Molecular cloning and development of RAPD-SCAR markers for Dimocarpus longan variety authentication

As an edible fruit and source of traditional medicine, D. longan is grown in most areas of Southern China. Identification of D. longan cultivars by using molecular markers is important genetically. In this study, we cloned fragments from improved randomly amplified polymorphic DNA (RAPD), and developed stably diagnostic sequence-characterized amplified region (SCAR) markers. The specific RAPD bands of D. longan cultivars from Guangxi, with size ranging from 500 bp to 900 bp were gel-purified, cloned and sequenced. Four clones named LY2-1, LY4-7, LY4-8 and LY5-2 were identified. In order to investigate whether the fragments were specific for the species, four pairs of SCAR primers were then designed. PCR amplifications were conducted to analyze 18 samples including different D. longan cultivars and other species. The specific bands with expected sizes were amplified in five D. longan samples but not in others. To identify and characterize the difference between D. longan and D. confinis, PCR amplifications were performed again. The specific bands with expected sizes were found in D. longan but not in D. confinis by SCAR markers LY2-1, LY4-7 and LY5-2, respectively. These results showed that our developed SCAR markers could be very useful as a specific D. longan variety authentication. Therefore, our study provides an effective and precise PCR-based diagnostic method and markers to identify D. longan species.


Introduction
Dimocarpus longan Lour (D. longan), also called longan or dragon eye, is a commercially attractive fruit that is widely distributed in subtropical areas. China is the world's largest cultivator of D. longan, with more than 2000 years history of cultivation. As a traditional medicine, D. longan fruit is used for enhancing memory, promoting blood metabolism, relieving insomnia, and preventing amnesia. Its secondary metabolic products have been also shown to have anti-oxidative, anti-obesity, anti-cancer, anti-tyrosinase, and immunomodulatory activities (Park et al. 2010;Prasad et al. 2010;Zhong et al. 2010). A number of researches have been conducted to give an insight of genetic characterization and authentication in D. longan samples.
RAPD markers are DNA fragments from PCR amplification of the genomic DNA's random segments with single primer of arbitrary nucleotide sequence (Williams et al. 1990). It is a relatively easy, inexpensive and rapid technique because of its simplicity and requirement for minimal amounts of genomic DNA (Micheli et al. 1994).
It has been widely used in the identification and genetic relationship analysis of a number of plant and animal species. The improved RAPD can improve the resolution of the PCR products and its repeatability (Fu et al. 2000;Fu et al. 2013;Mei et al. 2013). The sequence characterized amplified region (SCAR) marker is one of the stable markers, generally derived from random amplified polymorphic DNA (RAPD) (Dnyaneshwar et al. 2006;Li et al. 2010;Rajesh et al. 2013). The basic principle is to convert the dominant markers into co-dominant markers to reduce the tedious procedures of RAPD (Li et al. 2010;Rajesh et al. 2013). These markers generally reveal higher levels of polymorphism owing to higher annealing temperatures and longer primer sequence specificity (Kumla, et al. 2012). With SCAR marker, analysis is reduced to a simple PCR analysis using PCR primers designed from the sequence of the amplicon of RAPD (Kumla, et al. 2012;Rajesh et al. 2013).
Dimocarpus confinis is a species of plant in the genus Dimocarpus, which is grown in a range of Southern China to Southeast Asia. It is mainly grown as ornamental plants but cannot be used as a food source due to its poisonous nature. D. confinis produces ovalshaped drupe fruits, which is very similar to D. longan, causing difficulty in distinguishing it from D. longan in the market, if anyone ever sells D. confinis as longan [Source: http://www.people.com.cn/GB/paper503/13144/ 1179090.html]. In this study, we aimed to distinguish D. longan from D. confinis by genetic characterization using RAPD and SCAR. In order to increase the reproducibility and reliability of PCR assays in D. longan and distinguish it from D. confinis, SCAR markers have been developed from clones of RAPD fragments. In the present study, the DNA fragments were amplified with the DNA template of D. longan from Guangxi Province, using an improved RAPD (Fu et al. 2000;Fu et al. 2013;Mei et al. 2013) followed by DNA ligation, cloning, and sequencing. After a series of experiments, four new specific longan DNA fragments had been confirmed. According to the sequencing results, 4 pairs of primers (SCAR markers) had been designed to converted, which detected specificity of D. longan varieties. Three of SCAR markers were useful to distinguish D. confinis from D. longan. Therefore, the SCAR primers can be used to assess the genetic diversity and population structure of D. longan from D. confinis.

