Rice Science, 2012, 19(1): 1−7
Copyright © 2012, China National Rice Research Institute
Published by Elsevier BV. All rights reserved
Identification and Genetic Mapping of a Lesion Mimic Mutant in
Rice
MA Jian-yang1, 2, CHEN Sun-lu2, 3, ZHANG Jian-hui1, 2, DONG Yan-jun1, TENG Sheng2
(1Laboratory of Plant Genetics and Functional Gene, College of Life and Environment Science, Shanghai Normal University,
Shanghai 200234, China; 2Institute of Plant Physiology & Ecology, Shanghai Institute for Biological Sciences, Chinese Academy of
Sciences, Shanghai 200032, China; 3National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural
University, Nanjing 210095, China)
Abstract: A lesion mimic stripe mutant, designated as lms1 (lesion mimic stripe 1), was obtained from the
M2 progeny of a 60Co γ-radiation treated japonica rice variety Jiahua 1. The lms1 mutant displayed
propagation type lesions across the whole growth and developmental stages. Physiology and
histochemistry analysis showed that the mutant exhibited a phenotype of white stripe when grown under
high temperature (30 °C), and the lesion mimic caused by programmed cell death under low temperature
(20 °C). The genetic analysis indicated that this lesion-mimic phenotype is controlled by a single locus
recessive nuclear gene. Furthermore, by using simple sequence repeat markers and an F 2 segregating
population derived from two crosses of lms1 × 93-11 and lms1 × Pei’ai 64S, the lms1 gene was mapped
between markers Indel1 and MM0112-4 with a physical distance of 400 kb on chromosome 6 in rice.
Key words: rice; lesion mimic mutant; gene mapping
Some plants can exhibit spontaneous spots on leaves in
the absence of environmental stress, agrochemical
damage, physical damage and pathogen attack. This
kind of spots is similar to lesions caused by the
infection of pathogens. We term this kind of mutants
as lesion mimic mutants (Wang, 2005). Some lesion
mimic traits are controlled by recessive genes, and
some by dominant genes (Wang Z H et al, 2004).
Lesion mimic mutants can be divided into two types
according to the phenotype: propagation type and
initiation type (Neuffer and Calver, 1975; Dangl et al,
1996; Shirasu and Schulze-Lefert, 2000). The
distribution and the size of the propagation-type lesion
mimics are comparatively constant, whereas the
initiation- type ones are diffused to other parts of
leaves and also to leaf sheaths and culms after formed.
The lesion mimics in plants were mainly resulted from
the programmed cell death led by hypersensitive
response and the abnormal burst of reactive oxygen
species in the apoptosis pathway (Ryerson and Heath,
1996; Dietrich et al, 1997; Lamb and Dixon, 1997). In
addition, plant hormones and overexpression of
exogenous genes took part in the lesion-mimic
formation (Beffa et al, 1995; Mittler et al, 1995; Asai
et al, 2000), and the environmental factors such as
light, temperature and humidity also had influence on
the formation of lesion mimics (Yamanouchi et al,
2002). To date, many lesion mimic mutant genes have
been identified and cloned, such as Lsd1, acd2, acd11,
Mod1, Cpn1 in A. thaliana; les22, lls1, RP1 in maize;
mlo in barley; and spl7, spl11, OSlsd1, spl28 in rice
(Collins et al, 1999; Jiang et al, 2003; Wang J J et al,
2004; Zeng et al, 2004; Wang et al, 2005; Qiao et al,
2010). Cloning of these genes will help to demonstrate
the molecular mechanism of the occurrence and formation
of the lesion mimics in plants, and to understand the
genes involved in plant cell development and apoptosis
and signaling pathways for stress tolerance of disease
resistance in plants (Wu et al, 2008; Matin et al,
2010).
A lesion mimic stripe mutant (lms1) was selected
from the progeny of a γ-radiation treated japonica rice
variety Jiahua 1. Morphological characterization, genetic
analysis and molecular mapping were conducted to
the mutant, which will benefit for future cloning,
functional analysis and application of this gene.
