Improving germinability leads to enhanced seedling establishment at low temperatures in rice through QTL pyramiding

doi:10.21203/rs.3.rs-4826007/v1

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Improving germinability leads to enhanced seedling establishment at low temperatures in rice through QTL pyramiding

https://doi.org/10.21203/rs.3.rs-4826007/v1

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Low-temperature germinability is a crucial trait for seedling establishment at low temperatures, which is essential for the direct-sowing culture of rice. We pyramided four quantitative trait loci (QTLs) for germinability at low temperatures in the genetic background of the Japanese cultivar Akitakomachi. In the QTL pyramiding lines, the accumulated QTLs additively increased the low-temperature germination rate, despite the weak effects of the individual QTLs. This can be observed when considering the QTL qLTG3-2, which alone did not increase the germination rate at low temperatures but further enhanced the germination rate when combined with either qLTG3-1 or qLTG11-1. Low-temperature germinability remarkably improved in proportion to the number of accumulated QTLs. Thus, QTL pyramiding was an effective method to enhance this trait. Furthermore, the improved low-temperature germinability in the lines that accumulated the four QTLs also enhanced the seedling establishment compared to Akitakomachi. Seedling establishment at low temperatures is a complex trait that involves continuous growth from seed germination to seedling development. The remarkable improvement in low-temperature germinability is likely to contribute to the improved seedling establishment at low temperatures. However, the four QTL pyramiding line exhibited a reduced brown rice appearance quality. To avoid the introduction of undesirable traits, pyramiding using new QTLs for low-temperature germinability is expected to develop a superior rice variety suitable for direct-sowing cultivation.

rice

Oryza sativa L.

low-temperature germinability

seedling establishment

quantitative trait loci

pyramiding

direct-sowing cultivation

Direct-sowing culture can reduce the labor and cost of rice production compared to the transplanting culture technique. Germination and seedling growth are often suppressed by low temperatures, which makes germinability and seedling establishment at low temperatures essential traits for varieties suited to direct sowing. This is particularly important in high-altitude and cold-water irrigated areas, where low temperatures are common during the early stages of rice growth (Fujino et al. 2004, Ji et al. 2008, Li et al. 2013). However, many Japanese varieties lack these traits because transplanting cultivation is well-established in Japan, and thus these traits have not been selectively bred.

Germinability and seedling establishment at low temperatures are quantitative traits that involve multiple genes, and quantitative trait loci (QTLs) have been identified via various genetic resources to improve these traits (Biswas et al. 2019, Najeeb et al. 2021). In general, each of these QTLs exerts a small effect and is insufficient to improve quantitative traits alone. Therefore, it is necessary to accumulate multiple QTLs to achieve a substantial improvement. Furthermore, to enhance the adaptability of superior varieties for direct sowing, it is important to introduce only the target QTLs related to these traits into the superior varieties to avoid any accompanying undesirable traits.

The development of near-isogenic lines (NILs) using marker-assisted selection (MAS) allows for the introduction of desired QTLs into the superior varieties. The accumulation of desired QTLs by intercrossing NILs using MAS is an effective breeding method known as QTL pyramiding (Mackill et al. 1999, Ashikari and Matsuoka 2007). Previously, QTL pyramiding has been effective in improving cold tolerance at the reproductive stage (Shinada et al. 2014). For example, pyramiding two QTL alleles has improved cold tolerance at the reproductive stage of Hitomebore, an elite variety in Japan (Endo et al. 2016). Moreover, it has been reported that pyramiding QTLs for low-temperature germination in a backcrossed inbred line resulted in rapid and uniform germination in low-temperature environments (Li et al. 2018).

Previously, we identified four QTLs for low-temperature germinability using an F₂ population between the Eastern European cultivar Maratteli and the Japanese cultivar Akitakomachi. Furthermore, the qLTG1-1 allele from Akitakomachi and the qLTG3-1, qLTG3-2, and qGLT11-1 alleles from Maratteli have been presumed to enhance low-temperature germinability (Satoh et al. 2016).

