Cheng, Y.‐C., D.‐J. Zhang, Z.‐Y. Lu, X.‐S. Ye, J.‐G. Wang, P. Sun, and B.‐W. Zhang. 2018 Isolation and characterization of microsatellite loci for *Prunus mongolica* (Rosaceae). Applications in Plant Sciences 6(6): e1158.

*Prunus mongolica* Maxim. (Rosaceae) is a relict flora that is endemic to arid areas of the Asian interior. *Prunus mongolica* was listed in the China Plant Red Data Book as endangered; this has resulted from mining, firewood collection, industrialization, and urbanization, which have led to habitat loss and fragmentation (Fu, [1992](#aps31158-bib-0002){ref-type="ref"}). Scientific conservation requires a full understanding of genetic diversity, phylogeny, and population structure. Genetic studies are limited in *P. mongolica*; therefore, it is necessary to develop microsatellite loci for use in the species.

Microsatellites, also known as simple sequence repeats (SSRs), have been widely used as DNA markers in studies of population genetics, phylogeography, landscape genetics, and parentage analysis due to their high level of polymorphism, putative neutrality, codominant inheritance, and ease of scoring (Moriguchi et al., [2015](#aps31158-bib-0006){ref-type="ref"}). In this study, we isolated and characterized 51 polymorphic microsatellite loci for *P. mongolica* by transcriptome sequencing and bioinformatic screening.

METHODS AND RESULTS {#aps31158-sec-0005}
===================

Total RNA was extracted from fresh leaves of one individual of *P. mongolica* using the EasyPure Plant RNA Kit (TransGen Biotech Inc., Beijing, China) following the manufacturer\'s protocol. A cDNA library was prepared using the TruSeq Stranded Total RNA Sample Prep Kit (Illumina, San Diego, California, USA) and sequenced on a HiSeq 3000 sequencing platform (Illumina).

Extra (adapters and other Illumina‐specific sequences) and low‐quality sequences were removed from the raw data using the software Trim Galore version 0.4.4 (Li et al., [2015](#aps31158-bib-0005){ref-type="ref"}). FastQC version 0.11.5 software (Yang et al., [2013](#aps31158-bib-0012){ref-type="ref"}) was used to analyze the quality control of the pre‐processed data. A total of 53,866 contigs were obtained by de novo assembly using Trinity version 2.3.2 (Grabherr et al., [2011](#aps31158-bib-0003){ref-type="ref"}). SSR detection was performed with MISA version 1.0 (Thiel et al., [2003](#aps31158-bib-0008){ref-type="ref"}) with the following criteria: ≥6 repeat units for dinucleotides, ≥5 for tri‐, and ≥4 for tetra‐, penta‐, and hexanucleotides. Primer sets were designed with Primer3 using the default parameters (Koressaar and Remm, [2007](#aps31158-bib-0004){ref-type="ref"}; Untergasser et al., [2012](#aps31158-bib-0009){ref-type="ref"}). A total of 19,498 SSRs were identified from 15,837 contigs, and 6069 primer sets were designed. Two hundred primer sets that amplified di‐, tri‐, and tetranucelotide repeats with ≥6, ≥5, and ≥4 repeat units, respectively, were selected to test for polymorphism. Raw transcriptome data were deposited in the National Center for Biotechnology Information Short Read Archive (BioProject no. PRJNA418876, BioSample no. SAMN08038438).

