Development of submergence tolerance introgression lines (ILs)-F 5 using major Sub1QTL on chro9 from BRRI dhan52 in rice (Oryza sativa L.) through Marker-Assisted Selection (MAS)

doi:10.21203/rs.3.rs-7306050/v1

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Development of submergence tolerance introgression lines (ILs)-F 5 using major Sub1QTL on chro9 from BRRI dhan52 in rice (Oryza sativa L.) through Marker-Assisted Selection (MAS)

https://doi.org/10.21203/rs.3.rs-7306050/v1

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Rice is a highly polymorphic crop species with a wide geographical distribution. Molecular markers are important tools for breeding selection, genotype detection, and studying the organization and evolution of plant genomes. The main objective of this study was to introgress Sub1 QTL into high yielding rice cultivars for developing submergence tolerance with Sub1 gene fix in selected lines. The band corresponded to an allele for F₁ confirmation from the susceptible parent Binadhan-17 and the tolerant one BRRI dhan52, as 159 bp and 169 bp bands, respectively, at locus SC32/RM23668. The lowest survival% (32.75 %) was recorded in F₂-6 and the highest (68.25 %) in F₂-12 plant numbers from Binadhan-17 x BRRI dhan52. In F₃generation, the highest survival rate (%) at vegetative stage was obtained from Binadhan-17 x BRRI dhan52 (F₃) 94.25% in F₃-15, then the other crosses were 93.25% in F₃-11 from Binadhan-7 x BRRI dhan52 (F₃) and Guti swarna x BRRI dhan52 (F₃) in F3-10 of 85.25%. All F₁ confirmation plants with Sub1 introgression had significantly lower survival rates than the original parent and survival rates and Sub1 gene expression were significantly higher with homozygous tolerant selected lines than heterozygote plants. The introgressed lines of the crosses at F₄ generations were designated as BPRLs BPR21 (P7 = BPR21-1-S, P9 = BPR21-2-S, P11 = BPR21-S-S, P12 = BPR21-S-M), BPR22 (P12 = BPR22-1-S, P13 = BPR22-2-S, P15 = BPR22-S-S) and BPR17 (P2 = BPR17-S-M, P10 = BPR17-S-17) showed homozygous amplification of Sub1 gene. As the tolerant allele at Sub1C on chromosome 9 was fixed as Sub1C173 and ERF3 in this selected population, additional QTL responsible for submergence tolerance were expected to be revealed. The antioxidants such as SOD, POD, CAT, APX and GPX activities increased under submergence stress compared to respective controls. BPR17-S-M, BPR21-S-M and BPR22-S-S showed higher antioxidant activity compared to the control. Higher SOD, POD, CAT, APX and GPX enzyme activities indicate greater mitigating levels of O₂•- and H₂O₂ under submergence stress. Importantly, under stress conditions, three breeding lines, BPR21-S-M (19.33 g), BPR22-S-S (20.23 g) and BPR17-S-M (15.26 g), produced the most grain yield per plant, which was significantly higher than the donor parent of BRRI dhan52 (12.71 g) and also was found to be 3.00 g to 8.00 g more than the parent and 20 g to 25 g less than the non-flooded condition. However, three Sub1 lines; BPR21-S-M, BPR22-S-S and BPR17-S-M showed better yield contributor with very good recovery ability after submergence stress under field conditions. It could be concluded that these three newly developed promising Sub1 lines will be used to develop high yielding submergence tolerant (21 days) cultivars for flood prone areas in Bangladesh. It will be ensured the food security of the nation by continuing rice production cold prone areas in Bangladesh

DNA

F1 hybrid

Sub1

marker-assisted selection

submergence tolerance

SSR primer

antioxidant

The lines of Binadhan-7-Sub1, Binadhan-17-Sub1 and Guti swarna-Sub1 showed homozygous for Sub1 gene in the F₄ generation as confirmed by the gene specific primers Sub1C173.
It was confirmed that three promising lines as BPR21-S-M, BPR22-S-S and BPR17-S-M showed better performance in yield with very good recovery ability against submergence stress in field conditions.
Maturity duration of three lines as BPR21-S-M, BPR22-S-S and BPR17-S-M was shorter than their respective submergence tolerant parents.
Limited increase in H₂O₂ and O₂•- content and higher activities of SOD, POD, CAT, APX & GPX were found to improve submergence stress tolerance in rice.

The 'Vision 2041' initiative has been established to fulfill the developmental aspirations of the Government of Bangladesh. Its objective is to eliminate poverty and transform Bangladesh into a developed nation free from hunger by the year 2041, thereby embodying the concept of 'Golden Bangladesh'. Rice serves as a crucial element in the fight against poverty and hunger, being the staple food of the nation, often referred to as a 'global grain' [1–3] and encapsulated in the phrase 'Rice is life' [4–6]. However, its production is experiencing a gradual decline due to factors such as submergence, climate change, and infrastructure development [7]. By 2035, it will be necessary to achieve a 26% increase in rice production to sustain the growing population [8]. Submergence represents an unavoidable abiotic stress impacting 22 million hectares of lowland rainfed rice farms globally, which constitutes 18% of the worldwide rice supply [9–10]. Furthermore, economic losses attributed to this issue have reached up to one billion US dollars [11–13]. In South and Southeast Asia, submerged stress regularly impacts over 15 million hectares of rain-fed lowland rice fields [14, 15]. In Bangladesh, flash floods have affected more than 2.0 million hectares of land, resulting in an average yield loss of approximately 5% [6, 16, 76]. Consequently, it is imperative to develop high-yielding mega varieties that exhibit early maturity and submergence tolerance, as well as to assess the performance of Sub1 lines across various genetic backgrounds through Marker Assisted Selection (MAS).

The Sub1QTL located on chromosome 9 was responsible for approximately 70% of the phenotypic variation observed for survival in submerged conditions and was fine-mapped to gene clusters on chromosome 9 and QTL clones [3, 6, 17]. The genetics of submergence tolerance in rice identified three genes (Sub1A, Sub1B, and Sub1C) within the Sub1 (Submergence 1) QTL region, which is situated near the centromere of chromosome 9. The Sub1 QTL plays a crucial role in conferring the submergence tolerance phenotype during the seedling stage in rice. The Sub1 genes are known to encode ethylene response factors. The accumulation of Sub1A and Sub1C mRNA was significantly but temporarily induced by submergence, and this effect was diminished in the seedling leaves of the tolerant indica cultivar FR13A. Importantly, stable transformation of japonica rice with Sub1A-1 resulted in the down-regulation of Sub1C, which conferred ectopic expression and submergence tolerance [19–21]. The cloning of the major Sub1QTL has provided an exceptional opportunity to enhance our understanding of the molecular mechanisms and to reveal the pathways that underlie submergence tolerance, as well as to develop gene-based or closely linked markers for more accurate genotyping. A significant QTL, derived from Ciherang-Sub1 and designated qSUB8.1, was identified on chromosome 8, exhibiting a LOD score of 10.3 and a phenotypic variance of 27.5%. Furthermore, a minor QTL, also derived from Ciherang-Sub1, was discovered on chromosome 2, with a LOD score of 3.5 and a phenotypic variance of 12.7% [10]. Submergence tolerance is governed by a single major quantitative trait locus (QTL) located on chromosome 9, alongside several minor QTLs [17, 22, 23]. This research utilized the traditional genotype FR13A, recognized as one of the most submergence-tolerant donor varieties. The primary QTL, referred to as Sub1, exhibited a LOD score of 36 and an R 2 value of 69% [17], which provided tolerance to complete submergence for a duration of up to 2 weeks. Fine-mapping of Sub1 involved 2950 F₂ segregating individuals. More recently, this gene was effectively introgressed through marker-assisted backcrossing (MAB) from the Swarna genotype (India) into a widely cultivated high-yielding variety within a span of 2 years [14]. Despite the low recombination rate in this region, Sub1 was confined to a genomic area of approximately 0.06 cm [18]. Prior research has indicated the creation of submergence-tolerant cultivars by incorporating the Sub1 locus through marker-assisted selection [24]. Nevertheless, these initiatives have not been substantiated to leverage marker-assisted selection specifically and efficiently for gene transfer. Utilizing BC₂F₂ segregation data for submergence SSR analysis, it was demonstrated that the gene is associated with a single copy DNA clone, RM23805, located on chromosome 9 at a distance of 0.06 cM [18]. Therefore, it is essential to consider the opportunity to commence marker-assisted selection. In this article, we present the development of a PCR-based DNA marker derived from the RM23805 clone [3, 60]. (Fig. 1). These findings illustrate the effectiveness of marker-assisted backcross selection as a supplementary method to conventional breeding.

