Accurate Cultivar Authentication of Jujube Fruits Using Nano-Fluidic Genotyping of Single Nucleotide Polymorphism (SNP) Markers

Abstract

Chinese jujube (Ziziphus jujuba Mill.) is an economically important fruit tree that is highly adapted to marginal crop lands and can be grown under a very broad range of climate conditions. Jujube fruits produced from several traditional cultivars in Ningxia, China have enjoyed a premium market price due to their unique flavor and quality attributes. One problem with the high-end jujube market is the adulteration of premium cultivars. The accurate identification of the genetic identity of single jujube fruits is essential for cultivar traceability and authentication. The multi-locus SNP barcoding approach offers an effective solution for cultivar authentication of jujube fruits. To identify variant SNP sequences a nanofluidic array approach was used to test the efficacy of this method with DNA extracted from the fruit pulp of eight jujube cultivars from Ningxia, China. The SNP marker profiles identified the genetic identity of each fruit unambiguously based on multilocus matching and ordination analysis. Results for repeated sampling of multiple fruits from the same tree (using independent DNA extractions) showed a high level of concordance, which demonstrated the reliability of SNP based genotyping platform. This method can handle 100 s to 1000 s of samples per day (based on the platform used). It is robust and cost-effective and has a high potential for its practical application in the jujube industry. The developed method and protocol can be readily applied for botanical authentication of other stone fruits in general.

1. Introduction

The Chinese jujube (Ziziphus jujuba Mill.) is an economically important fruit crop in the Rhamnaceae family. The species is native to China, with its putative center of origin located in the Yellow River basin in northern China [1,2,3,4]. Chinese jujube has wide adaptability to different agro-ecologies in the subtropical and temperate regions [5,6]. This fruit tree is becoming increasingly popular for its adaptability, multi-purpose utilization, and positive impacts on human health [7]. Today jujube has been introduced into more than 50 countries throughout Asia, Europe, Africa, the Americas, and Oceania [8]. The market value of the jujube industry in China was estimated at $14–16 billion in 2017 [1,7].

Jujube is one of the earliest domesticated fruit trees, with an estimated cultivation history of 7000 years [2]. There are over 800 different jujube cultivars, which can be classified from the perspectives of postharvest and consumption. Some are table or fresh-use cultivars for the fresh fruit market; some are best for drying and processing as dry dates (‘red dates’); while others have dual-purpose usage [6]. For the dry dates (or red dates), the fruits are harvested when fully ripen. The harvested fruits are sun-, air-, or heat-dried to reduce the water content to approximately 25% in the fruits [6]. The dry dates can be consumed directly as snacks or used in porridges, soups, or teas, stewed, or processed further into various foods and medicinal products [7]. For the fresh fruit cultivar types, good shelf life, desirable texture, and taste are the main quality attributes and the fruits are typically harvested just before they are fully ripened [6].

The jujube industry is a pillar of the local agricultural economy in Ningxia, China. Fresh fruits produced from several traditional cultivars in Ningxia have enjoyed a premium market price due to their unique quality attributes [9]. Ningxia has started using geographical indications (GIs) as a separate type of intellectual property (IP). Several traditional cultivars, such as “Lingwu Changzao” and “Zhongning Yuanzao” (Figure 1) have been granted as GI-registered products. GIs are an important type of IP used for enhancing quality policy in agriculture. However, efficient and practical methods for cultivar authentication of fresh and dry jujube dates have not yet been developed, which hinders the implementation of GIs regulations in the jujube industry. Traditional methods for jujube fruit identification recognize the botanical origins by their morphological characteristics. This type of method largely depends on human skill and expertise thus it is not practical for large-scale applications. Instrumental methods, such as Inductively Coupled Plasma Mass Spectrometry (ICP-MS) and Thermal Ionization Mass Spectrometry (TIMS) have been used to study the traceability of dried jujube fruit based on the composition of stable isotopes [10]. Based on a similar principle, Isotopic Ration Mass Spectrometry(IRMS) was applied for traceability studies of fresh jujube [11]. In both cases, the authors reported that it is feasible to differentiate the geographical origin of jujube fruit produced from different regions, using isotopic analysis technology. However, the instrumental analysis does not have the capacity to determine the cultivar identity of fresh or dry jujube fruits. Positive identification of botanical origin for jujube fruits requires a complementary approach based on genetic testing of single jujube fruits. The advantages of the DNA-based fingerprinting method are well recognized for agricultural and food products [12,13,14]. DNA barcodes have been reported as successful in the identification of cultivar authenticity for cacao [15], tea [16], coffee [17], and olive oil [18]. Moreover, SNP markers for the accurate identification of jujube varieties have also been developed [9], which can be used for the detection of the genetic identity of jujube fruits.

