in the present study. The analysis of variance for yield and agro-morphological traits displayed that the mean for all characters were at par with Pratikshya in most of the lines, many lines performed to a greater degree in terms of seed yield per plant, and the majority of the lines were close to the recurrent parent, in the present study. However, various studies have revealed that the high seed yield of some pyramided lines may be attributable to the transmission of yield features from the donor source to the recipient parent [30, 34]. Meanwhile, in the background of Pratikshya, pyramided lines had no antagonistic effect due to the introgression of resistance genes. High degrees of resistance to BLB, the lack of a penalty on yield, and good recovery of recurrent parent genome point towards a successful selection approach at both the molecular and phenotypic levels in the preset study. However, some scientists also found similar results from their study [11, 22, 34].

Due to the inadequacy of a single resistance gene in providing resistance against different pathogen races of BLB in Odisha, India, a wide range of resistance against BLB is critical. However, some scientists, reached a similar conclusion, observing a broad spectrum of resistance against bacterial leaf blight when multiple genes were introgressed into an elite genetic background rather than a single gene [44]. However, in the present study, pyramiding three resistance genes in a single genetic background was found to be an effective barrier against prevalent pathogen races of BLB in Odisha, India conditions, and expected to provide durable resistance.

Conclusion

Marker-assisted foreground and background selection along with phenotypic selection was found to be the best way to breed resistant varieties, making the breeding program much more effective. The current research concludes that, the marker-assisted backcross breeding program was successful in introducing three BLB resistance genes (xa5, xa13, and Xa21) into the high-yielding rice variety Pratikshya. The resulting pyramided lines exhibited a high level of resistance to BLB infection and were similar to Pratikshya in terms of morphological features and yield parameters. This study suggests that, the three-gene pyramided lines could offer a significant yield advantage over the recipient parent Pratikshya. The pyramided lines can further be used for testing at multilocation, so as to be released as a variety or can be used as a potential donor for BLB resistance genes. The present study also suggests the deployment of multiple resistance genes can be more effective than the use of a single resistance gene. The BLB pyramided lines are proven to be successful in providing long-lasting and durable resistance in the genetic background of Pratikshya, which is an accomplishment for developing BLB-resistant pyramided lines bearing three resistance genes.