Uniparental Inheritance of Salinity Tolerance and Beneﬁcial Phytochemicals in Rice

: Salinity stress is one of the most problematic constraints to signiﬁcantly reduce rice productivity. The Saltol QTL (quantitative trait locus) has been known as one among many principal genes / QTLs responsible for salinity tolerance in rice. However, the introgression of the Saltol QTL from the donor (male) into the recipient (female) cultivars induces great recessions from the progeny generation, which results in heavy ﬁeldwork and greater cost and time required for breeding. In this study, the F 1 generation of the cross TBR1 (female cultivar, salinity tolerant) × KD18 (male cultivar, salinity susceptible) was preliminarily treated with N-methyl-N-nitrosourea (MNU) to induce the mutants M 1 . Results on physiological traits show that all the M 2 (self-pollinated from M1) and M 3 (self-pollinated from M2) individuals obtain salinity tolerant levels as the recurrent TBR1. Twelve SSR (simple sequence repeat) markers involved in the Saltol QTL (RM493, RM562, RM10694, RM10720, RM10793, RM10852, RM13197, RM201, RM149, RM508, RM587, and RM589) and other markers related to yield-contributing traits and disease resistance, as well as water and nitrogen use, have e ﬃ cacy that is polymorphic. The phenotype and genotype analyses indicate that the salinity tolerant Saltol QTL, growth parameter, grain yield and quality, pest resistance, water and nitrogen use e ﬃ cacy, and beneﬁcial phytochemicals including antioxidants, momilactone A (MA) and momilactone B (MB) are uniparentally inherited from the recurrent (female) TBR1 cultivar and stabilized in the M 2 and M 3 generations. Further MNU applications should be examined to induce the uniparental inheritance of other salinity tolerant genes such as OsCPK 17, OsRMC , OsNHX 1, OsHKT 1;5 to target rice cultivars. However, the mechanism of inducing this novel uniparental inheritance for salinity tolerance by MNU application needs elaboration.


Introduction
Salt is one of the main causes of land degradation worldwide with approximately 2000 million ha affected land being recorded every year, according to a study by Economics of Salt-Induced Land Degradation and Restoration (unu.edu/media-relations/releases). Salinity stress can reduce 70% yield loss of wheat, maize, rice and barley and the total cost of such loss in crop productivity can reach and it shows low quality parameters as compared with common commercial rice [11]. Our laboratory observed that >97% kernel color of the FL478 was similar to that of Pokkali (Supplementary Table S1). In addition, the use of DNA molecular markers such as RFLP, RAPD, AFLP, ISSR, SSR, SNP, DarT and Retrotransposons are useful to reduce the time for selection, but they can not exhibit all QTLs/genes interactions involved in the selected traits [12,25].
Our group has developed a protocol using N-methyl-N-nitrosourea (MNU) to treat rice seeds at low concentrations for three months prior to germination [12,25]. The MNU application induced uniparental inheritance of the yield attributing traits, including plant height, semi-dwarfism, amylose content, protein content, gel consistency, grain yield, and spikelet fertility [12,25]. In this study, we continuously examine whether the salinity tolerant Saltol QTL can also be uniparentally inherited by MNU treatment. The influence of the uniparental inheritance on induction of beneficial phytochemicals including total phenols and flavonoids, antioxidant activities, and momilactones A and B in the offspring lines were also evaluated.

