Introduction
============

Chinese jujube (*Ziziphus jujuba* Mill.; 2*n* = 2*x* = 24) is an economically important species in the Rhamnaceae that is native to China ([@ref-2]). One of the world's oldest cultivated fruit tree crops, according to archaeological evidence jujube was used at least 7,700 years ago in China ([@ref-13]). Fruits of jujube have high nutritional value as well as medicinal properties, and are consumed fresh, dried or in processed form ([@ref-8]).

Over a long period of natural evolution and artificial selection, Chinese jujube has acquired a wide range of variation, with more than 800 cultivars reported ([@ref-12]). These cultivars are distributed across China and are propagated either by rootstock grafting or as rooted cuttings ([@ref-29]). The origins of most of these cultivars are obscure because of the frequent exchange of plant material between different cultivation areas and the lack of cultivar historical documentation ([@ref-30]). During the frequent exchanges, the gene flow among cultivars and the gene flow between cultivars and their wild relatives are not reported. The evolutionary history of jujube cultivars thus remains unclear.

However, genetic studies based on molecular markers could offer new clues to the evolutionary history of cultivars ([@ref-23]; [@ref-26]; [@ref-20]). Simple sequence repeats (SSRs) are tandemly repeated nucleotides in DNA sequences. Because of their high polymorphism, codominance and stability, SSR markers are widely used as genetic markers to analyze genetic diversity, phylogenetic relationships and the biology of populations ([@ref-25]; [@ref-1]). The development of SSR markers for jujube is therefore useful for reconstruction of phylogenetic relationships among cultivars, assessment of genetic diversity, and for breeding and genetic analysis of this species.

Several studies have reported the development of SSR markers for jujube. For example, 66 expressed sequence tag-SSRs were developed from a Chinese jujube fruit cDNA library and amplified in six cultivars, but without evaluation of polymorphism ([@ref-15]). Using selectively amplified microsatellites, [@ref-17] constructed a set of 25 primer pairs, and an additional 31 primer pairs were developed from SSR-enriched genomic libraries ([@ref-30]). Seventy-one polymorphic tri-nucleotide SSR markers were developed from assembly fruit transcriptome of jujube ([@ref-10]). A set of 551 primer pairs were developed from jujube genome and tested in 6 jujube cultivars ([@ref-32]). These primer pairs were polymorphic among different jujube cultivars and were used to infer genetic relationships among major Chinese jujube cultivars, but few of these are validated at the level of jujube populations.

Nine microsatellites were validated on the level of jujube populations in the study about the genetic diversity and population structure of *Z. acidojujuba*, which is considered as the ancestor of cultivated jujube ([@ref-34]). However, study of population structure of jujube cultivars is rare. Cultivars often have lower genetic polymorphism levels than wild species, such as peanut ([@ref-18]), soybean ([@ref-9]), apple ([@ref-33]), due to diversity loss during artificial selection and vegetative propagation. Markers with high polymorphism levels in jujube cultivar populations are thus still needed for genetic and evolutionary research on Chinese jujube. The recent availability of the jujube genome sequence ([@ref-16]) allows rapid identification of highly polymorphic markers for population genetics and facilitates SSR primer development.

For complementing previous data sets, we develop highly polymorphic markers in jujube cultivar populations and test them in populations as well. In this study, we examined the abundance of microsatellites with repeated unit lengths of 1--6 bp in jujube, characterized their distribution in the jujube genome, and developed 70 SSR markers from the genome sequence. After screening using a series of criteria, we finally used 20 polymorphic primer pairs to genetically characterize three jujube populations. The validated microsatellites could be used in research about the genetic structure and evolutionary history of Chinese jujube.

Materials and Methods
=====================

SSR identification and primer design
------------------------------------

The final assembly of Chinese jujube genome spans 437.65 Mb (98.6% of the estimated size) with 321.45 Mb anchored to the 12 pseudo-chromosomes ([@ref-16]). The 12 pseudo-chromosomes sequences were downloaded from GenBank (accession number: [CM003114](CM003114)-- [CM003125](CM003125)) and scanned using MSDB v2.4 software (<http://msdb.biosv.com>) to identify all perfect microsatellites ([@ref-31]). The identification criteria used for mono-, di-, tri-, tetra-, penta- and hexanucleotide repeats were default in MSDB, with a minimum of 12, 7, 5, 4, 4 and 4 repeats, respectively. After analyzing each chromosome independently, all 12 chromosomes were analyzed together. Genome sequences of *Arabidopsis thaliana* were downloaded from GenBank (accession number: [CP002684](CP002684)-- [CP002688](CP002688)) and comparatively analyzed against the jujube genome.

