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Article

Genetic Enhancement of Blast and Bacterial Leaf Blight Resistance in Rice Variety CO 51 through Marker-Assisted Selection

by
Samuthirapandi Subburaj
1,†,
Thiyagarajan Thulasinathan
2,†,
Viswabharathy Sakthivel
1,
Bharathi Ayyenar
2,
Rohit Kambale
2,
Veera Ranjani Rajagopalan
2,
Sudha Manickam
2,
Raghu Rajasekaran
2,
Gopalakrishnan Chellappan
3,
Kalaimagal Thiyagarajan
1,
Manonmani Swaminathan
3 and
Raveendran Muthurajan
2,*
1
Department of Genetics and Plant Breeding, Centre for Plant Breeding and Genetics, Tamil Nadu Agricultural University, Coimbatore 641 003, Tamil Nadu, India
2
Department of Plant Biotechnology, Centre for Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultural University, Coimbatore 641 003, Tamil Nadu, India
3
Department of Rice, Centre for Plant Breeding and Genetics, Tamil Nadu Agricultural University, Coimbatore 641 003, Tamil Nadu, India
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agriculture 2024, 14(5), 693; https://0-doi-org.brum.beds.ac.uk/10.3390/agriculture14050693
Submission received: 2 February 2024 / Revised: 6 April 2024 / Accepted: 11 April 2024 / Published: 28 April 2024
(This article belongs to the Special Issue Feature Papers in Genotype Evaluation and Breeding)
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Abstract

:
The increased use of chemicals in rice farming poses significant issues regarding the emergence of pesticide/fungicide resistance and environmental sustainability concerns. This study was aimed at the genetic improvement of blast, bacterial leaf blight (BB) and gall midge resistance in a popular rice variety CO 51 which already harbours a blast resistance gene Pi54. Efforts were made to pyramid an additional blast resistance gene Pi9 along with two BB resistance genes (xa13 and Xa21) and two gall midge resistance genes (Gm1 and Gm4) into an elite rice variety CO 51 to enhance the resistance level to biotic stresses. The superior lines were selected using functional markers conferring resistance to blast (NBS4 and Pi54MAS linked to Pi9 and Pi54 genes, respectively) and BB [(xa13Prom (xa13) and pTA248 (Xa21)] and SSR markers linked to Gm1 (RM1328) and Gm4 (RM22550) for phenotypic screening and agronomic evaluation. The genotyping and phenotyping of F6 and BC2F6 progenies of CO 51 X 562-4, for agronomic traits and resistance to BB and blast, identified ten superior progenies in F6 and five superior progenies in BC2F6. The breeding lines harbouring both xa13+Xa21 exhibited high levels of resistance to BB (score ≤ 1 cm) and Pi9+Pi54 exhibited strong resistance to blast (score ≤ 2). Identified lines can be evaluated further for varietal improvement or utilised as genetic stocks in breeding programs.

