Xu, Z.‐C., Y.‐C. Jin, R. I. Milne, Z.‐Y. Xiahou, H.‐T. Qin, L.‐J. Ye, L.‐M. Gao, J. Liu, and D.‐Z. Li. 2020 Development of 32 novel microsatellite loci in *Juglans sigillata* using genomic data. Applications in Plant Sciences 8(3): e11328.

*Juglans sigillata* Dode (Juglandaceae), the iron walnut, is a woody perennial nut tree endemic to southwestern China (Lu et al., [1999](#aps311328-bib-0008){ref-type="ref"}). As one of the most important edible walnut species, *J. sigillata* is significantly different from the common walnut (*J. regia* L.) in morphology, fruit quality, and environmental adaptability (Lu et al., [1999](#aps311328-bib-0008){ref-type="ref"}; Gunn et al., [2010](#aps311328-bib-0004){ref-type="ref"}). There are more than 100 local cultivars and superior individuals of this species, which vary in seed quality and ecological adaptation. Therefore, *J. sigillata* is an important component of the genetic resources within *Juglans* L., given its great value for both cultivar development and scientific research. However, accurate determination of *Juglans* species requires both taxonomic expertise and complete specimens, and even then, identification can be difficult between closely related species or infraspecific taxa (e.g., cultivars), due for example to effects of age and environment. The scenario presents a major problem for improvement and conservation because accurate identification and delimitation of taxa are at the core of crop genetic breeding and genetic resource conservation (Cooke, [1995](#aps311328-bib-0002){ref-type="ref"}). Moreover, the precise taxonomic determination of specimens at the marketplace is rendered difficult or impossible; for example, the methods available to local people and authorities might not distinguish among dried nuts or seedlings of different *Juglans* taxa usually sold in markets.

As a traditional molecular marker, simple sequence repeats (SSRs or microsatellites) have been widely used in genetic diversity research. SSRs are abundant, genome‐ and locus‐specific, codominantly inherited, highly reproducible, and usually highly polymorphic (Powell et al., [1996](#aps311328-bib-0012){ref-type="ref"}). They have extensive applications, including but not limited to forensics, population and conservation genetics, identification of species and cultivars, and phylogeography (Hodel et al., [2016](#aps311328-bib-0006){ref-type="ref"}). However, SSR development typically requires a substantial input of time, funds, equipment, and expertise, and the biggest challenge involved is the identification or development of high‐quality primers that will amplify across the taxonomic group of interest (Zane et al., [2002](#aps311328-bib-0016){ref-type="ref"}). Nonetheless, once suitable primers are available, taxon identification via DNA fingerprinting becomes relatively straightforward and circumvents the need for taxonomic expertise.

A large number of *Juglans* microsatellite primers have been developed using traditional approaches (e.g., Woeste et al., [2002](#aps311328-bib-0015){ref-type="ref"}; Hoban et al., [2008](#aps311328-bib-0005){ref-type="ref"}; Ikhsan et al., [2016](#aps311328-bib-0007){ref-type="ref"}), and some of them have been employed in previous population genetic studies (e.g., Wang et al., [2008](#aps311328-bib-0014){ref-type="ref"}; Gunn et al., [2010](#aps311328-bib-0004){ref-type="ref"}). However, our preliminary cross‐species amplification results showed that most of the previously developed primers are inefficient for cultivar identification in *J. sigillata* (J. Liu et al., unpublished data). Therefore, we set out to design and screen a novel set of high‐quality primers using recently published genomic data for *J. regia* (e.g., Martínez‐García et al., [2016](#aps311328-bib-0009){ref-type="ref"}; Bai et al., [2018](#aps311328-bib-0001){ref-type="ref"}), with the goal of producing high‐quality SSR markers capable of distinguishing taxa and cultivars within *Juglans*.

