Development of Heat Tolerant Two-Line Hybrid Rice Restorer Line Carrying Dominant Locus of OsHTAS

Mehmood Jan, Gulmeena Shah, Huang Yuqing, Liu Xuejiao, Zheng Peng, Du Hao, Chen Hao, Tu Jumin

Research Paper

Development of Heat Tolerant Two-Line Hybrid Rice Restorer Line Carrying Dominant Locus of

Mehmood Jan#, Gulmeena Shah#, Huang Yuqing, Liu Xuejiao, Zheng Peng, Du Hao, Chen Hao, Tu Jumin

(Zhejiang Key Laboratory of Crop Germplasm Resources, Institute of Crop Science, Zhejiang University, Hangzhou 310058, Zhejiang, China; These authors contributed equally to this study)

In order to create novel germplasm resources for breeding heat tolerant variety, we transferred a dominant allele, previously characterized and cloned from a high-temperature tolerant local variety HT54, which was collected from the rice production area of southern China, into a high- temperature sensitive intermediate breeding line HT13 through six rounds of successive backcross by using marker-assisted selection. The molecular analysis showed that the recovery of genetic background of a resultant near isogenic line (NIL), MHT13, was around 99.8%. Thegene introduced in the MHT13 expressed normally in the HT13 genetic background, mediating heat tolerance and phenotype similar to those of the donor parent HT54. The major agronomic traits of MHT13 resembled those of the recurrent parent HT13. Moreover, MHT13 had high general combining ability and its rice quality reached the grade 3 standard of edible high-quality rice issued by Ministry of Agriculture of the People’s Republic of China, which greatly improved its application value in rice production.

rice; heat tolerance; high temperature; yield; near isogenic line; marker-assisted selection; grain quality;gene

The extreme alteration in temperature as a result of global warming has seriously declined the production of grain crops over the past few decades (Cheng et al, 2012). According to IPCC (2013) report, the mean global temperature seems to increase by 1.0 oC to 3.7 oC at the end of 20th century. Since 1950, 11 warm years out of 13 were from 2001 to 2011, the year 2011 being the warmest (Ye et al, 2015a). This change in temperature results in extreme weather events like heat waves and other adverse effects on grain crops (Challinor et al, 2014). Therefore, breeding crop varieties with improved heat tolerance is essential to feed the world in the era of global warming.

Although rice is a thermophilic crop, the optimum temperature for its growth and reproduction ranges from 24 oC to 29 oC during night and day, and above optimum temperature can be considered as stress (Figueiredo et al, 2015; Fahad et al, 2016). Like other crops, rice can’t stand severe high temperature. Generally, when the field temperature reaches 35 oC or higher, it will damage the growth and reproduction of rice (Feng et al, 2019). In Southeast Asia, the main rice producer including the middle and lower reaches of the Yangtze River and the southern rice production region of China, continuing high temperature over 35 oC of long duration (8–15 d or even longer) occurs almost every year recently (Xu et al, 2018; Cao et al, 2020). This heat stress causes great loss to rice production, its seed-setting dropping to less than 20% at the year of extremely heat stress (Shi et al, 2017).

Rice response varies with the temperature magnitade and duration of heat stress. Continuing high temperature adversely affects rice growth and development (Aghamolki et al, 2014; Fang et al, 2015; Kilasi et al, 2018) by altering the hormonal and biochemical responses (Li X M et al, 2015; Liu et al, 2016), which ultimately leads to decreased yield and productivity (Kunimitsu et al, 2014; Li X M et al, 2015; Cheabu et al, 2019). In previous studies, a wide range of phenotypic and molecular variations have been documented in rice under high temperature stress at different growth stages (Chen et al, 2008; Lei et al, 2013; Li X M et al, 2015; Liu et al, 2016).is the first major quantitative trait locus (QTL) cloned from African cultivated rice by map-based cloning, which controls these thermo-tolerance phenotypic and molecular variations (Li X et al, 2015). This gene protects cells from heat stress through more efficient elimination of cytotoxic denatured proteins and effective maintenance of heat-response processes than achieved with(Li X et al, 2015).is another major dominant locus controlling the variation of heat tolerance at seedling stage discovered by map-based cloning, which enhances heat tolerance through modulation of abscisic acid (ABA) biosynthesis and hydrogen peroxide-induced stomatal closure (Liu et al, 2016). In addition, other heat tolerance genes in rice were reported including(Liu et al, 2019),(El-Esawi and Alayafi, 2019),(Zhu et al, 2017),(Liu et al, 2018),(Mishra et al, 2018),(Zhao et al, 2016),(Fang et al, 2015),(Sato et al, 2016),(Ye et al, 2015b) andreceiptor- like kinase ERECTA (Shen et al, 2015). However, no successful breeding of new varieties using the above mentioned genes as resistance sources has been reported although the breeding effect of one of them has been studied (Ye et al, 2015b).

