ORIGINAL PAPER

Production and molecular characterization of diploidand tetraploid somatic cybrid plants between male sterileSatsuma mandarin and seedy sweet orange cultivars

Shi-Xin Xiao • Manosh Kumar Biswas •

Meng-Ya Li • Xiu-Xin Deng • Qiang Xu •

Wen-Wu Guo

Received: 25 July 2013 / Accepted: 19 September 2013 / Published online: 1 October 2013

� Springer Science+Business Media Dordrecht 2013

Abstract Seedlessness, an important economic trait for

fresh fruit, is among the prior goal for all citrus breeding

programs. Symmetric somatic hybridization provides a

new strategy for citrus seedless breeding by creating cy-

brids transferring mitochondrial DNA (mtDNA) controlled

cytoplasmic male sterility (CMS) from the callus parent

Satsuma mandarin (C. unshiu Marc.) to seedy cultivars. In

this study, protoplast fusion was adopted to transfer CMS

from C. unshiu Marc. cv. Guoqing No. 1 (G1) to three

seedy sweet oranges (C. sinensis L. Osb.), i.e. ‘Early gold’,

‘Taoye’ and ‘Hongjiang’. Flow cytometry analysis showed

that 12 of 13 regenerated plants from G1 ? ‘Early gold’, 9

of 12 from G1 ? ‘Taoye’ and both two plants from

G1 ? ‘Hongjiang’ were diploids, while the remaining

regenerated plants were tetraploids. Molecular analysis

using 23 simple sequence repeat (SSR) markers previously

proven to map to the citrus genome showed that the nuclear

DNA from all recovered diploid and tetraploid plants

derived from their corresponding leaf parent, while cleaved

amplified polymorphic sequence analysis showed that the

mtDNA of all regenerated plants derived from the callus

parent, indicating that the regenerated 2X and 4X plants

from all these three combinations are authentic cybrids.

Furthermore, the Chloroplast SSR analysis revealed that

somatic cybrid plants from the three combinations

possessed either of their parental chloroplast type in most

cases. These results demonstrated that mtDNA of G1 Sat-

suma mandarin was successfully introduced into the three

seedy sweet orange cultivars for potential seedlessness via

symmetric fusion.

Keywords Citrus � Cytoplasmic male sterility �Molecular markers � Protoplast fusion � Seedlessness �Somatic cybrid

Abbreviations

CAPS Cleaved amplified polymorphic sequence

CMS Cytoplasmic male sterility

cp-SSR Chloroplast simple sequence repeat

DAPI 40-6-Diamidino-2-phenylindole

G1 C. unshiu Marc. cv. Guoqing No. 1

mtDNA Mitochondrial DNA

SSR Simple sequence repeat

Introduction

For citrus, especially for seedy species such as those of

sweet orange (C. sinensis) and pummelo (C. grandis),

seedlessness has always been a prior goal of breeding.

Seedless Satsuma mandarin with cytoplasmic male sterility

(CMS) trait is a superior material for seedless breeding

(Yamamoto et al. 1997). However, it is difficult to transfer

its CMS to other seedy citrus cultivars by conventional

breeding, as breeders may encounter barriers such as

nucellar polyembryony and long juvenility of most citrus

cultivars (Cai et al. 2009; Grosser and Gmitter 2011),

which may be overcome by somatic hybridization. For

citrus, by the highly successful model of fusing

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11240-013-0384-1) contains supplementarymaterial, which is available to authorized users.

S.-X. Xiao � M. K. Biswas � M.-Y. Li � X.-X. Deng � Q. Xu �W.-W. Guo (&)

Key Laboratory of Horticultural Plant Biology

(Ministry of Education), Huazhong Agricultural University,

Wuhan 430070, China

e-mail: guoww@mail.hzau.edu.cn

123

Plant Cell Tiss Organ Cult (2014) 116:81–88

DOI 10.1007/s11240-013-0384-1

embryogenic suspension derived protoplasts with leaf-

derived protoplasts, somatic hybrid plants have been cre-

ated from nearly 500 different parental combinations to

date (Grosser and Gmitter 2011). Somatic hybridization

played an important role in citrus seedless breeding

because allotetraploid somatic hybrids are superior breed-

ing parents in interploid crosses for seedless triploid

breeding (Dambier et al. 2011; Grosser and Gmitter 2011).

