3.1 Molecular identification
In this study, the two cultivated rice varieties with the different major Wx alleles (Table S2) were selected firstly, QLD (Wxa) and YSZ (Wxb), which are widely grown in the rice region of Southwest China. We designed a CRISPR/Cas9 (Fig. 1a) construction accurately targeting the second exon (87-109 bp) of the Wx gene with the expectation to generate a null mutation (Fig. 1b). Based on this vector, the results suggest four major mutant types of QLD (Fig. 1c) and five major mutant types of YSZ (Fig. 1d) were obtained. This experiment observed high mutagenesis efficiency in 80% T0 transformants mutating in QLD plants and 82.35% YSZ (Table S2). Finally, our results again proved this method did not change the main agronomic traits (Fig. S1).
3.2 The quality of wx
We further identified two single-base homozygous mutations, YSZwx1 and QLDwx1, and performed quality analysis on them (Table S3). The results show no significant difference in the major grain quality of the two wx seeds except that the colour of endosperm changed into milky white (Fig. 1e) and the kernel weight (Fig. S2). The cooking and eating quality of YSZwx1 rice was soft but not sticky, while the QLDwx1 rice was very sticky (Fig. 1f), and both their gel consistency was a significant increase (Fig. 1g).
This study observed that the AAC significantly decreased to 1.16% and 2.36% (Fig. 2a) (reduced by 91.01% and 89.80%), leading to increased gel consistency (Fig. 2b). The other qualities were also experimented. For example, the total protein was increased 26.01% and 6.37%, respectively (Fig. 2c), but the total starch showed no regularity, increasing by 8.11% and decreasing by 6.19% compared to WT (Fig. 2d). The above result suggests that the other rice quality would be significantly changed by editing the Wx gene, not only the AAC，and wx mutant does not always have the same quality trend. Breeders can cultivate the different qualities of waxy rice by this method.
Differential scanning calorimetry (DSC) is widely used to study grain gelatinization temperature (GT), and the GT is usually represented by peak temperature (Tp) (Sasaki et al. 2000). The GT of rice is generally divided into three types: low (<70℃), intermediate (70-74℃) or high (>74℃) (Kongseree and Juliano 1972). In this experiment, the wx mutants showed a slightly higher GT range (To, Tp and Tc) than the WT (Fig. 2e). The GT ranges of WT starch was 66.08℃ (YSZwx1) and 73.46℃ (QLDwx1) (onset temperature, To) to 76.03℃ and 82.06℃ (conclusion temperature, Tc) with an enthalpy (ΔH) of 11.6 J/g and12.55 J/g (Table S4). The results suggest that To, Tp, Tc and ΔH from wx mutants were increased, which lead to them being gelatinized later compared with WT, but they still belonged to the same GT types as the WT. Here, we propose a hypothesis that editing the Wx gene does not change the GT, and subsequent experiments were to verify this hypothesis with knocked other rice varieties’ Wx gene.
3.3 Pasting properties
The wx mutants and WT pasting properties were significantly variable (p<0.05) (Fig. 3a). For example, the PKV, HPV, BDV, CPV, SBV, peak time and PT of QLD and QLDwx1 ranged from 2647 to 2775 cp, from 1729 to 1362 cp, from 918 to 1413 cp, from 4080 to 1757 cp, from 1333 to -1018 cp, from 6.38 s to 4.57 s and from 80.7℃ to 81.5℃, respectively (Table S5). The results show that QLDwx1 and YSZwx1 have more waxy rice pasting properties (Fig. 3b).
3.4 Chain length distribution of amylopectin
The chain length distributions of amylopectin in wx mutants and WT were distinctly different (Fig. 4a). At chain length degree of polymerization (DP) 7-20, wx mutants increases compared to WT, while at DP > 20, wx was decreased (Fig. 4b).According to the value of ΣDP≤10/ΣDP≤24 (amylopectin chain ratio, ACR value), amylopectin structure from cultivated rice can be classified into three types: L-type (ACR≤0.200), S-type (ACR≥0.240) and M-type (ACR 0.201-0.239) (Nakamura et al. 2002). Interestingly, even although the ACR value of the wx mutants were significantly lower than that of WT, they still belonged to the same amylopectin structure type. The amylopectin structure QLDwx1 and QLD belonged to the L-type, and YSZwx1 and YSZ belonged to the M-type (Fig. 4c).
3.5 Data Analysis
We applied the same method to three other rice varieties with the same material treatment to find a potential link between wx mutants and WT (Fig. S3 and Table S6). The other three rice varieties belonged to the Wxb genotype (Table S6). The results showed that WT (Fig. 5a) with higher AAC and the AAC of corresponding its wx mutant (Fig. 5b) was also higher, and some wx mutants AAC were more than 2%, which means they may not belong to high-quality waxy rice. The same results were observed that the Tc and ΔH of wx mutants were increased, however, their GT types were not changed (Table 1). Similarly, a result showed the ACR value was altered but not beyond specified value such as M-type (ACR 0.201-0.239), therefore, wx and WT have the same amylopectin structure type (Fig. 5b). Further data analysis showed a direct linear correlation between ACR and GT (Fig. 5c), and the ACR value depended on GT. Here, there are two new types be proposed to identify the structure of amylopectin, respectively, which were HGT-type (high gelatinization temperature type, ACR < 0.18) and LGE-type (low gelatinization temperature type, ACR > 0.18). Based on these findings, we demonstrated that GT type and ACR type would not be changed by editing the Wx gene and if WT with a higher AAC, its wx mutant would be endowed a higher AAC.
In this study, we also separately analyzed the correlation between the related physicochemical properties of WT or wx mutants. Pearson's correlation analysis suggested that WT and wx mutants had dissimilar correlations between rice quality, amylopectin structure and physicochemical properties (Fig. 6). The data of WT reflected that GT was negatively correlated with DP6-12 and ACR and positively correlated with DP13-24. The AAC was positively correlated with peak time, CPV, HPV, SBV, and was negatively correlated with BDV and GC. However, the wx mutantsdata reflected that GT was negatively correlated with DP6-12, DP6-24 and ACR and positively correlated with DP13-24, peak time, CL, and DP25-100. The AAC was negatively correlated with GC and positively with peak time HPV and CPV.
The above results implied that amylopectin structure and AAC in diverse rice varieties determines different physicochemical properties. So, the quality data of wild and waxy varieties added together in statistics analyzing may not be an excellent way to find the relationship between the physicochemical properties. Here, we suggest that if there are accurately diagnoses of the physicochemical properties of waxy rice, it is best to use glutinous rice data only.
3.6 Suggestion of specific quality breeding of waxy rice
It is generally believed that the AC of waxy rice should be less than 2% (Juliano 1992). We recommend that if you want to obtain high-quality waxy rice, you should first choose rice varieties with low AAC, otherwise, these wx varieties may not be called waxy rice. On the other hand, GT also can be selected before waxy rice breeding, breeders only have to choose a rice variety with the targeted GT. Finally, any new waxy rice variety breeders want can be obtained based on the ACR value, AAC and agronomic traits.