Jujube is a typical drupe (or stone fruit) thus the edible outer layer is the pericarp, which is developed from the ovary wall of the flower. Therefore, the fleshy tissue shares the same diploid genotype with the mother tree and can be directly used for DNA-based cultivar authentication of jujube fruit. In this study, we investigated the efficacy of using a nano-fluidic SNP genotyping method for the verification of cultivar authenticity in fresh jujube fruits. The objective of this work was to develop a DNA-based barcoding method that can accurately verify the botanical authentication of jujube fruits. The resulting information can serve as a scientific baseline for the implementation of the GIs regulations in the jujube industry. The study was conducted with a focus on eight jujube cultivars from Ningxia, China, but the developed method and protocol can be readily applied for botanical authentication of jujube cultivars from other regions, as well as for other stone fruits in general.

2. Materials and Methods

2.1. Jujube Fruit Samples and DNA Extraction

Plant materials used in the present study include eight jujube cultivars from the Ningxia autonomous region of China (

Table 1. List of names, origins, and sources of eight Chinese jujube cultivars used for testing fruit authenticity in the present experiment.

#	Cultivars	Type	Origin	No. of Samples Analyzed Fruits
1	Lingwu Changzao	Traditional cultivar	Ningxia, China	3
2	Zhongning Yuanzao	Traditional cultivar	Ningxia, China	3
3	Lingwu Suanzao	Traditional cultivar	Ningxia, China	3
4	Longzhu #1	Bred cultivar	Ningxia, China	3
5	Longzhu #2	Bred cultivar	Ningxia, China	3
6	Suanzao	Traditional cultivar	Ningxia, China	3
7	Zaoqiuhong	Bred cultivar	Ningxia, China	3
8	Tongxin Yuanzao	Traditional cultivar	Ningxia, China	3
	Total			24

). The fresh fruits were collected from the Horticulture Research Station of Ningxia University, Yinchuan, China. A single fruit was independently sampled from each of the three clones per cultivar.

DNA was extracted, from approximately 500 mg of pulp tissue from each of the fresh fruits. Total genomic DNA was extracted using a modified Cetyl Trimethyl Ammonium Bromide (CTAB) procedure described by Porebski et al. [19] with the extraction buffer (2.5% CTAB, 0.1 M Tris-Cl [pH 9.5], 0.025 M EDTA, 1.5 M NaCl, 4% PVP-10, 0.5% (v/v) 2-β-mercaptoethanol). DNA quality was evaluated on 0.8% agarose gel stained with ethidium bromide and then visualized with a UV trans-illuminator model M-20 (Upland, CA, USA). The DNA samples were then sent to the Sustainable Perennial Crops Laboratory, USDA Beltsville Agricultural Research Center, Maryland, USA for genotyping. Prior to genotyping the DNA concentrations (at 260 nm) and purity (A260/280 ratio) were measured, using a NanoDrop spectrophotometer (Thermo Scientific™, Wilmington, DE, USA).

2.2. SNP Markers and Genotyping

The jujube SNP marker genotyping panel used in this study (Fluidigm Corp) was previously reported by Song et al., (Song Lihua, unpublished data). The level of polymorphism and the pairwise linkage relationships was used to select the 96 SNPs markers. When two SNP markers showed significant linkage disequilibrium, the one with the lower value of Polymorphic Information Content (PIC) was excluded. The selected SNP markers were used to assemble a SNPtype™ genotyping panel (Fluidigm Corp). These SNPs and their flanking sequences are shown in

Table 2. The top 24 selected SNP markers recommended as a genotyping kit for jujube fruit authentication. The full panel of 96 markers was listed in Supplementary Table S1.