1. Introduction

Nearly half of India’s population and one-third of the world’s population rely on rice for calorie and carbohydrate intake [1]. Despite enormous production and resolving global hunger, a 30% increase in rice production is required by 2030 from the present level, and rice production needs to be boosted by 160 million tonnes [2] and should increase by 70% in 2050 [3] to ensure global food security and nutritional security. Due to modern civilisation, there is a reduction in cultivatable land and water resources for irrigation [4]. Continuous changes in the ecosystem, and pests and pathogens have evolved for their survival in an adverse environment. Due to several biotic and abiotic stresses, rice production can be affected by unfavourable climate changes. The productivity of rice continues to be under threat by biotic stresses, viz., blast and bacterial leaf blight (BB), which cause significant losses in rice production [5]. Among fungal diseases affecting rice crops, blast disease ranked first among the top 10 diseases [6,7]. Rice yield losses are severe in the southern state of India due to blast and BB [5,8,9]. Blast, a fungal disease caused by the ascomycete fungus Magnaporthe grisea Barr., poses a significant threat to global rice production, resulting in yield losses of 70–80% [10,11]. Bacterial blight, caused by X. o. pv. oryzae, is considered the oldest and most destructive disease affecting rice [12]. It can lead to significant crop yield losses, up to 80% [13,14]. In India, during the Kharif season, the gall midge leads to a substantial yield loss of approximately USD 80 million, while the estimated loss on the Asian continent amounts to USD 550 million [15,16].
By way of releasing high-yielding varieties, food demand can be mitigated to meet the demand of the growing population [17]. In the modern era of genetics and biotechnology, various techniques are available to increase rice production and productivity by enhancing the host plant resistance against biotic stresses, through the pyramiding/stacking of multiple resistance genes/alleles into elite cultivars which provides strong and broad-spectrum resistance against many pathogens. So far, 102 resistance (R) genes [18,19] and nearly 500 QTLs [20] have been identified for rice blast disease. Among them, 38 R genes have been characterised at a molecular level and eight genes, namely Pi9, Pi54, pi21, Pi50, Pi7, Pi57, Pigm, and Ptr, have been reported as broad-spectrum resistance genes against blast disease [21]. The R genes were distributed to all 12 chromosomes in the rice genome except chromosome 3 [22]. In rice, Pi-b is the first R gene that was identified in the japonica variety [23]. Pi9 is a major resistance gene, isolated from the wild species Oryza minuta [24,25], and pi-kh, which was renamed to Pi54 [26] and isolated from the Tetep cultivar mapped in chromosome 11L [27], has resistance to major isolates of M. oryzea [28].
Another important and destructive disease is bacterial leaf blight (BB) caused by (X.o pv., oryzae), which resulted in up to 40% of yield losses at the tillering stage and up to 50% of yield losses in the initial stage [29]. So far, 46 R genes have been identified for BB resistance and 13R genes, viz., Xa1, Xa3/Xa26, Xa4, xa5, Xa7, xa8, Xa10, xa13, Xa21, Xa23, xa25(t), Xa27(t), and xa41(t), have been cloned and characterised at the molecular level [30]. In total, 29 genes are dominant resistant, viz., Xa1, Xa3/Xa26, Xa4, xa5, Xa10, xa13, Xa21, Xa23, xa25(t), Xa27(t), and xa41(t), whereas 17 genes are recessive resistant, viz., xa5, xa8, xa13, xa15, xa19, xa20, xa24, xa25(t), xa26(t), xa28(t), xa31(t), xa32(t), xa34(t), xa41(t), xa42, xa44(t), and xa45(t) [30]. A major dominant R gene, ‘Xa21’, confers broad-spectrum resistance against many virulent isolates in India and was initially identified in wild species, O. longistaminata [31]. Another R gene xa13 also confers broad-spectrum resistance against many virulent isolates in India and possesses mutation in the promoter region and is homologous to nodulin [5,32].
In 1978, Nelson proposed the concept of integrating specific QTLs/genes for resistance to abiotic and biotic stresses into crop varieties via gene pyramiding or introgression. Gene pyramiding involves combining two or more genes from different donor parents, each controlling multiple traits, into the genetic background of a desired variety, known as multi-trait introgression. This can be accomplished through three methods described by Singh [33]. 1. Separate backcross programs: Each donor parent is used as a pollen parent and crossed with the recurrent parent as a seed parent. The goal is to introgress the target gene from each donor parent into the genetic background of the recurrent parent. The resulting F1 lines are then crossed with the recurrent parent to produce Backcross Inbred Lines (BILs) with homozygous or heterozygous conditions for the targeted QTLs/genes [33]. Subsequently, the BILs carrying the targeted QTLs/genes are intercrossed to combine all desired QTLs/genes into a single genetic background. 2. Single backcross: Symmetrical mating- Each donor parent serves as a pollen parent and is crossed with the recurrent parent as a seed parent. The resulting F1 lines, genotyped with targeted QTLs/genes, are selected and intercrossed to combine all desired QTLs/genes into a single genetic background [33]. These intercrossed progenies carrying targeted QTLs/genes are then backcrossed with the recurrent parent to the Recover Recurrent Genome (RRG) and develop BILs. 3. Single backcross: Tandem mating. In this approach, the recurrent parent is first introgressed with targeted QTLs/genes by crossing with one donor parent (improved recurrent parent). Then, the improved recurrent parent, now containing targeted QTLs/genes, is crossed with a second donor parent (also called the improved recurrent parent) to further introgress targeted QTLs/genes [33]. These methods allow for the development of crop varieties with improved stress tolerance and resistance by combining multiple beneficial traits from different donor parents into a single genetic background.
Das and Rao [34] developed an Improved Lalat variety that is tolerant to multiple stresses by incorporating four bacterial blight resistance genes (Xa4, Xa21, xa13, and xa5), as well as QTLs/genes for blast, gall midge, salinity, and submergence. In another study, Das et al. [35] successfully combined several QTLs/genes into a cultivar named enhanced Tapaswini (xa13 and Xa21), which exhibits resistance to submergence (Sub1), salinity (Saltol), blast (Pi9, Pi54), and gall midge (Gm1, Gm4). This groundbreaking research involved the stacking of ten distinct genes/QTLs (six previously identified and four newly introduced) and their expression at desired levels in a new genetic background, showcasing the potential for a new era of molecular plant breeding. Dixit et al. [36] achieved the pyramiding of genes for blast (Pi9), bacterial leaf blight (BLB) (Xa4, xa5, xa13, and Xa21), brown plant hopper (BPH) (Bph3, Bph17), gall midge (Gm4 and Gm8), and QTLs for drought tolerance (qDTY1.1 and qDTY3.1) in the Swarna variety, combining a total of 11 genes with existing traits.
Through the utilisation of MAS, researchers introduced five biotic stress resistance genes (Pi40, Xa4, Xa5, Xa21, and Bph18) into a Korean japonica rice variety Jinbubyeoa, resulting in the creation of gene-pyramided breeding lines specifically targeting bacterial blight, blast, and brown plant-hopper [37]. These gene-pyramided lines exhibited impressive resilience against biotic stresses. SSR graphical mapping indicated that gene-pyramided lines retained 93% of the recurrent parent Jinbubyeo’s genome. The researchers evaluated the impact of these QTLs/genes in a novel genotype using phenotypic screening methods, demonstrating enhanced levels of resistance/tolerance against the targeted stresses. Furthermore, through marker-assisted backcrossing, two significant QTLs (Sub1 and Qbph12), associated with abiotic and biotic tolerance/resistance, were introduced into traditional jasmine rice cultivar KDML105. Positive progenies carrying both QTLs exhibited tolerance to both abiotic and biotic stresses, showcasing notable differences in Days to Flowering, Plant Height, and grain yield [38].
This present study focused on the development of a durable resistant cultivar against gall midge, blast, and BB pathogens through the stacking of six R genes. The closely linked markers, viz., xa13Prom for the xa13 gene, pTA248 for Xa21, NBS4 for the Pi9 gene, Pi54MAS for Pi54 genes, RM1328 for Gm1, and RM22550 for Gm4, were used to confirm the presence of the target genes against blast, BB, and gall midge in rice cultivar CO 51.

2. Materials and Methods

2.1. Genotypes Used

A popular rice variety CO 51 was used as a recurrent parent in this study. It is a short-duration (110–115 days), fine-grain, and high-yielding rice genotype cultivated in 14 states in India [39,40]. CO 51 is moderately resistant to blast due to the presence of the Pi54 allele and is severely susceptible to BB disease. An intermittent genetic stock #562-4 derived between CO 43 X VRP 1 harbouring Pi9, xa13, Xa21, Gm1, and Gm4 was used as a donor and was developed in the Department of Plant Biotechnology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu India (Table 1. The details of markers listed in Supplementary Table S1) [41].

2.2. Development of Pyramided Lines to Enhance Biotic Stress Tolerance of CO 51

For marker-assisted selection (MAS), leaf samples from all progenies and parent lines were collected 3 weeks after transplanting. DNA extraction was performed with the CTAB method [42]. Molecular markers NBS4, Pi54MAS, xa13Prom, pTA248, RM1321, and RM22550 were used for foreground selection which are closely linked with Pi9, Pi54, xa13, Xa21, Gm1, and Gm4, respectively.
The rice cultivar CO 51 was used as a female parent and the donor parent 562-4 was used as a male parent to develop F1. The identification of true F1 was performed using closely linked markers for the target genes, viz., NBS4, Pi54MAS, xa13Prom, pTA248, RM1328, and RM22550. Then, these true F1s were crossed with recurrent parent CO 51 to generate BC1F1, and selfing was allowed for generating F2. The positive BC1F1 plants were identified with foreground markers and crossed with CO 51 to generate BC2F1. Then, BC2F1 was allowed to BC2F6 and each generation was genotyped with foreground markers.
Then, simultaneously, the selected F2 was allowed to generate F3 and genotyped with linked markers. Superior F3 progenies were selected based on grain type and advanced to F4 in the field conditions. In F4:5, selections were based on grain type and high yield over the recurrent parent CO 51. Selected progenies in F5:6 and BC2F6 were used for blast screening in natural hotspot areas and BB screening in field conditions.

