Genetic Authentication of Gardenia jasminoides Ellis var. grandiflora Nakai by Improved RAPD-Derived DNA Markers

The evergreen shrub, Gardenia jasminoides Ellis var. grandiflora Nakai is one of the most popular garden-plants, with significant ornamental importance. Here, we have cloned improved random amplified polymorphic DNA (RAPD) derived fragments into T-vector, and developed sequence-characterized amplified region (SCAR) markers. These markers have been deposited in GenBank database with the accession numbers KP641310, KP641311, KP641312 and KP641313 respectively. The BLAST search of database confirmed the novelty of these markers. The four SCAR markers, namely ZZH11, ZZH31, ZZH41 and ZZH51 can specifically recognize the genetic materials of G. jasminoides from other plant species. Moreover, SCAR marker ZZH31 can be used to distinguish G. jasminoides Ellis var. grandiflora Nakai from other G. jasminoides on the market. Together, this study has developed four stably molecular SCAR markers by improved RAPD-derived DNA markers for the genetic identification and authentication, and for ecological conservation of medicinal and ornamental plant G. jasminoides.

The improved RAPD amplification results are shown in Figure 2, where red arrows indicate the bands labeled with by primer N5 (Figure 2A) and Q12 ( Figure 2B). The arrow-indicated bands were cut from an agarose gel and further purified, and then ligated to T-vector by TA cloning. The blue/white screening method in LB agar plate was adopted firstly to screen the positive clones (data not shown). The white clones were then identified by polymerase chain reaction (PCR) amplification using SP6/T7 primer pair that is mentioned in the Experimental section. In Figure 3A the clone ZZH11 is shown in lane 7 as an expected inserted DNA-fragment with ~800 bp in size, whereas the clones ZZH31, ZZH41 and ZZH51 are shown in Figure 3B-D, as three inserted DNA-fragments with right length in sizes, respectively. Clones ZZH11, ZZH31, ZZH41 and ZZH51 were finally selected for Sanger sequencing.   The improved RAPD amplification results are shown in Figure 2, where red arrows indicate the bands labeled with by primer N5 (Figure 2A) and Q12 ( Figure 2B). The arrow-indicated bands were cut from an agarose gel and further purified, and then ligated to T-vector by TA cloning. The blue/white screening method in LB agar plate was adopted firstly to screen the positive clones (data not shown). The white clones were then identified by polymerase chain reaction (PCR) amplification using SP6/T7 primer pair that is mentioned in the Experimental section. In Figure 3A the clone ZZH11 is shown in lane 7 as an expected inserted DNA-fragment with~800 bp in size, whereas the clones ZZH31, ZZH41 and ZZH51 are shown in Figure 3B-D, as three inserted DNA-fragments with right length in sizes, respectively. Clones ZZH11, ZZH31, ZZH41 and ZZH51 were finally selected for Sanger sequencing. The improved RAPD amplification results are shown in Figure 2, where red arrows indicate the bands labeled with by primer N5 (Figure 2A) and Q12 ( Figure 2B). The arrow-indicated bands were cut from an agarose gel and further purified, and then ligated to T-vector by TA cloning. The blue/white screening method in LB agar plate was adopted firstly to screen the positive clones (data not shown). The white clones were then identified by polymerase chain reaction (PCR) amplification using SP6/T7 primer pair that is mentioned in the Experimental section. In Figure 3A the clone ZZH11 is shown in lane 7 as an expected inserted DNA-fragment with ~800 bp in size, whereas the clones ZZH31, ZZH41 and ZZH51 are shown in Figure 3B-D, as three inserted DNA-fragments with right length in sizes, respectively. Clones ZZH11, ZZH31, ZZH41 and ZZH51 were finally selected for Sanger sequencing.     6 7 8 9 10 11 12 13  bp  2000  1000  750  500  250  100   A   Fragment 1   M 6 7 8 9 10 11 12 13  bp  2000  1000  750  500  250  100   A   Fragment 3   M 31 32 33 34 35 36 37 38 39  bp  2000  1000  750  500  250  100   B   Fragment 3   M 31 32 33 34 35 36 37 38 39  bp  2000  1000  750  500  250