Our previous study reported that the locus ofwas located within an interval of 420 kb between two closely linked markers InDel 5 and RM7364 on the long arm of chromosome 9 (Wei et al, 2013). In this study, we attempted to apply marker- assisted backcrossing to introduce this heat-tolerance locus into a intermediate breeding line HT13, sensitive to high temperature, for creating germplasm resource for heat stress resistant hybrid rice breeding.

Results

Development of OsHTAS near isogenic line (NIL) and its derivative hybrids

The procedure used to develop NILs is illustrated in Fig. 1-A. Female parent HT13 was crossed with HT54, and the resultant F1was further crossed with HT13 to produce BC1F1seeds. All 24 BC1F1plants were crossed again with HT13 to produce BC2F1seeds. The foreground screening of BC2F1plants was performed by the PCR-RFLP marker RBsp1407 (Wei et al, 2012), and the positive plants were crossed with HT13 to produce BC3F1seeds. BC3F1plants were again screened for foreground and again crossed with HT13 to produced BC4F1seeds. Further background and foreground selections were done for 94 BC4F1plants to identify the occurrence of heterozygotes in the non-targeted chromosomal parts through the analysis of 144 polymorphic SSR markers plus RBsp1407 (Fig. S1). The resultant 8 BC4F1plants with high background recovery rate (79%–84%) in the non-targeted chromo- somal regions were crossed again to produce 84 BC5F1seeds. All BC5F1plants developed from these seeds were again screened by SSR markers. Among them, 18 plants with high recovery rate (96.5%) in the non-targeted regions were crossed with HT13 to produce BC6F1seeds. Then, 120 BC6F1plants were used for foreground and background selection with molecular markers, and 22 plants with heterogeneous foreground and cleaned HT13 background were self-pollinated to get BC6F2populations, which were further used for producing homozygote NILs in, the BC6F3. The one with the highest genetic background recovery was selected and named as MHT13. The genotype of MHT13 is presented in Fig. 1-B, and the genetic background recovery calculated based on the proportion of genome segments of recurrent parent to whole genome was around 99.8%.

The backcross-derived MHT13 was then crossed with different self-bred photo-thermo-sensitive genic male sterile (PTGMS) lines 208S, 218S and 228S to produce their derivative hybrids for further assessment.

Fig. 1. Schematic diagram of marker-assised backcross breeding and graphical genotype of near isognic line MHT13.

A, Flow chart of marker-assisted backcross breeding for developing near isogenic line with the dominant locus of. FS, Foreground selection; BS, Background selection. B, Graphical genotype of MHT13. The purple solid bar represents the foreground fragment containingintroduced from HT54, while the hollow vertical bar represents the background genotype inherited from HT13. The background selection was carried out using 144 SSR markers. Chr, Chromosome.

Evaluation of MHT13 and its derivative hybrids under field conditions

We evaluated seven agromorphological traits, ie. growth duration, plant height, number of tillers per plant, panicle length, number of grains per panicle, 1000- grain weight and seed-setting rate, among HT54, HT13 and MHT13 under field conditions in Hangzhou, 2017 (Table 1). HT54 was a traditional semi-dwarf variety with shorter growth duration, broad and drooping leaves, higher 1000-grain weight and shorter spikelet. Although HT13 was also a semi-dwarf line, its growth period is longer, and its flag leaf is narrow and straight with lower 1000-grain weight and longer spikelet. The agronomic traits of the bred variety MHT13 were highly similar to the recurrent parent line HT13, with respect to the seven agromorphological traits (Table 1).

Table 1. Agronomic performance of MHT13 under normal growth conditions (Hangzhou, 2017).

Table 2. Agronomic performance of MHT13-derivative hybrids under field conditions in 2017 and 2018, respectively.

Different lowercase letters indicate significant differences at< 0.05.