Genomic DNA extraction
The DNAs were extracted from fresh young leaves of D. longan and D. confinis by using previously described slightly modified Cetyl trimethylammonium bromide (CTAB) method ) and stored at −20°C till to use. The fresh young leaves of D. confinis were provided by Dr. Jiechun Pan from Agricultural College of Guangxi University in Guangxi Province. Leaves were first fixed in fixing solutions containing chloroform, (without liquid nitrogen), and then grinded into tiny pieces by silica (SiO 2 ) for the extraction of DNA with CTAB method. DNA quality was determined after electrophoresis on 1% agarose gels. DNA concentration and quality was measured by spectrophotometry at 260 and 280 nm and normalized to a concentration of 10 ng/μl, then stored at −20°C for further study Fu, 2012).

Improved RAPD amplification
The D. longan DNAs were initially screened with three random primers . The PCR reactions were performed with Tiangen reagents (Beijing, China). The improved RAPD reaction solution consisted of 7.5 μl 2 × Taq PCR MasterMix, 1.5 μl 2.5 μM primer and 1.5 μl genomic DNA, to a total volume of 15 μl. Amplification reactions were performed in an Eppendorf Authorized Thermal Cycler (Mastercycler 5331 system, Eppendorf, Germany) under the following program, which involves an initial pre denaturation at 95°C for 90 s. It was then followed by 40 cycles of denaturation at 94°C for 40 s, annealing at 36°C for 60 s, and extension at 72°C for 90 s. The final extension step was performed at 72°C for 5 min. During the procedure, the temperature rose at the rate of 0.3°C /s, and declined at the rate of 3°C /s. The amplified products were detected with electrophoresis on 1.5% agarose gel.

Cloning and sequencing of DNA fragments
Four different bright bands were excised from agarose gel and purified with TIANgel Midi Purification Kit (DP209, China) according to the company provided protocol. Purified DNA fragments were ligated into pGM-T vector (No. VT202) (Tiangen reagents, Beijing, China), and transformed in DH5α E. coli complement cells and the recombinant clones were selected on LB agar plates containing 100 μg/μl of ampicillin, 40 mg of X-gal and 160 μg of IPTG. The blue white screening was Table 1 Sequences of SCAR primers, PCR condition and product size  adopted to find white colony firstly. Then presence of the appropriate insert was verified by PCR with T7/SP6 primer pairs (T7 primer: 5′-TAA TACGACTCACTATA GGG -3′, SP6 primer: 5′-ATTTAGGTGACACTATAGA A-3′), or EcoRI digestion, which is located at pGM-T vector nearly to the ligation ends, and then for DNA sequencing (Fu, 2012).

Sequence homology searches and bio-informatics analysis
Homology searches were performed by online program BLAST from NCBI (http://www.ncbi.nlm.nih.gov/BLAST/) in different species.

SCAR analysis
The nucleotide sequence of each of the cloned RAPD fragment was used to design pairs of SCAR primers using Primer 3 software from the website (http://bioinfo. ut.ee/primer3-0.4.0/primer3/). Sequences of the SCAR primers, amplification length and PCR condition were shown in Table 1 PCR amplification and development of SCAR markers.
The PCR reaction solution consisted of 7.5 μl 2 × Taq PCR MasterMix, 1.5 μl of 2.5 μM each pair of SCAR primers and 1.5 μl genomic DNA (15 ng), to a total volume of 15 μl. Amplification reactions were performed in an Eppendorf Authorized Thermal cycler with an initial pre-denaturation for 90 s at 95°C followed by 35 cycles of denaturation at 94°C for 40 s, annealing at 60°C for 30 s, and extension at 72°C for 40 s. The final extension step was performed at 72°C for 5 min. The amplified PCR products were resolved by electrophoresis on 1.0% agarose gel in 1 × TAE buffer. Gels were visualized by 0.5 μg/mL ethidium bromide staining and the images were documented using the ChemiDoc XRS (Bio-Rad, USA). To distinguish the difference between D. longan and D. confinis, PCR amplifications were performed by using above mentioned 4 pairs of SCAR primers and amplification conditions (Table 1).

