Identification of WA-type three-line hybrid rice with real-time polymerase chain reaction (PCR) method.

Abstract

A real-time fluorescent PCR (RTF-PCR) was developed to detect and quantify wild abortive (WA)-type three-line hybrid rice (Oryza sativa L.). The mitochondrial R₂₋₆₃₀ WA gene was reported to be closely related to male sterility in plants, and developed as a molecular maker to identify the cytoplasmic male sterility system of hybrid rice. First, we got the DNA sequence of R₂₋₆₃₀ WA gene in 17 rice species with traditional PCR. Then, a pair of specific primers (P₃, P₄) and TaqMan fluorescence probe (P₃₋₁₄) were designed based on the R₂₋₆₃₀ DNA sequence. The following RTF-PCR was performed on the 17 rice species finally. The results indicate that the probes used here are specific for three-line hybrid rice F₁ and male sterile lines. We can even identify a single hybrid seed using the probes, which confirmed that the probes can be applied to the identification and quantification of the WA-type three-line hybrid rice. In addition, the RFT-PCR system can be optimized when the annealing temperature is 60 °C and the Mg²⁺ concentration is 3.5 mmol/L.

Full text

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Identification of WA-Type Three-Line Hybrid Rice
with Real-Time Polymerase Chain Reaction
(PCR) Method
Y. Cheng & B. D. Gao & H. Y. Chen & J. J. Mao &
A. X. Cao & J. G. Zhu & S. F. Zhu
Received: 6 July 2011 /Accepted: 24 October 2011
# Springer Science+Business Media, LLC 2011
Abstract A real-time fluorescent PCR (RTF-PCR) was developed to detect and quantify
wild abortive (WA)-type three-line hybrid rice (Oryza sativa L.). The mitochondrial R2-630
WA gene was reported to be closely related to male sterility in plants, and developed as a
molecular maker to identify the cytoplasmic male sterility system of hybrid rice. First, we
got the DNA sequence of R2-630WA gene in 17 rice species with traditional PCR. Then, a
pair of specific primers (P3, P4) and TaqMan fluorescence probe (P3-14) were designed
based on the R2-630DNA sequence. The following RTF-PCR was performed on the 17 rice
species finally. The results indicate that the probes used here are specific for three-line
hybrid rice F1and male sterile lines. We can even identify a single hybrid seed using the
probes, which confirmed that the probes can be applied to the identification and
quantification of the WA-type three-line hybrid rice. In addition, the RFT-PCR system
can be optimized when the annealing temperature is 60 °C and the Mg2+concentration is
3.5 mmol/L.
Keywords Real-timefluorescentPCR.Hybridrice.Molecularidentification.Male
sterility.mtDNA.R2-630WA
Appl Biochem Biotechnol
DOI 10.1007/s12010-011-9471-0
Y. Cheng (*)
Institute of Bast-Fiber crops, Chinese Academy of Agricultural Sciences, Xianjiahu West Road 348,
Changsha, China
e-mail: charles_chengyi@yahoo.com.cn
H. Y. Chen:J. J. Mao:A. X. Cao:S. F. Zhu (*)
Institute of Animal and Plant Quarantine, Chinese Academy of Inspection and Quarantine, Huixin West
Road 241, Beijing, China
e-mail: zhushf@netchina.com.cn
Y. Cheng:B. D. Gao:J. J. Mao
College of Bio-safety Science and Technology, Hunan Agricultural University, Furong District,
Changsha, China
J. G. Zhu
Hunan Entry-Exit Inspection and Quarantine Bureau,
Xiangfu Middle Road 188, Changsha, China

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Rice is a principal food crop over 110 different countries and feeds more than half of the
world’s population. The development of hybrid rice with improved yield characteristics is
vital to meet the food needs of increasing world population. Comparing to routine varieties,
hybrid rice is 10–20% higher in yield. Up to now, hybrid rice has been large scale cultivated
in China, India, Vietnam, and the Philippines and has been commercially generalized by
more than 20 countries [5, 23]. To keep pace with the present population growth and the
consumption pattern, the trade market of hybrid rice seed increased greatly. In 2007, more
than 2,500 tons of hybrid rice seeds were exported to other Asian countries from China.