Numerous research studies have pinpointed quantitative trait loci (QTL) associated with submergence tolerance from various populations, as illustrated in Fig. 2 [3, 22–27]. The identification of significant quantitative trait loci (QTL), particularly the Submergence 1 (Sub1) locus, has marked a significant advancement in breeding for submergence tolerance, addressing a critical challenge. The cloning and isolation of the Sub1 locus from the well-known submergence-tolerant variety FR13A led to the discovery of the ethylene-responsive factor (ERF) gene SUB1A-1, which plays a crucial role in submergence tolerance. The Submergence 1 (Sub1) locus comprises a cluster of three ethylene-responsive factor (ERF) genes: Sub1A, Sub1B, and Sub1C. The identification of the Sub1 gene has facilitated the implementation of active marker-assisted selection (MAS) for enhancing submergence tolerance. Certain resistant rice cultivars preserve their vigor by suppressing plant elongation in fully flooded conditions, with their submergence response regulated by Sub1A, which encodes the Ethylene response factor. Research indicates that rice varieties possessing the Sub1A-1 allele, such as FR13A, exhibit greater resistance to water damage, surviving for up to two weeks under complete submersion, whereas those with the Sub1A-2 allele do not possess this capability.

The physiological mechanism was synthesized in the rice-tolerant varieties containing Sub1A Ethylene was synthesized in the plants under drowning conditions. On the one hand, ethylene promoted the degradation of ABA and promoted the expression of Sub1A. Sub1A promoted the accumulation of inhibitors SLR1 and SLRL1 in GA signaling and inhibited it. The response of GA inhibits the elongation of plants above the ground under water, reduces the consumption of carbohydrates, and ultimately improves the ability to withstand mites. The submerged sensitive rice varieties that do not contain the Sub1A gene are synthesized in the plants under drowning conditions. On the one hand, Ethylene encourages ABA's deterioration and prevents the transmission of SLR1 and SLRL 1 inhibitory factor in GA signaling, so that the plant reacts to GA. The plants prolongs under the water and consumes sugar, finally sinking. Sensitive phenotype. The Sub1 genes in rice encodes three ethylene response factor ERF and the Sub1A allele carrying rice is more resistant to the floods. To elucidate the molecular mechanism of Sub1A-1 mediated flood tolerance, the authors analyzed and compared the M₂0₂ variety with Sub1A-1 and Compared with the submerged transcriptome of the sub1A-free genus, 898 genes were found to be regulated by Sub1A-1, and the existing published metabolic pathway data were integrated, and the flood-tolerant pathway was compared with anaerobic respiration and phytohormone response. The antioxidant system is involved in an attractive group of AP2/ERF protein transcript families that are involved in the SubA-1 mediated flood tolerance. The analysis of the phylogenetic tree concerning the expression model of the visible AP2/ERF gene superfamily can categorize 12 flood-controlled AP2/ERF genes into three distinct groups; the first group pertains to the accumulation of ethylene under flooded conditions, which leads to anaerobic respiration and cytokinin-mediated delays in cell senescence, involving three ERFs. The second group consists of five ERFs that are regulated by the negative control of ethylene. Lastly, the third group is associated with gibberellin's negative control related to stem longevity. The findings confirmed that the SubA-1 gene influences various metabolic pathways that respond to flood stress [64] as illustrated in (Fig. 3). During the submergence stress experienced by rice, reactive oxygen species (ROS) such as superoxide (O₂-), hydrogen peroxide (H₂O₂), and hydroxyl radicals are rapidly generated. These species inflict oxidative damage on lipids, proteins, and nucleic acids, disrupting common metabolic processes [28]. To mitigate the effects of oxidative stress and reactive oxygen species, plants have evolved several active oxygen scavenging systems, which include both antioxidant enzymes and non-enzymatic antioxidants. Among these, superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), glutathione reductase (GR), and dehydroascorbate reductase (DHAR) are crucial in protecting plants from oxidative stress-induced damage [55]. Superoxide (O₂-) is primarily scavenged by SOD through the dismutation of O₂- into H₂O₂ [29].

The identification of the Sub1 gene led to the development of eight enhanced mega varieties (Swarna-Sub1, Samba Mahsuri-Sub1, IR64-Sub1, BR11-Sub1, Thadokkam1-Sub1, CR1009-Sub1, PSBRc18-Sub1, and Ciherang-Sub1) at the International Rice Research Institute (IRRI) utilizing a precise marker-assisted backcrossing (MABC) approach [10,14,30–34]. BRRI dhan52 (BR11-Sub1) possesses the Sub1QTL, which enables it to withstand flash floods for two to three weeks in Bangladesh, and this QTL has been introgressed into several high-yielding varieties (HYV) at IRRI [3,6, 10,31]. However, in the northern regions of Bangladesh, such as the Rajshahi and Rangpur divisions, the duration of flash floods can extend to approximately three to four weeks. Currently, the varieties that have been released in Bangladesh are unable to endure such stress levels (3–4 weeks). Consequently, it is essential to introduce new submergence-tolerant QTLs alongside high-yielding varieties. Binadhan-17 possesses a native allele that may confer tolerance to submergence for a few days (up to 7 days). Therefore, it is anticipated that complementary or supplementary gene actions will be achieved for an extended duration of submergence tolerance in the pyramided lines. Binadhan-17 is an early-maturing rain-fed rice variety with a growth duration of 114 days; however, it lacks the Sub1QTL and is susceptible to submergence stress. The main objective of this study was to introgress Sub1QTL into high yielding rice cultivars for developing their submergence tolerant genotypes. This study was conducted to convert the early-maturing Binadhan-17 into submergence-tolerant genotypes by introgressing the Sub1QTL from BRRI dhan52 through Marker Assisted Selection (MAS). It is expected that the high-yielding Binadhan-17, specifically the Binadhan-17-Sub1 lines will be better suited for flood-prone areas and favored by farmers in the northern regions of Bangladesh.

2.1 Development of plant population

Four rice (Oryza sativa L.) cultivars were used in indica and the population was technically improved in this study from three cross combinations. BRRI dhan52 was selected as a donor for submergence because it is a high yielding, flash flood tolerant, rainfed lowland rice variety with bearing Sub1QTL. Female parents like Binadhan-7 and Binadhan-17 are improved high yielding varieties developed by BINA with low nitrogen fertilizers, with short duration of 110–120 days. Guti Swarna is a local high yielding lowland condition and tall tree type variety. F₁ heterozygous plants for Sub1 were obtained from three crosses Binadhan-7 × BRRI dhan52, Binadhan-17 × BRRI dhan52 and Guti Swarna ×BRRI dhan52 with pedigree number BPR21, BPR22 and BPR17 respectively. F₁ plants were phenotypically and genetically confirmed using especially polymorphic primers. One thousand fifty F₂ seeds were produced in each confirmed F₁ plant from each cross combination. About 600 F₂ seeds were planted in trays. One head panicle per plant was picked and collected for the F₃ generation (Fig. 4).

2.2 Imposition of submergence in artificial tanks.

Three indica F₂ populations were generated from three crosses as Binadhan-7 × BRRI dhan52, Binadhan-17 × BRRI dhan52 and Guti Swarna × BRRI dhan52. About 600 F₂ plants were used for submerged screening with parents and some checks in artificial submergence tanks at Bangladesh Institute of Nuclear Agriculture (BINA). To determine the level of submergence tolerance of recombinants and F₂ hybrids. Submergence assays and expression studies were conducted for both recombinant and F₂ progeny as described by [19]. The F₂ lines and the parent are soaked until the shoots. They were sown in plastic trays. Ten seeds from each F₂ and parents were completely submerged in 30-day-old seedlings with their checks. When checks show 50% damage, usually about 14–21 days after complete submergence, trays were de-submerged and recovery was scored after 7 days. For the vegetative stage, the F₃ generation from each combination was transplanted at twenty-one-day old seedlings and submersed 48 days after transplanting (DAT) in artificial tanks (Figure S5). The water level should be raised rapidly and a depth of at least 80–100 cm maintained for 21 days as the standard period of treatment. Plant survival was scored 21 days after desubmergence and percent survival was calculated for data analysis.

