HWA1- and HWA2-Mediated Hybrid Weakness in Rice Involves Cell Death, Reactive Oxygen Species Accumulation, and Disease Resistance-Related Gene Upregulation

Hybrid weakness is a type of reproductive isolation in which F1 hybrids of normal parents exhibit weaker growth characteristics than their parents. F1 hybrid of the Oryza sativa Indian cultivars ‘P.T.B.7′ and ‘A.D.T.14′ exhibits hybrid weakness that is associated with the HWA1 and HWA2 loci. Accordingly, the aim of the present study was to analyze the hybrid weakness phenotype of the ‘P.T.B.7′ × ‘A.D.T.14′ hybrids. The height and tiller number of the F1 hybrid were lower than those of either parent, and F1 hybrid also exhibited leaf yellowing that was not observed in either parent. In addition, the present study demonstrates that SPAD values, an index correlated with chlorophyll content, are effective for evaluating the progression of hybrid weakness that is associated with the HWA1 and HWA2 loci because it accurately reflects degree of leaf yellowing. Both cell death and H2O2, a reactive oxygen species, were detected in the yellowing leaves of the F1 hybrid. Furthermore, disease resistance-related genes were upregulated in the yellowing leaves of the F1 hybrids, whereas photosynthesis-related genes tended to be downregulated. These results suggest that the hybrid weakness associated with the HWA1 and HWA2 loci involves hypersensitive response-like mechanisms.


Introduction
The traits of existing crop cultivars can be improved by crossing cultivars or lines to introduce beneficial traits, such as resistance or tolerance to disease or stress, to susceptible cultivars. However, because reproductive isolation mechanisms can hinder the production of hybrids, methods must be developed to overcome the underlying mechanisms of such reproductive isolation.
One type of post-zygotic reproductive isolation, namely hybrid weakness (i.e., hybrid lethality or hybrid necrosis). F 1 hybrids that exhibit this phenomenon are characterized by weaker growth than their parents, and the phenomenon has been reported to occur in the offspring of crosses involved a number of species, including Oryza sativa [1][2][3][4], Nicotiana spp. [5], Capsicum spp. [6], Arabidopsis thaliana [7], Triticum spp. [8,9], Gossypium spp. [10], and Phaseolus vulgaris [11]. The genetic mechanisms of hybrid weakness are explained by the Bateson-Dobzhanzky-Muller model [12][13][14], which posits that the reduced hybrid vigor is driven by deleterious interactions between genes at different loci. In many cases, one of the causal genes is related to disease resistance (R), and interactions between the R gene and other causal gene cause autoimmune responses in the hybrid offspring [2,7]. The autoimmune responses include the accumulation of reactive oxygen species such as H 2 O 2 , cell death, upregulation of disease resistance-related genes, and downregulation of photosynthesis-related genes [2,7,15,16].
In rice, hybrid weakness has been reported to result from interactions between the HWI1 locus, which encodes the LRR-RLK gene (R gene), and the HWI2 locus, which encodes a subtilisin-like protease, and hybrids have been reported to exhibit localized programmed cell death (PCD), the high accumulation of salicylic and jasmonic acids, and amplified heat-related weakness symptoms [2]. These results demonstrate that the interaction of causal genes can activate downstream immune responses, such as hypersensitive response-like mechanisms [2,7].
The hybrid weakness that results from the interaction of Hwa1-1, a dominant allele of the HWA1 locus, and Hwa2-1, a dominant allele of the HWA2 locus, was firstly reported in rice by Oka [4]. In that study, F 1 hybrid seedlings that exhibited hybrid weakness were reported to exhibit normal germination and seedling growth until developing three to four leaves, after which plant growth halted and the leaves yellowed. Then, unless the environment was particularly favorable, the plants died before reaching anthesis. The distributions of the Hwa1-1 and Hwa2-1 alleles were limited to Indian cultivars [4], and both the HWA1 and HWA2 loci were located in a 1637-kb region of the long arm of chromosome 11 [17]. However, the causal genes have not been identified, and the molecular mechanism underlying the hybrid weakness associated with the HWA1 and HWA2 loci remain unclear.
Accordingly, the aim of the present study was to characterize the phenotypes of the hybrid weakness that is associated with the HWA1 and HWA2 loci, in order to understand the system's underlying mechanisms. The effectiveness of SPAD values, an index correlated with chlorophyll content [18], for determining the progression of the hybrid weakness was also evaluated. The occurrence of cell death and H 2 O 2 accumulation was also evaluated, and the expression of disease resistance and photosynthesis-related genes in the leaves of F 1 hybrids exhibiting hybrid weakness were analyzed.