Received: 21 February 2011; Accepted: 12 May 2011
Corresponding authors: TENG Sheng (steng@sippe.ac.cn);
DONG Yan-jun (dong@shnu.edu.cn)
The mutant lms1 was isolated from the M2 progeny of
Jiahua 1, which was treated with 60Co γ-radiation. All
MATERIALS AND METHODS
Rice materials
2
agronomic traits of lms1 were stable and uniform after
self-pollination for several generations in Shanghai and
Hainan, China.
Methods
Phenotypic observation
The characteristics of the mutant lms1, including the
occurrence period, shape, colour and distribution of
the spots on leaves were investigated across the whole
growth period in the greenhouse. The agronomic traits
such as plant height and seed-setting rate of the
mutant lms1 in fields were also recorded in Hainan
Province, China. To investigate the dependence of the
phenotype of the lms1 mutant on growth temperature,
germinated seeds of lms1 and its wild type were
transferred into growth chambers (GXZ Zhineng-Type,
Ningbo Jiangnan Instrument Ltd, China) with the
same light intensity of 180 μmol/(m2·s) (12 h light/12
h dark) but two different temperatures (20 and 30 °C).
Ultrastructural analysis of chloroplasts of lms1 leaves
The white parts of lms1 leaves and the same parts of
Jiahua 1 were sampled from 12-day-old seedlings
grown under 30 °C, and fixed in the solution of 2.5%
glutaraldehyde and 1% osmic acid (prepared with 0.2
mol/L phosphoric acid buffer solution, pH 7.2) at 4 °C.
After 5 h, they were dehydrated by 50%, 70%, 80%,
95% and 100% ethylalcohol and acetone, respectively,
then embedded into epoxy resins. After spliced, the
samples were dyed with uranyl acetate, observed and
photographed by a Hitachi-7650 transmission electron
microscope (Hitachi).
Measurement of leaf pigment content
Chlorophyll was extracted with 80% acetone from 0.2
g fresh leaves of 20-day seedlings of the lms1 mutant
and the wild type grown in the greenhouse,
respectively. The extract was measured at 470, 645
and 663 nm by a spectral-photometer (BCEKMAN
COULTER-DU720). Contents of total chlorophyll,
chlorophyll a, chlorophyll b and total carotenoids
were determined according to the method of Gao
(2006). The experiments were repeated three times.
Trypan blue staining
The mutant and wild type leaves were stained with
lactic acid-phenol-trypan blue solution (2.5 mg/mL
trypan blue, 25% lactic acid, 23% water-saturated
phenol and 25% glycerol) at boiling water bath for 10
min and kept at room temperature for 12 h before
replacing the lactic acid-phenol-trypan blue solution
with a chloral hydrate solution (25 mg in 10 mL of
H2O) for 3−4 d for destaining (Yin et al, 2000). Then
Rice Science, Vol. 19, No. 1, 2012
leaf tissues were observed under an SZ2-1LST
stereoscope (Olympus) and photographed with a
PC1200 digital camera (Canon). The experiments
were repeated three times.
DAB (diamino benzidine) staining
DAB staining to directly localize H2O2 in rice leaf
tissues was performed as described by Thordalchristansen (1997). The leaves of mutant and wild
type rice were incubated in 1 mg/mL DAB (pH 3.8)
and lighted at 25 °C for 8 h, then put into 96% ethanol
and boiled 10 min to destain. Finally, the leaves were
submerged in fresh ethanol at 25 °C for 4 h and
observed under an SZ2-1LST stereoscope (Olympus)
and photographed with a PC1200 digital camera
(Canon). The experiment was repeated three times.
Genetic analysis and construction of mapping
population
For genetic analysis, three F2 populations were
developed from the crosses between lms1 with 93-11,
Pei’ai 64S and Guangzhan 63S, respectively. The
population from the cross between lms1 and 93-11
was used for preliminary mapping the lms1 locus, and
the population from the cross between lms1 and Pei’ai
64S was used for fine mapping of the lms1 gene. F2
seeds of the crosses were germinated at 37 °C for 3 d,
sown in plastic containers with rice paddy soil, and
grown in the greenhouse for 14 d. The seedlings with
mutant phenotype were transferred into the growth
chambers for further observation.
Genomic DNA extraction
Genomic DNA was extracted from young leaves of
each parent and F2 individuals by the modified CTAB
method (Muray and Thomoson, 1980).
