Genetic Enhancement for Biotic Stress Resistance in Basmati Rice through Marker-Assisted Backcross Breeding

Pusa Basmati 1509 (PB1509) is one of the major foreign-exchange-earning varieties of Basmati rice; it is semi-dwarf and early maturing with exceptional cooking quality and strong aroma. However, it is highly susceptible to various biotic stresses including bacterial blight and blast. Therefore, bacterial blight resistance genes, namely, xa13 + Xa21 and Xa38, and fungal blast resistance genes Pi9 + Pib and Pita were incorporated into the genetic background of recurrent parent (RP) PB1509 using donor parents, namely, Pusa Basmati 1718 (PB1718), Pusa 1927 (P1927), Pusa 1929 (P1929) and Tetep, respectively. Foreground selection was carried out with respective gene-linked markers, stringent phenotypic selection for recurrent parent phenotype, early generation background selection with Simple sequence repeat (SSR) markers, and background analysis at advanced generations with Rice Pan Genome Array comprising 80K SNPs. This has led to the development of Near isogenic lines (NILs), namely, Pusa 3037, Pusa 3054, Pusa 3060 and Pusa 3066 carrying genes xa13 + Xa21, Xa38, Pi9 + Pib and Pita with genomic similarity of 98.25%, 98.92%, 97.38% and 97.69%, respectively, as compared to the RP. Based on GGE-biplot analysis, Pusa 3037-1-44-3-164-20-249-2 carrying xa13 + Xa21, Pusa 3054-2-47-7-166-24-261-3 carrying Xa38, Pusa 3060-3-55-17-157-4-124-1 carrying Pi9 + Pib, and Pusa 3066-4-56-20-159-8-174-1 carrying Pita were identified to be relatively stable and better-performing individuals in the tested environments. Intercrossing between the best BC3F1s has led to the generation of Pusa 3122 (xa13 + Xa21 + Xa38), Pusa 3124 (Xa38 + Pi9 + Pib) and Pusa 3123 (Pi9 + Pib + Pita) with agronomy, grain and cooking quality parameters at par with PB1509. Cultivation of such improved varieties will help farmers reduce the cost of cultivation with decreased pesticide use and improve productivity with ensured safety to consumers.