METHODS AND RESULTS {#aps311328-sec-0005}
===================

SSR detection, primer design, and validation {#aps311328-sec-0006}
--------------------------------------------

Genomic data of two *J. regia* individuals were downloaded from the National Center for Biotechnology Information (NCBI) database (i.e., PRJNA291087 \[Martínez‐García et al., [2016](#aps311328-bib-0009){ref-type="ref"}\] and PRJNA356989 \[Bai et al., [2018](#aps311328-bib-0001){ref-type="ref"}\]). Comparing these genomes, we used QDD_v3 (Meglécz et al., [2009](#aps311328-bib-0010){ref-type="ref"}) to detect the SSRs with default parameters and found 279,712 loci containing microsatellites, 39,741 of which were polymorphic between the two genomes. In total, primers were designed for 14,217 of the polymorphic SSRs detected, using QDD with default parameters. After considering the PCR product size (ca. 100--400 bp), repeat type (perfect repeat), motif length (3--6 bp), and repeat frequency (\>6), we selected 434 of these primers for downstream laboratory validation. We adopted a three‐step pipeline to screen polymorphic primers.

### Step 1: Amplification screening {#aps311328-sec-0007}

Four *J. sigillata* individuals from three different populations were used to test the success of PCR amplification (Appendix [1](#aps311328-app-0001){ref-type="app"}). The PCR reaction comprised 18 μL of Golden Star T6 Super PCR Mix (TsingKe Biological Technology, Beijing, China), with 0.5 μL each of forward and reverse primers, plus 1 μL of DNA template (\~50 ng DNA), in a final reaction volume of 20 μL. The PCR protocol was: 95°C for 5 min; followed by 30 cycles of 95°C for 10 s, primer‐specific annealing temperatures (55--58°C, Table [1](#aps311328-tbl-0001){ref-type="table"}) for 3 min, 72°C for 1 min; and a final extension at 72°C for 5 min. PCRs were carried out on a Veriti 96‐Well Thermal Cycler (Applied Biosystems, Foster City, California, USA). The obtained PCR products were detected using 6% polyacrylamide gel (PAGE), and each primer was judged to be genuine if observed products were close to the expected size (ca. 20 bp fluctuation). One hundred and eleven primers successfully amplified all individuals and were further evaluated in step 2.

###### 

Characteristics of 32 microsatellite loci isolated from *Juglans sigillata*.