However, in 2010, the number rose rapidly to 38,000 tons since more and more
professionals accepted the hybrid rice [3, 4]. Meanwhile, development of a rapid and
accurate identification system of hybrid rice comes out to be a serious challenge to the
research scientists as well as inspection personnel.
Currently, the production of hybrid rice is primarily based on the three-line hybrid system,
which involves a CMS line or “A” line, a corresponding isonuclear maintainer (B) line, and a
genetically diverse restorer (R) line. In addition, A line is maintained by being crossed with B
line and hybrid seed is produced by crossing A line with R line. In consequence, after several
generations of hybridization, variations between hybrids and their parents become complicated
due to the increasing genetic complexity [17, 19]. The grow out test is a common method to
identify hybrid rice. It consists of growing a representative sample of F1seed and then
classifying it using descriptors of differences as true hybrid seed or off types. It is a time-
consuming and space-occupying method, and often interacted with the environment in which
the variation is grown and thus makes the variety identification subjective. In contrast,
molecular markers provide an unbiased means of identifying crop seeds based on DNA
sequence variation and have a direct impact on the development of new markers, which are
more suitable to plant breeding, food inspection, disease detection, and variety identification
[15]. Researchers have developed various DNA-based methods, including RFLP, RAPD, as
well as AFLP [13, 18]. But their disadvantages such as time consuming, low reproducibility,
marker available limitation, and high cost limited their commercial application. With
advantages of high polymorphism, reliability, and codominance, SSR has become the major
method for variety identification and seed purity test in the recent years [6, 14, 16]. However,
the high development cost, long development period and efforts required to obtain working
primers for a given species limited the wide use of SSR markers [11].
A less time consuming and more effective PCR-based assay, referred as RTF-PCR, has
recentlyshownpromiseinthespecificandrapiddetectionofharmfulmicroorganismsandplant
variety recognition [2, 8, 10]. This technique applies a combined primer set and an additional
dual-labeled fluorogenic probe to monitor the amplicon synthesis consistently during
thermocycling. Moreover, this technique requires no PCR handing for target quantification.
The primary objective of this study is using RTF-PCR to identify wild abortive (WA)-type
three-line hybrid rice and then develop an identification and quantification system for hybrid rice
seed which can be applied in the scientific research as well as international trade. Moreover,
differentDNApreparationmethodswerealsocomparedtodevelopanidealidentificationsystem.
Materials and Methods
Plant Materials
For the purpose of molecular characterization, hybrids F1sample 1 to 6 and their parental
lines sample 7 to 13 were used (Table 1). Combination 1∼3 of hybrid seeds were kindly
Appl Biochem Biotechnol

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provided by the National Rice Research Institute (Hangzhou, China). Other samples were
obtained from the National Hybrid Rice Research and Development Center (Changsha,
China). Combination 4 and japonica rice sample 17 were used for comparison purpose. For
the purpose of RTF-PCR, seeds were stored at 4 °C until used [12]. Samples used in the
study were numbered from 1 to 17.
DNA Extraction
Two methods, i.e., method of McDonald et al. [12] and a plant genomic DNA isolation kit,
were compared for genomic DNA extraction. The protocol based on the method of
McDonald et al. was slightly modified, and RNAase treatment of isolated DNA was
included. The plant genomic DNA isolation kit (Shanghai Waston bio-engineer, China) was
also used. Total DNA was isolated from dry seeds directly by grinding seed tissue,
according to the manufacturer’s instructions. DNA concentration was determined by
measuring the absorbance at 260 nm with a Smart Spec 3000 spectrophotometer (Bio-Rad
Laboratories, Hercules, CA). If necessary, DNA was diluted with ultrapurified water.
