IC4R011-GWAS-2016-26381647

Project Title

• New insights into the genetic basis of natural chilling and cold shock tolerance in rice by genome-wide association analysis

The Background of This Project

• As one of the major crops, rice is widely grown in tropical, subtropical and temporal regions, and temperature is one of the major environmental factors limiting its geographic distribution. The optimal temperature for rice growth is 25–30°C (Kim et al. 2014).Previous studies on low-temperature stress in rice mainly concentrated on chilling stress (temperature around 10°C), which was frequently used to distinguish it from freezing stress (temperature around 0°C); however, there is no clear definition of chilling or cold/freezing stress, and the treatment temperatures were often different for the same term in many reports (Cheng et al. 2007;Guo et al. 2006; Ma et al. 2015; Wang et al. 2013; Wang et al.2014; Yang et al. 2012). To date, no report has compared different low-temperature stresses such as natural chilling stress with the acute freezing stress in rice. Recent studies have revealed some mechanisms and signalling networks involved in the cold stress response in rice (Knight & Knight 2012; Ma et al. 2015; Wang et al. 2014; Yang et al. 2012).

Plant Culture & Treatment

• A total of 529 rice accessions including 202 from the China Core Collection and 327 from the World Core Collection were used for the association analysis (Supporting Information Table S1). This panel of rice accessions is essentially the same as the panel of 533 accessions as previously described except three accessions (C126, W196 and W232) with severe heterozygosity and one (W190) with a low mapping rate (10%) omitted.
• After germination for 7 d, the seedlings with uniform growth were transplanted to 10 cm × 10 cm pots each containing nine seedlings. Each accession was planted in three pots as three biological repeats, and the layout of the pots followed a completely randomized block design.
• In this study, the researchers designed two treatments to mimic the natural chilling (temperature gradually declined to a range of 5–12 °C) and cold shock (constant temperature at 4 °C) stresses, respectively. For the natural chilling stress treatment, the seedlings were grown in a greenhouse for 3 weeks, at which time the plants went into the 4-leaf-stage. The natural chilling treatment was carried out in the greenhouse in winter (Wuhan, China) with the heating and light turned off and the natural low-temperaturefluctuating between 5 and 12 °C depending on the outside temperature. The temperature in the greenhouse was recorded every half hour by a weather station (Spectrum Technologies,Inc. WatchDog 2000), and a portion of the record is provided in Supporting Information Fig. S1. For the cold shock treatment, the 4-leaf-stage rice plants were performed in a growth chamber set at a constant 4 °C with 14/10 h of light/darkness.

Research Findings

• The results of seven electrolyte leakage indices (ELN,ELC1,ELC2, ELR, ELSN, ELSC1 andELSC2)indicatedthat with the prolonged time duration of stress treatment, large variation was observed for EL and the relevant ratio traits including ELR1, ELR2, ELR3, ELSR1 and ELSR2 (Table 1). The range of ELN and ELSN was not exactly the same, which may be due to the slight differences of seedling growth state inthetwoexperiments.Among thesetraits,thevariationrange of ELC1 is similar to ELN, but the variations of ELC2 and ELR were clearly increased, suggesting slight cell membrane damage at the early stage of natural chilling stress treatment but that the damage became serious and irreversible at the later stage of the stress treatment. The situation was different in the cold shock stress treatment, in which ELSN, ELSC1 and ELSC2 were increased since the onset of the stress treatment (Table 1), indicating that the cell membrane damage was faster and more significant.

• A quantile–quantile plotof all 16 traits isprovided in Supporting Information FigsS3,S4andS5.Manhattan plots for the association analysis of these traits are displayed in Fig. 1.

'Figure 1. Manhattan plots for six traits: (a) ELR2, (b) ELC2, (c) RLC, (d) ELSR2, (e) ELSC1 and (f) SRC. The negative log10 transformed P-values of genome-wide scan are plotted against the marker position in the genome. Dotted line: P = 1E�06. The positions of five reported genes are indicated with black arrow. Examples of some loci are indicated; L27, L104 and L63 in red were detected by EL-related traits under the two cold stress conditions, while locus L18 and L79 in blue were identified by two traits under natural chilling stress condition. L is short for locus.'

