Detection of the 28-bp deletion and phenotyping of indica
Based on the reported 28-bp deletion that leads to different reactions to BBC among rice cultivars (Maeda et al., 2019), an InDel marker was developed to detect this variation in 631 indica accessions commonly used in rice breeding in China (Table S1, Figure S1). The results showed that the 28-bp deletion was widely distributed among China’s indica accessions, with an average rate of 28.1%. Among them, the deletion rate of the 3-line restorer was the highest, reaching 50.7%, but no deletion was found in the 87 3-line cytoplasm male sterile (CMS) lines. Forty (18.1%) conventional lines, 24 (40.7%) 2-line restorers, and five (9.8%) 2-line genic male sterile (P/TGMS) lines harbored the 28-bp deletion (Table 1). In general, there were more than 28-bp deletions in the restorer lines (48.5%), followed by conventional rice and then the sterile lines (3.6%).
Table 1
Indica accession numbers of the 28-bp deletion, T1510G mutation and lines susceptible to BBC
| Total | 28-bp deletion | HS | MS | T1510G |
Conventional varieties | 221 | 40 (18.1%) | 55 (24.9%) | 1 (0.5%) | 16 (7.2%) |
3-line restorer | 213 | 108 (50.7%) | 129 (60.6%) | 3 (1.4%) | 24 (11.3%) |
2-line restorer | 59 | 24 (40.7%) | 29 (49.2%) | 2 (3.4%) | 7 (11.9%) |
CMS | 87 | 0 | NA | NA | 14 (16.1%) |
P/TGMS | 51 | 5 (9.8%) | NA | NA | 3 (5.9%) |
ALL | 631 | 177 (28.1%) | NA | NA | 64 (10.1%) |
CMS, 3-line cytoplasm male sterility; P/TGMS, 2-line genic male sterile; HS, highly susceptible; MS, moderately susceptible; NA, no phenotype |
In order to determine whether the BBC-sensitive phenotype is completely related to the 28-bp deletion, we selected 493 accessions including conventional and restorer lines (sterile line seeds are more valuable and we did not have enough seeds) to receive BBC. At first, Nipponbare (NB) (HIS1) and IR64 (his1) were used to determine suitable BBC concentrations. The results showed that IR64 started to show injury when the concentration was 0.03 g/l. When the concentration reached 0.15 g/l, the difference between IR64 and NB was already very obvious, and when the concentration reached 1.2 g/l, NB also started to show susceptibility (Fig. 1A, B). Finally, a concentration of 0.4 g/l was selected to evaluate the resistance of indica accessions to BBC. According to the different degrees of rice responses to BBC, we divided the phenotypes into six grades: grades 0 and 1 showed complete resistance, grades 4 and 5 were completely sensitive, and grades 2 and 3 were intermediate types (Fig. 1C). The BBC spraying results showed that most indica accessions showed high resistance or high sensitivity, and only a few were of the intermediate type, which is probably related to the HIS1 gene as the major BBC resistance gene (Maeda et al., 2019) (Fig. 1D). Among the three different types of rice accessions, the BBC-sensitive trait had a certain relationship with the 28-bp deletion, but the number of BBC-sensitive accessions obviously exceeded the number of 28-bp deletions (Table 1). Therefore, in addition to the 28-bp deletion, there may be other mutation(s) of the HIS1 gene that lead to BBC sensitivity.
T1510G mutation in the HIS1 gene leads to the loss of function
In order to find out whether BBC sensitivity is caused by mutations of the HIS1 gene, we performed PCR cloning and sequencing of the HIS1 gene in 493 phenotyped indica accessions. Sequencing results showed that there were only five mutation sites in exons of HIS1, which divided the HIS1 gene into five haplotypes (Fig. 2A), among which H2 and H5 are alleles of the HIS1 gene in Nipponbare and IR64, respectively. Compared with H2, H1 had two mutations (C9T, G1055A), H3 had only one mutation (T1510G), H4 had two mutations (G61C, G1055A), and the 28-bp deletion was found only in H5. When the phenotype of each indica accession was correlated to the haplotype of the HIS1 gene, we found that accessions containing H1, H2, and H4 showed full resistance to BBC, while H3 and H5 showed complete susceptibility to BBC (Fig. 2A). Studies have shown that non-functional H5 is due to the 28-bp deletion (Maeda et al., 2019). Because H3 had only one single base mutation compared with functional H2, it was initially believed that this T1510G mutation was the cause of the loss of function.
The T1510G mutation in H3 caused an amino acid change (V286G) at the protein level (Figure S2), located in the region of the putative substrate pocket that is very important for the function of Fe(II)/2OG-dependent oxygenases (Maeda et al., 2019) (Fig. 2B). In order to rule out that non-functional H3 was due to different expressions of HIS1, we randomly selected five accessions harboring H2 and H3 to detect the HIS1 mRNA level, using NB and IR64 as controls. The results showed that HIS1 was normally expressed in all accessions with H3, and there was no obvious correlation between HIS1 gene expression and anti-susceptibility to BBC (Fig. 2C). An F2 population of Nongxiang42 (containing H2) and Xianhui207 (containing H3) was constructed to analyze the relationship between susceptibility to BBC and the T1510G mutation. The results of spraying BBC on the F2 population showed that among the 968 individual plants, 737 individual plants showed resistance, and 231 individual plants showed sensitivity to BBC. The R:S ratio was 3:1 (X2 = 0.607 < X(0.05)2=3.84), which demonstrated that there was only one dominant resistance gene in Nongxiang42. Next, we designed a dCAPS marker based on the T1510G mutation to distinguish H3 from other haplotypes (Fig. 2D, E). Using this H3-specific marker to detect individual plants of the F2 population, we found that the T1510G mutation was completely co-segregated with the susceptibility to BBC (Figure S3). In summary, our results showed that the T1510G mutation caused the loss of function of the HIS1 gene.