#	SNP Code	Chr.	SNPs and Flanking Sequences
1	Zj002	1	GCCTTGTTCCTGACGAGTTAGAGATTCCCTGGTAAATTAGGGTAGTGGAATGAGACCTAGGCAAACCAATATGCATCGGC[T/C]AGCAGGGCCAGAGAGCGGGTTCCCCACCTCGAAAAACACACACACACACATACATATATATATATATATATATATGTAAT
2	Zj018	1	GTCGAGCTTTAGTTCCAAAAGTTATTCGGATTCAGATGATTGGGCATCCTATAATATTACCAATTGTAGATATGCCTTTG[T/C]GATGGAAGAAAGCAAATTCAAGTTCTCTACAAGTTTCAAAGAAAAGGTTCCAATGGTTATAAATTGGGCAATTGGGAAAG
3	Zj028	2	TCTTTTTCTTTTTAAACTAAGTATAACCTTAAGAAACCTACCTTCTTATTATAAAAACAAATAAATAAAAATATATATAA[C/T]TTTTTTAAAAACACACACACACACACACACACACACACACACACACACATATATTGGTTTATAATTTAAAATATCCAAAT
4	Zj041	2	AAAATAAAAAAAACCAAAATTTTCTTTTCTTTTTCAAAAACAATAAAAAAGTTTTCCAAATGCTTCAAAAAAAAATAAAA[A/C]AAATCAAGCACCACCATGATCAAAACAAAATGGGTTATCTTTCAATGTTCCAAAAAAAACTTCACCAAGAGAAAAAATGT
5	Zj056	3	AATTTTTGAAGTAATCGAAAGGCTAATAAGGGAGGTTTTGAACTAAGAATTTATAAATTAGTTTGGAAGAATCATGCTGA[T/C]ATAACAAATGCTTTTGGTGATATCTCATGAAATATTTAAAAACTAAAAAATTAGTGGGACAAGTAACAACCATTAGCACT
6	Zj062	3	CTGAAATTGTAAGATACTTCAACTTTTGTTTCAGATTCTCTGCATTCCATGGTTTAAATCTTTTGTTGAGATCATTACGT[T/C]GTGTTGTTGTTGTTATATTTATTTATTATTTTTTTAAATGTATTCTGGTTGTAGGGTTGTTCATTGGTTGTACTTGATCA
7	Zj089	4	TTCTCCCTAACAATCTAAAACCTAAACAAATAAAAAGGGGGCATCCAAGTTCAATTCTTGTTAATACGTGTACTTCATAC[C/T]TATGCTGCTTTTTAATTATATTTTCAATTAAGTAAAATAAATTTACATGCTATTGCAAGGCGTTATCGATGATTTAAATT
8	Zj090	4	TAATATATATAAATCTTCATGGTGTATCTAATTAATTACCATCTAATCATGATCATGATCAACTATAATGAGCGACATCT[C/A]CCACCATATTGTGCCAAACAGAGCTAGATTCTTCGTCCCTTCTTGTGTGTCCCCAAGTCTATCTCATCAACCAATTAATG
9	Zj106	5	TTCTTTATATATTTATTGGAAGAGTTTTTTCATTTTTTGTCTGAATATGGTTGTTATGGTTTTGTGCTCTTATTTGTTTT[T/C]CTTAACCCTGCACGTTTCTTTTTTCTTTAAAAAGAGACCCTGCATGTTTAGTTGCATATGAATTCCCTAGCTTTAGTGAT
10	Zj124	5	CTGCTTCGTTTGATGCTGCAGAAATTTTAAAAGTAGAGACATGTTGAGGAGGTCTGGTACTTGGTGTGAATATATCTATC[T/G]CTTGTGGAGCTGTAGAGACTGAAAGCCATATTGTTCATCATCATTGTCAAAGACAACAATGTTCAAGGCAAATACAATAA
11	Zj151	6	AATTTGAGAAAATTGAGTGCTTGAATTTTGCTATCTCATATCTTATTAATTAAAAGTCCCTAGAAAGATAGCTATAGTAG[T/C]ATTGTAAATAAACATTTTTTTTCGATTTTAATATAGTTCTTCACTGGTAAAGCGAGGAATTGGAAACAACATTTATTGAA
12	Zj157	6	CACCACATACCAATAATATGCAAAGCACAGAATACACACATTTCTAAAGAAAAATAAAGCAATCCAAGGCGATTAGGGGG[C/T]TATCGATATCCTCCATAGGAATAGAGTCGATGTCATGGTCATGAAGTGGAGCCAGCGCTTCGCCAACCGCAATCTCGTCG