Conventional PCR Validation, Sequencing, and TaqMan Probe Design
The R2-630WA gene was selected from rice male sterility-related genes, which is found by
Xu et al. [20] through arbitrarily primed-polymerase chain reaction. To verify R2-630WA
that can be used in this study, conventional PCR was performed first to get the DNA
sequence of R2-630 WA in the 17 samples, and the primers P1–P2 (Table 2) used in
Table 1 List of the rice species used for RTF-PCR
No. AbbreviationRice variety Lines (combinations)Geographical location
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
VY46
XY63
XYG99
SY63
XFY103
XXY80
V20A
ZX97A
V20B
ZX97B
MY46
MH63
G99
LYP9
PA64
R288
ZH8
Weiyou 46
Shanyou 63
Xianyougui 99
Siyou 63
Xiangfengyou 103
Xinxiangyou 80
V20A
Zhenshan 97A
V20B
Zhenshan 97B
Miyang 46
Minghui 63
Gui 99
Liangyoupei 9
Pei'ai 64
R288
Zhonghua 8
F1(1)
F1(2)
F1(3)
F1
F1
F1
Sterile line (1)
Sterile line (2/3)
Maintainer line (1)
Maintainer line (2/3)
Restorer line (1)
Restorer line (2)
Restorer line (3)
F1(4)
Sterile line (4)
Maintainer line (4)
O. sativa japonica
Hangzhou, Zhejiang, China
Hangzhou, Zhejiang, China
Hangzhou, Zhejiang, China
Changsha, Hunan, China
Changsha, Hunan, China
Changsha, Hunan, China
Hangzhou, Zhejiang, China
Hangzhou, Zhejiang, China
Hangzhou, Zhejiang, China
Hangzhou, Zhejiang, China
Hangzhou, Zhejiang, China
Hangzhou, Zhejiang, China
Hangzhou, Zhejiang, China
Changsha, Hunan, China
Changsha, Hunan, China
Changsha, Hunan, China
Changsha, Hunan, China
VY46, V20A, V20B, and MY46 consist of combination 1; XY63, ZX97A, ZX97B, and MH63 consist of
combination 2; XYG99, ZX97A, ZX97B, and G99 consist of combination 3; LYP9, PA64, and R288 consist
of combination 4. Combination 1, 2, and 3 are three-line hybrid, however combination 4 is a two-line hybrid.
Sample 17 is O. sativa japonica, other samples are O. sativa indica
Appl Biochem Biotechnol

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conventional PCR were designed based on the sequences reported before. The 50-μL
conventional PCR mixture consists of 5 μL of 10× reaction buffer [1 mM Tris–HCl
(pH 9.0), 5 mM KCl, and 0.01% Triton X-100], 1 mM Mg2+, 0.3 mM dNTP, 1 unit of
Ampli Taq DNA polymerase, 100 nM of each primer, and 80 ng of template DNA.
Chemicals were purchased from Promega (Madison, WI). Amplifications were carried out
in a thermal cycler GeneAmp PCR system 9700 (Applied Biosystem) programmed as
follows: initial denaturation step at 94 °C for 4 min, followed by 35 cycles of denaturation
at 94 °C for 1 min, annealing at 56 °C for 30 s and extension at 72 °C for 1 min, and a final
extension step at 72 °C for 10 min. The amplified products were resolved by
electrophoresis in 1.5% agarose gels in 1× Tris–acetate–EDTA buffer and photographed
under UV light (302 nm) using Polaroid 667 film after being stained with ethidium
bromide. The amplified PCR products were also cloned and sequenced. The sequences
were aligned with the sequences reported by Xu et al. [20].
Based on the sequences we got from the 17 samples, the primers and TaqMan probe
(Table 2) were designed for RFT-PCR with Primers Express (version 2.0, PE Applied
Biosystems). Primers and probe were also synthesized by PE Applied Biosystems.