In this study, we developed NILs with the Akitakomachi genetic background harboring each QTL and then developed QTL pyramiding lines by intercrossing the NILs using MAS. We demonstrated that low-temperature germinability improved with the number of pyramided QTLs. Moreover, seedling establishment at low temperatures was also improved in the lines pyramided with all the QTLs for low-temperature germinability.

Plant materials

The Japanese cultivar Akitakomachi, the Eastern European cultivar Maratteli, three NILs of Akitakomachi, and QTL pyramiding lines with the Akitakomachi genetic background were used in this study. Akikei770, Akikei771, and Akikei775 are NILs harboring the Maratteli alleles of qLTG3-1, qLTG3-2, and qLTG11-1, respectively, in addition to the qLTG1-1Akitakomachi allele. These NILs are also two QTL pyramiding lines. Akikei791, Akikei792, and Akikei793 are QTL pyramiding lines harboring a combination of Maratteli alleles (qLTG3-1 and qLTG3-2, qLTG3-1 and qLTG11-1, and qLTG3-2 and qLTG11-1, respectively) as well as the qLTG1-1Akitakomachi allele. Akikei812, Kei2161, Kei2162, Kei2171, and Kei2172 are QTL pyramiding lines that possess the Maratteli alleles of qLTG3-1, qLTG3-2, and qLTG11-1, in combination with the qLTG1-1 Akitakomachi allele (Fig. 1).

The European cultivars Italica Livorno and Arroz da Terra, which exhibited vigorous seedling establishment in a field evaluation system (Iwata et al. 2010), were used in an experiment to evaluate seedling establishment at low temperatures.

Selection of QTL pyramiding lines

Plants harboring the QTL alleles from Maratteli were selected using two PCR markers flanking each QTL: RM3436 and RM6329 for qLTG3-2 and RM206 and RM7277 for qLTG11-1. For qLTG3-1, plants were selected using G1320, which identifies an In/del within the qLTG3-1 gene (Table S1, Fig. 1).

Preparation of seeds for germination and seedling establishment test

Plants were grown in a paddy field at the Akita Agriculture Experiment Station in 2013 and 2014, with a planting density of 20.7 plants m^− 2. Mature rice plants were harvested and dried in a well-aerated room for one month. High-quality seeds were selected by removing the immature, shriveled, and unfilled seeds using an automatic seed selection machine (PAT453922, Yamamoto Co., Ltd.) and then stored in a seed storage cabinet at 10°C with 10% relative humidity.

Evaluating germinability under low-temperature conditions

Low-temperature germinability was evaluated according to the method previously described by Satoh et al. (2016). The seeds were surface-sterilized by immersing them for 15 min in a 10% solution of sodium hypochlorite and rinsing them three times with tap water. Each sample set of 25–35 seeds was placed in a glass Petri dish (60 mm diameter) with 5 mL of tap water and incubated at 15°C in a growth chamber (LH301, Tomy Seiko Co., Ltd.). Germinating seeds were counted every day for 14 days, and the tap water was renewed daily. The germination potential of seeds that did not germinate at low temperatures was evaluated by further incubation at 25°C for 24 h. The germination test was repeated three times for each line, and the germination rate (%) was calculated using the following formula:

Germination rate (%)

$$\:\:=\:\frac{\text{n}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\text{s}\text{e}\text{e}\text{d}\text{s}\:\text{t}\text{h}\text{a}\text{t}\:\text{g}\text{e}\text{r}\text{m}\text{i}\text{n}\text{a}\text{t}\text{e}\text{d}\:\text{d}\text{u}\text{r}\text{i}\text{n}\text{g}\:\text{t}\text{h}\text{e}\:\text{t}\text{e}\text{s}\text{t}\:\text{p}\text{e}\text{r}\text{i}\text{o}\text{d}}{\text{t}\text{o}\text{t}\text{a}\text{l}\:\text{n}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\text{s}\text{e}\text{e}\text{d}\text{s}\:\text{t}\text{h}\text{a}\text{t}\:\text{g}\text{e}\text{r}\text{m}\text{i}\text{n}\text{a}\text{t}\text{e}\text{d}}\:\times\:\:100$$

