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Acta Agronomica Sinica ›› 2018, Vol. 44 ›› Issue (03): 332-342.doi: 10.3724/SP.J.1006.2018.00332

• CROP GENETICS & BREEDING · GERMPLASM RESOURCES · MOLECULAR GENETICS • Previous Articles     Next Articles

Identification and Gene Mapping of a Lesion Mimic Mutant spl34 in Rice (Oryza sativa L.)

Bao-Yu LIU(), Jun-Hua LIU, Dan DU, Meng YAN, Li-Yuan ZHENG, Xue WU, Xian-Chun SANG, Chang-Wei ZHANG*()   

  1. Rice Research Institute of Southwest University / Chongqing Key Laboratory of Application and Safety Control of Genetically Modified Crop, Chongqing 400716, China
  • Received:2017-05-22 Accepted:2017-11-21 Online:2018-03-12 Published:2017-12-04
  • Contact: Chang-Wei ZHANG E-mail:673325435@qq.com;603519375@qq.com
  • Supported by:
    This study was supported by the National Major Project for Developing New GM Crops (2016ZX08001002-002).

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Abstract:

A mutant spotted leaf 34 (spl34) was screened from the progeny of indica restorer line Jinhui 10 treated with ethyl methane sulfonate (EMS). Brown lesions in spl34 exhibit on the sheath of lower leaves at the late tillering stage, then spred from the midrib to entire leaf and finally throughout the whole plant at maturity stage. Compared with the wild type, the plant height, ear length, grain number per panicle, seed setting rate and thousand-grain weight as well as the activities of protective enzymes (CAT, POD, and T-SOD) were all significantly decreased while the content of reactive oxygen species (ROS) increased in spl34. The shading assay showed that the formation of lesions in spl34 was induced by light. Histochemical analysis showed that spl34 had excessive hydrogen peroxide (H2O2) deposition and programmed cell death in the position of lesions. In addition, the chlorophyll fluorescence was weaker in spl34 than in the wild type under the fluorescence microscopy. There was no significant difference in blast resistance between spl34 and the wild type. Genetic analysis suggested that the phenotype of spl34 was controlled by a single recessive nuclear gene, which was mapped between InDel markers LR49 and LR52 on chromosome 4 with an interval of 200 kb. Sequencing analysis revealed that a single base substitution (G to T) occurred at 3449 bp in the DNA sequence of LOC_Os04g56480, resulting in an amino acid change from tryptophane to cysteine. The qRT-PCR results showed that the transcriptional level of LOC_Os04g56480 was down-regulated in spl34, while that of some pathogenesis-related genes was highly up-regulated when compared with the wild type.

Key words: rice, lesion mimic mutant, spl34, gene, fine mapping

Fig. 1

Phenotype of the wild type (WT) and the spl34 mutant at tillering stage and mature period A: plants of the wild type (WT) and the spl34 mutant at tillering stage; B: plants of the wild type (WT) and the spl34 mutant at mature period; C: leaves of the wild type (WT) and the spl34 mutant at mature period."

Table 1

Agronomic traits of the wild type (WT) and spl34 mutant"

材料
Material		 株高
Plant height
(cm)		 有效穗数
Effective panicle		 穗长
Panicle length (cm)		 每穗粒数
Grain number per panicle		 结实率
Seed-setting rate (%)		 千粒重
1000-grain weight (g)
WT 107.12±3.35 8.60±1.20 25.26±1.40 180.90±12.48 78.41±4.45 25.57±0.81
Spl34 100.32±3.59* 8.80±0.82 22.34±1.22* 135.42±8.66** 53.65±3.96** 22.68±0.30**

Fig. 2

Effects of shading on the wild type and the spl34 mutant leaves A: leaf of the wild type after shading; B: leaf of wild type regained normal light for one week after shading; C: leaf of spl34 after shading; D: leaf of spl34 regained normal light for one week after shading."

Fig. 3

Photosynthetic pigments contents of the wild type and the spl34 mutant at heading stage A-C: photosynthetic pigments contents of the flag leaves, second leaves, third leaves respectively in the wild type and the spl34 mutant at heading stage. * Significantly different at P<0.05; ** significantly different at P<0.01."

Fig. 4

Autofluorescence of the wild type and the spl34 mutant A, B: microstructure in cross section of wild type under natural light and UV light; C, D: microstructure in cross section of spl34 mutant under natural light and UV light; Bar=100 μm. The positions pointed by white arrows are the lesion formation sites, those with yellow arrows are the sites without lesion mimic."

Fig. 5

Histochemical analysis of the wild type and the spl34 mutant A, B: leaves of the wild type (WT) and the spl34 mutant stained by trypan blue; C, D: leaves of the wild type (WT) and the spl34 mutant stained by DAB."

Fig. 6

Physiological indices of the wild type (WT) and the spl34 mutant at heading stage * Significantly different at P<0.05; ** significantly different at P<0.01."

Table 2

Resistance spectrum of the wild type (WT) and the spl34 mutant to rice blast (%)"

材料
Material		 对稻瘟病各生理群的抗病频率
Resistance frequency to each physiological group		 总群抗病频率
Resistance frequency to total
population
ZA ZB ZC ZD ZE ZG
WT 39.39±5.25 74.75±1.76 0 100 100 100 70.37±1.85
spl34 45.45±5.45 72.04±4.10 0 100 100 100 70.37±3.20

Table 3

Disease indices of the wild type and the spl34 mutant to rice blast (%)"

材料
Material		 苗瘟病情指数
Disease indexes of leaf blast
at seedling stage		 叶瘟病情指数
Disease indexes of leaf blast
at tillering stage		 穗颈瘟发病率
Disease incidence of neck blast
WT 32.00±2.23 71.43±3.31 3.94±0.61
spl34 36.56±2.01 77.14±2.22 4.19±0.52

Table 4

Newly designed InDel markers"

标记
Marker		 正向引物
Forward primer (5°-3°)		 反向引物
Reverse primer (5°-3°)
LR7 TTCCTACACGGTCGAAGATCAT TCAAGTACCGGATTGAGTTTGAG
LR14 GAGATTATCCTGCGGTCTTAATC TTGATGATACGATGACCAAGTTC
LR25 ATATTCACATGCGCCGTTTG CTCCAACAGATCCATCTCAACC
LR31 GATCCAATGCGATGCTACTCC GCGATTAACTGGAGACATCACG
LR33 TTCAATGTGCTGCATTTAGAAAT AAAGATGGTTCAAGTCTTGAGGA
LR49 GCCAAACAGATTGGTTAGCG TTAGCGTAGCTGGCATAACATC
LR52 TTGATCTCGACTTCGATCACTAG GATGTCTGGACGTACACACACG

Fig. 7

Gene mapping of the spl34 mutant"

Fig. 8

Relative expression analysis of the target gene spl34"

Fig. 9

Relative expression analysis of the pathogenesis-related genes and the rice blast resistance genes The pathogenesis-related genes: PAL, PR1a, PR1b, POX22.3, PR5, NPR1, POC1; the rice blast resistance genes: Pi-d3, Pish, Pi9, Pit, Pi5, Pia."

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