Because of habitat loss, only 32 *P. mongolica* individuals were sampled from three populations (Appendix [1](#aps31158-app-0001){ref-type="app"}). Total genomic DNA was extracted from 0.15 mg of leaf tissue using a modified cetyltrimethylammonium bromide (CTAB) method (Wang et al., [2010](#aps31158-bib-0011){ref-type="ref"}). All PCRs were performed in total volumes of 15 μL: 1 μL of DNA, 5.5 μL of sterilized deionized water, 7.5 μL of 2× EasyTaq PCR Supermix (TransGen Biotech Inc.), and 0.5 μL each of forward and reverse primers (each forward primer was fluorescently labeled with FAM, HEX, or TAMRA). PCR amplification was carried out using the following thermal conditions: initial denaturation for 5 min at 94°C; then 35 cycles of denaturation for 30 s at 94°C, annealing for 30 s at the annealing temperature (Table [1](#aps31158-tbl-0001){ref-type="table"}), and 20 s at 72°C; followed by a final extension for 10 min at 72°C. PCR products were genotyped on an ABI PRISM 3730 genetic analyzer (Applied Biosystems, Waltham, Massachusetts, USA) with a GeneScan 500 size standard (Applied Biosystems). Allele peaks were scored using GeneMarker version 1.3 (SoftGenetics, State College, Pennsylvania, USA). MICRO‐CHECKER version 2.2.3 (van Oosterhout et al., [2004](#aps31158-bib-0010){ref-type="ref"}) was used to detect genotyping errors due to null alleles, stuttering, or allele dropout. The number of effective alleles and levels of observed and expected heterozygosity were calculated using GENETIX version 4.0 (Belkhir et al., [2001](#aps31158-bib-0001){ref-type="ref"}). Deviation from Hardy--Weinberg equilibrium and linkage disequilibrium was tested with GENEPOP version 3.4 (Rousset, [2008](#aps31158-bib-0007){ref-type="ref"}).