  Locus                              Primer sequences (5′--3′)          Repeat motif   Allele size range (bp)   *T* ~a~ (°C)   Fluorescent dye   GenBank accession no.
  ---------------------------------- ---------------------------------- -------------- ------------------------ -------------- ----------------- -----------------------
  JS01                               `F: AGGGATTGTGTTCAAATTGACCT`       (CACGA)~5~     79--94                   56             FAM               MN604168
  `R: GCTGGAGTGACAAATTCGAGT`                                                                                                                     
  JS02                               `F: GCATGATTAGTGACTTGAATTAGAAGC`   (AAAG)~8~      87--115                  57             HEX               MN604165
  `R: CATGACATGCTCGACACGC`                                                                                                                       
  JS03                               `F: TGACGAGGTTTACCAGATGGG`         (GAA)~5~       90--105                  58             TAMRA             MN604150
  `R: CGTTCTTCTTTCAGAGTGCTGTT`                                                                                                                   
  JS04                               `F: GAGGAAGAAGAGATGAAGACGA`        (GAA)~6~       93--111                  56             HEX               MN604155
  `R: CCCTTTCTCTATAATCCAATGAAATCC`                                                                                                               
  JS05                               `F: CGGCATTACAGTCGGCAGTA`          (GAA)~10~      93--120                  57             FAM               MN604159
  `R: ACAATTCCCGTGCTGCATCT`                                                                                                                      
  JS06                               `F: CCCTGCATGCAATCAATCACA`         (AGT)~5~       96--111                  55             TAMRA             MN604154
  `R: ATGGGACGAGTGATGGACTC`                                                                                                                      
  JS07                               `F: ACCAGCAGTTCCATGTACGG`          (GAG)~9~       111--132                 57             TAMRA             MN604158
  `R: GCTCATGCCATTATCTGCTTCG`                                                                                                                    
  JS08                               `F: GCATGCATTGGAACCTACCC`          (AAGA)~6~      112--122                 56             TAMRA             MN604166
  `R: TCAAAGCATTAACGTTAGCGAC`                                                                                                                    
  JS09                               `F: TTCGACCGCGTTTCCAGTTA`          (TTC)~7~       116--131                 56             FAM               MN604156
  `R: CCAGACTCACGGTCAGTTCC`                                                                                                                      
  JS10                               `F: GCTCCAGGCACATGAAAGAG`          (GGA)~7~       121--142                 57             HEX               MN604163
  `R: AATCCACCATTGCCCAACGA`                                                                                                                      
  JS11                               `F: CCAGACGGGTCCAGGTAT`            (AGC)~8~       123--141                 57             FAM               MN604149
  `R: GCCTTAGAGGTCAAGAATCCGA`                                                                                                                    
  JS12                               `F: TCAACATTGGCGAGGTGACA`          (TTA)~7~       128--155                 55             TAMRA             MN604152
  `R: AGGCAAGTCTACTTCTTTCCCT`                                                                                                                    
  JS13                               `F: TCTTGTCAGCATACTAAGCTTGTT`      (TTCT)~5~      129--158                 56             HEX               MN604164
  `R: ACTAACTGCATATAGGATCAACCA`                                                                                                                  
  JS14                               `F: CACATCGAGTGTTTCAAGTGACA`       (TGC)~6~       134--149                 57             FAM               MN604162
  `R: TGCACATGAGGAATTAACTGCTT`                                                                                                                   
  JS15                               `F: TGACACCTCGAGATTGTGCC`          (TTG)~5~       135--150                 57             TAMRA             MN604161
  `R: CCCGCCACCATCTTCATCTA`                                                                                                                      
  JS16                               `F: CGTTAACCTGCTGCATGCTC`          (CTC)~5~       193--223                 56             HEX               MN604157
  `R: CCTGAAGAAGAGTCATGTGCTG`                                                                                                                    
  JS17                               `F: GCCACTGTCTTCACTGGGAG`          (CTT)~5~       197--221                 55             HEX               MN604148
  `R: CGCAAAGGACAACAGCCAAT`                                                                                                                      
  JS18                               `F: GGGAAGTTGTTGAAGGAGCG`          (GAT)~6~       199--211                 57             HEX               MN604160
  `R: ATCACATGCGCGTTTCCTTT`                                                                                                                      
  JS19                               `F: AGATGATTTATGGCAGCCAATGA`       (AAG)~7~       216--252                 56             FAM               MN604153
  `R: TGCTGGGTAAACGCATGAGT`                                                                                                                      
  JS20                               `F: GCAACTTGACAGTTTCTCTTCTTTG`     (CCTCT)~5~     234--259                 57             FAM               MN604167
  `R: CTTCTCGTCATCGTCCGCAT`                                                                                                                      
  JS21                               `F: ACGAACCCTGGAAGCCAAAT`          (CGA)~5~       242--254                 55             HEX               MN604151
  `R: ACCACGAAGGCCTACAACTG`                                                                                                                      
  JS22                               `F: AAAGTTGCTCCTCAGCTTGG`          (ATC)~7~       266--293                 56             FAM               MN604147
  `R: TAATTAGCAATGAACAGATGGTGG`                                                                                                                  
  JS23                               `F: TGATGGATTTCGGTTGCAACTG`        (CTTGGT)~5~    286--304                 57             HEX               MN604169
  `R: ACGCCCGAGTAGTACTCCTT`                                                                                                                      
  JS24                               `F: CGGGTCTTGCTAACTGATTTCC`        (TAGGAA)~5~    287--305                 56             TAMRA             MN604170
  `R: TGGGAGCTGGATTTGATCTGTC`                                                                                                                    
  JS25                               `F: TAGGTTTGCCGGTGTTGGAA`          (TAT)~8~       310--334                 55             HEX               MN604171
  `R: CCATCTCATCTTCCAAACAACCC`                                                                                                                   
  JS26                               `F: ACATTTGAAAGAAGACGTGTACTG`      (AAAT)~6~      312--316                 57             HEX               MN604175
  `R: GAGATGCAGGGATGTTTGGGA`                                                                                                                     
  JS27                               `F: TCTTGGCAATATGCTTCCAATCG`       (TTTA)~5~      312--336                 55             HEX               MN604177
  `R: TGTGTATGCTGAAGATGCTCTT`                                                                                                                    
  JS28                               `F: AAAGGGTGAAGGAAGAAATTAGGAT`     (TCTCT)~5~     316--332                 57             HEX               MN604178
  `R: CCAAATTAAGCCAAACATGGTTGC`                                                                                                                  
  JS29                               `F: TGCTCATTAGGAACTTACAGCT`        (TCT)~8~       318--330                 57             FAM               MN604172
  `R: TGGTCTTATTCGGGTTGACACA`                                                                                                                    
  JS30                               `F: GGACCCACGAGTGCCAAATA`          (CCAA)~6~      319--327                 55             FAM               MN604176
  `R: GCTTAAAGAAGTGGTAAAGTAGGTAAT`                                                                                                               
  JS31                               `F: AATCACTGTTAGCCAAGCCT`          (TTA)~6~       321--345                 57             HEX               MN604173
  `R: TTTGCAAATATAGAACGCCAAGA`                                                                                                                   
  JS32                               `F: GTAGCTCTTTCAAACCGATCG`         (GAT)~6~       418--433                 57             FAM               MN604174
  `R: ATCAGGAATTAGACTGAGATATTCACA`                                                                                                               