2.3. Screening of Selected RILs and BILs against Blast Pathogen

Selected progenies of F5:6 and BC2F6 were raised in the Uniform Bed Nursery (UBN) in the Hybrid Rice Evaluation Centre, Gudalur, Tamil Nadu, India. Plants were raised along with recurrent and donor parents. The susceptible check CO 39 was sown on both sides of UBN and one in every five rows to ensure a continuous supply of blast inoculum. The disease infection was measured at an interval of 15 days up to 45 Days After Sowing (DAS) in all the test entries. Scores 0–3 were considered resistant (R), 4–5 moderately resistant (MR), and 6–9 susceptible (S) [1].

2.4. Screening of Selected RILs and BILs against BB Pathogen

Twenty-one day-old seedlings of selected progenies of F5:6 and BC2F6 were transplanted in the main field along with parents and the susceptible check TN 1. TN 1 was transplanted once every five rows to ensure a continuous supply of BB pathogen. A virulent bacterial blight pathogen X. o p.v. oryzae was collected from the Department of Plant Pathology, Tamil Nadu Agricultural University (TNAU), Coimbatore. The pathogen X. o p.v. oryzae strain was grown in peptone sucrose agar media for the production of inoculum. The bacterium was scraped from all plates and suspended in sterilised double-distilled water. The maximum tillering stage (40–45 days) of rice was inoculated with BB pathogen X. p.v., oryzae using the clip inoculation method with sterilised scissors [47]. A total of ten plants were inoculated and approximately ten upper leaves/plants were measured for lesion length. The lesion lengths were measured 14 and 21 days post inoculation when the lesion was stable. The average lesion length was calculated from 10 maximum lesion lengths per entry. Based on lesion length, plants were classified as resistant (R) when the length was 0–3 cm, moderately resistant (MR) when the length was more than 3–6 cm, moderately susceptible (MS) when the length was more than 6–9 cm, and susceptible (S) when the length was more than 9 cm [36,48].

2.5. Agronomic Performance and Grain Quality Parameters of RILs and BILs

Selected superior progenies in F5:6 and BC2F6 populations were raised under field conditions. Twenty-one day-old seedlings were transplanted in the main field. Those selected RILs were grown in 3 rows of 2 m in length along with recurrent and donor parents with two randomised replication plots. The row-to-row distance was 0.2 m and the plant-to-plant distance was 0.15 m. Standard agronomic practices were followed. Phenotypic traits including Plant Height (PH), Number of Tillers (NT), Number of Productive Tillers (NPT), Days to First Flowering (DFF), Days to 50% Flowering (D50%F), Flag Leaf Length (FLL), Flag Leaf width (FLW), Panicle length (PL), and Grain Length and width (GL and GW) were recorded.

3. Results

3.1. Introgression of Pi9, xa13, Xa21, Gm1, and Gm4 into CO 51 Harbouring Pi54

Marker-assisted breeding was followed for the stacking of genes in the background of CO 51 (Figure 1). The cultivar CO 51 already possesses the Pi54 gene, giving broad-spectrum resistance against blast pathogens. This study focused on the stacking/pyramiding of Pi9, xa13, Xa21, Gm1, and Gm4 genes in the CO 51 background to enhance strong and stable durable resistance against blast pathogens, BB pathogens, and the gall midge biotype in the southern part of India.
Initially, donor parent 562-4 was crossed with CO 51 to generate F1 plants. The markers which were closely linked to genes Pi9, Pi54, xa13, Xa21, Gm1, and Gm4 were used to identify true F1 plants. A total of 17 plants were raised in a pot in greenhouse conditions and four were identified as true F1 plants, viz., #2, #7, #9, and #14. The true F1 was identified using the foreground markers linked with the target genes (NBS4, Pi54MAS, xa13Prom, pTA248, RM1328, and RM22550 for Pi9, Pi54, xa13, Xa21, Gm1, and Gm4, respectively). The F1 progeny (#9) was forwarded into F2, and six progenies out of thirty in F2 were found to harbour all four genes, viz., Pi9, Pi54, xa13, Xa21, Gm1, and Gm4 (Supplementary Table S2). Those six selected progenies of F2 were harvested and raised as F3 in a greenhouse. Those F3 progenies were genotyped with linked markers of Pi9, Pi54, xa13, xa21, Gm1, and Gm4. A total of 26 F3 were selected based on the grain type and forwarded into F4. In the segregating population of F4:5, five plants/progeny were tagged in all 26 families and genotyped with foreground markers for Pi9, Pi54, xa13, Xa21, Gm1, and Gm4. Out of five plants per progeny, one plant was selected that harboured the maximum QTLs/genes. In F5:6, 26 progenies were selected with different QTL/gene combinations of Pi9, Pi54, xa13, Xa21, Gm1, and Gm4 with different zygotic statuses of target QTLs/genes (Figure 2 and Table 2).
In F1, progeny (#9) was used in backcrossing, and the evaluation of seven BC1F1 progenies identified one BC1F1 progeny, namely Plant# 9-2 possessing all six target genes, namely, Pi9, Pi54, xa13, Xa21, Gm1, and Gm4. Then, BC2F1 was generated from crosses between #9-2 of BC1F1 and CO 51. A true BC2F1 plant was identified with foreground selection and BC2F1 was forwarded into BC2F5. Seventy-seven progenies were selected in BC2F5. The presence of target genes, viz., Pi9, Pi54, xa13, Xa21, Gm1, and Gm4, was confirmed with SSR markers and functional markers. Out of 77 progenies, 16 progenies harbouring all six genes with a homozygous condition were selected for phenotyping for blast and BB screening (Figure 3 and Table 3).