13	Zj171	7	GACACATTCGGTCATATGCTTACAATCTTTAAAATTTTCTGTTCAAAGTTTCAAGAACTAATATTCTGATGCAGAATCAA[C/T]AACATAACAATTTTATGTTGGTCTTAAACAACAAAGATTCAGCAGACCTTGCATTTTAAATGCTTATATAAAATTATGAA
14	Zj179	7	TGGATCAAAATATGCAATGATCCAATATTTTTTTGACTGGTGTTGCTTAAAAACACATTTTTGCAAGTGAACTGAAATTT[A/G]CCCAAGGTGGTCACTTGATAGTTTACTGCTCTTTCTTCTTGCTTCAACTGAACAAGTTTACCGGGGAATATAATCAAGCA
15	Zj190	8	ACTTATAGTATATGCTACAAACCAGAAAAGTTCTAAGTAGTTTAATGAAAATTTAGGACCTTACATAACTTTTTAGATTA[T/C]ATTTAAATCATTTTGAGAAGCAATGCCAATATCAGCATATTTCTTACTATTTCCTAAAAATTGATTTTTATTTTTATTTA
16	Zj204	8	AAAATAGTAACTTTCTTAAACTTATTTGTCCAGCAAACAAATAAATAAAAACCAAAAGTAAAGGTAATTTCCTAAAACAA[A/G]AAGTAGAAATCAAATTAGAAAACTAAGATACTATCTATTTTACTAATTTGACTTTGACCAATATTTGATCATGAAGTCCC
17	Zj216	9	ATTGATTGTAGTTTATTGACTAGTAGTCCACCAAAATCGGAGCTGCAATTAAAAAGTTATGATCAAAACAATTTGTGATC[T/C]AGTCTAATTGAGCAAGCTCGAATTTGATTTTTTATTTGCATGAATTTTGGTTTTGAATTGTATACCATTATATAGTACTC
18	Zj220	9	TTTAGTGATTGTTAGATTTGTTTATTTAAGTTGCAAGATTTTTAATACCCCAAATCAGAATTTTTTCGATTTTTAGATCC[A/G]AGTCTTGTCTTAAAGGGAGAGATTAAGATATATGAGCCATTTACATGTCTGAATCTTGACACTCTGAGAAGTTGTAGAGC
19	Zj225	10	GAAAGACATGCAAATTTTGTAACGAATAAATGTGTACTTGAAAAACAGAAAAATAAACGGCAATCTTCAATATTTTGCTT[C/T]GCATTTTTAAGAAATATTTTCAGTTTTTATTTGTAGATGAAAAGTCAAAAATTAATCAAAGAAAAAAAAATGTGCGTTTT
20	Zj242	10	AGTTTAGAAACAATCTTGGTGAAGCCTATGAAGAAGTGATCATAAAGAGTCATCTTGTTGTAGCTCTCCATCCAAGACCT[G/A]GTAACTAGCTTGACACTGTCACTGAATGCCTTTTCCAATGTATTGAAGAGATCATTTCATAGCATTGATGGTTATGTTGC
21	Zj271	11	TCATCATTGGATGTCACACCGACACGACACTCGTAACCATTCATTTCTTTGCAACTTTCTTCTTCATTTTCTACTATGGC[T/C]GCGTGCTTTAATGATGTTGACATGGCCTGAGACAATACCACTGGATTCTTTTCCACACATCTTACACCATCTTCTGTAGG
22	Zj275	11	AGTATGAGAATCCACAGAATTGCAAGCAGCTCTGTAGCAGACATGTAAACGGCAGAAAGCATTGCAGTGTGTATGTTCTG[C/T]AATAACCAGCCAGACCAGGATTATTTTTTCATCTCTTGTAATACTTATATGCAGTTTCCCATCCAAAAAAAGATTTAATT
23	Zj290	12	GTTCATGTGTCACTGTTCACACGTACTGTACACTATTCATTATCACTGTTCATTGCCGGGTGTCGCACTAAAGACCGACA[C/T]GTGGCACGTGCCACACTCTCGAACGGCCACGTGTCATCTGCTACAGACGACATGTGGATCATCCTTAGCACATTTGAACG
24	Zj304	12	TTGGTCAAAGAATGGTCAAAGTCAACTTAGTCAAAAAGCTAGTATTTTAGTTTCCTAATTTGATTTTACTTTTTGTTTTA[A/G]GAAATTACCATTACTTTTTGGATTCGATTTACTTATTTTATGGACAAATAAGTTGAGGAAAATTATTATTTTATTATTTC

