Marker-Assisted Improvement for Durable Bacterial Blight Resistance in Aromatic Rice Cultivar HUR 917 Popular in Eastern Parts of India

Bacterial blight (BB) is a devastating disease of rice in the tropics of Indian sub-continent, where the presence of Xoo races with varying levels of genetic diversity and virulence renders disease management extremely challenging. In this context, marker-assisted improvement of plant resistance has been proven as one of the most promising approaches for the development of sustainable rice cultivars. The present study demonstrates the marker-assisted introgression of the three BB resistant genes (Xa21 + xa13 + xa5) into the background of HUR 917, a popular aromatic short grain (ASG) rice cultivar in India. The performance of the resulting improved products (near isogenic lines (NILs), HR 23-5-37-83-5, HR 23-5-37-121-10, HR 23-5-37-121-14, HR 23-65-6-191-13, HR 23-65-6-237-2, HR 23-65-6-258-10 and HR 23-65-6-258-21) establishes the utility of marker-assisted selection (MAS) approach for accelerated trait introgression in rice. The MAS-bred lines carrying three introgressed genes showed broad spectrum BB resistance (lesion length, LL of 1.06 ± 1.35 cm to 4.61 ± 0.87 cm). Besides, these improved lines showed the complete product profile of recurrent parent HUR 917 along with the enhanced level of durable BB resistance. The improved introgression lines with durable BB resistance would contribute to sustainable rice production in India, particularly in the Indo-Gangetic plane that has substantial acreage under HUR 917.


