Documente Academic
Documente Profesional
Documente Cultură
DOI 10.1007/s11032-015-0394-2
Introduction
In the twentieth century, food security is still a huge
challenge in the world. As has been proven in
agricultural production, heterosis utilization is one of
the most effective ways for increasing yield of crop
plants (Yuan and Peng 2005). As one of the most
important approaches, cytoplasmic male sterility
(CMS) has been widely used for commercial hybrid
rice breeding programs (Wise and Pring 2002; Tan
et al. 2012). Exploration of novel CMS will greatly
enforce the basis for heterosis utilization (Wang et al.
2013). Since discovery of the first rice CMS line
(Shinjyo 1969), over 60 rice CMS lines had been
developed through widecross within or between
species (Luan et al. 2013). However, only a small
part of which have been used for hybrid rice breeding,
which limits diversity of the CMS mitotypes, and will
hamper to breed of desirable rice varieties with high
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(Supplementary Table S1). Among them, seven commercial sporophytic CMS lines, including Maxie A, II32 A, K17 A, Yue4 A, Xieqingzao A, Gang46 A, and
Zhenshan97 A and their maintainer lines were used as
controls for screening of CMS-inducing cytoplasm,
and elite maintainer lines, respectively. The rice
accessions were kindly provided by IRRI, Hunan
Agricultural University, Jiangxi Academy of Agricultural Sciences, and Hubei Academy of Agricultural
Sciences. All the accessions were planted in the
experimental field within Wuhan University campus
in the summer and Hainan Island in the winter during
20092013. The hybrid rice combinations and their
corresponding parents used for combining ability
analysis were planted in the experimental field within
Hunan Agriculture University campus in the summer
of 2014.
DNA extraction and PCR amplification
Total genomic DNA was extracted from young leaves
using the CTAB method (Murray and Thompson
1980). The mitotyping of O. glaberrima and wild rice,
and nuclear genotyping of O. sativa were referenced to
the methods of Xie et al. (2014), and Li et al. (2012),
respectively. For the analysis of CMS and restorer
related genes, PCR were performed with the primers
listed in Supplementary Table S2.
PCR was carried out in a total volume of 10 ll
containing 50 ng of total genomic DNA, 1 ll
10 9 PCR buffer, 0.25 lM of each primer, 75 lM
of each dNTP and 0.25 units of Taq DNA polymerase
(Fermentas). The PCR procedure was as follows:
denaturation at 94 C for 5 min, followed by 32 cycles
of 94 C for 30 s, 5057 C (dependent on the
primers) for 30 s, 72 C for 1 min, and a final
incubation at 72 C for 5 min. All the PCR products
were analyzed by electrophoresis on agarose gel.
Phylogenetic analysis
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Results
Screening of new CMS-inducing cytoplasm
from wild rice and O. glaberrima
Eleven O. glaberrima and 50 wild rice accessions
were screened with orf(H)79, a specific gametophytic
CMS marker, to rule out gametophytic cytoplasm.
Analysis revealed that there are eight wild rice
accessions carrying orf(H)79 alleles (Fig. 1a). The
rest 53 accessions were further screened using orf352
genes, of which, 22 accessions were detected carrying
orf352, and selected for further mitotype analysis
(Fig. 1a).
In order to select differential mitotype of carring
CMS-inducing cytoplasm in the wild rice and O.
glaberrima accessions, the mitochondrial genomes of
the 22 accessions were further investigated using 27
MSS markers which cover the whole mitochondrial
genome (Xie et al. 2014). PCR analysis shows that,
relative to the seven sporophytic CMS control, the
selected 22 CMS lines show clearly polymorphic
profiles (Fig. 1b). Then, a dendrogram was constructed based on the data matrices derived from the
profiling of the 27 MSS markers using the UPGMA
method (Fig. 1c). From the dendrogram, two monophyletic groups are identified at the similarity
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Table 1 Effects of the general combining ability of the five CMS lines
CMS Lines
Plant height
Rongfeng A
-0.21
3.67
-6.69
3.02
4.00
8.08
-1.97
10.13
-5.64
2.38
3.17
Xie A
Zaofeng A
1.30
-6.18
-2.69
3.73
4.51
-4.91
-0.11
0.99
-2.92
-0.73
-0.58
-1.37
Wufeng A
-2.56
-2.83
-3.14
1.80
-2.93
-6.14
Luofei A
Effective panicle
Grain number/panicle
the six investigated characters (Table 2). In comparison, Luofei A ranks the third for the special combining ability of grain yield, which means that Luofei A
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1000-grain weight
Grain yield/plant
5.10
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Table 2 Variance of the special combining ability of the five CMS lines
CMS Lines
Rongfeng A
Plant height
Effective panicle
Grain number/panicle
1000-grain weight
Grain yield/plant
3.05
103.28
39.52
5.94
6.73
48.42
12.08
6.54
21.84
9.87
8.58
50.66
Xie A
Zaofeng A
4.46
5.52
51.06
91.84
10.52
22.99
48.03
15.13
8.42
14.95
32.48
65.82
Wufeng A
1.52
32.08
30.94
10.65
3.89
63.79
Luofei A
Discussion
Hybrid rice has been made a great progress, and
contributed greatly to the increase of rice production in
China during the last decades (Cheng et al. 2007; Rao
et al. 2014). Exploitation of new CMS resource is one
of the important means to ensure high yield and avoid
the potential threat of genetic vulnerability of the CMS
lines, which will help to enforce the basis for hybrid
rice sustainable development (Fujii and Toriyama
2009). Traditional breeding is difficult to create new
CMS by random outcrossing, because it is hard for us
by naked eyes and experiences to estimate which rice
carries new CMS-inducing mitotype or an expectant
genotype with recessive restorer genes. Thus, it is
eventually impossible to avoid being homogenization
for the commercial CMS lines, just as shown in our
results that the seven commercial CMS lines show
very similar PCR profiles, and are clustered in the
same subgroup (Fig. 1c). However, our results indicate that a great many of mitotypes actually exist in the
wild rice and O. glaberrima accessions (Fig. 1b), and
can be well differentiated by MSS molecular markers
(Xie et al. 2014), Which highlights the dominant and
significant role of MSS-based selection on new CMS
development from rice wild relatives.
Concomitantly, elite maintainer lines which do not
carry any restorers for a certain CMS, and have a
different genotype from the other maintainers can also
be effectively detected based on the SCAR and Rfspecific markers (Fig. 2). Comprehensively use both
of the mitotype- and Rf-specific molecular markers
make MAS-based selection directional and high
efficiency in the development of novel CMS. This is
agreement with the results of combining ability
analysis. It is well known that combining ability for
a new CMS line depends mainly on the nucleus, a
certain level of distance between parents can make
expectant heterosis of a hybrid (Makumbi et al. 2011).
References
Ahmadikhah A, Karlov GI (2006) Molecular mapping of the
fertility-restoration gene Rf4 for WA-cytoplasmic male
sterility in rice. Plant Breed 125:363367
Cheng SH, Zhuang JY, Fan YY, Du JH, Cao LY (2007) Progress
in research and development on hybrid rice: a super-domesticate in China. Ann Bot 100:959966
Fujii S, Toriyama K (2009) Suppressed expression of retrograde-regulated male sterility restores pollen fertility in
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