. The genotyping analysis was performed on the Fluidigm EP1™ system, using Fluidigm SNPtype Genotyping Reagent Kit, per the manufacturer’s instructions, and a nanofluidic 96.96 Dynamic Array™ IFC (Integrated Fluidic Circ.uit; Fluidigm Corp.). Fluorescent intensity was measured with the EP1™ reader and plotted in two axes. Genotypic calls were made using the Fluidigm SNP Genotyping Analysis Program (Fluidigm Corp, South San Francisco, CA, USA).

2.3. Data Analysis

To evaluate the authenticity of fresh and dried jujube fruits, multilocus matching was used to assess whether multiple fruits of the same cultivar were produced from the same clone. The computer program GenAlEx 6.5 [20,21] was used to match multi-locus SNP profiles. Samples that matched at all 96 SNP loci were considered identical and therefore derived from the same clone. To determine the genotyping capacity of the jujube SNP panel, the siblings probability of identity (PID-sib) [22] was computed, which is the probability that two sibling individuals drawn at random from a population will have the same multilocus genotype. The overall PID-sib is the upper limit of the possible ranges of PID in a population and provides the most conservative number of loci required to resolve all individuals, including relatives [22].

To understand and visualize the relationship between the eight jujube cultivars from Ningxia, the genetic distances for each possible pair of tested cultivars was computed. The matrix of genetic distances was then visualized using principal coordinates analysis (PCoA), implemented in GenAlEx 6.5 [20,21]. Cluster analysis, based on the Neighbor-Joining method, was used to further illustrate the genetic relationship among the eight cultivars. The identical SNP profiles of the three fruits were condensed and only one SNP profile was retained for cluster analysis. The distances between individual cultivars were calculated using the shared proportion of alleles as described in the program Microsatellite Analyser, with 100 bootstraps [23]. The distance matrices derived from the results were used to generate a consensus tree using the Neighbor-joining algorithm [24] in the program PHYLIP [25]. Once generated the dendrogram was visualized using the FigTree program version 1.4.0 [26].

3. Results

3.1. DNA Extraction from the Pulp Tissue of Jujube Fruits

DNA concentration ranged from 29.3 to 67.5 ng μL⁻¹ among the 24 fruit samples representing the eight jujube cultivars, with an average of 47.4 ng μL⁻¹ per fruit sample. The average ratio of absorbance at 260 nm and 280 nm by Nanodrop measurement was 1.86 among, the 24 samples. The lowest (1.74) was found in the fruit of “Zhongning Yuanzao”, whereas the highest was for fresh fruit of “Lingwu Suanzao” (2.01;

Table 3. Concentration and quality of DNA samples extracted from the pulp tissue of fresh and dried jujube fruits.

Name of Cultivar	Concentration (ng µL−1)		A260/280
	Range	Mean	Range	Mean
Lingwu Changzao	29.3–57.6	44.4	1.75–1.96	1.86
Zhongning Yuanzao	32.5–65.8	48.8	1.74–1.89	1.81
Lingwu Suanzao	42.7–57.5	49.4	1.82–2.01	1.95
Longzhu #1	40.4–58.6	52.6	1.77–1.89	1.81
Longzhu #2	38.8–67.5	46.5	1.79–1.99	1.85
Suanzao	32.6–55.4	38.8	1.83–2.00	1.93
Zhaoqiuhong	44.2–62.3	54.5	1.75–1.95	1.85
Tongxin Yuanzao	42.1–47.9	44.1	1.78–1.88	1.80
Mean		47.4		1.86

).