Hybrid Weakness Phenotypes
The Oryza sativa Indian cultivars 'A.D.T.14 and 'P.T.B.7 carry homozygous Hwa1-1 and Hwa2-1 alleles, respectively. All the F 1 hybrids of a cross between 'A.D.T.14 and 'P.T.B.7 exhibited dwarfism, reduced tiller number, and leaf yellowing ( Figure 1). Increases in the height of the F 1 hybrids nearly halted at 50 days after sowing (DAS), whereas that of the parents continued increasing (Figure 2A). The progression of plant age in leaf number in the F 1 hybrids was the same as that in both parents ( Figure 2B). The tiller number of both parents continued increasing and reached >35 tillers at 70 DAS, whereas that of the F 1 hybrids increased little and only reached five tillers by 70 DAS ( Figure 2C). In addition, both parents headed by 80 DAS, whereas none of the F 1 hybrids had started heading after 140 DAS (Figure 1). the lower leaves were lower than those of the upper leaves (fourth vs. fifth and sixth leaves and fifth vs. sixth leaves), and within each leaf, the SPAD values of the leaf tips were lower than those of the leaf bases ( Figure 3D-F).   the lower leaves were lower than those of the upper leaves (fourth vs. fifth and sixth leaves and fifth vs. sixth leaves), and within each leaf, the SPAD values of the leaf tips were lower than those of the leaf bases ( Figure 3D-F).

Cell Death and H2O2 Accumulation
The physiological changes that accompanied leaf yellowing were surveyed by analyzing F1 hybrid leaves that had been classified into four stages based on degree of yellowing ( Figure 4A). Chlorophyll content was assessed by SPAD analysis and spectrophotometry. The SPAD values of Stage-1, -2, and -3 leaf tips, Stage-3 leaf middles, and Stage-3 leaf bases were lower than those of Stage-0 leaves ( Figure 4B). The SPAD values of Stage-3 leaf tips, Stage-3 leaf middles, and Stage-3 leaf bases were lower than those of Stage-1 leaves ( Figure 4B). The SPAD values of Stage-3 leaf middles and Stage-3 leaf bases were lower than those of Stage-2 leaves ( Figure 4B). Total chlorophyll content also decreased in all leaf parts (tip, middle, and base) as yellowing progressed ( Figure 4C). Because of the usefulness of SPAD value as discussed later, the progression of yellowing of leaves used in the subsequent experiments were evaluated based on SPAD value.
To determine whether cell death occurred in F1 hybrid leaves, cellular ion leakage, owing to ion permeability by cell death, was measured. Ion leakage increased slightly and significantly in the tips of Stage-2 and Stage-3 leaves, respectively ( Figure 4D). Cell death in F1 hybrid leaves was also evaluated using trypan blue staining, which is used to identify the highly permeable membranes of dead cells. Only Stage-3 leaves contained dead cells ( Figure 5A), and the analysis of transverse sections of Stage 3 revealed that the dead cells were located around vascular and epidermal cells ( Figure 5B).
Meanwhile, 3,3-diaminobenzidine (DAB) staining revealed the presence of H2O2, which, as a reactive oxygen species, is an important regulator of cell death. Plant tissue is stained brown when DAB is oxidized by H2O2 into an insoluble polymer. Hydrogen peroxide (H2O2) was detected in the leaves of all stages, except Stage 0 ( Figure 6).

Cell Death and H 2 O 2 Accumulation
The physiological changes that accompanied leaf yellowing were surveyed by analyzing F 1 hybrid leaves that had been classified into four stages based on degree of yellowing ( Figure 4A). Chlorophyll content was assessed by SPAD analysis and spectrophotometry. The SPAD values of Stage-1, -2, and -3 leaf tips, Stage-3 leaf middles, and Stage-3 leaf bases were lower than those of Stage-0 leaves ( Figure 4B). The SPAD values of Stage-3 leaf tips, Stage-3 leaf middles, and Stage-3 leaf bases were lower than those of Stage-1 leaves ( Figure 4B). The SPAD values of Stage-3 leaf middles and Stage-3 leaf bases were lower than those of Stage-2 leaves ( Figure 4B). Total chlorophyll content also decreased in all leaf parts (tip, middle, and base) as yellowing progressed ( Figure 4C). Because of the usefulness of SPAD value as discussed later, the progression of yellowing of leaves used in the subsequent experiments were evaluated based on SPAD value.
To determine whether cell death occurred in F 1 hybrid leaves, cellular ion leakage, owing to ion permeability by cell death, was measured. Ion leakage increased slightly and significantly in the tips of Stage-2 and Stage-3 leaves, respectively ( Figure 4D). Cell death in F 1 hybrid leaves was also evaluated using trypan blue staining, which is used to identify the highly permeable membranes of dead cells. Only Stage-3 leaves contained dead cells ( Figure 5A), and the analysis of transverse sections of Stage 3 revealed that the dead cells were located around vascular and epidermal cells ( Figure 5B).
Meanwhile, 3,3-diaminobenzidine (DAB) staining revealed the presence of H 2 O 2 , which, as a reactive oxygen species, is an important regulator of cell death. Plant tissue is stained brown when DAB is oxidized by H 2 O 2 into an insoluble polymer. Hydrogen peroxide (H 2 O 2 ) was detected in the leaves of all stages, except Stage 0 ( Figure 6).

