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Acta Agronomica Sinica ›› 2021, Vol. 47 ›› Issue (1): 50-60.doi: 10.3724/SP.J.1006.2021.92069

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

Physiological characters and gene mapping of a dwarf and wide-leaf mutant osdwl1 in rice (Oryza sativa L.)

HUANG Yan1(), HE Huan-Huan1, XIE Zhi-Yao1, LI Dan-Ying1, ZHAO Chao-Yue1, WU Xin1, HUANG Fu-Deng2, CHENG Fang-Min1, PAN Gang1,*()   

  1. 1College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, Zhejiang, China
    2Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, Zhejiang, China
  • Received:2019-12-22 Accepted:2020-09-13 Online:2021-01-12 Published:2020-09-25
  • Contact: PAN Gang E-mail:869163852@qq.com;pangang12@126.com
  • Supported by:
    National Natural Science Foundation of China(31771688);National Natural Science Foundation of China(31971819);National Major Project for Developing New GM Crops(2016ZX08001-002)

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Abstract:

Plant height is one of the important factors affecting rice lodging. The semi-dwarf rice varieties possess high level of lodging resistance, and could reduce yield loss and improve grain quality. Thus, it is very important to study the molecular and physiological mechanism of dwarf formation in rice. In this study, a stable hereditary dwarf and wider-leaf mutant osdwl1 was obtained from 60Co γ-radiated indica restore line Zixuan 1, and its morphological and physiological characteristics, cytological observation, genetic analysis and gene mapping were investigated. Under field condition, the mutant osdwl1 exhibited dwarf and wider-leaf after the tillering stage due to shorter length of the parenchyma cells, and its panicle length and all internodes length were significantly shorter compared with wild type plants at mature stage. Paraffin sections and scanning electronic microscopy (SEM) observation revealed that the number of small vascular (SV) bundles and the distance between SVs increased significantly, resulting in wider-leaf blade in osdwl1. Moreover, the number of microhairs on the abaxial and adaxial epidermis were also increased significantly in osdwl1. In addition, starting at the 3-4 leaf seedling stage, yellowing was visible at the upper middle parts of old leaves in osdwl1. Physiological analysis and transmission electron microscopy (TEM) observation indicated that the lamellar structure of chloroplast was distorted and began to collapse in some mesophyll cells, which led to the reduction of total chlorophyll contents, net photosynthetic rate and Fv/Fm ratio of the second and third leaves from top in osdwl1 at the heading stage. Relative to the wild type plants, the soluble protein content, catalase (CAT) and superoxide dismutase (SOD) activities were significantly decreased, which in turn resulting in the accumulation of H2O2 and O2-, and a steady increase of malondialdehyde (MDA) contents in the mutant leaves. Genetic analysis and gene mapping showed that osdwl1 was controlled by a single recessive nuclear gene, located in a region of 333 kb between SSR marker RM19297 and the InDel marker ID269-2 on the short arm of chromosome 6. The results would further facilitate the cloning and functional analysis of OsDWL1 gene.

Key words: rice, osdwl1, dwarf and wider-leaf, physiological analysis, gene mapping

Fig. 1

Phenotypes of osdwl1 and its wild-type (WT) plants at the different growth stages A: seedling stage; B: early-flowering stage; C: leaves at the early-flowering stage, and F means flag leaf and 2-4 means 2nd to 4th leaf from top in order, respectively; D: mature stage; E: panicle and the internodes at the mature stage, P means panicle, 1-4 means the 1st to 5th internode from top, respectively; F: the length of different internodes at the mature stage in 2019; G-H: longitudinal sections of the 2nd internode from top in wild-type plants (G) and osdwl1 (H); Bar = 20 cm in A-F; Bar = 20 μm in G-H. Values marked with * and ** indicate significant differences at P < 0.05 and P < 0.01 by Student’s t-test, respectively."

Table 1

Main agronomic traits of osdwl1 and its wild-type plants"

性状
Trait		 2018 2019
野生型
WT		 突变体
osdwl1		 野生型
WT		 突变体
osdwl1
株高 Plant height (cm) 110.74±2.16 65.90±3.62** 82.91±4.96 58.40±2.38**
穗长 Panicle length (cm) 24.35±0.31 14.87±1.26** 23.07±0.82 15.67±1.25**
有效穗数 Effective panicle number 6.20±1.64 3.60±1.55** 11.80±3.56 5.20±2.49**
每穗粒数 Grain number per panicle 165.96±7.64 97.56±12.67** 177.72±10.51 117.74±21.21**
结实率 Seed-setting rate (%) 90.92±3.19 55.07±1.97** 79.57±3.01 43.57±8.01**
千粒重 1000-grain weight (g) 23.17±1.04 26.91±1.31** 22.42±0.67 26.06±1.06**
单株产量 Yield per plant (g) 19.44±1.34 3.27±0.91** 20.89±2.13 4.76±1.00**

Fig. 2

Phenotypic characteristics of leaf blade in osdwl1 and its wild-type plants A: leaf blade width of the osdwl1 and its WT plants; B: transverse cross-sections of the flag leaf blade of the osdwl1 and its WT plants, blue triangles indicate small vascular bundles (SV), and LV means large vascular bundle; C: number of SVs in the whole flag leaves of osdwl1 and its WT plants; D: the distance between the two SVs in the osdwl1 and its WT plants; E: adaxial epidermal cell numbers between two SVs; F: adaxial epidermal cell width; G: SEM analysis of the abaxial and adaxial epidermis of the flag leaf blade in the osdwl1 and its WT plants, double arrow means the distance between two SVs; H, I: number of macrohairs and microhairs on the abaxial (H) and adaxial epidermis (I) of the flag leaf in the osdwl1 and its wild-type plants. Bar = 200 μm. Values marked with * and ** indicate significant differences by Student’s t-test at P < 0.05 and P < 0.01, respectively."