*T* ~a~ = annealing temperature.

John Wiley & Sons, Ltd

### Step 2: Polymorphism identification {#aps311328-sec-0008}

PCR amplification was performed using 20 *J. sigillata* individuals and four *J. regia* individuals from five populations (Appendix [1](#aps311328-app-0001){ref-type="app"}). Once again, the quality of the amplification products was visually evaluated using PAGE gels. Thirty‐two primers showed results that were specific, reproducible, and revealed Mendelian polymorphisms and were taken forward to step 3.

### Step 3: Population genetic characterization {#aps311328-sec-0009}

Forward primers were 5′‐end fluorescently labeled with FAM, HEX, or TAMRA (Optimus Bio, Kunming, China). Ninety‐five individuals were used for the analysis, including three populations of *J. sigillata* (*n* = 60), two of *J. regia* (*n* = 29), and one each of *J. cathayensis* Dode (*n* = 3) and *J. mandschurica* Maxim. (*n* = 3) (Appendix [1](#aps311328-app-0001){ref-type="app"}). The fragment size of PCR products was determined by ABI 3730xl (Applied Biosystems). All of the 32 primer pairs selected for this stage were carried forward for data analysis.

Data analysis {#aps311328-sec-0010}
-------------

Based on PCR products from step 3, genotyping was carried out using GeneMarker version 2.2.0 (SoftGenetics, State College, Pennsylvania, USA). GenAlEx version 6.5 (Peakall and Smouse, [2012](#aps311328-bib-0011){ref-type="ref"}) was used to calculate the number of alleles and levels of observed and expected heterozygosity. Departures from Hardy--Weinberg equilibrium were determined at locus level and population level using GENEPOP (Rousset, [2008](#aps311328-bib-0013){ref-type="ref"}). Finally, FSTAT version 2.9.3 (Goudet, [2001](#aps311328-bib-0003){ref-type="ref"}) was used to detect any linkage disequilibrium between primers. Significance levels were adjusted using a sequential Bonferroni correction for multiple comparisons.

A total of 399 primer pairs (92%) successfully amplified the target loci. The majority of loci (74.4%) were monomorphic, and 111 primer pairs (25.6%) were selected for step 2, whereas only 32 primer pairs (7.4%) were selected for population genetic screening in step 3. Of the 32 loci selected, 25 had \>95% PCR amplification success rate, whereas the rate ranged from 55% to 90% for the remaining seven loci (Table [2](#aps311328-tbl-0002){ref-type="table"}). The number of alleles per locus ranged from two to eight, with an average of four, whereas mean levels of observed and expected heterozygosity ranged from 0.000 to 1.000 and 0.078 to 0.724, with average values of 0.436 and 0.470, respectively (Table [2](#aps311328-tbl-0002){ref-type="table"}). Significant deviations of Hardy--Weinberg equilibrium in terms of heterozygosity deficiency were detected in 30 of 96 locus--population pairs (Table [2](#aps311328-tbl-0002){ref-type="table"}). Linkage disequilibrium (*P* \< 0.05) was detected between loci JS23 and JS24.