After searching the complete jujube genome, primers were designed in PRIMER v5.0 ([@ref-35]) using loci with repeat numbers greater than 25, for possible high polymorphism, and less than 100 for fitting with the scoring size standards. Microsatellites with mononucleotide repeats were removed when primer were designed. All primers were designed according to the following parameters: (1) product size from 100 to 400 bp and (2) primer size from 18 to 22 bp with an optimum size of 20 bp. All primers were synthesized by Tsingke Biological Technology Co. (Beijing, China). After screening using a series of criteria, parts of forward primer 5′ ends were labeled with a fluorescent dye (FAM or HEX) from Tsingke Biological Technology Co.

Plant materials and genomic DNA isolation
-----------------------------------------

In this study, we tested the amplification and polymorphism of all primers using three populations: two populations (17 individuals each) of jujube cultivars 'Bianhesuan' and 'Huizao' and one population (14 individuals) of *Z. acidojujuba* C.Y. Cheng et M.J. Liu, the ancestor of Chinese jujube ([@ref-12]). We treated all plants of a cultivar collected from its major planting regions as a population. In addition, 10 main jujube cultivars were obtained from the Xinzheng Jujube Research Institute, Henan, China, to test the amplification feasibility of SSR markers in different cultivars. One individual was collected for each cultivar. Sample information is provided in [Table S1](#supp-1){ref-type="supplementary-material"}. Total genomic DNA was extracted from dried leaves with a modified CTAB method ([@ref-3]), quantified using a NanoDrop 2000c spectrophotometer (Thermo Scientific, Waltham, MA, USA), and checked for quality by gel electrophoresis.

Polymerase chain reaction (PCR) and fragment analysis
-----------------------------------------------------

PCR amplifications were carried out in 15-µl reaction volumes containing 10--20 ng template DNA, 1× PCR Buffer, 1.5 mM MgCl~2~, 0.4 µM of each dNTP, 0.2 µM of each primer and 1 unit of *Taq* DNA polymerase (Takara, Dalian, China). The PCR cycling profile consisted of an initial step of 95 °C for 5 min, followed by 35 cycles of 95 °C for 45 s, 50--55 °C (annealing temperature of each primer) for 45 s and 72 °C for 30 s, with a final extension step of 72 °C for 7 min. The PCR products were subsequently detected using a 3730XL Genetic Analyzer Sequencer (Applied Biosystems, Foster City, California). Allele sizing was performed in GENEMAPPER v4.0 (Applied Biosystems) by comparing alleles with a GeneScan-500LIZ size standard (Applied Biosystems).

Analysis of genetic diversity
-----------------------------

The presence of null alleles, scoring errors and large allele dropout was checked using MICROCHECKER version 2.2.3 ([@ref-28]).The null allele frequency (*r*), inbreeding coefficient (*Fis*) and linkage disequilibrium (LD) between all pairs of polymorphic loci were calculated using program GENEPOP 4.0 ([@ref-21]; [@ref-22]). The LD was tested with 10,000 permutations. The number of alleles per locus (*N*~*a*~), observed (*H*~*o*~) and expected heterozygosity (*H*~*e*~), deviations from Hardy-Weinberg equilibrium (HWE) were calculated using ARLEQUIN version 3.5 ([@ref-4]). The HWE was tested with 1,000,000 steps in Markov chain.