The yield performance of hybrids produced from the crosses between the self-bred PTGMS lines 208S, 218S, 228S, and HT13 or its derivative MHT13 were also observed under field conditions in two places in 2017 and 2018 (Table 2). The yields of hybrid produced from 218S/MHT13 and 208S/MHT13 in Hangzhou in 2017, recorded 65.90 and 70.36 g per plant, which were 12.0% and 6.3% higher than those from 218S/HT13 and 208S/HT13, respectively. Similarly, the yields of hybrids produced from 218S/MHT13 and 228S/MHT13 in Changxing in 2018 recorded 72.03 and 63.77 g per plant, which were also 11.2% and 3.7% higher than those from 218S/HT13 and 228S/HT13, respectively. Further analysis showed that the improvement of seed-setting due to the introduction of the heat tolerance genewas probably the fundamental cause of the yield increases of all MHT13-derived hybrids over the HT13-derived hybrids in different years and different locations (Table 2). The above genetic background selection and agronomic trait assessment results suggested that MHT13 restores the agronomic traits of HT13 in addition to obtaining a small fragment containinglocus from the donor line HT54, and can be used as two line hybrid rice restorer line.

Expression pattern verification of OsHTAS in MHT13 and its derivative hybrids

The previous dynamic expression analysis conducted by quantitative RT-PCR showed thatdetected in the HT54 seedlings had the strongest peak expression at 6 h after 48 oC high temperature treatment, which was significantly higher than that of the allele detected in the HT13 under the same stress condition (Wei et al, 2013). In order to verify whetherin MHT13 has the same expression features, we checked its dynamic expression levels by qRT-PCR at different time intervals according to the previous high temperature treatment program. Results showed that the dynamic expression pattern ofin the MHT13 was the same as that in HT54, and the peak expression also appeared at 6 h (Fig. 2-A), which was significantly higher than that of HT13.

To further confirm if this expression pattern works in the MHT13-derived hybrid, we first verified the polymorphism of PCR-RFLP marker RBsp1407 existing between NIL-derived hybrids and control HT13- derived hybrids, and then measured the dynamic expression level ofin them. The results showed that the data obtained are consistent with that obtained between MHT13 and HT13, indicating that the dynamic expression pattern ofis working in the genetic background of MHT13-derived hybrids (Fig. 2-B).

Phenotypic verification of heat tolerance of OsHTAS in MHT13 and its derived hybrids

In order to test the high temperature tolerance of MHT13 at seedling stage, 21 day seedlings were subjected to 48 oC treatment for 79 h according to the previous standard procedure (Wei et al, 2013). Thedonor parent HT54 and high temperature sensitive parent HT13 as well as negative BC6F3line without theloci from HT54, named as Neg, were used as positive and negative controls. The data showed that MHT13, like itsdonor parent HT54, survived completely after 79 h of high temperature treatment and 10 d of normal temperature recovery, while the sensitive control parents HT13 and Neg withered and died (Fig. 3). These results therefore demonstrated that MHT13 has indeed acquired the characteristics of high temperature tolerance at the seedling stage.

In order to test the high temperature tolerance of HT54 and MHT13 at flowering stage, we treated them at 38 oC for 6 h on the day of flowering, and compared them with sensitive materials HT13 and Neg. The results showed that HT54 and MHT13 showed better tolerance and more seed-setting than HT13 and Neg, although all the materials showed different degrees of decline in seed-setting rate (Fig. 2-C). Seed-setting rates of HT54, MHT13, HT13 and Neg were 94%, 96%, 93% and 94%, respectively, at 29 oC control temperature. However, after 38 oC high temperature treatment for 6 h, the seed-setting rates of HT54 and MHT13 decreased only by 20% and 44%, while those of HT13 and Nrg decreased by 78%. Similarly, we also treated the hybrid plants of 208S/MHT13 and 208S/HT13 with the same high temperature stress at flowering stage, and results revealed that both hybrids showed reduced seed-setting rates with much more decline in hybrid of 208S/HT13 as compared to 208S/MHT13 (Fig. 2-D). These results suggest that, compared with HT13, seed-setting of MHT13 has been greatly improved, especially the hybrids produced from MHT13 at the high temperature.