2.3 DNA marker analysis

For the marker assay, a tightly-linked primer (RM8300) and/or two gene specific markers (ERF3 and Sub1C173), a polymorphic marker (RM23668) [14,20] and 54 SSR markers for polymorphic markers between the donor and recurrent parents and identified through a primer survey (Table S1) were used for genotype selection at the Biotechnology Lab, BINA. Using sub1 specific primers such as ERF3 and Sub1C173, which are amplified in genotypes containing the Sub1 QTL. Thus, all parental selection was done for Sub1QTL and then F₄ plants from each cross combination were confirmed at Sub1QTL. Leaf segment (2–3 cm pieces) of each genotype were used for DNA extraction using the Cetyl Trimethyl Ammonium Bromide (CTAB) mini-prep method [36] carried out at the Biotechnology Laboratory, Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh. Bangladesh. The CTAB method described by [37] is modest and rapid compared to other methods and does not require liquid nitrogen [77]. PCR was performed using the method of [38]. PCR conditions were 94°C for 2 min, 30 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, and a final extension at 70°C for 5 min and finally hold at 10°C for infinity time. The PCR product was mixed with bromophenol blue gel loading dye and analyzed by electrophoresis on a 6% polyacrylamide gel (PAGE) for all SSR primers using mini vertical polyacrylamide gels for high throughout manual genotypes. All SSR markers were selected from Gramene database (http://www.gramene.org/). Genotypic data and phenotypic data were analyzed using IciMapping4.0 software. The size of each allele of the SSR primer was quantified bymolecular weight using Alpha-Ease 5.5 software. Allele sizes of SSR markers were tested using the program Power Marker version 3.25[38].

2.3 Biochemical Analysis

For detection and determination of different types of ROS, 21-day-old seedlings were transplanted and submerged at 48 DAT in artificial tanks. Plants grown under normal conditions were used as controls. Leaf tissue from desubmerged or nonsubmerged control plants was excised for this Biochemical analysis. For H₂O₂ estimation, the method was followed as described by [39] with some modifications at Molecular Breeding Laboratory, BARI, Gazipur. Afresh sample of 0.5 g was homogenized in 3 ml of 5% TCA solution using an ice-cold mortar and pestle. The homogenized sample was centrifuged at 11,500 rpm for 15 min and 500 µl of supernatant was collected in a Falcon tube. An assay mixture containing 500 µl 10 mM potassium phosphate buffer (pH 7) and 1 ml 1 M potassium iodide (KI), solution and 500 µl supernatant was added. The solution was kept in a dark condition for 1 hour and finally the absorbance of the supernatant was measured at 390 nm using a spectrophotometer. Calculate the data from the standard curve (0–90 µM). Prepare blank sample 0.1% TCA instead of leaf extract. Superoxide radicals (quantification of O₂•-) were determined according to the method of [40] with slight modifications. Leaves (0.3 g) were homogenized in 3 ml of extraction buffer (EB) made consisting of 2.6 ml of 65 mM (K-P) buffer (pH 7.8) with 17.4 ml of distilled water. The mixture was then centrifuged at 5,000 × g for 10 min at 4°C. The resulting supernatant (750 µl) was vortex mixed with 675 µl extraction buffer (EB) and 70 µl 10 mM hydroxylamine hydrochloride in a plastic tube with a green cork and incubated for 25 min at 25°C. Added 375 µl of 17 mM sulfanilamide and 37.5 µl of 7 mM α -naphthylamine were added to 337.5 µl extraction buffer were vortexed. The mixture was then combined with 2.25 ml of diethyl ether and cover it otherwise the ether will evaporate. Vortex and keep it for 10 minutes (cover it) taken the super clean part and absorb immediately. Autozero was made with ether and its absorbance was measured at 530 nm. From each sample, 0.5 g of leaf tissue was homogenized in 1 ml of 50 mM ice–cold K–P buffer (pH 7.0) containing 100 mM KCl, 1 mM ascorbate, 5 mM mercaptoethanol and 10% (w/v) glycerol. The homogenates were centrifuged at 11, 500 ×g for 10 min and the supernatants were transferred to a tube which was used for determination of enzymes (SOD, POD, CAT, APX and GPX) activity.

2.4 Seedling performance

Seedling height (cm): Seedling height of five plants was measured at 30 days only seedling stage.

Seedling weight (g): Seedling weight was measured from 5 plants taken just before planting.

Seedling strength (g/cm): The parameter was measured by a formula as follows:

Recovery ability: It was recorded 5 and 30 days after desubmergence based on tillering ability, % survivability, vegetative growth etc. following [35].

2.5 Field evaluation under submergence prone areas

Eleven lines with the chick variety were evaluated in the fields of these farmers and the experimental design was a randomized complete block design with three replications. Each plot (5.4 m x 10 row plot) was planted at 20 x 20 cm spacing. Thirty-day-old seedlings of each genotype were planted in 2–3 seedlings/hill. Fertilizer dose will be 133:112:75: 60:11 kg/ha of urea, TSP, MP, gypsum and ZnSO4. All fertilizers except urea were applied as basal before final land preparation. Urea fertilizer was applied as top dressing with 3 equal splits at 15-20DAT, 30-35DAT and 45–50 DAT. Furadan 5G was applied twice with second and third top dressing of urea fertilizer. Standard crop management practices such as weeding, disease control and pest control were followed when necessary. Experimental plots were submerged for 21 days starting at 30 days after transplanting. The average water depth was 90 cm. The average water temperature recorded was 30°C, and the water pH was 7.1. After complete submergence, water was completely drained from the plot. The following variables were measured plant height (measured from the soil line to the tip of the flag leaf), date of 50% tillering and date of maturity. Five panicles per plant and five plants per plot were harvested at physiological maturity. Total seed weight, 1,000-grain weight, and total seed number were also determined. Total tillers/plant, Effective tillers/plant, panicle length (cm), unfilled grain/plant, yield/plant (g) were recorded for each population in the trial. Data were analyzed using igetintopc.com_Minitab_18.1 software for yield and yield contributing characters of rice genotypes.

3.1 Molecular Screening to Identify Sub1QTL

The initial phase in discovering new QTL for submergence tolerance involves identifying novel germplasm accessions that exhibit the same level of submergence tolerance as the Sub1 line, yet do not carry the FR13A-derived Sub1QTL allele. In Bangladesh, there is a scarcity of molecular-level information regarding submergence-tolerant germplasm. Molecular markers can highlight variations among accessions at the DNA level, serving as a more direct, dependable, and efficient instrument for crop enhancement in plant breeding. This research was conducted to pinpoint new sources of submergence-tolerant donors by characterizing the Sub1 region utilizing the Sub1C173 DNA marker genotype across a selection of newly characterized submergence-tolerant accessions from Bangladesh. The findings of this study are anticipated to facilitate the discovery of new submergence tolerance QTL linked to Sub1, which can be utilized to breed for enhanced levels of submergence tolerance. Molecular screening of submergence-tolerant landraces indicated that FR13A is comparable to the submergence-tolerant high-yield variety (HYV) BRRI dhan52 and possesses Sub1QTL. Local cultivars such as Suman swarna, Ranjit swarna, Rangina swarna, Mamun swarna, Sada Guti swarna, Guti swarna, Nepali swarna, along with HYVs like BRRI dhan49, Binadhan-7, and Binadhan-17 do not contain Sub1QTL and are dissimilar to BRRI dhan52, which served as a submergence-susceptible check variety, as Sub1C173 was not amplified (Figure S1).

