Marker assisted introgression of bacterial blight resistance gene into NR 9 (Sairam) rice variety

Introduction

Rice (Oryza sativa L.) is a major staple crop that supports the food security of more than half of the global population. It is especially crucial in Asia and Africa, where it forms the foundation of daily diets and contributes significantly to economic stability (Khatun et al., 2024) [17] . Bacterial Blight (BB) caused by the Xanthomonas oryzae pv. oryzae (Xoo) is a widespread and devastating disease of rice affecting crops in both temperate and tropical regions and is particularly destructive result in heavy yield loss. (Yugander et al., 2017) [35] . It spreads throughout the vascular system often resulting in reduced yield and quality under BB infection (Chukwu et al., 2019; Mew et al., 1993) [6,22] . Infections during the tillering stage result in total crop failure. resistant varieties, natural cultural practices, biological control and chemical measures are way to tackle BB infection in rice. It has been shown that the bacterial blight gene Xa21 originally derived from Oryza longistaminata is on 11 number chromosome of rice with pTA248 as gene linked marker confers resistance against multiple bacterial blight races by activating defense signaling pathway (Ronald et al., 1992; Peng et al., 2015; Khan et al., 2023) [29,24,15] . The most impactful method for tackling BB is use of resistance varieties (Khush et al., 1989) [18] . Because of the masking effect of genes, linkage drag, and epistasis (i.e., interallelic gene interaction), it would be challenging to choose rice crops with many resistance genes using the traditional method alone. Gene from resistance varieties transferred to susceptible one using marker assisted breeding method and many BB resistant rice varieties and hybrid's parents have been developed using this method that typically reducing varietal development duration upto 4-5 years in compassion with conventional breeding (Chen et [4,9,31,7,1,2,30,25] . NR-9 is a long duration (140-145 days), mid-tall (110-120 cm), non-lodging, high yielding and premium short slender variety released during 2005 and is popular among farmers. Despite its popularity, NR-9 is highly susceptible to bacterial blight disease and sporadic occurrence of BB results in considerable loss to farmers who cultivate NR-9. Therefore, introgression of wide range resistant gene against BB in NR-9 offers an valid solution. In current work, we have successfully introgressed gene (Xa21) into the background of NR-9 to develop near isogenic lines (MAS-14-283 named as NR-1033) that has combined resistance for BB disease with all other features similar to NR-9

Materials and methods

Crossing and marker assisted backcross breeding

In Rabi-2016 crosses taken between NR-9 and MR-6 (Xa21 donor line) at NSPL Paddy Research Station, Dhasai, Murbad, Dist-Thane, Maharashtra, India and the F 1 s were grown during the Kharif 2017 season. True F 1 s were confirmed with the help of foreground marker for the gene Xa21.The true F 1 s were then backcrossed with the recurrent parent NR-9 to generate BC 1 F 1 population developed from cross F 1 with NR-9 were subjected to foreground selection using gene linked marker and background selection using polymorphic markers. A set of 101 SSR markers evenly distributed across 12 chromosomes was used for parental polymorphism analysis to identify informative markers for background selection. BC 2 F 1 population obtained by backcrossing selected BC 1 F 1 plants with Xa21 gene and maximum RPG recovery with NR-9 and the MABB process was continued up to the BC 3 generation (Fig. 1). Selected plants from BC3F1 generation with Xa21 and highest RPG ere selfed to generate BC 3 F 2 s and plants that possessed Xa21 gene in the homozygous condition were identified from BC 3 Inoculation and evaluation of bacterial blight culture response: Donor line, recurrent parent, susceptible check and backcross populations derived from parents were screened using Xanthomonas oryzae pv. oryzae (Xoo) culture which received from IIRR, Hyderabad, India (Supp.table1). The bacterium was grown on wakimotos medium at 27 °C. The growth was scrapped off the plates, resuspended in sterile water and adjusted OD 600 by using colorimeter (CL223, Make-Elico) and the suspension was used as inoculum. At the maximum tillering stage (45 to 55 days after transplanting), the rice plants were clip-inoculated with a bacterial suspension of Xoo strains (Kauffman et al., 1973) [14] . Scissor was dipped in the inoculum (bacterial suspension) and one/fourth of top leaves was clipped through it. Each plant received an inoculation of five to ten leaves, and fourteen days after the inoculation, the disease response was recorded (Fig. 2). The disease incidence was evaluated using the IRRI Standard Evaluation System (SES) scale (table1).

Screening for agro-morphological traits

The agronomic performance was conducted at NSPL Paddy Research Station, Dhasai, Murbad, and Dist-Thane, Maharashtra India (19.260N, 73.390E, and 83 m above MSL). Improved lines of NR-9 were field evaluated for the different agro-morphological traits by reordering data from 5 competitive plants chosen at random. Different traits evaluated as involving days to 1000 grain weight (g), grain number per panicle (no.), plant height (cm), spikelet fertility (%), number of productive tillers (no.), 50% flowering (days), panicle length (cm), grain type and yield (quintal/acre).

