*2.5. Fine Mapping and Candidate Gene Prediction for qLTG6*

To identify the possible novel genes for low germination within the QTLs, *qLTG6* was thoroughly analyzed because it has a high LOD value and relatively small genetic intervals (Figure 4A, Table 2). *qLTG6* was first identified within a 400 kb interval. Then, further high-resolution mapping of *qLTG6* was carried out using four key homozygous recombination lines from 1,569 segregating population plants and newly developed markers, as shown in Table 3. Finally, *qLTG6* was localized to a region of a 45.8 kb physical interval between markers M002 and M008 via the progeny testing of key homozygous recombinant lines (Figure 4B). Overall, there was the prediction of about seven genes in the target region (Table 4). A re-sequencing data analysis of the delimited region only detected differences in the gene *LOC\_Os06g01320* (Figure 4C). To confirm and estimate these differences, PCR-based sequencing was conducted to analyze the gene body and the 3 kb promoter of *LOC\_Os06g01320*, which revealed a 32 bp deletion and a T–C transition in the promoter. In the gene body, a C–A transition was also detected in the 18th exon, causing a substitution of Thr to Asn in LTH (Figure 4D).

**Figure 4.** Identification of the candidate gene of the QTL, *qLTG6*. (**A**). Linkage analyses of the QTL, *qLTG6*; (**B**). Graphical representation of recombinants in the RILs of germinability under low temperatures, refining the location of *qLTG6* in an interval defined by bin markers; (**C**). The identification of the candidate gene, *LOC\_Os06g01320*, which encodes a chromodomain, helicase/ATPase, and DNA-binding domain (CHD)-related (CHR) proteins, CHR723; (**D**). Validation of the mutation of *LOC\_Os06g01320* by PCR sequencing.


**Table 3.** The markers developed for the fine mapping of *qLTG6* on chromosome 6.

**Table 4.** Candidate genes in the 45.8 kb target region corresponding to *qLTG6.*


#### *2.6. Expression Analysis of LOC\_Os06g01320*

Both an SNP and deletion were detected at the promoter of *LOC\_Os06g01320* in LTH, which suggests that expression might vary between parents. For further investigations on these variants, qPCR analyses were executed to evaluate the expression patterns of *LOC\_Os06g01320* in two parents. During seed germination under optimal conditions at 28 ◦C, the expression levels of *LOC\_Os06g01320* decreased in the LTH and SN265 from 0 h to 1 d (Figure 5), which indicates that *LOC\_Os06g01320* is necessarily required to be repressed during seed germination. Under the conditions of low-temperature stress with a temperature range around 15 ◦C, the expression levels of *LOC\_Os06g01320* in LTH were significantly lower than SN265, both at 12 h and at 1 d. After conducting a further refinement, these results showed that LTH has lower expression levels of *LOC\_Os06g01320* than SN265, which could reduce the negative regulation of gene expression and promote seed germination under low-temperature stress.

**Figure 5.** The expression analysis of the candidate gene of *qLTG6*. Relative expression of LOC\_Os06g01320 at the germination stage under 15 ◦C and 28 ◦C in the LTH and SN265, respectively. \*\* *p* < 0.01, using Student's *t*-test. Bar SEM, *n* = 3.

#### **3. Discussion**

In recent years, direct-seeded rice has received much attention in Asia because of its time and labor savings and low input demand as an alternative to conventional rice systems. However, the long-term cultivation method of seedling-transplantation has led to a loss of expressions of some low-temperature-tolerant genes, which are usually expressed at the germination stage. Poor germinability remains one of the major problems [5,21,29,30]. Therefore, screening cultivars with high germination abilities under low-temperature stress is necessary to sustain rice yield and ensure the application of direct seeding cultivation in the NEC region. In the current study, the *japonica* landrace variety, LTH, a locally well-adopted cultivar originating from the far Southwest province of China, was selected since it shows a high germination rate under low-temperature conditions. The germination of LTH began two days after the start of incubation at a temperature of 15 ◦C and took five more days to reach a germination rate of almost 90%. Screening of the low-temperature germination ability of 135 cultivars from the NEC was then done, which revealed that no cultivar was more tolerant to low-temperature stress than LTH. Therefore, the identification of tolerant genes during germination under low-temperature stress in LTH has important scientific value for improving the low-temperature germination ability of rice in the NEC.