Rice Materials and Salinity Treatment
TBR1 and KD18 are commercial rice in Vietnam. Both are Indica subtypes, where TBR1 performs a higher yield, protein content and lipid content compared to KD18. TBR1 is resistant and KD18 is susceptible to pests and diseases. In this study, TBR1 was used as the recurrent (female) cultivar, whilst KD18 was the male variety. They were provided by the Agricultural Genetics Institute, Hanoi, Vietnam. The field experiment was conducted near Hiroshima University, Higashi-Hiroshima, Hiroshima, Japan (270-280 m elevation; 33/25 • C day/night; humidity: 60%-65%; precipitation average: 1485 mm). The fertilizers, weeding, watering, and pesticides were provided by conventional methods in Japan. The original F 1 rice seeds (total 300 seeds) were obtained by crossing TBR1 (female) and KD18 (male) cultivars. Subsequently, the F 1 seeds were treated with the MNU as described previously [12,25] to induce the first mutated generation M 1 (200 seeds). The M 1 seeds were kept in the dark for 3 months in a hermetic condition and stored at 4 • C before self-pollinating in a paddy field to obtain the second generation (M 2 ) seeds. The M 2 population (200 seeds) was continuously self-pollinated in rice fields to provide the third generation (M 3 ). After gemination, TBR1, KD18, F 1 , F 2 (self-pollinated from F 1 ), M 2 , and M 3 seeds were grown in a 0.5% agarose media supplied by Yoshida nutrient and placed in a plant growth chamber (28 • C day: 25 • C night; 12 h light: 12 h dark). Salinity was applied after five days of transplanting with a concentration of 12 dSm −1 NaCl to evaluate the salinity tolerant of the parental cultivars and progenies. The treatment without NaCl was considered as control (0 dSm −1 ). During treatment, EC and pH (5.5) were checked and maintained daily.

Physiological Analysis of Salt Tolerance
After 21 days of treatment, salinity tolerant rice materials were scored by a standard evaluation score (SES) [26]. Fifty plants from the five replications were randomly selected to evaluate the phenotypic characteristics. Survivability was determined by percentage of survived plants. Root length and plant height of rice samples were measured in milimeters. Rice plants were weighed twice. Fresh weight was measured right after treatment and dry weight was determined after drying in hot air oven for 5 days at 40 • C.

Total Phenols, Total Flavonoids, and Antioxidant Activities
The roots, stems, and shoots of rice seedlings were harvested and transferred directly to laboratory. They were cleaned by tap water and rinsed many times with distilled water. After drying for 5 days at 40 • C, the mixture of roots, stems, and shoots of rice samples were ground into powder. This powder was then extracted by methanol after 3 days using a magnetic stirrer. After that, the extract was separated by hexane and finally dried by evaporator at 50 • C. The obtained powder was kept in methanol at 4 • C in the dark for further measurements. Total phenolic content (TPC) and ABTS•+ decolorization measurement were conducted following a method described by Quan et al. [27]. Total flavonoid content (TFC) was estimated as detailed in Xuan et al. [28]. 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) and nitric oxide (NO) scavenging assays were evaluated by a method described in Govindarajan et al. [29]. The antioxidant activity was calculated by the following formula: where A sample is the absorbance of reaction with sample and A control is the absorbance of reaction with pure methanol. IC 50 value is determined as the essential concentration to obtain 50% radical reduction.

Identification and Quantification of Momilactones A and B
Momilactone A (MA) and momilactone B (MB) were identified and quantified by UPLC-ESI-MS following a protocol described in Quan et al. [30] using MA and MB purified from rice husk as the standards. The quantification of each momilactone was measured using a linear model through peak areas and retention times. Detection and quantitation limits of MA and MB were 0.68 and 2.05 ng/mL and 0.27 and 0.83 ng/mL, respectively.

Genetic Analysis
The total DNA in rice samples was extracted by cetyl-trimethyl ammonium bromide (CTAB) method [12]. Before applying for polymerase chain reaction (PCR), 0.8% agarose gel electrophoresis was used to check the quality of DNA. The DNA was amplified using a Thermal Cycler Gen Atlas S machine [12]. The amplification products were resolved in 3% agarose gel in tris borate EDTA (TBE) buffer (0.5 X) and 2.5 µl of safe view under room temperature at a constant voltage of 50 volts for 75 min. After running, the gel was visualized by AMZ System Science limited STAGE system ECX-F15M (Vilber Lourmat, Eberhardzell, Germany).