Meanwhile, citrus somatic cybrids were regenerated from

over 40 parental combinations by standard symmetric

fusion (reviewed by Guo et al. 2013).

A cybrid is a cytoplasmic hybrid possessing the nucleus

of one species and the mitochondria and/or chloroplast of

another species (Bassene et al. 2008). Cytoplasm may alter

the expression of nuclear genes through a communication

termed retrograde signaling (Bassene et al. 2011) and

cytoplasm substitution could have positive effects on

important agronomic traits such as CMS (Yamamoto et al.

1997), aroma (Fanciullino et al. 2005), fruit taste and

nutritional quality (Bassene et al. 2008). In current culture

conditions for citrus protoplasts, since the regenerated cy-

brids usually possess the mitochondria DNA of the sus-

pension parent (Saito et al. 1993; Grosser et al. 1996;

Moriguchi et al. 1996; Moreira et al. 2000; Cabasson et al.

2001; Fanciullino et al. 2005; Bassene et al. 2011; Guo

et al. 2006, 2013), targeted cybridization to create cybrids

with transferred mitochondrial DNA (mtDNA) controlled

CMS to seedy citrus cultivars from Satsuma mandarin (C.

unshiu Marc.) as the embryogenic callus parent was put

forward and realized (Guo et al. 2004, 2013; Cai et al.

2007, 2009). The cybrid of G1 ? HBP (Citrus grandis cv.

Hirado Buntan Pink pummelo) reported by Guo et al.

(2004) has recently overcome juvenility, and encourag-

ingly male sterile character was realized (Zheng et al.

2012). It indicated that transferring mtDNA controlled

CMS to seedy citrus cultivars by simply performing

symmetric fusion with Satsuma mandarin as the embryo-

genic callus parent could realize male sterility and create

seedless fruit (Fig. 1).

Sweet orange is the most popular and large-scale planted

elite species in the world, but most cultivars of ordinary

sweet orange are seedy. It is tricky to obtain sexual hybrids

between Satsuma mandarin and sweet orange because of

their polyembryony (An et al. 2008). However, somatic

cybrids with potential seedlessness between these two cit-

rus species have been created by protoplast fusion (Cai

et al. 2007). The aim of this work was to continuously

obtain more cybrids with potential seedless trait for citrus

scion improvement by fusing seedy sweet oranges with

Satsuma mandarin.

Materials and methods

Materials

A callus line of ‘Guoqing’ NO.1 Satsuma mandarin (G1)

was maintained on solid MT (Murashige and Tucker 1969)

basal medium containing 40 g/L sucrose and 8 g/L agar

(pH 5.8). Suspensions cultures were established and

maintained as previously described (Fu et al. 2004), with

protoplast isolation during days 4–10 after three subculture

cycles. Seeds of three sweet orange cultivars (C. sinensis)

i.e. ‘Early gold’, ‘Taoye’ and ‘Hongjiang’ were germinated

on MT basal medium containing 25 g/L sucrose and 8 g/L

agar (pH 5.8), and the fully expanded leaves were used for

mesophyll protoplast isolation.

Protoplast fusion and plant regeneration

Protoplast isolation was carried out according to Grosser

and Gmitter (1990). After being purified by 13 % mannitol/

Fig. 1 The strategy to create seedless cybrid by transferring Satsuma CMS to seedy varieties by symmetric fusion

82 Plant Cell Tiss Organ Cult (2014) 116:81–88

123

25 % sucrose gradient centrifugation, protoplasts of G1

callus were mixed with those of ‘Early gold’, ‘Taoye’ or

‘Hongjiang’ sweet oranges respectively at a one:two ratio.

Protoplast fusion was mediated using a somatic hybridizer

SSH-2 instrument (Shimadzu Somatic Hubridizer-2, Shi-

madzu, Japan) equipped with a 1.6 ml FTC-04 type elec-

trofusion chamber. The protoplasts of the three fusion

combinations were fused electrically following the proce-

dure of Cai et al. (2007). After electrofusion, protoplasts

from G1 ? ‘Early gold’, G1 ? ‘Taoye’ and G1 ? ‘Hon-

gjiang’ were resuspended in BH3 medium (Grosser and

Gmitter 1990) and adjusted to a density of 1 9 105/ml,

then cultured in BH3 medium. Protoplast culture and plant

regeneration were carried out according to Guo and Deng

(1998).