Introduction
Basmati rice is uniquely characterized by very-long cooked grains, delicious taste and exquisite aroma.It is a high-quality specialty rice from the Indo-Gangetic Plains, appreciated by consumers all over the world.The export of Indian Basmati rice earns more than US$ 4.7 billion annually [1].However, this crop is constantly threatened by various biotic stresses of bacterial and fungal origin.Of them, bacterial blight (BB) disease caused by the Xanthomonas oryzae pv.oryzae (Xoo), a Gram-negative bacterium, and the rice blast caused by the heterothallic ascomycete fungal pathogen Magnaporthe oryzae, cause yield losses up to 50 to 90% [2].These diseases are usually managed by the application of chemical pesticides.Pesticide residue in Basmati rice is one of the major concerns for the rejection of Basmati rice consignments.Furthermore, the application of chemical pesticides is not an ecologically friendly approach [3].
One of the leading Basmati rice varieties, Pusa Basmati 1509 (PB1509), with sturdy stem, semi-dwarf plant height (95-100 cm), early maturity (115-120 days), and nonshattering and exceptional grains [4], has contributed significantly to the financial reserves of the country through its export earnings.Further, due to early maturity, it provides enough opportunity to take up the timely sowing of subsequent crops.However, PB1509 is a potential target for incorporating genes for BB and blast resistance as it is highly susceptible to these diseases.
The BB resistance gene xa13 was identified in the genotype BJ1 and mapped onto the chromosome [15].This gene interacts strongly with other resistance genes, namely, Xa4, xa5 and Xa21 [16].Studies based on the pathogenicity analysis have revealed that xa13 activates unique defense genes as compared to other major dominant R genes, namely Xa4, Xa10 and Xa26, suggesting a unique resistance mechanism of the gene [17].Another important broad-spectrum BB resistance gene Xa21 was discovered in an accession of Oryza longistaminata.The Xa21 gene was mapped to the long arm of chromosome 11 and a functional marker, pTA248 was developed for its effective deployment through marker-assisted selection [18].Another dominant BB resistance gene Xa38, located on the long arm of chromosome 4, was identified from an accession of Oryza nivara, and a tightly linked InDel marker (marker-LOC_Os04g53050-1) co-segregating with the BB resistance was developed [19,20].Moreover, pyramiding two or more BB resistance genes is considered effective as it increases the spectrum of resistance and reduces the speed of evolution of isolates virulent against the deployed genes.A combination of xa13 and Xa21 has been widely utilized in rice breeding programs and several varieties have been released [21,22].The importance of another BB resistance gene, Xa38, in rice breeding programs, is highlighted by its utilization in the development of several BB-resistant genotypes [13,23].Considering the resistant spectrum of xa13, Xa21 and Xa38, the present study was targeted to introgress them into the genetic background of PB1509.
Another profoundly serious and devastating disease of rice is blast.Yield losses due to blast disease are variable depending on the genotype and environment, reaching up to 100% under disease-friendly conditions [24].Similar to BB, the development of host plant resistance is the most effective strategy for the management of blast disease [25].So far, more than 100 resistance genes have been identified and 37 of them have been cloned [26].Although several blast resistance genes have been identified, only a few of them were used in breeding programs for blast disease management in India [27].The major blast resistance gene, Pi9, mapped to the Piz locus on chromosome 6 confers resistance to 43 M. oryzae isolates prevalent in 13 countries.Of