Heat tolerance mechanism verification of OsHTAS in MHT13

In order to prove thatin MHT13 improves the heat tolerance of rice through the same mechanism under the HT13 genetic background, we measured the ABA content and water loss in both parents and MHT13 seedlings before and after high temperature treatment for 24 h. The results demonstrated that HT54 and MHT13 had higher ABA contents as compared to HT13 in control conditions (Fig. 2-E). However, after 24 h of high temperature treatment, the ABA content of HT54 and MHT13 seedlings increased significantly by 16% and 18%, respectively, while that of HT13 seedlings increased slightly by 4% (Fig. 2-E). Meanwhile, the results also showed that the water losses of HT54 and MHT13 seedlings after high temperature treatment were significantly less than those of HT13 at all four sampling time points of 1, 2, 4 and 6 h (Fig. 2-F). These results thus suggested that MHT13 inherits the heat tolerance mechanism of the donor parents through the introduction ofgene.

Fig. 2.Dynamic expression, phenotype and heat tolerance mechanism ofin MHT13 and its derivative hybrids.

A, Dynamic expression features ofunder 48 oC high temperature. B, Expression changes ofin hybrids of 208S/HT13 and 208S/MHT13 before and after 48 oC high temperature treatment. C, Changes of seed-setting rate of heat resistant and sensitive parents and their continuously-backcrossing obtained MHT13 and negative materials before and after 38 oC high temperature treatment. D, Changes of seed-setting rate of heat-sensitive HT13 and its improved MHT13-formulated hybrids before and after 38 oC high temperature treatment.E, ABA content of MHT13 and its parental lines before and after 48 oC high temperature treatment. F, Changes of water loss before and after 48 oC high temperature treatment. High temperature (48 oC) treatment lasting 79 h was carried out at the seedling stage, and heat stress response was then scored and photographed after end of treatment and recovery for 10 d. High temperature (38 oC) treatment was carried out on the day of flowering, the treatment lasted 6 h and the seed-setting rate was counted after maturity. Error bars indicate SE based on three biological replicates. Significant differences between lines were determined by the Duncan’s multiple range test at the 0.05 level.

Fig. 3. High temperature tolerance of MHT13 at seedling stage.

Neg represents the negative line obtained by marker-assisted breeding. The treatment used was heat stress at 48 oC for 79 h.

Grain quality of MHT13

Rice quality is important to measure whether a germplasm has significant production and utilization value. In order to comprehensively examine the utilization value of MHT13, we measured the processing quality, appearance quality, cooking and eating quality, and nutritional quality of MHT13. The results indicated that among the criteria tested, head milled rice rate, alkali spreading value and amylose content reached level 1, whereas processing quality of brown rice rate and appearance quality of transparency degree reached level 2, and chalkiness degree and gel consistency was at level 3 (Table 3). Therefore, it was comprehensively evaluated as edible quality rice at level 3 according to the quality standard of ediblerice issued by Ministry of Agriculture of China.

Discussion

The continuing high temperature from global warming is becoming a serious constraint to rice production (Driedonks et al, 2016; Lesk et al, 2016; Zhao et al, 2017). It has been proved that screening and identifying genetic resources, exploring potential genes and breeding new varieties with high temperature tolerance is one of the most effective ways to cope with the continuous warming of climate (Driedonks et al, 2016). In this study, we reported the successful breeding of MHT13, with a dominant high temperature tolerance allele(Wei et al, 2013) by the marker-assisted backcross breeding. The molecular analysis showed that the genetic background recovery of MHT13 was around 99.8% (Fig. 1). Thegene introduced in MHT13 expressed normally in the new genetic background (Fig. 2-A), mediating heat tolerance and phenotype similar to those of the donor parent HT54 (Figs. 2-E, -F and 3). The agronomic traits of MHT13 resembled those of the recurrent parent HT13 (Table 1). In addition, because of its high general combining ability (Table 2) and its rice quality reaching the grade 3 standard of edible high-quality rice issued by Ministry of Agriculture of China (Table 3), MHT13 had a wide application in rice production.

Table 3. Evaluation results of grain quality of MHT13 according to quality standard of edible indica rice issued by the Ministry of Agriculture of China.

Grain lengths for Grade III standard are > 6.5 mm for long grains, 5.6?6.5 mm for middle grains and < 5.6 mm for short grains. BRR, Brown rice rate; HMRR, Head milled rice rate; GL, Grain length; CD, Chalkiness degree; TD, Transparency degree; ASV, Alkali spreading value; GC, Gel consistency; AC, Amylose content; MRR, Milled rice rate; GLWR, Grain length-width rate; CGR, Chalky grain rate; PC, Protein content.