Ploidy analysis

The ploidy level of all regenerated plants was determined

by a flow cytometer (Cyflow space, Munster, Germany)

following the protocol described by Guo et al. (2007). The

root-tips of regenerated plants were collected for chromo-

some counting by staining with 40-6-diamidino-2-pheny-

lindole (DAPI) according to Xu et al. (2013). Image

acquisition was carried out with a fluorescent microscope

(Olympus BX 61, Japan) fitted with a CCD camera DP70,

with the excitation wavelength set as 360–370 nm, and

emission wavelength as 420 nm.

Molecular analysis of nuclear genome and cytoplasmic

DNA

Total DNA was isolated from the young leaves of regen-

erated plants and their corresponding parents following an

efficient protocol described by Cheng et al. (2003). The

PCR reaction was performed in a MJ-PTC-200 thermal

PCR cycler (MJ Research, Waltham Mass) according to

Cheng et al. (2006). Amplification products of SSR and

cpSSR analysis were denatured at 94 �C for 5 min, and

then an aliquot (4 ll) of each sample was analyzed by

6.0 % (w/v) denaturing polyacrylamide gels, followed by

silver staining of the gel as the protocol described in the

technical manual of Promega Corporation (USA).

Molecular analysis was performed using 126 SSR

primers mapping to the genome of sweet orange (http://

citrus.hzau.edu.cn/orange). Finally, six previously pub-

lished and 17 new primers from our program (Supple-

mental Table 1) that revealed polymorphism among the

corresponding parents were used to amplify nuclear DNA

according to Cheng et al. (2003). Locations of TAA15 and

the 17 new primers on sweet orange chromosomes were

confirmed by electronic PCR. At least two primer pairs

from each chromosome as well as some markers located in

multiple chromosomes (Supplemental Table 1) that

revealed polymorphism among the corresponding parents

were selected. To determine the inheritance pattern of

chloroplast DNA (cpDNA), two pre-screened primer pairs

NTCP9 and ARCP5 (Supplemental Table 2) were used for

cpSSR analysis.

For CAPS analysis of the mitochondrial genome of

regenerated plants and their corresponding parents,

mtDNA amplification using three mitochondrial primers

(Supplemental Table 2) was performed in a MJ-PTC-200

thermal PCR cycler (MJ Research, Waltham Mass).

Amplification fragments were digested as described by Cai

et al. (2007), and four different restriction endonucleases

AluI, MobI, TaqI and TasI were used.

Results

Protoplast culture and plant regeneration

After 40–90 days of protoplast culture, abundant callus

masses and a few viridescent embryoids appeared from the

three fusion combinations. Embryos were recovered from

all three combinations and transferred to EME1500 solid

medium to expedite growth and development. A large

number of green and vigorous shoots were developed from

the three fusion combinations after one to 2 months of

shoot-induction culture, and only one shoot from each

embryoid generated from the three fusion combinations

was transferred to root induction medium. Finally, 13

plants were recovered from the combination of

G1 ? ‘Early gold’, and they were classified into two

groups according to their leaf morphology, i.e., one of the

plants had round and thick leaves (Fig. 3c); while the

remaining 12 plants had leaf morphology similar to ‘Early

gold’ (Fig. 3c). As for the combination G1 ? ‘Taoye’, 12

plants were recovered, in which three had round and thick

leaves (Fig. 4c), and nine had leaf morphology similar to

‘Taoye’ (Fig. 4c). Only two plants with leaf morphology

similar to ‘Hongjiang’ were recovered from G1 ? ‘Hon-

gjiang’ (Table 1). All recovered plants were transferred to

greenhouse where they grow normally and vigorously

(Figs. 3a, b, 4a, b, 5a).

Ploidy level of recovered plants

Flow cytometry analysis confirmed that, of the regenerated

plants of the G1 ? ‘Early gold’, the regenerated plant with

round and thick leaves was tetraploid (Fig. 2c), the

remaining 12 plants had leaf morphology similar to ‘Early

gold’ were diploid. Similarly, of the regenerated plants of

G1 ? ‘Taoye’, three plants having round and thick leaves

were tetraploid (Fig. 2d), the remaining nine plants with

Plant Cell Tiss Organ Cult (2014) 116:81–88 83

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leaf morphology similar to ‘Taoye’ were diploid. As

expected, the two regenerated plants of G1 ? ‘Hongjiang’

were diploid (Fig. 2b). Moreover, the root-tips of regen-

erated plants from G1 ? ‘Hongjiang’ were collected for

chromosome counting, which confirmed their diploidy

(2n = 2x = 18) (Fig. 5b).