Real-Time PCR Assay
Real-time PCR was performed in 30-μL reactions using MicroAmp Optical 96-well plates
and MicroAmp Optical Caps (PE Applied Biosystems). The 30-μL PCR mixture consists
of 3 μL of 10× reaction buffer [1 mM Tris–HCl (pH 9.0), 5 mM KCl, and 0.01% Triton X-
100], 1 mM Mg2+, 500 μM dNTP, 0.5 units of Ampli Taq DNA polymerase, 100 nM of
each primer (P3and P4, Table 2), 50 nM TaqMan probe P3-14(Table 2), and 50 ng of
template DNA. As a negative control, ultrapure water rather than template DNA was
included in each PCR run. The ABI Prism 7700 Sequence Detection System (PE Applied
Biosystems) was used for amplification and fluorescence measurement at each temperature
step and cycle during the reaction. Thermal cycling conditions consisted of 5 min at 95 °C
followed by 40 cycles of 15 s at 95 °C and then 25 s at 60 °C. The ABI Prism Sequence
Detector Software (version 1.6) was used to collect and analyze data of normalized
fluorescence (ΔRn) and Ct. The ΔRn value represents a difference in fluorescence intensity
between two cycles; Ct refers to the specific cycle number at which the ΔRn value becomes
greater than the base line. The Ct value is inversely proportional to the log of the initial
template of DNA and has been shown to be the most reliable parameter to detect and
quantify target DNA with real-time PCR. Triplicates were performed in each experiment,
and each experiment was repeated at least three times.
Optimal conditions were determined by performing the real-time PCR with concentra-
tion gradients of primers (50 to 800 nM) and Mg2+(0.4 to 6.5 mM) at different annealing
and extension temperature (55, 57, 59, 60, 61, 63, and 65 °C). Conditions were considered
Table 2 The primers used in the test
PrimersSequences of primerGC/%Tm/°C Bases/bp
P1
P2
P3
P4
P3-14
5′-AATGCTACCCGAAGACGA-3′
5′-GGAAAGCCCATCCCTACT-3′
5′-GGCGGAGAGGGATTCATTAGT-3′
5′-GGTTAGTGTTTCATTTCGATAGGTGA-3′
5′-FAM-CCGGGTACTGGTACCGCTTAAGCCC-TAMRA-3′
50.0
55.6
52.4
38.5
68.6
56.7
56.5
58.2
58.6
64.0
18
18
21
26
25
Appl Biochem Biotechnol

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optimal when they produced the smallest Ct values and the most fluorescence. Fifty
nanograms of genomic DNA of Shanyou 63 was used for the optimization of conditions.
Specificity and Sensitivities of Three-Line Hybrid Rice Real-Time PCR Assay
The specificity of primers and probe were examined based on the DNA templates (50 ng each)
from the 17 samples under the optimal PCR conditions. To test sensitivity and accuracy, five
samples (100 grains of seed each) were prepared and each contained one, two, three, four, and
five grains of hybrid seed Shanyou 63, respectively, and other seeds were MH63, conventional
rice;threesamplesofwhichcontained100grainsofriceseed,including20,50,and70grainsof
Shanyou 63seed,respectively,werealsoprepared.DNAofeachsingle seedwasextracted,and
real-time PCR test was performed on each seed of these three samples. Triplicates were
performed ineach experiment,and each experimentwas repeatedat leastthree times.Ultrapure
water was included in each PCR run as a negative control.
Application of Real-Time PCR to Purity-Known samples
Eight conventional rice samples, MH63, well mixed with 10%, 30%, 50%, 70%, 90%, 95%,
99%, and 100% hybrid seed Shanyou 63, respectively, were prepared for real-time PCR assay.
One hundred-milligram seeds were chosen from each sample, and real-time PCR test was
carried outone byone. The Ctvaluewas recorded,andthiswas repeatedat least threetimes.Ct
values for each standard reaction were calculated on three replications, performed in three
separate runs. Standard curves were devised by plotting the Ct values versus the hybrid seed
concentration, and the correlation equation was obtained. Twenty purity-known samples with
5% concentration ladder were tested using the obtained correlation equation.