the six NBS-LRR domains present in this locus, Nbs2-Pi9 is the functional blast resistance gene Pi9 [28].The Pib gene in rice is known to confer resistance to a wide range of races of the rice blast pathogen, including race IE1k [29].Another blast resistance gene Pita located on chromosome 12 adjacent to the centromere in the genotype 'Tetep' [30] was found to encode a cytoplasmic protein-containing NBS-LRR domain [31,32].The pyramiding of Pi9, Pib and Pita is a promising strategy to widen the resistant profile of genotypes as well as to achieve substantial resistance.There have been several reports of the successful incorporation of blast resistance genes into the genetic background of different rice varieties [33,34].
Considering the prevailing diseases in the Basmati growing regions of the country, developing resistance to both BB and blast diseases can prove to be an effective strategy as it can significantly reduce the use of chemical pesticides while reducing the cost of cultivation.Therefore, we attempted to incorporate BB (xa13, Xa21 and Xa38) and blast (Pi9, Pib and Pita) resistance genes into the genetic background of PB1509 through MABB.

Generation of PB1509-NILs Carrying BB and Blast Resistance Genes
For the development of NILs with resistance to biotic stresses, four donor parents (DPs), namely, PB1718, P1927, P1929 and Tetep, were crossed with the RP (PB 1509) to generate F 1 s, which were designated as Pusa 3037, Pusa 3054, Pusa 3060 and Pusa 3066, respectively.The list of polymorphic markers obtained in all the cross combinations is presented in Supplementary Tables S1-S4.
The BC 1 F 1 plants generated from the cross PB1509/PB1718 carrying the target genes xa13 and Xa21 in a heterozygous state were identified through foreground selection using the markers xa13prom and pTA248, respectively, and the recurrent parent genome (RPG) recovery estimated using 74 polymorphic SSR markers ranged from 64.8% to 84.2% (Table 1).Similarly, in BC 1 F 1 s from the cross PB1509/P1927, foreground selection with marker-Os04g53050 led to the identification of plants carrying Xa38 in a heterozygous state and RPG recovery estimated using 83 polymorphic SSR markers ranged from 73.8% to 88.6%.In the BC 1 F 1 s from the cross PB1509/P1929, the plants carrying Pi9 and Pib were found to have RPG recovery of 70.7% to 81.0% with 84 polymorphic SSR markers.In the case of PB1509/Tetep, the BC 1 F 1 s carrying Pita in a heterozygous state had RPG recovery ranging from 57.3 to 87.5%.Further, based on RPG and RP phenotype recovery, the best plants in each of the cross combinations were identified for backcrossing to generate BC 2 F 1 s.During BC 2 F 1 generation, background selection with the remaining polymorphic markers and the markers that were heterozygous in the BC 1 F 1 generation revealed RPG recovery of up to 94.4%, 93.3%, 91.9% and 86.4% in the cross combinations PB1509/P1927, PB1509/ PB1718, PB1509/P1929 and PB1509/Tetep, respectively.In each of the combinations, a BC 2 F 1 plant with maximum resemblance to the phenotype of RP coupled with maximum RPG recovery was selected and backcrossed to generate BC 3 F 1 seeds.The best BC 3 F 1 plant was selfed to generate BC 3 F 2 populations.A total of 12, 15, 14 and 15 plants homozygous for Pusa 3037, Pusa 3054, Pusa 3060 and Pusa 3066, respectively, were identified through foreground selection using gene-linked markers.Stringent phenotypic screening for RP phenotype was carried out till BC 3 F 5 generation.A total of four NILs for Pusa 3037 and 5 NILs each for Pusa 3054, Pusa 3060 and Pusa 3066 were identified and subjected to multi-location evaluation for yield and its component traits, grain and cooking quality parameters.
subjected to multi-location evaluation for yield and its component traits, grain and cooking quality parameters.

