IC4R010-GWAS-2016-26612069

Project Title

• Uncovering novel loci for mesocotyl elongation and shoot length in indica rice through genome-wide association mapping

The Background of This Project

• Direct-seeding cultivation is becoming popular in rice(Oryza sativa L.)-planting countries because it is labor- and time-efficient. However, low seedling establishment and slow seedling emergence have restricted the application and popularity of the technique. Mesocotyl elongation and shoot length are two important traits that can enhance rice seedling emergence. A single nucleotide polymorphism(SNP) is a genome sequence variation caused by a single base within a population, and SNPs evenly distributed throughout the genomes of plant species.
• In this study, a genome-wide association study (GWAS), based on 4136 SNPs, was performed using a compressed mixed linear model that accounted for population structure and relative kinship to detect novel loci for the two traits.

Plant Culture & Treatment

• To reduce spurious associations caused by population structure, a set of 469 global diverse indica accessions with rich genetic diversity (supplementary Table S1), representing the major rice-growing regions, was collected to construct a large association mapping population. All the samples were genotyped using a custom-designed array containing 5291 single nucleotide polymorphisms (SNPs)following the Infinium HD Assay Ultra Protocol (Illumina,Inc. San Diego, CA), with the minor allele frequency \5 % eliminated. Finally, 4136 SNPs were used in our GWAS analyses (supplementary Table S2). These accessions were planted in a randomized complete block design with three replications in a six-column 9 six-row area in Lingshui(LS; N18�320, E110�010) and Hangzhou (HZ; N30�150,E120�120) in 2014.

Research Findings

• In this study, 469 indica accessions collected from 20 countries (Fig. 1a) were used as the GWAS panel The germplasm resources maintain rich mesocotyl elongation and shoot length variations (Fig. 1b–d), and the phenotypic variations are summarized in Table 1and supplementary Table S4.

'Fig. 1 Material distribution and mesocotyl elongation diversity.a The worldwide distribution of 469 indica accessions. The black dots represent the country-specific distribution. b Different mesocotyl lengths in the germplasm. Bar 3 cm. c Histogram of mesocotyl lengths in Hangzhou and Lingshui. d Histogram of shoot lengths in Hangzhou and Lingshui'

• In total, 52 accessions, which were divided into two groups, one with long and the other with short mesocotyl lengths, were selected to analyze the POE and SOE. SOE was the average POE per day before the germination rate stopped significantly increasing. The sowing depths of 2 and 5 cm represent shallow overburden and deep seeding,respectively. The results are summarized in Fig. 2.The mesocotyl lengths between the two groups were highly different independent of the sowing depth (Fig. 2a). For the 2-cm sowing depth, the POE of the long mesocotyl accessions was significantly greater than that of the short mesocotyl accessions, reaching 90.76 % (Fig. 2b).For the SOE trait, regardless of the sowing depth, the long mesocotyl group had a higher emergence rate than that of the short one (Fig. 2c).

'Fig. 2 Direct-seeding emergence of long and short mesocotyl length accessions at two sowing depths. a Two groups of mesocotyl lengths in two sowing depths. Error bar standard error. b Cumulative percentage of emergence in 18 days. The pink area represents'

• Additionally, the correlations among POE, SOE, mesocotyl length and shoot length were analyzed, and the results showed that POE and SOE were highly positively correlated with mesocotyl length at 2- and 5-cm sowing depths (Fig. 3a, b).Moreover, in the deep soil condition,the POE and SOE were also positively correlated with shoot length, but the correlation coefficients were lower than those of the mesocotyl length (Fig. 3b).

'Fig. 3 Correlation of four direct-seeding traits at two sowing depths.The upper panel contains the correlation coefficients, and the lower panel contains the distributions of the two traits. The diagonal represents the density line of the traits. Single asterisk and Triple asterisk represent significant at 0.05 and 0.001, respectively. POE percentage of emergence, SOE speed of emergence, ML mesocotyl length and SL Shoot length'

• The PCA results showed that PC1, PC2 and PC3 accounted for 17.8, 8.7 and 6.4 % of the genetic variation, respectively (Fig. 4a). Moreover, the top 10 PCs explained *53.5 % of the total variation in population structure (Fig. 4b).

'Fig. 4 Population structure of 469 indica accessions. a Different colors represent different ingredients, and PC1, PC2 and PC3 are the top three PCs. b Cumulative variations explained by the top 10 PCs'

Labs working on this Project

• State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, China

Corresponding Author

• Xinghua Wei:weixinghua@caas.cn & Qing Lu:luqing2016@126.com