Evaluation of low-temperature seedling establishment

The seedling establishment was evaluated following the method previously described by Matsumoto et al. (2001). A seedling box (60 × 30 × 3 cm) was divided into 72 sections (5 × 5 cm) using paper pots, and 20 dried seeds were sown in each section. The seedling box was submerged in a pool to a depth of 2 cm, and the seedlings were grown in running tap water from April 21 to June 4, 2014. The change in the water temperature in the pool during this period is presented in Fig. S1. Plant height, root length, and leaf age were measured. The experiment was replicated four times for each line. The seedling establishment (%) was evaluated according to the method described by Furuhata et al. (2005) using the following formula:

Seedling establishment (%)

$$\:\:=\:\frac{\text{n}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\text{p}\text{l}\text{a}\text{n}\text{t}\text{s}\:\text{t}\text{h}\text{a}\text{t}\:\text{a}\text{c}\text{h}\text{i}\text{e}\text{v}\text{e}\text{d}\:\text{s}\text{e}\text{c}\text{o}\text{n}\text{d}\:\text{l}\text{e}\text{a}\text{f}\:\text{e}\text{m}\text{e}\text{r}\text{g}\text{e}\text{n}\text{c}\text{e}}{\text{t}\text{o}\text{t}\text{a}\text{l}\:\text{n}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\text{s}\text{e}\text{e}\text{d}\text{s}\:}\:\times\:\:100$$

DNA extraction and PCR

Crude DNA was extracted from the leaf tips of seedlings as previously described (Kato et al., 2007). The leaves were dried at 70°C for 2 h and then ground to a powder with stainless steel balls (φ3 mm) using a Micro Smash (MS-100, Tomy Seiko Co., Ltd.). Extraction buffer (Edwards et al., 1991) was added to the powder, and the mixture was incubated at room temperature for 1 h. DNA was precipitated from the mixture by adding an equal volume of 2-propanol.

DNA markers were amplified using Takara Taq HS (Takara Bio Inc.) with the primer pairs shown in Table S1. PCR was performed with 35 cycles of 10 s at 94°C, 30 s at 55°C, and 60 s at 72°C using a Thermal Cycler 9700 (Thermo Fisher Scientific Inc.). Polymorphisms for the amplified DNA markers were determined via electrophoresis using 1.8% UltraPure Agarose (Thermo Fisher Scientific Inc.) or 2.0% MetaPhor Agarose (LONZA).

Statistical analysis

The obtained data for the replicates were used to test for significant differences with the Student’s t-test, Dunnett’s, or Tukey’s test using the statistical analysis software Excel Toukei Ver.6.0 (ESUMI Co., Ltd.).

Pyramiding of four QTLs for low-temperature germinability using MAS

The QTL pyramiding lines with the Akitakomachi genetic background were developed by crossing NILs carrying each of the qLTG3-1, qLTG3-2, and qLTG11-1 Maratteli alleles using MAS. Akikei770, carrying qLTG3-1, was crossed with Akikei793, carrying qLTG3-2 and qLTG11-1. The resulting F₂ population of 576 individuals was subjected to MAS. Initially, 137 individuals homozygous for the Maratteli allele of qLTG3-1 were selected using the G1320 DNA marker. From these, 36 individuals homozygous for the Maratteli allele at RM7277 were selected, and subsequently, 28 individuals homozygous for the Maratteli allele qLTG11-1 were identified using RM206. The remaining eight individuals were recombinants between RM7277 and RM206. Finally, eight individuals homozygous for the qLTG3-2 Maratteli allele, flanked by RM6329 and RM3436, were selected from the 28 individuals. There were four recombinants between these markers. The following year, eight lines carrying the Maratteli alleles of qLTG3-1, qLTG11-1, and qLTG3-2, along with the qLTG1-1 Akitakomachi allele, were cultivated in the field. From these, the QTL pyramiding lines Kei2161, Kei2162, Kei2171, and Kei2172 were selected. Finally, Akikei812, harboring all four QTLs for low-temperature germinability, was established from Kei2161 (Fig. 1, Table 1).