###### 

Characteristics of 51 microsatellite loci developed in *Prunus mongolica*

  Locus                       Primer sequences (5′--3′)   Repeat motif   Allele size range (bp)   *T* ~a~ (°C)   Function                                                                      GenBank accession no.
  --------------------------- --------------------------- -------------- ------------------------ -------------- ----------------------------------------------------------------------------- -----------------------
  TR3320                      F: CGCCTCCTCTCTCTCTCTA      (TC)~19~       127--175                 55             EIN3‐binding F‐box protein 1                                                  [MG682092](MG682092)
  R: CCCAGAAAAGATTAAGGCTAC                                                                                                                                                                     
  TR5622                      F: AAACGAGAGCAGGATCAATA     (CAA)~8~       127--179                 55             B3 domain‐containing transcription factor                                     [MG682093](MG682093)
  R: CGTAGTTTGGGTTGGAGTC                                                                                                                                                                       
  TR9296                      F: GCAGAGAACTTATCGCTTATG    (TTTA)~6~      133--201                 55             Glyoxylate/hydroxypyruvate reductase HPR3                                     [MG682094](MG682094)
  R: CATCTGGAAAGTGAATCTGAG                                                                                                                                                                     
  TR10339                     F: ACTCTTCTCTCCATTGAGTCC    (CT)~20~       105--141                 55             Protein MIZU‐KUSSEI 1                                                         [MG682095](MG682095)
  R: GTCCTCATCTGGGAAATTTAG                                                                                                                                                                     
  TR10936                     F: AGAAGATAGAGGGATGGGAGT    (AG)~21~       153--181                 56             GATA transcription factor 11                                                  [MG682096](MG682096)
  R: CTCTCTGCAACAACCAAAAC                                                                                                                                                                      
  TR12256                     F: GAGAGAGAAAAATTGCAGTCC    (TCT)~7~       131--177                 55             Unknown                                                                       [MG682097](MG682097)
  R: CCATGGAGATTAAGAGGAAGA                                                                                                                                                                     
  TR12298                     F: CAATAGCCAAAACCCTCTC      (TC)~6~        123--169                 54             DNA‐directed RNA polymerases II, IV, and V                                    [MG682098](MG682098)
  R: GCCTTCCATTTCTCTATGTCT                                                                                                                                                                     
  TR12728                     F: AAATCTAAGCCCACGTACATC    (GA)~18~       153--193                 56             Protein‐tyrosine‐phosphatase MKP1                                             [MG682099](MG682099)
  R: CACCCTGGTAACTCATAATCA                                                                                                                                                                     
  TR14394                     F: TGAGCTACAGACAGGTGAACT    (GAA)~7~       143--169                 55             Transcription factor bHLH25                                                   [MG682100](MG682100)
  R: ACACAACACACCACTTTTCTC                                                                                                                                                                     
  TR16484                     F: GTGTTGGCTAGAGTGACTTTG    (TC)~8~        157--195                 56             UDP‐glycosyltransferase 74F2                                                  [MG682101](MG682101)
  R: CGCCACACCTAAGTTTTT                                                                                                                                                                        
  TR17663                     F: TCATCTCCTCTACCTTTTTCC    (CT)~19~       115--169                 55             Unknown                                                                       [MG682102](MG682102)
  R: GGTCATGATCTCTGCTTGATA                                                                                                                                                                     
  TR17745                     F: CCTTCCATTTCTCTGTTCTCT    (TC)~21~       111--149                 55             Aquaporin PIP1‐2‐like                                                         [MG682103](MG682103)
  R: CACTCAACCAAATCATAGCTC                                                                                                                                                                     
  TR18640                     F: AACTAGAGAGAGAGACGCACA    (GA)~16~       91--139                  55             Succinate‐CoA ligase \[ADP‐forming\] subunit beta, mitochondrial              [MG682104](MG682104)
  R: GTTCTTTCGCATTCTCAGAC                                                                                                                                                                      
  TR18685                     F: CAGAAGTCAGAGCAGAGTCAG    (ACAG)~6~      139--169                 55             VQ motif‐containing protein 9                                                 [MG682105](MG682105)
  R: CTACCGGGAGATATTTCAGAT                                                                                                                                                                     
  TR18807                     F: GTCCTTAAGCAGCCCAGTA      (CT)~20~       121--173                 55             Probable acyl‐activating enzyme 17, peroxisomal                               [MG682106](MG682106)
  R: AGAGCTTCGATGTCAGAGAC                                                                                                                                                                      
  TR19174                     F: CTTGAGAGGAGGAAAAAGAAG    (AGA)~8~       145--217                 55             Transcription factor PRE6‐like                                                [MG682107](MG682107)
  R: GCTGTCTGGGGTTATAAATTC                                                                                                                                                                     