Hybrid Weakness-Related Gene Expression
At 70 DAS, the 11th (Stage 3) and 13th (Stage 0) leaves of the parents and F1 offspring were collected for gene expression analysis (Figure 7). The 13th leaves of both the parents and hybrids were entirely green, as indicated by high SPAD values, even though the SPAD values of the F1 hybrids were somewhat lower than those of either parent ( Figure 7A,C). Meanwhile, the 11th leaves of the F1 hybrids exhibited significant yellowing, as indicated by low SPAD values, whereas those of both parents were entirely green, as indicated by high SPAD values ( Figure 7B,D).

Hybrid Weakness-Related Gene Expression
At 70 DAS, the 11th (Stage 3) and 13th (Stage 0) leaves of the parents and F1 offspring were collected for gene expression analysis (Figure 7). The 13th leaves of both the parents and hybrids were entirely green, as indicated by high SPAD values, even though the SPAD values of the F1 hybrids were somewhat lower than those of either parent ( Figure 7A,C). Meanwhile, the 11th leaves of the F1 hybrids exhibited significant yellowing, as indicated by low SPAD values, whereas those of both parents were entirely green, as indicated by high SPAD values ( Figure 7B,D).

Hybrid Weakness-Related Gene Expression
At 70 DAS, the 11th (Stage 3) and 13th (Stage 0) leaves of the parents and F 1 offspring were collected for gene expression analysis (Figure 7). The 13th leaves of both the parents and hybrids were entirely green, as indicated by high SPAD values, even though the SPAD values of the F 1 hybrids were somewhat lower than those of either parent ( Figure 7A,C). Meanwhile, the 11th leaves of the F 1 hybrids exhibited significant yellowing, as indicated by low SPAD values, whereas those of both parents were entirely green, as indicated by high SPAD values ( Figure 7B,D).
The expression of 11 disease resistance-related genes and four photosynthesis-related genes were surveyed (Table 1 and Table S1). The PR1 genes (PR1A and PR1B), the expression of which is induced by salicylic acid [19,20], were upregulated in the 11th leaves of the F 1 hybrids (Figure 8), as were several PR2 genes (Gns5, Gns2, and OsEGL2), which encode glucanase-related proteins that degrade fungal cell walls [21] (Figure 8). Meanwhile, of several genes that encode chitinase-related proteins (PR4, CHT9, CHT11, and RIXI), which also degrade fungal cell walls [22], PR4, CHT9, and RIXI were all upregulated in the 11th leaves of the F 1 hybrids; however, only PR4 was upregulated significantly (Figure 8). The expression of ACO2, which encodes an enzyme related to ethylene production [23], was similar in the 11th and 13th leaves of the F 1 hybrids (Figure 8). Finally, PDC1, the expression of which is induced by jasmonic acid [24], was upregulated in the 11th leaves of the F 1 hybrids, although not significantly (Figure 8).
Of the four photosynthesis-related genes, PSAF, LHCB, and OsRbcL were somewhat downregulated in the 11th leaves of the F 1 hybrids; however, none of these trends were significant (Figure 8). The expression of 11 disease resistance-related genes and four photosynthesis-related genes were surveyed (Table 1 and S1). The PR1 genes (PR1A and PR1B), the expression of which is induced by salicylic acid [19,20], were upregulated in the 11th leaves of the F1 hybrids (Figure 8), as were several PR2 genes (Gns5, Gns2, and OsEGL2), which encode glucanase-related proteins that degrade fungal cell walls [21] (Figure 8). Meanwhile, of several genes that encode chitinase-related proteins (PR4, CHT9, CHT11, and RIXI), which also degrade fungal cell walls [22], PR4, CHT9, and RIXI were all upregulated in the 11th leaves of the F1 hybrids; however, only PR4 was upregulated significantly (Figure 8). The expression of ACO2, which encodes an enzyme related to ethylene production [23], was similar in the 11th and 13th leaves of the F1 hybrids ( Figure 8). Finally, PDC1, the expression of which is induced by jasmonic acid [24], was upregulated in the 11th leaves of the F1 hybrids, although not significantly (Figure 8).
Of the four photosynthesis-related genes, PSAF, LHCB, and OsRbcL were somewhat downregulated in the 11th leaves of the F1 hybrids; however, none of these trends were significant (Figure 8).