The segregation ratios for each of qLTG3-1, qLTG11-1, and qLTG3-2 fit the theoretical 1:2:1 ratio (Table 1). Eight plants carrying three QTLs were observed among the 576 F₂ individuals, which fits the 1:63 theoretical segregation ratio for three independent genes, possibly due to the distant location of qLTG3-1 and qLTG3-2 on chromosome 3 (Table 1, Fig. 1). This result suggests that no segregation distortion occurred during the pyramiding of QTLs for low-temperature germinability. While stepwise DNA marker selection reduced the number of analyses, increasing the number of pyramided QTLs or segregation distortion may considerably increase the number of plants analyzed, thus potentially limiting the QTL accumulation via MAS.

Improvement of low-temperature germinability of Akitakomachi through QTL pyramiding

Figure 2 shows the germination rate on day five after sowing at 15°C for the QTL pyramiding lines with the Akitakomachi genetic background. The germination rates of Akitakomachi and Maratteli were 3% and 81%, respectively. The germination rates of the two QTL pyramiding lines, Akikei770 (qLTG1-1 + qLTG3-1) and Akikei775 (qLTG1-1 + qLTG11-1) were 8% and 13%, respectively, which was significantly higher than that of Akitakomachi at the 1% level, as determined by the Student’s t-test. Conversely, the germination rate of Akikei771 (qLTG1-1 + qLTG3-2) was significantly lower than that of Akitakomachi at the 5% level, as determined by the Student’s t-test. These results indicate that qLTG3-1 and qLTG11-1 from Maratteli increased the germination rate of Akitakomachi at low temperatures, while qLTG3-2 did not.

The three QTL pyramiding lines, Akikei791 (qLTG1-1 + qLTG3-1 + qLTG3-2), Akikei792 (qLTG1-1 + qLTG3-1 + qLTG11-1), and Akikei793 (qLTG1-1 + qLTG3-2 + qLTG11-1) had germination rates of 47%, 40%, and 41%, respectively, at 15°C on day five after sowing, which was significantly higher than those of the two QTL pyramiding lines, Akikei770 and Akikei775. In addition, the germination rates of the four QTL pyramiding lines, Akikei812 and Kei2171 (qLTG1-1 + qLTG3-1 + qLTG3-2 + qLTG11-1), were 73% and 72%, respectively, which was significantly higher than those of the three QTL pyramiding lines. Another four QTL pyramiding line, Kei2161, exhibited a slightly higher germination rate than the three QTL pyramiding lines (Fig. 2).

These results demonstrated that the germination rate at low temperatures increases proportionally with the number of accumulated QTLs. Consequently, the low-temperature germination rate of Akitakomachi was remarkably improved by pyramiding QTLs for low-temperature germinability, which approached the level of Maratteli (Fig. 3).

Germination profile at low temperature of the QTL pyramiding lines

Figure 4 shows the time course of the germination rate at 15°C for the QTL pyramiding lines. Maratteli began germinating 4 days after sowing, with a rapid increase in germination rate, reaching 100% by day seven. Akitakomachi began germinating five days after sowing, completing germination by day 10. The germination rates of the three QTL pyramiding lines, Akikei791, Akikei792, and Akikei793, increased faster than Akitakomachi but later than Maratteli. The four QTL pyramiding lines, Akikei812 and Kei2171, exhibited a similar germination profile to that of Maratteli. The germination profile of another four QTL pyramiding line, Kei2161, was similar to that of the three QTL pyramiding lines. These results demonstrate that the low-temperature germination profile of Akitakomachi was enhanced to be closer to that of Maratteli by pyramiding four QTLs for low-temperature germinability.

Low-temperature seedling establishment of the QTL pyramiding lines

There were no significant differences in plant height, root length, and leaf age at low temperatures between Akitakomachi and the pyramiding lines with two or three QTLs for low-temperature germinability. However, in Akikei2161 and Akikei2162, which harbor four QTLs, these three traits were significantly increased compared to Akitakomachi. Similarly, these traits in Akikei2172 were also higher than those in Akitakomachi. In contrast, there was no difference in these traits between another four QTL pyramiding line, Akikei2171, and Akitakomachi (Fig. 5a-c). Since Akikei2171 and Akikei2172 are sibling lines with the same genotypes, these differences might be caused by differences in the physiological status of the harvested seeds.

