Botanical Studies (2008) 49: 295-300.

*

Corresponding author: E-mail: guoww@mail.hzau.edu.cn;

Tel: +86-27-87281543; Fax: +86-27-87280016.

INTRODUCTION

Somatic hybridization has been an effective and

successful tool for plant improvement (Davey et al., 2005)

and is still being applied in several crops such as cotton

(Sun et al., 2004), potato (Trabelsi et al., 2005), rapeseed

(Wang et al., 2005) and wheat (Chen et al., 2004). The

somatic hybridization technique is especially useful in

circumventing the natural polyembryony and pollen/ovule

sterility of perennial citrus crops. During the past two

decades, numerous somatic hybrids have been produced

and evaluated for cultivar improvement (Guo et al., 2004a;

Grosser and Gmitter, 2005).

These novel germplasm resources are also valuable for

plant somatic cell genetics research by molecular markers.

Molecular analysis could not only help us understand

the nucleus-nucleus, nucleus-cytoplasm, and cytoplasm-

cytoplasm interaction between both fusion parents, but it

could also be conducive to the correlation of phenotypic

performance or specific traits with the concrete nuclear

and cytoplasmic composition of these novel hybrids. For

their molecular evaluation, RAPD (random amplified

polymorphic DNA) and RFLP (restriction fragment length

polymorphism) were previously widely applied, but newly

developed simpler and more efficient molecular markers

such as SSR (simple sequence repeat), CAPS (cleaved

amplified polymorphic sequence), and chloroplast SSR

are currently utilized (Lotfy et al., 2003; Guo and Grosser,

2005; Takami et al., 2005; Wu et al., 2005). Genetic

information of plant mitochondria has been the subject of

intensive research work (Cheng et al., 1997; Cheng and

Dai., 2000; Huang et al., 2003; Lo et al., 2003; Wang et al,

2007). Herein, we report the molecular characterization of

somatic hybrids between sweet orange and rough lemon by

novel markers where recombination of the mitochondria

genome was revealed for the first time by CAPS analysis

and DNA sequencing.

MATERIALS AND METHODS

Plant materials

The protoplast fusion and regeneration of somatic

hybrid plants between Bonanza sweet orange (Citrus

sinensis Osbeck) and rough lemon (C. jambhiri) were

described and detailed by Guo and Deng (2000). These

plants are six-years-old and are maintained in the

germplasm field of the National Citrus Breeding Center,

Huazhong Agricultural University, Wuhan.

Analysis of mitochondrial genomes in Citrus

interspecific somatic hybrids produced by protoplast

fusion

Wen-Wu GUO*, Reng-Chao WU, Gai-En FAN, and Yun-Jiang CHENG

National Key Laboratory of Crop Genetic Improvement, National Center of Crop Molecular Breeding, Huazhong

Agricultural University, Wuhan 430070, P.R. China

(Received December 14, 2006; Accepted April 9, 2008)

ABSTRACT.

Somatic hybrids produced via protoplast fusion are valuable germplasm for citrus improvement.

Herein, four randomly selected six-year-old interspecific somatic hybrid plants between Bonanza sweet orange

(Citrus sinensis Osbeck) and rough lemon (C. jambhiri), were thoroughly analyzed. Flow cytometry and

nuclear SSR analysis confirmed them as true tetraploid somatic hybrids. Chloroplast SSR analysis showed

the random inheritance nature of chloroplast DNA. PCR amplification of mitochondrial genome by universal

primer pair showed that the somatic hybrids had the specific band from rough lemon (the leaf parent) while

all samples shared a common band. CAPS analysis by further Ta sI restriction endonuclease cut of the PCR

products, however, revealed the band specific to Bonanza orange, the embryogenic callus parent, was also

present in all these analyzed hybrids. Further sequencing of the common band and searching for restriction

endonuclease recognition sites well explained the banding pattern by CAPS analysis. It was concluded that

mitochondrial recombination in citrus somatic hybrids occurred as revealed by CAPS analysis and DNA

sequencing.

Keywords: Citrus; Cleaved amplified polymorphic sequence (CAPS); Cytoplasmic genome; DNA sequencing;

Simple sequence repeat (SSR); Somatic hybrids.