Results
=======

Characterization of ssr distribution
------------------------------------

The frequency and density of SSRs on each of the 12 jujube chromosomes ranged from 818.29 (chromosome 5) to 920.85 (chromosome 12) loci/Mb and from 16,035.53 (chromosome 5) to 17,750.21 (chromosome 12) bp/Mb, respectively. Analysis of 321.56 Mb of sequence data from the assembled jujube genome uncovered 280,596 SSRs covering 5.37 Mb. These SSRs included 142,795 mono-, 87,362 di-, 29,900 tri-, 15,827 tetra-, 3,237 penta- and 1,475 hexanucleotide repeats, which respectively corresponded to 46.88%, 35.12%, 10.87%, 5.22%, 1.27% and 0.73% of total SSRs. There were 373 SSRs with more than 100 repeat motifs in the jujube genome, the longest of which comprised 2,446 AT repeats. Analysis of 119.15 Mb of the *A. thaliana* genome revealed only two SSRs with more than 100 repeat motifs (201 AAG and 117 TCT). Average SSR sizes in jujube and *A. thaliana* genomes were 19.14 and 17.40 bp, respectively. Jujube genome SSR frequency and density were 872.60 loci/Mb and 16,700.42 bp/Mb, respectively, with corresponding values of 221.78 loci/Mb and 3,858.19 bp/Mb in *A. thaliana*.

SSR frequency and density varied with motif length. As motif length increased (from mono- to hexanucleotide repeats), the frequency of repeats decreased from 444.06 to 4.59 loci/Mb and density decreased from 7,829.71 to 121.64 bp/Mb ([Fig. 1](#fig-1){ref-type="fig"}). The 10 most frequent motif types in the jujube genome were two mononucleotide (A/T, G/C), three dinucleotide (AT/TA, AG/CT, GT/AC), four trinucleotide (ATT/AAT, AAG/CTT, ATC/GAT, AAC/GTT) and one tetranucleotide (ATTT/AAAT) repeats ([Table 1](#table-1){ref-type="table"}). When calculating the frequency of each motif type, we considered all possible types in a repeat sequence, such as two potential types (AG and GA) for the AG motif repeat and three potential types (ATT, TTA and TAT) for the ATT motif repeat. As for *A. thaliana*, no tetranucleotide repeats were observed among the 10 most frequent motif types. The average length, frequency and density of the 10 most frequent microsatellite motif types are provided in [Table 1](#table-1){ref-type="table"}.

![The density (bp/Mb) of microsatellite with repeated unit lengths of 1--6 base pairs in genome of Chinese jujube (*Ziziphus jujuba* Mill.) and *Arabidopsis thaliana*.](peerj-04-1735-g001){#fig-1}

10.7717/peerj.1735/table-1

###### The ten most frequency microsatellite motif type in genome of Chinese jujube (*Ziziphus jujuba* Mill.) and *Arabidopsis thaliana*.

![](peerj-04-1735-g002)

  No.   Motif       Average length (bp)   Frequency (loci/Mb)   Density (bp/Mb)                               
  ----- ----------- --------------------- --------------------- ----------------- -------- -------- --------- ---------
  1     A/T         A/T                   17.64                 14.98             436.39   112.63   7699.69   1686.89
  2     AT/TA       AT/TA                 20.37                 22.60             201.50   30.68    4103.91   693.38
  3     ATT/AAT     AAG/CTT               19.59                 18.63             59.10    21.13    1157.87   393.79
  4     AG/CT       AG/CT                 25.04                 20.86             50.39    16.95    1261.91   353.66
  5     ATTT/AAAT   ATG/CAT               17.54                 18.51             39.07    7.34     685.46    135.76
  6     GT/AC       AAC/GTT               24.39                 17.96             19.73    5.81     481.22    104.31
  7     AAG/CTT     GT/AC                 20.07                 16.72             17.72    4.30     355.69    71.84
  8     G/C         AAT/ATT               16.94                 19.24             7.68     3.31     130.12    63.63
  9     ATC/GAT     AGG/CCT               19.19                 17.19             5.16     2.93     99.02     50.36
  10    AAC/GTT     ACC/GGT               18.90                 16.80             5.05     1.99     95.52     33.41

**Notes.**

Each motif type contained all the possible cases in a repeat sequence; for example, AG contained AG and GA, ATT contained ATT, TTA and TAT.