Data Analysis
Data were statistically analysed by analysis of variance (ANOVA). A linear model was used to calculate correlation between parental and progeny data. Reduction proportions of agronomical and chemical data were calculated by following formula: where P sample is value of sample growing in salt concentration 12 dSm −1 and P control is the value of sample in non-salinity.

Salt Tolerance, Agronomical, and Phytochemical Performances of Rice Population
The results in Table 1 show that phenotypic performances among F 1 and F 2 , and M 2 and M 3 are not remarkably different. In control conditions, no significant difference was observed in injury score, survivability, and growth parameters among TBR1, KD18, F 1 , F 2 , M 2 and M 3 . In salinity stress condition, the injury score and survivability of the M 2 and M 3 were not significantly different from the recurrent parent TBR1. On the contrary, those of the F 1 and F 2 were between the values of the parents. The other growth parameters of the M 2 and M 3 including root length, shoot length, fresh weight, and dry weight were neither markedly different from TBR1 nor less affected from salinity than KD18 (Table 1), as they showed greater performances than those of the parents. In the control population (F 1 and F 2 ), these values were between the values of parental cultivars. Findings of Table 1 indicate that the TBR1 cultivar was salinity tolerant whilst KD18 was salinity susceptible. The response of M 2 and M 3 against salinity was as tolerant as that of the recurrent parent TBR1, thus, it was concluded that the M 2 and M 3 are also salinity tolerant. Therefore, the salinity tolerant characteristic of M 2 is uniparentally inherited from TBR1. To date, all reports in literature, such as Mishra et al. [46] and Mohammadi et al. [47], reported that the salinity tolerance trait in rice is polygenic, principally inherited from father cultivar following the Mendelian rules. However, in contrast, findings in Table 1 show that the treatment of MNU can uniparentally control the salinity tolerant Saltol QTL in rice, conversely different from all researches in literature.   Table 2 shows the changes of chemical components including TPC, TFC, and contents of MA and MB, and antioxidant activities (DPPH, ABTS, and NO levels). In the untreated conditions, the phytochemical contents and antioxidant activities vary among TBR1, KD18, F 1 , F 2 , M 2 and M 3 , whilst no trace of MB was detected. Similar with phenotypic responses, no significant disparity was recorded in chemical compositions between F 1 and F 2 , and M 2 and M 3 . However, in the salinity stress condition, antioxidant activities of TBR1, F 1 , F 2 , M 2 and M 3 were both significantly stronger than that of KD18, of which F 1 and F 2 show weaker antioxidant properties than TBR1. 1 No remarkable difference in antioxidant capacities between TBR1 and M 2 was observed ( Table 2). The TPC and TFC values of the M 2 , M 3 , F 1 , and F 2 individuals were either significantly higher or lower than that of its parent KD18 and recurrent parent TBR1 (Table 2). Subsequently, the content of MA in TBR1 was higher than KD18, but M 2 and M 3 show much greater content of MA than their parental cultivars do (104.7 and 102.5 ng/g, respectively). Interestingly, no trace of MB was found in KD18, but the salinity stress induced MB in TBR1 (46.3 ng/g), M 2 (34.9 ng/g), and M 3 (20.5 ng/g) ( Table 2). Findings of Table 2 indicate that the MNU treatment also caused the uniparental inheritance of beneficial phytochemicals, including antioxidant activity and MA and MB from TBR1 to F 2 generation.

Correlation of Physiological Parameters between Progeny and Parental Lines
The distributions of examined traits of the M 2 population are shown in Figure 1. A high correlation of 0.81 (R 2 = 0.6494) was observed between M 2 and TBR1, while in contrary a much lower correlation of 0.45 (R 2 = 0.2059) was found between M 2 and KD18. The evidence indicates that agronomical and chemical properties of progenies are uniparentally inherited from the female cultivars.