Discussion
Oka [4] reported that F1 hybrids that exhibit hybrid weakness associated with the HWA1 and HWA2 loci exhibit growth termination and leaf yellowing after the seedlings developed three or four leaves. However, the plant growth phenotypes were not described in detail. In contrast, the present study determined that F1 hybrids from the cross of 'A.D.T.14′ and 'P.T.B.7′ rice exhibited limited growth and tiller number, as well as and leaf yellowing (Figures 1-3). Even though leaf yellowing was also reported by Oka [4], the timing of the yellowing process was different [4]. In the present study, leaf yellowing was observed in F1 hybrids that had developed seven or eight leaves at 30 DAS and, furthermore, was associated with the downregulation of photosynthesis-related genes ( Figure 8).

Discussion
Oka [4] reported that F 1 hybrids that exhibit hybrid weakness associated with the HWA1 and HWA2 loci exhibit growth termination and leaf yellowing after the seedlings developed three or four leaves. However, the plant growth phenotypes were not described in detail. In contrast, the present study determined that F 1 hybrids from the cross of 'A.D.T.14 and 'P.T.B.7 rice exhibited limited growth and tiller number, as well as and leaf yellowing (Figures 1-3). Even though leaf yellowing was also reported by Oka [4], the timing of the yellowing process was different [4]. In the present study, leaf yellowing was observed in F 1 hybrids that had developed seven or eight leaves at 30 DAS and, furthermore, was associated with the downregulation of photosynthesis-related genes (Figure 8).
In O. sativa, three other gene sets have been reported to cause hybrid weakness, and the phenotypes associated with each system are different. More specifically, the hybrid weakness associated with the HWC1 and HWC2 loci is characterized by short stature, short roots, and rolled leaves [26], whereas that associated with the HWI1 and HWI2 loci is characterized by short stature and impaired root formation [2], and that associated with the HW3 and HW4 loci is characterized by short culms, fewer panicles, pale green leaves, and chlorotic leaf spots [3]. Remarkably, leaf yellowing has only been reported for the hybrid weakness associated with the HWA1 and HWA2 loci. Together, these reports suggest that either the causal genes of each system have different functions or the processes downstream of the causal gene interactions are different.
In the present study, the usefulness of SPAD value for determining the progression of leaf yellowing during hybrid weakness associated with the HWA1 and HWA2 loci were evaluated. SPAD values generally corresponded with leaf yellowing (Figure 4A,B) but failed to identify significant differences between the chlorophyll content of Stage-0 leaves and that of either the bases of Stage-1 leaves or the middles or bases of Stage-2 leaves. These results indicate that spectrophotometry is more sensitive than SPAD values to changes in chlorophyll content ( Figure 4B,C). However, it is important to note that, because SPAD value accurately reflected degree of leaf yellowing and because spectrophotometry requires leaf destruction (Figure 4), SPAD value measurement is an effective and nondestructive method that can be used to quickly and easily evaluate hybrid weakness associated with the HWA1 and HWA2 loci.
The hybrid weakness phenotype that was studied by the present study also exhibited cell death in the yellow leaves ( Figures 4D and 5). Similarly, the hybrid weakness associated with the HWI1 and HWI2 loci involved cell death at the basal nodes [2], and the hybrid weakness associated with the HW3 and HW4 loci involved cell death in leaves [3]. Cell death has been also detected in the leaves of intraspecific Arabidopsis hybrids that exhibit hybrid necrosis [7,27] and in the leaves, stems, and roots of interspecific Nicotiana hybrids that exhibit hybrid lethality [28,29]. Therefore, despite differences in localization, cell death appears to be a common feature of hybrid weakness in plants.
In the present study, cell death was only detected in Stage-2 and Stage-3 leaves, which indicates that the timing of cell death does not coincide with that of either leaf yellowing or reductions in SPAD value or chlorophyll content (Figure 4). On the other hand, H 2 O 2 was detected in Stage-1, -2, and -3 leaves that exhibited yellow leaf tips and low SPAD values ( Figure 6). H 2 O 2 commonly triggers plant cell death during hypersensitive reactions, senescence, abiotic stress responses, and development [30][31][32]. We detected that reactive oxygen species would lead to cell death on hybrid weakness by HWA1 and HWA2.
In many cases of hybrid weakness, one of the causal genes encodes an R gene, and the interaction of the R gene with another causal gene triggers an autoimmune response [7,27,33,34]. In the hybrid weakness associated with the HWI1 and HWI2 loci, the causal genes include the LRR-RLK gene (R gene) and a subtilisin-like protease gene, respectively, and the interaction of the causal genes results in an autoimmune response [2]. Meanwhile, in the hybrid weakness associated with the HW3 and HW4 loci, HW3 encodes a calmodulin-binding protein, and even though the gene is a defense-response gene, not an R gene, the interaction of HW3 with HW4 results in an autoimmune response [3]. Furthermore, in the hybrid weakness associated with the HWA1 and HWA2 loci, a candidate region, which harbored both loci, also contained 12 R genes, along with many other genes [17]. During hypersensitivity reactions, reactive oxygen species are produced, thereby mediating cell death, chloroplast disruption, and the upregulation of defense-related genes [35,36]. In the present study, cell death was detected in the F 1 leaves after H 2 O 2 generation and leaf yellowing (Figure 3, Figure 5, and Figure 6), the yellow leaves of the F 1 hybrids exhibited upregulated defense-related genes (Figure 8). These results suggest that the hybrid weakness associated with the HWA1 and HWA2 loci involves hypersensitive reaction-like responses. However, because leaf senescence is also associated with H 2 O 2 production, cell death, the upregulation of certain defense genes, and leaf yellowing [37], it is possible that the hybrid weakness associated with the HWA1 and HWA2 loci involves premature senescence. Additional molecular studies will reveal the exact mechanism underlying the hybrid weakness associated with the HWA1 and HWA2 loci.

