Molecular profiling and phenotypic evaluation of thermo-sensitive genic male sterility genes for high-yielding rice hybrids (Oryza sativa L.)

Observations recorded

One-month-old seedlings were transplanted in a randomized block design (RBD) with three replications during winter 2022 at TNAU and monsoon 2023 at HREC, with a spacing of 20 × 20 cm. Fertilizer application was carried out at the rate of 150:60:60 kg/ha of NPK, with nitrogen and potassium applied in three split doses during the basal, active tillering and panicle initiation stages, adhering to the recommended field management guidelines from the TNAU Agritech portal (http://www.agritech.tnau.ac.in/). Yield-related traits such as days to 50% flowering, plant height, productive tillers, panicle length, pollen fertility, spikelet fertility, and single plant yield were recorded in the fertility-inducing environment (HREC). Floral traits, including pollen sterility, stigma length, stigma exertion and glume angle, were measured under sterility-inducing conditions (TNAU). Pollen fertility was assessed by staining mature anthers from five plants with a 1% potassium iodide solution. Fertile pollen appeared dark blue under the light microscope, while sterile pollen remained colourless. Pollen fertility percentage was calculated using the formula: ${\displaystyle \text{Pollen sterility percentage}=\frac{\text{Number of sterile pollens}}{\text{Total number of pollens}}\times 100.}$

The environmental conditions were carefully monitored to capture the natural transition between fertility and sterility. Minimum and maximum temperatures at critical stages were documented following the protocol established by Vinodhini et al. (2019). TGMS lines were exposed to varying environmental conditions, ranging from 13 °C to 38 °C, reflecting the spectrum of natural cycles. The average minimum and maximum temperatures at which the shift from fertility to sterility occurred were defined as the critical fertility temperature (CFT) and critical sterility temperature (CST), respectively. Observations were taken from five randomly selected plants per genotype, ensuring representative data. These values were then averaged to provide a comprehensive understanding of the response of TGMS lines under dynamic environmental conditions, bridging the relationship between temperature sensitivity and the expression of sterility and fertility traits.

DNA extraction

Two-week-old seedlings from each line were selected from the nursery and young leaves were collected for DNA extraction using the protocol given by Doyle & Doyle (1987). The plant tissues were flash-frozen in liquid nitrogen to preserve their integrity and ground into a fine powder using a mortar and pestle. The pulverized tissue was transferred into a tube containing an extraction buffer (CTAB) and incubated at 65 °C for 30 min with periodic gentle mixing to ensure thorough processing. Following incubation, an equal volume of chloroform: isoamyl alcohol (24:1) was added to the mixture, which was then gently mixed and centrifuged at 12,000 rpm for 10 min at room temperature. The aqueous phase, containing the extracted DNA, was carefully transferred to a fresh tube. DNA was precipitated by adding 0.6 volumes of isopropanol or 2 volumes of 100% ethanol, followed by incubation at −20 °C for a minimum of one hour or overnight to encourage precipitation. The DNA was pelleted by centrifugation at 12,000 rpm for 10 min. The supernatant was discarded, and the pellet was washed with 70% ethanol and centrifuged again for 5 min at the same speed. The DNA pellet was allowed to air dry for 10–15 min before being dissolved in TE buffer or a similar solution. The dissolved DNA was stored at 4 °C, typically overnight, to ensure complete dissolution. RNA contamination was removed by treating the solution with RNase A. The quality and concentration of the extracted DNA were evaluated using a spectrophotometer. Absorbance readings were taken to confirm the purity, with an optimal 260/280 ratio of ∼1.8 indicating high-quality DNA suitable for downstream analyses.

Molecular assay

The polymerase chain reaction (PCR) was performed using eight SSR primers (Table 2) specific to the tms4, tms5, tms8 and tms10 genes. Each reaction was carried out in a 10 µl mixture containing 2 µl of template DNA (20 ng/µl), 0.5 µl of forward primer (10 µM), 0.5 µl of reverse primer (10 µM), 4 µl of 2X master mix and 3 µl of sterile water. The PCR thermal cycling profile included an initial denaturation step at 95 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 45 s, and extension at 72 °C for 30 s. A final extension step was performed at 72 °C for 10 min. Amplified samples were stored at 4 °C until further analysis (Nivedha et al., 2024). The PCR products were separated on a 3% agarose gel prepared in 1X TBE buffer, alongside a 100 bp DNA ladder (Bio-Helix) for size reference. The gel was visualized using Bio-Rad imaging equipment under UV trans illumination, enabling clear differentiation of the amplified fragments.

