SRG1, Encoding a Kinesin-4 Protein, Is an Important Factor for Determining Grain Shape in Rice

doi:10.1016/j.rsci.2018.06.008

Abstract

Abstract:

Grain shape is one of the important agronomic traits, which is closely related to the yield of rice. A new rice mutant, named small and round grain (srg1), was isolated from an indica cultivar Zhenong 34 by ethyl methane sulfonate (EMS) mutagenesis. The microscopic analysis showed that the cell length of spikelet in srg1 was decreased compared with that in wild type (WT), which caused the grain length short. Meanwhile, the grains of srg1 were wider than those of WT because of the increased cell layers in spikelet in the lateral direction. Therefore, the inhibition of cell expansion and increased cell proliferation collectively led to the small and round grain. By map-based cloning, the gene SRG1 was located on the short arm of chromosome 9, which encodes a kinesin-4 protein, represented by the gene LOC_Os09g02650. A single nucleotide polymorphism, occurred in the 16th exon of SRG1, led to premature translation stop in mutant. The cell cycle-related genes were up-regulated in srg1, which conferred that SRG1 controlled grain width through the cell proliferation. Since the role of SRG1 in regulating grain shape was not clarified well before, it is valuable to explore the mechanism of grain development. This study could, hence, provide a morphogenesis and molecular basis for elucidating the function of SRG1, as well as a new germplasm resource for the further study of grain development.

Key words: small and round grain, kinesin-4 protein, gene mapping, rice

Ran Qin, Akhter Delara, Chengcong Yang, Kumar Nath Ujjal, Eshag Jamal, Xiaoli Jin, Chunhai Shi. SRG1, Encoding a Kinesin-4 Protein, Is an Important Factor for Determining Grain Shape in Rice[J]. Rice Science, 2018, 25(6): 297-307.

Figures/Tables 11

Table 1 Agronomic traits of wild type and srg1 mutant.

Trait	Wild type	srg1 mutant
Grain length (mm)	7.51 ± 0.37	6.08 ± 0.29**
Grain width (mm)	3.24 ± 0.14	3.53 ± 0.18*
Grain length/width	2.32 ± 0.17	1.72 ± 0.13**
Roundness value	0.46 ± 0.12	0.61 ± 0.21**
No. of grains per panicle	204.4 ± 10.5	114.0 ± 12.0**
1000-grain weight (g)	25.35 ± 0.86	21.44 ± 0.63*
Plant height (cm)	83.90 ± 1.45	50.35 ± 1.22**
Seed-setting rate (%)	92.6 ± 1.1	87.1 ± 0.4*
Panicle length (cm)	20.21 ± 0.98	13.29 ± 0.44**

Supplemental Table 1 Primer list for gene mapping.

Table 2 Functional predications of candidate genes of srg1.

ID	Gene function annotation
LOC_Os09g02650	Kinesin motor domain containing protein, putative, expressed
LOC_Os09g02670	Expressed protein
LOC_Os09g02680	Retrotransposon protein, putative, unclassified
LOC_Os09g02690	Expressed protein
LOC_Os09g02700	Polyadenylate-binding protein, putative, expressed
LOC_Os09g02710	CBS domain containing membrane protein, putative, expressed
LOC_Os09g02729	Phospholipase C, putative, expressed
LOC_Os09g02750	Expressed protein
LOC_Os09g02760	Retrotransposon protein, putative, Ty1-copia subclass, expressed
LOC_Os09g02770	Translation initiation factor IF-2, putative, expressed
LOC_Os09g02780	OsMADS77-MADS-box family gene with M-alpha type-box, expressed

Supplemental Table 2 Primer list for quantitative real-time PCR.

Fig. 1. Phenotypes of wild type (WT) and srg1 mutant. A, Grain length. B, Grain width. C, Plant height. D, Internode. The numbers 1, 2, 3, 4 and 5 refer to the 1st, 2nd, 3rd, 4th and 5th internodes from the top, respectively. E, Culm brittleness. Scale bars in A, B, C and D are 1.0 cm, 0.1 mm, 10.0 cm and 2.5 cm, respectively.

Supplemental Fig. 1. The scanned images of the grain shape of WT(A) and srg1(B). Using the images the roundness was obtained by the rice appearance quality inspection system(SC-E).

Fig. 2. Effect of SRG1 on grain shape. A-D, Scanning electron microscope analysis of outer and inner surfaces of wild type (WT, A and C) and srg1 mutant (B and D) lemmas, respectively. Square frame in C and D indicates the cell length. E and F, Cross-sections of the central parts of WT and srg1 lemmas. G, Expression levels of cell cycle genes in WT and srg1 spikelets from young panicles. H, Expression levels of genes related to grain shape in WT and srg1 spikelets from young panicles. Rice Actin gene was used as an internal control. Scale bars in A, B, C, D, E and F are 50 µm, 50 µm, 25 µm, 25µm, 100 µm and 100 µm, respectively. Values represent mean ± SD (n = 3); **, P ≤ 0.01.

Fig. 3. Alterations of structure and components in cell wall of culm in wild type (WT) and srg1 at heading stage. A-D, Transverse electric and magnetic observation of sclerenchyma cell walls in wild type (WT) (A and C) and srg1 culms (B and D). C and D were the amplification of cell wall structure in A and B, respectively. E, Cell wall main components in culm of WT and srg1. PCW, Primary cell wall; SCW, Secondary cell wall.Scale bars in A, B, C and D are 2.0 µm, 2.0 µm, 0.5 µm and 0.5 µm, respectively. Value represents the mean ± SD (n = 3); **, P ≤ 0.01.

Fig. 4. Map-based cloning of SRG1.A, Primary mapping of srg1 using 94 F2 individuals. The recombinant number was listed under the corresponding marker. B, SRG1 was fine-mapped to a 121.2 kb interval between markers gs18 and gs16 with 372 F2 individuals. C, Total 11 genes were annotated in the region according to the database of Rice Genome Annotation Project. LOC_Os09g02650 with the green color was presumed to be the locus of SRG1. D, The sequencing analysis of LOC_Os09g02650. There was 1 bp substitution (G to A) occurred in the 16th exon (+1 925 bp) of the srg1, which caused the tryptophan (+642 aa, TGG) changing to a stop codon (TAG). E, The structure domains of SRG1 obtained from the National Center for Biotechnology Information.

Fig. 5. The expression pattern of SRG1.A, Expression levels of SRG1 in different organs; B, Expression levels of SRG1 in spikelets of different panicle lengths. Rice Actin gene was used as an internal control. Value represents mean ± SD (n = 3); **, P ≤ 0.01.

Fig. 6. Phylogenetic tree of homologous proteins of SRG1. Homologs were obtained by a BLASTP in the National Center for Biotechnology Information against the amino acid sequence of SRG1. This analysis was performed by MEGA 6.0 with Neighbor-Joining (NJ) method. The SRG1 is marked with green color and the proteins from Oryza are indicated by red line.

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