Results
Different Methods for DNA Extraction
DNA with high quality and good condition can be obtained by DNA extraction methods
and the method developed by McDonald et al. However, the fast DNA Kit from Shanghai
Waston only costs 1 to 2 h to extract the DNA. Moreover, better results of PCR
amplifications could be obtained if the yellow seeds were grown in dark at 37 °C for 3–5 days.
Therefore, the fast DNA Kit was used to obtain the DNA in the following fluorescent
PCR assay.
Conventional PCR Results and Sequence Analysis
Agarose gel electrophoresis of conventional PCR products displayed that an indicative
bright band of 430 bp could be found in the PCR product of sample 1 to 8 which
demonstrated that the primer pair P1and P2works for these samples. However, this primer
pair failed to amplify samples 9 to 13 even if the PCR conditions were modified (Fig. 1).
As a result, hybrid rice F11 to 6 and sterile line 7 and 8 could be distinguished easily from
their maintainer line 9 and 10 and restorer line 11,12, and 13 in the combination 1 to 3 by
RTF-PCR with the primer pair P1–P2. Then after DNA cloning and sequencing, we got the
DNA sequences of the gene we amplified. It confirmed the gene to be a R2-630WA
homologue after an alignment with the reported DNA sequence of R2-630WA gene.
Appl Biochem Biotechnol

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Primers and TaqMan Probe Design
PCR amplification from the six hybrid seed samples and two sterile line seeds in
combination 1 to 3 got about a 430-bp product. DNA sequences of the PCR products from
these eight samples were aligned with R2-630WA gene, and no variation was found (100%
similarity). So the specific primers and a TaqMan probe reporter for the following RTF-
PCR were designed based on the R2-630WA gene we got. Primers Express (version 2.0, PE
Applied Biosystems) was used here for the primer designing. The forward primer (P3) and
the reverse primer (P4) were respectively 5′-GGCGGAGAGGGATTCATTAGT-3′ and 5′-
GGTTAGTGTTTCATTTCGATAGGTGA-3′ (Fig. 2). The TaqMan probe P3-14was 5′-
FAM-CCGGGTACTGGTACCGCTTAAGCCC-TAMRA-3′ (Fig. 2) and was labeled at the
5′ end with fluorescent reporter dye 6-carboxy-fluorescein (FAM) and the 3′ end with
quencher dye 6-carboxy-tetramethhylrhodamine (TAMRA; TaKaRa Bio Inc., Shiga, Japan).
The normalized fluorescent intensity of the reporter dye (ΔRn) was plotted against cycle
number to graph PCR amplification and to determine the Ct values. The smallest Ct values
(and maximum fluorescence) were obtained with 100 nM forward primer, 100 nM reverse
primer, 3.5 mM Mg2+, and an extension temperature of 60 °C (Fig. 2).
Specificity, Sensitivity, and Accuracy of TaqMan PCR Primers and Probe
The specificity of the primers and dual-labeled fluorescent probe was examined with
genomic DNA extracted from sample 1 to 8, including six hybrid rice seeds and two sterile
Fig. 1 Agarose gel electrophoresis of P1–P2PCR products. Hybrids F1(VY46, XY63, XYG99, SY63,
XFY103, and XXY80) and their sterile lines (V20A and ZX97A) of rice located at line 1 to line 8. Their
maintainers (V20B and ZX97B) and restorers (MY46, MH63, and G99) are located at line 9 to line 13. DNA
was isolated from single seedling listed in Table 1. Molecular weight marker (lane M) is a 2-kilobase DNA
ladder. The detail of electrophoresis was described in the “Materials and Methods” section
Fig. 2 Sequences of primer P3and P4and probe P3-14
Appl Biochem Biotechnol

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line seeds. PCR was conducted with 40 thermal cycles and the optimized conditions. All
DNA extracts from sample 1 to 8 yielded significant increase of fluorescence, whereas the
DNA extracts from other rice samples produced no detectable fluorescence (Fig. 3). Thus,
specificity of both primers and probe was confirmed.