Discussion
PB1509 is an elite Basmati rice variety and is popular among farmers owing to its early maturity, semi-dwarfness, exquisite grain and cooking qualities.Furthermore, it contributes immensely to the national exchequer due to its export potential.However, it suffers adversely due to biotic stresses imposed by disease-causing organisms such as Xoo and M. oryzae.Choosing pesticides wisely and their timely application is essential for the management of this disease.However, there are several concerns associated with environmental hazards and consumer safety.Additionally, the rise in the stringency of pesticide residue limits by importing nations has led to concerns of the rejection of Basmati rice consignments.Therefore, developing inbuilt genetic resistance is the only viable option for the maintenance of healthy international trade, the environment, and satisfied consumers.MABB is considered an effective approach for arming the Basmati rice cultivars with inbuilt disease resistance [35].To ameliorate the issue of susceptibility to diseases, the present study was aimed at incorporating three bacterial blight resistance genes, namely, xa13, Xa21 and Xa38, and blast resistance genes, namely, Pi9, Pib and Pita, in various combinations, into the genetic background of PB1509.
The Xoo pathogen is known to secrete transcription-activator-like effectors (TALEs), which target promoters for induction of one of the SWEET genes (SWEET11, SWEET13 and SWEET14).The major BB resistance gene xa13 provides race-specific resistance and it is also referred to as SWEET11.The other dominant gene Xa21 is known to encode a receptor kinase with the NBS-LRR domain, which recognizes a conserved determinant present in multiple races of Xoo [36].The combination of xa13 and Xa21 has been proven to provide synergistic action in imparting elevated levels of resistance to the Xoo isolates.Therefore, xa13 + Xa21 has been widely deployed into rice varieties such as Pusa Basmati 1, Ranbir Basmati, PRR78, Pusa 6B, etc. [37,38].However, recently there have been reports of the breakdown of resistance governed by xa13 + Xa21 [39].Subsequently, a major dominant BB resistance gene Xa38 with broad-spectrum resistance mapped on chromosome 4 [19] was incorporated into a series of elite rice varieties, viz., Pusa Basmati 1121, Super Basmati and Improved Samba Mahsuri [13,23,40].The NILs carrying Xa38 were resistant to a greater number of races of Xoo as compared to the NILs carrying xa13 + Xa21, indicating a wider spectrum of resistance governed by the gene Xa38 [13].The pyramiding of multiple genes improves the resistance spectrum of a genotype as well as reduces the speed of evolution of pathogens.Therefore, incorporating xa13 + Xa21 and Xa38 into the genetic background of PB1509 was considered a better choice to have prolonged resistance against multiple races of Xoo.We used PB1718 and P1927, carrying BB resistance genes xa13 + Xa21 and Xa38, respectively, as DPs.
Marker-assisted backcross breeding is the effective approach for defect correction in the otherwise agronomically superior varieties.Three rounds of backcrossing were employed to ensure sufficient recovery of the RP genome.In each of the backcross generations, foreground selection ensured the incorporation of the target gene, and background and phenotypic selection identified the plant with maximum recovery for the RP genome and phenotype, respectively.The current strategy has led to the development of five NILs carrying Xa38 (Pusa 3054) and four NILs carrying xa13 + Xa21 (Pusa 3037), which exhibited an RP genome similarity of more than 97%.The NILs carrying xa13 + Xa21 were highly resistant with BB lesion lengths ≤ 3.5 cm, while the NILs carrying Xa38 possessed lesion lengths of ≤3.0 cm.The NILs carrying xa13 + Xa21 + Xa38 possessed lesion lengths of ≤1.0 cm.This indicates that xa13 + Xa21, Xa38 and xa13 + Xa21 + Xa38 were effective in conferring resistance against the virulent isolates dominant in Basmati-producing regions of the country.The successful introgression of bacterial blight resistance genes xa13, Xa21, Xa4, xa5, Xa33, Xa40, etc., has been earlier reported [14,[45][46][47].Similarly, five NILs both for Pi9 + Pib (Pusa 3060) and Pita (Pusa 3066) were developed, which were highly resistant to the prevalent isolates Mo-ei-MB20, Po-RML21, Po-RML29, Po-HP5-2, Po-NWI-102 and Po-NWI-14 under artificial inoculated conditions.
Background selection using SSR markers during early backcross generations has led to maximizing RPG recovery, which ranged from 86.1% to 99.6% during BC 3 F 1 generation.SSR markers were cost-effective, accessible, and with better ease of handling in the laboratory [48].Therefore, SSRs were suited for background selection during backcross generations.Further, high-density SNP assays aid in having accurate estimates of background recovery as well as DP introgressions [49].Therefore, RPGA comprising 80K SNPs was used for background analysis in the advanced generations for identifying the lines with maximum similarity to RP [38].The effectiveness of the approach was evident from the genome similarity values of more than 96% observed in NILs as compared to RP, with a maximum similarity of 98.92% in the NIL, Pusa 3054-2-47-7-166-24-261-2 (G16).
Considering the mean vs. stability analysis plot, the arrow sign on the AEC line ranks the genotypes in increasing order.The NILs, G16, G13 and G18 were high-yielding in Env1 and Env3, while G11 and G17 were the genotypes with higher yield in Env2.Pusa 3037-1-44-3-164-21-255-1 (G12) and Pusa 3060-3-55-17-157-4-124-1 (G1) were less stable but with a high yield.Therefore, this genotype can be a potential line for the crop improvement program.A genotype placed on the horizontal axis with zero vertical projection is considered more stable, while a genotype with a lengthy direction from the AEC abscissa is treated as an unstable genotype [51].In the 'which-won-where' pattern plot, the environmental indicators are positioned into two segments, confirming the presence of distinct interaction between genotype and environment.The genotype that is attached to the vertex of polygon where the environmental marker drops suggests that the genotype gives a high yield and performs best in that environment [52].Therefore, G11 was the best performer in Env 1 and 3, whereas G1 was best for Env 2.
Several rice varieties with resistance to either bacterial blight or blast have been developed and released for cultivation, which provides farmers an opportunity to choose according to the disease prevalence in their growing environment.However, in the majority of the Basmati growing regions, BB and blast co-exist, which raises the need for combining bacterial blight and blast resistance into high-yielding varieties [53].Therefore, PB1509 NILs carrying xa13 + Xa21 + Xa38, Pi9 + Pib + Pita and Xa38 + Pi9 + Pib were developed.These lines were effective against the BB and blast isolates prevalent in the Basmati growing regions of the country.The adoption of multi-disease resistant NILs would reduce the application of chemical pesticides, paving the way for eco-friendly rice cultivation, which is safe for consumers, while strengthening the international trade of Basmati.

