Molecular marker analysis of ‘ Shatangju ’ and ‘ Wuzishatangju ’ mandarin ( Citrus reticulata Blanco )

1 College of Horticulture, South China Agricultural University, Guangzhou, Guangdong, 510642, P. R. China. 2 State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou, Guangdong, 510642, P. R. China. 3 Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences, Danzhou, Hainan, 571737, P. R. China.


INTRODUCTION
Citrus is one of the world's most important fruit crops which is widely grown in most areas with suitable climates between latitude 35°N to 35°S.With the rapid development of Citrus industry and taste for better quality, the demand for desirable cultivars has been increased to meet the challenge of process industry and particular consumer preferences.Cross hybridization as a genetic improvement strategy for citrus cultivar development have led to the production of a large number of improved cultivars.
Unfortunately, cross hybridization faces many serious impediments such as highly genetic heterozygosity, longer juvenility, nucellar embryo interference, sexual or incompatibility of many species.The fact that Citrus cultivars were maintained by vegetative propagation; the large number of cultivars originated from bud sport events.Therefore, bud sport selection is one of the most important breeding approaches in Citrus.In the past 20 years, the cultivars presently grown mainly originated from bud sport selection (Deng et al., 1996;Deng, 2005;Liu and Deng, 2007) .However, most characteristics of a bud sport are identical to the original variety from which they are derived.Accurate characterization of bud sport and their original cultivar is crucial for the protection of future intellectual property rights over new cultivars.
Traditional methods for identification of genetic variability based on morphological, physiological and agronomic traits are often laborious and time-consuming because these traits are not all available for sampling at a single time (Fang et al., 2001).
Furthermore, these methods are inherently weak since they are limited by environmental factors and subjectivity of observations.With the rapid development of modern biotechnology, the advent of molecular markers has made it possible to detect genetic difference between genotypes at DNA level.Molecular markers overcome some of these limitations and have been widely used in Citrus assisted-selection breeding, genetic diversity analysis, population genetics and molecular evolutionary genetics (Wang et al., 2000;Gong et al., 2008).
'Wuzishatangju' (Citrus reticulata Blanco), derived from a bud sport of a seedy 'Shatangju' cultivar, is seedless, very tasty and easy-to-peel and has become one of the newly grown varieties during the last decade in China (Ye et al., 2006).Our previous studies showed that 'Wuzishatangju' results from gametophytic selfincompatibility which caused seedlessness by blocking fertilization in the ovary (Ye et al., 2009).Except for seed number, no difference was observed between 'Shatangju' and 'Wuzishatangju' cultivar in term of sprout appearance, growth habit, leaf type, flower color, fruit shape and size (Ye et al., 2006;2009).Therefore, rapid and accurate identification of the new variety is of great significance for further extension and application.
In this study, RAPD, ISSR, SSR, SRAP, IRAP and retrotransposon-microsatellite amplified polymorphism (REMAP) markers were used to study the genetic variations between 'Shatangju' and 'Wuzishatangju'.The results presented herein aimed to determine whether it is possible to detect molecular markers that distinguish bud sport variety and its original cultivar in their early stage of development.

MATERIALS AND METHODS
Five-year-old trees of 'Wuzishatangju' (six trees) and 'Shatangju' mandarin (four trees) are planted in an orchard of South China Agricultural University.

Genomic DNA extraction
Genomic DNA was extracted from young leaves of 'Shatangju' and 'Wuzishatangju' using a cetyltrimethyl ammonium bromide (CTAB) method (Xiong et al., 2002).The quality and concentration of DNA were examined by ethidium bromide (EB)-staining 0.7% (w/v) agarose gel electrophoresis and spectrophotometer (Bio-RAD, USA) analysis.The working DNA solutions were prepared at 10 ng/μl.

RAPD analysis
1196 RAPD primers were used to detect the genetic variations between 'Shatangju' and 'Wuzishatangju' based on establishing an optimization of RAPD-polymerase chain reaction (PCR) reaction system and procedures (Qin et al., 2011).PCR products were examined by EB-staining 1.5% (w/v) agarose gel electrophoresis.

ISSR analysis
An orthogonal experimental design was used to optimize ISSR-PCR system (Table 1) using DNA from 'Shatangju' as template.100 ISSR primers were synthesized according to the sequences from University of British Columbia and used to detect the genetic variations between 'Shatangju' and 'Wuzishatangju' based the optimized ISSR-PCR reaction system.The PCR parameters were followed by the method of Qiao et al. (2009).PCR products were examined by EB-staining 1.5% (w/v) agarose gel electrophoresis.