3.2. Introgression of Pi9 to Enhance the Resistance to Blast in CO 51

A total of 26 RILs and 16 BILs were pyramided with Pi9, Pi54, xa13, Xa21, Gm1, and Gm4 and were subjected to screening against blast and BB. The blast screening was carried out in the Hybrid Rice Evaluation Centre, Gudalur, Tamil Nadu, India. Blast scoring was performed on the 30th and 45th Days After Sowing in UBN at 15-day intervals. The susceptible check CO 39 had scales of 6.8 and 9.9 in the first and second scores with an average of 8.35 showing a susceptible reaction to pathogens (Figure 4a and Supplementary Table S3). CO 39 acted as an inoculum source and the blast pathogen spore reproduced in CO 39, which ensures a continuous supply of spores in UBN. The donor parent (562-4) recorded scales of 1.3 and 2.3 in the first and second scores with an average score of 1.8 showing resistance, whereas the recurrent parent CO 51 exhibited scales of 4.4 and 5.9 in the first and second scores with an average of 5.15, showing moderate resistance to blast pathogens.
All 26 progenies of CO 51 RILs harbouring Pi9 and Pi54 genes recorded scores of blast ranging from 1.2 to 3.6 with an average of 2.24 in the F6 population, and they exhibited a resistance reaction to blast pathogens. RIL #9-10-8-23 recorded 1.8 and 1.8 in scores I and II, respectively, with constant resistance against blast pathogens. RIL # 9-10-8-25 recorded scores of 2.2 and 1.4 in scores I and II with an average of 1.8. RILs #9-20-2-99 and #9-20-2-100 recorded scores of 1.0 and 2.2 in scores I and II with an average of 1.6. RIL #9-20-7-102 recorded scores of 1.0 and 2.6 in scores I and II with an average of 1.8, whereas RIL #9-20-9-112 recorded scores of 0.6 and 1.8 in scores I and II with an average of 1.2. All the RIL populations of CO 51 showed better resistance than the recurrent parent against blast disease.
Sixteen BC2F6 progenies of CO 51 X 562-4 harbouring Pi9 and Pi54 ranged from 1.3 to 2.8 with an average resistance score of 2.0 to blast pathogens. BILs #6-17-5-18-29-1-1, #6-17-5-18-29-1-2, #6-17-5-21-19-4-1, and #6-17-5-18-5-1-5 all recorded scores of 1.4 and 1.8 in scores I and II, respectively, with an average of 1.6 as strong resistance. BIL #6-17-5-18-5-1-6 recorded scores of 1.2 and 1.4 in scores I and II, respectively, with an average of 1.3 as strong resistance. The BILs harbouring Pi9 and Pi54 recorded scores of 0 < 2 against blast (Figure 4b and Table 4).

3.3. RILs Harbouring Xa13 and Xa21 Exhibited Enhanced Resistance to BB

A total of 26 RILs and 16 BILs of CO 51 X 562-4 along with recurrent parent CO 51, donor parent 562-4, and TN 1 (susceptible check) were sown in the nursery and then transplanted into the main field. TN 1 recorded 15 cm in score I and 19.82 cm in score II with an average of 17.41 cm. The recurrent parent CO 51 recorded 7.49 cm in score I and 12.32 cm in score II with an average of 9.91 cm, whereas donor parent 562-4 recorded 0.23 cm in score I and 0.34 cm in score II with an average of 0.29 cm (Figure 5a).
The RILs of the population ranged from 0.24 cm to 0.43 cm in score I with a mean of 0.31 cm and 0.36 cm to 0.79 cm with a mean of 0.54 cm in score II (Supplementary Table S3). The grant means ranged from 0.29 cm to 0.58 cm with an average of 0.42 cm. RIL #9-10-8-23 recorded 0.32 cm and 0.42 cm in scores I and II with a mean of 0.37 cm. RIL #9-10-8-25 recorded scores of 0.37 cm and 0.79 cm in scores I and II with an average of 0.58 cm. RILs #9-20-2-99 and #9-20-2-100 recorded scores of 0.28 cm and 0.29 cm in score I and scores of 0.38 cm and 0.39 cm in score II with an average of 0.33 cm and 0.34, respectively. RIL #9-20-7-102 recorded scores of 0.24 cm and 0.34 cm in scores I and II with an average of 0.29 cm, whereas RIL #9-20-9-112 recorded scores of 0.26 cm and 0.36 cm in scores I and II with an average of 0.31 cm. All the RIL populations of CO 51 pyramided with resistance genes showed better resistance over the recurrent parent in BB disease. The graphical representation for the blast and BB scoring of CO 51 and 562-4 and the 10 selected superior progenies is given in Figure 6 and Table 5.
Sixteen BC2F6 progenies of CO 51 X 562-4 harbouring xa13 and Xa21 ranged from 0.30 to 0.50 cm with an average of 0.37 cm against blight pathogens (Figure 5b and Table 4). BIL #6-17-5-18-29-1-1 recorded scores of 0.28 and 0.45 cm in scores I and II with an average of 0.37 cm. BIL #6-17-5-18-29-1-2 recorded scores of 0.32 cm and 0.53 cm in scores I and score II with an average of 0.43 cm. BIL #6-17-5-21-19-4-1 recorded scores of 0.24 and 0.39 cm in scores I and II with an average of 0.32 cm, and BIL #6-17-5-18-5-1-5 recorded scores of 0.24 and 0.38 cm in scores I and II with an average of 0.31 cm. BIL #6-17-5-18-5-1-6 recorded scores of 0.23 cm and 0.38 cm in score I and score II with an average of 0.31 cm. The BILs harbouring Pi9 and Pi54 recorded a score of 0 < 0.5 against the BB pathogen (Figure 7).

3.4. Performance of RILs and BILs of CO 51 X 562-4 Lines for Important Traits

The 10 superior progenies of F6 of CO 51 X562-4 were selected based on resistance against blast and BB diseases. Those selected progenies were evaluated for their agronomic performance for yield and its attributing traits. CO 51 single plant yield (SPY) was recorded as 29.58 g, whereas those for RILs and F6 ranged from 29.97 to 36.45 g with an average of 32.94 g. The 1000-grain weight was recorded as 19.02 g in CO 51, whereas those for RILs ranged from 15.50 g to 23.28 g with an average of 19.63 g (Supplementary Table S4). The yield in improved CO 51 lines ranged from 1.32 to 42.70% over CO 51 (Figure 8). Maximum yields were recorded in RILs #9-10-9-146 (42.70%), #9-10-8-23 (26.27%), #9-20-2-98 (23.23%), and #9-10-8-25 (19.10%).
The BILs of CO 51 X 562-4 recorded single plant yields of 33.29 to 43.74 g with an average of 37.64 g, whereas the 1000-grain weight recorded in RILs ranged from 18.50 to 23.92 g with an average of 21.16 g (Supplementary Table S4). The yield in improved CO 51 lines ranged from 12.54 to 47.87% compared to CO 51 (Figure 9). Maximum yields were recorded in BILs #6-17-5-18-5-1-5 (47.87%), #6-17-5-18-29-1-5 (45.17%), #6-17-5-18-5-1-4 (43.91%), and #6-17-5-18-29-1-6 (42.06%).