Molecular analysis of recovered plants

All regenerated plants of the three fusion combinations

were selected for analysis of nuclear and cytoplasmic

genomes. Polymorphisms between the corresponding

fusion parents of the three combinations were detected

using SSR analysis with the 23 selected primer pairs. First,

TAA15 was applied to analyze the nuclear genome of the

regenerated plants of the three fusion combinations. The

result showed that among the recovered plants of any of the

three fusion combinations, irrespectively of their ploidy

level, all possess the same banding pattern as their meso-

phyll leaf parent, with no specific band of their suspension

parent G1 (data not shown), indicating that the nuclear

genome of hybrid plants obtained in our study derive from

their mesophyll leaf parent at TAA15 locus.

To further confirm their nuclear genome inheritance, 22

primers that had been mapped to the genome of sweet

orange were used for SSR analysis. According to their

distribution, at least two primer pairs locating on each

chromosome while some markers located in multiple

chromosomes (Supplemental Table 1) were applied in

nuclear genome analysis on the regenerated plants and their

parents. The results showed that all the recovered plants

from the three fusion combinations exhibited the same

banding pattern as their corresponding mesophyll parent

(Figs. 3f–h, 4e–h, 5e–h) using any of the 22 primer pairs.

Thus, our previous conclusion that all the recovered plants

in our study inherited their nuclear genome from their

corresponding mesophyll parent was confirmed.

The mtDNA primer pair/enzyme combination of 18S

rRNA-5S rRNA/TasI was used to analyze the mtDNA of

all the recovered plants as this primer pair/enzyme com-

bination revealed polymorphisms among the corresponding

parents. As a result, all the recovered plants (both diploid

and tetraploid plants) from the three combinations showed

the same banding pattern as their common suspension

parent G1 (Figs. 3d, 4d, 5c).

For the inheritance analysis of cpDNA, the primer pair

NTCP9 was used for cp-SSR analysis. The results showed

that the cpDNA of recovered plants from the three com-

binations possessed a random segregation pattern. For the

combination of G1 ? ‘Early gold’, four of twelve diploid

plants analyzed derived their cpDNA from ‘Early gold’,

while the other eight diploid plants and the tetraploid plant

analyzed derived their cpDNA from G1 (Fig. 3e). For the

combination of G1 ? ‘Taoye’, six of nine diploid plants

and all the three tetraploid plants analyzed derived their

cpDNA from ‘Taoye’, the remaining three diploid plants

Table 1 Parental combinations and number of recovered plants

Suspension

parent

Leaf parent Number of

recovered plants

Ploidy

level

G1 ‘Early gold’ sweet orange 12 2X

1 4X

‘Taoye’ sweet orange 9 2X

3 4X

‘Hongjiang’ sweet orange 2 2X

Fig. 2 Ploidy analysis by flow cytometry. a G1 (as a control);

b regenerated plants of G1 ? ‘Hongjiang’ sweet orange (PK1);

c regenerated plants of G1 ? ‘Early gold’ sweet orange, diploid

(PK1) and tetraploid (PK2); d regenerated plants of G1 ? ‘Taoye’

sweet orange, diploid (PK1) and tetraploid (PK2)

84 Plant Cell Tiss Organ Cult (2014) 116:81–88

123

analyzed derived their cpDNA from G1 (Fig. 4i). For the

combination of G1 ? ‘Hongjiang’, both diploid plants

derived their cpDNA from ‘Hongjiang’ (Fig. 5d).

In summary, all the recovered plants (both diploid and

tetraploid) from the three combinations derived their

nuclear DNA from their mesophyll parents, and mtDNA

from their common suspension parent G1, but the cpDNA

possessed a random pattern of segregation. The results

indicated that all the recovered plants from the three

combinations are true cybrids.