  TR19201                     F: TATTGCACTCTCCTATGAACC    (CAG)~7~       139--167                 55             Auxin response factor 7 (LOC110749255), transcript variant X2                 [MG682108](MG682108)
  R: GTTGAGACTCTTGCTGTTGTT                                                                                                                                                                     
  TR20374                     F: CTTCCATCCACACTTACAT      (GA)~8~        149--207                 55             Uncharacterized protein At1g04910                                             [MG682109](MG682109)
  R: GAGGGACTGAATAAACCTGAG                                                                                                                                                                     
  TR21328                     F: AAGGTGAAGGGTGAAGATACT    (GA)~12~       141--195                 55             Magnesium‐chelatase subunit ChlH, chloroplastic                               [MG682110](MG682110)
  R: AAACACAATCACAGAGAGAGC                                                                                                                                                                     
  TR21918                     F: CCTCCTTCTGTCTCTCTCTTC    (CT)~12~       125--187                 55             E3 ubiquitin‐protein ligase RNF144A                                           [MG682111](MG682111)
  R: CAGTTAAACCACCAGCACTAC                                                                                                                                                                     
  TR20030                     F: ATAACCCCAAGCTCTCTCTC     (CTC)~8~       131--161                 55             *Prunus persica* receptor‐like protein 51                                     [MG682112](MG682112)
  R: AGGAGGTAGGAGATGGAGTG                                                                                                                                                                      
  TR22071                     F: CAGAAGAAAAAGAAGGAGAGC    (GAA)~7~       131--161                 55             *Prunus avium* cysteine proteinase inhibitor 12‐like, transcript variant X6   [MG682113](MG682113)
  R: TGTCAACCATACTCACCTCAT                                                                                                                                                                     
  TR23600                     F: CAGCTCTCTCTTTCTCTCTCC    (TC)~8~        141--183                 55             Probable serine/threonine‐protein kinase DDB_G0282963                         [MG682114](MG682114)
  R: ACTTCCACTACCAGAATCCAT                                                                                                                                                                     
  TR24122                     F: GGTCTGTTAATGGAGGTTCTT    (CT)~13~       133--169                 56             UDP‐*N*‐acetylglucosamine--dolichyl‐phosphate                                 [MG682115](MG682115)
  R: CACCAATATTGAGAGAGAGAC                                                                                                                                                                     
  TR25043                     F: AACCAACCCAACCCACAC       (ACAT)~6~      111--165                 57             *N*‐acetylglucosaminephosphotransferase                                       [MG682116](MG682116)
  R: GGGGGAGAACCTATCTTATATT                                                                                                                                                                    
  TR25166                     F: CAGGCTCTGTAGTTCGATTC     (TCT)~8~       147--179                 55             Gibberellin 20 oxidase 1                                                      [MG682117](MG682117)
  R: TGTATGTTAGTAAGGCGAAGG                                                                                                                                                                     
  TR25414                     F: CAGCAGAGAGACCAGAATAGA    (TCCA)~6~      103--125                 55             Uncharacterized protein At2g17340‐like                                        [MG682118](MG682118)
  R: GAGCATTATAGCTGTGTGTGG                                                                                                                                                                     
  TR26908                     F: TATCTTCATCGCTCTCTTCTG    (CAT)~10~      111--149                 55             Galactomannan galactosyltransferase 1                                         [MG682119](MG682119)
  R: CTGGGGTCTTAATTAGACGTT                                                                                                                                                                     
  TR27527                     F: CACCAAACCTTTCTCTCTCTT    (CT)~27~       117--165                 54             Transcription factor HY5‐like, transcript variant X1                          [MG682120](MG682120)
  R: ACAAAGTCGATCAGCATCTC                                                                                                                                                                      
  TR29327                     F: CTGCCGTCTATCTCTCTCTCT    (CT)~6~        113--145                 56             Probable fructokinase‐4                                                       [MG682121](MG682121)
  R: CAAGTTTGGTGCTCGTATCTA                                                                                                                                                                     
  TR30962                     F: CTATGGGTCTGGTTATGGAA     (GGA)~11~      101--161                 55             Cytosolic 5′‐nucleotidase 3, transcript variant X5                            [MG682122](MG682122)
  R: CTCCCAGCCAATATCTCAG                                                                                                                                                                       
  TR31156                     F: CTCTGCCTCTTTCTCTTCTCT    (AG)~14~       117--155                 55             Putative glycine‐rich cell wall                                               [MG682123](MG682123)
  R: ATCCCCCTACTTTCTTGC                                                                                                                                                                        
  TR31399                     F: GAAGAACAAGAAGGGCAAG      (AG)~20~       117--167                 55             Structural protein 1                                                          [MG682124](MG682124)
  R: CAACACTAACACCACCAGATT                                                                                                                                                                     
  TR31622                     F: AAGCCAGAACCCTTATCTCTA    (AAC)~5~       125--179                 55             Unknown                                                                       [MG682125](MG682125)