• For a better view of the comprehensive association results,all of the detected association loci in the whole panel were marked on the 12 chromosomes according to their physical distance on the rice genome (Fig. 2), and the loci for natural chilling stress (57 loci) and cold shock stress (63 loci) are shown in blueand green, respectively.

'Figure 2. Distribution of 132 loci on 12 chromosomes according to physical distance. The loci and their associated traits were marked on the right of chromosomes with the relative position of each locus (200 kb) shown by its own front physical position; the overlapping known QTL are shown in grey column on the left, while the markers of QTL are shown on the right. The 57 loci in blue were identified under natural chilling stress condition, the 63 loci in green were identified under cold shock stress condition and the 12 loci in red were detected under both stress conditions.'

• Further haplotype analysis was focused on the reported gene OsMYB2 because it was reported to participate in abiotic stress (including cold, salt and drought) response at the seedling stage(Yang et al. 2012), and the L33 containing OsMYB2 was overlapped with the known QTL qLVG3 (Han et al. 2006). The SNP data were referred to RiceVarMap including its intragenic region and 2 kb upstream (Zhao et al. 2014a). Four major haplotypes were observed, with low frequency haplotypes (less than five accessions) being omitted (Fig. 3a).The reserchers noticed that two non-synonymous SNPs led to changes in amino acids(one at 11325395 caused a C in Hap1 to a Y in the other three haplotypes, and the other at 11325747 caused a W in Hap1 to an R in the other three haplotypes), which suggests that these SNPs may be associated with the gene function to a certain degree. The researchers further checked the latitude distribution of the haplotypes by a scatter diagram of the latitude of origin of these accessions (Fig. 3b). The accessions in Hap1 were from higher latitude regions compared with the accessions in the other three haplotypes (P<0.05; (Fig. 3c).

'Figure 3. Haplotype analysis of OsMYB2. (a) Haplotypes in 412 accessions (haplotypes with less than five accessions were omitted) according to SNP data from RiceVarMap based on MSU6.1 annotation. The region contained coding region and 2 kb upstream of the gene. Letters on the right of the average are ranked by Duncan test at P < 0.05; different letters indicate significant difference. (b) Scatter diagram for the latitudes (ascending sorted) of the origins of accessions of the four haplotypes at OsMYB2 locus. (c) Comparison of latitude distribution between accessions of the four haplotypes. Letters above the bars are ranked by Duncan test at P < 0.05; different letters indicate significant difference.'

• The researchers also checked the subpopulation and geographical distribution of all 412 accessions in relation to the four haplotypes of OsMYB2 (Fig. 4). It was noticed that 97.75% of the accessionsinHap1groupbelongto japonica,while89.03%oftheaccessions in Hap2 belong to indica. Meanwhile, 77.27% of the accessions in Hap3 group belong to aus subgroup, and 67.81% and 23.29% of the accessions in Hap4 group belong to indica and japonica, respectively (Fig. 4b), which may partially explain why Hap4 exhibits an intermediate type in terms of cold sensitivity. The researchers also found that accessions in Hap1 in red distributed widely no matter around the world or China(Fig. 4a,c). These results suggest that the rice accessions in different haplotypes of OsMYB2 had indica–japonica differentiation and differential latitudinal distribution tendency.

'Figure 4. Geographic and subpopulation distribution of accessions of the four OsMYB2 haplotypes. Geographic distribution of accessions in the four haplotypes on the world map (a) and map of China (c), the solid circles in different colours (red, light blue, dark blue and green) represent accession numbers of the four haplotypes (Hap1–4, respectively), except for five accessions in Hap1, 16 accessions in Hap2 and 14 accessions in Hap4 withunknowngeographiclocation.(b)Thesubpopulationdistributionof89accessionsinHap1,155accessionsinHap2,22accessionsinHap3and146 accessions in Hap4.'

Labs working on this Project

• National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China
• National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China

Corresponding Author

• Lizhong Xiong:lizhongx@mail.hzau.edu.cn