Evaluation of the grain quality characters

A laboratory dehuller was used to dehull 200 grams of raw rice kernels in order to assess head rice recovery (HRR) by taking ratio for the whole polished grains weight to raw grains weight (Lapis et al., 2019) [20] . Ten grains from each entry was used to record observations for various grain quality parameter such as Kernel Length After Cooking (KLAC), Kernel Width Before Cooking (KWBC), Elongation Ratio (ER) and length-width ratio (Khanna et al., 2015). The whole milled kernels were selected and soaked for 30 min in 10 ml of distilled water taken in test tubes. The lower portion of the tube containing the rice kernels was then fully submerged for eight to ten minutes after the tubes were dropped into a boiling water bath. Data was recorded after the fried kernels were transferred into a petri plate and allowed to cool at room temperature. Alkali Spreading Value (ASV) is an indicator of gelatinization temperature and cooking texture. Six ground entire kernels were spread out in a petri-plate and immersed in 1.7% KOH for 23 hours at 30°C in order to calculate ASV. The ASV per sample was recorded as per the 17 score scale (Little et al., 1958) [21] . The amylose content of the improved lines was estimated as per standard protocol (Juliano, 1971). All measurements were replicated appropriately to ensure reliability.

Statistical analysis

The experiment was laid out using a Randomized Complete Block Design (RCBD), and the resulting data were subjected to statistical analysis. Agro-morphological observations and phenotypic BB screening data were analyzed following standard statistical procedures, including the calculation of means, significant Standard Error of the Mean (SEM ±), Analysis of Variance (ANOVA), and Least Significant Difference (LSD) at the 5% significance level. All computations related to the significant SEM (±) were performed using MS Excel.

Results and Discussion

BB is the main obstacle to rice cultivation and results in significant yield loss. Given the need for greater rice output in a rapidly expanding population, molecular breeding is important in the fight against BB disease in rice, even though it cannot be used in place of conventional breeding. Through MAS, a number of BB resistance genes have been found, described, integrated, and pyramided. (Fiyaz et [8,6,34,19] . By [34,31,32] . Although Xa21 confers broad-spectrum resistance, monitoring pathogen evolution and pyramiding with additional genes in future programs will be important for durability. BB resistance gene Xa21 was added to the genetic background of NR-9 by combining MABB with phenotype selection. Total 35 F 1 seeds produced from cross between NR-9 and MR-6. The most successful breeding strategy was found to be combining markerassisted foreground and background selection with phenotypic selection, which significantly improved the breeding program's efficiency. Nine true F 1 s heterozygous for the Xa21 gene were found using foreground selection, and these were backcrossed with NR-9 to produce 62 BC 1 F 1 plants. Foreground selection using the markers pTA248, for Xa21 gene on the BC 1 F 1 s found 30 plants with gene of interest (Fig. 3). Out of total 101 marker used for parental polymorphic study identified total 36 polymorphic markers for background selection (Fig. 4; table 2).

The selected phenotypically desirable plants from BC 1 F 1 were genotyped for background selection with 36 polymorphic markers which showed RPG between 57% to 71%. the BC 1 F 1 plants was backcrossed with NR-9 to produce 81 BC 2 F 1 s. A similar selection approach was adopted and 43 BC [31,23] . RPG levels above 92% indicate successful introgression with minimal donor genome retention, reducing linkage drag and ensuring recovery of NR-9 agronomic characteristics. In present study introgressed line MAS-14-283 showed the highest recurrent parent genome recovery. Twelve Xa21 gene introgressed lines and two parent's agromorphological data showed that the improved lines had superior recurring parent traits and were resistant to bacterial blight. In green house under artificial inoculation with BLB culture an average lesion length of 18.56 ± 3.30 cm for NR-9 as against MR-6 that showed average lesion length of 1.5 ± 0.24 cm. NR-9 improved lines showed an average lesion length of 1.5-2.8 cm that showed good level of BB resistance over NR-9 (Supplementary table1; Fig. 5). The BB screening also carried out for Xa21 gene introgressed lines using bacterial blight culture from IIRR Hyderabad and symptoms clearly observed for susceptible and resistance lines (Abhilash Kumar et al., 2017; Balachiranjeevi et al., 2017) [1,2] . Evaluating DUS features lastly in the improved lines is not preferable as there is no way to get the desirable RP characters back. Therefore, foreground-positive individuals should be chosen first, followed by those with a higher yield index and those chosen for higher recovery of RP traits (Nandi et al., 2023) [23] . MABB programs also use careful visual selection to make sure the new lines that improved keep the plant traits and yield compared to the original parent variety (Joesph et al., 2004; Hari et al., 2013) [12,11] . Various studies mentioned that some gene introgressed lines had higher yields than the recurrent parent; this could be because the recurrent parent inherited certain yield features or QTLs from the donor parent ( [26, 27, 34, 33] . All of the introgressed lines were on at par with the recurrent parent, according to an analysis of variance for several morphological and grain quality characteristics. viz. days to 50% flowering, plant height, 1000 grain weight (g), grains per panicle (No.), number of productive tillers (No.), spikelet fertility (%), panicle length (cm) & yield per acre (q). In our study, two improved lines were performed well over the NR-9 in terms of number of productive tillers per plant, spikelet fertility (%) and grain yield per acre (quintal/acre). During the Kharifseason, the chosen improved lines of NR-9 at the BC 3 F 4 generation along with parents were assessed for specific agro-morphological traits.