3.2 Marker specificity and gene diversity

A total of 350 microsatellite or simple sequence repeat (SSR) primers were utilized for the parental survey [41-43]. All data regarding SSR primers were obtained from the Gramene database (www.gramene.org). A polymorphism survey was conducted using 350 microsatellite markers, including RM286, SSR1, RM495, RM1178, RM126, RM316, RM153, RM23770, RM23668, RM5799, RM1115, RM296, RM2367, ERF3, Sub1C173, RM8300, RM23901, RM231, RM518, RM587, RM234, RM5799, RM5708, RM4112, RM313, RM296, RM3609, SC36, RM23778, RM23679, RM23805, RM23843, RM23902, RM23915, RM219, RM23917, RM23958, RM23843, and RM23915. For the primer survey, the DNA bands of all genotypes, such as FR13A, BRRI dhan52, Guti swarana, Mamun swarna, Rongina swarna, Bilati swarna, Suman swarna, Nepali swarna, Sadaguti swarna, Ronjit swarna, Binadhan-17, and Binadhan-7, were scored based on their molecular weight using polyacrylamide gel electrophoresis. Bands produced by primers that amplified across multiple levels were classified as polymorphic markers, while primers yielding bands at the same level were designated as monomorphic markers. Conversely, primers that did not yield any bands were categorized as non-amplified. The polymorphic flanking markers of Sub1QTL, namely SC32/RM23668, RM495, RM1115, and RM153, located on chromosomes 1, 5, and 9, were employed to assess the F₁ generation between the recipient and donor parent BRRI dhan52 by amplifying the DNA sequence associated with submergence tolerance. A total of 139 alleles were identified from 16 primers across 16 genotypes. The number of alleles per locus varied from 7 to 11, with an average of 8.688 alleles per locus. RM316, RM153, and ERF3 exhibited the highest allele count (11), while SC11/RM23770 and RM1115 produced the lowest (7). The average allele frequency (%) was 0.118, and gene diversity values ranged from 0.766 (SSR1) to 0.891 (RM316). The polymorphism information content (PIC) values differed among loci, ranging from 0.735 to 0.881, with an overall average of 0.830 across all primers (Table S3).

3.3 Wide compatibility and hybrid potency

Analyzing the success rate of F₁ seeds resulting from crosses between various varieties of two ecotypes and assessing the compatibility of these combinations. Notable differences were noted in the success rates of F₁ seed production across different cross combinations. The combination of Binadhan-7 and BRRI dhan52 demonstrated a comparatively higher success rate of 78.26% than the other combinations. Among the three combinations, all exhibited negative heterosis, with the mid-parent combination of Bandhan-17 and BRRI dhan52 (F₂) showing a significant negative heterosis of -7.06%, while Binadhan-7 and BRRI dhan52 (F₂) recorded the least negative heterosis at -4.62% from mid-parent to maturity day. All crosses with negative heterosis were observed to be better than their respective parents. The heterobeltiosis ranged from -17.33% to -5.33%. Notably, the cross of Binadhan-7 and BRRI dhan52 (F₂) displayed a higher negative heterosis of -17.33% compared to the better parent (heterobeltiosis). When considering mid-parent heterosis and heterobeltiosis, excellent heterotic combinations for grain yield per plant were identified. All combinations, with the exception of the Guti Swarna and BRRI dhan52 (F₂) cross, exhibited positive heterosis relative to the mid-parent. Consequently, the highest positive heterosis was recorded in 45.71% of all crosses, while significantly positive heterotic combinations accounted for 37.29% of the total combinations. In terms of heterobeltiosis, heterosis varied from -21.43% to 35.71% in comparison to the better parent. The most significant positive value of 35.71% was noted in the Binadhan-7 and BRRI dhan52 (F₂) cross, followed by the Binadhan-17 and BRRI dhan52 (F₂) cross at 28.31% (Table 1).

3.4 Genotypic confirmation of F₁by locus SC32/ RM23668 on the top of chromosome 9

The development of the population Binadhan-7, Binadhan-17, and Guti Swarna served as susceptible parents in the cross, while BRRI dhan52 was utilized as a donor due to its possession of Sub1QTL. This led to the confirmation that F₁ plants were cultivated and self-pollinated to produce BC₁F₁ and F₂ populations for marker-assisted selection (MAS). The F₁ plants exhibiting heterozygous bands for SC32/RM23668 were validated as true F₁s. Gel images indicated that the heterozygous bands included bands from both parental lines, confirming the authenticity of the F₁s. Leaf samples from F₁ plants (1–15) and their respective parents were collected for DNA extraction and marker assay. The confirmation of the F₁ population from the cross of Binadhan-7 and BRRI dhan52 (F₁) was achieved using the polymorphic primers SC32/RM23668. Plant numbers 1, 8, 10, and 14 did not exhibit heterozygous bands, whereas plant numbers 2, 3, 4, 5, 6, 7, 9, 11, 12, 13, and 15 displayed heterozygous bands, confirming their status as F₁s and were selected (Figure S2). In the case of the Binadhan-17 and BRRI dhan52 cross, the confirmed F₁ population included plant numbers 2, 3, 4, 5, 6, 7, 10, 11, 12, 13, and 15, while BC₁F₁ and F₂ seeds were produced from the selected plants, with plant numbers 1, 8, 9, and 14 being excluded. Furthermore, for the Guti Swarna and BRRI dhan52 cross, the F₁ population confirmed plant numbers 2, 4, 5, 7, 9, 10, 13, and 14, while plant numbers 1, 3, 6, 8, 11, 12, and 15 did not exhibit heterozygosity. The estimated genotypes at the SC32/RM23668 locus on chromosome 9 were determined using various banding scores, with P1 representing the female parent (homozygous band = 1), P2 as the donor parent (homozygous band = 1), heterozygous plants (3), and no band = 0, respectively. The amplicon sizes for all genotypes concerning the RM23668 marker allele were measured and are presented in Table S2.

3.5 Phenotypic and Genotypic variation of F₂

The F₂ progeny resulting from the crosses of Binadhan-7 x BRRI dhan52, Binadhan-17 x BRRI dhan52, and Guti Swarna x BRRI dhan52 exhibited significantly lower tolerance compared to the tolerant parent, BRRI dhan52. Furthermore, the F₂ plants derived from Guti Swarna x BRRI dhan52 demonstrated less tolerance than those from the other two cross combinations involving Sub1. It was also observed that the breeding line of Guti Swarna x BRRI dhan52, which contained a heterozygous Sub1 locus, showed reduced tolerance to submergence stress. A tolerated version of Guti Swarna x BRRI dhan52 was developed using a method similar to that employed for Binadhan-17 x BRRI dhan52. Among the 45 total F₂ plants genotyped from the three crosses, line #29 was selected. These initial studies indicated that for maintaining a high level of tolerance in hybrids, the tolerant allele of Sub1 must be present in both parental lines. Conversely, the phenotypic performance of the Binadhan-7 x BRRI dhan52 (F₂) cross was superior, yielding 38.00 g, compared to the Guti Swarna x BRRI dhan52 (F₂) cross combination, which yielded 22.00 g. The Binadhan-7 x BRRI dhan52 (F₂) cross exhibited the highest grain yield per plant at 38.00 g, significantly surpassing both parental lines. Other high-yielding cross combinations, such as Binadhan-17 x BRRI dhan52 (F₂), yielded 31.00 g, which was also significantly higher than their respective parents. Therefore, the high-yielding cross combination of Binadhan-7 x BRRI dhan52 (F₂) achieved the desired grain yield per plant. Both the Binadhan-7 x BRRI dhan52 (F₂) and Binadhan-17 x BRRI dhan52 (F₂) cross combinations were early maturing, taking 124 days and 125 days, respectively. In the initial applications of Marker-Assisted Breeding (MAB) to develop submergence-tolerant cultivars, the diagnostic marker RM8300 was utilized. However, in certain instances, RM8300 failed to distinguish between tolerant and non-tolerant varieties within the F₂ population. Nevertheless, a moderate tolerance for heterozygous hybrids can prove advantageous in certain circumstances where a blend of moderate submergence tolerance and moderate elongation capacity is sought. This is particularly relevant in scenarios where prolonged stagnant partial flooding is anticipated either prior to or following inundation.