Genetic Segregation of F2, M2 and M3 Populations
A total of 228 rice plants were used to analyze the segregations in the F2, M2 and M3 generations (Figures 2,3). The results show that in the amplification of all the polymorphic SSR markers, the F2 population possess both the male parent allele KD18 and the female parent allele TBR1 (following the Mendelian theory). However, both M2 and M3 generations are completely inherited (100%) from the recurrent parent TBR1 cultivar (Figure 3a,b). It was observed that the preliminary treatment of MNU on the original seeds from the cross TBR1 × KD18 induced the salinity tolerance in the M2 and M3 genotypes, which are completely inherited from the recurrent parent TBR1 cultivar. The uniparental inheritance of salinity tolerance in the M2 generation was completely stabilized in M3 generation, with no segregation observed (Figure 3b).

Genetic Segregation of F 2 , M 2 and M 3 Populations
A total of 228 rice plants were used to analyze the segregations in the F 2 , M 2 and M 3 generations (Figures 2 and 3). The results show that in the amplification of all the polymorphic SSR markers, the F 2 population possess both the male parent allele KD18 and the female parent allele TBR1 (following the Mendelian theory). However, both M 2 and M 3 generations are completely inherited (100%) from the recurrent parent TBR1 cultivar (Figure 3a,b). It was observed that the preliminary treatment of MNU on the original seeds from the cross TBR1 × KD18 induced the salinity tolerance in the M 2 and M 3 genotypes, which are completely inherited from the recurrent parent TBR1 cultivar. The uniparental inheritance of salinity tolerance in the M 2 generation was completely stabilized in M 3 generation, with no segregation observed (Figure 3b).

Genetic Segregation of F2, M2 and M3 Populations
A total of 228 rice plants were used to analyze the segregations in the F2, M2 and M3 generations (Figures 2,3). The results show that in the amplification of all the polymorphic SSR markers, the F2 population possess both the male parent allele KD18 and the female parent allele TBR1 (following the Mendelian theory). However, both M2 and M3 generations are completely inherited (100%) from the recurrent parent TBR1 cultivar (Figure 3a,b). It was observed that the preliminary treatment of MNU on the original seeds from the cross TBR1 × KD18 induced the salinity tolerance in the M2 and M3 genotypes, which are completely inherited from the recurrent parent TBR1 cultivar. The uniparental inheritance of salinity tolerance in the M2 generation was completely stabilized in M3 generation, with no segregation observed (Figure 3b).