Mapping of lms1 gene
The polymorphism between the lms1 and 93-11 was
tested using 231 pairs of SSR markers, which were
released by http://www.gramene.org/. The polymorphic
SSR markers were applied for genetic linkage analysis
using a mapping population of 81 mutant individuals
from the F2 population. A total of 513 mutant individuals
of the F2 population from lms1 × Pei’ai 64S and new
SSR markers developed by the Watson Institute of
Genome Science, Zhejiang University, China (http://
www.dnaresearch.oxfordjournals.org) were used for
fine mapping of lms1. The volume of PCR system was
10 μL, including 100 mmol/L Tris-HCl (pH 9.0), 100
mmol/L KCl, 20 mmol/L MgSO4, 80 mmol/L (NH4)2SO4,
2.5 mmol/L dNTPs, 10 μmol/L primers, 5 U/uL Taq
polymerase, 20 ng of template DNA. PCR amplification
proceeded by the Bio-Rad PCR cycler and the primers
MA Jian-yang, et al. Identification and Genetic Mapping of a Lesion Mimic Mutant in Rice
3
Fig. 1. Phenotypes of the lesion mimic stripe mutant lms1 and its wild type.
A, Adaxial view of leaves of wild type (left) and lms1 mutant (right); B, Abaxial view of leaves of wild type (left) and lms1 mutant (right); C,
Mature plants of lms1 mutant (left) and wild type (right).
were synthesized by the Shanghai SBS Genetech Co.
Ltd, China. The PCR program was as follows: 4 min
initialization at 94 °C; 50 s denaturation at 94 °C, 45 s
annealing at 55 °C, and 30 s extension at 72 °C for 35
cycles; 10 min final extension at 72 °C. After
electrophoresis on agarose gels (2.5%–3.5%) and
dying with ethidium bromide, the products were
imaged on a UVP Bioimaging system.
Linkage analysis
Linkage analysis was conducted with the phenotypes and
genotypes using MAPMAKER/EXP 3.0 (Lincoln et al,
1992) and Mapplotter (Shen et al, 2000).
leaves of the lms1 mutant at 30 °C, and white stripes
and brown necrotic lesions appeared at 20 °C (Fig. 2),
which suggests that low temperature accelerates the
formation of lesion mimics in the lms1 mutant.
Photosynthetic pigment content in lms1
By measuring the photosynthetic pigment content in
the leaves of the wild type and mutant grown in the
greenhouse, we found that the total chlorophyll,
chlorophyll a, chlorophyll b and carotenoid contents
were decreased by 32.2%, 28.9%, 40.9% and 46.0%,
RESULTS
Phenotypes of lms1
The rice lms1 mutant grown in the greenhouse
exhibited brown-yellow stripes before the first true
leaf unfolding at the seedling stage. With the leaf
expansion, the stripes enlarged and grew into yellowwhite streaks, where the necrotic lesion located (Fig.
1-A and 1-B). A leaf generally displayed two or three
groups of lesion mimics, which were mostly located
on the upper and middle parts of leaves. The lesion
mimics were also observed on culms during the whole
growth period as well as leaves. The mature mutants
planted in the fields were dwarfed and premature
senescence with lower seed-setting rate (Fig. 1-C) as
compared with the wild type under the same conditions,
which was also observed in the greenhouse.
To investigate the effect of temperature on the
phenotype, the phenotypes of lms1 plants grown under
different temperatures (20 and 30 °C) were surveyed.
The wild type of Jiahua 1 had normal green leaves at
two temperatures, whereas white stripes emerged on the
Fig. 2. Phenotypes of lms1 mutant and wild type seedling grown at
different temperatures.
A, Wild type (left) and lms1 mutant seedlings (right) grown at 30 °C;
B, Wild type (left) and lms1 mutant seedlings (right) grown at 20 °C; a
and b, Magnification of the white frame in A and B, respectively.
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Rice Science, Vol. 19, No. 1, 2012
respectively (Fig. 3), suggesting that the biosynthesis
of chlorophyll was interfered in the lms1 mutant.
Ultrastructure of leaf chloroplasts of lms1 seedlings
To further understand the influence on chloroplast
development exerted by the mutation, a transmission
electron microscope (TEM) was used to observe the
chloroplasts of the wild type and lms1. The chloroplasts
of the wild type had a characteristic structure in the
ellipse shape, with normal granum composed of neatly
stacked thylakoids, and the grana were intimately
connected by stroma thylakoids (Fig. 4-A to 4-C).
