Rice is the most extensively studied crop among cereals because of its global significance as a food crop being the main staple food for more than half of the world’s population. In Asia, 60% of the earth’s people live where 90% of the world’s rice is grown and 3 billion Asians get 35-75% of their calories from rice [1]. As the global population is anticipated to increase to 9.77 billion by 2050, rice production needs to be increased by twofold from the current levels to ensure both global food and nutritional security. Either expanding the area of cultivation, and productivity or by reducing the yield losses can enhance the total rice production. According to global research, the unavailability of arable land shows no possibility of expanding the area. Therefore, the only practicable way to boost productivity and overall production is by avoiding losses due to adverse environmental factors and post-harvest losses [2].

Rice productivity is constrained by several factors among which diseases play a significant role in limiting rice yields. Among the biotic stress, Bacterial Leaf Blight and Blast are the two major diseases causing considerable yield losses in rice. Bacterial Leaf Blight caused by the gram-negative bacterium Xanthomonas oryzae pv. oryzae (Xoo) is a devastating disease of rice. It infects rice at the maximum tillering stage, leading to water-soaking lesions (blighting) on the leaves, which enlarge and constrict the photosynthetic area of the crop leading to incomplete grain filling resulting in poor yield.  Earlier studies reported that it results in yield losses ranging from 74 to 81% [3]. A total of 46 BLB resistance genes (R genes) have been reported [4], 11 of which have been cloned, characterized, and designated in series from Xa1 to Xa46 [5]. Among these broad-spectrum resistance genes, Xa21 belongs to the nucleotide-binding site leucine-rich repeat (NBS-LRR) class identified in the Wild species of rice Oryza longstaminata. Through inter-specific hybridization Xa21 located on chromosome 11 was transferred to Oryza sativa background [6]. Broad spectrum BB-R gene Xa21 was introgressed in the background of Basmati-385, an indica rice cultivar using IRBB59 as donor parent [7].

Among the fungal diseases of rice, blast caused by the fungus Magnaporthe oryzae (anamorph Pyricularia oryzae), is a destructive disease, which leads to significant yield losses up to 70-80% depending on its severity. Pathogens can infect at all growth stages causing symptoms on leaves, panicles, and culm reducing the photosynthetic efficiency and yield. More than 101 blast resistance (R) genes and about 500 quantitative trait loci (QTLs) have been identified. Out of them, 38 resistant genes have been cloned and characterized. Among these thirty-eight genes, eight genes viz., Pi9, Pi54, Pi50, Pi7, pi21, Pigm, Pi57, and Ptr were reported to have broad-spectrum blast resistance [2]. The first blast-resistant gene to be cloned was Pib [8] then Pita [9] and the third gene to be cloned was Pi54 at NRCPB, New Delhi [10]. Pi54 was identified in a highly resistant variety Tetep located on chromosome 11 has shown broad-spectrum resistance against predominant races found in India [11]. Three BB resistance genes xa5, xa13, and Xa21, and two blast resistance genes Pi9 and Pi54, were combined in the genetic background of the elite Indian rice cultivar Krishna Hamsa [12].

Though chemical control methods are available for the management of both diseases, this method not environmentally friendly. The long-term usage of chemicals stimulates tolerance and evolution of pests and pathogens. Marker-assisted breeding is an effective strategy to develop lines that can offer durable resistance in less time compared to conventional breeding. The present study was undertaken to develop breeding lines similar to RNR 15048 (Telangana Sona) with durable resistance to BB (Xa21) and blast (Pi54) diseases through marker-assisted pedigree breeding.