Introduction
Rice has the global identity as staple food crop for more than half of the world population with substantial acreage (167.13 mha) and production (782.0 million tons) [1]. In India, it is grown on~44.5 mha area producing 172.5 million tons with an average productivity of 3.88 tons per hectare [1] which is directly linked to the livelihood and economy. Global rice production should increase by 40% to feed the world population by 2030 [2]. The sustainability of the global rice production is challenged by several factors, of which growing vulnerability of the crop to changing pest pathogen dynamics remains crucial [2,3]. More than 40 types of biotic and abiotic stresses threaten rice cultivation around the globe. The key biotic stresses of rice include diseases caused by various fungi, bacteria, nematode and viruses that impact upon almost every stage of the crop growth and development [3]. Amongst these diseases, bacterial blight (BB) (Xanthomonas oryzae pv. Oryzae), blast caused (Magnaporthe oryzae) and sheath blight (ShB) (Rhizoctonia solani) substantially reduce rice yield in addition to deteriorating the quality of the rice grain [3].
Indian sub-continent is well known for its native wealth of basmati and aromatic nonbasmati rice, of which aromatic short grain (ASG) types remain important with respect to aroma, cooking and quality traits. These traits are responsible for greater consumer preference globally and fetching premium prices in domestic as well as in global markets [4,5]. Unfortunately, low yielding capacity and susceptibility to various pests and diseases render the deployment of ASG types in rice breeding challenging. Non-basmati scented rice varieties are primarily grown in the Indo-Gangetic region of India including states like Uttar Pradesh, Bihar, and Chhattisgarh. The HUR 917, a popular ASG type rice variety of medium duration, has raised much hope amongst farmers and rice-exporters owing to its superiority over other preferred aromatic non-basmati rice [5,6]. However, the susceptibility of this rice variety to BB contributes to inconsistent yield.
BB is a devastating disease across rice ecology (temperate to tropical) and territory, causing substantial losses (20-100%) in rice yield [7,8]. Disease symptoms appear in the form of leaf blight and Kresek (wilt phage). The warm temperatures (25-30 • C), high humidity, heavy rainfall and water stagnation coupled with heavy nitrogen dose [9] promote disease occurrence. Besides, luxuriant crop growth, close spacing and winds injury also make rice crop prone to BB spread [10]. The Xoo inoculum enters through injured plant epidermis and perpetuates in the xylem vessels. The pathogenic Xoo races, highly dynamic and diverse in nature [11][12][13], produce a variety of toxic secretions including extracellular polysaccharides, extracellular enzyme, iron chelating siderophores and the type III-secretion dependent effectors [14,15] that compromise plant's immune response [16]. To date, globally, 30 races of Xoo have been documented [13,17,18].
Improving plant resistance through incorporation of resistance genes (R genes) is an effective strategy for managing BB in rice [19]. Modern genomic tools and techniques have facilitated mapping and more importantly, the stacking of R genes into elite agronomic backgrounds having susceptibility the disease [11,[20][21][22][23]. More than 40 genes have been reported in rice that confer BB resistance with varying range of effectiveness against diversified Xoo races [22,24]. Of these, the genes, Xa4, xa5, Xa7, xa13, Xa21 and Xa38 have been most frequently deployed to incorporate BB resistance in rice cultivars [20][21][22]. For instance, the xa5 gene encoding gamma subunit of transcription factor IIA5 (TFIIA) in combination with the genes Xa7 and Xa21 improved BB resistance of the recipient genotypes [20,25,26]. Similarly, the gene xa13 encoding a plasma membrane protein has been reported to confer broad spectrum BB resistance, in complementation with Xa21 against majority of the Indian virulent Xoo races [20,27]. The Xa21 gene coding for an NBS-LRR protein remains the most effective for imparting broad spectrum BB resistance and therefore the Xa21 gene has been extensively used in rice breeding programs [20][21][22]28,29].
The functional complementation of these gene(s) is found to be more effective, and results in comparatively broader spectrum of durable resistance in comparison to the deployment of the single resistance gene [20][21][22]30]. The eastern region of India is the hotspot for BB disease, and the R genes with functional complementarities including Xa21 + xa13 + xa5 + Xa4, Xa21 + xa13 + xa5 and Xa21 + xa13 have been proven successful against the Xoo races [20][21][22]. Therefore, deployment of functional R gene combinations is imperative to extend the durability and resistance levels to counter the evolving virulent races of the pathogen [31,32]. Recent advancements in molecular breeding techniques make it convenient to improve the varieties/lines for desirable traits with great precision and efficiency. Molecular breeding techniques such as MAS, marker-assisted backcrossing (MABC), and genomic selection (GS) have provided strong evidence in support of their efficient use in indirect selection/or to trace the trait of interest in plant breeding programs [20,33].
In view of the above, the current study was undertaken with the objective to transfer R gene combinations with the greatest functional compatibility (Xa21 + xa13 + xa5) into a BBsusceptible yet popular short grain aromatic rice variety HUR 917. The study incorporated broad spectrum BB resistance into the rice variety HUR 917 that is still grown in sizeable acreage in Indo-Gangetic planes of India (Figure 1). + xa5 + Xa4, Xa21 + xa13 + xa5 and Xa21 + xa13 have been proven successful against the Xoo races [20][21][22]. Therefore, deployment of functional R gene combinations is imperative to extend the durability and resistance levels to counter the evolving virulent races of the pathogen [31,32]. Recent advancements in molecular breeding techniques make it convenient to improve the varieties/lines for desirable traits with great precision and efficiency. Molecular breeding techniques such as MAS, marker-assisted backcrossing (MABC), and genomic selection (GS) have provided strong evidence in support of their efficient use in indirect selection/or to trace the trait of interest in plant breeding programs [20,33].
In view of the above, the current study was undertaken with the objective to transfer R gene combinations with the greatest functional compatibility (Xa21 + xa13 + xa5) into a BB-susceptible yet popular short grain aromatic rice variety HUR 917. The study incorporated broad spectrum BB resistance into the rice variety HUR 917 that is still grown in sizeable acreage in Indo-Gangetic planes of India (Figure 1).