MOLECULAR BIOLOgy

296

Botanical Studies, Vol. 49, 2008

Flow cytometry analysis

Ploidy analysis of these plants was conducted by

Partec flow cytometry (D-48161 Munster Germany).

Approximately 1 cm

2

of young leaf was chopped in a

plastic Petri dish containing 0.4 ml Partec HR-A buffer

(Partec high resolution nuclei extraction solution). After

being filtered, the samples were stained with 0.8 ml

of HR-B buffer (Partec high resolution DAPI staining

solution), and the relative fluorescence of total DNA was

measured. Each histogram was generated by analyzing at

least 3000-5000 nuclei.

DNA extraction, nuclear SSR and chloroplast

SSR analysis

Total DNA was extracted following a modified CTAB

procedure (Cheng et al., 2003b). SSR analysis of nuclear

genome was conducted using primer pairs TAA1 (primer

1: 5.-GAC AAC ATC AAC AAC AGC AAG AGC-3.;

primer 2: 5.-AAG AAG AAG AGC CCC CAT TAG

C-3.), and TAA3 (primer 1: 5.-AGA GAA GAA ACA

TTT GCG AGC-3.; primer 2: 5.-GAG ATG GGA CTT

GGT TCA TCA CG-3.) (Kijas et al., 1997). One pre-

screened chloroplast SSR primer pair SPCC1 (primer 1:

5.-CTT CCA AGC TAA CGA TGC-3.; primer 2: 5.-CTG

TCC TAT CCA TTA GAC AAT G-3.) was also applied to

reveal chloroplast inheritance (Cheng et al., 2005). SSR

and chloroplast SSR reaction conditions were according to

Cheng et al. (2005). The products were analyzed on 6.0%

(w/v) denaturing polyacrylamide gels, then the gels were

silver-stained according to the protocol of the technical

manual on silver sequence DNA staining reagents

(Promega, Madison, Wis.).

Mitochondrial DNA analysis by CAPS

For CAPS analysis, one prescreened mitochondrial

universal primer pair, i.e. 18S rRNA-5S rRNA (forward:

5.-GTG TTG CTG AGA CAT GCG CC-3., reverse: 5.

-ATA TGG CGC AAG ACG ATT CC-3.) (Cheng et al.,

2003a) was selected, and PCR reaction was performed in

a PTC-200 thermocycler (Bio-Rad). The PCR reaction

mixture (50 £gl) consisted of 67 mM Tris-HCl (pH 8.8), 16

mM (NH

4

)

2

SO

4

, 0.01% Tween 20, 0.2 £gM of each primers

and 300 £gM of dNTP, 1.5 to 2.5 mM MgCl

2

, 1.5 unit of

Ta q DNA polymerase and 100 ng of sample DNA. The

amplification parameters were: 1 initial denaturing cycle at

94¢XC for 3 min, 32 cycles of 1 min denaturing at 94¢XC, 40

s annealing at 55¢XC, 2 min elongation at 72¢XC, a final step

of 10 min at 72¢XC, then storage at 4¢XC. DNA digestion: 5

to 8 £gl of the PCR products were digested with 5 units of

the HinfI, MspI and Ta sI restriction endonucleases (MBI

Fermentas) at 37¢XC or 65¢XC for 4 h. For DNA analysis,

the digested DNA samples were electrophoresed in 2.0%

agarose gel with 1

¡Ñ

TBE and 5 £gg/ml ethidium bromide at

2 V/cm for 3 to 4 h, then photographed under UV light.

DNA sequence analysis

The common band of the PCR products generated by

mitochondrial universal primer pair 18S rRNA-5S rRNA

was extracted with an EZNA Gel Extraction Kit (Omega

Biotek, Doraville, GA). The DNA samples obtained were

ligated into the pMD 18-T Vector (Takara Biotech, Dalian,

China) and transformed into Escherichia coli strain

DH5

£\

, and then sequenced by the United Gene Company

(Shanghai, China). DNA sequence comparison was

analyzed by using ClustalW Multiple Sequence Alignment

(dot.imgen.bcm.tmc.edu). Number and sites of restriction

endonuclease Ta s I cut of these sequence samples were

compared.

RESULTS

These somatic hybrid plants had been growing in the

field for more than six years. They were verified to be

tetraploids by chromosome counting (Guo and Deng,

2000). To evaluate their ploidy stability, flow cytometry

analysis was conducted, and they were confirmed as stable

tetraploids (Figure 1).

