Marker-assisted enhancement of bacterial blight (Xanthomonas oryzae pv. oryzae) resistance in a salt-tolerant rice variety for sustaining rice production of tropical islands

Introduction Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae is a major disease of rice, specially in the tropical regions of the world. Developing rice varieties with host resistance against the disease is the most effective and economical solution for managing the disease. Methods Pyramiding resistance genes (Xa4, xa5, xa13,and Xa21) in popular rice varieties using marker-assisted backcross breeding (MABB) has been demonstrated as a cost-effective and sustainable approach for establishing durable BB resistance. Here, we report our successful efforts in introgressing four resistance genes (Xa4, xa5, xa13, and Xa21) from IRBB60 to CARI Dhan 5, a popular salt-tolerant variety developed from a somaclonal variant of Pokkali rice, through functional MABB. Results and discussion Both BB and coastal salinity are among the major challenges for rice production in tropical island and coastal ecosystems. Plants with four, three, and two gene pyramids were generated, which displayed high levels of resistance to the BB pathogen at the BC3F2 stage. Under controlled salinity microplot environments, the line 131-2-175-1223 identified with the presence of three gene pyramid (Xa21+xa13+xa5) displayed notable resistance across locations and years as well as exhibited a salinity tolerance comparable to the recurrent parent, CARI Dhan 5. Among two BB gene combinations (Xa21+xa13), two lines, 17-1-69-334 and 46-3-95-659, demonstrated resistance across locations and years, as well as salt tolerance and grain production comparable to CARI Dhan 5. Besides salinity tolerance, five lines, 17-1-69-179, 46-3-95-655, 131-2-190-1197, 131-2-175-1209, and 131-2-175-1239, exhibited complete resistance to BB disease. Following multilocation testing, potential lines have been identified that can serve as a prospective candidate for producing varieties for the tropical Andaman and Nicobar Islands and other coastal locations, which are prone to BB and coastal salinity stresses.