###### 

Population genetic summary statistics of three *Juglans sigillata* populations.[a](#aps311328-note-0003){ref-type="fn"}

  Locus   Population                                  Total (*n* = 60)   Mean    PCR amplification rate (%)                                                                                                                  
  ------- ------------------------------------------- ------------------ ------- ------------------------------------------- ------- ------- ------------------------------------------- ------- ------- --- ------- ------- -----
  JS01    2[b](#aps311328-note-0004){ref-type="fn"}   0.950              0.499   2[b](#aps311328-note-0004){ref-type="fn"}   0.950   0.499   2[b](#aps311328-note-0004){ref-type="fn"}   0.950   0.499   2   0.950   0.499   100
  JS02    4                                           0.650              0.545   3                                           0.632   0.536   2                                           0.500   0.480   5   0.594   0.520   98
  JS03    5                                           0.650              0.731   5                                           0.400   0.445   5                                           0.750   0.560   6   0.600   0.579   100
  JS04    5                                           0.550              0.696   6                                           0.950   0.775   5[b](#aps311328-note-0004){ref-type="fn"}   0.400   0.700   6   0.633   0.724   100
  JS05    3                                           0.650              0.565   5                                           0.750   0.518   3                                           0.550   0.526   6   0.650   0.536   100
  JS06    3                                           0.700              0.599   3                                           0.600   0.595   3                                           0.550   0.511   3   0.617   0.568   100
  JS07    2[b](#aps311328-note-0004){ref-type="fn"}   0.000              0.495   3[b](#aps311328-note-0004){ref-type="fn"}   0.000   0.395   2[b](#aps311328-note-0004){ref-type="fn"}   0.000   0.180   3   0.000   0.357   100
  JS08    4                                           0.550              0.443   4                                           0.400   0.341   5                                           0.500   0.415   5   0.483   0.400   100
  JS09    2                                           0.350              0.499   2                                           0.700   0.495   3                                           0.550   0.545   3   0.533   0.513   100
  JS10    3                                           0.600              0.454   4                                           0.500   0.516   4                                           0.500   0.530   4   0.533   0.500   100
  JS11    3                                           0.600              0.585   5                                           0.500   0.619   6[b](#aps311328-note-0004){ref-type="fn"}   0.500   0.686   6   0.533   0.630   100
  JS12    4                                           0.200              0.303   4[b](#aps311328-note-0004){ref-type="fn"}   0.450   0.671   3[b](#aps311328-note-0004){ref-type="fn"}   0.400   0.559   4   0.350   0.511   100
  JS13    4                                           0.650              0.546   4                                           0.800   0.636   3[b](#aps311328-note-0004){ref-type="fn"}   0.550   0.659   4   0.667   0.614   100
  JS14    3[b](#aps311328-note-0004){ref-type="fn"}   0.450              0.421   3                                           0.700   0.579   3                                           0.550   0.629   3   0.567   0.543   100
  JS15    2                                           0.100              0.095   4                                           0.600   0.670   5                                           0.600   0.574   5   0.433   0.446   100
  JS16    3[b](#aps311328-note-0004){ref-type="fn"}   0.250              0.529   3[b](#aps311328-note-0004){ref-type="fn"}   0.250   0.485   3                                           0.550   0.551   5   0.350   0.522   100
  JS17    2                                           0.200              0.320   2                                           0.350   0.469   2                                           0.450   0.489   2   0.333   0.426   100
  JS18    4[b](#aps311328-note-0004){ref-type="fn"}   1.000              0.569   2[b](#aps311328-note-0004){ref-type="fn"}   1.000   0.500   3[b](#aps311328-note-0004){ref-type="fn"}   1.000   0.545   4   1.000   0.538   98
  JS19    4[b](#aps311328-note-0004){ref-type="fn"}   0.368              0.499   6[b](#aps311328-note-0004){ref-type="fn"}   0.368   0.716   6[b](#aps311328-note-0004){ref-type="fn"}   0.400   0.713   6   0.379   0.642   97
  JS20    3[b](#aps311328-note-0004){ref-type="fn"}   0.118              0.431   5[b](#aps311328-note-0004){ref-type="fn"}   0.188   0.691   4                                           0.588   0.678   5   0.298   0.600   83
  JS21    2                                           0.150              0.139   3                                           0.250   0.301   3                                           0.300   0.266   3   0.233   0.235   100
  JS22    6[b](#aps311328-note-0004){ref-type="fn"}   0.650              0.785   4                                           0.600   0.606   7                                           0.600   0.718   8   0.617   0.703   100
  JS23    4                                           0.400              0.431   4                                           0.550   0.674   3                                           0.750   0.661   4   0.567   0.589   100
  JS24    4                                           0.400              0.431   4                                           0.550   0.674   3                                           0.750   0.661   4   0.567   0.589   100
  JS25    2                                           0.050              0.049   4                                           0.316   0.359   3                                           0.200   0.261   4   0.189   0.223   87
  JS26    4                                           0.588              0.673   4[b](#aps311328-note-0004){ref-type="fn"}   0.474   0.669   5[b](#aps311328-note-0004){ref-type="fn"}   0.333   0.699   7   0.465   0.680   90
  JS27    2                                           0.000              0.180   1                                           0.000   0.000   2[b](#aps311328-note-0004){ref-type="fn"}   0.000   0.278   2   0.000   0.153   55
  JS28    2                                           0.444              0.346   3                                           0.250   0.271   4[b](#aps311328-note-0004){ref-type="fn"}   0.063   0.279   5   0.252   0.299   82
  JS29    1                                           0.000              0.000   2[b](#aps311328-note-0004){ref-type="fn"}   0.000   0.095   2                                           0.050   0.139   5   0.017   0.078   100
  JS30    2                                           0.222              0.198   2                                           0.313   0.264   2                                           0.250   0.219   2   0.262   0.227   82
  JS31    4[b](#aps311328-note-0004){ref-type="fn"}   0.150              0.303   4                                           0.200   0.269   6[b](#aps311328-note-0004){ref-type="fn"}   0.150   0.455   2   0.167   0.342   100
  JS32    1                                           0.000              0.000   2                                           0.235   0.208   2                                           0.100   0.095   3   0.112   0.101   85