3.6 Performance of heterozygous Sub1 plants

The submergence tolerance of F₁ hybrids from three cross combinations was assessed in comparison to their parent plants. The heterozygous individuals carrying the tolerant allele exhibited significantly lower tolerance than those that were homozygous for the same allele. Furthermore, the expression of the Sub1 allele in heterozygotes was found to be less than that in homozygotes. These findings suggest that the Sub1 locus does not demonstrate dominance and that tolerance is closely associated with the expression levels of the Sub1 allele. The evaluation included BRRI dhan52, Binadhan-7, Binadhan-17, and Guti Swarna, along with 29 F₂ lines, to determine their survival scores. This evaluation facilitates improved recombination for the submergence response within the population. A considerable variation in survival percentage was noted among the populations under flooded conditions, ranging from 0.00% to 69.50%. The results of the evaluation are presented in Table 2. A broad variation in survival percentage (31.50% to 67.50%) was observed under flooded conditions. Among the 11 F₂ plants derived from the Binadhan-7 x BRRI dhan52 cross, the survival percentage in response to flooding was recorded at 67.50% for the F₂-7 plants. The lowest survival percentage of 32.75% was noted for the F₂-6 plant, while the highest survival percentage of 68.25% was observed in the F₂-12 plant from the Binadhan-17 x BRRI dhan52 cross. This indicates a favorable recombination for plant submergence within the populations. However, in the case of the Guti Swarna x BRRI dhan52 cross combination, the highest survival percentage was 63.25% for the F₂-2 plant.

3.7 Seedling performance and submergence screening

The screening and performance of seedlings from local germplasm under complete submergence were conducted for all swarna varieties, including Suman Swarna, Ranjit Swarna, Rangina Swarna, Mamun Swarna, Sada Guti Swarna, Guti Swarna, Nepali Swarna, as well as high-yielding varieties (HYV) such as BRRI dhan52, Binadhan-7, and Binadhan-17, in an artificial tank. The height of the seedlings increased, and the seedling height of various rice cultivars was influenced by complete submergence (Table 6). Among all swarna varieties, HYV, and three F₂ crosses, Rongina Swarna exhibited the tallest plant height at 47.00 cm, while Binadhan-7 recorded the shortest at 25.00 cm. Across all levels of submergence, the maximum final seedling height of 75.23 cm was observed in the cross Binadhan-17 x BRRI dhan52 (F₂), surpassing both parent varieties. The final seedling height increased with higher levels of submergence from both parent varieties. The highest percentage of elongation (40.83%) was noted in Binadhan-17 x BRRI dhan52 (F₂), whereas the lowest was recorded from its parent varieties at 5.66% and 6.63% for Binadhan-7 and Binadhan-17, respectively. The weight and strength of seedlings from different rice genotypes were impacted by submergence stress. Among all genotypes, Binadhan-17 x BRRI dhan52 (F₂) had the highest weight at 1.75 grams, while Binadhan-7 had the lowest at 0.26 grams. Under tank conditions with prolonged submergence (7 days), the screening results indicated a range of 1.01–4.82 g/cm for seedling strength and 11.00–98.00% for survival across all tested accessions. All local germplasm and three F₂ cross combinations (Binadhan-7 x BRRI dhan52 (F₂), Binadhan-17 x BRRI dhan52 (F₂), Guti Swarna x BRRI dhan52 (F₂) demonstrated their performance at the seedling stage, serving as a tolerant check against BRRI dhan52. The survival rate (%) during the recovery stage of the three cross combinations, namely Binadhan-17 x BRRI dhan52 (F₂), Binadhan-7 x BRRI dhan52 (F₂), and Guti Swarna x BRRI dhan52 (F₂), was recorded at 98.00%, 96.00% (indicating very good recovery ability), and 89.00% (indicating good recovery ability), respectively. In comparison, the survival rate (%) of the tolerant check, BRRI dhan52, was 87.00% (considered good). Other germplasm accessions exhibited reasonable performance, albeit with poor recovery ability following de-submergence, and their tolerance levels were not as high as that of BRRI dhan52. The survival rates of these accessions exceeded 50%. This report presents the results of screening and testing for selected tolerant accessions during the Aman season.

3.8 Submergence Screening at Vegetative Stage

During the vegetative phase, the F₃ generation from each combination was transplanted as 21-day-old seedlings and submerged in artificial tanks 48 days post-transplanting (DAT). The water level increased swiftly and was sustained at a minimum depth of 88 cm for a standard treatment duration of 21 days. Plant survival was assessed 21 days following de-submergence, and the percentage of survival was computed for QTL analysis. The artificial tank exhibited average turbidity, with water temperatures ranging from 29 to 31°C, a pH level between 7.2 and 7.5, dissolved O₂ levels of 2.9 to 3.8 mg/L, and light intensity measured at 400 to 61 μmol/m²/s at the mid-tank level. A significant variation in survival percentage under flooded conditions was noted, with population ranges varying from 55.75% to 94.25%. A broad variation (55.75% - 93.25%) in survival percentage under flooded conditions was also observed among 11 F₃ plants of the Binadhan-7 x BRRI dhan52 combination. Among all F₃ plants, the highest survival percentage of 93.25% was recorded for the F3-11 plants in response to flooding. The lowest survival percentage (59.25%) was noted in F2-6, while the highest (94.25%) was observed in F₃-15 from the Binadhan-17 x BRRI dhan52 combination (Figure S6). This combination demonstrated the highest recombination for plant submergence across populations. Conversely, in the Guti Swarna x BRRI dhan52 cross combination, the highest survival percentage was 84.25% for the F₃-10 plant. However, the highest survival rate (%) during the vegetative stage of the F₃ generation was achieved from the Binadhan-17 x BRRI dhan52 (F₃) combination at 94.50%, surpassing the other cross, Binadhan-7 x BRRI dhan52 (F₃) at 93.25%. The Guti Swarna x BRRI dhan52 (F₃) combination recorded a survival rate of 85.25% (Table-3).

3.9 Confirmation of Sub1 gene in selected lines of F₄ generation

Sub1QTL in three F₂ populations exhibited a discrete distribution and co-segregated with DNA markers located in the target region. Segregation regions were identified on chromosome 9, where Sub1QTL were recognized in these common segregation areas across the three populations, namely BPR17, BPR21, and BPR22 in the F₂ generation. The SC32/RM23668 marker at the upper end of chromosome 9 in all three populations demonstrated a significant impact on submergence tolerance. The principal Sub1QTL, which had the highest LOD score of 12.17, accounted for a phenotypic variance explained (R2) of approximately 36.12%, enabling tolerance to complete submergence for up to three weeks. The presence of the Sub1 gene in the F4 generation was confirmed using gene-specific primers ERF3 and Sub1C173, located at the top of chromosome 9 (Figure S3). Nine selected lines from the F4 generation (BPRLs) were evaluated for Sub1 gene-specific markers alongside submergence-tolerant Indel markers, which were potential candidate markers for the Sub1 gene. The results indicated that binadhan-17 did not amplify, while the resistant allele of BRRI dhan52 was distinctly amplified (Figure S4). The BPRLs from the three crosses included BPR21 (P7=BPR21-1-S, P9=BPR21-2-S, P11=BPR21-S-S, P12=BPR21-S-M), BPR22 (P12=BPR22-1-S, P13=BPR22-2-S, P15=BPR22-S-S), and BPR17 (P2=BPR17-S-M, P10=BPR17-S-17), which were homozygous and exhibited the same banding pattern as the resistant allele from the donor parent of the Sub1 gene. Ultimately, it was confirmed that all selected plants possessed the Sub1 gene and were classified as submergence-tolerant lines. Nine selected BPRLs were established with additional background markers. No phenotypic or genotypic segregation was observed in these plants when utilizing the additional background markers. The head-to-row process was also implemented on the selected plants to achieve phenotypically homogenous plants for validation in submergence-prone regions of Bangladesh.

