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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (6): 1389-1400.doi: 10.3724/SP.J.1006.2022.12035

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

Phenotypic characterization and gene mapping of an early senescence leaf H5(esl-H5) mutant in rice (Oryza sativa L.)

ZHENG Chong-Ke1(), ZHOU Guan-Hua1, NIU Shu-Lin1,2, HE Ya-Nan1, SUN wei1, XIE Xian-Zhi1,*()   

  1. 1Shandong Rice Research Institute / Shandong Academy of Agricultural Sciences, Jinan 250100 Shandong, China
    2College of Life Sciences, Shandong Normal University, Jinan 250014, Shandong, China
  • Received:2021-05-27 Accepted:2021-10-19 Online:2022-06-12 Published:2021-12-05
  • Contact: XIE Xian-Zhi E-mail:zhengck1983@163.com;xzhxie2010@163.com
  • Supported by:
    Fund:The study was supported by the Strategic Priority Research Program of Chinese Academy of Sciences(XDA24030101-6)

Abstract:

A stable mutant esl-H5 (early senescence leaf H5) was identified from the mutant library of japonica rice Huaidao 5 population induced by ethyl methane sulfonate (EMS) treatment. The mutant was normal at seedling stage. However, the lower leaves in esl-H5 mutant displayed premature senescence at about 50 days after sowing. Compared with the wild type (WT), the heading date of the esl-H5 mutant was delayed, while agronomical traits including plant height, panicle length, grain number per panicle, effective tiller numbers, and 1000-grain weight were significantly reduced. Moreover, chlorophyll content was also decreased in esl-H5 mutant. Genetic analysis indicated that the early senescence trait in esl-H5 mutant was controlled by a single recessive gene. ESL-H5 gene was localized on chromosome 1 using molecular marker. MutMap analysis further revealed that one nucleotide G to A replace occurred in the last exon of Os01g0533000 gene which encodes callose synthase. The G to A replace in the ESL-H5 introduced a premature stop codon. Phylogenetic analysis showed that ESL-H5 was homology with Arabidopsis AtGSL7 (Glucan Synthase-Like 7). At tillering stage, the contents of soluble sugar and starch were significantly increased in the leaves of the esl-H5 mutant compared with those of the WT. These results implied that the mutation of ESL-H5 affected the transport of photosynthetic products, resulting in premature leaf senescence phenotypes. The qRT-PCR analysis revealed that the expression levels of disease resistance-related genes PR1a, PR1b, PR2, PR4, PR5, and PR10 in esl-H5 mutant were higher than those in WT, which was consistent with the observation that esl-H5 mutant improved bacterial blight resistance. The present results lay the foundation for studying the roles of sugar signal in regulating rice senescence and disease resistance.

Key words: rice (Oryza sativa L.), early senescence, gene mapping, callose synthase

Table 1

Primers for qRT-PCR"

引物名称
Primer name
正向引物序列
Forward sequence (5'-3')
反向引物序列
Reverse sequence (5'-3')
OseEF-1α TTTCACTCTTGGTGTGAAGCAGAT GACTTCCTTCACGATTTCATCGTAA
OsESL-H5 GATTGGGCGACAGAAGTTTG CAGCCATCACAGAGACAAAAC
qPR1a TTCATCACCTGCAACTACTCG TGCATAAACACGTAGCATAGCAT
qPR1b GTGTGCGGGCACTACACG CGGCTTATAGTTGCATGTGA
qPR2 ACAACTCGGAGAAGCATCAG GAAACATAATCCTCGCCAAAGC
qPR4 TGGGACCTGAACAAAGTGAG GGATACACTTGCCACACGAG
qPR5 ATCGACGGCTACAACGTC GTGTCTTGGTGTTGTCTTCG
qPR10 CACCATCTACACCATGAAGC AGCACATCCGACTTTAGGAC
qOsWRKY23 TCCAGTTCCTCTCCCAGTTCTAA CACATTGTTCTCCTTTTCTTCCC
qOsWRKY72 CACCACAAATCACATCTACTCCG GCTGAAGGGAAGAGAGGTGAG
qOsSGR AGGGGTGGTACAACAAGCTG ATCCTGGATTTGGCAAGAAC
qOsNAP CAAGAAGCCGAACGGTTC GCTCCTTGCGGAAGATGTAG
qOsh36 GAACTACAGGAAGGGTCGGT GTTAGAGTGGAGCAGCAT
qOsl2 GCAGACAACAAATCGCCAAAT AGTATCCTGGTGCCAGTTCC
qOsl30 AACCTTTTTCTTGGAGATGATACGA TCTCCAGCAACTCTAACCAGCAT
qOsl85 TGGGCAAAGGAGTTACTGAA CTTGAACTGTAGGGGCTTGCTT
qOsl43 TGTGACAAGTGCTAATAATACATACGA ATCCTGGATTTGGCAAGAAC
qrbcS TCCGCTGAGTTTTGGCTATTT GGACTTGAGCCCTGGAAGG
qlhcA GTCTGTGGTTTGACCCGCT GAGCCGCCCGTTCTTGA
qlhcB GCTCAAGGTGAAGGAGATCAAGA ATGCGTTGTTGTTGACGGG
qrbcL CTTGGCAGCATTCCGAGTAA ACAACGGGCTCGATGTGATA
qPsaA GCGAGCAAATAAAACACCTTTC GTACCAGCTTAACGTGGGGAG
qPsbA CCCTCATTAGCAGATTCGTTTT ATGATTGTATTCCAGGCAGAGC
qpetD TAATGGTTTCTGTGCCGACG CCTAAAGTTAAGGATTTTTCAATGGG
qndhA CAAAGCGATATCCCAAAGGAAT CCGCATCGATACAACAACGTAT
qatpA GATCTCTCCAAACAGGCACAAG CGGCTCTTTCTAAAAGGCGTG