Primer design and evaluation
----------------------------

Taking no account of mononucleotide repeats, a total of 664 SSRs with repeat numbers greater than 25 and less than 100 were detected in jujube. We randomly selected 100 SSRs from the 664 loci and successfully designed 70 primer pairs. Screened in 2% agarose electrophoresis, 42 of the 70 primer pairs produced clear, unique amplification products of the expected size. All the 42 SSR markers showed their ability to detect polymorphisms across jujube cultivars and wild species. A set of 20 SSR markers that amplified the most easily scorable fragment polymorphisms was chosen to label with a fluorescent dye and evaluate polymorphism in the three populations. Characteristics of these 20 SSR loci are listed in [Table 2](#table-2){ref-type="table"}. All 20 primer pairs were also amplified successfully in the 10 jujube cultivars. Among the 20 loci, *N*~*a*~ ranged from 6 to 21, with an average of 12.8 alleles per locus ([Table 2](#table-2){ref-type="table"}). The mean value of *r* among the three populations ranged from 0 to 0.261. Loci Juju18 presented too many null alleles (*r* \> 0.15) in all populations and was eliminated from further analysis. Average *H*~*o*~ and *H*~*e*~ across the three populations were 0.909 and 0.701, respectively. The *N*~*a*~ had the highest mean values (8.5) in population 1 (*Z. acidojujuba*) among the three populations ([Table 3](#table-3){ref-type="table"}). Population 2 ('Bianhesuan') has the highest *H*~*o*~ (0.967) but lowest *H*~*e*~ (0.578). The *H*~*o*~ and *H*~*e*~ were zero at locus Juju64 in population 2. Of the 20 loci, 16 (all except Juju18, Juju57, Juju63, Juju64 and Juju68) showed significant deviation from HWE in two cultivar populations ([Table 3](#table-3){ref-type="table"}). None loci showed significant deviation from HWE in population 1. The *Fis* of the three populations were −0.066, −0.490 and −0.305, respectively. Three locus pairs (Juju11 & Juju23, Juju11 & Juju25 and Juju25 & Juju42) showed signficant LD (*P* \< 0.001) across the three populations.

10.7717/peerj.1735/table-2

###### Characteristics of 20 microsatellite loci and primer pairs developed from Chinese jujube (*Ziziphus jujuba* Mill.) genome.

![](peerj-04-1735-g003)