4. Discussion

The present study aimed to pyramid or stack genes against blast, BB, and gall midge disease into the CO 51 background to improve the existing cultivar, which can be quickly achieved using marker-assisted selection (MAS) and marker-assisted backcross breeding (MABB) through closely linked markers to targeted genes. The pyramiding of multiple genes/alleles in a single background gene-based marker or tightly linked markers paved the way for achieving the introgression of target QTLs/genes and saved time and resources [43,44,45]. In MAS, foreground selection, background selection, and recombination selection played a vital role in the selection of RILs harbouring pyramided QTLs/genes with a maximum of recurrent genomes and a minimum of donor segments. The recurrent parent, CO 51, was recorded as having an average yield of 29.58 g, a blast score of 5.2 as moderately resistant, and a BB score of 9.91 cm. RIL #9-10-9-146 recorded a single plant yield of 42.21 g, a blast resistance score of 1.4, and a BB resistance score of 0.57 cm. RIL #9-10-8-23 was recorded as having an average yield, average blast score, and average BB score of 37.35 g, 1.8, and 0.37 cm, whereas RIL #9-20-2-98 recorded 36.45 g, 1.8, and 0.34 cm, respectively. RIL #9-10-8-25 recorded an average yield of 35.23 g, a blast score of 1.8, and a BB score of 0.58 cm. Transgressive segregation in the phenotypic traits of the RILs, viz., PH, NT, NPT, PL, total grain, FG, 1000-grain WT, and SPY, were recorded and compared to the recurrent parent, whereas DFF was recorded as earlier genotypes as compared with the recurrent parent [49,50,51]. BIL #6-17-5-18-5-1-5 recorded an SPY of 43.74 g, a blast score of 1.6 as a resistant reaction to blast pathogens, and a BB score of 0.31 cm as a resistant reaction to BB pathogens, and the SPY recorded in BIL #6-17-5-18-29-1-5 was 42.94 g, its blast score was 2.0 as a resistant reaction to blast pathogens, and its BB score was 0.30 cm as a resistant reaction to BB pathogens.
Among several R genes against diseases, the Pi9 gene conferring resistance was reported in previous studies [9,46,52] and is resistant against a broad-spectrum range of Indian blast isolates [43]. Among the R gene, the Pi54 gene confers resistance against M. oryzea [28]; Pi1, Pi2, and Pi9 are the most effective fungus races; and Pi9 is a major resistance gene, isolated from wild species Oryza minuta [24,25] which shows broad-spectrum resistance against a vast isolate of M. oryzea. In this study, Pi9 was stacked along with the Pi54 gene. The resistance level of CO 51 (plus Pi54 gene) showed a mean of 5.2 as moderate resistance, and donor parent 562-4 (plus Pi9 gene) showed 1.8 as a high-resistance reaction to blast pathogens (Table 4 and Table 5). The RILs harbouring Pi9 and Pi54 genes together, viz., RIL #9-10-8-23, #9-10-8-25, #9-20-2-98, #9-20-7-102, #9-20-9-112, #9-10-9-145, #9-10-9-146, and #9-10-9-152, and all BILs harbouring Pi9 and Pi54 genes together showed high resistance to blast pathogens in UBN (Table 4 and Table 5). The stacking of more than one R gene into the recurrent parent also revealed a strong and durable resistance being imparted to diverse isolates of blast pathogens [43]. The improved lines with R genes combinations of Pi54+Pi1+Pita, Pib+Pi9+Pi5, and Pi2+Pib+Pi5 were shown to impart a high level of resistance to a wider range of isolates of blast pathogens [43,53]. The RIL lines, viz., RIL #9-10-8-23 and RIL #9-10-8-25, harbouring both R genes Pi9 and Pi54 were recorded as highly resistant to blast pathogens. The RIL line harbouring Pi9 alone, viz., RIL #9-20-2-98, RIL #9-20-7-102, RIL #9-20-9-112, RIL #9-10-9-145, RIL #9-10-9-146, and RIL #9-10-9-152, also imparted high levels of resistance to blast pathogens over the recurrent parent CO 51. RILs harbouring Pi9 monogenic genes and exhibiting high resistance to blast than the RIL and BILs harbouring Pi9 and Pi54 genes were identified [54,55]. The Pi2 and Pi54 broad-spectrum resistance genes were stacked in Pusa Basmati 1509 against blast disease and can confer high resistance to various isolates of pathogens [45]. The genes Pi9 and Pi54 trigger an effector-induced immune response to pathogen infection through nuclear binding site leucine-rich repeats (NBS-LRR) and have a synergistic effect on the pathogen, to enhance resistance for a wider range of pathogen isolates [54]. In this study, Pi9 and Pi54 together conferred a highly resistant reaction to leaf blast and panicle blast and the additive effect was recorded in a leaf blast-pyramided line that harboured either Pizt or Pi9 and Pi54 [54]. However, together, these genes exhibited lower resistance likely due to the presence of incompatible reactions of the pathogen for Pi9 and Pi54 genes [56].