Sequencing and Characterization of G. jasminoides Ellis var. grandiflora Nakai Specific RAPD Fragments
After sequencing of the above mentioned four RAPD fragments clones of G. jasminoides, BLAST searches of the nucleotide sequences in GenBank database were performed and indicated that these clones have no significant identity to that of any species. The sequencing results revealed that clone ZZH11, consisting of 700 nucleotides, was deposited into GenBank with accession number KP641310 ( Figure 4A); cloneZZH31, consisting of 699 nucleotides, was deposited into GenBank with accession number KP641311 ( Figure 4B); clone ZZH41, consisting of 942 nucleotides, was deposited into GenBank with accession number KP641312 ( Figure 4C); and clone ZZH51, consisting of 457 nucleotides, was deposited into GenBank with accession number KP641313. In this study, we designed and synthesized four pairs of primers (for ZZH11, ZZH31, ZZH41 and ZZH51) to generate more stable G. jasminoides Ellis var. grandiflora Nakai-specific diagnostic SCAR markers based on our cloned sequences ( Figure 4). The primers used in the RAPD analysis are listed in Table 2. The designed SCAR primer pairs were used to amplify the genomic DNA collected from 22 samples to test amplification species-specificity. The PCR amplification results are shown in Figure 5. The PCR results by SCAR markers ZZH11, ZZH31, ZZH41 and ZZH51 ( Figure 5) indicated that the PCR products with expected size were observed only in samples of G. jasminoides, and no amplification in other species we tested. This indicates that the SCAR marker ZZH11, ZZH31, ZZH41 and ZZH51 are specific for G. jasminoides Ellis var. grandiflora Nakai. The lack of this specific amplicons in the samples from other species indicates the efficacy of this marker in distinguishing the samples of G. jasminoides Ellis var. grandiflora Nakai from other species. Negative controls without DNA template did not show any PCR product (data not shown). Therefore, we confirm that G. jasminoides-specific SCAR markers were successfully developed, which can be used for the authentication of these plants.

Sequencing and Characterization of G. jasminoides Ellis var. grandiflora Nakai Specific RAPD Fragments
After sequencing of the above mentioned four RAPD fragments clones of G. jasminoides, BLAST searches of the nucleotide sequences in GenBank database were performed and indicated that these clones have no significant identity to that of any species. The sequencing results revealed that clone ZZH11, consisting of 700 nucleotides, was deposited into GenBank with accession number KP641310 ( Figure 4A); cloneZZH31, consisting of 699 nucleotides, was deposited into GenBank with accession number KP641311 ( Figure 4B); clone ZZH41, consisting of 942 nucleotides, was deposited into GenBank with accession number KP641312 ( Figure 4C); and clone ZZH51, consisting of 457 nucleotides, was deposited into GenBank with accession number KP641313.