3.2. Jujube Fruit Authentication Using SNP Fingerprints

All 96 polymorphic SNPs were reliably scored across the DNA samples extracted from 24 fruits, representing eight cultivars (

Table 4. Examples of individual fruit SNP profiles based on DNA extracted from fresh jujube fruits (showing truncated profiles). The full data for all 24 fruit samples across 96 SNPs were presented in Supplementary Table S1.

#	Cultivar	Zj002	zj018	zj028	zj041	zj056	zj062	zj089	zj090	zj106	zj124	zj151	zj157	zj171
1	Lingwu Changzao	TT	CT	CT	AC	CT	CT	CT	CC	CT	GT	CC	CT	CT
2	Zhongning Yuanzao	CC	TT	CC	CC	CT	CT	TT	CC	CT	GT	CC	CT	TT
3	Lingwu Suanzao	CC	TT	CT	AC	CC	TT	TT	TT	AC	CT	GT	CT	CT
4	Longzhu#1	TT	CC	CC	CC	CT	TT	TT	AC	CT	GT	CC	CT	CT
5	Longzhu#2	CC	TT	CC	CC	CT	CT	CT	AC	CT	GG	CC	CT	CT
6	Dasuanzao	CC	TT	CC	CC	CT	TT	TT	AC	TT	GG	TT	CT	CC
7	Zaoqoiuhong	TT	CT	CT	CT	CT	TT	TT	CC	TT	GT	TT	CT	CT
8	Tongxin Yuanzao	TT	CT	CT	CT	CT	TT	TT	CC	CT	GT	CC	CT	CT

and Table S1), which enables the unambiguous differentiation of all eight jujube cultivars. The reliability of the 96 SNPs was demonstrated by the result of repeated genotyping of three fruits from the same cultivars (Table S1). The overall repeatability reached 99.8% if no calls were scored (as 0) in some repeated samples. The fruit-based SNP profiles fully matched (with identical SNP profiles excluding missing data) with the leaf-based results (Lihua Song, unpublished data), thus confirming the true-to-type authenticity of these cultivars.

PID-sib, calculated from the 24 fruit samples used in this study, predicted that the probability of two unrelated samples having the same genotype at all 96 SNP loci was 5.98 × 10⁻¹⁵. Furthermore, these results showed that multiple clones sampled from these cultivars were from the same mother tree. The matrix of genetic distance among the eight cultivars was presented in Table S2. Results of Principle Coordinate Analysis (PCoA) revealed the genetic diversity within the eight Ningxia jujube cultivars, which had a total of 81.1% of genetic variation explained by the top three axes (Figure 2). Each of the eight cultivars was clearly separated from the other cultivars, which confirmed that these cultivars are genetically distinguishable from each other.

The Neighbor-joining tree based on the 96 SNP markers showed that the eight cultivars could be separated into three groups (Figure 3). The first group included the cultivar from “Lingwu Suanzao” alone, which is likely a domesticated cultivar from indigenous wild jujube germplasm in Ningxia (Song Lihua, unpublished data). The second group included “Tongxin Yuanzao”, “Dasuanzao”, and “Zaoqiuhong”. The third group included “Longzhu #1”, “Longzhu #2”, “Zhongning Yuanzao”, and “Lingwu Changzao”. Since each cultivar was based on condensed samples of three fruits showing 100% similarity, this clear cultivar difference suggested that this SNP panel is highly effective for cultivar authentication of jujube fruits (Figure 3).

3.3. Selection of top 24 SNP Markers for Jujube Fruit Authentication

From the 96 SNP loci, a subset of 24 SNPs that has high polymorphism informative content (PIC) and balanced chromosome representation was further selected and recommended for jujube fruit authentication (Table 2 and

Table 5. Expected number of individuals with the same multilocus genotype for increasing locus combinations (number of loci = 24). PI = Probability of Identity; PID-sib = Sibling probabilities of identity; PIC = polymorphism information content.