Natural allelic variations of HIS1 in cultivated rice
In order to fully understand which indica rice accessions can tolerate BBC herbicide, we tested all P/TGMS and CMS lines with the H3-specific marker. The results showed that there were three P/TGMS and 14 CMS lines that harbored the T1510G mutation (Table 1). By combining the detection results of the 28-bp deletion in the early stage, we can conclude that about 40% of the indica rice materials and about 25% of conventional rice should be sensitive to BBC (Table 1). Although the proportion of 3- and 2-line restorers that are sensitive to BBC is very high (60% and 52.6%, respectively), due to the low proportion of P/TGMS and CMS lines, the ratio of the indica hybrids that are sensitive to BBC should be very low, considering that HIS1 is a major dominant resistance gene.
In order to clarify the distribution of non-functional HIS1 alleles in cultivated rice, we used the 3,000 sequenced rice genomes (Wang et al., 2018) to conduct a comprehensive analysis of the 28-bp deletion and T1510G mutation of the HIS1 gene. The results showed that among 1716 indica rice accessions, 117 harbored the 28-bp deletion and 292 accessions contained the T1510G mutation; among 825 japonica rice accessions, no 28-bp deletion was found, and only three accessions had the T1510G mutation. Non-functional alleles of the HIS1 gene are rarely found in japonica rice, and this phenomenon could explain why the BBC herbicide is registered and can be used safely in the japonica rice area. In the two other types of cultivated rice, aus and aro, there was no 28-bp deletion, and only one T1510G mutation was found in aus (Fig. 3A). Unlike the 3,000 rice accessions, which are mainly landraces with wide genetic variations (Wang et al., 2018), the 631 indica accessions we tested were mainly Chinese indica rice breeding materials. Non-functional alleles of HIS1 have increased significantly in our 631 indica accessions, especially in the restorer lines (Fig. 3B), compared with the 3,000 rice accessions, which should be the result of artificial selection during the rice breeding process.
BBC Is Applicable For Two-line Hybrid Rice Seed Purification
In order to evaluate the possibility of the HIS1 gene in solving 2-line hybrid rice seed purity issues, we sprayed BBC on the eight P/TGMS lines that harbored the 28-bp deletion or the T1510G mutation. The results showed that all eight P/TGMS lines were susceptible to BBC (Fig. 4A). In addition, a BBC concentration gradient experiment was conducted on P/TGMS lines SE21S (T1510G) and MingS (28-bp deletion) with Y58S as a control. The results showed that SE21S and MingS were injured at a low BBC concentration (0.05 g/l) while Y58S exhibited no obvious damage until the BBC concentration reached 1.2 g/l (Fig. 4B). Finally, we chose a commercialized 2-line hybrid rice variety Mingliangyou143 from China, of which the female and male parents are MingS and P143 (containing HIS1 H2), respectively, to examine the effect of BBC on the purification of 2-line hybrid rice seeds. The results showed that at least in the range of 0.05–0.6 g/l, BBC can be very effective in killing the female parent MingS but is safe for the Miangliangyou143 hybrid (Fig. 4C). At high concentrations, the hybrid containing the heterozygous HIS1/his1 gene seemed to be less resistant to BBC than the homozygous male parent, and there were sufficient BBC concentration intervals for the purification of the 2-line hybrid seeds.
To figure out whether BBC was still effective in solving 2-line hybrid rice seed purity issues in the field, we sprayed BBC on Mingliangyou143 and its parents in the field and found that under direct seeding cultivation method, when the BBC dosage was 300 g/ha (the recommended dosage was 150–225 g/ha for weeding in the field), all seedlings of the female parent MingS showed obvious bleaching, and growth stopped completely, while Mingliangyou 143 and the male parent P143 grew normally (Fig. 5). These results showed that BBC could solve the problem of 2-line hybrid rice seed purity while weeding in the field.
Potential Applications Of Other β-triketone Herbicides
The commonly used bTHs are BBC, mesotrione, sulcotrione, tembotrione, and tefuryltione. Currently, mesotrione is the world's most widely used HPPD-inhibitor herbicide (Hua, 2015). In order to identify whether other bTHs are the same as BBC, i.e., can be used in indica rice areas and 2-line hybrid rice seed purification, we chose mesotrione for further study. As shown in Fig. 6A–D, IR64 and SE21S started to be susceptible to mesotrione when the concentration exceeded 0.01 g/l. However, NB and Y58S only started to show obvious injury when the mesotrione concentration reached 0.45 g/l. In addition, the results of mesotrione concentration gradient spraying on Mingliangyou143 and its parents showed that mesotrione can also be used for purification of 2-line hybrid rice seed, at least when the application concentration ranges from 0.01 g/l to 0.1 g/l (Fig. 6E). Since the HIS1 gene has been proved to be a broad-spectrum resistance gene for bTHs (Maeda et al., 2019) and mesotrione is similar to BBC in terms of the phytotoxicity responses of different indica rice varieties, we can determine that by detecting the HIS1 gene in indica rice, there is potential to use bTHs in indica rice areas and in the purification of 2-line hybrid rice seeds.