Development of Specific SCAR Markers for G. jasminoides Ellis var. grandiflora Nakai, and Analysis of the PCR Amplicons at Different Species
In this study, we designed and synthesized four pairs of primers (for ZZH11, ZZH31, ZZH41 and ZZH51) to generate more stable G. jasminoides Ellis var. grandiflora Nakai-specific diagnostic SCAR markers based on our cloned sequences ( Figure 4). The primers used in the RAPD analysis are listed in Table 2. The designed SCAR primer pairs were used to amplify the genomic DNA collected from 22 samples to test amplification species-specificity. The PCR amplification results are shown in Figure 5. The PCR results by SCAR markers ZZH11, ZZH31, ZZH41 and ZZH51 ( Figure 5) indicated that the PCR products with expected size were observed only in samples of G. jasminoides, and no amplification in other species we tested. This indicates that the SCAR marker ZZH11, ZZH31, ZZH41 and ZZH51 are specific for G. jasminoides Ellis var. grandiflora Nakai. The lack of this specific amplicons in the samples from other species indicates the efficacy of this marker in distinguishing the samples of G. jasminoides Ellis var. grandiflora Nakai from other species. Negative controls without DNA template did not show any PCR product (data not shown). Therefore, we confirm that G. jasminoides-specific SCAR markers were successfully developed, which can be used for the authentication of these plants.  4 5 6 7 8 9 10 11 12 1314 15 16 17 18 19 20 21 22 2 3 4 5 6 7 8 9 10 11 12 1314 15 16 17 18 19 20 21 22     Then, to further test whether these four markers can be amplified in the sample of G. jasminoides we collected in the market from Luzhou of Sichuan (Table 1), the results in the Figure 6 showed that only SCAR marker ZZH31 were amplified in the sample from G. jasminoides Ellis var. grandiflora Nakai, not from G. jasminoides, whereas other three SCAR markers ZZH11, ZZH41and ZZH51 were amplified in the samples of both G. jasminoides Ellis var. grandiflora Nakai and G. jasminoides. Again, four G. jasminoides samples and four G. jasminoides Ellis var. grandiflora Nakai samples were collected from different localities ( Table 1, Nos. 8~15), all four makers can be amplified in samples of G. jasminoides and G. jasminoides Ellis var. grandiflora Nakai, except that marker ZZH31 can't amplified in the samples of G. jasminoides, further supporting that SCAR marker ZZH31 is specific for G. jasminoides Ellis var. grandiflora Nakai (Figure 7).

Discussion
This investigation aimed to improve the authentication process of G. jasminoides Ellis var. grandiflora Nakai using a RAPD-derived SCAR molecular method. Authentication and characterization of any living organisms has been revolutionized with the development of molecular marker technologies. Over the last three decades, numerous molecular marker techniques have been developed, including RAPD, amplified fragment length polymorphism (AFLP) analysis, simple sequence repeat (SSR) analysis, inter-simple sequence repeat (ISSR) analysis and etc. [13,14,20,21]. It has been reported that the genetic identification and authentication of any species or cultivars become more specific and stable, when another bio-technique SCAR is combined with RAPD [16,18,19,[22][23][24][25].
To develop the SCAR markers, in this study, we have at first collected six cultivars of G. jasminoides Ellis var. grandiflora Nakai, from different geographically isolated regions of China. The DNA materials of these plant leaves were extracted and amplified by an improved RAPD, which revealed the significant genetic variations among the cultivars [12]. We have then focused on the development of SCAR markers specific to G. jasminoides Ellis var. grandiflora Nakai, which can identify this species or cultivars from others. The RAPD fragments were cloned and then four SCAR markers ZZH11, ZZH31, ZZH41 and ZZH51 were developed, which are specific to all of the cultivars of G. jasminoides Ellis var. grandiflora Nakai species used in this study. Notably, DNA material from other species was not recognized by these four markers ( Figure 5). Further investigation demonstrated that only SCAR marker ZZH31 were amplified in the sample from G. jasminoides Ellis var. grandiflora Nakai, not from G. jasminoides when tested by collecting five samples from different localities, whereas other three SCAR markers ZZH11, ZZH41 and ZZH51 were amplified in the samples of both G. jasminoides Ellis var. grandiflora Nakai and G. jasminoides samples. In line with previous studies, this clearly indicates that these three SCAR markers ZZH11, ZZH41 and ZZH51 are specific to Gardenia family, whereas SCAR marker ZZH31 is strictly specific to G. jasminoides Ellis var. grandiflora Nakai cultivars. Thus, we could use SCAR marker ZZH31 to distinguish G. jasminoides Ellis var. grandiflora Nakai from G. jasminoides in our markets (Figures 6 and 7).
Expectedly, in GenBank database, the BLAST searches of these four nucleotide sequences did not show significant identity to that of any species. This indicates that ZZH11, ZZH31, ZZH41 and ZZH51 are the novel biomarkers for the identification and authentication of G. jasminoides, particularly G. jasminoides Ellis var. grandiflora Nakai. In some previous studies, RAPD-SCAR marker techniques have been successfully developed for genetic identification of some other plants, like Lonicera japonica, Dimocarpus longan, and other etc. [18,19,[21][22][23][24][25][26], however, as our best knowledge, this is the first study, which developed molecular SCAR markers for the authentication of G. jasminoides species. Although microsatellite markers for the studying species G. jasminoides have been developed very recently [27][28][29]. The results highlighted that development of these SCAR markers will be helpful for genetic and ecological preservation and conservation of this plant. We suggest that RAPD-SCAR technology could be useful for the identification and characterization of different Chinese herbal materials in the plant pharmaceutical industry or of ornamental importance.
Molecules 2015, 20, page-page 7 [27][28][29]. The results highlighted that development of these SCAR markers will be helpful for genetic and ecological preservation and conservation of this plant. We suggest that RAPD-SCAR technology could be useful for the identification and characterization of different Chinese herbal materials in the plant pharmaceutical industry or of ornamental importance.  (Table 1). Lane 7 is a sample of G. jasminoides from Luzhou in Sichuan (No. 7 in Table 1). Lane M indicates the DNA molecular weight marker DL600 with the fragment size (bp).    (Table 1). Lane 7 is a sample of G. jasminoides from Luzhou in Sichuan (No. 7 in Table 1). Lane M indicates the DNA molecular weight marker DL600 with the fragment size (bp).
Molecules 2015, 20, page-page 7 [27][28][29]. The results highlighted that development of these SCAR markers will be helpful for genetic and ecological preservation and conservation of this plant. We suggest that RAPD-SCAR technology could be useful for the identification and characterization of different Chinese herbal materials in the plant pharmaceutical industry or of ornamental importance.  (Table 1). Lane 7 is a sample of G. jasminoides from Luzhou in Sichuan (No. 7 in Table 1). Lane M indicates the DNA molecular weight marker DL600 with the fragment size (bp).