Materials and Methods

A high-yielding variety of Telangana i.e., RNR 15048 (Telangana Sona) having 125 days duration developed from the cross between MTU1010 and JGL3855 by Rice Research Center, Agricultural Research Institute, Rajendranagar, Hyderabad, PJTSAU, Telangana. It is possessing a desirable, short-slender grain type having a low glycemic index with good cooking quality but no durable resistance to blast disease, moderately susceptible to bacterial blight disease and weak culm hence attempts were made to improve this variety using as female parent and using MTU1010 NIL with strong culm and Improved Samba Mahsuri (ISM) NIL possessing Xa21 and Pi54 as donor parent. Near isogenic lines of Improved Samba Mahsuri were developed from the cross between Improved Samba Mahsuri (xa5, xa13, and Xa21) and RPBio Patho-1(Pi2) and RPBio Patho-2 (Pi54) possessing Xa21 and Pi54 genes by ICAR-Indian Institute of Rice Research, Hyderabad[13] and MTU1010 NIL having long-slender grain type possessing strong culm were used as male parents for transferring resistance genes against bacterial leaf blight and blast resistance along with culm strength to RNR 15048 through limited marker-assisted pedigree breeding.

During Rabi, 2020-2021, RNR 15048 was crossed with MTU1010 NIL, possessing a strong culm trait and F₁s were generated. During Kharif, 2021 the generated F₁s were checked for culm strength using a prostrate tester and culm diameter using vernier calipers, those found phenotypically superior with strong culm (RNR-15048 x MTU1010 NIL) were selected and were crossed with ISM NIL possessing Xa21+Pi54 genes (RNR-15048 x MTU1010 NIL//ISM NIL) for BB and blast resistance respectively. The true F₁s were confirmed for Xa21 and Pi54 andevaluated for culm strength using a prostrate tester during Rabi, 2021-2022. True F₁s were selfed and forwarded to the F₂ generation.

 Staggered sowings of the parents were undertaken at an interval of ten days to ensure synchronous flowering to produce adequate crossed seed. Thirty days old seedlings were transplanted to the field at a spacing of 15×15 cm. Emasculation was done by clipping method. The top 1/3^rd portion of each floret was clipped with scissors and anthers were removed using forceps without damaging stigma and pollination was done the next day morning. Foreground selection was done for the targeted genes i.e., bacterial blight resistance gene, Xa21, using co-dominant marker, pTA248 [14], and the blast resistance gene, Pi54, using co-dominant marker Pi54MAS[15]. In F₁s and F₂s artificial inoculation was carried out using clipping method[16] and plants were screened for BB resistance. The F₁ and F₂ lines along with parents were screened for culm strength and culm diameter [17]. At the fully ripened stage prostrate tester (Daiki Rika Kogyo Co., Tokyo, Japan) was set perpendicular to the middle of the plant from 20 to 25 cm above the ground and the plant was pushed at an angle of 45˚ to measure the pushing resistance and culm diameter was measured at 2nd internode from the bottom at 20 days after heading using vernier calipers. 

Plant DNA was isolated using CTAB (Cetyl Tri Methyl Ammonium Bromide) method[18]. The genomic DNA was extracted from individual plants in F₁, and F₂ generations along with the parents. The fresh, healthy, and young leaf tissue from seedlings was collected from the plants in a labelled plastic cover kept in a freezer pack and stored at -80℃ until used for DNA extraction. The quality and quantity of the genomic DNA was assessed by using 0.8% agarose gel.

PCR amplification was carried out using 10 µl volume having 2 µl Template DNA having 40-50ng of DNA, 3 µl of PCR Master mix (Taq DNA polymerase, dNTPs, MgCl2, optimized buffer, gel loading dye (green), and a density reagent), 0.5 µl of Forward primer and 0.5 µl of Reverse primer and 4 µl of sterile water. The PCR condition was with one cycle of denaturation at 94˚C for 5 min, followed by 35 cycles at 94˚C for 30 sec, 55˚C for 30 sec, and 72˚C for 1 min, and with a final extension at 72˚C for 7 minutes. The PCR product was resolved in 1.5% Agarose gel using a standard DNA ladder (100-1000 bp). The banding pattern was observed and recorded using a gel documentation unit (Alpha Innotech, USA).

