SHORT COMMUNICATION

Selection of reference genes for quantitative real-time PCRduring flower bud development in CMS7311 of heading Chinesecabbage (Brassica rapa L. ssp. pekinensis)

Xiaoyong Xu • Zeping Yang • Xilu Sun •

Lugang Zhang • Zhiyuan Fang

Received: 4 May 2013 / Revised: 31 October 2013 / Accepted: 31 October 2013 / Published online: 20 November 2013

� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2013

Abstract The accuracy of quantitative real-time PCR

(qRT-PCR) depends on the stability of the reference gene

used for normalization. In heading Chinese cabbage (Bras-

sica rapa L. ssp. pekinensis), the most stable reference genes

for qRT-PCR during flower bud development have not been

elucidated. In this study, the statistical software geNorm was

used to test eight candidate reference genes during flower

bud development in male sterile (Ms) and fertile (Mf) plants.

The result revealed that the stability order was Tub/GAP-

DH [ Cyp [ EF1a [ U34559 [ BrTip41 [ Apr [ 18S

rRNA, Tub and GAPDH were the most stable genes [average

expression stability (M) 0.614], and the combined use of six

reference genes [pairwise variation (V) 0.15] was suggested

to be the optimal reference gene for qRT-PCR during flower

bud development. Furthermore, the expressions of

BcPME31 during flower bud development normalized with

the combined use of six reference genes and with GAPDH or

Tub alone were compared; the various results also suggested

that selection of the optimal reference gene was necessary for

gene expression analysis.

Keywords Flower bud development � Heading

Chinese cabbage � Male sterility � qRT-PCR �Reference gene selection

Abbreviations

CMS Cytoplasmic male sterility

GAPDH Glyceraldehyde-3-phosphate dehydrogenase

qRT-PCR Quantitative real-time polymerase chain

reaction

Ms Male sterile

Mf Male fertile

Rf Restorer gene

Introduction

The use of gene expression analysis is very important in

functional research, and this has led to the development of

various methods to quantify gene expression. Compared

with traditional methodologies such as Northern hybrid-

ization and quantitative competitive polymerase chain

reaction (PCR), the use of quantitative real-time PCR

Communicated by P. Sowinski.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11738-013-1437-0) contains supplementarymaterial, which is available to authorized users.

X. Xu � Z. Yang � X. Sun � L. Zhang (&) � Z. Fang

College of Horticulture, State Key Laboratory of Crop Stress

Biology in Arid Areas, Northwest A&F University, Yangling

712100, Shaanxi, People’s Republic of China

e-mail: lugangzh@163.com

X. Xu

e-mail: xuxy7926@163.com

Z. Yang

e-mail: zepingyang@qq.com

X. Sun

e-mail: xilusun2011@163.com

X. Xu

College of Horticulture, Shanxi Agriculture University, Taigu

030801, Shanxi, People’s Republic of China

Z. Fang (&)

Institute of Vegetables and Flowers, Chinese Academy of

Agricultural Sciences, Beijing 100081, People’s Republic of

China

e-mail: mengyf@mail.caas.net.cn

123

Acta Physiol Plant (2014) 36:809–814

DOI 10.1007/s11738-013-1437-0

(qRT-PCR) is more responsive, rapid, and provides high-

throughput results. This has leaded to its being widely used

to identify gene expression in various fields of biological

research. However, the accuracy of qRT-PCR is dependent

on the choice and stability of reference genes used for

normalization. Presently, a number of reference genes have

been widely used for qRT-PCR, including b-actin, glyc-

eraldehyde-3-phosphate dehydrogenase (GAPDH), 18S

rRNA, EF-1-a, ubiquitin, and a or b-tubulin (Gu et al.

2011; Qi et al. 2010). However, more and more studies

have suggested that no single reference gene is universal

for all experiments (Gutierrez et al. 2008; Tong et al. 2009;

Dong et al. 2012). This makes it important to choose the

optimal reference genes in each biological system before

using qRT-PCR to investigate gene expression.

Heading Chinese cabbage (Brassica rapa L. ssp. pekin-

ensis) is one of the most important leafy vegetables in Asia.

With the completion of the Chinese cabbage Chiifu-401-42

genome sequencing project, it follows that functional

genomics will become more significant in this species,

making transcript analysis both necessary and important.