In this study, high-throughput genotyping was employed through whole-genome resequencing, and bin map construction was carried out for QTL mapping. A total of 11 QTLs were identified on Chr.1, Chr.3, Chr.4, Chr.6, Chr.7, Chr.9, Chr.10, and Chr.12. By comparing the positions of the QTLs, 19 previously identified LTG QTLs were found to be near 10 QTLs, except for *qLTG9a*, as presented in Table 5. The *qLTG-1* was mapped near the *qCTGERM1-5* region for LTG [31]. The QTL location of *qLTG-3* was very close to that of the cloned QTL *qLTG3-1* [9]. The QTLs of *qLTG4*, *qLTG6*, and *qLTG10* were as also found by Teng et al. [32], Ji et al. [33] and Shakiba et al. [31] to enhance low-temperature germinability. *qLTG7a* was mapped near *qCTGERM7-1* [31], *qLTG-7* [34], *qGR-7*, and *qGI-7* [35]. Furthermore, *qLTG7b* was found to be mapped near *qCTGERM7-4* [31], *qLTG-7* [36], and *OsSAP16* [10]. At the germination stage, there was a correlation between the cold tolerance of *qLTG9b* and the *qLTG-9* region [32,33]. *qLTG12a* was mapped near *qLTG-12a* [37] and *qLTG-12* [38]. *qLTG12b* was identified near the region of *qCTGERM12-1* [31], *qGR-12* [10], *qLTG-12* [36], and *qLTG-12* [39]. The above comparison results reflect the accuracy of this study, as well as the complexity of cold tolerance during germination. Moreover, this study also suggests that LTH's strong low-temperature germination ability was acquired by accumulating more cold-tolerant genes. These results not only strengthen the findings of previous studies but also reflect the complexity of low-temperature germination in accelerating the breeding programs for enhanced cold-tolerance among rice cultivars.

Among the 11 QTLs identified in this study, *qLTG-6* was ultimately narrowed down to the 45.80 kb region (Figure 4B). Currently, seven genes have been observed in the target region (Table 5) where *LOC\_Os06g01250* is found to encode the protein named cytochrome P450. *LOC\_Os06g01260* has functional activities in encoding Glutathione gamma-glutamyl cysteinyl transferase-1. Moreover, *LOC\_Os06g01280* encodes a retrotransposon protein. *LOC\_Os06g01270* and *LOC\_Os06g01290* encode a protein that has yet to be discovered. *LOC\_Os06g01304* encodes Spotted Leaf 11, whereas *LOC\_Os06g01320* encodes chromodomain, helicase/ATPase, and DNA-binding domain proteins. Considering the organ specificity in gene expression and molecular function information, it is difficult to ensure that a gene is a target gene. According to the sequencing data, this study identified only the C–A transition in the 18th exon of *LOC\_Os06g01320*, which is predicted to result in the substitution of Thr to Asn in LTH. We also found a 32 bp deletion and a T–C transition in the promoter region. In addition, the expression level of *LOC\_Os06g01320* in LTH was found to be lower than that of SN265 under lower temperatures. This lower expression level could be associated with seed germination at low-temperature stress. The sequence and gene expression data suggest that *LOC\_Os06g01320* might be the most plausible prospect for *qLTG-6*, but the current evidence remains insufficient and will require us to carry out genetic modification complementation or gene editing verification.


**Table 5.** Comparison of QTL positions on the rice genome.

<sup>a</sup> Chr., chromosome.