DNA Extraction from G. jasminoides
Firstly, six fresh young leaves of different cultivars of G. jasminoides Ellis var. grandiflora Nakai which was previous described [12], five more G. jasminoides Ellis var. grandiflora Nakai and five G. jasminoides were collected (Table 1, Figure 1). All the samples of G. jasminoides Ellis var. grandiflora Nakai were collected from gardens from different localities and all the samples of G. jasminoides were purchased in the pharmacy stores which indicated their originations clearly. The samples of G. jasminoides are dried fruits. Plant specimens were identified by the authors and all voucher specimens have been deposited at the Medicinal Botanical Association of Zhongshan Mountain (MBAZM), Sichuan Medical University. Then the plant genomic DNA materials were extracted as previously described slightly modified cetyltrimethylammonium bromide (CTAB) method [29]. DNA was then diluted with 1ˆTE buffer to make the final concentration of 10 ng/µL, and stored at´20˝C until usage.

Amplification of DNA by Improved RAPD
DNA materials of different cultivars of G. jasminoides Ellis var. grandiflora Nakai samples were amplified with primers SBC-N5 and SBS-Q12 using Tiangen Biotech reagents (Beijing, China) according to the manipulation protocol. A total 10 µL PCR reaction system consisted of 5 µL 2ˆTaq PCR MasterMix, 1 µL 2.5 µM primer, 1.5 µL genomic DNA, and 2.5 µL ddH 2 O. Amplification reactions were performed in an Applied Biosystems Veriti 96-Well Thermal Cycler (Life Technology, Carlsbad, CA, USA) using the following program: initial denaturation at 95˝C for 90 s, followed by 40 cycles of denaturation at 94˝C for 40 s, annealing at 36˝C with the RAMP rate from annealing to extension adjusted to 0.125˝C/s (5% ramp rate) for 60 s, extension at 72˝C for 90 s, and a final extension step at 72˝C for 5 min [12]. PCR products were loaded into a 1.5% agarose gel for electrophoresis, along with 1 kb or 100 bp DNA ladder markers (Tiangen Biotech). Measurements were repeated three times in each well.