A challenge that appears in stacking a gene against BB is the distinct virulence of X.o pv., oryzae strains in different geographical regions [57]. Hence, F6 line with high resistance to BB strain, stacking more than one R genes, in contrast to single R genes to overcome the pathogenicity of X.o pv., oryzae strains that become virulent to stacked R genes [58]. In this study, xa13 and Xa21 genes were stacked in the RIL lines. The recurrent parent recorded a mean of 9.91 cm for BB symptoms, whereas donor parent 562-4 (harbouring xa13 and Xa21) recorded a mean of 0.29 cm for BB symptoms against X.o pv., oryzae. The RILs harbouring monogenic Xa21 alone recorded a mean of 0.37 cm to 0.79 cm for BB symptoms, whereas RILs harbouring monogenic xa13 alone recorded 0.37 cm for BB symptoms (Table 3). The RILs harbouring digenic xa13 and Xa21 together recorded BB symptoms ranging from 0.29 cm to 0.48 cm. Also, BIL BC2F6 harbouring digenic xa13 and Xa21 recorded 0.30 to 0.50 cm with an average of 0.37 cm against blight pathogens. RIL #9-20-7-102 showed symptoms of 0.29 cm on par with donor parent 562-4 (Supplementary Table S3). The R genes, viz., xa13 and Xa21 digenic or together, are resistant to broad-spectrum strains and impart resistance against the X.o pv., oryzae races in the Basmati growing region of India [44,45,59]. This study confirmed that, together, the R genes, viz., xa13 and Xa21, revealed the genetic potential of stacked genes at a phenotypic level in the southern part of India. Those selected R genes xa13 and Xa21 have been used widely to improve popular rice varieties such as Improved Pusa Basmati 1 [59], Pusa Basmati 1728, and Pusa Basmati 1718 [60]. The R-gene-stacked plant is invaded by BB strains X.o pv., oryzae, which possesses a gene pthXo1 for its virulence; gene xa13 is a recessive allele of gene Os8N3, a Nodulin family gene; and the transcript of xa13 is unresponsive to pthXo1, which reslts in resistance [61]. The other genes, Xa21, activated by RaxX protein, and tyrosine-sulphated protein from X.o pv., oryzae [62] encode receptor-like protein kinase [62,63]. Tyrosine-sulphated protein from X.o pv., oryzae triggers the immune defence responses in rice [64]. In plant immune response, WRKY transcriptional factors act as key regulators [65] and WRKY comprises a superfamily of mostly plant-specific transcriptional factors and a highly conserved WRKYGQK sequence at their N-termini [66,67]. A total of over 80 WRKY gene families have been identified in the rice genome [68] and have a region of approximately 60 amino acids containing a conserved WRKY amino acid sequence adjacent to a zinc-finger-like motif [66]. The RIL lines have been stacked with broad-spectrum resistance genes, viz., Pi9, Pi54, xa13, and X21, and have shown strong resistance to blast and BB pathogens. When plants are invaded by M. oryzae and X.o pv., oryzae, around 45 OsWRKYs are induced against early responsive genes to confer resistance reaction [69,70]. The resistance level of RILs could overexpress OsWRKY genes due to an immune response triggered by pathogens. Different studies revealed that the overexpression of OsWRKY71 and OsWRKY13 showed enhanced resistance to the X.o pv., oryzae pathogen [71,72], whereas a reduced expression of OsWRKY45 compromised resistance to the X.o pv., oryzae pathogen [73].
The superior RIL lines harbouring Pi9+xa13+Xa21 genes together (RIL #9-9-4-30, #9-20-2-98, #9-20-2-99, and #9-20-7-102) and BIL lines harbouring Pi9+Pi54+xa13+Xa21in (BIL #6-17-5-18-29-1-1, #6-17-5-18-29-1-2, #6-17-5-21-19-4-1, #6-17-5-18-5-1-5, and #6-17-5-18-5-1-6) imparted resistance (Supplementary Table S3 and Table 4) against biotic stress (M. oryzae and X.o pv., oryzae), which is probably a complete expression of OsWRKY genes in pyramided/stacked lines and was an additive effect of stacked genes. On the other hand, RIL lines, viz., RILs #9-10-10-6, #9-10-10-8, #9-10-9-145, #9-10-9-146, and #9-10-9-149, harbouring Pi9+Xa21 and #9-10-7-17 harbouring Pi9+Pi54+xa13 showed moderate resistance to M. oryzae and X.o pv., oryzae. It may have a reduced expression of OsWRKY genes in pyramid/stacked lines. The R gene Pi9+Pi54-pyramided lines, viz., RILs #9-10-7-17, #9-10-7-19, #9-10-8-21, #9-9-2-31, and #9-9-2-32, showed blast resistance scores of 2.8, 2.2, 4.0, 2.4, and 2.6, whereas RILs harbouring Pi9 alone, viz., RILs #9-20-2-98, #95-2-99, #9-20-2-99, #9-20-2-100, #9-20-7-102, and #9-10-9-145, recorded 1.8, 1.6, 1.6, 1.8, 1.2, and 1.7, respectively. RILs harbour Pi9 monogenic genes and exhibit high resistance to blast than the RILs harbouring Pi9 and Pi54 digenic genes [54].
The RILs and parental lines were screened against blast, bacterial leaf blight, and agro-morphological performance. The RILs were recorded as superior progeny compared to their parent. As compared with agronomic performance and phenotypic screening, 10 promising progenies in F6 (viz., RILs #9-10-8-23, #9-10-8-25, #9-20-2-98, #9-20-2-99, #9-20-2-100, #9-20-7-102, #9-20-9-112, #9-20-9-145, #9-10-9-146, and #9-10-9-152) and 5 promising progenies of BC2F6 (viz., BIL #6-17-5-18-5-1-5, #6-17-5-18-29-1-5, #6-17-5-18-29-1-6, #6-17-5-18-5-1-4, and #6-17-5-18-5-1-1) were selected as superior RILs and BILs as compared with their recurrent parent CO 51 (Supplementary Tables S2 and S3, Figure 6). In this study, RILs harbouring both genes, Pi9+Pi54 (RIL#9-10-7-17, #9-10-7-19, and #9-10-8-21) and xa13+Xa21 (RIL#9-20-2-96), displayed a moderate level of resistance to blast and BB, respectively. This may be due to the recombination between genes and markers that were used in this study [74], the interaction among QTLs/genes, and the possibility of antagonistic or synergistic interaction among QTLs/genes in the recurrent plant [1,35,75,76]. Because of climate change, different variants of pathogens still evolve across the environment and appear active, and immediate attention should be given to new biotypes in developing varieties with resistance to concerned biotic stress. From the successful introgression of Pi9, xa13, Xa21, Gm1, and Gm4 into CO 51+Pi54 through MAS in early generation and stringent phenotypic selection in advanced generation, pyramided lines impart resistance to blast and BB pathogens.