Moreover, the present study also found that the heat tolerance of MHT13 was slightly lower than that of its donor parents (Fig. 3); especially, its performance at flowering stage was more different from that of the donor parent (Fig. 2-C). This variation in the performance may be attributed to the different genetic backgrounds. It has been reported previously that the function of bacterial-blight resistance genein rice is influenced by genetic background (Zhou et al, 2009). They found thatL. ssp.background can increaseexpression, which results in an enhanced disease resistance. Alberio et al (2018) studied the effect of genetic background on the stability of sunflower fatty acid composition in different high oleic mutations. They found that the NM1-NILs show an oleic level higher than 910?g/kg and those are more stable across environments with a zero or low dependence on the genetic background; on the other hand, high oleic materials bearing the P mutation show lower levels of oleic acid, with a higher variation in fatty acid composition and a highly significant dependence on the genetic background. Galois et al (2018) also summarized the role of the genetic background in resistance to plant viruses. They pointed out that a large part of virus resistance phenotype, conferred by a given QTL, depends on the genetic background. Shandil et al (2017) investigated RB gene mediated late blight resistance in potato, and demonstrated that high degree of resistance (< 25% infection) was observed in KJ × SP951 derived seedlings (85.2%), whereas level of resistance in KB × SP951 (36.4% infection) derived seedlings was of low order. This study corroborated the fact that efficacy of R gene is largely dependent on the genetic background of the recipient. All these reports confirmed the influence of different genetic backgrounds on the expression of specific genes.

The inconsistent performance of the same gene in seedling and flowering stages are also supported in many other reports. For instance, the prototypical variation ranging from 2% to 50.11% in high temperature stress tolerance at seedling and flowering stage has been identified on different loci covering twelve chromosomes (Zhao et al, 2006; Jagadish et al, 2010; Ye et al, 2012). Interestingly, a recent study on N22 has identified 10 QTLs on chromosomes 1–6 and 10 that were responsible for high temperature tolerance at the seedling stage (Kilasi et al, 2018), which are similar to QTLs identified in N22 at flowering stage by other research groups (Jagadish et al, 2010; Ye et al, 2015b; Shanmugavadivel et al, 2017). Similarly, another study reported five QTLs contributing to thermo- tolerance and among them one major QTL () on chromosome 3 was further characterized and verified through NILs, which were tolerant to high temperature at both seedling and flowering stages. These data indicated the complexity of high temperature resistance of plants. Strikingly, all MHT13-derived hybrids have better performance of yield under natural high temperature conditions as compared with HT13- derived hybrids (Table 2). After thorough analyzing of the data, we found that the superior yield performance and improved spikelet fertility of all hybrids derived can be attributed to the introduction of the dominant heat-resistance gene,, into the NIL of MHT13 (Table 2). Compared with the hybrids derived from HT13, although three quarters of the above- mentioned hybrids derived from MHT13 showed no statistically significant difference in grain yield, other agronomic traits of the MHT13-derived hybrids sometimes also showed significant increasement/ enhancement. For example, the tiller number per plant of 218S/MHT13 hybrid was significantly higher than that of 218S/HT13 hybrid when planted in Changxing in 2018; however, the increase of its tiller number per plant did not reach significance level when planted in Hangzhou in the previous year (Table 2). In 2018, the number of tillers per plant of 228S/MHT13 hybrid planted in Changxing was even 0.6 less than that of 228S/HT13 hybrid (Table 2). Similar results were also observed in the traits of plant height, number of grains per panicle and 1000-grain weight. These results hence indicated that the increasement/enhancement of these traits is a random event for MHT13 derived hybrids.

methods

Plant materials

Tworice cultivars, HT54 (local variety) tolerant to high temperature and HT13 (intermediate breeding line) sensitive to high temperature, collected from Guangdong Province, China, were used as parental lines for developing positive NIL MHT13 carryingheat tolerance locus and negative line (Neg) not carrying. MHT13 and Neg were produced from the procedures of marker-assisted backcross breeding using HT13 as recurrent parent (Fig. 1).

Other photo- and thermo-sensitive genic male sterile lines, such as 208S, 218S and 228S, used in this study are bred by our research groups, whose sterilities are stable under normal field temperature, and their fertility conversion temperatures below or slightly above 23 oC meets the production and application standards.