Fig. 3

Photosynthetic characteristics and chloroplast ultrastructure of leaves in the osdwl1 and wild-type plants at the booting stage A-D: Chl a (A), Chl b (B) and total Chl contents (C), and Chl a/b ratio (D) of leaves in osdwl1 and wild-type plants; E-H: ultrastructure of chloroplast in osdwl1 (G, H) and wild-type plants (E, F), nu means nucleus, Cl means chloroplast, og means osmiophilic granule; I, J: Net photosynthesis rate (I) and Fv/Fm ratio (J) of leaves in osdwl1 and wild-type plants. 1: flag leaves; 2: 2nd leaves from top; 3: 3rd leaves from top. Values marked with * and ** indicate significant differences by Student’s t-test at P < 0.05 and P < 0.01, respectively."

Fig. 4

Accumulation analysis of O2?and H2O2 contents, and CAT and SOD activities in the osdwl1 and its wild-type plants at booting stage 1: flag leaves; 2: 2nd leaves from top; 3: 3rd leaves from top. Values marked with * and ** indicate significant differences by Student’s t-test at P < 0.05 and P < 0.01, respectively."

Fig. 5

MDA and soluble protein contents of osdwl1 and the wild-type plants at booting stage 1: flag leaves; 2: 2nd leaves from top; 3: 3rd leaves from top. Values marked with * and ** indicate significant differences by Student’s t-test at P < 0.05 and P < 0.01, respectively."

Table S1

Molecular markers used for OsDWL1 gene mapping"

标记
Marker		 正向引物
Forward primer (5'-3')		 反向引物
Reverse primer (5'-3')
RM7399 CAGATATGATGTTCTTGCCCTTGC GCTTGCCAGATCACCTACCTACC
RM19288 CGGAGCTGTTGCCGTTCTGC CGATGTGCCATGTCAGGATGACC
RM19297 ATTTGCTCCGCTTCCAAATCACC AGCGGCCAACAGAGACAACTGG
ID269-2 AGGGTGTGTTTAGTTCACGA AAAATTTGTCATGGCTGTTG
RM3805 ACACCACCATCAACGTACCAACC AAGTCGAGAGGAAGAAGCCAAGG
RM19549 CCTGGTACTAACCATGTGATTGAGC AACGTCAGAGTCTCACCACAAGC

Fig. 6

Molecular mapping of OsDWL1 gene on the short arm of chromosome 6"

Table 2

Gene names and their functional annotations in the target interval"

基因号
Locus identifier		 功能注释
Functional annotation
LOC_Os06g03390 Expressed protein
LOC_Os06g03486.1 Expressed protein
LOC_Os06g03514.1 Expressed protein
LOC_Os06g03520.1 DUF581 domain containing protein, expressed
LOC_Os06g03530.1 Pentatricopeptide, putative, expressed
LOC_Os06g03540.1 Oligopeptide transporter, putative, expressed
LOC_Os06g03560.1 Oligopeptide transporter, putative, expressed
LOC_Os06g03570.1 Pentatricopeptide, putative, expressed
LOC_Os06g03580.1 Zinc RING finger protein, putative, expressed
LOC_Os06g03600.1 Transcriptional corepressor SEUSS, putative, expressed
LOC_Os06g03610.1 The CrRLK1L-1 subfamily has homology to the CrRLK1L homolog, expressed
LOC_Os06g03640.1 BAG domain containing protein, expressed
LOC_Os06g03660.1 Peroxisomal biogenesis factor 11, putative, expressed
LOC_Os06g03670.1 Dehydration-responsive element-binding protein, putative, expressed
LOC_Os06g03676.1 CAMK includes calcium/calmodulin dependent protein kinases, expressed
LOC_Os06g03682.1 Calcium-dependent protein kinase isoform AK1, putative, expressed
LOC_Os06g03690.1 RNA recognition motif containing protein, putative, expressed
LOC_Os06g03700.1 Oligopeptide transporter, putative, expressed
LOC_Os06g03710.1 DELLA protein SLR1, putative, expressed
LOC_Os06g03720.1 Ribonucleoside-diphosphate reductase small chain, putative, expressed
LOC_Os06g03750.1 Dehydration response related protein, putative, expressed
LOC_Os06g03760.1 LMBR1 integral membrane protein, putative, expressed
LOC_Os06g03770.1 ABC transporter, putative, expressed
LOC_Os06g03780.1 NUC153 domain containing protein, expressed
LOC_Os06g03790.1 39S ribosomal protein L47, mitochondrial precursor, putative, expressed
LOC_Os06g03800.1 Pollen ankyrin, putative, expressed
LOC_Os06g03810.1 Expressed protein
LOC_Os06g03820.1 Expressed protein
LOC_Os06g03830.1 Retinol dehydrogenase, putative, expressed
LOC_Os06g03840.1 Bric-a-Brac, Tramtrack, Broad Complex BTB domain with H family conserved sequence, expressed
LOC_Os06g03850.1 Impaired sucrose induction 1, putative, expressed
LOC_Os06g03860.4 Uncharacterized membrane protein, putative, expressed
LOC_Os06g03890.1 Alpha-L-fucosidase 3 precursor, putative, expressed
LOC_Os06g03910.1 Hydrolase, NUDIX family, domain containing protein, expressed
LOC_Os06g03920.1 Expressed protein
LOC_Os06g03930.1 Cytochrome P450 86A1, putative, expressed
LOC_Os06g03940.1 Spastin, putative, expressed
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