5. Conclusions

The present study was successful in stacking broad-spectrum resistance and durable genes against blast (M. oryzae), BB (X.o pv., oryzae), and gall midge (Orseolia oryzae) through marker-assisted selection in early generation. From the phenotypic selection of blast and BB in advanced breeding lines, the genes with broad-spectrum and durable resistance can reveal the complete expression of the gene in the CO 51 elite cultivar. The selected superior improved CO 51 can be used as a genetic stock in future breeding programs.

Supplementary Materials

The following supporting information can be downloaded at https://0-www-mdpi-com.brum.beds.ac.uk/article/10.3390/agriculture14050693/s1, Table S1: List of foreground markers and primer sequences; Table S2: Genotyping of F2 of CO 51 X 562-4 with FGS; Table S3: Phenotypic scoring of RILs against blast and BLB disease; Table S4: Agro-morphological performance of RILs population.

Author Contributions

S.S. and T.T., research, writing—original draft preparation; V.S. and B.A., data compilation and analysis; G.C., B.A., V.R.R. and R.K., technical support, supervision, screening; S.M., R.R., G.C., K.T., M.S. and R.M., conceptual editing of the manuscript; R.M., conceptualization, funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

Funding support from the Department of Biotechnology (grant number BT/PR13454/COE/34/43/2015), Government of India, New Delhi, India, is sincerely acknowledged. The first author S.S. sincerely acknowledges the Department of Biotechnology-Junior Research Fellowship Programme (DBT JRF) (Fellow Id. Number: DBT/2020/TNAU/1434), Department of Biotechnology, Government of India.

Data Availability Statement

The details of primer sequences, genotypic, phenotypic scoring data of Blast and BLB resistance and Agro-morphological performance of RILs population is given as Data Availability Statement.

Acknowledgments

We acknowledge the Department of Rice, Centre for Plant Breeding and Genetics (CPBG), Tamil Nadu Agricultural University (TNAU), Coimbatore, Tamil Nadu, India, for providing human resources and land resources for conducting field trials and also acknowledge the Department of Plant Biotechnology, Centre for Plant Molecular Biology and Biotechnology (CPMB&B), TNAU, Coimbatore, Tamil Nadu, India, for the support of laboratory research and technical support.

Conflicts of Interest

The authors declare no conflicts of interest.