  R: GCCTGAATTGAGTGAACTGTA                                                                                                                                                                     
  TR32795                     F: CAAGTTGATTTCTTCCTCTCC    (AGA)~13~      119--155                 55             Peamaclein‐like                                                               [MG682126](MG682126)
  R: TATAACGCCTCTCTCTGTTGA                                                                                                                                                                     
  TR33107                     F: AAAACAGAGAGGACGATGG      (TTGT)~6~      139--171                 55             BEL1‐like homeodomain protein 9, transcript variant X1                        [MG682127](MG682127)
  R: GCTCTAGGTGTTGGGTTAAAT                                                                                                                                                                     
  TR35128                     F: GCCTGAATTGAGTGAACTGTA    (TTG)~8~       129--181                 56             Glutamine--fructose‐6‐phosphateaminotransferase \[isomerizing\] 2             [MG682128](MG682128)
  R: AAGCCAGAACCCTTATCTCTA                                                                                                                                                                     
  TR35536                     F: CTCTTGTGTTCATCGGCTAC     (TGA)~6~       153--175                 55             Calcium‐dependent protein kinase 13                                           [MG682129](MG682129)
  R: ATCTCACCCGTCTCTTTATTC                                                                                                                                                                     
  TR37605                     F: CTTTCTAACCCCTCTGTGTCT    (TC)~21~       117--153                 55             BEL1‐like homeodomain protein 9, transcript variant X2                        [MG682130](MG682130)
  R: TATGAACAGCTTACCTTCGTC                                                                                                                                                                     
  TR37669                     F: CCTCTTTGATCTCCTTCCATA    (TCT)~8~       141--181                 55             Glutamate synthase \[NADH\], amyloplastic, transcript variant X3              [MG682131](MG682131)
  R: GAAGACGACCTTTTTCATACC                                                                                                                                                                     
  TR37811                     F: GATCAAGGAGGAAGACAAAGT    (AG)~26~       105--145                 55             Heterogeneous nuclear ribonucleoprotein 1                                     [MG682132](MG682132)
  R: GCAGAGAGAGAGAAAGAGGAA                                                                                                                                                                     
  TR38169                     F: GATCATGAAGTTGCAGAAGAG    (TGG)~10~      143--185                 56             Probable WRKY transcription factor 12                                         [MG682133](MG682133)
  R: ATGTGCCCTTCCTGTGAG                                                                                                                                                                        
  TR38361                     F: GGATGTGCTTTACTGGAGAA     (TGA)~8~       127--155                 56             Probable phospholipid‐transporting ATPase                                     [MG682134](MG682134)
  R: CTCCTTCTAATCCCTTCAGC                                                                                                                                                                      
  TR41171                     F: TAGAAACTATGTGCGTGTGTG    (GA)~15~       123--175                 55             4, transcript variant X2                                                      [MG682135](MG682135)
  R: CTTCAGGGAGGCTATAAATTC                                                                                                                                                                     
  TR42254                     F: ATTGCTTGCTCTGTCTCTGTA    (AAG)~5~       141--181                 55             Homeobox‐leucine zipper protein HOX11‐like, transcript variant X2             [MG682136](MG682136)
  R: CTTATTACCCACTACCCCAAC                                                                                                                                                                     
  TR42785                     F: TCTCTGAAATTCTCTCTGCTC    (GTGC)~6~      117--147                 55             Formin‐like protein 4                                                         [MG682137](MG682137)
  R: TCTTTCTTCCTCCTCTCTCAC                                                                                                                                                                     
  TR46482                     F: CCACCTTCTTATAGCGACTG     (TATG)~6~      133--193                 55             Probable E3 ubiquitin‐protein ligase RZFP34, transcript variant X4            [MG682138](MG682138)
  R: AAGTACGATACAAGCACTCCA                                                                                                                                                                     
  TR47534                     F: CGATCTCCTTCCAGACCT       (GTG)~6~       119--157                 55             Auxin efflux carrier component 1                                              [MG682139](MG682139)
  R: CGACATCCTCAAGAACATCTA                                                                                                                                                                     
  TR47552                     F: ACAATCTCTTCGTCTCTGGTT    (AG)~19~       121--177                 54             UDP‐glycosyltransferase 76E2‐like, transcript variant X2                      [MG682140](MG682140)
  R: GCTCCAGTGTACGTTGTAAG                                                                                                                                                                      
  TR50321                     F: AAGACCACTCACAACTCCAC     (CTT)~8~       263--293                 55             Cyclic nucleotide‐gated ion channel 1‐like, transcript variant X1             [MG682141](MG682141)
  R: CTTTGAAACACTGTTCTGGTC                                                                                                                                                                     
  TR52495                     F: TGATGAGCTTGCCATTCT       (GAA)~8~       147--173                 55             G‐box‐binding factor 4, mRNA                                                  [MG682142](MG682142)
  R: CTTCTTGTCCGCTAATTCAC                                                                                                                                                                      