Fig. 1

Phenotypes of esl-H5 mutant A: phenotypes after being sowed for 10 d, bar: 1 cm; B: plant phenotypes at tillering stage, bar: 5 cm; C: plant phenotypes at filling stage, bar: 5 cm; D: phenotypes of upper three leaves at heading stage, bar: 5 cm; E: spike phenotypes at mature stage, bar: 1 cm; F and G: phenotypes of grain length and grain width, bar: 1 cm."

Table 2

Comparison of agronomic trains between esl-H5 mutant and wild type"

农艺性状
Agronomic trait
esl-H5 野生型
Wild type
抽穗期 Heading date 124.20 ± 0.28** 120.80 ± 0.23
株高 Plant height (cm) 60.80 ± 0.42** 87.95 ± 0.62
分蘖数 Tiller number 5.75 ± 0.32** 11.65 ± 0.42
穗长Main panicle length (cm) 15.04 ± 0.23** 15.23 ± 0.21
每穗实粒数 Grain number per panicle 64.50 ± 2.65** 158.60 ± 4.80
一级枝梗数No. of primary branch 12.28 ± 0.25** 13.00 ± 0.37
二级枝梗数No. of secondary branch 20.08 ± 0.86** 27.00 ± 1.23
粒长 Grain length (mm) 7.82 ± 0.25 7.84 ± 0.21
粒宽Grain width (mm) 3.76 ± 0.17** 3.88 ± 0.11
千粒重 1000-grain weight (g) 20.90 ± 0.01** 27.20 ± 0.01

Fig. 2

Relative expression levels of senescence and photosynthesis related genes in WT and esl-H5 mutant A: relative expression levels of senescence related genes in WT and esl-H5 mutant; B: relative expression levels of photosynthesis related genes in WT and esl-H5 mutant."

Fig. 3

Chlorophyll content in esl -H5 mutant and wild type at seedling and tillering stages A: the chlorophyll content of the newly developed leaves after being sowed for 40 days; B: the chlorophyll content of the second leaves from the uppermost after being sowed for 40 days; C: the chlorophyll content of the newly developed leaves after being sowed for 60 days; D: the chlorophyll content of the second leaves from the uppermost after being sowed for 60 days; **: P < 0.01; *: P < 0.05."

Table 3

Separation ratio of three F2 segregation population"

杂交组合
Cross combinations
F1表型 F1 phenotype F2表型 F2 phenotype
野生型
Wild type
突变型
Mutant
野生型
Wild type
突变型
Mutant
χ2 (3:1)测验
χ2 (3:1) text
WT/esl-H5 15 0 109 35 0.01
NPB/esl-H5 23 0 672 217 0.14
9311/esl-H5 19 0 105 31 0.24

Table 4

Information of markers displayed polymorphism between the pools"

标记名称
Marker name
引物序列
Primer sequence (5'-3')
使用的酶
Enzymes used
ID1-9F GTACGCAAGCGATCAAAC
ID1-9R AGTCCCGTCTAAAATTCACA
ID1-24F ATACTGGGTGTGCTCATTCT
ID1-24R TTTGTTTATTCTTGTCCGTTT
dSNP1F TGATGAAGGTAAGATTAGAGGATGAAGGAATTC EcoR І
dSNP1F TCAGTTAGAAATTACTCCCT
dSNP2F AGGGATCCGAATAGGAGTCCTACGGGCCTTGGGTACC Kpn І
dSNP2R GCACCGCAAACCAGCGTTGCCT
dSNP3F CCTGTTGTTTGTTGTTTCTTCCATCTAGA Xba І
dSNP3R GCACCAACCAGTAGGCATGA

Fig. 4

Mapping and cloning of ESL-H5 genes A: fine mapping of ESL-H5 (the marker above the horizontal line represents the InDel or SNP marker used in fine mapping, and the number below represents the number of recombinants); B: changes of G to A in the genome of Os01g0533000; C: SNP3 is converted to dCAPS marker; D: plants with early senescence phenotype was detected using dCAPS markers. E: relative expression level of Os01g0533000 in WT and esl-H5."

Fig. 5

Protein structure prediction and phylogenetic tree analysis of ESL-H5 A: protein structure prediction of wild-type and esl-H5 mutant, VTA1, FKS1, and Glucan synthase refer to protein domains; B: ESL-H5 transmembrane region prediction; C: phylogenetic tree analysis of callose synthase in Arabidopsis and rice; D: alignment of amino acid sequences of ESL-H5 and AtGSL7."

Fig. 6

Relative expression patterns of ESL-H5 genes"

Fig. 7

Starch and soluble sugar contents in wild type and mutant leaves at tillering stage A: soluble sugar contents at 6:00; B: soluble sugar contents at 18:00; C: starch contents at 6:00; D: starch contents at 18:00. *: P < 0.05; **: P < 0.01."

Fig. 8

Identification of resistance to bacterial blight of wild type and mutant at tillering stage A: phenotype of bacterial blight resistance; B: statistics of lesion length; C: relative expression levels of pathogenesis-related protein. **: P < 0.01."

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