  Locus    Primer sequences(5′--3′)    Repeat motif   Size (bp)   *T*~*a*~ (°C)   Total no. of alleles   Fluorescent dye   Location           Accession in Genebank
  -------- --------------------------- -------------- ----------- --------------- ---------------------- ----------------- ------------------ --------------------------
  Juju2    F: ACATGGAGAAATGGGATC       (AG)~87~       220--256    53              15                     FAM               add_scaffold 107   [KN813357.1](KN813357.1)
           R: GGTTGATAGGTGGTTTGC                                                                                                              
  Juju5    F: GGCGACGATTAGAGGAAA       (AG)~36~       202--226    53              7                      FAM               add_scaffold 247   [KN813805.1](KN813805.1)
           R: CTGCTTGTACGGCCAGTT                                                                                                              
  Juju10   F: AAGCGGTTGTGGTATGGG       (AG)~108~      147--169    53              11                     FAM               add_scaffold 290   [KN813315.1](KN813315.1)
           R: TTTCTGCCACCTGCTCCT                                                                                                              
  Juju11   F: CCACTTGCGTTACTTCTC       (CT)~92~       192--222    53              8                      FAM               chromosome 6       [CM003119.1](CM003119.1)
           R: AATCTCGCTTTGCTCTAT                                                                                                              
  Juju13   F: TAGGCATTTGCATGGTAT       (TC)~32~       190--208    53              8                      HEX               chromosome 3       [CM003116.1](CM003116.1)
           R: TTGTCCGCTTTCTTGAGT                                                                                                              
  Juju17   F: AATCGGTTACATTGCTGC       (GA)~25~       118--252    53              12                     HEX               chromosome 6       [CM003119.1](CM003119.1)
           R: TTTCGGAGGTTACCACAT                                                                                                              
  Juju18   F: GATGTACGGGAAAGACGG       (CT)~52~       268--334    53              21                     FAM               scaffold 959       [KN812918.1](KN812918.1)
           R: ATCACTCCTGGTTGCTTG                                                                                                              
  Juju23   F: CCATCCGACCACTGAAAT       (AG)~29~       107--163    53              20                     FAM               chromosome 10      [CM003123.1](CM003123.1)
           R: CGTAAAGCACCAGCAAAA                                                                                                              
  Juju25   F: GTACGGTATGACTCCACA       (CA)~48~       116--192    53              17                     FAM               chromosome 3       [CM003116.1](CM003116.1)
           R: CATCCAATCACTGAAAAT                                                                                                              
  Juju30   F: AAATGACCATCGAATCCC       (GA)~25~       224--260    53              11                     HEX               chromosome 11      [CM003124.1](CM003124.1)
           R: CTTTGTTGTTACCCCAGA                                                                                                              
  Juju35   F: TTGGATTAGTGTACTTGG       (TC)~47~       109--219    53              12                     HEX               chromosome 6       [CM003119.1](CM003119.1)
           R: ACATGAGGAAACCTGGAA                                                                                                              
  Juju42   F: CTTCAGGACGGACCAAAT       (AG)~25~       150--230    50              21                     HEX               chromosome 8       [CM003121.1](CM003121.1)
           R: GAATGCTTCAATAAACTC                                                                                                              
  Juju43   F: CCAAATTGCCAGGTCTAG       (AG)~27~       164--192    53              11                     HEX               chromosome 2       [CM003115.1](CM003115.1)
           R: AACTGATCCTCCTTCGTC                                                                                                              
  Juju52   F: TTGAAAAGGAAGGAAGAG       (TG)~28~       203--229    50              10                     HEX               chromosome 6       [CM003119.1](CM003119.1)
           R: TGAGGATTATGAAGGGTT                                                                                                              
  Juju54   F: GAATCCTTACATCCAATA       (TACA)~37~     86--158     53              6                      HEX               chromosome 4       [CM003117.1](CM003117.1)
           R: ACTTACCATAATCTGTGC                                                                                                              
  Juju57   F: ATTTATTCCTTATTGCTAGTAG   (CA)~27~       122--210    55              18                     HEX               scaffold 844       [KN812877.1](KN812877.1)
           R: CAACCTTCTTGTAGTTATTTT                                                                                                           
  Juju63   F: ATCAGCCAGCGTCACAAA       (TA)~35~       155--205    55              10                     FAM               chromosome 11      [CM003124.1](CM003124.1)
           R: ATCCAAATAAGCCCACCT                                                                                                              
  Juju64   F: ATATTGGAAACTTTCTGATC     (TC)~97~       108--138    53              6                      FAM               chromosome 11      [CM003124.1](CM003124.1)
           R: CTGTAATACTGGGATGCT                                                                                                              
  Juju66   F: TGGATACCGTGAAGGAAC       (GA)~68~       178--216    53              13                     HEX               chromosome 7       [CM003120.1](CM003120.1)
           R: AGCCCATTAGAAAGCAAC                                                                                                              
  Juju68   F: AGGCTTCAACTCTTATCC       (TAA)~61~      128--204    53              19                     FAM               chromosome 1       [CM003114.1](CM003114.1)
           R: CCAAAACCACCACAAAAT                                                                                                              

**Notes.**

*T*~*a*~annealing temperatur

10.7717/peerj.1735/table-3

###### Results of initial primer screening in three populations.

One population is *Ziziphus acidojujuba*. CY Cheng et MJ Liu and the other two are cultivars "Bianhesuan" and "Huizao."

![](peerj-04-1735-g004)

