Fig. 1. Phenotypes of trichomes in Nipponbare and Suwangwasnger (SWWR). A, Adaxial leaf of Nipponbare. B, Adaxial leaf of SWWR. C, Leaf sheaths of Nipponbare (Left) and SWWR (Right). D, Grain hulls of Nipponbare (Left) and SWWR (Right). E, Adaxial leaf of Nipponbare under scanning election microscope. F, Adaxial leaf of SWWR under SEM. Scale bars in A, B, C and D, 1 mm. Scale bars in E and F, 500 μm.

Supplemental Fig. 1. Trichomes at different leaf positions in the tillering stage under stereoscope. A represents SWWR -0.5 leaves. B represents SWWR 0.5 leaves. C represents SWWR 1.5 leaves. D represents SWWR 2.5 leaves. Scale bar, 1mm.

Supplemental Fig. 2. Genetic linkage map of HL6 SWWR on chromosome 6. The numerals in left column represent the genetic distance. The numerals in right column represent SSR marker names. The HL6SWWR gene is located between markers RM528 and RM5988.

Fig. 2. Map-based cloning and mutation site analysis of HL6SWWR. A, Primary mapping of HL6SWWR locus using 288 F2 plants. B, Fine mapping of HL6SWWR locus using 4 250 plants. C, High-resolution linkage analysis of the HL6SWWR locus and open reading frame in the target region on Rice Genome Annotation Project (RGAP). D, Different sites of HL6 coding sequence and amino acid sequence (in brackets) between Nipponbare and Suwangwanger (SWWR). E, Promoter sequence of 1 500 bp upstream of HL6SWWR transcription initiation site comparing with that of Nipponbare. The relative positions of the variations are given with respect to the start codon (ATG).

Fig. 3. Trichome characteristics of HL6SWWR complementary transgenic plants. A to C, Adaxial leaves of wild type (WT) Nipponbare, complementary transgenic line 1 (CP1-1) and complementary transgenic line 2 (CP1-2), respectively. Scale bars, 1 mm. D to F, Leaf sheaths of WT, CP1-1 and CP1-2, respectively. Scale bars, 1 mm. G, Grain hulls of WT (Left), CP1-1 (Middle) and CP1-2 (Right), respectively. Scale bar, 1 mm. H to J, Adaxial leaves under scamming electron microscopy of WT, CP1-1 and CP1-2, respectively. Scale bars, 500 μm.

Supplemental Fig. 3. Relative expression of HL6 in leaf, leaf sheath and root in three leaf stage of two parents. Asterisks indicate significant difference at P < 0.01 by t test. Error bars represent SD with triplicates.

Supplemental Fig. 4. Expression level of HL6SWWR complementary transgenic plants. WT represent wild type, CP1-1, CP1-2 are the transgenic line number. The macro-hair of CP1-1 is longer than CP1-2. Asterisks indicate significant difference at P < 0.01 by t test. Error bars represent SD with triplicates.

Supplemental Fig. 5. Leaf phenotype of adaxial leaf surface in transgenic plants under stereoscope. A and C represent adaxial leaf surfaces of PROSWWR:HL6NIP and PRONIP:HL6SWWR transgenic plants, respectively. B and D represent adaxial leaf surfaces of wild type. Scale bar, 1mm.

Supplemental Fig. 6. Relative expression of HL6 in the transgenic lines. CP1 represents PROSWWR:HL6SWWR complementary transgenic plant. CP2-1 and CP2-2 refer to two transgenic lines with the vector PRONIP:HL6SWWR. CP3-1 and CP3-2 refer to two transgenic lines with the vector PROSWWR:HL6NIP.Asterisks indicate significant difference at P < 0.01 by t test. Error bars represent SD with triplicates.

Fig. 4. Trichome characteristics of knock-out (KO) mutant hl6SWWR under Suwangwanger (SWWR) background. A, Schematic diagram of the target site in HL6SWWR genome sequence and HL6 sequence alignment between KO mutant and wild type (WT). B and D, Adaxial leaf and leaf sheath of WT. Scale bars, 1 mm. C and E, Adaxial leaf and leaf sheath of KO mutant hl6SWWR. Scale bars, 1 mm. F, Grain hull of KO mutant hl6SWWR (right) and its wild type (left). Scale bar, 1 mm. G and H, Adaxial leaf under scanning electron microscopy of WT and KO mutant hl6SWWR, respectively. Scale bars, 500 μm.

Fig. 5. Subcellular localization, expression pattern and transcriptional activity analysis of HL6SWWR. A, Subcellular localization of HL6SWWR in nuclei. Tobacco leaves were used for subcellular localization. 35S:HL6SWWR:GFP represents HL6SWWR and GFP fusion protein. 35S:GFP represents the control. B, Real-time PCR analyses of HL6SWWR in SWWR different tissues. Actin was used as the internal control. Bars represent SD (n = 3). C, HL6SWWR has transcription-activation activity in yeast. HL6SWWR coding sequence was cloned into the vector pGBKT7. pGBKT7-53 was used as a positive control. Transformed yeast was serially diluted and placed on SD screening plates.

Fig. 6. Scatter-plot of enriched KEGG pathways for differentially expressed genes (DEGs). A, DEGs in the groups of hl6SWWR (TA) / wild type Suwangwanger (SWWR) (TB) and complementary line HL6SWWR (CA) / wild type Nipponbare (CB). B, DEGs shared by the two groups. C, Scatter-plot of enriched KEGG pathways for DEGs in TA/TB and CA/CB. Q value is the P value after multiple hypothesis test correction. The value of Q is [0, 1]. The closer to zero, the more significant the enrichment. Rich-factor refers to the ratio of the number of DEGs enriched in the pathway to the number of genes annotated into the pathway gene. The larger the rich-factor, the greater the degree of enrichment.

Supplemental Fig. 7. GO enrichment analysis for differentially expressed genes. A, Gene Ontology enrichment for differentially expressed genes in hl6SWWR mutant and wild type SWWR plants. B, Gene Ontology enrichment for differentially expressed genes in HL6SWWR complementary transgenic and wild type Nipponbare plants. The x-axis refers to the number of genes annotated by the GO term. The y-axis represents GO classification including Biological Process, Cellular Component and Molecular Function.

Fig. 7. Analysis of differential expression gene (DEGs) in different pathway. A, DEGs in the hl6SWWR (TA) / Suwangwanger (SWWR) (TB) group. B, DEGs in the HL6SWWR complementary transgenic plant (CA) / wild type Nipponbare (CB). C, DEGs involved in different phytohormone signal transduction pathway. IAA, Auxin; CK, Cytokine; GA, Gibberellic acid; ABA, Abscisic acid; ET, Ethylene; BR, Brassinosteroid; JA, Jasmonic acid; SA, Salicylic acid.

Fig. 8. Validation of RNA-seq results by qRT-PCR. A, qRT-PCR was conducted in hl6SWWR mutants and WT plants. B, qRT-PCR was conducted in HL6SWWR complementary transgenic (CP1) line and WT plants. Ten DEGs were selected to be tested by qRT-PCR. Actin gene was used as the internal control. Bars are SD of three independent replicates. * and ** represent significant differences at the 0.05 and 0.01 levels, respectively.