The sensitivity of the real-time PCR assay was examined with five samples, including
one, two, three, four, and five grains hybrid rice seeds, with their Ct values 19.954, 19.486,
18.455, 18.418, 17.724, and 17.354, respectively, even a single hybrid seed could be
detected by fluorescent PCR assay (Fig. 4). The accuracy of the TaqMan probe P3-14could
even reach as high as 100% (Table 3).
Applications of Real-Time PCR to Purity-Known Samples
For testing purity of the known samples, the standard curve was obtained by plotting Ct
values versus the hybrid rice concentration, with eight hybrid samples. The standard
curve was linear over 8 log units of initial quantities of template DNA, with whose
hybrids purity gradient spanning from 10% to 100% (Fig. 5). Regression equation of
standard curve is Y=−0.0358X+20.338; the coefficient of determination (R2) is 0.938.
Therefore, the Ct values were closely correlated with hybrid seeds purity, indicating the
RTF-PCR assay could be used for quantifying hybrid rice seed.
Using protocol of this standard curve, the purity of hybrid rice in 20 samples, well mixed
with conventional rice seed in advance, could be easily calculated as displayed in Table 4.
Table 4 showed that the deviation between practical purity and test data is approximately
1.5% when the purity of hybrid rice ranged from 10% to 45%. However, the deviation
increased to 3.5% when samples were mixed with 50% to 100% hybrid rice seed.
Discussion
Fast and accurate identification system of hybrid rice is crucial in the international trade of
rice as well as in the breeding of the hybrid rice. Identification based on morphology is
difficult because merely no morphologic difference was detected between the hybrid seeds
Fig. 3 Specificity of the real-time PCR assay for hybrids. The assays were conducted with 50 ng of DNA
template from all the samples listed on the Table 1 and using the specific primer set P3and P4and TaqMan
probe P3-14under optimized conditions. CK control without DNA template. To distinguish each sample from
each other, different symbols were used for every treatment
Appl Biochem Biotechnol

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and the nonhybrid ones. Many different molecular ways have been developed as a result.
However, it is hard for us to find a reliable nuclear marker gene due to the very complicated
genetic background of hybrid seeds [18]. However, since the report that CMS is relevant to
mitochondria DNA (mtDNA) [9], which is maternally inherited and stays comparatively
stable after millions of years evolution, the researches on CMS started to focus on mtDNA.
Recent studies revealed differences between sterile lines and maintainer lines on
restriction enzyme banding patterns and microstructure of mtDNA in hybrid rice. Some
mitochondrial genes were found to be associated with male sterility, such as chimeric gene
urf-rmc found by Kadowaki, orf-79 found by Akagi, R2-630WA found by Xu, and PHW-17
reported by Yang (Kadowaki [7], Akagi [1], Xu et al. [20], Yang et al. [21]). The new but
powerful real-time PCR assay was applied in this study. Specific TaqMan probe was
designed based on CMS-related-gene R2-630WA after several times of tests, which can
accurately identify any R2-630WA-containing plants, such as hybrid rice and its sterile lines.
Therefore, this special probe can be used to identify and quantify three-line hybrid rice.
The real-time PCR assay developed in this study could significantly improve the
working efficiency. It is approved that 96 samples can be analyzed at one time
without post-PCR gel electrophoresis. The fluorescent PCR technology also provides
more robust results than other PCR methods when small amplicons of 20 to 100 bp
need to be analyzed. The assay was sensitive and proved to work in all WA-type
three-line hybrid rice samples in this study. Furthermore, based on Xu’s reports on the
R2-630WA’s application of Bt-type and DA-type CMS [20], we can also predict that this
assay may be used to distinguish these two type three-line hybrid rice as a molecular
marker. Moreover, this assay was also applied to the two-line hybrid rice Liangyoupei 9.
