Chin Bull Bot ›› 2019, Vol. 54 ›› Issue (5): 606-619.doi: 10.11983/CBB19053

• EXPERIMENTAL COMMUNICATIONS • Previous Articles     Next Articles

Gene Mapping and Candidate Gene Analysis of Rice Early Senescence Mutant LS-es1

Zhou Chun1,Jiao Ran1,Hu Ping2,Lin Han1,Hu Juan1,Xu Na1,Wu Xianmei2,Rao Yuchun1,*(),Wang Yuexing2,*()   

  1. 1. College of Chemistry and Life Science, Zhejiang Normal University, Jinhua 321004, China
    2. State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, China
  • Received:2019-03-20 Accepted:2019-06-20 Online:2020-03-10 Published:2019-09-01
  • Contact: Rao Yuchun,Wang Yuexing E-mail:ryc@zjnu.cn;wangyuexing@caas.cn

Abstract:

Senescence is an autonomous and irreversible adaptive response at the end of plant development. The molecular mechanism related to premature senescence of leaves is important for rice genetic improvement and breeding of anti-aging varieties. LS-es1 is a stable hereditary premature early senescence mutant obtained by EMS mutagenesis of indica variety TP309. Phenotypic observation, physiological and biochemical analysis of LS-es1 and its wild type TP309 found that LS-es1 accumulated a large amount of reactive oxygen species and more cell death, while the yield-related agronomic traits of LS-es1 were significantly decreased compared to wild type TP309, which also verified the early senescence characteristics of LS-es1. Exogenous hormone treatment of LS-es1 and TP309 seedlings showed that LS-es1 was more sensitive to salicylic acid (SA), abscisic acid (ABA) and methyl jasmonate (MeJA). The LS-es1 gene was mapped to the 46.2 kb region of the long arm of rice chromosome 7 by map-based cloning, which included 8 open reading frames (ORFs). Bioinformatics analysis of the genes in this interval revealed that two candidate functional genes, Os07g0275300 and Os07g0276000, were associated with the early senescence pathway, and the expression levels of these two genes were significantly different between wild type and mutant. The results laid the foundation for further cloning of the LS-es1 gene and in-depth study of its biological function.

Key words: rice, early senescence, physiological characteristics, fine mapping, candidate genes

Table 1

Primers used for gene mapping and qRT-PCR"

Primer name Forward primer (5'-3') Reverse primer (5'-3')
InDel-1 AGCGGGGATGGAGATGATG CTTGCCTCACACCAGATCTG
InDel-2 GGCGCCTTTGTTCCATAGTT GAGGAGCCAGTGGTAGCAG
InDel-3 CGTTTTTACAACCAATTTTGGAA CCATCTTCTACCTCCGGACA
InDel-4 GATTGGATTGGTTGCTCGCT AACAGCGAATCGAGATGCAC
InDel-5 TTACTGCTGCCGTTGTTTCA TTGTGGACCTCCAGGATCAG
SGR AGGGGTGGTACAACAAGCTG GCTCCTTGCGGAAGATGTAG
Osh36 GCACGGAGGCGAACGA TTGAGCGGTAGCACCCATT
Osl85 GAGCAACGGCGTGGAGA GCGGCGGTAGAGGAGATG
OsNAP CAAGAAGCCGAACGGTTC GTTAGAGTGGAGCAGCAT
Actin CAGGCCGTCCTCTCTCTGTA AAGGATAGCATGGGGGAGAG

Figure 1

Phenotypes of rice wild type (WT) TP309 and mu- tant LS-es1 (A) Phenotypes at seedling stage; (B) Phenotypes at tillering stage; (C) Phenotypes at maturity stage. Bars=6 cm"

Table 2

The comparison of agronomic traits between rice wild type TP309 and mutant LS-es1"

Agronomic traits TP309 LS-es1
Effective number of panicle 13.40±5.41 8.20±0.84
Flag leaf length (cm) 39.46±8.16 35.68±4.08
Secondary branch number 29.80±6.87 18.00±4.47*
Tiller number 16.40±5.46 11.40±2.70
Filled grain number per panicle 169.60±8.08 125.60±24.83**
Seed-setting rate (%) 81.01±6.98 64.76±17.23

Figure 2

The comparison of agronomic traits between rice wild type (WT) TP309 and mutant LS-es1 (A) Panicle length; (B) Plant height; (C) Primary branch number; (D) 1000-grain weight. * and ** indicate significant differences between TP309 and LS-es1 at 0.05 and 0.01 level, respectively."

Figure 3

Histochemical analysis of rice wild type (WT) TP309 and mutant LS-es1 leaves DAB staining of wild-type and LS-es1 leaves (Bar=2 cm);(B) NBT staining of wild-type and LS-es1 leaves (Bar=2 cm); (C), (E) Tunel detection of wild-type leaves (Bars=100 μm); (D), (F) Tunel detection of LS-es1 leaves (Bars=100 μm)."

Figure 4

Catalase (CAT) (A), peroxisome (POD) (B), and superoxide dismutase (SOD) (C) activities and malondialdehyde (MDA) (D) and H2O2 (E) contents of LS-es1 and wild type (WT) at heading stage of rice * and ** indicate significant differences between TP309 and LS-es1 at 0.05 and 0.01 level, respectively."

Table 3

The comparison of SPAD value and photosynthetic rate between rice TP309 and LS-es1"

Net photosynthetic rate Stomatal conductance Intercellular CO2 concentration Transpiration rate SPAD
TP309 9.4±0.961 0.110±0.01 263±3 4.63±0.289 40.633±1.206
LS-es1 2.225±1.407** 0.0403±0.007** 312.5±10.606** 2.3±0.283** 27.65±2.333**

Figure 5

Transmission electron microscopy (TEM) analysis of chloroplast in rice wild type (WT) TP309 and mutant LS-es1 (A) TP309 leaf cells in 6000X; (B) TP309 leaf cells in 25000X; (C) TP309 leaf cells in 40000X; (D) LS-es1 leaf cells in 6000X; (E) LS-es1 leaf cells in 25000X; (F) LS-es1 leaf cells in 40000X. N: Cell nucleus; C: Chloroplast; Thy: Thylakoid; S: Starch granule; Og: Eosinophil. Bars=1 μm"

Figure 6

Scanning electron microscopy (SEM) analysis of seeds in rice wild type TP309 and mutant LS-es1 (A), (B) TP309 seed; (C), (D) LS-es1 seed. (A), (C) Bars=1 mm; (B), (D) Bars=100 μm"

Figure 7

Inhibition of exogenous hormone treatment on the growth of seedlings in rice wild type (WT) TP309 and mutant LS-es1 (A) Salicylic acid (SA) (top), abscisic acid (ABA) (middle), methyl jasmonate (MeJA) (bottom) (Bars=2 cm); (B) Comparison of shoot length of TP309 and LS-es1 seedlings treated with hormones; (C) Comparison of root length of TP309 and LS-es1 seedlings treated with hormones. ** indicate significant differences at 0.01 level between TP309 and LS-es1."

Table 4

Genetic analysis of rice early senescence phenotypes of LS-es1"

Hybrid combination (male/female) F1 pheno-
type
Namber of F2 normal phenotype Namber of F2 mutant phenotype Number of F2 population χ2 (3:1)
LS-es1/TN1 Normal 1381 471 1852 0.1842
LS-es1/ZF802 Normal 526 178 704 0.0303

Figure 8

Fine mapping of rice mutant LS-es1 genes"

Figure 9

Expression of LS-es1 candidate genes and senescence-related genes in rice ** indicate significant differences at 0.01 level."

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