Introduction
Rice (Oryza sativa L.) is the world's primary carbohydrate source, feeding nearly half of the global population.India must produce 135-140 million tons of rice by 2030 to maintain selfsufficiency and meet future food grain demands (Gujja and Thiyagarajan, 2009).This goal must be achieved while considering the challenges posed by limited agricultural lands, depleting water supplies, declining soil productivity, emerging pests and pathogens, and the potential adverse effects of climate change.Historically, rice has also been the principal grain crop of the Andaman and Nicobar Islands, situated in the Bay of Bengal, despite its poor average yield.The farmers of the Andaman and Nicobar Islands belonging to diverse socioethnic groups and tribes mostly cultivate traditional rice varieties.The warm and humid conditions prevalent almost throughout the year in tropical islands facilitate the growth and multiplication of biotic stress factors, including diseases and insect pests.Among the biotic stresses, bacterial blight (BB) caused by Xanthomonas oryzae pv.oryzae is one of the most important diseases of rice, which spreads epidemically in susceptible cultivars and causes yield losses ranging from 20% to 50%, depending on weather conditions (Singh et al., 2011).The favorable temperature for the development of disease is between 25 and 34°C, with a relative humidity above 70%, which commonly coincides with the weather conditions of the islands.The problem is further exacerbated by susceptible varieties, poor management practices, and closed farming system scattered across different islands.Chemical disease management is not a recommended practice in these islands due to its high cost and negative environmental impact on terrestrial and marine ecosystems.The prevalence of BB has a visible and direct detrimental effect on the Paddy-cum-fish and duck-type farming systems, as well as an indirect impact on the coastal mangrove ecology (Chukwu et al., 2019).The most effective approach to minimizing losses is to develop resistant/tolerant rice varieties.Additionally, the Andaman and Nicobar Islands face challenges from coastal salinity and tsunami effects, which affect about one-third of the total area and are compounded by the region's vulnerability to climate change.While salt stress poses a worldwide threat to agricultural production (Hopmans et al., 2021), the effects of salinity are exacerbated in high rainfall coastal and islands' ecosystems due to the presence of acid sulfate soils (Sarangi et al., 2022).
The development of resistant cultivars carrying resistant genes has been the most effective and economical strategy for controlling BB disease without causing environmental pollution (Huang et al., 1997;Singh et al., 2001;Jena and Mackill, 2008;Sundaram et al., 2008;Rajpurohit et al., 2011;Dokku et al., 2013;Suh et al., 2013).To confer resistance against BB, more than 46 disease-resistance (R) genes have been identified and designated with a series from Xa1 to Xa46 (Fiyaz et al., 2022).Many of these genes are tagged with DNAbased markers, and some have been cloned and characterized (Kumar et al., 2020).The marker-assisted introgression of multiple resistance genes into a single genetic background is invariably preferred because the probability of simultaneous mutation at multiple loci to overcome the resistance is low (Koide et al., 2010).
Pokkali is a traditionally popular rice variety cultivated in the coastal saline areas of Kerala, India (Gregorio et al., 2002).It has been identified as a universal donor of salt tolerance genes, of which Saltol is the most prominent (Thomson et al., 2010).The analysis of phenotypic responses, genomic composition, and QTLs present in the salt-tolerant introgressed lines (ILs) developed from a salttolerant rice landrace "Pokkali" using SSR and SNP markers has revealed potential salt tolerance mechanisms in these ILs, including Na + dilution in leaves, vacuolar Na + compartmentalization, and possibly the synthesis of compatible solutes (De Leon et al., 2017).Besides salt tolerance, Pokkali has also been found to be nutritionally rich in various micronutrients, bran oil, and antioxidants such as oryzanols, tocopherols, and tocotrienols (Shylaraj et al., 2018).In a quest to develop an agronomically desirable plant type, induction of somaclonal variation in Pokkali led to the development of an elite variety called CARI Dhan 5 (Mandal et al., 1999), which is now an increasingly popular and successful salt-tolerant variety in Andaman and Nicobar Islands due to its wide seed dissemination and larger-scale adoption (Singh et al., 2020).However, it is important to note that CARI Dhan 5 is susceptible to BB, which remains a major disease of rice in the tropical Andaman Islands.
Through our systematic research, we have evaluated the relative efficacy of various resistance genes for managing BB disease, further complemented by analyzing the diversity, distribution, and virulence of the pathogen.This has provided a deeper understanding of the disease dynamics and has opened up new opportunities through the host pathogen approach under Bay Islands (Gautam et al., 2015;Gautam et al., 2016a;Gautam et al., 2016b;Sakthivel et al., 2016;Sakthivel et al., 2017;Sakthivel et al., 2021).Our previous study proved the effectiveness of four gene combinations (Xa4, xa5, xa13, and Xa21) carried by IRBB60 under Andaman Islands conditions (Gautam et al., 2013).This led us to pyramid these resistance genes into the popular salt-tolerant variety CARI Dhan 5 using marker-assisted backcross breeding, considering the effectiveness of these genes and the susceptibility of CARI Dhan 5 to BB.

Plant materials and crossing scheme
The donor parent, IRBB 60, possessed an impressive lineage of four BB resistance genes, namely, Xa4, xa5, xa13, and Xa21, which were incorporated into the background of IR24 through development at the International Rice Research Institute (IRRI), Philippines.On the other hand, the recurrent parent, salt-tolerant CARI Dhan5, was developed from the somaclonal variant selection from Pokkali (Mandal et al., 1999) and successfully notified for commercial cultivation in coastal saline and acid sulfate soils of the Andaman and Nicobar Islands.The crossing scheme for this hybridization was executed at Bloomsdale Research Farm, ICAR-CIARI, Port Blair, which is illustrated in Figure 1.

Bioassay against BB disease resistance
Following 40 days of planting, both the parents and hybridization-derived plants were inoculated with a virulent isolate from the Andaman Islands using the clipping procedure as outlined by Kauffman (1973).After a period of 15 days postinoculation, the lesion length of the inoculated leaves was meticulously measured.Disease scoring was carried out as per the standard evaluation system (SES, IRRI, 1996;Sakthivel et al., 2017).Based on the mean lesion length, the lines were categorized into four distinct groups, namely, resistant (<5 cm), moderately resistant (5-10 cm), moderately susceptible (10-15 cm), and susceptible (>15 cm).A BB-resistant and -susceptible reaction after inoculation is presented in Figure 2.