Plant Material
PB1509, an elite Basmati rice variety was used as RP; PB1718 carrying BB resistance genes xa13 +Xa21, P1927 carrying BB resistance gene Xa38, P1929 carrying blast resistance genes Pi9 + Pib, and Tetep possessing blast resistance gene Pita were used as the DPs.

Breeding Strategy
The breeding methodology adopted is presented in Figure 7. RP was crossed with four different DPs to generate F 1 seeds.The F 1 plant was selected based on a test of hybridity and backcrossed with RP to produce BC 1 F 1 seeds.Foreground selection was then carried out on BC 1 F 1 plants to identify the heterozygous plants, which were then subjected to background selection followed by phenotypic selection for agro-morphological and grain quality parameters.A BC 1 F 1 plant with maximum resemblance to recurrent parent was backcrossed to generate BC 2 F 1 seeds.Subsequently, same strategy was followed to generate BC 3 F 1 seeds.The BC 3 F 1 plant with superiority was selfed, and the plants homozygous for genes of interest were identified in BC 3 F 2 populations.These lines were tested for grain and cooking quality traits and advanced through pedigree selection till BC 3 F 5 generation.Simultaneously, intercrosses were made between the best BC3F1s plants to gener intercross F1s carrying xa13 + Xa21 + Xa38, Xa38 + Pi9 + Pib and Pi9 + Pib + Pita.The pla homozygous for the respective genes were identified in the intercross F2 populations a Simultaneously, intercrosses were made between the best BC 3 F 1 s plants to generate intercross F 1 s carrying xa13 + Xa21 + Xa38, Xa38 + Pi9 + Pib and Pi9 + Pib + Pita.The plants homozygous for the respective genes were identified in the intercross F 2 populations and were advanced till F 6 generation through pedigree selection.The developed NILs were evaluated before being nominated into the national system of varietal release.

DNA Extraction and PCR
The genomic DNA was extracted from 15-day-old seedlings using CTAB buffer with slight modification to the protocol [54].PCR of 10 µL volume was set up using 20-30 ng template DNA, 5 pmol of each primer and EmeraldAmp Max PCR Master Mix (2X Premix, Takara) using the Biorad T100TM thermal cycler with the standard PCR program and electrophoresis was carried out using Metaphor™ agarose gel and visualized on Gel DocTM XR+ documentation System.

Foreground Selection
Foreground selection for the genes xa13, Xa21 and Pi9 was carried out using the gene-based markers, namely, xa13prom, pTA248 and Pi9STS1, respectively.The selection for the genes Xa38, Pib and Pita was conducted using gene-linked markers, viz., marker-Os04g53050-1, RM535 and RRS12, respectively.

Recurrent Parent Genome Recovery Using SSR and SNP Markers
The RP and the DPs were surveyed for polymorphic markers using 735 genome-wide SSR markers from the URL link http://www.gramene.org(accessed on 22 August 2016) and the primers were synthesized.A final set of developed NILs were subjected to estimation of RPG similarity using Affymetrix based 80K RPGA [55].The genome similarity was visualized using the Phenogram software from the URL link http://visualization.ritchielab.psu.edu/phenograms/plot (accessed on 12 February 2023).The similarity to the genome of RP was obtained using the formula, (R + 1/2H) X 100/P, where R = Number of markers amplifying homozygous allele for recurrent parent, H = number of heterozygous markers and P = total number of markers.