Statistical analysis methodology

Statistical analyses were performed using R software to ensure rigorous evaluation and enhance the accuracy of the results. The “aov” package was utilized for analysis of variance (ANOVA), and the “cor” package was employed for correlation analysis. To calculate descriptive statistics, including the mean and standard error of the mean (SEM), the psych package was used in R-Studio 4.3.2.

Morphological characterization

A detailed evaluation of 57 TGMS rice lines was undertaken to profile the genes governing male sterility and to explore their associations with floral traits that enhance outcrossing potential, an essential element in optimizing hybrid seed production. Significant genotypic variation in yield and floral characteristics was observed through analysis of variance (Supplemental Information 1). Traits sensitive to temperature changes, including pollen sterility, stigma length, stigma exertion and glume angle, were evaluated under sterility-inducing conditions at the Paddy Breeding Station, Coimbatore, while agro-morphological traits were recorded at the Hybrid Rice Evaluation Centre (HREC), Gudalur, under fertility-inducing conditions.

The TGMS lines exhibited flowering durations ranging from 62.3 to 152.0 days (Supplemental Information 2). Among short-duration genotypes, TNAU 112S (62.3 days), TNAU 15S (72.3 days), TNAU 39S (86.0 days), TNAU 16S (91.0 days) and TNAU 93S (91.0 days) showed the earliest flowering. Plant heights varied between environments, ranging from dwarf to tall, with TNAU 16S and TNAU 93S recording the shortest height (66.67 cm). Productive tiller counts were highest in TNAU 16S (27.67), TNAU 95S (25.00) and TNAU 19S (22.33), while TNAU 132S exhibited the fewest productive tillers (10.0). All TGMS lines demonstrated panicle exertion greater than 80%, with TNAU 143S (106.35%), TNAU 83S (103.45%) and TNAU 82S (100.00%) achieving the highest values. TNAU 103S recorded the longest panicle length (26.33 cm), while TNAU 93S had the shortest (15.00 cm). Grain counts ranged from 34.0 in TNAU 112S to 326.0 in TNAU 114S. The highest single-plant yields were observed in TNAU 114S (43.0 g), TNAU 131S (42.3 g) and TNAU 34S (41.0 g), whereas TNAU 101S had the lowest yield (7.7 g). Temperature sensitivity was critical for regulating sterility and fertility transitions. The critical fertility temperature (CFT) at Gudalur ranged from 15.5 °C to 19.0 °C, while the critical sterility temperature (CST) at Coimbatore ranged from 26.5 °C to 31.0 °C (Supplemental Information 3, Fig. 2). During sterility-inducing conditions, most accessions displayed complete pollen sterility (Table 3, Fig. 3). Under fertility-inducing conditions, pollen fertility ranged from 31.9% in TNAU 93S to 98.6% in TNAU 95S, with TNAU 16S (97.1%) and TNAU 19S (97.0%) also demonstrating high fertility. Conversely, poor fertility was observed in TNAU 112S (50.6%) and TNAU 111S (50.8%). High spikelet fertility was noted in TNAU 15S (86.1%), TNAU 126S-1 (84.4%) and TNAU 16S (82.9%), while low fertility was observed in TNAU 113S (14.7%), TNAU 93S (16.6%) and TNAU 31S (24.8%).

Floral traits measured under sterility-inducing conditions revealed stigma lengths ranging from 1.2 mm in TNAU 71S to 2.48 mm in TNAU 113S. Additional lines with significant stigma lengths included TNAU 112S (2.44 mm), TNAU 100S (2.32 mm) and TNAU 4S (2.22 mm) (Fig. 4). Glume angles varied from 10.0° in TNAU 135S to 31.7° in TNAU 126S-1 and TNAU 126S-2, with notable values also observed in TNAU 2S (30.0°) and TNAU 16S (28.3°). Stigma exertion, a key trait for enhancing outcrossing potential, was highest in TNAU 112S (85.9%), TNAU 92S (84.6%) and TNAU 142S (76.6%), while the lowest values were recorded in TNAU 98S (10.9%), TNAU 46S (14.8%) and TNAU 30S (19.3%) (Fig. 5).