Evaluation of yield and other component traits of the pyramided lines
In the study, a total of 35 pyramided lines (BC 3 F 3/4 ) and a single check (CARI Dhan5) were transplanted in both normal and saline environments in microplots and experimental fields located at ICAR-CIARI, Port Blair, in the Andaman and Nicobar Islands.Various agronomic traits were recorded, including plant height, days to 50% flowering, number of tillers per plant, panicle length, leaf length, leaf width, grain length, grain width, 100-seed weight, and phenotypically acceptability score (Table S4).To evaluate the distinctness, uniformity, and stability (DUS) of the 15 pyramided lines in normal soil, 41 agro-morphological traits were recorded at Bloomdale Farm of ICAR-CIARI, Port Blair, in the Andaman and Nicobar Islands, during the kharif season of 2020.The chosen advanced generation pyramided lines were phenotyped for their grain yield and related traits performance in both salinity stress (EC 5.0 dSm −1 ) and normal soil conditions in a controlled micro-plot facility at ICAR-CIARI, Port Blair, Andaman and Nicobar Islands.Here, it is pertinent to mention that the 30-day-old nursery seedlings were transplanted in microplots.After 7 days of seedling establishment, artificially prepared saline water (EC 5.00 dS/m) was applied throughout the crop season to maintain this level of salt stress until maturity.On the other hand, only normal or fresh water (EC ~0.00 dS/m) was applied to the normal microplots.The pyramided lines were also screened for BB resistance along with their evaluation for grain yield and related traits.

DNA isolation and PCR amplification
The CTAB method described by Doyle and Doyle (1990) was used to isolate high-quality genomic DNA from rice.In brief, this method involves the use of CTAB, a cationic detergent, to extract DNA from the plant tissues, followed by precipitation and washing steps to purify the DNA.The resulting DNA was quantified and used as a template for PCR amplification.The PCR reaction mixture consisted of 50 ng of template DNA, 10 pmol of each of the primers, Back crossing scheme used to develop BB-resistant lines of CARI Dhan 5.
200 mM of each dNTP, 1 × PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl), 1.5 mM MgCl 2 , and 0.5 units of Taq DNA polymerase in a volume of 20 ml.The cycling conditions for the PCR reaction involved an initial denaturation step at 94°C for 5 min, followed by 35 cycles of amplification at 95°C for 30 s, 55°C for 30 s for primer annealing (Chen et al., 1997;Panaud et al., 1996), and 72°C for 1 min for extension.The final extension was carried out at 72°C for 5 min.Template DNA was followed with the following cycling conditions: 30 s primers annealing at 55°C.The PCR products and the DNA fragments produced were separated by gel electrophoresis using an agarose gel, and gel images were analyzed on an Alpha Imager (Alpha Innotech, USA).

Marker analysis
Sequence tagged site (STS) markers as well as functional PCRbased markers, Xa4F/Xa4R, for the Xa4 gene (Ma et al., 1999;Sundaram et al., 2011); RG556 and xa5SF/xa5SR/xa5FM-RF/ xa5FM-RR for the xa5 gene (Hajira et al., 2016); RG136 and xa13-prom for xa13 gene (Chu et al., 2006); and pTA248 for the Xa21 gene (Song et al., 1995) were used for the foreground selection of resistance allele of each gene during each backcross generation (Table 1).In case of Xa4, the resistant allele is of size 150 bp while the susceptible allele is of size 120 bp.We have marked the identity of these bands in both the susceptible parent (SP) and the resistant parent (RP) to prevent any ambiguity.In case of the xa5 gene, the 424-bp band is common to all while a 313-bp band marks susceptible allele and a 134-bp band marks the resistant allele.We have marked the identity of these bands in both the susceptible parent (SP) and the resistant parent (RP) to prevent any ambiguity.The detection of Xa13 and Xa21 was performed as per Chu et al. (2006) and Song et al. (1995), respectively.Parental polymorphism survey was done between donor (IRBB60) and recipient (CARI Dhan 5) varieties using 200 HvSSR markers to select polymorphic markers associated with the recipient parents for background selection in later generations.The genomic contribution of the parent was determined by the assessment of SSR marker data in the selected recombinants.The marker-assisted backcross breeding approach was implemented from the F 1 stage up to the BC 3 F 1 generation.At each stage, foreground selection was carried out to identify plants carrying the BB resistance genes.Only progenies with the resistance alleles were advanced to the next generation (Figure 1).