Currently, several candidate reference genes have been

compared under drought stress and downy mildew infection

(Qi et al. 2010), but the stability of these genes in different

flower bud stages has not been verified. Furthermore, male

sterility is an unusual phenomenon in higher plants, and there

is no difference in phenotype from normal plants except for

the presence of dysfunctional or degenerate stamens. It was

reported that the expression of thousands of genes was

changed during flower stamen development (Alves-Ferreira

et al. 2007). Therefore, it is necessary to define the reference

gene for qRT-PCR before assessing the expression of genes

of interest during flower bud development in the male sterile

plant of heading Chinese cabbage.

In this study, the statistical software geNorm (Vande-

sompele et al. 2002) was used to evaluate the expression

stability of eight candidate reference genes during flower

bud development in male sterile (Ms) and male fertile (Mf)

plants of heading Chinese cabbage. Based on the statistical

result, the expression level of BcPME31 during flower bud

development was also quantized. This is the first report

about the selection of reference genes during flower bud

development and will be of special interest in the field of

male sterility relative research.

Materials and methods

Plant material preparation

Ms and Mf plants used in this study have been described

previously (Xu et al. 2013). The flower buds of Ms and Mf

plants were collected and sorted into four groups based on

the bud size length (\1.0, 1.0–2.0, 2.0–3.0, and

4.0–5.0 mm in length). Buds of analogous size were mixed

together and stored at -80 �C until required.

Total RNA isolation and cDNA synthesis

Total RNA was isolated from the floral bud samples uti-

lizing Bizol (BIOER, China) according to the manufac-

turer’s instructions. Recombinant DNase I (Takara, Japan)

was used at 37 �C for 30 min to remove trace DNA. The

quality of total RNA was determined by agarose gel elec-

trophoresis and an ultraviolet spectrophotometer (Nano-

Drop 2000C, Thermo Scientific, Wilmington, DE).

Equal amounts of total RNA (about 2 lg) in all samples

were used for first-strand cDNA synthesis by Prime-

ScriptTM RT reagent kit (TaKaRa, Japan), according to the

manufacturer’s instructions. Purified cDNA was dissolved

in ddH2O and stored at -20 �C for use. RNA extraction

and cDNA synthesis from all of the different samples were

performed for three biological replicates.

Primer design

The optimal reference gene was determined from nine

candidate genes in all samples. The designs of primers for

six candidate genes including EF1a, GAPDH, Ubc, 18 s

rRNA, Apr, and Cyp were excerpted from previous studies

(Qi et al. 2010), with the exception of Tub where primers

were redesigned to amplify a fragment about 110 bp. Two

candidate genes, BrTIP41 and U34559, were accorded to

the gene TIP41 in Brassica napus and AT4G33380 in

Arabidopsis, respectively (Chen et al. 2010; Czechowski

et al. 2005), and their sequence annotations were referred

to as Bra011516 and Bra034559 in the Brassica Databases

(BRAD http://brassicadb.org/brad/). The primers were

designed with a Tm value of approximately 58–62 �C

using the software Primer Premier 5 (www.PremierBiosoft.

com). The specificity of each primer was confirmed by

BLAST searching against the Brassica Database. The pri-

mer sequences are listed in Table S1.

QRT-PCR and data analysis

QRT-PCR was carried out in an optical 8-well plate (Ax-

ygen, USA) using the Bio-Rad IQ5 System (Bio-Rad,

America). SYBR Green II (Takara, Japan) was used to

monitor dsDNA synthesis. The reactions were performed

in triplicate in a final volume of 20 ll (SYBR� Premix Ex

TaqTM (29) 10 ll; forward and reverse primers (10 lM)

0.4 ll each; tenfold diluted cDNA 2 ll; sterile ddH2O

7.2 ll) using the following program: initial denaturation at

95 �C for 3 min, followed by 40 cycles (95 �C for 20 s,

60 �C for 30 s, 2 �C for 30 s). Melting curve analysis was

810 Acta Physiol Plant (2014) 36:809–814

123

performed to check for the occurrence of primer dimers

and non-specific PCR products by using cycles of 95 �C for

15 s followed by a constant increase of temperature

between 60 and 95 �C. The amplification specificity was

also determined by electrophoresis and sequence analysis

of their PCR products.