The nuclear origin of these regenerates was determined

by SSR analysis with two primer pairs (TAA1, TAA3),

which could distinguish both parents. All the four analyzed

plants had specific bands from both parents (Figure 2).

Flow cytometry and nuclear SSR analysis confirmed these

plants were true allotetraploid somatic hybrids.

To reveal the cytoplasmic genome composition of

these hybrid plants, chloroplast SSR and mitochondria

CAPS analysis were conducted. Chloroplast SSR analysis

indicated that primer pair SPCC1 distinguished the parents

well. Among the four analyzed plants, No. 2 hybrid had the

chloroplast DNA from Bonanza orange, and that of Nos.

1, 3 and 4 was from rough lemon (Figure 3), suggesting

chloroplast DNA was randomly inherited in these hybrids.

Mitochondria CAPS analysis of these four hybrid plants

with mitochondria primer pair, i.e. 18S rRNA-5S rRNA

was performed. Polymorphism was detected even without

Figure 1. Ploidy determination by flow cytometry analysis. A:

diploid control (leave mixture of Bonanza sweet orange and rough

lemon); B: tetraploid somatic hybrids between sweet orange and

rough lemon.

GUO et al. ¡X Mitochondrial genome analysis of

Citrus

somatic hybrids

297

Figure 2. SSR analysis of nuclear genome in somatic hybrids and their parental genotypes. Lane 1: rough lemon, 2-5: somatic hybrids No.

1 to No. 4, 6: Bonanza navel orange. Primer pair for A: TAA1, and B: TAA3. Arrows indicate specific bands.

Figure 3. Chloroplast SSR analysis by chloroplast primer pair

SPCC1. Lane 1: rough lemon, 2-5: somatic hybrids No. 1 to No. 4,

6: Bonanza navel orange. Arrows indicate specific bands.

restriction endonuclease digestion, where the banding

pattern of these somatic hybrids was identical to rough

lemon, the leaf parent; meanwhile all samples shared

a common band of about 1,100 bp (Figure 4A). After

the PCR product was further digested with restriction

endonuclease Ta s I, the band specific to Bonanza sweet

orange, the embryogenic callus parent, was revealed and

was present in all four analyzed hybrid plants (Figure 4B).

To further explain the mtDNA banding pattern

following Ta sI cut, the approximately 1,100 bp common

band was extracted and sequenced for both parents and

somatic hybrids No. 1 and No. 2. It showed that the band

is 1,069 bp in size for Bonanza navel orange, somatic

hybrids No. 1 and No. 2 while that for rough lemon is

1,064 bp. There are some single nucleotide mutations in

these sequences. Searching for Ta sI recognition site of 5.-

.

AATT-3. in the DNA sequence revealed that Bonanza

navel orange, somatic hybrids No. 1 and No. 2 had three

recognition sites producing four fragments of 81 bp,

412 bp, 173 bp and 403 bp while rough lemon, with one

Figure 4. Mitochondrial DNA analysis using universal primer pair 18S rRNA-5S rRNA. M: 100 bp DNA ladder, Lane 1: rough

lemon, 2-5: somatic hybrids No. 1 to No. 4, 6: Bonanza navel orange. A: before restriction endonuclease Ta s I cut; B: after restriction

endonuclease Ta s I cut. The boxed bands in Figure 4A are the same as the boxed bands in Figure 4B. The size of DNA ladder (left of

Figure 4A) and that of the specific bands after Ta sI cut (right of Figure 4B) were indicated.

298

Botanical Studies, Vol. 49, 2008

Ta s I recognition site mutated, had two recognition sites

producing three fragments of 81 bp, 412 bp and 571 bp,

respectively (Table 1; DNA sequences not shown). The

sequencing information explained the banding pattern of

CAPS analysis well since the 412 bp and 403 bp fragments

were too close in size to be apart, and they appeared as

one band by agarose gel electrophoresis (Figure 4B). DNA

sequencing combined with CAPS analysis suggested that

mtDNA recombination occurred in these somatic hybrids.