Data analysis
The experiments were performed in two to three replications and repeated twice for confirmation.The results were expressed as the mean ± SE of different independent replicates.An analysis of variance was performed in WASP software (version 2.0).P values of ≤0.05 were considered as statistically significant.The PCR amplification was performed in a Bio-Rad thermocycler (C1000 BB resistance testing in resistant introgression lines (A) and in the recurrent parent (B).
Thermal Cycler, Hong Kong, China).The images of the gel were analyzed on a Bio-Rad Gel Doc XR+System (California, USA).

Results
4.1 Pyramiding of BB resistance genes into CARI Dhan 5 The introgression of four BB resistance genes into CARI Dhan 5 was achieved via a crossing scheme (Figure 1) wherein IRBB60 (Xa4, xa5, xa13, and Xa21) was used as the donor parent.The results of the parental polymorphism survey revealed a significant level of polymorphism (23.5%) between CARI Dhan 5 (recurrent) and IRBB60 (non-recurrent) based on the analysis of 200 microsatellite markers.Among the markers surveyed, 47 were found to exhibit parental polymorphism.In addition, parental polymorphism was observed for all four target resistance genes, which was confirmed through the use of linked markers pTA248 (Xa21); RG136 (xa13); RG556, xa5SF/xa5SR (xa5); and Xa4F/Xa4R (Xa4).
In 2013-2014, F 1 seeds were generated by crossing CARI Dhan 5 with IRBB 60.The BB-resistant F 1 plants were confirmed based on hybridity for morphological and molecular markers of four resistance genes and were then pollinated by CARI Dhan 5 during the period 2014-2015 and 356 seeds of BC 1 F 1 were obtained (Table 2).The progenies were thoroughly screened for BB resistance and the presence of linked genes through foreground selection, and BC 2 F 1 crosses were attempted accordingly.In the Kharif season of 2015-2016, a total of 79 BC 2 F 1 plants were screened using foreground selection, and only the plants exhibiting the presence of BB resistance genes were again pollinated by CARI Dhan 5 to obtain BC 3 F 1 seeds.In the year 2016-2017, a total of 3,505 BC 3 F 1 plants were transplanted in the field, where foreground selection was carried out in conjunction with selection for CARI Dhan 5 phenotypes.Subsequently, the selected plants were self-pollinated to develop BC 3 F 2 seeds.

Disease screening and marker genotyping of the BC 1 F 1 population
To introgress BB resistance genes into CARI Dhan 5, single F 1 plants were selected and further backcrossed with the recurrent parent.
A total of 356 BC 1 F 1 plants were produced and artificially inoculated with virulent Xoo isolates.Out of these, 24 plants scored highly for resistance to BB disease (Table 2).These twenty-four plants were subjected to foreground selection for the presence of BB resistance genes, namely, Xa4, xa5, xa13, and Xa21, by using corresponding linked markers.The molecular detection of the respective BB resistance genes, Xa4, xa5, xa13, and Xa21, in the BC 1 F 1 plants was carried out, and specific bands were obtained as 19 (150 bp), 24 (300 bp), 16 (490 and 530 bp), and 18 (1,000 bp), respectively.Furthermore, the number of BC 1 F 1 plants with different target resistance gene combinations obtained were as follows: (1) xa5, xa13, and Xa21 ( viz., 15, 17, 46, 86, 88, 113, 118, 131, and 134).Out of the nine BC 1 F 1 plants carrying all four target resistance genes, three plants (17, 46, and 131) exhibited the highest similarity with CARI Dhan 5 and were selected for further back-crossing with the recurrent parent to obtain BC 2 F 1 seeds.