A standard curve for each gene was generated using ten-

fold serial dilutions of pooled cDNAs with equal templates of

every sample. The efficiency of these primers was calculated

using LinRegPCR (Ramakers et al. 2003). The statistical

software program geNorm (Vandesompele et al. 2002) was

employed to compare the expression stability of these can-

didate genes in eight samples from Ms and Mf plants.

Results and discussion

Primer specificity and efficiency

To evaluate the amplification specificity, agarose gel

electrophoresis was performed for amplicons of the can-

didate reference genes derived from pooled cDNA and

genomic DNA templates (Fig. S1). All primer pairs gen-

erated only single amplicon band in Ms and Mf cDNA

pools. Except BrTip41 whose forward primer was com-

posed of parts of two exons and could not amplify any

band, the rest of the primer pairs could amplify the specific

band with DNA templates; especially four genes including

GAPDH, Ubc, Apr, and U34559 showed amplification

differences in amplicon size between DNA and cDNA

templates, as their primers span an intron in the genome.

As a result, these four genes could be used for RNA

extraction quality assay. All these interest bands were

cloned and sequenced; except non-specific amplification of

genomic DNA by Ubc primers, the sequences of the

remaining bands showed high identities with expected

sequences (data not shown).

Before carrying out qRT-PCR, all of the RNA samples

of high quality were verified by PCR results of GAPDH

and Apr. There was no genomic DNA contamination in

cDNA templates. Neither primer dimers nor unexpected

products were found (data not shown). Amplification effi-

ciency ranged from 93.9 % in U34559 to 124.3 % in Ubc,

and coefficient of determination (R2) varied from 0.994 to

0.998 (Table S1). Except Ubc, which was abolished for the

amplification efficiency over 120 %, the remaining genes

were demonstrated to be efficient and specific for the target

gene amplification.

Expression dynamics of the candidate reference genes

RNA transcription levels were measured to evaluate the

expression stability of the remaining eight candidate genes

(Fig. S2). A relatively wide range of Ct values was

observed from 7.06 to 34.03 in eight tested samples, and

most Ct values were between 20 and 30. The least abun-

dant transcripts were of BrTip41 with Ct value 34.03. The

transcript level of 18S rRNA was expressed highly with the

minimum Ct value of 7.06.

Statistical analysis of gene expression stability

The Ct datasets of eight candidate genes were transformed

into relative quantification data using Microsoft Excel with

the 2-DCT method and then imported into geNorm for

comparison analysis (Fig. 1). Among these eight genes,

Tub and GAPDH were found to be the optimal reference

genes during flower bud development, with an average

expression stability (M) of 0.614. The gene order from

the greatest stable to the least stable was Tub/GAP-

DH [ Cyp [ EF1a [ U34559 [ BrTip41 [ Apr [ 18S

rRNA (Fig. 1a). Because the expression of the 18 s rRNA

gene was much higher than other genes, we further cal-

culated gene expression stability without 18s rRNA, and

found that the stability order was not changed (data not

Fig. 1 Gene expression stability and validation of candidate refer-

ence genes by geNorm analysis. a Average expression stability of

eight genes among different samples. b Determination of the optimal

number of candidate reference genes for normalization corresponding

to (a). M average expression stability, V pairwise variation

Acta Physiol Plant (2014) 36:809–814 811

123

shown). The Ct values of eight candidate genes were also

analyzed by BestKeeper (Pfaffl et al. 2004) (Table S2).

After the geNorm analysis, Tub showed the best value

among all candidate genes, with GAPDH next and 18S

rRNA the least stable gene. The main difference was that

the stability order of the other five genes was not consistent

with that of geNorm analysis.

Although Tub was proved to be one of the most variable

genes in considering male and female reproductive tissues,

spikelets, roots, and leaves of apomictic and sexual Bra-

chiaria brizantha (Silveira et al. 2009) and in leaves, roots,

and stems of two soybean cultivars exposed to cadmium

(Wang et al. 2012), it was also regarded as one of the most

stable genes in all developmental stages and under all stress

conditions of Platycladus orientalis (Chang et al. 2012)

and in our experiment. All these results illustrate that ref-

erence gene Tub was not suited for all experiments.

GAPDH was regarded as one of the most stable genes in

various stress environments and in different tissues during

plant development (Qi et al. 2010), and has been widely

used as reference gene in Chinese cabbage (Zhang et al.

