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

doi:10.11983/CBB19053

Abstract

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

Chun Zhou,Ran Jiao,Ping Hu,Han Lin,Juan Hu,Na Xu,Xianmei Wu,Yuchun Rao,Yuexing Wang. Gene Mapping and Candidate Gene Analysis of Rice Early Senescence Mutant LS-es1[J]. Chinese Bulletin of Botany, 2019, 54(5): 606-619.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB19053

https://www.chinbullbotany.com/EN/Y2019/V54/I5/606

Figures/Tables 13

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	χ^{2 (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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