DISCUSSION

Nuclear and cytoplasmic inheritance analysis

was conducted on these plants produced by somatic

hybridization. In our previous research, primer pairs

showing good polymorphism for nuclear SSR, chloroplast

SSR and CAPS analysis were screened (Cheng et al.,

2002, 2003a, 2005), some of which showed good results

in this research. Compared with previously routinely used

RFLP analysis with labeled probes, CAPS is simpler, more

rapid, and less expensive (Bastia et al., 2001; Guo et al.,

2004b). Meanwhile, the results by CAPS technique were

proved reliable and agreeable to that of RFLP (Cheng et

al., 2002). Furthermore, compared with CAPS analysis,

chloroplast SSR is even more convenient and efficient

since enzyme cutting following PCR reaction is not

needed (Provan et al., 2001; Cheng et al., 2005).

Herein, DNA sequencing revealed novel information

for mitochondrial genome variation in citrus somatic

hybrids. DNA sequencing combined with searching for

restriction endonuclease recognition sites explained the

banding pattern of CAPS analysis. DNA sequencing can

provide direct and reliable genetic information while

CAPS is based on fragment length polymorphism and

much work as well as money is needed to screen for

restriction endonucleases that can produce polymorphic

fragments. Since DNA sequencing is currently quite cheap

and convenient, for CAPS analysis, it might be better

to sequence the common band and search for restriction

endonuclease recognition sites that could produce

polymorphic fragments before conducting restriction

endonuclease cutting and agarose gel electrophoresis,

thus circumvent the tedious and expensive work of

screening for restriction endonucleases. DNA sequencing

combined with further CAPS analysis could also be done

in confirmation of each other.

The fusion model of "embryogenic callus protoplasts +

mesophyll protoplasts" was widely adopted in citrus

protoplast fusion. Previous molecular analysis on many

citrus somatic hybrids revealed that their mitochondrial

genome was usually non-randomly inherited from their

corresponding embryogenic callus parent while their

chloroplast genome was randomly inherited (Guo et al.,

2004a). The result presented herein was consistent with

this rule for chloroplast genome; but for the mitochondrial

genome, the result of this fusion was an exception since

mitochondrial recombination was revealed in all four

analyzed hybrid plants. This is also the first report on

mitochondrial recombination in citrus somatic hybrids

as revealed by CAPS markers and DNA sequencing.

Mitochondrial recombination was only revealed previously

by RFLP analysis in a few plants from three intergeneric

(Motomura et al., 1995; Cheng et al., 2003a) and two

interspecific fusions (Moriguchi et al., 1997; Olivares-

Fuster et al., 2005) in citrus.

For mitochondrial interaction after protoplast fusion

at the subcellular level, by fusing protoplasts containing

either green fluorescent protein or MitoTracker-labelled

mitochondria, Sheahan et al. (2005) reported the

phenomenon of massive mitochondrial fusion (MMF)

which leads to near-complete mixing of the mitochondrial

population within 24 h. MMF appears specific to

dedifferentiation, since it also occurs in mesophyll

protoplasts of Arabidopsis and Medicago but not in

protoplasts from already dedifferentiated cells such as

tobacco BY-2 or callus cultures. These results by Sheahan

et al. (2005) allow a clearer interpretation of how novel

mitochondrial genotypes develop following cell fusion.

Such research is also currently underway in our group

to reveal the mitochondria interaction at the subcellular

level following various kinds of protoplast fusion in

citrus, which may explain why dominantly non-random

mitochondrial inheritance and a few novel mitochondrial

genotypes occurred, following cell fusion in citrus.

Table 1. DNA sequence analysis of the approximately 1100 bp common band for both parents and two hybrids after Tas I restriction

endonuclease cut.

Fragment Nos.

Sample Nos. and size (bp)

Rough lemon Somatic hybrid No. 1 Somatic hybrid No. 2 Bonanza navel orange

Common band

1064

1069

1069

1069

Fragment 1

81

81

81

81

Fragment 2

412

412

412

412

Fragment 3

571

173

173

173

Fragment 4

/

403

403

403

GUO et al. ¡X Mitochondrial genome analysis of

Citrus

somatic hybrids

299

Acknowledgements. The research was financially

supported by the National Natural Science Foundation

of China (Nos. 30771481, 30571288), the Ministry

of Education of China (Nos. 705037, 20060504014,

IRT0548), and the 863 High Technology Program of

MOST (No. 2006AA100108).

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