Allelic diversity of rice TGMS genes

The molecular profiling of TGMS lines identified the presence or absence of four major rice TGMS genes tms4, tms5, tms8 and tms10 using SSR markers. Detailed results are presented in Table S1, and the electrophoresis patterns of SSR markers linked to these genes are shown in Fig. 6. The genetic frequencies of the four TGMS genes ranged from 8.92% to 69.64%. Among these, tms8 was the most widely distributed, occurring in 69.64% of the genotypes, followed by tms10 (57.14%), tms5 (16.07%) and tms4 (8.92%). The rice marker RM27 (158 bp) linked to the tms4 gene was detected in five of the 57 accessions. Nine accessions exhibited amplification for RM174 (208 bp), RM5897 (141 bp), RM6247 (103 bp) and RM7575 (118 bp), markers associated with tms5. Positive bands for RM21 (157 bp) and RM224 (157 bp), markers connected to tms8, were observed in 39 genotypes. The marker RM300 (121 bp), associated with tms10, was amplified in 32 accessions.

Two accessions, TNAU 38S and TNAU 60S, demonstrated the presence of all four TGMS genes, showing positive amplification for all associated markers. Six accessions, including TNAU 23S, TNAU 39S, TNAU 59S-1, TNAU 82S, TNAU 83S and TNAU 85S, carried three TGMS genes. Additionally, 22 lines possessed two TGMS genes, 26 lines carried three genes, and 12 lines had only one gene, either tms8 or tms10. These findings illustrate the diverse genetic composition of TGMS lines, with varying combinations of major TGMS genes contributing to their sterility characteristics. The presence of multiple genes in some accessions, particularly TNAU 38S and TNAU 60S, highlights their potential for use in breeding programs aimed at developing stable and high-performing rice hybrids.

Genetic diversity between the tms genes

Genotyping of TGMS lines for the tms4, tms5 and tms10 genes on chromosome 2, and tms8 on chromosome 11, revealed seven distinct gene combinations (Table 4). The combination of tms8 and tms10 was the most commonly observed across the lines. Notably, two lines, TNAU 60S and TNAU 38S, contained all four tested genes. Among the lines carrying three TGMS genes, two distinct combinations were identified. TNAU 59S-1, TNAU 82S, TNAU 83S and TNAU 85S carried tms5, tms8, and tms10, while TNAU 23S and TNAU 39S contained tms4, tms8 and tms10. Additionally, tms8 and tms10 were found together in 18 lines, reflecting their strong co-occurrence. The tms10 gene was observed alone in four lines, while the tms8 gene was present independently in eight lines. Lines carrying multiple gene combinations from diverse genetic backgrounds exhibited robust and effective sterility, demonstrating the significance of gene interactions in controlling sterility expression. These findings emphasize the genetic diversity and adaptability of TGMS lines, highlighting the potential of specific gene combinations, particularly those involving tms8 and tms10, to enhance sterility stability for hybrid rice breeding programs.

Correlation of male sterility with floral characters and its implications for enhanced outcrossing in hybrid seed production in Coimbatore

The TGMS lines studied here reveal their deep connection to the natural cycles of fertility and sterility, offering a profound potential to harmonize with breeding programs aimed at male sterility and enhanced cross-pollination traits for hybrid seed production. Among them, TNAU 23S and TNAU 39S, carrying the tms4, tms8 and tms10 gene combination, demonstrated complete pollen sterility (100%) and strong panicle exertion, measured at 85.1% and 76.2%, respectively (Table S5). These lines also exhibited favourable floral traits, with stigma lengths of 2.1 mm and 1.92 mm, stigma exertion of 20% and 70.8%, and glume angles of 16.7° and 25° respectively. Their single plant yields, recorded at 18.7 g and 16.5 g, reflect their resilience and adaptability. This combination of sterility and robust panicle exertion helps maintain the natural balance by effectively preventing self-pollination while encouraging outcrossing. TNAU 38S and TNAU 60S, which carry the tms4, tms5, tms8 and tms10 gene combination, similarly aligned with nature’s design for hybrid seed production. Both lines expressed complete pollen sterility (100%) and exhibited floral traits enhancing outcrossing. TNAU 38S achieved 79.4% panicle exertion, with a stigma length of 1.93 mm, stigma exertion of 43.6%, glume ∠ of 11.7° and single plant yield of 21.2 g. TNAU 60S, with 77.3% panicle exertion, stigma length of 1.78 mm, stigma exertion of 41.1%, glume ∠ of 20°, and single plant yield of 31.3 g, stands as a testament to the harmonious integration of genetic diversity and environmental responsiveness. These lines embody the essence of consistent sterility, ensuring the balance needed for controlled environments.