*T* ~a~ = annealing temperature.

John Wiley & Sons, Ltd

A total of 51 primer sets produced consistent amplifications in all samples and were polymorphic (Table [1](#aps31158-tbl-0001){ref-type="table"}). These 51 loci had high numbers of effective alleles and high levels of observed and expected heterozygosity, ranging from seven to 11, 0.545 to 1.000, and 0.600 to 0.989, respectively (Table [2](#aps31158-tbl-0002){ref-type="table"}). Cross‐amplification of these 51 primer sets was tested for in *P. triloba* Lindl., *P. davidiana* (Carrière) Franch., *P. persica* (L.) Batsch, *P. cerasifera* Ehrh., and *P. serrulata* Lindl.; most primers amplified in these five species except *P. triloba* (*P. triloba*: 35/51; *P. davidiana*: 43/51; *P. persica*: 46/51; *P. cerasifera*: 45/51; *P. serrulata*: 41/51; Table [3](#aps31158-tbl-0003){ref-type="table"}).

###### 

Genetic diversity of 51 microsatellite loci in three populations of *Prunus mongolica*.[a](#aps31158-note-0003){ref-type="fn"}

  Locus     Wulanchabu population (*n* = 10)   Chifeng population (*n* = 10)   Eerduosi population (*n* = 12)                                             
  --------- ---------------------------------- ------------------------------- -------------------------------- -------- ------- ------- -------- ------- -------
  TR3320    8.500                              0.889                           0.922                            8.500    0.889   0.869   9.000    1.000   0.925
  TR5622    11.000                             1.000                           0.928                            10.500   1.000   0.963   9.500    0.833   0.880
  TR9296    7.000                              1.000                           0.967                            8.000    1.000   0.970   7.000    1.000   0.925
  TR10339   11.000                             1.000                           0.900                            11.000   1.000   0.832   10.000   1.000   0.935
  TR10936   10.500                             1.000                           0.922                            10.000   1.000   0.889   9.500    0.909   0.926
  TR12256   10.000                             0.889                           0.837                            9.500    0.800   0.600   9.500    0.800   0.847
  TR12298   11.000                             1.000                           0.941                            10.500   1.000   0.858   9.500    1.000   0.906
  TR12728   10.000                             1.000                           0.948                            10.000   1.000   0.954   9.000    1.000   0.874
  TR14394   11.000                             0.778                           0.895                            10.500   0.900   0.758   9.500    0.750   0.699
  TR16484   10.500                             1.000                           0.908                            10.000   1.000   0.921   9.500    1.000   0.909
  TR17663   10.500                             1.000                           0.980                            10.500   1.000   0.961   9.000    1.000   0.960
  TR17745   11.000                             1.000                           0.974                            10.500   1.000   0.953   9.500    1.000   0.928
  TR18640   10.500                             1.000                           0.989                            10.500   1.000   0.953   10.000   1.000   0.939
  TR18685   11.000                             0.875                           0.908                            10.000   0.800   0.858   9.000    0.833   0.790
  TR18807   9.500                              1.000                           0.942                            10.500   1.000   0.967   9.000    1.000   0.939
  TR19174   11.000                             0.889                           0.810                            10.500   1.000   0.853   9.500    1.000   0.938
  TR19201   11.000                             1.000                           0.858                            11.000   0.900   0.884   10.000   0.917   0.888
  TR20030   11.000                             1.000                           0.941                            10.500   1.000   0.953   9.500    0.917   0.928
  TR20374   10.500                             1.000                           0.974                            10.000   1.000   0.963   9.500    1.000   0.952
  TR21328   11.000                             1.000                           0.963                            11.000   1.000   0.958   10.000   1.000   0.960
  TR21918   11.000                             1.000                           0.954                            10.500   1.000   0.921   9.500    1.000   0.960