However, the chloroplasts from the white-stripe part
of lms1 leaves were aberrant. The thylakoid discs
were loose and assembled into abnormal grana, which
slackly connected by stroma thylakoids (Fig. 4-D to
4-F). These results indicate that the mutation of lms1
affects the development of chloroplasts.
Programmed cell death (PCD) and H2O2
accumulation in leaves of lms1
Trypan blue staining of leaves displayed that the
leaves of lms1 presented dark blue spots under a light
blue background, indicating that a mass of PCD
Fig. 3. Comparison of the leaf chlorophyll and carotenoid contents
in leaves between the lms1 mutant and wild type.
occurred in the leaves of lms1 and then led to visible
lesion mimics (Fig. 5-A). The emergence of dark blue
spots around the lesions suggested the expansion of
lesion mimics. Contrary to those of lms1, PCD was
not observed in the leaves of wild type, since the
leaves were hardly stained (Fig. 5-A). These results
imply that the formation and development of lms1
lesion mimics might be actually the process of PCD in
leaves.
Fig. 4. Transmission election microscopy pictures of the chloroplasts of wild type and white-stripe part of lms1 mutant.
A, B and C, Wild type; D, E and F, lms1 mutant.
MA Jian-yang, et al. Identification and Genetic Mapping of a Lesion Mimic Mutant in Rice
5
Chr. 6
Fig. 5. Staining of leaves of the mutant lms1 and wild type.
A, Trypan blue staining of the leaves of wild type (left) and lms1
mutant (right); B, DAB (diamino benzidine) staining of the leaves of
wild type (left) and lms1 mutant (right).
When H2O2 accumulated in plant cells, the exogenous
diamino benzidine (DAB) reacted with H2O2 via
peroxidase and rapidly engendered rufous polymer,
thus becoming a method for detecting the accumulation
of H2O2. After DAB staining, the leaves from lms1
appeared a great deal of rufous spots compared with
those of wild type (Fig. 5-B), suggesting that lms1
bred the burst of reactive oxygen species (ROS) which
led to hypersensitive response.
Genetic analysis of lms1
The lms1 mutant was crossed with three wild-type rice,
Pei’ai 64S (an indica variety with the maternal origin
of japonica), Guangzhan 63S (an indica sterile line)
and 93-11 (a typical indica variety). All F1 plants had
normal phenotypes, which indicated that the mutation
was controlled by recessive nuclear genes. In the
further survey of F2 generations of lms1 × Pei’ai 64S
and lms1 × Guangzhan 63S, the progenies of both
crosses produced normal and lesion-mimic plants in a
ratio of 3:1 (χ2 < χ0.052 = 3.84, Table 1). Furthermore,
the 2 174 F3 progenies from the F2 generations that
were heterozygous at lms1 locus displayed the phenotypic
segregation ratio of 3:1, namely 1 622 normal plants
and 552 mutational plants (χ2 < χ0.052 = 3.84). Hence,
our statistical results suggest that the lesion-mimic
phenotype is controlled by a single locus recessive
nuclear gene.
Gene mapping of lms1
Out of 231 simple sequence repeat (SSR) markers from
the Gramene Genome Browser (http://www.
gramene.org), which cover 12 rice chromosomes, 95
were polymorphic between lms1 and 93-11. Eighty-one
Fig. 6. Molecular linkage map of rice lesion mimic stripe gene lms1
on rice chromosome 6.
mutant plants showing lesion-mimic phenotype were
obtained in the F2 generation of lms1 × 93-11, and were
then used for linkage analysis. Consequently, the lms1
locus was delimited between RM469 and MM0135 on
chromosome 6. Subsequently, by using of a newlydeveloped SSR marker MM0112-4 (5′-CCTGGAGC
AACTGTGGTA-3′, 5′-GCCAGATAAAAGTATGAA
ATG-3′) and an Indel marker Indel1 (5′-GTAAACCC
TATCCCTATGAGTATC-3′, 5′-TTCCTTCCTGGTAC
AGCTCTTC-3′), and 513 mutant plants of the F2
generation of lms1 × Pei’ai 64S, the lms1 locus was
further identified within 400 kb between Indel1 and
MM0112-4 with genetic distances of 2.5 and 0.7 cM,
respectively (Fig. 6).