Experimental Materials and Breeding Strategy
We selected IRBB66 carrying five BB resistance genes (Xa21 + xa13 + Xa7 + xa5 + Xa4) and having closest genetic relationship (0.77) with the recurrent parent (RP) for markeraided improvement of BB resistance level of the popular variety HUR 917 (Tables 1 and S1, Supplementary Figure 1) [34]. Hybridity of the individual F1 plants generated from the cross HUR 917/IRBB66 was confirmed with the DNA markers (pTA248, xa13prom and RM122) [3,7,20,35,36,37] (Table 2). The true hybrids were backcrossed with the RP to generate BC1F1 seeds. Advancement of target gene(s)/traits with maximum RP genome recovery was assessed in every back-cross (BC) generation with linked and informative DNA markers, followed by phenotyping. The best BC1F1 plants with maximum RP genome and phenome were advanced to the next generation. The BC2F1 plants were also subjected to foreground selection (FS), background selection (BS) and phenotyping to identify the plants with maximum recovery for RP genome and phenome (Tables 3 and S2). Selection differential (Δd) analysis for the product profile traits, days to fifty percent flowering

Experimental Materials and Breeding Strategy
We selected IRBB66 carrying five BB resistance genes (Xa21 + xa13 + Xa7 + xa5 + Xa4) and having closest genetic relationship (0.77) with the recurrent parent (RP) for markeraided improvement of BB resistance level of the popular variety HUR 917 (Table 1 and  Table S1, Supplementary Figure S1) [34]. Hybridity of the individual F 1 plants generated from the cross HUR 917/IRBB66 was confirmed with the DNA markers (pTA248, xa13prom and RM122) [3,7,20,[35][36][37] ( Table 2). The true hybrids were backcrossed with the RP to generate BC 1 F 1 seeds. Advancement of target gene(s)/traits with maximum RP genome recovery was assessed in every back-cross (BC) generation with linked and informative DNA markers, followed by phenotyping. The best BC 1 F 1 plants with maximum RP genome and phenome were advanced to the next generation. The BC 2 F 1 plants were also subjected to foreground selection (FS), background selection (BS) and phenotyping to identify the plants with maximum recovery for RP genome and phenome (Table 3 and Table S2). Selection differential (∆d) analysis for the product profile traits, days to fifty percent flowering (DFF), plant height (PH), grain L/B ratio, head rice recovery (HRR) and aroma content was performed in BC and segregating generation (Supplementary Table S3). The percent disease index (PDI) and area under disease progress curve (AUDPC) analysis was done to recover BB resistant NILs (Supplementary Table S3). The positive BC 2 F 1 with perfect product profile traits were advanced to BC 2 F 2 generation, and FS was repeated in order to identify the plants that were homozygous for Xa21 + xa13 + xa5 gene combination (Table 3). Further, desirable plants were advanced with single seed descent (SSD) under field using rapid generation advance (RGA) strategy to accelerate development of the NILs [36].

PCR and Marker Analysis
The genomic DNA from the leaf sample of each plant (20-22 days old seedling) was extracted and purified using CTAB method [39]. The PCR reaction was carried out using 15 ng of template DNA, 1× Taq assay buffer, 0.3 mM of MgCl 2 , 133.0 µM of dNTPs, 1 U/µL of Taq DNA polymerase (Thermo-scientific, Life Science Products, Mumbai, Maharashtra, India) and 1.25 µM of each primer (Eurofins, Genomics, Hyderabad, India). The PCR was carried out in an Eppendorf Thermo Cycler with the following program: (1) initial denaturation at 94 • C for 3 min; (2) 39 cycles for denaturation for 30 s at 94 • C, annealing for 30 s at 56 • C, extension for 1 min at 72 • C; and (3) final extension at 72 • C for 5 min. The amplified products were resolved with 2.5% Metaphor TM Agarose gel (Typhoon FLA 700, Alpha Innotech, MA, USA) and visualized under a UV light source in photographed gel documentation (Gel-Doc) system (Gel Doc TM XR, Bio-Rad Laboratories Inc., Hercules, CA, USA).