Seed germination

To facilitate germination and break dormancy, seeds were placed in incubator at 40 oC for one week, and then surface-sterilized with 5% sodium hypo chloride (NaClO) for 5 min and washed three times with distilled water. Finally, seeds were soaked in double distilled water for 2 to 3 d at 30 oC and germinated at 37 oC for one day.

Seedling cultivation and high temperature treatment

The uniformly-germinated seeds were then transferred to seedling trays (16 cm × 32 cm × 45 cm) containing 16 kg clay loam soil. The designed layout has 12 rows and 10 seeds per row containing 3 rows of parental lines HT54 and HT13 and MHT13. The extra two layers of HT54 (tolerant line) were sown on all sides of the trays to protect seedlings from marginal overheating. All materials were grown at normal temperature 29 oC/24 oC and with 12 h light-dark cycle rotation. After germination for 21 d, trays were transferred to a climate chamber, in which temperature was gradually increased from 29 oC to 48 oC within 5 h and maintained constant for 79 h. Relative humidity (RH) was set at 70%. After treatment, trays were moved out to the normal growth conditions for 10 d recovery. The heat stress response was then scored and photographed.

Heat stress at flowering stage

Pot-planted plants (one plant per pot) were grown in the greenhouse, and the growth conditions were set at temperatures of 29 oC / 24 oC at day/night with the RH of 70%. Pot-planted plants in the greenhouse were placed 25–30 cm apart to avoid overcrowding effects. In order to ensure that the growth conditions of each pot of plants were the same, the order of pot placement were adjusted every two weeks.

For high temperature treatment at the flowering stage, pot-planted plants were moved from greenhouse to an automated growth room at 8:00 am on the day of flowering. The temperature was gradually increased from 29 oC to 38 oC until 8:30 am and maintained at 38 oC until 2:30 pm (for 6 h) with a constant RH of 70%. Once the processing was completed, the opened treated spikelets were marked with red acrylamide paint, and the pot-planted plants were transferred back to previous normal growth conditions (29 oC /24 oC). As flowering in rice does not occur on the same day, different sets of plants were used for treatments. During treatment, the plants were placed 50–60 cm apart for uniform treatment.

Genome screening and DNA extraction

In the previous study, 322 SSR markers were identified to be polymorphic between the two parents (Wei et al, 2013), of which, 144 markers were used for the whole genome survey of backcross plants and lines in the present study. Similarly, for foreground selection, primer pair used was RBspF: 5′-CC ATCCAAACACGCCCTAA-3′ and RBspR: 5′-ATTGCCCCTT GCTATGGT-3′. DNAs from fresh leaves were extracted using the procedures as described previously by Dellaporta et al (1983). For polymerase chain reaction (PCR), a mixture of 20 μL containing 2 μL of 10 ng template DNA, 0.5 μmol/L forward and reverse primers, 2 μL 10× Mg2+free buffer, 1.5 μL of 2.5 mmol/L dNTPs, 4 mmol/L MgCl2and 0.1 μL of 1 U rDNA polymerase (Takara, Japan) was prepared. The thermocycling procedures were: initial denaturation at 95 oC for 5 min, followed by 35 cycles at 95 oC for 30 s, 55 oC for 30 s, and 72 oC for 60 s for denaturation, annealing and extension, respectively, with a final extension at 72 oC for 7 min. The PCR products were run on 3%–4% agarose or 40% polyacrylamide gel and were visualized under ultraviolet light (UV) after staining with silver staining or GelRed (Panaud et al, 1996).

RNA extraction, cDNA and qRT-PCR

Fresh leaves were harvested and ground into fine powder with liquid nitrogen. For RNA extraction, Trizol reagent (Invitrogen, USA) was used according to manufacturer’s instructions. RNA was reverse transcribed by Prime Script TMRT kit (Takara, Japan) according to the manufacturer’s instructions. The cDNA samples from different treatments were assayed by quantitative real-time PCR in 96 well plates with light cycler 96 well real-time PCR (Roche, Switzerland) using SYBER Premix Ex(Takara, Japan). RiceI was used as an internal reference (Zhang et al, 2012). Data were analyzed according to Livak and Schmittgen (2001). Primers used for qPCR was qOsHTAS-F: 5′-GATTCTCAGGAAGCTCCCAAT-3′ and qOsHTAS-R: 5′-TTAAGCATGGAAAGCAGCAG-3′. The thermal cycling protocol used was 95 oC for 15 s, followed by 40 cycles at 95 oC for 5 s and 60 oC for 30 s.