3.10 Biochemical Analysis

Expression of H₂O₂ and O ₂-

The findings indicated a significant overexpression of H₂O₂ and O₂- within the ranges of 1.345 to 3.463 and 1.353 to 4.124, respectively, when compared to the control. This suggests that the concentration of H₂O₂ and O₂- in recovery plants exhibited elevated levels across all rice genotypes in contrast to the control conditions (Figure 10). On average, the variations in H₂O₂ and O₂- were noted to be 2.204-fold and 2.180-fold, irrespective of the genotypes under submerged conditions. Notably, BRRI dhan52 demonstrated a greater tendency for H₂O₂ accumulation compared to the other genotypes. The increase in H₂O₂ concentration under submerged conditions ranged from 34.50% to 246.28% for the genotypes GS-M4-P-2 and BRRI dhan52, respectively. Among the other genotypes, H₂O₂ levels rose by 62.63% in recovery plants under stress conditions compared to their respective controls. Similarly, for O₂-, the maximum accumulation was recorded in the Binadhan-7 x BRRI dhan52 (F₃) combination among all genotypes. The results indicate that the Binadhan-7 x BRRI dhan52 (F₃) combination maintained a higher level of O₂- akin to the tolerant genotypes after recovery. The elevated accumulations of O₂- in stress-tolerant genotypes suggest a protective mechanism against oxidative damage through better regulation of O₂- formation. However, tolerant genotypes such as BPR17-S-M, BPR21-S-M, and BPR22-S-S exhibited significantly higher levels of O₂•– generation (98.90%, 312.42%, and 94.07%, respectively) compared to their respective controls. The generation of superoxide anion under submerged conditions showed an upward trend in GS-M4-P-2 (102.16%), MS-M4-P-8 (35.25%), and BRRI dhan52 (65.06%).

Activities of antioxidant enzymes

The rice genotypes exhibited markedly different levels of SOD activity in the control group. As a result of the treatment, submergence led to a significant increase in activity compared to the control. In terms of interaction, when compared to their respective controls, submergence enhanced SOD activity by 20.51% in BPR17-S-M, 22.22% in BPR21-S-M, 18.18% in BPR22-S-S, 20.69% in Binadhan-7, and 32.26% in BRRI dhan52. Furthermore, in terms of interaction, the POD enzyme activity in BPR17-S-M, BPR21-S-M, and BPR22-S-S was elevated under submergence stress. The POD activity saw increases of 111.11%, 196.92%, 123.19%, 21.33%, and 20.59% in BPR17-S-M, BPR21-S-M, BPR22-S-S, Binadhan-7, and BRRI dhan52 respectively. Under conditions of submergence stress, CAT activity rose by 34.82%, 31.30%, 39.42%, 32.65%, and 33.33% in BPR17-S-M, BPR21-S-M, BPR22-S-S, Binadhan-7, and BRRI dhan52 respectively, in comparison to their respective controls. The three lines BPR17-S-M, BPR21-S-M, and BPR22-S-S demonstrated higher activity than the others. APX activity was increased by 27.22%, 11.70%, and 37.37% for the tolerant genotypes BPR17-S-M, BPR21-S-M, and BPR22-S-S respectively. The GPX activity also showed a slight increase in the susceptible genotype Binadhan–7, although it remained comparatively lower than that of the tolerant genotypes (BRRI dhan52). The GPX activity increased by 9.34%, 8.74%, 12.71%, 9.78%, and 15.84% in BPR17-S-M, BPR21-S-M, BPR22-S-S, Binadhan-7, and BRRI dhan52 respectively (Figure 11).

3.11 Adaptability of the Sub1 lines

3.11.1 Non-stress conditions

Genetic variability exists among all genotypes concerning validated traits such as duration, plant height, filled grain, unfilled grain, number of panicles, 1000-grain weight, survival rate, and yield. The grain yield and other yield-contributing characteristics of all introgression lines were significantly greater than those of all parent lines, indicating satisfactory agronomic performance. Under non-stress conditions from three trials at the BINA Mymensingh, the average growth duration of Sub1 lines from the Binadhan-7×BRRI dhan52 (F₅) cross combination, namely BPR21-1-S, BPR21-2-S, BPR21-S-S, and BPR21-S-M, was 123, 124, 122, and 123 days, respectively, with average grain yields per plant of 25.64 g, 27.00 g, 40.20 g, and 42.00 g. These yields are significantly higher than that of the recipient parent, Binadhan-7 (25.76 g), and the donor, BRRI dhan52 (30.16 g). Sub1 lines from the Binadhan-17×BRRI dhan52 (F₅) cross combination, specifically BPR22-1-S, BPR22-2-S, and BPR22-S-S, exhibited average growth durations of 126, 133, and 131 days, with average grain yields per plant of 37.83 g, 45.53 g, and 42.69 g, respectively, which were significantly higher than the recipient parent, Binadhan-17 (21.80 g), and the donor parent, BRRI dhan52 (30.16 g). Sub1 lines from the Guti Swarna × BRRI dhan52 (F₅) cross combination, identified as BPR17-S-M and BPR17-S-17, had average growth durations of 145 and 142 days, with average grain yields per plant of 34.83 g and 36.78 g, respectively, which were significantly higher than the recipient parents of Guti Swarna (29.47 g) and the donor parent, BRRI dhan52 (30.16 g) (Table 4). The average growth durations of mutant lines GS-M5-P-2 and MS-M5-P-8, derived from Guti Swarna and Mamun Swarna, were 145 and 142 days, with average grain yields per plant of 31.46 g and 30.20 g, respectively, which were significantly higher than those of their parent lines.

3.11.2 Stress conditions

A validation test was performed at the three trails in the natural submergence field in the submergence prone areas to evaluate the performance of yield and yield-contributing traits for eleven entries alongside standard checks, specifically BRRI dhan52 in the farmer’s field. A control submergence of twenty-one days was implemented with a water depth of 78 cm using an artificial tank at BINA HQ. The average performance of the breeding lines under flooded conditions in the farmer’s field indicated that the growth duration varied from 137.77 to 160.33 days among all Sub1 lines, whereas it was 161.66 days for the donor parent BRRI dhan52. The shortest growth period recorded was 137.77 days, while the highest average grain yield per plant was achieved by BPR21-S-M (19.33 g) among four Sub1 lines derived from the Binadhan-7 × BRRI dhan52 (F₅) cross combination, which was significantly greater than that of the donor parent BRRI dhan52 (12.71 g). The minimum growth duration was 141.66 days, and the highest average grain yield per plant was noted in BPR22-S-S (20.23 g) among the three sub1 lines from the Binadhan-17×BRRI dhan52 (F₅) cross combination, which also significantly surpassed the donor parent BRRI dhan52 (12.71 g). The lowest growth duration recorded was 148.33 days, with the highest average grain yield per plant found in BPR17-S-M (15.26 g) among the two Sub1 lines (BPR17-S-M and BPR17-S-17) from the Guti Swarna × BRRI dhan52 cross combination, which was significantly higher than the donor parent BRRI dhan52 (12.71 g) (Table 5). Among the two mutant lines, MS-M5-P-8 exhibited the shortest growth duration and the highest mean grain yield per plant, measuring 158.33 days and 13.41 g, respectively. Furthermore, significant differences were observed between all sub1 lines and BRRI dhan52 across all other parameters, including plant height (cm), panicle/plant, filled grain/panicle, sterility, and thousand grain weight. Notably, three breeding lines, BPR21-S-M (19.33 g), BPR22-S-S (20.23 g), and BPR17-S-M (15.26 g), yielded the highest grain per plant, significantly exceeding the donor parent BRRI dhan52 (12.71 g) and demonstrating values that were 3.00 g to 8.00 g higher than the respective donor parents. The maturity duration for the three lines, namely BPR21-S-M, BPR22-S-S, and BPR17-S-M, is approximately 8 to 13 days shorter than that of their submerged condition and 20 to 25 grams lower than the non-flooded condition (Figure S7). Among the various genotypes, farmers selected these three lines (BPR21-S-M, BPR22-S-S, and BPR17-S-M) due to their tall plant structure and superior yield, as evidenced by the increased average plant height, which provided more straw for livestock and enhanced overall productivity to promote food security. The final result of this research was achieved the highest grain yield per plant at 20.23 g and growth duration 141 days as a short duration a submergence tolerant line for 21 days.