2010, 2009). This gene was also identified as the second

stable gene in our experiment. These results suggest that

GAPDH had a wide range of adaptability.

Additionally, geNorm also recommended the minimum

number of reference genes required for determining a

reliable normalization factor. Vandesompele et al. (2002)

have proposed 0.15 as the threshold value for the pairwise

variation (V), below which, the additional reference gene is

not required. The least pairwise variation in our samples

was 0.148 (Fig. 1b), which suggested that the combined

use of six reference genes may be necessary for normal-

izing the expression of the interest gene.

Expression analysis of BcPME31 in eight samples

Pectin methylesterase (PME/PE) plays important roles in

pollen development and pollen tube growth (Tian et al.

2006). Reducing PME expression led to abnormal or

shrunken pollen, and lower pollen vigor in Chinese cab-

bage (Liu et al. 2006). As a example, the expression of

BcPME31 in these samples was quantized with the com-

bined use of six reference genes including Tub, GAPDH,

Cyp, EF1a, U34559, and BrTip41 in this study. The

expression of BcPME31 in Mf buds over 2.0 mm (F3 and

F4) was about twofold to threefold higher than that in Mf

buds\2.0 mm (F1 and F2), while there were no significant

differences in its expression among the four samples of Ms.

BcPME31 expression in the flower buds of Ms was sig-

nificantly lower than that in the flower buds of Mf

(Fig. 2a). Compared with the combined use of six refer-

ence genes, using GAPDH and Tub alone as the internal

control did not substantially change the expression level of

BcPME31 in these samples except for the sample F4 with

bud length from 4.0–5.0 mm (Fig. 2a). The expression

level of BcPME31 in F4 normalized by GAPDH was

higher than that by the combined use of six reference

genes, but an opposite result was obtained when using Tub

gene as the internal control (Fig. 2b). These variable results

indicated that additional reference genes may be necessary

for the normalization of the expression of targeted gene and

that selection of the optimal reference gene plays an

important role in gene expression assays.

So far, three statistic programs GeNorm (Vandesompele

et al. 2002), BestKeeper (Pfaffl et al. 2004), and Norm-

finder (Andersen et al. 2004) have been used for the

identification of reference genes (de Oliveira et al. 2012;

Fig. 2 The BcPME expression normalized by different reference

genes. a The BcPME gene expression normalized by GAPDH, Tub,

and the combination of six reference genes. b The histogram of the

BcPME expression at the F3 and F4 samples of (a). Six means six

reference genes recommended by GeNorm including Tub, GAPDH,

Cyp, EF1a, U34559, and BrTip41 were used as the internal control in

combination. S1–S4 represent male sterile samples no. 1–4; F1–F4

represent male fertile samples no. 1–4. Error bars on each point

indicate ±SE from three biological repeats

812 Acta Physiol Plant (2014) 36:809–814

123

Han et al. 2010; Mallona et al. 2010; Wan et al. 2010). Of

these, GeNorm was widely used for its additional function

regarding the minimum number of reference genes

required for determining a reliable normalization factor. In

this study, GeNorm was employed to compare eight can-

didate reference genes in Ms and Mf plants of heading

Chinese cabbage during flower bud development. The

statistical result revealed that the stability order was Tub/

GAP-

DH [ Cyp [ EF1a [ U34559 [ BrTip41 [ Apr [ 18S

rRNA, and the combined use of six reference genes was

optimal for qRT-PCR. Furthermore, the expressions of

BcPME31 normalized with the combined use of six refer-

ence genes and with GAPDH or Tub alone were compared;

the results also suggested that the selection of the optimal

reference genes was necessary in gene expression-related

research.

Author contribution For this paper, Zhiyuan Fang and

Lugang Zhang designed the study, gave direction, and

provided research facilities. Xiaoyong Xu performed most

of the experiments and wrote the manuscript. Zeping Yang

helped with statistics analysis. Xilu Su helped with qRT-

PCR test. All authors reviewed and commented on the final

manuscript. The authors declare that no conflict of interest

exists.

Acknowledgments This work was supported by grants from the

National Natural Science Foundation of China (No. 30871717) and

the National Science & Technology Pillar Program

(2012BAD02B01).

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