Characterization of a Novel Weak Allele of RGA1/D1 and Its Potential Application in Rice Breeding

doi:10.1016/j.rsci.2022.03.001

Abstract

Abstract:

Semi-dwarfing improves the lodging resistance and yield of rice, and the vast majority of modern rice varieties harbor the sd1 allele to decrease plant height, resulting in reduced genetic diversity and negative agronomic traits. Thus, exploring alternative sources of dwarfism is imperative for rice breeding. Here, we identified a novel RGA1 allele, d1-w, from a local indica variety Xiaolixiang (XLX) using a map-based cloning approach. Compared with other rice varieties, RGA1 in XLX contained a unique single nucleotide polymorphism that resulted in an additional transcript and reduced functional RGA1 transcript level. The RGA1 from Nipponbare was introduced into XLX to estimate the value of d1-w in rice breeding. Compared with transgenic XLX plants (XLX^D1), XLX exhibited reduced plant height, increased stem strength, lower reactive oxygen species accumulation, delayed senescence, stronger photosynthesis, higher grain yield and quality (including external, milling and nutritional qualities), and enhanced resistance to drought and Rhizoctonia solani. Therefore, we proposed that the d1-w allele has potential as an excellent dwarfism resource for rice breeding.

Key words: rice, weak allele, RGA1, dwarf germplasm resource, yield, grain quality, drought resistance, sheath blight

Liu Yantong, Li Ting, Jiang Zhishu, Zeng Chuihai, He Rong, Qiu Jiao, Lin Xiaoli, Peng Limei, Song Yongping, Zhou Dahu, Cai Yicong, Zhu Changlan, Fu Junru, He Haohua, Xu Jie. Characterization of a Novel Weak Allele of RGA1/D1 and Its Potential Application in Rice Breeding[J]. Rice Science, 2022, 29(6): 522-534.

Figures/Tables 10

Fig. 1. Phenotypic characterization of XLX, HZ, CHT025 and their F1 plants. A and B, Phenotypes of XLX, HZ, CHT025 and their F1 plants at the filling stage. Scale bars, 20 cm. C, Grain shapes of XLX, HZ, CHT025 and their F1s. Scale bar, 5 mm. XLX, Xiaolixiang; HZ, Huazhan; CHT025, Changhui T025.

Table 1. Agronomic characteristics of XLX and three common varieties as well as their F1 plants.

Rice material	PH (cm)	EPN	PL (cm)	PBN	SBN	GNPS	SSR (%)	TGW (g)	YPP (g)
XLX	112.1 ± 3.5	15.7 ± 2.7	23.5 ± 0.6	14.0 ± 0.4	53.6 ± 1.6	298.2 ± 19.6	90.6 ± 1.2	16.3 ± 0.1	70.1 ± 5.9
TN1	108.1 ± 2.2	12.4 ± 1.7	25.9 ± 0.6	12.3 ± 0.4	24.5 ± 3.3	177.8 ± 16.7	83.3 ± 1.8	26.2 ± 0.6	48.0 ± 4.0
CHT025	106.3 ± 1.9	9.1 ± 0.7	29.0 ± 1.2	13.0 ± 0.2	58.2 ± 5.4	386.0 ± 6.0	95.5 ± 0.7	17.6 ± 0.2	59.4 ± 4.1
HZ	110.5 ± 1.8	11.8 ± 0.7	24.1 ± 1.1	12.4 ± 0.3	26.5 ± 2.3	183.9 ± 20.2	87.3 ± 0.3	24.6 ± 0.8	46.8 ± 2.0
XLX/TN1 F₁	183.0 ± 3.8	20.2 ± 1.9	30.2 ± 0.5	13.8 ± 0.6	71.7 ± 3.2	346.3 ± 17.6	94.6 ± 0.7	21.2 ± 0.2	140.0 ± 7.1
XLX/CHT025 F₁	169.0 ± 2.3	16.0 ± 0.2	31.3 ± 0.4	13.7 ± 0.7	72.0 ± 5.1	344.8 ± 22.3	93.7 ± 0.5	18.0 ± 0.1	103.1 ± 5.3
XLX/HZ F₁	164.6 ± 1.5	18.0 ± 1.2	31.1 ± 0.4	14.4 ± 0.5	71.9 ± 4.5	334.3 ± 18.2	94.2 ± 0.1	21.0 ± 0.2	119.1 ± 6.4

Fig. 2. Map-based cloning of DEP-SRS gene. A, Fine mapping of DEP-SRS gene. The gene was mapped to the interval between molecular markers RM18410 and RM18448 on chromosome (Chr.) 5 and further delimited to a 125-kb genomic region between markers M4 and M5 containing 14 open reading fragments (ORFs). Numbers below the markers indicate the number of recombinants. B, The first intron sequences of XLX and NPB. Black boxes indicate exons, black lines indicate introns, white box with arrow indicates promoter, and white box without arrow indicate 3′-UTR. Letters with yellow background indicate exon sequences, while those with red background indicate a stop code in the new transcript. Red letter with underline indicates the divergent sequence and black letter with underline indicates the splice site. C, The divergence (A to G) in the first intron produced a new splice site in XLX. Red star indicates the SNP, and red arrow indicates a stop code in the new transcript. D, Relative expression levels of functional RGA1/D1 transcripts. The two-week-old seedlings of XLX, NPB and TN1 were collected for qRT-PCR, and the OsACT gene was used as a control. Mean and SD values were obtained from three biological replicates. **, P < 0.01 (the Student’s t-test). XLX, Xiaolixiang; NPB, Nipponbare; TN1, Taichung Native 1; SNP, Single nucleotide polymorphism.