進(jìn)入新世紀(jì),黨中央國(guó)務(wù)院提出以人為本、全面協(xié)調(diào)可持續(xù)的科學(xué)發(fā)展觀。2003年,胡錦濤在中央人口資源環(huán)境工作座談會(huì)上強(qiáng)調(diào),環(huán)保工作要著眼于人民喝上干凈的水,呼吸清潔的空氣和吃上放心的食物,在良好的環(huán)境中生產(chǎn)生活,集中力量先行解決危害人民群眾健康的突出問題。截至2005年的中央人口資源環(huán)境座談會(huì),中央一直將民生作為環(huán)境保護(hù)的目標(biāo)。2008年國(guó)務(wù)院機(jī)構(gòu)改革,原國(guó)家環(huán)保總局升格為環(huán)境保護(hù)部,主要職責(zé)為“擬訂并組織實(shí)施環(huán)境保護(hù)規(guī)劃、政策和標(biāo)準(zhǔn),組織編制環(huán)境功能區(qū)劃、監(jiān)督管理環(huán)境污染防治、協(xié)調(diào)解決重大環(huán)境問題等”。

Determination of ABA contents and water loss

Endogenous ABA was determined by ELISA Kit (Rapiobio, USA) according to the manufacturer’s instructions. Frozen leaves were crushed and powdered in liquid nitrogen and extracted with HPLC grade methanol. The extract was then cleaned by centrifuge at room temperature for 10 min at 4000 ×. Transfer the resulting supernatant to a new clean tube and evaporate in vacuum to one-tenth of the initial volume. The remaining residue was dissolved in one percent acetic acid solution and filtered with 0.20 μm filter. Samples were extracted with a C18 SPE column as described previously by Nakurte et al (2012). HPLC grade methanol was used to elute analysates from the column which was evaporated to dryness and was re-dissolved in TBS buffer. The standards and samples were added in the wells of micro-titer plates with horseradish peroxide conjugated reagent, after mixing and incubating for 30 min at 37 oC. Then, HPR conjugated ABA was added to it to stop the reaction. The absorbance was measured at 450 nm for all studied hormones.

Water loss was measured according to Zhang et al (2008). Briefly, fresh leaves were detached from the plants and loss in fresh weight was measured at the indicated time.

Field evaluation and grain quality determination

Multisite plus multiyear field trials of HT13 and MHT13 and their derivative hybrids were carried out in the fields under normal rice growing seasons in Hangzhou and experimental station, Zhejiang University in 2017 and 2018. Thedonor parent HT54 was used as positive control. A sequential block design was adopted with three replications for the field trials of both parental lines and their derivative hybrids. Each plot consisted of 5 rows with 8 plants per row. Plant spacing and row spacing were 19.8 and 26.6 cm, respectively. Each parental line and hybrid were sampled from three replicates, 10 plants were taken from each replicate, and altogether 30 plants were used for the measurement of agronomic traits, which include plant height, growth duration, number of tillers per plant, panicle length, number of grains per panicle, 1000-grain weight, seed-setting rate and yield per plant. The mean value of each trait was based on three replications with ten plants each.

After MHT13 was harvested, the milling, appearance, cooking, edible and nutritional qualities were analzed. The quality grade was classified according to the standard of ediblerice quality issued by Ministry of Agriculture of China (NY/T2013).

Statistical analysis

Morphological and physiological/biochemical data were analyzed using a statistical package, SPSS Version 19.0. One way ANOVA was employed, followed by Duncan’s multiple range test to determine the significant differences among means of the treatments at< 5%. Gene expression data were presented as mean values of three biological replicates (two technical replicates for each biological replicate).

AcknowlegementS

We sincerely thank Dr. Qi Zhenyu and Mr. Zhang Zhenzhong for their assistance in climate chamber and field management for cultivation of rice materials. This work was supported by the funds from the National Key Research and Development Plan of China (Grant No. 2017YFD0100305).

supplemental data

The following material is available in the online version of this article at http://www.sciencedirect.com/science/journal/16726308; http://www.ricescience.org.

Fig. S1. Molecular marker linkage map for this study.

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http://dx.doi.org/10.1016/j.rsci.2020.11.011

14 February 2020;

8 August 2020

Chen Hao (haochen@zju.edu.cn)

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