Disease Bioassay Analysis
Each BC generations and NILs (BC 2 F 3 ) carrying effective hetero/homo-alleliec combinations of R genes were grown in field condition along with parents and bio-assayed with eight virulent Xoo pathotypes/races (Xa17, Xa7, xa2, Xb7, Xc4, xd1, xa1 and xa5) prevalent in the eastern region of the country. The pathotypes were maintained in peptone sucrose agar (PSA) medium [42] and single spore culture with 10 8 cfu/mL bacterial Xoo suspension density was used for inoculation. The top five leaves of each plant were clipped off and inoculated [43]. Post inoculation, the plants were observed after every 24 hours' time interval to note the appearance of disease symptoms. The LL were measured at 14, 21 and 28 days after inoculation (DAI) [13] using a disease score index of 0-9 [44,45]. The LL of <5 cm was considered resistant (R), 5-10 cm was considered moderately resistant (MR), 10-15 cm was moderately susceptible and >15 cm was considered highly susceptible. The epidemiological parameters like PDI and AUDPC which indicates overtime disease accumulation were calculated to assess the disease severity [9].

Agro-Morpho Evaluation of NILs
The BC 2 F 3 and BC 2 F 4 NILs along with RP and donor parents were evaluated for yield and other agro-morphological traits in randomized complete block design (RCBD) with three replications. Standard agronomic management practices were followed for raising the rice crop at two locations (ICAR-National Rice Research Institute, Cuttack and Banaras Hindu University, Uttar Pradesh) in India. The data were recorded on five plants from each of the entries for the characters namely: days to panicle initiation (DPI), days to first panicle emergence (DFPE), days to 50% flowering (DFF), days to maturity (DM), plant height (PH), number of effective tillers per plant (NETPP), panicle length (PL), number of grains per panicle (NGP), test weight (TW), grain yield per plant (GYPP), and disease severity (DS). Further, the lines were also analyzed for grain and cooking quality parameters such as head rice recovery (HRR) [46], kernel length before cooking (KLBC), kernel breadth before cooking (KBBC), length/breadth ratio (L/B), amylose content (AC), GC content [47] and aroma as described by [48]. The statistical analysis was performed using DARwin-6.0, XLSTAT and SPSS packages. Use of plant material complies with relevant institutional, national, and international guidelines and legislation.  Tables S1 and S2). The diversity analysis revealed varying degrees of genetic relatedness (0.06 to 0.77) among the parents (Supplementary Table S1 and Figure S1). The donor genotype, IRBB66 (Xa21 + xa13 + Xa7 + xa5 + Xa4) showed closest genetic relation with RP (0.77) followed by IR 64-MAS (Xa21 + xa13 + xa5 + Xa4) (0.76) and CRMS 31B-MAS (Xa21 + xa13 + xa5 + Xa4) (0.50). The bioassay analysis of IRBB 66 also showed incompatible or HR disease reaction (PDI of 3.65 ± 0.110, AUDPC of 74.13 and LL of 0.57 ± 0.21 cm to 1.83 ± 0.35 cm) (Tables 4 and S3). To avoid undesirable linkage drag and allow a rapid fixation of segregating loci, the genotype IRBB66 [22] was used as a donor in the present backcross scheme.