TNAU 59S-1, TNAU 83S and TNAU 85S, with the tms5, tms8 and tms10 gene combination, demonstrated harmony between yield potential and floral characteristics. TNAU 59S-1 recorded a yield of 32 g, glume ∠ of 11.7°, stigma length of 1.49 mm, stigma exertion of 49.7%, complete pollen sterility and 78.6% panicle exertion. TNAU 83S showed a yield of 22.7 g, glume ∠ of 18.3°, stigma length of 2.05 mm, stigma exertion of 51.7% and 81.4% panicle exertion, balanced by 100% pollen sterility. Similarly, TNAU 85S achieved a yield of 19 g, glume ∠ of 28.01°, stigma length of 2.19 mm, stigma exertion of 75%, and 75.1% panicle exertion, maintaining complete pollen sterility. The wider glume angles and higher stigma exertion percentages in these lines echo nature’s intent, facilitating cross-pollination and enabling the union of diverse genetic material. By carrying the tms5, tms8 and tms10 combination, these lines reflect an evolutionary harmony that enhances hybridization potential, aligning with the cycles of nature and the needs of humanity. These findings underscore the sacred interdependence between genetic diversity and environmental interaction, providing a path forward for sustainable and adaptive hybrid rice breeding. TGMS genotypes ranked by composite scores highlight TNAU 85S as the top performer with balanced traits: 75% panicle emergence, 2.19 mm stigma length, and 28.01° glume angle (score: 0.8). TNAU 39S ranks second (PE: 76.2%, score: 0.67) due to shorter stigma length (1.92 mm) and single plant yield (16.5 g). TNAU 83S achieves the highest PE (81.4%) but lower stigma exertion (51.7%) places it third (score: 0.58). Composite scoring effectively identifies genotypes for enhancing outcrossing efficiency (Fig. 7).

Correlation analysis of genetic markers and pollen sterility patterns in TGMS rice lines

The analysis of genetic markers in TGMS rice lines reveals a dynamic interplay between specific allele combinations and pollen sterility as shown in the Table S4. Certain lines, such as TNAU 1S, TNAU 2S and TNAU 4S, exhibit 100% sterility associated with uniform allelic patterns across markers RM174, RM5897, RM6247, RM7575, RM21, RM224, and RM300. These patterns suggest that the cumulative contribution of these markers aligns with complete sterility, making them valuable tools for selecting lines with strong sterility potential. This alignment highlights the harmony within the genetic network, reinforcing the connection between marker combinations and sterility outcomes. However, the journey toward sterility in some lines deviates from this uniformity, reflecting the broader complexity of genetic and environmental interactions. Lines such as TNAU 30S (8.2% sterility), TNAU 82S (24.3% sterility), TNAU 86S (23.4% sterility) and TNAU 93S (34.8% sterility) illustrate that additional forces whether unexamined genetic elements, environmental conditions or epistatic interactions contribute to their incomplete sterility. These variations emphasize the interconnectedness of genetic factors and environmental cycles, suggesting that sterility arises not from isolated markers but from a symphony of interacting elements.

Interestingly, near-complete sterility is observed in lines such as TNAU 53S (93.4% sterility), TNAU 114S (92.8% sterility), TNAU 115S (92.8% sterility), TNAU 135S (95.5% sterility) and TNAU 136S (91.4% sterility). Although these lines share allelic patterns with fully sterile lines, they do not achieve complete sterility. This distinction suggests that subtle variations whether minor genetic differences, incomplete gene penetrance or environmental interactions disrupt the balance needed for absolute sterility. Such outcomes reflect the adaptive harmony between genetic potential and environmental forces, underscoring nature’s capacity for variation within broader patterns of order. Further adding to this complexity, lines such as TNAU 59S-2, TNAU 98S and TNAU 143S achieve 100% sterility despite having a “0” allele at marker RM21, which is typically associated with sterility. This observation suggests that RM21’s variation may play a lesser role in sterility under specific genetic or environmental contexts, highlighting the flexibility and resilience of sterility mechanisms. Conversely, TNAU 113S, with only 11.7% sterility despite a similar allelic pattern to highly sterile lines, underscores the influence of additional genetic interactions or environmental factors that suppress sterility. These exceptions reflect the intricate web of connections that govern sterility, where each thread influences the whole.