Stability analysis

3.12.1 Stability analysis by AMMI model

Biplot analysis is arguably the most effective interpretive tool for AMMI models. There are two fundamental types of AMMI biplots: the AMMI 1 biplot, which plots the main effects and IPCA1 scores for both genotypes and environments against one another. The AMMI 1 biplot demonstrated a model fit of 98.7% (Figure 5). The relative or ordinate axis indicated that a genotype positioned on the right side of the midpoint of this axis yielded more than those on the left side. As a result, among the genotypes, BPR21-S-M, BPR22-S-S, BPR17-S-M, and GS-M5-P-2 displayed high yields with low IPCA scores; however, the genotype GS-M5-P-2 exhibited a positive IPCA1 score near zero, while the genotypes BPR21-S-M, BPR22-S-S, and BPR17-S-M showed negative IPCA1 scores close to zero, suggesting that these genotypes were stable and less affected by environmental factors. Conversely, the remaining genotypes experienced greater G × E interaction effects. The AMMI-2 biplot, which illustrates the interaction of PC1 and PC2, presented the grain yield of the tested genotypes across three environments (Figure 6). In this model of environmental effects, genotypes situated near the origin were less sensitive to environmental interactions, whereas those positioned further from the origin were more sensitive and exhibited significant interactions with the environment. The findings indicated that the first principal component axis accounted for PC1 (98.7%) and the second PC2 (1.3%) variation. Together, the two IPCA axes accounted for 100% of the genotype by environment interaction mean square. This suggests that the interaction of 12 rice genotypes with their environments was effectively predicted by the first two components of IPCA. In this investigation, the genotypes BPR21-S-M, BPR22-S-S, BPR17-S-M, and BPR22-1-S were located close to the origin, indicating they were less interactive with environmental variations and were situated very near the polygon region. Consequently, these three genotypes, BPR21-S-M, BPR22-S-S, and BPR17-S-M, were selected for their high yield across various environments.

3.12.2 GGE biplot, Genotype Ranking and Heatmap

The GGE biplot illustrating the yield of 12 genotypes revealed a significant PC1 score of 91.53% and a minor PC2 score of 8.33%. The values for the first principal component (PC1) and PC2 were calculated to create a GGE biplot graph (Figure 7). This biplot graph facilitated the identification of the best-performing genotypes suited for a specific location or stable genotypes across multiple locations, and it also identified the most representative locations (mega environment) for a genotype. The small diamond represents the average environment, while ideal genotypes are characterized by the longest vector length and minimal G × E, indicated by the center of the bold diamond, which suggests the highest mean yield and stability. In this analysis, the high-yielding genotypes identified were BPR21-S-M, BPR22-S-S, and BPR17-S-M, while the remaining genotypes were classified as low-yielding. Consequently, these three lines were recognized as the most stable and high-yielding genotypes. The genotype ranking biplot (Figure 8) allows for the identification of an ideal genotype in comparison to the other evaluated genotypes. The genotypes BPR21-S-M, BPR22-S-S, and BPR17-S-M are highlighted as the leading genotypes due to their proximity to the circle arrowheads for yield. The first subgroup within the tolerant group consists of BPR21-S-M and BPR22-S-S, which is regarded as the most tolerant subgroup (Figure 9).

The current research demonstrated the application of marker-assisted selection to create recombinant lines (BPRLs) exhibiting various phenotypes distinct from the original mega variety. These new varieties were developed to ensure that farmers could easily recognize the new stress-tolerant variety. [45] indicated that the number of alleles per locus varied from 2 to 8, with an average of 3.8, specifically ranging from 0.51 to 0.99 and averaging 0.88, based on BRRI released varieties, which aligns with earlier microsatellite analysis estimates in rice [44], showing a range of 0.76 to 0.95 with an average of 0.855 [46], 0.26 to 0.65 with an average of 0.47 [80], 0.28 to 0.50 with a mean of 0.45 [47], and 0.239 to 0.765 with an average of 0.508 [48]. The highest PIC value was recorded for RM324 (0.8912), followed by RM152 (0.8502) and RM279 (0.8266). Conversely, the lowest PIC value was noted for RM234 (0.7535). This indicates the strong discriminatory power of the markers employed, thus justifying their application in genetic characterization studies [80]. [57] noted that PIC values of microsatellite markers exceeding 0.5 are regarded as highly polymorphic. [75] reported an average PIC value of 0.500, indicating 50% polymorphism, which corroborates the use of SSR markers in genetic research and in distinguishing the polymorphism rate of a marker at a specific locus.

The current findings indicate that waterlogging has triggered the production of H₂O₂, a non-radical reactive oxygen species (ROS), in the leaves of plants. An excess of ROS disrupts the balance between the over-accumulation of ROS and the activity of antioxidants, resulting in oxidative damage to plants [50]. Consequently, the overproduction of H₂O₂ leads to the peroxidation of membrane lipids, which in turn causes an increase in electrolyte leakage (EL) in plants following exposure to waterlogging [49]. Previous research has also documented the overproduction of ROS, including H₂O₂, and membrane lipid peroxidation in G. max under waterlogged conditions [51]. In this study, the activity of antioxidative enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), and glutathione peroxidase (GPX) was found to be significantly higher in selected genotypes that exhibit submergence tolerance (BPR17-S-M, BPR21-S-M, and BPR22-S-S). These findings align with those of [86], which suggest that increased activity of SOD isozymes aids in mitigating stress. Peroxidases (POD), located in the cytosol, vacuole, and extracellular space, effectively scavenge H₂O₂ through the oxidation of various substrates [85]. Catalase (CAT) is recognized as one of the most effective antioxidant enzymes for metabolizing H₂O₂, primarily found in peroxisomes, and exhibits a higher turnover rate than other enzymes [85]. APX demonstrates a greater affinity for H2O2 compared to CAT and POD, playing a vital role in managing ROS in plants during abiotic stress [84]. Additionally, GPX, a member of the non-heme peroxidase family, is another crucial enzyme involved in the scavenging of H₂O₂ [83].

[52] identified a notable positive heterobeltiosis in the majority of cross-grain yield per plant. [65] discovered hybrids exhibiting a 60% positive heterosis compared to the superior parent, while I observed positive heterobeltiosis in 80% of the combinations. [56], [32], [72], [74], and [79] reported significant positive heterosis for grain yield in most of the hybrids they studied. [65] found hybrids that were positively heterotic and also exhibited heterosis for panicle length, net assimilation rate, leaf area index, dry matter, harvest index, and 1000 grain weight. [79] noted that heterosis for yield was observed in tiller/plant, attributed to favorable heterosis in filled grain panicle-1. [81] indicated that spikelet panicle-1, panicle length, leaf area /plant, and number of panicles per m² contributed to positive heterosis for grain yield per plant. [66] recorded that favorable heterosis for grain yield per plant was associated with favorable heterosis for the number of productive tillers, panicle weight, panicle length, fertile spikelets per panicle, 1000 grain weight, and harvest index. [62] found diallel progeny in seeds from five rice genotypes, with heterosis ranging from 4.91–65.77%. [82] and [71] reported significant negative heterosis for days to maturity across numerous hybrids.

For over three decades, submergence tolerance has been a significant breeding objective [61]; [69]; [70]. In the mid to late 1970s, breeders at IRRI created hybrids between the submergence-tolerant donor FR13A and high-yielding varieties such as IR48 and IR36. By the early 1990s, submergence-tolerant lines with high yield potential were developed [66]. However, these tolerant prototypes did not gain widespread acceptance among farmers due to their inferior grain quality and other traits necessary for local adaptation. Conversely, mega varieties that exhibited most of the traits sought by farmers but lacked submergence tolerance began to proliferate in both irrigated and rainfed lowland areas of South and Southeast Asia [66]. The aim was to convert these varieties into submergence-tolerant types while preserving the desirable characteristics of the original parent through a specific molecular marker-assisted backcrossing (MAB) approach [69]. MAB is a technique designed to mitigate issues related to traditional plant breeding by shifting the selection criteria from phenotype selection to gene selection, either directly or indirectly. This study presents the localization of RM23805 on chromosome 9, which could serve as a foundation for the positional cloning of RM23805.

Further enhancement of submergence tolerance necessitates the identification of genes that provide increased levels of submergence tolerance, as well as the integration of the Sub1 gene with other traits essential for adaptation to flooding. It is established that additional QTLs in FR13A play a role in its significant submergence tolerance [22, 23]. Moreover, several moderately tolerant cultivars lacking the Sub1A-1 allele have been recognized. An F₃ population has been created, and submergence screening is currently in progress to ascertain whether there are novel QTLs that may function in conjunction with Sub1. The Sub1C gene, located between 6,404,482 and 6,406,039, at locus (LOC_Os09g11480.2), transcript variant (Os09t0287000-01), Gene Bank/cDNAs (AK106057) has a length of 1558bp (1.558kb region from chromosome 9) and a nucleotide length of 768 bp, with a predicted protein length of 256 from Chromosome 9. The Sub1 genes encode ethylene response factors, which are proteins that are encoded [19–21].