  TR22071   11.000                             0.889                           0.817                            10.500   0.900   0.942   9.500    0.750   0.928
  TR23600   10.500                             1.000                           0.908                            10.500   1.000   0.935   9.000    1.000   0.949
  TR24122   9.000                              1.000                           0.922                            8.500    0.900   0.937   9.500    1.000   0.925
  TR25043   10.500                             0.778                           0.882                            10.000   0.600   0.705   9.500    0.727   0.870
  TR25166   11.000                             1.000                           0.915                            10.500   1.000   0.963   9.500    1.000   0.920
  TR25414   10.500                             1.000                           0.733                            9.500    1.000   0.832   9.000    0.889   0.771
  TR26908   10.000                             0.900                           0.884                            10.500   0.889   0.791   9.500    0.909   0.905
  TR27527   11.000                             0.900                           0.863                            11.000   0.900   0.837   10.000   0.833   0.920
  TR29327   10.000                             1.000                           0.928                            10.000   1.000   0.876   9.000    1.000   0.952
  TR30962   9.500                              1.000                           0.867                            9.000    1.000   0.935   8.500    1.000   0.932
  TR31156   10.000                             0.889                           0.974                            10.000   1.000   0.915   9.000    1.000   0.931
  TR31399   10.500                             1.000                           0.961                            10.000   1.000   0.979   9.500    1.000   0.935
  TR31622   11.000                             0.800                           0.884                            11.000   1.000   0.916   10.000   0.833   0.851
  TR32795   11.000                             0.900                           0.921                            11.000   1.000   0.926   10.000   0.917   0.891
  TR33107   11.000                             0.900                           0.911                            11.000   0.900   0.874   10.000   0.833   0.909
  TR35128   11.000                             0.778                           0.817                            10.500   1.000   0.900   9.500    0.750   0.855
  TR35536   10.500                             0.778                           0.882                            10.000   0.900   0.837   9.500    0.909   0.900
  TR37605   11.000                             0.900                           0.926                            11.000   1.000   0.895   10.000   1.000   0.935
  TR37669   10.500                             0.889                           0.869                            10.500   0.889   0.889   9.000    1.000   0.899
  TR37811   10.500                             1.000                           0.935                            10.500   1.000   0.941   9.000    1.000   0.957
  TR38169   10.500                             1.000                           0.941                            10.500   1.000   0.961   9.000    0.917   0.935
  TR38361   10.000                             1.000                           0.858                            10.500   1.000   0.778   9.500    1.000   0.840
  TR41171   10.500                             1.000                           0.967                            10.000   1.000   0.980   8.500    1.000   0.931
  TR42254   11.000                             0.900                           0.921                            11.000   0.800   0.921   10.000   0.583   0.851
  TR42785   10.000                             0.750                           0.833                            10.000   0.875   0.867   8.000    0.833   0.866
  TR46482   9.500                              1.000                           0.941                            10.500   1.000   0.923   8.000    0.833   0.848
  TR47534   9.000                              1.000                           0.817                            8.500    0.778   0.680   8.500    0.556   0.804
  TR47552   10.000                             1.000                           0.967                            10.000   1.000   0.941   9.000    1.000   0.948
  TR50321   9.000                              0.800                           0.874                            10.500   0.857   0.868   8.500    0.545   0.827
  TR52495   9.500                              0.556                           0.843                            9.500    1.000   0.902   9.000    0.800   0.805