Genome Introgression on the Carrier and Non-Carrier Chromosomes
The success of the trait improvement strategy relies upon the extent of the RP genome recovery in the resulting products while retaining originality of the RP. Our results suggested an average of 95.17% RP genome recovery in the resulting pyramided lines, representing genomic segments from the chromosomes 1, 2, 4, 5, 6, 7, 9, 10 and 12 (Table 3 and

Morphological Evaluation of NILs
The phenotyping data of selected plants in backcross generations were recorded progressing but least selection differential (∆d) for most of the product profile traits like, days to 50% flowering (DFF) (2.12), plant height (PH) (1.68), grain L/B ratio (0.15) and head rice recovery (HRR) (1.53) (Supplementary Table S3). Analysis of variance (ANOVA) among NILs and parents showed substantial variances (at p ≤ 0.001) in all studied traits (  Figures 4, 5, S5 and S6). These selected BC 2 F 4 NILs were subjected to robust phenotyping for grain quality, and the results revealed similar quality as of RP with selection differentials: ∆d = 0.11 (Grain L/B ration), ∆d = 0.72 (HRR) and ∆d = 0.0 (Aroma) ( Table 6, Tables S3 and S6). The majority of the three resistance genes containing NILs were similar to the RP for the basic agro-morpho and quality traits ( Figure 6). Furthermore, genetic relatedness amongst parents and NILs were assessed based on similarity metrics data of 82 SSR markers, which ranged from 0.06 to 0.94 (data not presented). The entire 17 genotypes (including parents) could be distinguished into 2 two major clusters, cluster I-A consisted of only one genotype i.e., IRBB66 with similarity coefficient value of 0.77. Whereas cluster-II consisted of RP and 15 NILs derivatives with carrying triplet homoalleles of R genes (Xa21 + xa13 + xa5) and had more than 90% RP genome achieved complete product profile as of HUR 917 (Table 6 and Supplementary Figure S2). The HR 23-5-37-121-10, HR 23-65-6-258-21 and HR 23-5-37-121-14 were found to have similar 1000-grain weight (Test weight), HRR, AC content and aroma as of RP which are the most important product profile trait needed to be recovered in this experiment (Table 6, Supplementary Table S6). The quality assessment of NILs suggested that the introgressed R gene combination had similar quality parameters including grain type (Table 6 and Supplementary Figure S6) HRR (53.6% to 79.2%), AC content (20.09% to 24.12%), and GC content (31 to 59) ( Table 5). It is important to note that the aroma (BADH2), which was fixed in early generation, was retained in the NILs (Supplementary Figure S4). Besides, sensory evaluation results revealed that there was no significant difference between RP and NILs with respect to aroma. (Supplementary Table S5).

Genome Introgression on the Carrier and Non-Carrier Chromosomes
The success of the trait improvement strategy relies upon the extent of the RP genome recovery in the resulting products while retaining originality of the RP. Our results suggested an average of 95.17% RP genome recovery in the resulting pyramided lines, representing genomic segments from the chromosomes 1, 2, 4, 5, 6, 7, 9, 10 and 12 (Table 3 and Figure 5). The R genes (Xa21, xa13 and xa5)

Morphological Evaluation of NILs
The phenotyping data of selected plants in backcross generations were recorded progressing but least selection differential (Δd) for most of the product profile traits like, days to 50% flowering (DFF) (2.12), plant height (PH) (1.68), grain L/B ratio (0.15) and head rice recovery (HRR) (1.53) (Supplementary Table S3). Analysis of variance (ANOVA) among