Plant Materials and Growth Conditions
The Oryza sativa Indian cultivars 'A.D.T.14 (indica [17]), which is homozygous for the Hwa1-1 allele, and 'P.T.B.7 (aus [17]), which is homozygous for the Hwa2-1 allele, were crossed to generate F 1 hybrid offspring. The genotypes of the two cultivars were previously reported by Oka [4]. F 1 seeds were obtained by crossing 'P.T.B.7 (♀) and 'A.D.T.14 (♂) parents. Seeds of 'A.D.T.14 , 'P.T.B.7 , and the F 1 offspring were sown on 17 July 2011. After the seeds were germinated on moistened filter paper in Petri dishes, the seedlings were transplanted to soil (Sukoyaka-Jinko-Baido; Yanmar Co., Ltd., Osaka, Japan) in Wagner pots of 1/5000 a. The seedlings were grown under natural light conditions in a greenhouse at Osaka Prefecture University, Sakai, Japan. The temperature and humidity of the greenhouse were recorded using a data logger (Ondotori; T&D Co., Ltd., Matsumoto, Japan), and the plants were fertilized weekly using Otsuka-A prescription (OAT Agrio Co., Ltd., Tokyo, Japan), which contained 18.6 mM N, 5.1 mM P, 8.6 mM K, 8.2 mM Ca, and 0.4 mM Mg. The plants were cultivated for 140 DAS to survey plant height, plant age in leaf number, tiller number, days to heading, and SPAD value. The plant height was measured from the surface of the soil to the tip of the tallest leaves. To evaluate the relationship between leaf yellowing and physiology, the leaves were classified according to degree of leaf yellowing. Leaves in which 0%, 25%, 50%, or > 75% of the blade had turned yellow were assigned to Stages 0, 1, 2, and 3, respectively ( Figure 4A). These leaves classified according to degree of leaf yellowing were used to measured SPAD value and chlorophyll content, as well as to detect dead cells and H 2 O 2 . Parts of seedlings were cultivated in Wagner pots of 1/10,000 a in an incubator (14 h natural light and 10 h dark, 28 • C, light intensity: 512 µmol m −2 s −1 ), and at 70 DAS, these plants were used as material for gene expression analysis.