*A* ~e~ *=* number of effective alleles; *H* ~e~ = expected heterozygosity; *H* ~o~ = observed heterozygosity.

Locality and voucher information are provided in Appendix [1](#aps31158-app-0001){ref-type="app"}.

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###### 

Cross‐amplification results for the 51 microsatellite loci developed in *Prunus mongolica* in five *Prunus* species.[a](#aps31158-note-0005){ref-type="fn"} ^,^ [b](#aps31158-note-0006){ref-type="fn"}

  Locus     *P. triloba*   *P. davidiana*   *P. persica*   *P. cerasifera*   *P. serrulata*
  --------- -------------- ---------------- -------------- ----------------- ----------------
  TR10339   ---            163--195         115--147       133--171          117--151
  TR10936   ---            137--149         119--146       125--167          149--161
  TR12298   ---            103--115         ---            132--144          105--121
  TR12728   ---            110--122         105--117       105--127          120--132
  TR16484   ---            153--165         143--155       133--145          113--125
  TR17663   ---            131--140         130--142       ---               120--129
  TR17745   120--126       110--122         123--135       133--145          125--137
  TR18640   135--147       153--159         133--145       131--143          ---
  TR18807   140--149       143--155         133--145       113--119          131--143
  TR20374   115--127       151--163         157--169       153--165          151--159
  TR21328   111--131       115--136         113--125       117--129          ---
  TR21918   101--121       110--130         115--135       118--138          111--123
  TR23600   ---            ---              80--100        91--101           ---
  TR24122   ---            ---              130--150       139--159          133--153
  TR3320    ---            ---              ---            121--133          ---
  TR27527   140--149       145--160         132--144       141--153          135--147
  TR29327   133--145       135--147         130--142       132--138          ---
  TR31156   ---            ---              ---            ---               141--161
  TR31399   131--151       111--131         143--163       141--161          ---
  TR37605   125--145       126--146         121--141       122--142          123--143
  TR37811   131--161       121--151         111--141       146--176          132--162
  TR41171   131--191       112--142         123--153       119--149          145--175
  TR47552   121--141       122--142         111--131       123--143          101--131
  TR26908   113--129       133--149         113--125       115--127          131--143
  TR30962   111--127       131--143         127--139       123--139          135--147
  TR32795   125--137       123--126         147--159       131--161          126--138
  TR19174   103--123       121--133         116--128       113--129          132--144
  TR19201   142--154       131--161         125--137       126--138          141--171
  TR20030   115--135       117--139         123--135       134--146          137--149
  TR25166   113--125       132--144         115--135       162--182          133--145
  TR31622   151--163       131--143         133--163       142--154          125--137
  TR35128   117--129       146--158         135--155       132--144          172--184
  TR35536   137--157       117--129         153--165       135--155          127--167
  TR37669   123--138       121--133         125--137       122--134          115--127
  TR38169   ---            ---              125--137       ---               ---
  TR38361   133--149       131--147         115--127       124--136          125--137
  TR42254   122--134       117--129         115--127       123--135          116--128
  TR47534   123--135       115--127         132--144       124--136          125--137
  TR52495   117--129       112--124         113--125       117--129          123--135
  TR5622    121--151       124--136         142--154       141--153          123--135
  TR12256   ---            103--115         105--117       113--133          132--152
  TR14394   143--155       124--136         121--130       123--135          141--153
  TR22071   122--155       114--126         142--154       121--133          113--125
  TR50321   ---            ---              ---            123--143          114--124
  TR18685   113--125       121--133         135--147       123--135          141--153
  TR25043   ---            117--137         132--144       124--136          ---
  TR25414   133--145       123--139         121--133       142--158          152--164
  TR33107   121--133       143--155         117--139       ---               113--128
  TR42785   ---            ---              ---            ---               ---
  TR46482   253--265       221--236         232--244       ---               215--227
  TR9296    ---            ---              141--156       122--134          ---

--- = unsuccessful amplification.

Locality and voucher information are provided in Appendix [1](#aps31158-app-0001){ref-type="app"}.

*N* = 5 for all populations.

John Wiley & Sons, Ltd

CONCLUSIONS {#aps31158-sec-0006}
===========

In our study, a total of 19,498 SSRs were identified from 15,837 contigs, and 6069 microsatellite primers were designed. Among the identified SSR loci, 51 microsatellite primer pairs were polymorphic. The 51 novel microsatellite markers can be used to study the genetic diversity, landscape genetics, and phylogeography of the species. The microsatellite primers can also be used to genotype congeneric species (*P. triloba*,*P. davidiana*,*P. persica*,*P. cerasifera*, and *P. serrulata*).

The authors thank Xin Hong (Anhui University, Hefei, Anhui Province, China) for *Prunus* sampling. This work was supported by the National Key R&D Program of China (2016YFC0500705).

 {#aps31158-sec-0008}

Species*n*Collection localityGeographic coordinatesVoucher ID[a](#aps31158-note-1002){ref-type="fn"}Collector*Prunus mongolica* Maxim.10Wulanchabu, Inner Mongolia, China41°56′N, 110°28′E201607‐Pmon‐popI‐AHBZhanyuan Lu10Chifeng, Inner Mongolia, China43°23′N, 118°40′E201607‐Pmon‐popII‐AHBZhanyuan Lu12Eerduosi, Inner Mongolia, China39°35′N, 106°46′E201607‐ Pmon‐popIII‐AHBZhanyuan Lu*P. triloba* Lindl.5Hefei, Anhui, China31°52′N, 117°11′E201705‐Ptri‐AHBXin Hong*P. davidiana* (Carrière) Franch.5Hefei, Anhui, China31°52′N, 117°11′E201705‐Pdav‐AHBXin Hong*P. persica* (L.) Batsch5Wuhan, Hubei, China30°32′N, 114°24′E201705‐Pper‐AHBXin Hong*P. cerasifera* Ehrh.5Hefei, Anhui, China31°52′N, 117°11′E201705‐Pcer‐AHBXin Hong*P. serrulata* Lindl.5Hefei, Anhui, China31°52′N, 117°11′E201705‐Pser‐AHBXin HongNote[^1][^2]

[^1]: *n* = sample size.

[^2]: Vouchers are deposited at the herbarium of Anhui University (ANU), Hefei, Anhui Province, China.