Fig. 3. Phenotypes of complementation transgenic lines of Xiaolixiang (XLXD1). A, Morphology at the maturity stage. Scale bar, 10 cm. B, Morphology at the seedling stage. Scale bar, 5 cm. C, Panicle type. Scale bar, 5 cm. D and E, Grain shape. Scale bars, 5 mm. F, Plant height. G, Panicle length. H, Grain length. I, Grain width. J, Ratio of grain length to width. Mean and SD values were obtained from three biological replicates. **, P < 0.01 (the Student’s t-test).

Fig. 4. Lodging resistance of Xiaolixiang (XLX) and its transgenic XLXD1 plants. A, Morphologies of culm and panicles. Scale bar, 10 cm. B, Internode length. C, Culm strengths of internodes at the early (E)- and late (L)-filling stages. 1st to 5th indicate the first internode to the last internode. Mean and SD values were obtained from three biological replicates. **, P < 0.01 (the Student’s t-test).

Fig. 5. Photosynthesis of Xiaolixiang (XLX) and its transgenic XLXD1 plants. A, Morphologies of flag leaf (FL) and secondary leaf (SL) at the filling stage. Scale bar, 10 cm. B, Leaf lengths of FL and SL at the filling stage. C, Leaf widths of FL and SL at the filling stage. D, Pigment content of FL at the filling stage. Chl a, Chlorophyll a; Chl b, Chlorophyll b; Car, Carotenoid. E and F, Photosynthetic rate (E) and stomatal conductance (F) of FL at the heading and filling stages. G, Optimal/maximal quantum yield of PS II (Fv/Fm) of FL at the filling stage. H and I, Relative expression levels of Lhcb1 and Lhcb4 in FL at the filling stage. Mean and SD values were obtained from three biological replicates.*, P < 0.05; **, P < 0.01 (the Student’s t-test).

Fig. 6. Delayed leaf senescence in Xiaolixiang (XLX). A, Diaminobenzidine (DAB) staining and nitroblue tetrazolium (NBT) staining of leaves at the heading and filling stages. B, Changes in transcript levels of senescence-associated genes and ROS-scavenge enzyme genes in the leaves of XLX and XLXD1 plants at the filling stage. The flag leaves of XLX and XLXD1 plants at the filling stage were used for qRT-PCR. Two senescence-induced genes, SGR (LOC_Os09g36200) and Osh36 (LOC_Os05g39770); catalase gene CatB (LOC_Os06g51150); two peroxidase genes, POD1 (LOC_ Os01g22370) and POD2 (LOC_Os03g22010); two ascorbate peroxidase genes, APX1 (LOC_Os03g17690) and APX2 (LOC_Os07g49400) were detected, and the OsACT gene was used as a control. Mean and SD values were obtained from three biological replicates. The red line indicates the transcript level of these genes in XLX.

Table 2. Agronomic characteristics of Xiaolixiang (XLX) and its transgenic XLXD1 plants.

Rice material	Effective panicle number per plant	Primary branch number per plant	Secondary branch number per plant	Grain number per spikelet	Seed-setting rate (%)	1000-grain weight (g)	Theoretical yield per plant (g)
XLX	15.8 ± 0.7	14.0 ± 0.5	53.6 ± 1.6	298.2 ± 19.5	90.6 ± 1.2	16.3 ± 0.1	70.1 ± 5.9
XLX^D1-1	11.0 ± 0.9**	9.9 ± 0.6**	28.4 ± 1.3**	188.6 ± 6.1**	85.9 ± 3.9	21.2 ± 0.2**	37.7 ± 4.5**
XLX^D1-2	11.0 ± 0.7**	10.7 ± 0.2*	34.7 ± 2.3*	176.2 ± 8.6*	87.4 ± 1.6	21.8 ± 0.1**	36.9 ± 1.7**
XLX^D1-3	10.7 ± 1.1**	9.9 ± 0.4**	25.9 ± 1.3*	199.3 ± 13.0*	86.1 ± 1.5	21.0 ± 0.2**	38.7 ± 2.2**

Fig. 7. Grain quality of Xiaolixiang (XLX) and its transgenic XLXD1 plants. A, Head rice of XLX and its transgenic XLXD1 lines (XLXD1-1, -2 and -3). Scale bars, 1 cm. B, Chalk sizes of XLX and XLXD1. Scale bar, 1 mm. C, Chalky rice rates of XLX and XLXD1. D, Chalkiness degrees of XLX and XLXD1. E, Processing qualities of XLX and XLXD1, including brown rice rate (BRR), milled rice rate (MRR) and head rice rate (HRR). F and G, Protein contents (F) and nitrogen contents (G) of XLX and XLXD1 head rice. Mean and SD values were obtained from three biological replicates. **, P < 0.01 (the Student’s t-test).

Fig. 8. Xiaolixiang (XLX) exhibits stronger drought and sheath blight resistances. A and C, Representative images of XLX, XLXD1-2 and XLXD1-3. Scale bars, 20 cm. B and D, Thermal images of XLX, XLXD1-2 and XLXD1-3. Scale bars, 20 cm. E, Leaf temperatures of XLX, XLXD1-2 and XLXD1-3 in paddy field. F-H, Seedlings of XLX, XLXD1-2 and XLXD1-3 before 20% polyethylene glycol (PEG) treatment (F), PEG treatment for 13 d (G) and survival rate after PEG treatment (H). Scale bars, 5 cm. I, Seedlings infected with Rhizoctonia solani. Scale bar, 5 cm. J, Lesion positions on seedlings after inoculation with R. solani. Scale bar, 2 cm. K, Adult leaves in vitro inoculated with R. solani. The leaves were collected at the tillering stage. Scale bar, 1 cm. Mean and SD values were obtained from three biological replicates. **, P < 0.01 (the Student’s t-test).

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