SRAP analysis
Single factor test were used to optimize SRAP-PCR reaction system using DNA from 'Shatangju' as template.The 25.0 μl of reaction volume contained 1.5, 2.0, 2.5, 3.0 and 3.5 mM Mg 2＋ , 0.1, 0.15, 0.2, 0.25 and 0.30 mM dNTPs, 1.0, 1.25 and 1.5 U rTaq (5 U/µl).PCR amplification was carried out according to the procedure of Li and Quiros (2001).Differences in SSR fragments are often difficult to resolve on agarose gels and high resolutions can be achieved through the use of polyacrylamide gels in combination with AgNO3 staining.Therefore, SRAP-PCR products were separated on both agarose gels (1.5%) and polyacrylamide gel electrophoresis (PAGE) (10%), respectively.

IRAP analysis
IRAP primers were synthesized according to the sequences of Wei (2007) (Table 2).An orthogonal experimental design was used to optimize IRAP-PCR system (Table 3) using IRAP5 primer and 'Shatangju' DNA as template.The PCR parameters were carried out according to the method of Kalendar et al. (1999) and Wei (2007).

REMAP analysis
REMAP primers were from eight SSR primers combined with a forward or reverse IRAP primer (Table 2).REMAP-PCR system was optimized as described by IRAP.The 25.0 μl of reaction volume contained 30 ng DNA, 2.0 mM Mg 2+ , 0.2 mM dNTPs, 0.2 μM primers and 1.25 U rTaq (5 U/µl).The PCR parameters were: 94°C for 5 min then 35 cycles of 94°C for 1 min, 45°C for 1 min and 72°C for 1.5 min, with a final 72°C for 10 min.

RAPD analysis of 'Shatangju' and 'Wuzishatangju'
1196 RAPD primers were used to detect the genetic variations between 'Shatangju' and 'Wuzishatangju'.However, no specific band was obtained between 'Shatangju' and 'Wuzishatangju' (Figure 1).These results indicate that there was a very close genetic relationship between 'Shatangju' and 'Wuzishatangju' and RAPD could not distinguish them.

ISSR analysis of 'Shatangju' and 'Wuzishatangju'
A suitable ISSR reaction system for Citrus was established after screening various concentrations of Taq DNA polymerase, DNA template, Mg 2+ , primers and dNTPs (Figure 2).The optimum PCR reaction system (25 μl) was 20 ng DNA template, 1.5 mM Mg 2+ , 0.5 mM dNTPs, 1.0 μM primer and 0.5 U rTaq.The PCR parameters were: 94°C for 5 min then 35 cycles of 94°C for 1 min, 40 to 60°C for 1 min (different primers using different annealing temperature) and 72°C for 1.5 min, with a final 72°C for 10 min.

SSR analysis of 'Shatangju' and 'Wuzishatangju'
Seven pairs of SSR primers were used to identify the genetic variation between 'Shatangju' and 'Wuzishatangju'.However, no difference was observed between the two cultivars (Figure 4).

SRAP analysis of 'Shatangju' and 'Wuzishatangju'
Abundant, stable and clear strips were obtained using 153 pairs of SRAP primers.Agarose gel electrophoresis and PAGE were used to detect the PCR products.As shown in Figure 5, agarose gel electrophoresis was well separated for fragment sizes ranging from 100 to 2000 bp.However, no specific band was obtained between 'Shatangju' and 'Wuzishatangju' (Figure 5).Compared to agarose gel electrophoresis, PAGE were effectively separated; the fragment size ranged from 80 to 800 bp and 21 specific bands (shown by arrows) were obtained (Figure 6).The results indicate that SRAP could distinguish 'Shatangju' and 'Wuzishatangju'.

IRAP analysis of 'Shatangju' and 'Wuzishatangju'
A suitable IRAP reaction system for citrus was established after screening various concentrations of DNA template, dNTP, Mg 2+ , and Taq DNA polymerase (Figure 7).The optimum PCR reaction system (25 µl) was 1.5 mM Mg 2+ , 0.3 mM dNTP, 0.2 µM primers, 1.25 U Taq DNA polymerase and 25 ng/µl DNA templates.Based on the optimized reaction systems, abundant, stable and clear strips were obtained (Figure 7) and 22 pairs of specific primers from 28 primer combinations with good repeatability and polymorphism were further screened out.However, no specific band was detected between 'Shatangju' and 'Wuzishatangju' (Figure 8).

REMAP analysis of 'Shatangju' and 'Wuzishatangju'
56 pairs of REMAP primers were used to detect the genetic variations between 'Shatangju' and 'Wuzishatangju'.35 pairs of specific primers with good repeatability and polymorphism were further screened out.However, no specific band was detected between 'Shatangju' and 'Wuzishatangju' (Figure 9).