Locus	zjoo2	zj018	zjo38	zj041	zj056	zj062	zj089	zj090	zj106	zj124	zj151	zj157	zj171	zj179	zj190	zj204	zj216	zj220	zj225	zj242	zj271	zj275	zj290	zj304
PI	9.0000	7.0760	2.9547	1.1198	0.4199	0.1592	0.0665	0.0249	0.0098	0.0038	0.0015	0.0008	0.0003	0.0001	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000	0.0000
PI-Sib	14.2500	12.6535	8.0925	4.8446	2.8765	1.7220	1.1013	0.6539	0.4013	0.2462	0.1511	0.1103	0.0677	0.0405	0.0243	0.0149	0.0095	0.0058	0.0035	0.0021	0.0012	0.0007	0.0004	0.0003
PIC	0.37	0.29	0.26	0.37	0.38	0.23	0.38	0.31	0.37	0.37	0.37	0.36	0.37	0.37	0.29	0.37	0.30	0.38	0.37	0.38	0.22	0.37	0.37	0.37

). The idea is to recommend a small but essential number of SNP markers to reduce the genotyping cost. With the genotyping kit of 24 SNP markers, it could save the genotyping cost by 75% relative to the 96 SNP panel. The PIC value of the 24 SNPs ranged from 0.22 to 0.38 and the accumulated PID-sib value of these 24 SNPs reached 0.0003 (Table 5; Figure 4), which provides sufficient statistical power for accurate varietal authentication of jujube fruits. The generated SNP profiles can be converted into a simple bar code and serve as a labeling component for the GI-registered product, therefore enabling the implementation of GI product protection.

4. Discussion

Botanical authentication is an increasing concern in the jujube industry due to the rapid market differentiation and demand for high-quality jujube products, both for fresh and dry fruits. Many high-quality jujube cultivars now have been recognized as GI-registered products. This GI system provides a way for the jujube industry to leverage the value of their geographically unique products and attract consumers who are willing to pay premium prices for these products [27].

Ningxia is well-known for its unique jujube cultivars and jujube industry. In Ningxia, three traditional jujube cultivars have been recognized as GI-registered products [9]. Cultivar adulteration often happened when these products are exported to markets outside Ningxia. The adulteration was often found with local cultivars that have similar morphological characteristics but inferior quality attributes. To date, no methods have been developed to detect the genetic identity of jujube fruit by morphological and/or biochemical characteristics.

In the present study, SNP profiles were successfully generated for all tested jujube fruit samples from both Ningxia, China. DNA was extracted from the pulp of fresh fruits, which are genetically identical to the mother tree. SNP profiles generated by the selected SNP panel enabled accurate verification of clonality among multiple fruits and clear differentiation of all cultivars. So far, published studies on DNA-based fruit authentication remain scarce. Moreover, most of the reported studies were at the species level, such as Vaccinium bracteatum [28], Zanthoxylum species [29], and Garcinia spp. [30]. To the best of our knowledge, this is the first report on DNA-based fruit authentication at the cultivar level.

Compared with other DNA fingerprinting techniques, SNP genotyping offers an advantage, the positive confirmation of cultivar genotypes through multi-locus matching (without the need to compare genetic distances), thus it provides the most direct scientific evidence for cultivar authentication. In addition, this method can process and analysis many samples quickly and the results are highly robust and repeatable.

4.1. DNA Quality Extracted from Jujube Fruits

Jujube fruit is highly prized and contain large amounts of carbohydrates, polyphenols, proteins, minerals, vitamins, and organic acids [6,31,32]. Glucose, fructose, and sucrose are the main sugars found in jujube fruits, which contribute to the sweetness of the jujube fruit [6,32]. The average yields of genomic DNA from jujube fruit obtained in this study ranged from 29.2 to 65.4 ng μL⁻¹. The high concentration enabled further dilution of DNA samples, which is done to reduce contaminants such as polyphenolic and polysaccharide compounds in the DNA samples. The A260/A280 ratios were between 1.74 to 2.01, which is comparable to those obtained for DNA extractions for the jujube leaves (Lihua Song, unpublished data) indicating the good quality of DNA from these samples. In addition, the Specific Target Amplification (STA) reaction, utilized before genotyping reaction, is a multiplex PCR reaction that amplifies all the loci of interest, proportionally increasing them without targeting any specific alleles. This method significantly improves SNP call rates for genotyping and solves issues such as low DNA concentrations and high levels of polyphenolic compounds or other PCR inhibitory compounds.