Discussion
Non-basmati ASG rice with exclusivity to aroma, has excellent cooking quality traits, owing to which it attracts global consumer preference and premium prices in the international markets [4,5]. It is also a reservoir of nutritionally important traits. Research aiming to improve nutrients like Zn, Fe, vitamins and antioxidants by utilizing this genetic resource has opened the new avenues for rice export [50]. Despite having comparatively low amylose content, ASG lines provide better quality and aroma than basmati types [51]. In addition, this genetic resource retains substantial aroma even under warmer conditions [6]. Widespread preference for the ASG variety HUR 917 faces some challenges due to its less yielding capacity [52], vulnerability against stresses [53] and lodging [54]. The increased BB susceptibility of this variety has caused inconsistency in its yield. Enhancing resistance level of the host plant presents a standard practice to mitigate the risks of disease and pest attacks [20][21][22]54]. Evidence suggests that stacking of more than one R gene resulting in quantitative complementation or synergistic response, improves the resistance level of plants [55]. In recent years, advanced genomic tools [5,33,54,56] have made significant contributions to trait improvement in plant breeding. Several R genes conferring durable resistance against major biotic stresses have been successfully incorporated in basmati and non-basmati rice backgrounds [18,[20][21][22]48,57].
In this study, the genotype IRBB 66 (a derivative of IR 24) has high level of resistance against hyper-virulent Xoo pathotypes owing to the presence of five R genes (Table 4 and Figure 4). We selected IRBB 66 as a donor for MABC scheme because of its highest genetic proximity to the RP ( Figure 2) and similarity in quality traits of both [20]. The F1s with heteroalleles of effective R gene combinations (Xa21+ Xa7 + Xa4) had resistant reaction, however, backcross progenies with only Xa21 gene had moderately resistant reaction. The improved lines did not show any linkage drag from the donor genomic segments (Figures 5 and 6), which might be due to genetic closeness among donor and RP genotypes. DMRT and biplot analysis revealed perfect recovery of product profile and value-added traits in the derivatives (Table 6 and Figure 6) [36]. Stepwise BS analysis coupled with robust phenotyping led to a substantial increase in the RP genome (94.51% in BC 2 F 2 and 96.95% in BC 2 F 4 * ) [21,22]. The majority of the NILs carrying three R genes (Xa21 + xa13 + xa5), were found to be morphologically similar (duration, grain, yield and quality parameters) to RP with least selection differential (>0.90 similarity index, data not presented) ( Table 3, Table 6 and Table S6 and Supplementary Figure S6) and at par resistance with the donor, IRBB66 (Table 4). Phenotyping in combination with MAS in backcross generations was proven equally important as this strategy helped fast-track the transfer of genomic regions of functional relevance. Whereas SSR markers employed in the BS usually target the non-coding and heterochromatic regions and therefore may not be suitable to quantify the recovery of functional part of the genome [21]. (*data are not presented).
Notably, selection differentials for most of the desirable traits changed progressively throughout BC and segregating generations. No change in aroma, a key parameter, was reported in the BC 1 F 1 generation (Score-02) (Supplementary Table S5 and Supplementary Figure S4). Intensive selection caused fixing of the aroma trait (a key trait in ASG governed by the two recessive genes badh1 and badh2) during the early generations [58][59][60][61]. The sensory panel test revealed no substantial differences for aroma in RP and the derivatives, which validated the successful recovery of all major gene(s) responsible for fragrance i.e., 2-acetyl-1-pyrroline (2AP) in rice. The targeted product profile in NILs was attained since most of the traits considered in the study were attributed to a number of small effect genes/QTLs.
Adoption of limited backcrosses in this study found to have substantial edge over burden of more backcrosses as our rapid backcross scheme accelerated accumulation of desirable genes (minor) in derivatives. Thus, inclusion of genetically close parents in MAS not only reduces the breeding cycle, but it can also contribute to enhance response to selection for important traits in later stage of the population [21,62,63].
The bioassay of NILs confirmed the successful introgression of the R genes, and the lines manifested incompatible disease response against all virulent Xoo pathotypes considered in the study. The improved introgression lines with two or more effective R gene combinations showed resistance levels comparable to that of donor, which validated the efficacy of the introgression strategy followed in the current study.

Conclusions
Genomics-aided trait improvement to combat BB disease is an environmentally-sound and economically-viable strategy to achieve sustainable rice production worldwide. Prevalence of genetically diverse virulent Xoo strains over agro-climatic zone renders this disease extremely challenging to manage. The results of this study revealed that three R genes (xa5 + xa13 + Xa21) in combination could impart broad spectrum BB resistance. We demonstrated the precise stacking of R genes into the background of the popular rice variety HUR 917.