DISCUSSION
Bud sport selection has been widely used for creating novel cultivars in vegetatively propagated plants.The frequency of bud sports in Citrus is extremely high and many excellent Citrus cultivars such as 'Navel orange', 'Satsuma mandarin', 'Grapefruit', and 'Clementine' have been obtained through bud sport selection (Zhang and Deng, 2006) .However, it is very difficult to accurately discriminate between bud sport varieties and their original cultivar since they show very little variability in all characteristics.
RAPD marker is a simple method to detect DNA polymorphism (Welsh and McClelland, 1990;Williams et al., 1990) and has been widely used for analysis of plant genetic diversities, cultivar identification and assistedselection (Durham et al., 1992;Wang et al., 2000;Lei et al., 2009).Currently, RAPD marker has successfully been applied to identification of bud sport varieties in Citrus (Bretó et al., 2001;Luo et al., 2008;Qin et al., 2011), kiwifruit (Ning et al., 2003), pear (Gao et al., 2010) and grape (Wang et al., 2003).However, RAPD-PCR system is so sensitive that changes of any reaction component could significantly affect the results.In addition, bud sport is a kind of somatic mutations involved in chromosome number per cell, chromosome structure aberration and even a point mutation.Therefore, it is still controversial whether RAPD technology can be used to identify bud sport clones due to its limitation (Fang et al., 2001).
In this study, 1196 RAPD primers were used to detect the genetic variations between 'Shatangju' and 'Wuzishatangju' based on an optimized RAPD reaction system (Qin et al., 2011).However, no specific band was observed between 'Shatangju' and 'Wuzishatangju' (Figure 1) suggesting that RAPD marker could not distinguish the two cultivars.
SSR is an excellent molecular marker with the        However, no specific band was detected between 'Shatangju' and 'Wuzishatangju' (Figure 8).advantages of co-dominance, abundance, high reproducibility and simplicity.SSR has been considered one of the ideal molecular markers for diversity assessment of germplasm and marker-assisted selection in Citrus (Liu et al., 2005;Barkley et al., 2006;Jannati et al., 2009;Ollitrault et al., 2010).However, SSR analysis requires the construction of genomic library, the subse-quent hybridization with tandem repeated oligo-nucleotides and sequencing of the candidate clones to obtain working primers for a given study species.This is high cost, laborintensive and time-consuming which has restricted its application in cultivar identification.In the present study, seven pairs of SSR primers were used to detect the genetic variations between 'Shatangju' and 'Wuzishatangju' and no difference was observed (Figure 4) which may be due to the insufficient SSR primers.
ISSR overcomes the limitations of SSR and it is now the marker of choice to identify different individuals as they are abundant, very reproducible, highly polymorphic, highly informative and quick to use.To date, ISSR marker has been successfully applied in germplasm characterization, genetic diversity and breeding in Citrus species (Fang et al., 1997;Bretó et al., 2001).In this study, 100 ISSR primers were used to detect genetic variation between 'Shatangju' and 'Wuzishatangju' and two specific bands (> 2000 bp) were obtained (Figure 3).However, further research needs to confirm whether the two fragments are the characteristics of 'Shatangju' and 'Wuzishatangju'.
SRAP is a newly developed molecular marker with the advantages of simplicity, low cost, co-dominant makers, highly reproducibility and easy assay.SRAP is a more preferred technique for revealing genetic diversity among closely related cultivars than RAPD, SSR and ISSR markers (Budak et al., 2004).Currently, SRAP has been successfully used to identify bud sport variation in many plant species (Han et al., 2008;Zhang et al., 2010).In this study, 153 pairs of SRAP primers were used to identify 'Shatangju' and 'Wuzishatangju' and 21 specific fragments were obtained (Figure 6).The results demonstrate that SRAP marker was an effective method to detect genetic variation between original parents and their bud sports.
The mobility of transposon elements can be responsible for changes in bud sport of some species (Yao et al., 2001;Kobayashi et al., 2004;Tao et al., 2006).IRAP and REMAP markers are two new retrotransposonbased DNA fingerprinting techniques with the advantages of highly reproducibility and stability.In our study, 100 retrotransposon primers were used to detect genetic differences between 'Shatangju' and 'Wuzishatangju' and no difference was observed (Figures 8 and 9).The results suggest that retrotransposon may not cause the bud sport of 'Wuzishatangju'.

Figure 2 .
Figure 2. Optimization of ISSR reaction systems (optimum PCR reaction system was shown by an arrow).

Table 1 .
Orthogonal experimental design for ISSR.

Table 2 .
Primers used in this study.

Table 3 .
Orthogonal experimental design for IRAP.