The seedling establishment at low temperatures, which was evaluated by the percentage of individuals with a leaf age of 2.0 or more, was 8% for Akitakomachi. In contrast, this was 15.4% and 25.5% for Akikei770 and Akikei790, respectively, which accumulated two QTLs. The seedling establishment rates for the three QTL pyramiding lines ranged from 30.4–33.6%, which were higher than that of Akitakomachi. Furthermore, the seedling establishment rates at low temperatures for the four QTL pyramiding lines, Kei2161, Kei2162, Kei2171, and Kei2172, ranged from 38.6–54.5%, which were significantly higher than that of Akitakomachi (Fig. 5d).

The European varieties Italica Livorno and Arroz da Terra exhibited the highest plant height but did not significantly differ from Akitakomachi regarding the other traits. Plant height is a useful trait for evaluating low-temperature seedling establishment according to a previously used evaluation method (Matsumoto et al. 2001). The two European varieties were confirmed to be superior for low-temperature seedling establishment.

Conversely, Maratteli showed a tendency to be lower than Akitakomachi for all traits (Fig. 5). Maratteli has been reported to vary in plant height from 1999 to 2001 using the same evaluation system, which resulted in similar plant height to Akitakomachi (Matsumoto et al. 2001). Maratteli may be strongly influenced by the climatic conditions of the harvest year, possibly having characteristics that make seedling establishment unstable at low temperatures.

There was a high positive correlation (R² = 0.940) between the germination rate and seedling establishment at low temperatures among the QTL pyramiding lines with the Akitakomachi genetic background, which indicates that seedling establishment at low temperatures increased proportionally with the number of QTLs for low-temperature germinability (Fig. 6). The QTL pyramiding lines exhibited accelerated initiation and progression of germination at low temperatures (Fig. S2). This acceleration in germination was followed by faster seedling growth, resulting in higher seedling establishment. Therefore, accumulating QTLs that enhance low-temperature germinability is likely to improve low-temperature seedling establishment in elite varieties.

Improvement of low-temperature germination through QTL pyramiding

Low-temperature germinability is a quantitative trait that is controlled by multiple QTLs and their interactions. Improving such traits requires accumulating multiple QTLs, a process known as QTL pyramiding (Ashikari and Matsuoka 2006). Numerous QTLs for low-temperature germinability have been identified in various genetic backgrounds (Biswas et al. 2019; Najeeb et al. 2021), and pyramiding these QTLs is expected to enhance low-temperature germinability.

The effect of QTLs on low-temperature germinability is variable, and their effectiveness is also influenced by the genetic background (Iwata and Fujino 2010). We identified four QTLs in an F₂ population derived from the Eastern European variety Maratteli and the Japanese variety Akitakomachi (Satoh et al. 2016). We developed Akitakomachi NILs for each QTL and QTL pyramiding lines with the Akitakomachi genetic background by crossing these NILs using MAS, and evaluated the effect of the pyramided QTLs on low-temperature germinability.

Two QTLs, qLTG3-1 and qLTG11-1, increased the germination rate at low temperatures in Akitakomachi, although their effects were weak. The germination rate at low temperatures was significantly increased in the QTL pyramiding line with qLTG3-1 and qLTG11-1, which suggests that these two QTLs additively enhance low-temperature germinability. Surprisingly, qLTG3-2 significantly enhanced low-temperature germination when combined with either qLTG3-1 or qLTG11-1, despite being ineffective when present alone (Fig. 2).

Despite qLTG3-2 being identified as the most effective QTL in the F₂ population between Akitakomachi and Maratteli (Satoh et al. 2016; Biswas et al. 2019), it showed no effect alone on enhancing low-temperature germinability in Akitakomachi NILs. Theoretically, only 6.25% of individuals in the F₂ population harbor both qLTG3-1 and qLTG11-1 alleles from Akitakomachi. Since most of the F₂ individuals had a genetic background in which the Maratteli qLTG3-2 allele is effective, it likely demonstrated high efficacy in the F₂ population.