Precision introgression lines suggested by [58] contain very small donor segments on the carrier chromosome, such as BR11-Sub1 [30], which included only 800 kb of donor introgression. Sub1 was incorporated into BRRI dhan33 to create a short-duration, submergence-tolerant rice variety through MABC. To minimize expenses, we opted not to conduct background selection from the outset as previously practiced [16]; [14]; [20], but instead employed a combination of foreground markers and phenotypic selection, which entails selecting plants that closely resemble the recipient parent and exhibit the desired segregation for yield or other beneficial traits. [14] utilized 56 SSR markers as the primary background markers for the development of swarna-Sub1. The average distance between adjacent background primers varied from 13 to 29 cM for BR11 [16].

The recently developed BRRI dhan33-Sub1 line has demonstrated superior submergence tolerance and agronomic characteristics. The line BR9157-12-2-37-13-15-40 exhibited the highest submergence tolerance, achieving 87.7% survival and 11.4% elongation. The grain yield of the BRRI dhan33-Sub1 line was significantly greater than that of its parent variety, BRRI dhan33, in both flooded and non-flooded environments. A notably higher grain yield of BRRI dhan52 compared to its parent recurrent variety, BR11, was documented under controlled submergence conditions, as referenced in [31]. The maximum grain yield recorded for the BRRI dhan33-Sub1 line was 4.8 t/ha on-farm in non-flooded conditions and 3.8 t/ha on-farm in flooded conditions. However, the relatively lower grain yield of the BRRI dhan33-Sub1 line in both flooded and non-flooded scenarios can be partially attributed to the presence of bacterial blight and sheath rot.

The observed increase in grain yield compared to the original recurrent parent, ranging from 0.8 to 2.3 t/ha, was quite promising. The selected BRRI dhan33-Sub1 lines are suitable for use in participatory variety selection experiments, serving as either varieties or parental lines for the further development of high-yielding, short-duration, submergence-tolerant varieties through conventional breeding methods. The physicochemical properties of the BRRI dhan33-Sub1 line were found to be largely comparable to those of the recurrent parent, BRRI dhan33. Reference [16] indicated that BR11-Sub1 exhibited similar grain physicochemical characteristics when compared to its original recurrent parent, BR11. The plant height ranged from 94 to 120 cm. The highest recorded grain yield was 4.85 t/ha from BRRI dhan52, followed closely by BRRI dhan51 with a yield of 4.6 t/ha. The survival rate exceeded 90% across all genotypes, indicating that they were not significantly affected by the flash flooding experienced during this experiment [15,77, 78]. Reference [54] reported significant mean sum squares for all traits examined, with the exception of the 1000 grain weight. BRRI dhan51 and BRRI dhan52 demonstrated average performance among the genotypes, including two standard check varieties, with BRRI dhan52 exhibiting the shortest growth period of 148 days.

Under stress conditions, the shortest recorded growth duration was 137.77 days, while the highest average grain yield per plant was 19.33 g from BPR21-S-M, which was significantly higher than the donor parent BRRI dhan52 (12.71 g) among the four Sub1 lines. The minimum growth duration observed was 141.66 days among the three Sub1 lines, with BPR22-S-S achieving the highest average grain yield per plant at 20.23 g, which was also significantly greater than the donor parent BRRI dhan52 (12.71 g). The minimum growth duration was 148.33 days, and the highest average grain yield per plant was recorded from the Sub1 line BPR17-S-M at 15.26 g, which was significantly higher than the donor parent BRRI dhan52 (12.71 g) whereas it was 161.66 days for the donor parent BRRI dhan52. Only three lines exhibited superior performance in yield contribution, demonstrating excellent recovery abilities of 91.25%, 94.25%, and 84.75% as survival percentages under field conditions. The maturity duration of the three lines, namely BPR21-S-M, BPR22-S-S, and BPR17-S-M, was shorter compared to the other lines. Notably, additional information indicated that these three lines presented on the top of chromosome 9 confirmed the presence of gene-specific primers ERF3 and Sub1C173 for the Sub1 gene in the F₅ generation. It was established that the three promising lines, BPR21-S-M, BPR22-S-S, and BPR17-S-M, exhibit enhanced performance in yield contributions with commendable recovery abilities under field conditions. Ultimately, it was confirmed that all selected lines contained the Sub1 gene and were classified as submergence-tolerant lines for a duration of 21 days. The maturity duration of the three lines, BPR21-S-M, BPR22-S-S, and BPR17-S-M, was approximately 8–13 days shorter than under submerged conditions, and under stress conditions, the grain yield per plant was 20 to 25 g lower than in non-flooded conditions. These three newly developed promising Sub1 lines, BPR21-S-M, BPR22-S-S, and BPR17-S-M, are intended for use in developing a high-yielding submergence-tolerant (21 days) variety for flood-prone areas in Bangladesh.

Results

Under stress conditions, three breeding lines, BPR21-S-M (19.33 g), BPR22-S-S (20.23 g) and BPR17-S-M (15.26 g), produced the most grain yield per plant, and accordingly recorded growth duration was 137 days, 141 days, 148 days for 21 days submergence which was significantly higher than the donor parent of BRRI dhan52 (12.71 g) whereas it was 161 days for the donor parent BRRI dhan52

Limitations

The authors of a prior study posited that plants would not only experience stress during flooding but would also endure significant oxidative damage and rapid dehydration during submersion. The swift influx of oxygen into the plant following dehydration resulted in the production of excessive reactive oxygen species, which could lead to oxidative stress. Previous research has indicated that submergence stress exerts a specific physiological impact on rice. In this investigation, the influence of submergence stress on rice growth persisted until the recovery phase following the disaster, primarily evident as a decline in rice growth indices. This study concentrated solely on the visible growth characteristics of rice and did not track the alterations in physiological indices during the recovery phase after the alleviation of submergence stress. The response mechanism of rice plants to submergence stress requires further investigation to assess the submergence tolerance of rice and to analyze the underlying mechanisms of submergence, thereby providing a theoretical foundation for the breeding of submergence-resistant rice varieties. While rice varieties capable of withstanding flooding represent a vital solution for areas prone to floods, their implementation encounters specific challenges. These challenges include issues related to access to information, availability of seeds, and potential yield differences when compared to non-flood-tolerant varieties. Additionally, factors such as water quality, nutrient availability, and specific flood conditions (including duration, depth, and timing) can affect their efficacy. Challenges in the advancement of submergence-tolerant rice cultivation include the low survival rates of the majority of rice varieties.

BINA: Bangladesh Institute of Nuclear Agriculture; BRRI: Bangladesh Rice Research Institute; QTL: Quantitative Trait Loci; MAS: Marker Assisted Selection; ERF: Ethylene Responsive Factor; CTAB: Cetyltrimethyl ammonium bromide; SSR: Simple Sequence Repeats; PCR: Polymerase Chain Reactions; ANOVA: Analysis of Variance

Acknowledgement

Not applicable

Ethics Approval and Consent to Participate

Not applicable

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by the Bangladesh Agricultural University, Mymensingh, Bangladesh Institute of Nuclear Agriculture (BINA), and a project of the Rice Genes for Salinity and Submergence Tolerant. Fund: Project Director (PD) of R & D project from Ministry of Science and Technology (MoST), Bangladesh (Project id: R & D-2410078)

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Tables 1 to 6 are available in the Supplementary Files section

No competing interests reported.

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Development of submergence tolerance introgression lines (ILs)-F 5 using major Sub1QTL on chro9 from BRRI dhan52 in rice (Oryza sativa L.) through Marker-Assisted Selection (MAS)

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Version 1

Abstract

Figures

Key findings

1. Introduction

2. Materials and Methods

2.1 Development of plant population

2.2 Imposition of submergence in artificial tanks.

2.3 DNA marker analysis

2.3 Biochemical Analysis

2.4 Seedling performance

2.5 Field evaluation under submergence prone areas

3. Results

4. Discussion

5. Conclusion

Results

Abbreviations

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

Version 1