Discussion

Previous reports have demonstrated that in citrus cybrids

the nucleus always derives from their corresponding leaf

parent while mitochondria genome comes from their cor-

responding suspension parent (Guo et al. 2004; Fanciullino

et al. 2005; Guo et al. 2006; Cai et al. 2007; Bassene et al.

2011). However, few SSR primers were used to analyze the

nuclear genome preciously. In this study, 23 SSR primers

were used to analyze the nuclear genome. The results

showed that all the regenerated cybrids exhibited the same

banding pattern as their corresponding mesophyll parent to

all the SSR primers, indicating that all the regenerated

plants (both diploid and tetraploid) are true cybrids.

The molecular character of cybrids provides a novel

approach for citrus breeding. In citrus, cybridization can be

used as a strategy to transfer specific traits associated with

mitochondrial genome such as CMS without affecting the

main organoleptic and nutritional qualities (Bassene et al.

2008). Since most citrus species exhibit some level of

parthenocarpy, seedless fruits can develop normally. The

cybrid of G1 ? HBP (Guo et al. 2004) has male sterile

character and seedless fruits can set when planted in an

isolated area (Zheng et al. 2012). Sweet orange is a large-

scale planted elite species in the world’s citriculture, but

most varieties of ordinary sweet orange are seedy. Since

sweet orange is self-compatible and parthenocarpic, the

fusion of Satsuma mandarin with sweet orange will

regenerate cybrids with potential seedlessness. In this

study, 12, nine and two diploid cybrids were regenerated

Fig. 3 Leaf morphology and molecular analysis of regenerated plants

from G1 ? ‘Early gold’ sweet orange. a diploid cybrid plant;

b tetraploid cybrid plant; c Leaf morphology of diploid cybrid (right)

and tetraploid cybrid (left) of G1 ? ‘Early gold’ sweet orange; d mt-

CAPS analysis of regenerated plants from G1 ? ‘Early gold’ sweet

orange by universal primer of 18S rRNA-5S rRNA and digested with

TasI; e cp-SSR analysis of regenerated plants from G1 ? ‘Early gold’

sweet orange by NTCP9; f–h Nuclear SSR analysis of regenerated

plants from G1 ? ‘Early gold’ sweet orange by Csin.0349 (f),M1H2Si16887 (g) and M3H3Si763 (h). Lanes: M: 100 bp DNA

ladder; 1 ‘Early gold’ sweet orange; 2–13 diploid cybrids; 14

tetraploid cybrid; 15 G1 Satsuma mandarin. Bars 5 cm (a, b, c)

Plant Cell Tiss Organ Cult (2014) 116:81–88 85

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from G1 ? ‘Early gold’, G1 ? ‘Taoye’ and G1 ? ‘Hon-

gjiang’ respectively. These diploid cybrids hold great

potential for the development of new seedless variety.

To date, citrus cybrids have been created in many fusion

combinations, but the regeneration mechanism is still not

clear. A possible mechanism is that after one mesophyll

protoplasts fuses with one suspension protoplast, the

nucleus of the suspension protoplast was eliminated during

the regeneration progress (Grosser et al. 1996). Incorpo-

ration into mesophyll protoplasts of mitochondria released

from ruptured embryogenic cells may be the second

mechanism for cybridization (Grosser et al. 1996). Both

mechanisms are possible and none is conclusive. Previous

reports suggested that somatic hybridization with G1 as the

embryonic callus parent had potential to facilitate regen-

eration of diploid cybrids (Cai et al. 2007). In fact, there

have been six diploid cybrids regenerated from the com-

bination with G1 as suspension parent (Guo et al. 2004; Cai

et al. 2007, 2009). In this study, all three fusion

combinations between G1 and different sweet orange cul-

tivars regenerated diploid cybrids.