Furthermore, low-temperature germinability in Akitakomachi was remarkably improved in proportion to the number of accumulated QTLs, which resulted in the high level of this trait. In rice, the pyramiding effect was reported to increase with the number of accumulated QTLs for cold tolerance at the reproductive stage (Shinada et al. 2014). This indicats that QTL pyramiding, including minor QTLs, is essential for quantitative trait improvement. Therefore, it is concluded that pyramiding QTLs for low-temperature germination is an indispensable method to achieve high levels of low-temperature germinability in rice.

Pyramiding QTLs for low-temperature germination to improve low-temperature seedling establishment

The goal of increasing low-temperature germinability in superior varieties is to enhance their suitability for direct sowing. The QTL pyramiding lines of Akitakomachi for low-temperature germinability were further evaluated for traits related to seedling establishment at low temperatures. In the four QTL pyramiding lines for low-temperature germinability, we found that seedling establishment at low temperatures was also improved. Their low-temperature seedling establishment levels were similar to those of the European cultivars Italica Livorno and Arroz da Terra, which were superior in seedling establishment in the paddy field.

Seedling establishment at low temperatures is a complex trait that includes seed germination, budding, and seedling growth at low temperatures. Low-temperature germinability is important for seedling establishment in direct sowing, especially under low-temperature conditions (Fujino et al. 2004), and the germination speed affects seedling establishment (Furuhata 2015). In the Akitakomachi QTL pyramiding lines, the high correlation between germination rate and seedling establishment suggests that increasing the germination rate resulted in improved seedling establishment in the Akitakomachi genetic background.

There are QTLs for seedling establishment and growth at low temperatures near qLTG11-1 (Iwata et al. 2010; Ranawake et al. 2014; Xiao et al. 2018; Najeeb et al. 2020). Because seedling establishment involves continuous growth from seed germination to seedling development (Iwata et al. 2010), qLTG11-1 may encode genes that are involved in this process. In addition, OsGF14h, which was identified in the qLTG11-1 region of chromosome 11, has been reported to be involved in anaerobic germination and anaerobic seedling development (Sun et al. 2022). Therefore, the Maratteli qLTG11-1 allele that was introduced here may enhance such traits, which resulted in improved seedling establishment at low temperatures.

When improving elite varieties by pyramiding QTLs, it is important to avoid introducing undesirable traits from the QTL resources (Enodo et al. 2016). The appearance quality of brown rice from the four QTL pyramiding line Akikei812 was lower than that of Akitakomachi (Fig. S3), which suggests that the genes associated with the pyramided QTLs for low-temperature germinability may have reduced the appearance quality of brown rice. qLTG3-1, which encodes HyGRP (Fujino et al. 2008; Fujino et al. 2014), has pleiotropic effects that improve low-temperature germinability but reduce the appearance quality of brown rice (Miura et al. 2024). Therefore, qLTG3-1 itself probably reduced the brown rice appearance quality in Akikei812.

Pyramiding at least four QTLs for low-temperature germinability was necessary to improve seedling establishment at low temperatures. Therefore, a new QTL for enhancing low-temperature germinability in the Akitakomachi genetic background is required instead of qLTG3-1, which is known to be a major QTL. Many QTLs have been identified for low-temperature germinability in different genetic backgrounds (Biswas et al. 2019; Najeeb et al. 2021). Although the genetic background influences the QTL effects (Iwata and Fujino 2010), some QTLs are expected to be effective in Akitakomachi. Pyramiding such QTLs instead of qLTG3-1 could result in the development of new QTL pyramiding lines for direct sowing that maintain the high grain quality of Akitakomachi.

Author Contribution

H.A. designed the research. T.S., T.K. and H.A. prepared materials. T.S., H.T., A.W., K.U. and K.S. performed the experiments and analyzed. H.A. and K.S. wrote the manuscript. All authors reviewed the manuscript.

Acknowledgments

This research was supported by a grant from Akita Prefectural University in the Industry-Academia Joint Research Program.

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Table 1 is available in the Supplementary Files section

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Improving germinability leads to enhanced seedling establishment at low temperatures in rice through QTL pyramiding

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