Cloning, Identification and Sequencing of DNA Fragments
The DNA Cloning, identification and sequencing were described previously [16,18]. Briefly, four different bright bands were excised from the agarose gel, and purified by using TIANgel Mini Purification Kit (DP209, Tiangen Biotech). The purified DNA fragments were ligated into pGM-T vector (No. VT202) (Tiangen Biotech), and transformed into DH5α E. coli competent cells. The recombinant clones were seeded overnight at 37˝C on LB agar plates, containing 100 µg/µL of ampicillin, 40 mg of X-gal and 160 µg of IPTG and kept at 37˝C for overnight. The white colonies were screened out by blue white screening method. The presence of right insert was verified by PCR by using T7/SP6 primer pairs (T7 primer: 5 1 -TAATACGACTCACTATAGGG-3 1 , SP6 primer: 5 1 -ATTTAGGTGACACTATAGAA-3 1 ), then run on a 1% agarose gel electrophoresis [16,18]. The sequencing of the positive clones was performed by Sanger di-deoxy sequencing from Beijing ZiXi Biological Technology Co., Ltd. (Bejing, China) using ABI3500 sequencer (Applied Biosystems Inc., Foster City, CA, USA).

Bioinformatic Analysis by Online Program BLAST
To remove the vector sequences and verify whether the sequences of cloned RAPD fragments are novel, the online program BLAST [30] was used for the homology search of sequenced DNA from different species in GenBank database. Database searches of sequence homology were performed using the program BlastN set to general parameters (expect threshold was 10).

Design of SCAR Primers
The nucleotide sequence of each of the cloned RAPD fragment was used to design pairs of SCAR primers using online program Primer 3 v.0.4.0 [31]. The sequences of each primer, optimized PCR condition and amplification length are shown in Table 2.

Development SCAR Markers and SCAR Analysis
To develop SCAR markers, the PCR amplification was performed by using DNA template from 11 different species, including six cultivars of G. jasminoides Ellis var. grandiflora Nakai and another 10 medicinal plant species (16 samples in total) [12][13][14][15][16]18]. The content of 10 µL PCR reaction system was as follows: 5 µL 2ˆTaq PCR MasterMix, 1 µL of 2.5 µM each pair of SCAR primers, and 1 µL genomic DNA (10 ng), with the remaining volumes filled by ddH 2 O. PCR was performed in an Applied Biosystems Veriti 96-Well Thermal Cycler with an initial pre-denaturation for 90 s at 95˝C followed by 27~35 cycles of denaturation at 94˝C for 40 s, annealing at 60˝C or 64˝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 separated by electrophoresis on a 1.8% agarose gel in 1ˆTAE buffer. Gels were then visualized by 0.5 µg/mL ethidium bromide staining and the images were documented using the ChemiDoc XRS (Bio-Rad, Hercules, CA, USA) [29].

Conclusions
We have cloned and sequenced four improved RAPD derived fragments which were deposited in the GenBank database with the accession numbers KP641310, KP641311, KP641312 and KP641313 respectively, and developed SCAR markers, named ZZH11, ZZH31, ZZH41 and ZZH51 which can specifically recognize the genetic materials of G. jasminoides from other plant species. Moreover, SCAR marker ZZH31 can be used to distinguish G. jasminoides Ellis var. grandiflora Nakai from G. jasminoides at the DNA level. Thus, this study has developed four stably molecular SCAR markers by improved RAPD-derived DNA markers for the genetic identification and authentication, and for ecological conservation of medicinal and ornamental plant G. jasminoides. We suggest that RAPD-SCAR technology could be useful for the identification and characterization of different Chinese herbal materials in the plant pharmaceutical industry or of ornamental importance.