  Locus    *Z. acidojujuba* (*N* = 14)   'Bianhesuan' (*N* = 17)   'Huizao' (*N* = 17)                                                                                             
  -------- ----------------------------- ------------------------- --------------------- ------- -------- ----- ------- ------- --------- -------- ----- ------- ------- --------- --------
  Juju2    10                            0.000                     1.000                 0.846   −0.182   6     0.039   1.000   0.649\*   −0.568   3     0.298   0.727   0.685\*   −0.074
  Juju5    6                             0.000                     0.857                 0.757   −0.139   3     0.000   0.941   0.544\*   −0.772   3     0.000   0.667   0.665\*   0.059
  Juju10   6                             0.000                     0.800                 0.728   −0.059   7     0.000   0.941   0.695\*   −0.369   6     0.231   0.455   0.749\*   0.444
  Juju11   6                             0.000                     0.875                 0.827   −0.077   3     0.000   1.000   0.569\*   −0.801   4     0.051   1.000   0.720\*   −0.345
  Juju13   6                             0.076                     0.714                 0.765   0.068    5     0.000   1.000   0.386     0.000    5     0.139   0.538   0.706\*   0.276
  Juju17   6                             0.000                     0.769                 0.783   0.036    4     0.000   1.000   0.298     0.000    6     0.000   1.000   0.619\*   −0.649
  Juju18   10                            0.137                     1.000                 0.902   −0.113   4     0.400   0.667   0.363     0.273    7     0.246   1.000   0.820     −0.228
  Juju23   12                            0.000                     1.000                 0.913   −0.100   3     0.000   1.000   0.544\*   −0.889   4     0.000   1.000   0.697\*   −0.455
  Juju25   11                            0.000                     0.786                 0.884   0.115    4     0.000   1.000   0.644\*   −0.581   11    0.076   0.765   0.868\*   0.122
  Juju30   10                            0.000                     1.000                 0.889   −0.116   4     0.000   1.000   0.622\*   −0.639   3     0.000   0.909   0.500     −0.681
  Juju35   7                             0.159                     0.929                 0.857   −0.087   3     0.000   1.000   0.544\*   −0.889   3     0.161   1.000   0.544\*   −0.889
  Juju42   12                            0.037                     0.857                 0.915   0.066    7     0.000   1.000   0.647\*   −0.572   11    0.060   0.824   0.854\*   0.037
  Juju43   8                             0.000                     1.000                 0.863   −0.154   5     0.000   0.882   0.642\*   −0.391   5     0.000   1.000   0.748     −0.360
  Juju52   10                            0.000                     1.000                 0.913   −0.100   6     0.000   0.941   0.759\*   −0.249   3     0.035   0.647   0.611\*   −0.060
  Juju54   5                             0.000                     0.857                 0.754   −0.143   4     0.000   1.000   0.571\*   −0.795   2     0.000   0.938   0.511\*   −0.875
  Juju57   11                            0.000                     1.000                 0.909   −0.125   5     0.000   1.000   0.609     −0.395   9     0.055   0.833   0.778     −0.084
  Juju63   7                             0.100                     0.900                 0.844   −0.066   5     0.000   1.000   0.646     −0.440   7     0.000   0.929   0.802     −0.134
  Juju64   5                             0.000                     0.917                 0.726   −0.228   1     0.000   0.000   0.000     --       2     0.000   1.000   0.453     −1.000
  Juju66   11                            0.000                     0.833                 0.905   0.091    5     0.000   1.000   0.595\*   −0.716   2     0.000   1.000   0.515\*   −1.000
  Juju68   11                            0.060                     0.917                 0.895   −0.017   5     0.000   1.000   0.648     −0.507   6     0.048   0.923   0.779     −0.205
  Mean     8.5                           0.028                     0.901                 0.844   −0.066   4.6   0.022   0.967   0.578     −0.490   5.1   0.070   0.858   0.681     −0.305

**Notes.**

*N*~*a*~total number of alleles per locus*H*~*o*~observed expected heterozygosities*H*~*e*~expected heterozygosities*N*sample size for each population*r*null allele frequency*Fis*inbreeding coefficient

Significant departure from HWE at ^∗^*P* \< 0.01, respectively.

Discussion
==========

Characterization of SSR abundance
---------------------------------

The characterization of SSRs in our study was different from that reported by [@ref-32]. This difference was caused by two reasons. One was that their study was based on scaffold sequences and analysis 396.18 Mb of sequence data ([@ref-32]) and our study was based on assembled 12 chromosomes and analysis 321.56 Mb of sequence data. The other was the different identification criteria of defining SSR. Their identification criteria used for mono-, di-, tri-, tetra-, penta- and hexanucleotide repeats were a minimum of 10, 5, 5, 5, 5 and 5, respectively ([@ref-32]); however, ours were a minimum of 12, 7, 5, 4, 4 and 4. We used stricter criteria for mono- and dinucleotide repeats and therefore got less SSR loci. Although the results of two studies were different, they got consistent conclusions.

We calculated the frequencies of perfect SSRs composed of 1--6-bp long motifs in jujube and *A. thaliana*. The abundance of certain repeat types differed between the two plants. Jujube had a much higher SSR density than *A. thaliana*, as well as mulberry, peach, apple, pear, grape, strawberry and *Prunus mume* ([@ref-32]). SSRs containing A and T (A/T, AT/TA and ATT/AAT) were dominant in the jujube genome, consistent with its high AT content (66.59%, [@ref-16]). High proportions of (A+ T)-rich repeats ([@ref-32]) are also present in *A. thaliana* and other plants ([@ref-6]). In protein-coding regions, the high proportion of (A+ T)-rich repeats is due to the poly(A/T) tails of densely scattered retroposed sequences and processed pseudogenes ([@ref-27]).