Diploid cybrid regeneration via somatic hybridization in

citrus is not rare, but there were few reports on regeneration

of tetraploid cybrid-plants with doubled nuclear genome

from one parent and foreign cytoplasmic genome (Tusa

et al. 1990; Louzada et al. 1992; Grosser et al. 1996; Guo

et al. 2006; An et al. 2008; Grosser et al. 2010). The rareness

of tetraploid cybrids (that can be considered as autotetra-

ploid plants of the leaf parent with foreign cytoplasmic

genome) could be due to the fact that autotetraploids face

greater regeneration barriers than allotetraploid plants,

which showed some degree of hybrid vigor. In this study,

one and three tetraploid cybrids were regenerated from

G1 ? ‘Early gold’ and G1 ? ‘Taoye’, respectively. Inter-

estingly, for the combination of G1 ? ‘Taoye’, allotetra-

ploid somatic hybrids were also regenerated in our previous

fusion experiment (Fu et al. 2011) while in this study, both

diploid and tetraploid cybrids were regenerated. The

Fig. 4 Leaf morphology and molecular analysis of regenerated plants

from G1 ? ‘Taoye’ sweet orange. a diploid cybrid plant; b tetraploid

cybrid plant; c Leaf morphology of diploid cybrid (right) and tetraploid

cybrid (left) of G1 ? ‘Taoye’ sweet orange; d mt-CAPS analysis of

regenerated plants from G1 ? ‘Taoye’ sweet orange by universal

primer of 18S rRNA-5S rRNA and digested with TasI; e–h Nuclear SSR

analysis of regenerated plants from G1 ? ‘Taoye’’ sweet orange by

Csin.0191 (e), M1H2Si16887 (f), Csin.0349 (g) and M3H3Si763 (h);

i cp-SSR analysis of regenerated plants from G1 ? ‘Taoye’ sweet

orange by NTCP9. Lanes: M: 100 bp DNA ladder; 1 ‘Taoye’ sweet

orange; 2–10 diploid cybrids; 11–13 Tetraploid cybrids; 14 G1 Satsuma

mandarin. Bars 5 cm (a, b, c)

86 Plant Cell Tiss Organ Cult (2014) 116:81–88

123

regeneration mechanism of tetraploid cybrid remains poorly

understood. A hypothesis is that two mesophyll protoplasts

and one embryogenic protoplast were fused, and then the

nucleus of embryogenic protoplast was eliminated during

the regeneration progress (Grosser et al. 1996; Guo et al.

2006). But the most likely origin for tetraploid cybrid

regeneration might be that some seedlings for mesophyll

protoplast isolation are tetraploid themselves, since natural

nucellar autotetraploid development is common and exten-

sive across different citrus cultivars and species according to

Aleza et al. (2011). Similarly, our laboratory has recently

screened many natural nucellar autotetraploid plants from

over 20 different citrus cultivars and species (including

‘Early gold’ sweet orange used as leaf parent in this study)

by initial seedling morphology screening and subsequent

flow cytometry analysis (unpublished data).

Although the regeneration mechanism of diploid and

tetraploid cybrids is not clear, the cybrids have shown great

potential in citrus cultivar improvement, because cyto-

plasmic substitution could have positive effects on such

important agronomic traits as CMS (Yamamoto et al.

1997), aroma (Fanciullino et al. 2005), fruit taste and

nutritional quality (Bassene et al. 2008). Abbate et al.

(2012) analysed the fruits of hybrid plants obtained by

protoplast fusion of ‘Valencia’ sweet orange ? ‘Femmi-

nello’ lemon (embryogenic protoplast ? leaf protoplast),

and suggested that both diploid and tetraploid cybrids of

‘Valencia’ ? ‘Femminello’ could be new successful vari-

eties. In addition, the cybrid between Dancy mandarin and

Ruby grapefruit, which derived the nucleus from Ruby

grapefruit and the cytoplasm from Dancy mandarin, is very

late maturing by remaining firm with exceptional sweet-

ness and good flavor into August (Dr. Jude Grosser, per-

sonal commun.), indicating that cytoplasm substitution

could also have positive effects on such important agro-

nomic traits as harvest season. Thus, the cybrids of these

three combinations may show some attractive traits in

addition to seedlessness, and both diploid and tetraploid

cybrids could be new successful varieties.

In conclusion, the regenerated cybrid plants from these

three combinations provided precious materials for citrus

breeding. The diploid cybrids of these combinations hold

potential as new seedless varieties which will maintain the

good quality of their corresponding leaf parents. And the

tetraploid cybrids of G1 ? ‘Early gold’ and G1 ? ‘Taoye’

could also be new successful varieties or used as tetraploid

breeding parents.

Acknowledgments This research was financially supported by the

Ministry of Science & Technology of China (nos. 2011CB100606,

2011AA100205), the National NSF of China (nos. 31125024,

31221062), and the Fundamental Research Funds for the Central

Universities (No. 2013PY045).

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