SPAD and Chlorophyll Measurement
A SPAD meter (SPAD-502; Konica Minolta, Inc., Tokyo, Japan) was used to measure the SPAD values of the leaves without causing damage. SPAD values were obtained from the tip, middle, and base of each leaf. Meanwhile, total chlorophyll content was measured using a previously described spectrophotometric method [38]. Briefly, the leaves were cut into small pieces, weighed, treated with 20 mL 80% acetone, and ground using a pestle until bleached. The resulting solutions were transferred to 1.5 mL tubes and centrifuged at 10,000 g for 5 min. Each supernatant was transferred to a cuvette, and the absorbance of each supernatant was measured at 663.6 and 646.6 nm, after the spectrophotometer (V-530; JASCO Corp., Hachioji, Japan) was zeroed at 750 nm. Total chlorophyll concentration (mg g −1 FW) was calculated using the following equation: [(17.76 × OD 646.6 + 7.34 × OD 663.6 ) × extraction volume in a cuvette]/fresh weight (g).

Ion leakage Measurement
Ion leakage was measured, as described previously [39]. Leaf disks (3 cm 2 ) were taken from the tips, middles, and bases of the leaves, floated for 5 min in water that contained 0.2% (v/v) Tween 20 for removing ion generating on making leaf disks, transferred to Petri dishes that contained fresh water with Tween 20 (0.2%), and incubated for 3 h for leaking out ions by cell death. Their conductivity (value A) of the solutions was measured using a conductivity meter (Twin Cond B-173; Horiba, Ltd., Kyoto, Japan). The leaf disks were then incubated at 95 • C for 25 min, for leaking out ions of whole leaf disks by destroying whole organization, and cooled to room temperature, and their conductivity of the solutions was also measured (value B). Finally, ion leakage (%) was calculated using the following equation: (value A/value B) × 100%.

Trypan Blue Staining
Trypan blue staining was performed as described previously [40]. Detached leaves were stained by boiling for 8 min in a 1:1 (v:v) mixture of ethanol and lactophenol (i.e., alcoholic lactophenol) that contained 0.1 mg ml −1 trypan blue, cleared in 70% chloral hydrate solution overnight, and then preserved in 70% glycerol. Trypan blue stains dead cells. Transverse slices were prepared using a hand-section method and visualized using a light microscope (Olympus BX50; Olympus, Co. Ltd., Tokyo, Japan).

Detection of Hydrogen Peroxide Accumulation
Hydrogen peroxide was detected visually, using previously described methods [41]. Briefly, leaves were soaked in a 3,3-diaminobenzidine (DAB) solution for 24 h, transferred to boiling 96% ethanol until bleaching, and then visualized. The presence of H 2 O 2 was indicated by brown staining.

Real-Time qRT-PCR
Total RNA was isolated from leaves using an RNAiso PLUS kit (Takara Bio, Inc., Shiga, Japan), according to the manufacturer's protocol and then treated with RNase-free DNase (Promega Co., Madison, USA), and first-strand cDNA was synthesized from total RNA (2 µg) using oligo (dT) 18 primers and ReverTra Ace (Toyobo Co., Ltd., Osaka, Japan). Real-time RT-PCR was carried out to analyze the expression of 11 defense-related genes and four photosynthesis-related genes (Table 1), using Actin as an internal control. The primers used to amplify PR1A, PR1B, Gns5, PR4, and Actin had been reported previously [42], and the other primers were designed based on RAP-DB locus ID using the Primer-BLAST design tool [43] (Table S1). Real-time RT-PCR was performed in 20 µL reaction mixtures that contained 10 µL KAPA SYBR FAST qPCR Master Mix (2×) ABI PRISM (Takara Bio), 10 µM of each forward and reverse primer (0.4 µL each), and 1 µL cDNA template, and the real-time PCR amplification was performed under the following conditions: Initial denaturation at 94 • C for 10 min, followed by 40 cycles of 15 s at 94 • C and 1 min at 60 • C, with a final 30 s extension at 72 • C using an Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems, Foster, CA, USA). The results were analyzed using ABI Prism software (Applied Biosystems). Each gene expression level was divided by the expression level of Actin to calculate relative expression level.

Statistical Analysis
Data were analyzed using SPSS (version 22; IBM, Co., Armonk, USA). Tukey HSD tests were used to compare SPAD, chlorophyll content, and ion leakage values, and two-tailed Student's t-tests were used to compare mid-parental (mean of 'A.D.T.14 and 'P.T.B.7 ) and hybrid values of plant height, foliar age, and tiller number.