Disease screening and marker genotyping of the BC 2 F 1 population
Out of 79 BC 2 F 1 progenies produced, 18 plants showed a highly resistant reaction to bacterial leaf blight with a score of 1, while 14 plants showed resistant to moderate resistant reaction with a score of 3, as shown in Table 2.The 18 highly resistant plants were subjected to foreground selection for the presence of Xa4, xa5, xa13, and Xa21 genes by using corresponding linked markers.In BC 2 F 1 generation, the number of plants obtained with individual and different combinations of BB resistance genes were as [15 (Xa4), 17 (xa5), 12 (xa13), 4 (Xa21)] and [5 (Xa4, xa5, and xa13), 4 (Xa21, xa13, xa5 and Xa4)] (Figure S1).Out of four BC 2 F 1 plant progenies, three progenies exhibited the amplification of all four resistance genes, and therefore, BC 2 F 1 plants were pollinated by CARI Dhan 5 to generate BC 3 F 1 seeds.

Disease screening and marker genotyping of the BC 3 F 1 population
A total of 3,505 BC 3 F 1 backcross derivative progenies were produced by backcrossing the plants showing maximum recurrent genomes with the recipient parent, CARI Dhan5.Out of these, 62 plants showed high resistance to bacterial leaf blight with a score of 1 and were subjected to foreground selection for the presence of Xa4, xa5, xa13, and Xa21 genes to identify plants that were homozygous for different resistant genes or their combinations using resistant gene linked markers (Table 2).In the

Discussion
Salinity is a major abiotic stress for rice crops in coastal regions and the situation is expected to worsen due to climate change and the increasing number of irrigation networks.CARI Dhan 5, a somaclonal variant of the legendary rice landrace Pokkali, is a widely cultivated rice variety in the Andaman and Nicobar Islands due to its high yield and remarkable tolerance to coastal saline and acid sulfate soils (Mandal et al., 1999;Singh et al., 2020).However, CARI Dhan 5 is susceptible to BB disease caused by X. oryzae pv.oryzae, which is one of the most devastating diseases of rice.Being a tropical island, the hot and humid climate of the Andaman and Nicobar Islands provides congenial conditions for the completion of the disease triangle.In our previous studies, we conducted virulence profiling of Xoo isolates from the Andaman Islands, which revealed the prevalence of seven pathotypes of X. oryzae pv.oryzae belonging to two different clonal complexes (Sakthivel et al., 2021).Among these, pathotypes VI and VII were found to be highly virulent.Our analysis using multilocus sequence typing based on nucleotide sequence polymorphism in nine housekeeping genes, dnaK, fyuA, gyrB (two loci), rpoD, fusA, gapA, gltA, and lepA, further revealed that Xoo strains infecting rice in the Andaman and Nicobar Islands comprise a mixture of isolates representing mainland India and the islands, which might be due to trans-boundary movement of the BB pathogen through rice seeds (Sakthivel et al., 2021).
A combination of Xa4+xa5+xa13+Xa21 has been found to confer broad-spectrum resistance to BB disease caused by different strains of Xoo reported from different parts of the world.17-1-69-55 Moreover, studies conducted over the years and in multiple locations have demonstrated that the resistance imparted by this gene combination does not depict any sign of breakdown, making it a promising approach to combating the disease (Shanti and Shenoy, 2005;Pradhan et al., 2015).Therefore, in this study, we aimed to introgress these four BB resistance genes (Xa21, xa13, xa5, and Xa4) into the genetic background of CARI Dhan 5, a coastal salinitytolerant rice cultivar, using marker-assisted breeding.
The genetic background of the recipient parent is known to play a significant role in the successful integration and expression of gene pyramids.In this context, our previous research on five distinct genetic backgrounds carrying xa5+xa13+Xa21 combinations revealed that the rice lines with IMP ASD16/60 and Improved Samba Mahsuri imparted complete resistance against all pathotypes of Andaman (Sakthivel et al., 2017).Fortunately, there are several resistance genes that confer resistance against Xoo.Notably, Xa21, xa13, and xa5 have been reported to impart strong levels of resistance against Xoo strains prevalent in India (Goel et al., 1998;Shanti et al., 2001;Joseph et al., 2004).Xa21 and Xa4 are well-studied dominant resistance genes encoding receptorlike kinases (RLKs) (Song et al., 1995;Leach et al., 2001), while the Xa21 protein contains a nucleotide-binding site and leucine-rich repeat (NBS-LRR) domain (Song et al., 1995) and Xa4 protein belongs to the category of wall-associated kinases (WAK) family (Leach et al., 2001).These two genes exhibit complete dominance, function independently, and are cumulative in nature, indicating their crucial role in two distinct pathways of resistance (Li et al., 2001).On the other hand, the xa13 gene is a recessive resistance allele of the susceptibility gene Os-8N3 (the rice homolog of the nodulin MtN3) (Chu et al., 2006;Antony et al., 2010).The allele xa13 contains a mutation in the promoter region that inhibits binding of the transcription factor and subsequent expression of the susceptibility gene (Antony et al., 2010).Similar to xa13, xa5 is also a recessive resistance gene that encodes a variant form of the basal transcription factor TFIIAg.This variant form, resulting from a missense mutation, cannot bind to the promoter region of the susceptibility locus, providing resistance to Xoo (Han et al., 2020).Together, the two dominant resistance genes Xa4 and Xa21 confer vertical resistance, making the plant nearly immune, while the two recessive genes xa13 and xa5 offer strong horizontal resistance, which is durable in nature.
The DUS characterization of pyramided lines for 41 agromorphological and descriptor traits confirmed that the advanced elite lines recovered the genetic background of CARI Dhan 5 in addition to BB resistance (refer to Table S6 for details).It was imperative to confirm the retention of salinity tolerance in the ILs as compared to CARI Dhan 5. Based on the phenotypic scoring method, we found the ILs, viz., 17-1-69-55, 17-1-69-179, 17-1-69-334,131-2-175-1196, and131-2-175-1197, as BB resistant (disease score 1) as well as salt tolerant (salinity score 1).Further evaluation under the saline micro-plot facility (EC 5.0 dSm −1 ) revealed that the pyramided line, viz., 17-1-69-334, exhibited a consistent score of 1 (resistant/tolerant) for both BB and salinity stresses across years and locations, with comparable grain yield performance of CARI Dhan 5. Overall, the results indicate that the salt-tolerant lines, viz., 17-1-69-179, 46-3-95-655, 131-2-190-1197, 131-2-175-1209, and 131-2-175-1239, also demonstrated complete resistance to BB disease.In view of their potential for dual attributes, these promising derivatives are being nominated for multi-location yield trials, under the All-India Coordinated Rice Improvement Program with their potential for release as BB-resistant and salt-tolerant varieties for cultivation in BB-prone and coastal saline conditions.