4.2. The SNP Genotyping Kit for Jujube Fruit Authentication

In the present study, we used 96 SNP markers to perform the genotyping. These 96 SNPs were selected based on their high polymorphism informative content (PIC) and linkage equilibrium. However, to recommend a minimum essential number of SNP markers for unambiguous cultivar authentication and minimize the genotyping cost, we selected a subset of 24 SNP markers as a genotyping kit for jujube cultivar authentication, as well as for other downstream applications. The probability of identity (PI) [22] was used to determine the minimal number of loci required to identify all distinct individuals with a high confidence level. Multi-locus PI values were obtained by multiplying together single-locus PI values, with the assumption of independence of loci. A highly stringent PI value is needed for domesticated crop species because in many cases they share similar ancestors or parentage. PI calculations for sibs in the computation to provide a highly conservative boundary for jujube, to meet the threshold for a highly domesticated crop species.

In the present study, we demonstrated that these 24 SNP loci are sufficient to confirm the authenticity of the given jujube fruit. The chance of sampling identical genotypes from a random mating population is 0.0003 (Table 5; Figure 4). This predicts a high statistical power of this minimum essential set of SNP markers for jujube cultivar authentication. The SNP profiles generated can be converted into simple bar codes, which can be used as a labeling component for the GI-registered product, therefore allowing the implementation of GI product protection. This genotyping procedure and makers can also be used for nursery accreditation and quality control of jujube planting of clonal gardens. Nonetheless, it needs to be acknowledged that the testing was based on eight jujube cultivars from Ningxia only. The suitability of the 24 SNPs on another jujube germplasm still needs to be validated.

4.3. Advantage of Using SNP Markers for Jujube Cultivar Authentication

The feasibility of using DNA fingerprints for jujube cultivar authentication depends on whether the targeted cultivar has a uniform and distinguishable genetic profile. In the present study, all tested cultivars are highly uniform, as revealed by their SNP profiles. This was due to the wide adoption of clonal propagation for jujube cultivation in recent years, which has been used to improve yields and maintain the consistency of jujube quality [7]. The genotyping results of the multiple fruits from the different Ningxia cultivars demonstrated that the fruits were all sampled from clonal trees. Clonally propagated cultivars, authentication through SNP fingerprinting is straightforward. The genotyping results from the three clonal fruits (three independent DNA extractions were performed from the same clone) showed 100% concordance. The fresh fruits analyzed in this study fully matched with the leaf-based SNP profile of the mother tree, as presented in the previous study [1].

Compared with other DNA marker technologies such as SSR markers, the SNP-based cultivar authentication has clear advantages. Firstly, it enables data comparison and data compilation across multiple laboratories [33,34]. Despite the high polymorphism of SSR markers, the comparison and combination of SSR fingerprints generated by different laboratories or genotyping platforms (e.g., Applied Biosystems (ABI), Sequence (SEQ) or other gel box electrophoresis) is difficult. Secondly, SNP genotyping can be easily implemented in either low or high-throughput systems therefore it can be scaled to meet the demands of the jujube industry. Therefore, a small set of SNP markers will be highly useful for multiple downstream applications in the jujube value chain. Data generated by SNPs can be easily compared, regardless of the genotyping platform used.

5. Conclusions

We conducted a study that verified the authentication of jujube fruits based on the DNA extracted from the pulp. DNA fingerprints were generated using a nanofluidic array with SNP markers. This technology was tested on jujube fruits and generated high-quality SNP profiles for all. The pulp-based DNA fingerprints together with forensic statistical tools provided unambiguous identifications for all the tested jujube cultivars. This protocol used the nano-fluidic chip for SNP genotyping and enabled rapid cultivar authentication of jujube fruits. The same procedure can be applied for cultivar authentication of other stone fruits as well. To our knowledge, this is the first study using molecular makers to authenticate jujube. This practical application could be extremely useful for the verification of GI-registered jujube cultivars and thus has significant potential for cultivar authentication.