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Agriculture 14 00693 g001
Figure 1. Flowchart depicting the breeding method for the development of RILs of CO 51 harbouring blast and BB-resistant genes.
Figure 1. Flowchart depicting the breeding method for the development of RILs of CO 51 harbouring blast and BB-resistant genes.
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Figure 2. Progenies of CO 51 X 562-4 (F6) genotyped with foreground markers.
Figure 2. Progenies of CO 51 X 562-4 (F6) genotyped with foreground markers.
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Figure 3. Foreground selection of selected BILs of BC2F6 harbouring Pi9, Pi54, xa13, Xa21, Gm1, and Gm4.
Figure 3. Foreground selection of selected BILs of BC2F6 harbouring Pi9, Pi54, xa13, Xa21, Gm1, and Gm4.
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Figure 4. (a) Blast screening of F6 harbouring Pi9- and Pi54-gene-stacked lines. (b) Blast screening of BC2F6 harbouring Pi9- and Pi54-gene-stacked lines.
Figure 4. (a) Blast screening of F6 harbouring Pi9- and Pi54-gene-stacked lines. (b) Blast screening of BC2F6 harbouring Pi9- and Pi54-gene-stacked lines.
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Figure 5. (a) BB screening in F6 stacked with xa13 and Xa21 genes. (b) BB screening in BC2F6 stacked with xa13 and Xa21 genes.
Figure 5. (a) BB screening in F6 stacked with xa13 and Xa21 genes. (b) BB screening in BC2F6 stacked with xa13 and Xa21 genes.
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Figure 6. Disease reaction of F6 of CO 51 X 562-4 and progenies against blast and BB screening.
Figure 6. Disease reaction of F6 of CO 51 X 562-4 and progenies against blast and BB screening.
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Figure 7. Performance of BILs of BC2F6 against blast and BLB pathogen.
Figure 7. Performance of BILs of BC2F6 against blast and BLB pathogen.
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Figure 8. Graphical representation of RIL yield increased over CO 51.
Figure 8. Graphical representation of RIL yield increased over CO 51.
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Figure 9. Graphical representation of increase in BIL yield compared to CO 51.
Figure 9. Graphical representation of increase in BIL yield compared to CO 51.
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Table 1. Target region from donor parent and position.
Table 1. Target region from donor parent and position.
Target TraitsTarget QTLs/GenesDonor ParentsChromosomePosition in MbReferences
Blast resistancePi9562-4610.38[26,36,42]
Pi54CO 511124.2[43,44]
BB resistancexa13562-4826.0[44,45,46]
Xa21562-41120.5[44,45,46]
Gall midge resistanceGm1562-499.20[35]
Gm4562-485.45[35]
QTLs: Quantitative Trait Loci, BB: bacterial leaf blight.
Table 2. Genotyping of 26 F6 progenies using markers linked to BB, blast, and gall midge resistance.
Table 2. Genotyping of 26 F6 progenies using markers linked to BB, blast, and gall midge resistance.
S. NoRILsGm1 (RM138)Gm4 (RM22550)Pi9
(NBS4)		Pi54
(Pi54MAS)		xa13
(xa13Prom)		Xa21
(pTA248)
1RIL #9-10-1-1-+++-+
2RIL #9-10-10-6+++--+
3RIL #9-10-10-8+++--+
4RIL #9-10-7-17-++++-
5RIL #9-10-7-19H+++H-
6RIL #9-10-8-21++++-+
7RIL #9-10-8-23++++-+
8RIL #9-10-8-25++++-+
9RIL #9-9-4-30--+H++
10RIL #9-9-2-31++++-+
11RIL #9-9-2-32++++-+
12RIL #9-9-2-34+++-H+
13RIL #9-9-2-35+++-H+
14RIL #9-9-13-61H-+-++
15RIL #9-20-2-96H++-++
16RIL #9-20-2-97H++-++
17RIL #9-20-2-98H++-++
18RIL #9-20-2-99+++-++
19RIL #9-20-2-100+++-++
20RIL #9-20-7-102H++-++
21RIL #9-20-9-112+++-H+
22RIL #9-10-9-145+++--+
23RIL #9-10-9-146+++--+
24RIL #9-10-9-149+++--+
25RIL #9-10-9-150+++-++
26RIL #9-10-9-152--+-++
RIL: Recombinant Inbred Line, +: presence of QTLs/genes, -: absence of QTLs/genes, H: heterozygosity of concerned QTL/gene.
Table 3. Selected progenies of BC2F5 of CO 51 harbouring six QTLs/genes.
Table 3. Selected progenies of BC2F5 of CO 51 harbouring six QTLs/genes.
S. No.BC2F5Gm1Gm4Pi9Pi54xa13Xa21No. of QTLs/Genes
1BIL #6-17-5-18-29-1-1++++++6
2BIL #6-17-5-18-29-1-2++++++6
3BIL #6-17-5-18-29-1-3++++++6
4BIL #6-17-5-18-29-1-5++++++6
5BIL #6-17-5-18-29-1-6++++++6
6BIL #6-17-5-18-29-1-7++++++6
7BIL #6-17-5-18-29-1-9++++++6
8BIL #6-17-5-21-19-1-1++++++6
9BIL #6-17-5-21-19-4-1++++++6
10BIL #6-17-5-21-19-4-3++++++6
11BIL #6-17-5-21-19-4-9++++++6
12BIL #6-17-5-18-5-1-1++++++6
13BIL #6-17-5-18-5-1-2++++++6
14BIL #6-17-5-18-5-1-4++++++6
15BIL #6-17-5-18-5-1-5++++++6
16BIL #6-17-5-18-5-1-6++++++6
BIL: Backcross Inbred Line, QTL: Quantitative Trait Loci, +: positive allele.
Table 4. Blast and BB scores of 16 progenies of BC2F6.
Table 4. Blast and BB scores of 16 progenies of BC2F6.
Blast ScoreBLB Score (in cm)
BC2F6Score IScore IIAverageScore IScore IIAverage
CO 514.45.95.2 (MR)7.4912.329.91 (S)
BIL #6-17-5-18-29-1-11.41.81.6 (R)0.280.450.37 (R)
BIL #6-17-5-18-29-1-21.41.81.6 (R)0.320.530.43 (R)
BIL #6-17-5-18-29-1-31.61.81.7 (R)0.250.520.39 (R)
BIL #6-17-5-18-29-1-51.82.22.0 (R)0.280.320.30 (R)
BIL #6-17-5-18-29-1-61.62.01.8 (R)0.270.440.36 (R)
BIL #6-17-5-18-29-1-71.82.62.2 (R)0.330.480.41 (R)
BIL #6-17-5-18-29-1-91.52.42.0 (R)0.320.560.44 (R)
BIL #6-17-5-21-19-1-11.82.22.0 (R)0.280.410.35 (R)
BIL #6-17-5-21-19-4-11.41.81.6 (R)0.240.390.32 (R)
BIL #6-17-5-21-19-4-31.43.02.2 (R)0.210.450.33 (R)
BIL #6-17-5-21-19-4-92.02.82.4 (R)0.480.520.50 (R)
BIL #6-17-5-18-5-1-12.43.02.7 (R)0.310.380.35 (R)
BIL #6-17-5-18-5-1-22.43.22.8 (R)0.300.440.37 (R)
BIL #6-17-5-18-5-1-41.62.42.0 (R)0.350.460.41 (R)
BIL #6-17-5-18-5-1-51.41.81.6 (R)0.240.380.31 (R)
BIL #6-17-5-18-5-1-61.21.41.3 (R)0.230.380.31 (R)
Mean of BILs1.72.22.0 (R)0.290.440.37 (R)
562-41.32.31.8 (R)0.230.340.29 (R)
CO 396.89.98.4 (S)---
TN1---15.0019.8217.41 (S)
SD1.51.71.63.934.994.46
SE0.30.40.40.901.151.02
CD (at 5%)1.241.151.191.191.211.20
BIL: Backcross Inbred Line; BB: bacterial leaf blight; CO 39 and TN 1 were used as checks in blast and BB screening, respectively; SD: Standard Deviation; SE: Standard Error; CD: Critical Difference; R: resistant; MR: moderately resistant; S: susceptible.
Table 5. Blast and BB scoring of selected superior progenies of the F6 population.
Table 5. Blast and BB scoring of selected superior progenies of the F6 population.
Blast ScoreBB Score in cm
RILsScore IScore IIAverageScore IScore IIAverage
RIL #9-10-8-231.81.81.8 (R)0.320.420.37 (R)
RIL #9-10-8-252.21.41.8 (R)0.370.790.58 (R)
RIL #9-20-2-981.42.21.8 (R)0.290.390.34 (R)
RIL #9-20-2-991.02.21.6 (R)0.280.380.33 (R)
RIL #9-20-2-1001.02.21.6 (R)0.290.390.34 (R)
RIL #9-20-7-1021.02.61.8 (R)0.240.340.29 (R)
RIL #9-20-9-1120.61.81.2 (R)0.260.360.31 (R)
RIL #9-10-9-1451.61.81.7 (R)0.290.680.49 (R)
RIL #9-10-9-1461.01.81.4 (R)0.360.770.57 (R)
RIL #9-10-9-1521.41.81.6 (R)0.430.530.48 (R)
CO 514.45.95.2 (MR)7.4912.329.91 (S)
562-41.32.31.8 (R)0.230.340.29 (R)
CO 396.89.98.4 (S)---
TN 1---15.0019.8217.41 (S)
Mean of RILs2.102.402.20 (R)0.300.500.40 (R)
SD1.7372.3842.0514.3856.0505.211
SE0.4820.6610.5691.2161.6781.445
CD (at 5%)1.381.241.310.491.120.80
RIL: Recombinant Inbred Line; BB: bacterial leaf blight; CO 39 and TN 1 were used in blast and BB screening, respectively; SD: Standard Deviation; SE: Standard Error; CD: Critical Difference; R: resistant; MR: moderately resistant; S: susceptible.
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Subburaj, S.; Thulasinathan, T.; Sakthivel, V.; Ayyenar, B.; Kambale, R.; Rajagopalan, V.R.; Manickam, S.; Rajasekaran, R.; Chellappan, G.; Thiyagarajan, K.; et al. Genetic Enhancement of Blast and Bacterial Leaf Blight Resistance in Rice Variety CO 51 through Marker-Assisted Selection. Agriculture 2024, 14, 693. https://0-doi-org.brum.beds.ac.uk/10.3390/agriculture14050693

AMA Style

Subburaj S, Thulasinathan T, Sakthivel V, Ayyenar B, Kambale R, Rajagopalan VR, Manickam S, Rajasekaran R, Chellappan G, Thiyagarajan K, et al. Genetic Enhancement of Blast and Bacterial Leaf Blight Resistance in Rice Variety CO 51 through Marker-Assisted Selection. Agriculture. 2024; 14(5):693. https://0-doi-org.brum.beds.ac.uk/10.3390/agriculture14050693

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Subburaj, Samuthirapandi, Thiyagarajan Thulasinathan, Viswabharathy Sakthivel, Bharathi Ayyenar, Rohit Kambale, Veera Ranjani Rajagopalan, Sudha Manickam, Raghu Rajasekaran, Gopalakrishnan Chellappan, Kalaimagal Thiyagarajan, and et al. 2024. "Genetic Enhancement of Blast and Bacterial Leaf Blight Resistance in Rice Variety CO 51 through Marker-Assisted Selection" Agriculture 14, no. 5: 693. https://0-doi-org.brum.beds.ac.uk/10.3390/agriculture14050693

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