Discussion
The preliminary treatment of MNU on the original seeds from the cross TBR1 × KD18 induces the uniparental inheritance of salinity tolerant Saltol QTL from TBR1 to the M 2 and M 3 generations in both phenotypes (Tables 1 and 2) and genotypes (Figures 2 and 3). The genotype of the M 3 was completely similar to M 2 (Figure 3). Following the salinity tolerance, antioxidant activities and contents of MA and MB were also uniparentally inherited (Table 2), although the associated genetic markers with QTLs/genes determining antioxidant activities and biosynthesis of MA and MB were not examined in this study.
In this study, the F 1 seeds are the progenies of the cross between TBR1 (female variety) × KD18 (male variety), and the M 1 generation was induced by treating F 1 with MNU treatment. The M 1 was self-pollinated to obtain M 2 , and M 3 was induced by the M 2 self-pollination. The purpose of this was to check the stability of uniparental inheritance of the salinity tolerant Saltol QTL from the mother variety. In conventional breeding, the salinity tolerant cultivar should be the father cultivar. Because the salinity tolerant characteristics are determined by multiple genes/QTLs, they follow the Mendel rules with complicated segregation from the F 2 generation. Therefore, to stabilize the salinity tolerance, F 2 seeds are commonly crossed with the father variety (backcross) and repeated in many generations which requires huge amounts of fieldwork, time, and money [11,12,[48][49][50][51][52]. All previous work used the donor (male) cultivars having the Saltol QTL in the breeding to introduce the salinity tolerance to the target cultivars. For instance, Babu et al. [11] developed a rice line Pusa1734-8-3-3 having good salinity tolerance in the seedling stage compared with the FL478 donor (father cultivar). The salinity tolerance of Pusa1734-8-3-3 was the BC 3 F 4 + (>7 generations) from the cross Pusa Basmati 1121 (female cultivar) and FL478 (male cultivar). Similarly, Leon et al. in 2017 developed different salinity tolerant introgression lines (Ils) from the BC 4 F 4 (eight generations), but the salinity tolerance characteristic has not yet been stabilized [48]. The treatment of MNU in this study induced the uniparental inheritance for the Saltol QTL, providing a simple protocol to develop rice lines tolerant to salinity. To date, almost all known QTLs involved in salt-tolerance are located on chromosome 1 [7]. In this study, the selected nine SSR markers relevant to salinity tolerant are polymorphic and stationed on chromosomes 1, 2, 3, 4, 5, 7, 8, 10 and 11, in which the major SSR markers are located on the chromosome 1 (Supplementary Table S2). In this study, we did not carry out a vast screening on many SSR markers but selected only fifty known SSR markers related to important agronomic, pest resistant and salinity tolerant Saltol QTL, which have been already reported in previous research (Supplementary Table S2). These markers were used to distinguish the difference in parental genotypes associated with the phenotypes observed in Table 1. Subsequently, twenty-one SSR markers were found to be polymorphic and involved in salinity tolerant and elite agronomic traits (amylose content, plant height, spikelet fertility, brown plant hopper resistance, blast disease resistance) (Supplementary Table S2). Therefore, they were used to examine the F 2 , M 2 , and M 3 individuals. However, only nine SSR markers involved in Saltol QTL are polymorphic, including seven SSR markers locate on chromosome 1 (RM493, RM562, RM10694, RM10720, RM10793, RM10852, and RM13197), and two markers RM201 and RM149 located on chromosome 8 and 9, respectively [8][9][10] (Figure 2; Supplementary Table S2). Other polymorphic markers were identified relating to plant height (RM202, RM206, RM219, RM229) [12], leaf diameter (RM508) [43], spikelet fertility (RM202, RM206) [12] spikelet number (RM229) [12], grain yield (RM219, RM432) [12], amylose content (RM219, RM432, RM508, RM587, RM589) [12], gel consistency (RM589) [12], blast resistance (RM1233, RM206, RM207, RM213) [12,43], brown planthopper resistance (RM206) [12], and water and nitrogen use efficiency (RM518) [45] (Supplementary Table S2). Other SSRs relevant to the Saltol QTL are not polymorphic and they could not be used to identify uniparentally controlled by MNU treatment (Supplementary Figure S3). Further treatments of MNU should be applied to enable the uniparental inheritance of more salinity tolerant genes/QTLs, such as OsCPK17, OsRMC, OsNHX1, OsHKT1;5 genes [6], except the SalT gene derived from the Saltol QTL.
It is proposed that only these nine SSR markers relevant to the Saltol QTL are responsible for the salinity tolerance of the TBR1 × KD18 cross, and momilactones MA and MB are determined by five known biosynthesis genes (AK103462: dehydrogenase gene and CYP99A2 and CYP99A3: P-450 genes form a chitin oligosaccharide elicitor, together with OsKS4 and OsCyc1, the diterpene cyclase genes) clustered in chromosomes 2 and 4 [53]. Following the Mendelian genetic rules, because all these genes are located on chromosomes in the rice nucleus, the segregation ratios should be theoretically 1/1024 (5 genes) × 1/262,444 (9 genes) = 1/268,435,456 recombinants. Therefore, this study helps to reduce the huge laborious work, cost, and long breeding time that conventional breeding requires [12,25].