Screening for Disease Resistance
For evaluation of BB resistance, the parental lines and NILs were grown in the field conditions.The suspension of Xoo race 4 with a density of 10 9 cells/mL was prepared.Inoculation was performed with the isolate by clip inoculation method wherein top five leaves from every entry were clipped [56].The length of BB lesion was measured post-21 days of inoculation.The genotype with a lesion length of up to 5 cm was considered to be resistant, 5-10 cm was considered to be moderately resistant reaction, 10-15 cm was considered to be moderately susceptible response, and more than 15 cm to be as completely susceptible [57].
To screen for blast resistance, the NILs carrying blast resistance genes along with the RP and DPs were grown in pro-trays as per the protocol [58].The seedlings were grown under optimum conditions at temperature of 28 • C and 95% relative humidity.At three-leaf stage, the inoculum of M. oryzae isolate comprising of approximately 5 × 10 4 conidia per ml was mixed with 0.02% tween 20 and sprayed on the seedlings.At IARI, New Delhi, M. oryzae isolate Mo-ei-MB20 was used for screening.Mixture of five isolates, namely, Po-RML21, Po-RML29, Po-HP5-2, Po-NWI-102 and Po-NWI-141, constituted the inoculum for screening at Palampur.The seedlings were scored for the blast resistance 7 days postinoculation following Bonman's scale.Scores of 0-3 were considered resistant reactions, and scores of 4 and 5 were considered susceptible reactions.

Multi-Environment Agro-Morphological Evaluation of Developed NILs
The NILs along with the RP and DPs were evaluated for agronomic performance with three replications in RCBD at three different environments, namely, IARI-New Delhi (Env1), IARI Regional Station-Karnal (Env2), and Urlana (Env3), during kharif 2021 following recommended agronomic practices.Data for traits, viz., DF, PH, PL, NT, NFG, SF, TW and grain yield, were recorded.DF was recorded on a plot basis.A representative ten plants from each of the NILs were considered for measurement of other traits.

Grain and Cooking Quality Evaluation of Developed NILs
Traits related to quality, namely, HR, MR, HRR, KLBC, KBBC, KLAC, KBAC, ER, and aroma, were recorded following standard protocol.HR was calculated in percentage as the ratio of the weight of whole polished grains to the weight of the raw grains.Grain parameters such as KLBC and KBBC were recorded on a photo enlarger using ten grains from each of the NILs.For the recording of cooking quality characteristics, namely, KLAC and KBAC, ten whole milled kernels were selected and soaked for 30 min in 10 mL of distilled water taken in test tubes.The lower part of the tubes with rice kernels was then immersed in a boiling water bath for 8-10 min.The cooked kernels were cooled to room temperature after transferring the contents into a Petri plate, and data were recorded.The data were subjected to statistical analysis using the package CropStat 7.2 [59], and stability analysis was carried out using the Metan package v 1.18.0 [60].
Where DF = days to fifty percent flowering, PH = plant height, NT = number of tillers, PL = panicle length, FG = filled grains, SF = spikelet fertility and TW = test weight.* Significantly different from PB1509 at 5% level of significance.
Where DF = days to fifty percent flowering, PH = plant height, NT = number of tillers, PL = panicle length, TG = total grains, SF = spikelet fertility and TW = test weight.* Significantly different from PB1509 at 5% level of significance.

Figure 6 .Figure 6 .
Figure 6.The bacterial blight lesion length in NILs and parental lines.The values represented are the mean of replicated data over two seasons and the standard error is shown as error bars.

Table 2 .
Agronomic performance of the PB1509-NILs carrying different resistance genes for blast and bacterial blight at IARI, New Delhi along with recurrent parent similarity.

Table 3 .
Grain and cooking quality of the PB1509-NILs.

Table 4 .
Yield performance of PB1509 NILs at three different locations namely, New Delhi (Env1), Karnal (Env2) and Urlana (Env3).* Represents the NILs with a significant difference in yield compared to RP PB1509.

Table 5 .
Agronomic performance of the PB1509 NILs carrying different combinations for blast and bacterial blight resistance genes.

Table 5 .
Agronomic performance of the PB1509 NILs carrying different combinations for blast and bacterial blight resistance genes.
Where DF = days to fifty percent flowering, PH = plant height, NT = number of tillers, PL = panicle length, TG = total grains, SF = spikelet fertility and TW = test weight.* Significantly different from PB1509 at 5% level of significance.

Table 6 .
Grain and cooking quality of the intercrossed NILs.

Table 6 .
Grain and cooking quality of the intercrossed NILs.

Table 7 .
Blast reaction scores for the developed NILs with isolate Mo-ei-MB20 at New Delhi and mixture of five isolates at Palampur.