The findings showed that, in terms of yield components, most selections were similar to recurrent parents. Various traits as panicle length (cm), grains per panicle (No.), days to 50% flowering, spikelet fertility, plant height (cm), 1000 grain weight, number of productive tillers (No.) & yield per acre (q) found non significant. Days to 50% flowering of the improved lines ranged from 115 to 119 days compared with 118 days of NR-9. Pant height of the improved lines ranged from 109 cm to 118 cm compared with 116 cm of NR-9. The mean number of productive tillers per plant of the improved lines ranged from 12 to 14 compared with 13 of the recurrent parent NR-9. Panicle length for improved lines were in the range of 24.8 cm to 25.4cm. The mean grains per panicle (No.) of the improved lines ranged from 338 to 345 (No.) compared with 341 (No.) of the recurrent parent NR-9.

The entries such as MAS-14-283 and MAS-14-305 were promising with number of productive tillers/ plant ( 4; Fig. 6). Rice Grain Quality (RGQ) and yield represent the most critical and economically significant traits in rice cultivation, as they directly influence consumer preferences and contribute substantially to global food security (Cheng et al., 2022) [5] . Appearance, milling quality, cooking behavior, sensory traits, palatability and nutritional value are key factors that influence its market price. Rice physical quality includes traits such as grain shape, head rice yield, grain size, chalkiness, the amount of broken grains, grain breakage, color, and overall appearance (Handoka et al., 2022) [10] . Measuring different parameters such as texture traits, cooking time, grain expansion, amylose content, gelatinization temperature, gel consistency, the levels of protein, lipids, and micronutrients helps in product establishment tin market (Prodhan et al., 2024) [28] .

The selected BC 3 F₄ improved lines were subjected to rigorous phenotypic evaluation for grain quality and the results indicated that their quality attributes closely matched those of the recurrent parent. The improved lines have good level in of inbuilt resistance to BB and close features for agro morphological and grain related traits and yield at par with NR-9. All the improved versions possessed short slender grain type & showed similarity to recurrent parent in all the grain quality parameters analyzed, viz., kernel breadth (1.36 mm -1.43 mm), hulling (76.5-81.5%), milling (67.3-71.4%), HRR (57.9-63.2%), kernel length (5.34 mm -5.68 mm) , L/B ratio (3.79 -3.97), kernel length after cooking (9.0-10.20 mm), elongation ratio (1.66 -1.87), alkali spreading value (5.0 -6.0) and intermediate amylose content (21.33 -24.88%) (table.5). MAS-14-283 (NR-1033) and MAS-14-305 (NR-1047) were shown very good cooking quality of rice as comparable with the recurrent parent NR-9 (Fig. 7). To enhance yield stability and sustainability in BB prone regions where NR-9 is cultivated, advanced breeding lines developed through MAS and phenotypic selection offer effective and durable resistance. In this study, MAS enabled the successful introgression of the broad spectrum BB resistance gene Xa21, resulting in twelve promising improved lines with high RPG recovery and strong agronomic performance. The introgressed line MAS-14-283 [NR-1033 (Sairaj)] exhibited grain yield and quality comparable to NR-9 and demonstrated excellent disease resistance. These improved lines not only serve as superior cultivars for farmers but can also be effectively used as BB resistant donors in breeding programs aimed at enhancing resistance in other elite short slender or fine grain varieties. Overall, MAS enabled backcross breeding facilitated the efficient transfer of Xa21 gene into NR-9 while maintaining grain quality and agronomic performance. Several improved lines exhibited strong BB resistance and yields comparable or superior to NR-9, with MAS-14-283 emerging as the most promising line. This demonstrates that MAS in conjunction with phenotypic selection may be a dependable approach in backcross breeding initiatives.

Acknowledgement

AcknowledgementWe would like to express our sincere gratitude to all those who contributed to the successful completion of this research. We extend heartfelt thanks to the Board of directors of Nirmal seeds Pvt. Ltd. providing continuous guidance, support, and a conducive environment for conducting this study. We would like to acknowledge the support of internal funding by Nirmal seeds Pvt. Ltd. The financial assistance provided greatly facilitated data collection, analysis, and other essential components of this research. This support played a significant role in ensuring the timely and smooth execution of the project.

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