In our previous study, a total of 28 polymorphic SSR markers were selected from 200 markers relevant to plant height, semi-dwarfism, amylose content, protein content, gel consistency, grain yield, and spikelet fertility were genotyped on the second generation of similar cross TBR1 (mother) × KD18 (father) [12]. These markers are distributed on chromosomes 1-7, 9, and 11. All phenotypes and genotypes of the abovementioned growth and quality characteristics were uniparentally inherited in the second generation [12]. In addition, PCR results showed identical results of this study, indicating that all the 28 SSR markers were completely inherited from the recurrent TBR1 cultivar and no segregation was observed [12]. Combining the results obtained from this research, it can be concluded that by the prior treatment of MNU on F 1 seeds, important growth, quality traits, and the salinity tolerant Saltol QTL of the TBR1 cultivar can be uniparentally inherited to the F 2 and F 3 generations. Study on the ability of this promising rice source on other abiotic stresses is suggested. However, the subsequent generations should be strictly examined to ensure the stability of the uniparental inheritance. The mechanism of inducing this novel uniparental inheritance for salinity tolerance by MNU application needs to be further analyzed.
This study also shows that in salinity stress, rice plants promote the levels of antioxidant activities as well as content of MA and MB ( Table 2). In a non-treated control, no trace of MB was observed, but in the salinity treated condition, TBR1, M 2 , and M 3 induced MB as well as promoted MA content (Table 2). Although it is observed that phenotypically, antioxidant activities and induction of MA and MB in rice plants are also uniparentally inherited, polymorphic SSR markers related to antioxidant potentials and biosynthesis of MA and MB should be screened to evaluate whether their genotypes can be also uniparentally inherited or not. However, this study highlights that the preliminary treatment of MNU can also induce levels of antioxidant activities and important phytochemicals including MA and MB in rice, and the induction of antioxidant activities and MA and MB is also uniparentally inhibited to the second generation (Table 2).
MA and MB were first isolated and identified by Kato et al. [54] who reported that these two compounds are the growth inhibitors (allelochemicals) and phytoalexins [55,56] in rice. For more than 40 years since 1973, scientists worldwide have acknowledged that MA and MB are allelochemicals and conducted various experiments on examining quantities of MA and MB in different rice cultivars, as well as in rice organs and the release from roots [57,58]. However, the application of allelochemicals including MA and MB is questionable as the released amounts from the root leaches and plant parts are too low to inhibit growth of weeds, although they showed promising reduction on weed growth in laboratory experiments [28]. For MA and MB, we recently found that the two compounds are involved in drought and salinity tolerance in rice rather than allelochemicals [28,59]. In addition, MA and MB have the potential to control diabetes [60], cancers [61][62][63], and skin diseases [30]. In this study, MB was not found in the control condition, however the salinity stress induced MB in the recurrent parent (TBR1), M 2 , M 3 , and MA in all tested samples (Table 2). Therefore, it is concluded that treatment of MNU induced uniparental inheritance for both the salinity tolerant Saltol QTL, as well as beneficial phytochemicals, including antioxidants and MA and MB, in rice.

Conclusions
Findings of this study reveal that the principal salinity tolerant Saltol QTL trait in rice can be uniparentally controlled, along with beneficial phytochemical antioxidants and MA and MB in rice. The treatment of MNU aids to speed up and simplify the breeding of new rice cultivars, having both elite agronomic traits and tolerance to environment stresses. The question of whether QTLs/genes other than the Saltol QTL such as OsCPK17, OsRMC, OsNHX1, OsHKT1; 5 genes can also be uniparentally inherited by MNU treatment should be further examined.
Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4395/10/7/1032/s1, Table S1: Comparison of color between FL478 and Pokkali; Table S2: SSR markers used for distinguishing female parent (TBR1) and male parent (KD18); Figure S1: PCR products of parents and progenies with polymorphic SSR markers. Table S2: Polymorphic SSRs for screening progeny generations; Figure S2: PCR products of progenies with polymorphic SSR markers; Figure S3: Screening of parental DNAs with involved SSR markers. Funding: This research did not receive any external funding.