Cloning of RAPD amplification fragments
Three RAPD primers (SBS-I4, SBS-Q12, and SBS-Q19) were initially screened using DNA samples from Guangxi D. longan ). The characteristic DNA fragments with clear and polymorphic profiles were purified, ligated to T-vector by AT cloning. The blue and white screening method was adopted at first to screen positive cloning. The positive clones 2-1, 4-7, 4-8, 5-2 and 5-4 were identified by PCR amplification using SP6 primer and T7 primer (Figure 1 A and B) or by plasmid DNA digestion using EcoRI enzyme ( Figure 1C). In the Figure 1A, five clones are positive, but only clone 2-1 showed strongest PCR band with a similar inserted fragment and was selected for further sequencing, whereas in the Figure 1B, clones 4-4, 4-7 and 4-8 are PCR positive, and 4-7 and 4-8 with inserted frag-ments~600,~900 bp DNA-fragment respectively. In the Figure 1C, clones 5-2 and 5-4 with a same~500 bp inserted fragment digested by EcoRI, and clone 5-2 was selected for further sequencing.

Sequencing and characterization of D. longan-specific RAPD fragments
Sequencing of above four cloned RAPD fragments in D. longan showed that clone 2-1 consisted of 486 nucleotides and deposited into GenBank with accession number KC522607 (Figure 2A), clone 5-2 consisted of 486 nucleotides and deposited into GenBank with accession number KC522608 ( Figure 2B), clone 4-7 consisted of 556 nucleotides and deposited into GenBank with accession number KC522609 ( Figure 2C), clone 4-8 consisted of 903 nucleotides and deposited into GenBank with accession number KC522610 ( Figure 2D) (Note: the sequences information will not release till one year deposition in Genbank).
BLAST searches of the nucleotide sequences in GenBank showed that 202 nucleotides of clone 4-7 fragment (nucleotides 210 to 411) shared 67% identity to the Oryza sativa Japonica Group chromosome 10 clone OSJNBa0095J15 sequence (Sequence ID: gb|AC092173.3|) with an E value 8e-08 ( Figure 3A), and 78 nucleotides in the same region (nucleotides 304 to 409) shared 74% identity to the Silene vulgaris isolate 19 J19 retrotransposon putative Retand type sequence (Sequence ID: gb| JN624421.1|), with an E value 3e-07 ( Figure 3B). Sequences from other 3 clones didn't show any significant identity or similarity to any species.

Development of D. longan-specific SCAR markers
To generate stable longan-specific diagnostic SCAR markers from RAPD markers, four pairs of primers (LY2-1 L and LY2-1R; LY4-7 L and LY4-7R; LY4-8 L and LY4-8R; LY5-2 L and LY5-2R) ( Table 1) were designed and synthesized based on cloned sequences in Figure 2. The designed SCAR primer pairs were then used to amplify the genomic DNA from 18 of collected DNA samples to test the amplification species-specificity. PCR results indicated that the PCR products with expected size were observed only in five D. longan samples by SCAR marker LY2-1 (Figure 4), SCAR marker LY4-7 ( Figure 5), SCAR marker LY4-8 (Figure 6), and SCAR marker LY5-2 (Figure 7), without any amplification in other species we tested (Figures 4,5,6,7), which indicated that all four SCAR markers are longan-specific. The lack of this specific amplicon in the other species indicated the efficacy of these marker in distinguishing the longan group from the others.

Authentication of D. longan from D. confinis
To distinguish D. longan from D. confinis, PCR amplification were performed by using our developed four pairs of SCAR primers (Table 1)  confinis. LY2-1, LY4-7, LY4-8 and LY5-2 represent longan-specific SCAR markers for LY2-1, LY4-7, LY4-8 and LY5-2 which were used in the PCR amplification, respectively. D. longan samples by SCAR markers LY2-1, LY4-7 and LY5-2, without any amplification in the sample of D. confinis, which indicated that these three makers are useful for the identification D. longan from D. confinis (Figure 8). However, we still noticed the PCR product in D. confinis (LL) with same expected size from D. longan by SCAR marker LY4-8, which indicated that this marker can't be used in the authentication of D. longan from D. confinis (Figure 8).