Fig. 4 Sensitivity of the RTF-PCR assays for Shanyou 63 single-seed test. 1 based on five samples including
one, two, three, four, and five grains, respectively. CK stands for the negative control, with water instead of
DNA template in the PCR system. To distinguish one sample from the others, different symbols were used
for each treatment
No.OriginTotal
(grain)
Fact
(grain)
Test result
(grain)
Accuracy (%)
1
2
3
Hunan
Hunan
Hunan
100
100
100
20
50
70
20
50
70
100.0
100.0
100.0
Table 3 Results of accuracy test
detected by RTF-PCR
Appl Biochem Biotechnol

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However, no amplification has been obtained due to different male sterile mechanism
which is called photoperiod-sensitive genic male sterile.
Based on Table 4, we learned that the assay works fine when used in lower purity hybrid
samples while relatively larger deviations were found in higher purity samples. Three
reasons may be involved: first, more representative species and replicates should be
included in the establishment of curve equation for hybrid rice purity appraisal; second, the
quality and quantity of nucleic acid has great influence on the absolute quantification of
hybrids. Data would be more stable and accurate when more efficient probe labeled by
reference gene, which could be applied in implementation of relative quantification of
hybrids, is included [8, 22]. At last, as a highly sensitive protocol, real-time fluorescent
PCR was good at detecting some positive samples from large amount of negative samples.
It became easier to reach the reaction plateau phase as doses of probe and primers were
limited in the reaction system and deviation was generated. Therefore, the probe based on
specific DNA fragment or SNP sites of conventional rice was where the key problem lied
and would be more efficient in the hybrids purity test.
Table 4 Results of purity test of hybrid rice samples detected by RTF-PCR
No. OriginResult (%) Fact (%) No.OriginResult (%)Fact (%)
1
2
3
4
5
6
7
8
9
10
Hunan
Zhejiang
Hunan
Zhejiang
Hunan
Zhejiang
Hunan
Zhejiang
Hunan
Zhejiang
10.5
14.2
19.1
23.8
29.1
36.0
38.6
46.5
51.9
56.8
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
50.0
55.0
11
12
13
14
15
16
17
18
19
20
Hunan
Zhejiang
Hunan
Zhejiang
Hunan
Zhejiang
Hunan
Zhejiang
Hunan
Zhejiang
62.1
66.9
72.4
77.1
82.5
87.3
93.2
98.5
96.5
98.1
60.0
65.0
70.0
75.0
80.0
85.0
90.0
95.0
98.0
100.0
Fig. 5 Detection of Shanyou 63 in mixed samples using real-time polymerase chain reaction. Data are from
different samples prepared with 10%, 30%, 50%, 70%, 90%, 95%, 99%, and 100% hybrids Shanyou 63.
Minghui 63 was mixed with conventional rice seed in each sample. CK stands for control without DNA
template. In order to identify various samples, different colors were used to demonstrate different treatment,
respectively
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At present, though the whole rice genome sequencing and physical mapping has been
finished, the researches of comparative genomics and molecular taxonomy are still in their
initial stage. Failing to find out useful SNP sites in some genes, such as Nad1, Nad5, Nad7,
Atp6, and Cob [23], the author found that it is hard to find a credible intraspecific mutation
in the highly conserved mitochondrial genome of rice. However, with the development of
molecular tools and the mechanism of CMS, more powerful probe could be obtained, when
we find the corresponding fragments of conventional rice where male sterile-related
segments are located in the physical map of mitochondria genome. It can be forecasted that
the goal of establishing a rapid, accurate, and sensitive appraisal protocol will be achieved
in the near future, and real-time PCR assay may become a new way to differentiate and
quantify three-line hybrid rice.
Acknowledgments This work was partly funded by molecular marker system of bio-resources General
Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China. The
authors would like to thank Dr. H.L. Yang from the China National Hybrid Rice Research and Development
Center, Dr. W.X. Zhang from the Institute of Crop Germplasm Resource Chinese Academy of Agricultural
Sciences (CAAS) and Dr. S.W. Huang from the China National Rice Research Institute for the donation of
samples. We are grateful to Professor J. Leng and W. Li from the Institute of Bast-Fiber and L. Yu from
Hunan Agricultural University for their critical reading and editing of the manuscript.
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