Conclusion
High salt concentrations in soils can cause a significant decline in rice yield and quality, and in some cases, it may even lead to complete crop failure (Xiao et al., 2020).CARI Dhan 5, a somaclonal variationderived semi-dwarf variety from salt-tolerant rice landrace Pokkali in ICAR-CIARI, Port Blair, is a preferred rice variety in Andaman and Nicobar Islands due to its high yield, favorable grain quality, and high salinity tolerance.However, it is susceptible to BB disease, which is prevalent in the island's environmental conditions.The present study endeavored to use marker-assisted backcross breeding to pyramid four BB resistance genes (Xa4, xa5, xa13, and Xa21) in the background of CARI Dhan 5. Consequently, five stabilized rice lines combining salt tolerance with BB resistance have been developed in the genetic background of CARI Dhan 5.The development of these improved CARI Dhan 5 lines is expected to have a strong and favorable impact on eco-friendly rice cultivation in the geographically remote Andaman and Nicobar Islands, which are affected by coastal salinity and BB disease.

TABLE 3
Bacterial blight disease reaction of parental and pyramided lines against Xoo strains over the years and locations along with salinity score.

TABLE 1
Microsatellite markers used between CARI Dhan 5 and IRBB60 for BB resistance genes in foreground selection under study.

TABLE 2
Number and nature of breeding lines developed in a cross CARI Dhan 5 x IRBB60 over different filial generations.

Table
).The BC 3 F 1 derivative progenies carrying all four resistance genes, i.