Discussion
RAPD analysis can reveal high degrees of polymorphisms, does not require prior DNA sequence information of the species, and is easy to manipulate (Williams et al. 1990;Devaiah and Venkatasubramanian, 2008;Chen et al. 2010;Yazbeck et al. 2011;Bhat et al. 2012;Shakeel et al. 2013;Noormohammadi et al. 2013;Zhang et al. 2013). Therefore, researchers could explore its application for authentication of traditional Chinese medicines. RAPD needs less DNA template and is relatively easy to handle, but is poor in reproducibility and stability, leading to restrictions in practical application. However, after converting RAPD markers into SCAR markers, the specificity and stability can be greatly improved, which makes it more convenient and efficient in the testing of different alleles (Dnyaneshwar et al. 2006;Li et al. 2010;Rajesh et al. 2013). Since they can identify a single or a few bands instead of a complex pattern, SCAR markers are more straightforward than other molecular techniques, such as RAPD, SSR, ISSR and AFLP.
In this study, we selected the clear and bright amplified DNA bands from RAPD markers for SCAR marker development. In SCAR, pairs of 18-25 bp oligonucleotide primers specific to the sequence of polymorphic bands can be used to amplify the characterized regions from genomic DNA under stringent conditions, which makes these markers more specific and dependable as compared to RAPD markers. Based on the sequences of our cloned DNA fragments, four SCAR primer pairs (LY2-1, LY4-7, LY4-8 and LY5-2) were designed. Genomic D. longan DNAs collected from 5 different regions within China, contained the cloned DNA fragments. As shown in Figures 4,5,6,7, the primers were generated from 250-360 bp bands in all D. longan cultivars, while no amplicon was observed in other species. Thus, the genetic polymorphism observed among the cultivars is interesting and can be used to develop markers for D. longan-specific identification.
Traditionally, D. longan fruit is used for several diseases (Park et al. 2010;Prasad et al. 2010;Zhong et al. 2010;Mei et al. 2013). Recently it was found that the dried longan seed extracts also have potential inhibitory effects on cancer cell invasion (Panyathep et al. 2013). However, D. confinis, a species of plant in the genus Dimocarpus family, with an oval-shaped drupe fruit similar to D. longan (http://www.people.com.cn/GB/paper503/13144/1179090. html), is very hard to distinguish from D. longan in the market only by morphology. If people sell D. confinis as longan, and someone purchase mistakenly and eat, they will experience vomiting, diarrhea and psychiatric disorder including anxiety, depression, insomnia, apprehension, auditory and visual hallucination, and torpid reaction. To distinguish the D. longan and D.confinis, PCR amplifications were performed by using above mentioned SCAR primers from Table 1. The results showed that the PCR products with expected size were observed only in D. longan samples by SCAR markers LY2-1, LY4-7 and LY5-2, without any amplification in D. confinis (Figure 8), which suggests that we have developed RAPD-SCAR markers successfully for identification of D. longan from the other species with similar morphology. Although the PCR product in D. confinis (LL) with same expected size from D. longan samples by SCAR marker Y4-8 were detected, which indicates that this marker cannot be used in the identification of D. longan from D. confinis, we can demonstrate that the DNA quality extracted from D. confinis is excellent. Therefore, our results showed that the developed SCAR markers could be useful to assess the genetic diversity and population structure of D. longan from D. confinis.
In this study, we obtained four clones by improved RAPD and DNA sequencing in D. longan, and developed stably diagnostic Sequence-Characterized Amplified Region (SCAR) markers for DNA fingerprinting to distinguish the fruit of D. longan, as a species of plant in the genus Dimocarpus with similarity to that of D. confinis with poisonous nature. Our results have shown that these SCAR markers could be very useful as a specific D. longan variety authentication, particularly for distinguishing D. longan from D. confinis.