DISCUSSION
Natural and artificial lesion mimic mutants in plants
have been one of the research hotspots in plant
physiology, developmental biology, molecular biology,
agronomy, and so on. The identification and functional
characterization of lesion mimic related genes from
the mutants facilitate the elucidation of mechanisms
underlying PCD in plants (Huang et al, 2010).
Recently, a number of lesion-mimic related genes
were located and cloned, such as spl7, the first cloned
lesion-mimic gene using map-based method, which
encodes a transcription factor involved in the heat
shock response in rice (Yamanouchi et al, 2002). spl1,
Table 1. Segregation of mutants in F2 populations of the crosses between lms1 and normal rice lines.
Number of total plants
Number of mutant plants
Number of wild-type plants
χ2 (3:1)
P
lms1 × Pei’ai 64S
405
111
294
1.25
0.25−0.50
lms1 × Guangzhan 63S
1260
291
969
2.44
0.10−0.25
Cross
6
cloned by Zeng et al (2004), plays a role of negative
feedback regulation in cell apoptosis and defense
response of rice. The formation of plant lesion mimic
correlated with the regulatory disturbance of metabolic
pathway led by related genes mutation, and associated
with various environmental factors, including light,
temperature, humidity and nutrition. The expression of
necrotic leaf stripes was observed to be temperature
sensitive in the lesion mimic mutant Les1 of maize
(Zea mays) (Hoisington et al, 1982). Low temperature
stimulates the production of necrotic phenotype in a
maize lesion-mimic mutant rp1 (Hu et al, 1996), and
light is also noticed to regulate its disease lesion
mimicry (Johal et al, 1995). In the present study, the
lesion mimic mutant lms1 was controlled by a single
locus recessive nuclear gene, exhibiting lesion mimics
during the whole growth period. Through trypan blue
staining and DAB staining of mutant and wild type
rice leaves, a reaction resembling a hypersensitive
response (HR) without any external factor was found
to result in necrotic lesions, which suggests that the
formation and development of lms1 lesion mimics is
probably the process of PCD in leaves. The
temperature sensitive characteristic of lms1 plants was
similar to that of spl7 (Yamanouchi et al, 2002). The
white stripes were visible in lms1 leaves at 30 °C,
whereas necrotic lesions appeared at 20 °C, which
revealed that low temperature accelerated the lesion
formation of lms1. However, the lesion mimics
occurred in spl7 when temperature was above 35 °C,
indicating complicated mechanism for temperatureinduced leaf cell apoptosis to generate lesion mimics.
The accomplishment of rice genome sequencing
and the release of microsatellite database greatly
accelerate the locating and cloning of genes related to
various kinds of rice mutants. We primarily delimited
lms1 between the SSR markers RM469 and RM587
on the distal end of the short arm of chromosome 6
with 1.2 cM and 10.3 cM, respectively (data not show).
Subsequently, the interval genome sequence between
the two markers was used to develop new InDel markers
and SSR markers, and another F2 mapping population
was employed to narrow the lms1 locus to a 400-kb
DNA region between the molecular markers InDel1
and MM0112-4, which was spanned by a BAC contig
composed of four BAC clones, AP001552, AP001389,
AP002387 and AP003564 (http://ricegaas.dna.affrc.go.
jp). The lesion-mimic phenotype of lms1 is radically
differed from those of other three lesion-mimic
mutants of rice [bl2 (Jodon, 1957; Nagao et al, 1964),
bl3 (Nagao et al, 1964, 1966), spl4 (Mizobuchi R et al,
2002; Mizobuchi K et al, 2003)], whose loci were also
Rice Science, Vol. 19, No. 1, 2012
identified on rice chromosome 6. Furthermore, the lms1
locus is located on the distal end of the short arm, and
differs from the other three loci. Taken together, it can
be inferred that lms1 might be a new mutation gene
involved in lesion mimics. We are enlarging the
mapping population and developing new markers for
fine-mapping and cloning, and functional analysis of
lms1 in the further.
ACKNOWLEDGEMENTS
This research was supported by the National Basic
Research Program of China (Grant No. 2009CB119000),
the National Science Foundation of China (Grant Nos.
31000094, 31100188 and 30970246).
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