F₂ plants along with the parents were evaluated for resistance against bacterial blight using a virulent isolate of the bacterial blight pathogen, Xanthomonas oryzae pv. oryzae (Xoo) DX-020. The bacterial culture of the virulent isolate was maintained on Hayward’s agar media at 28℃ for 96 hours. After the incubation period, the bacterial cells were harvested and diluted with 10 ml of sterile distilled water to get a final concentration of approximately 10⁸ cfu/ml [19]. The leaf clipping method was followed for inoculation of bacterial culture at the maximum tillering stage (45-55 days after transplanting). The top 1-3cm of completely developed leaves were clipped off one by one by sterilized scissors dipped in the bacterial suspension. Approximately 5-10 leaves were inoculated per plant. The lesion lengths on the inoculated leaves were measured fifteen days after inoculation. The disease score was calculated as per IRRI standard evaluation system (IRRI-SES) scale, which is based on the percent diseased leaf area (IRRI 2014) (Table 1.)

Chi-square(χ2) analysis was done to analyze the segregation pattern of Xa21 and Pi54 in the F₂ population for an appropriate Mendelian ratio of 1:2:1 [20].

       χ²= (Observed number -Expected number)²/Expected number

Results and Discussion

 Rice is a significant staple food and supplies the main energy resource for almost   50% of the world’s population. RNR 15048 is a susceptible parent for BB and blast-resistant genes Xa21 and Pi54 with susceptible alleles. Polymorphism between parents was confirmed by using gene-linked markers pTA248 for the Xa21 gene and Pi54-MAS for the blast resistance gene, Pi54. RNR-15048 was crossed with MTU1010 NIL and 98 F₁s were developed during Rabi, 2020-2021. Culm strength observed in F₁s was 20.5-25.8, RNR 15048-24.0, and MTU 1010 NILs-27.68. 68 F₁s having strong culm were identified and crossed with a breeding line of the variety ISM NIL, possessing Xa21+Pi54 during Kharif, 2021. 92 F₁s were developed from the cross (RNR-15048 x MTU1010 NIL//ISM NIL) and seedlings were raised in the field during Rabi, 2021. Among them, 76 plants were identified to be true F₁sbased on analysis with the gene-specific markers. These heterozygous (Xa21xa21, Pi54pi54) plants were selfed to develop F₂ seed during Rabi, 2021-2022. A total of 2057 F₂ plants were grown in Kharif, 2022. F₂ plants to be subjected to marker-assisted selection was too many, phenotype and genotype-based combined approach was deployed for the identification of resistant F₂s for both the target genes [21] and this process saves time and resources as only a limited number of plants (which are phenotypically resistant) are needed to be subjected for marker-assisted selection. Plants were screened phenotypically for BB through clip inoculation procedure under field conditions using a local virulent isolate of the bacterial blight pathogen, DX020 (Fig. 1). In addition to the F₂ plants, a resistant check variety, ISM and the bacterial blight susceptible parent, RNR 15048 were also screened. Among the 2057 plants, 1428 were observed to be resistant to BB through phenotypic screening. These 1428 plants were then screened genotypically for the Xa21 gene using the pTA248 gene-linked marker and 382 plants were identified to possess the gene in a homozygous condition similar to a resistant parent and 412 plants were identified to possess the gene in a homozygous condition similar to a susceptible parent and 634 plants exhibited heterozygous nature for both alleles. 382 plants possessing the Xa21 gene in homozygous condition were screened for blast resistance gene (Pi54) using Pi54MAS gene-specific marker. 84 plants were identified to possess a gene in a homozygous condition similar to the resistant parent for both Bacterial blight and Blast (Xa21 Xa21Pi54Pi54) and 92 plants were identified to possess a gene in a homozygous condition similar to the susceptible parent (xa21xa21pi54pi54) and 206 plants exhibited heterozygous nature for both alleles (Xa21 xa21Pi54pi54) (Fig. 2 and 3). Chi-square (χ2) analysis indicated that both the genes Xa21 and Pi54 exhibited segregation ratio of 1 Xa21Xa21: 2 Xa21xa21 :1 xa21xa21 and 1 Pi54Pi54: 2 Pi54pi54:1 pi54pi54 respectively. The observations stated that at 2 df in comparison to chi-square critical value 5.991, calculated χ2 values were low representing a 1:2:1 mendelian segregation ratio for both the genes (Table 2). Earlier works reported that the Xa21 gene followed the Mendelian segregation ratio in F₂ population [22]. Similar results for Pi54 were shown for the segregation pattern of blast resistance in BC₃F₂ generation was non-significant and followed Mendelian ratio [23]. Lines with both Xa21 and Pi54 showed broad-spectrum durable resistance.