Microsatellites are associated with nonrepetitive DNA and have their highest frequencies in transcribed regions ([@ref-19]). Besides their utility as genetic markers, SSRs have important developmental, gene regulatory and evolutionary functions ([@ref-7]). The function of the high-density SSRs in the complex jujube genome ([@ref-16]) thus warrants further study.

The frequency of repeats in jujube decreased with increasing motif length, similar to sorghum, rice, *Medicago*, *Populus*, *Brachypodium* and *Brassica oleracea* ([@ref-5]; [@ref-24]). Mononucleotide repeats were the most abundant repeats. One of the 10 most abundant motif types in jujube were tetranucleotide motifs (ATTT/AAAT) which was not found in *A. thaliana*. This difference may be ascribed to the complexity of the jujube genome and its high SSR density, as well as its high AT content (66.59%, [@ref-16]).

Primer evaluation
-----------------

More alleles were detected in population 1 than in populations 2 and 3. This difference may be partly explained by the fact that population 1 consisted of a wild species whereas populations 2 and 3 were cultivars. It is relevant to the lower *Fis* in population 1 than in populations 2 and 3. Wild species often have higher genetic polymorphism levels than cultivars, such as peanut ([@ref-18]), soybean ([@ref-9]), apple ([@ref-33]). Using nine microsatellites, the average *H*~*o*~ and *H*~*e*~ of 31 *Z. acidojujuba* populations was 0.679 and 0.659, respectively ([@ref-34]). Comparing the *Z. acidojujuba* population in our study (from Luoyang) with their population from Luoyang (EYYC), our markers identified higher *N*~*a*~ (8.5 vs. 3.9) and higher *H*~*o*~ and *H*~*e*~ (0.901 and 0.844 vs. 0.683 and 0.625, respectively) ([@ref-34]). The higher values hinted that our marks could uncover subtler genetic structure. Locus Juju64 uncovered only one allele in population 2, causing *H*~*o*~ and *H*~*e*~ to be zero, and also uncovered least alleles in other two populations, thus revealing the low polymorphism of the locus.

Out of the 70 screened primer pairs, 42 (60.0%) produced clear, unique amplification products and 20 (28.6%) displayed high levels of polymorphism. Loci Juju18 presented too many null alleles thus was eliminated. After testing in three populations, all the remained 19 polymorphic SSRs were in HWE in at least one population. For our populations, especially population 2 and 3, many loci were not in HWE because cultivars are not in a random mating system. Taking LD into consideration, the removal of one locus (i.e., Juju11 and Juju25) from each linked pair would give 17 unlinked loci in HWE.

In conclusion, we developed 17 validated microsatellite primer pairs with applicability to jujube population genetics. These polymorphic markers will facilitate the study of jujube genetic structure and gene flow and aid investigations of the evolutionary history of this important fruit crop.

Supplemental Information
========================

10.7717/peerj.1735/supp-1

###### Information of plant samples used in this study

###### 

Click here for additional data file.

We thank Shan-shan Sun of the Wuhan Botanical Garden, Chinese Academy of Sciences, for providing laboratory support.

Additional Information and Declarations
=======================================

The authors declare there are no competing interests.

[Peng-cheng Fu](#author-1){ref-type="contrib"} conceived and designed the experiments, performed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables.

[Yan-Zhao Zhang](#author-2){ref-type="contrib"} performed the experiments, analyzed the data.

[Hui-yuan Ya](#author-3){ref-type="contrib"} reviewed drafts of the paper, revise the manuscript.

[Qing-bo Gao](#author-4){ref-type="contrib"} conceived and designed the experiments, reviewed drafts of the paper.

The following information was supplied regarding data availability:

[www.figshare.com/s/fd62f4687c5c11e595ff06ec4b8d1f61](www.figshare.com/s/fd62f4687c5c11e595ff06ec4b8d1f61)

[www.figshare.com/s/75aecd0a7c5f11e5a3dd06ec4b8d1f61](www.figshare.com/s/75aecd0a7c5f11e5a3dd06ec4b8d1f61).