Evaluation of Agro morphological traits

Nine Agro morphological traits were studied in 84 F₂ population, where considerable variation was observed and for plant height (98-131 cm, RNR 15048-101, ISM NILs-83), days to 50% flowering (91-105 days RNR 15048-93, ISM NILs-105), number of effective tillers per plant (8-13, RNR 15048-8, ISM NILs -11), panicle length (19-27cm, RNR 15048-24, ISM NILs-22), Number of grains per panicle (190-310, RNR 15048-251, ISM NILs-188), 1000-grain weight (14.2-28.9gms, RNR 15048-11.5, ISM NILs-18.6), Grain yield per plant (23-34gms, RNR 15048-20.8, ISM NIL-26.2) and culm strength (24.5-28.2, RNR 15048-24.0, MTU 1010 NILs-27.68) and culm diameter (6.71-5.21 mm, RNR 15048-5.11, MTU 1010 NILs-6.68). This large variation was observed due to the segregation of genes in the population. The superior progeny homozygous positive (Xa21 Xa21Pi54Pi54) for both the target traits Bacterial blight and Blast resistance were forwarded to F₃ generation.

Conclusion

In Telangana state, the PJTSAU released variety i.e., Telangana Sona (RNR 15048), with short slender grain type and higher yield levels became very popular among the farming community because of its good cooking quality and low glycemic index among all the central and state released varieties released so far. But this variety is often becoming susceptible to BB and blast and prone to lodging with weak culm in the majority of the areas. Though, this variety is possessing the pi2 gene conferring resistance to blast, showing no durable resistance in the state of Telangana, for which there is a need to transfer the Pi54 gene to achieve durable resistance to Blast. In this context, the present study was being taken up to develop durable resistant line/s with broad-spectrum resistance for BB and blast along with strong culm, without compromising the yield using the elite variety RNR 15048. Marker-assisted pedigree breeding was used to improve Telangana Sona by transferring two target genes viz., Xa21 for bacterial blight and Pi54 for the blast. These breeding lines with bacterial blight, blast resistance, and strong culm were forwarded to F₃ generation for further studies. In the future, these lines could be used as a donor in the breeding programs and could serve as a new improved variety of Telangana Sona with bacterial blight, blast resistance, and strong culm and perform well in disease-endemic areas of Telangana state.

Acknowledgments: We acknowledge Regional Agricultural Research Station, Jagtial and University authorities, PJTSAU, Telangana, India for providing all the support.

Conflicts of Interest: The authors have no conflicts of interest relevant to this study to disclose.

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Figure 1. Phenotypic screening for bacterial blight resistance using Xoo (DX020) culture. ISM-Resistant check; RNR 15048-Susceptible check; IL-1 to IL-8 -Improved breeding lines.

Figure 2. pTA248 marker segregation pattern in F₂ population for Xa21 gene. L=100bp Ladder, -ve=RNR 15048, +ve=ISM NIL (Xa21 and Pi54)

Figure 3. Pi54MAS marker segregation pattern in F₂ population for Pi54 gene. L=100bp Ladder, -ve=RNR 15048, +ve=ISM NIL (Xa21 and Pi54)

Table 1. Standard Evaluation System (SES) scale for evaluation of breeding lines for bacterial leaf blight of rice. (IRRI, 2014)

Table 2. Analysis of genetic segregation pattern of Xa21 and Pi54 gene in the F₂ population using chi-square (χ2) test.

     ns: non-significant at a 5% statistical level.