*A* = number of alleles per locus; *H* ~e~ = expected heterozygosity; *H* ~o~ = observed heterozygosity; *n* = number of individuals.

Locality and voucher information are provided in Appendix [1](#aps311328-app-0001){ref-type="app"}.

Significant deviation from Hardy--Weinberg equilibrium (*P* \< 0.05).

John Wiley & Sons, Ltd

To evaluate the transferability of the newly developed markers in congeneric species, amplification was tested across 35 individuals of *J. regia*,*J. cathayensis*, and *J. mandshurica* (Appendix [1](#aps311328-app-0001){ref-type="app"}). All primer pairs successfully amplified the target loci, with the exception of locus JS01 in *J. cathayensis* and loci JS18 and JS01 in *J. mandshurica* (Table [3](#aps311328-tbl-0003){ref-type="table"}).

###### 

Transferability of the 32 SSR markers developed for *Juglans sigillata* in *J. regia*,*J. cathayensis*, and *J. mandshurica*.[a](#aps311328-note-0006){ref-type="fn"}

  Locus   *J. regia*   *J. cathayensis*   *J. mandshurica*                                                                                                                                                                                                   
  ------- ------------ ------------------ ------------------ ----- ------------------------------------------- --------------------------------------------- ----- ------------------------------------------- --------------------------------------------- -----
  JS01    **+**        **+**              79--94             97    [\*](#aps311328-note-0007){ref-type="fn"}   [\*\*](#aps311328-note-0008){ref-type="fn"}   0     [\*](#aps311328-note-0007){ref-type="fn"}   [\*\*](#aps311328-note-0008){ref-type="fn"}   0
  JS02    ---          ---                103                100   ---                                         99                                            100   \+                                          91--99                                        100
  JS03    \+           \+                 90--102            97    \+                                          96--99                                        67    \+                                          93--99                                        100
  JS04    \+           \+                 93--105            100   \+                                          96--111                                       100   ---                                         105                                           100
  JS05    ---          \+                 102--117           100   ---                                         93                                            33    ---                                         93                                            33
  JS06    ---          \+                 102--108           97    ---                                         96                                            67    ---                                         96                                            100
  JS07    ---          ---                118                100   ---                                         120                                           67    \+                                          114--122                                      100
  JS08    ---          \+                 116--128           100   \+                                          119--131                                      100   ---                                         119                                           100
  JS09    \+           \+                 123--132           100   \+                                          111--117                                      100   ---                                         117                                           100
  JS10    ---          \+                 129--141           100   \+                                          111--132                                      100   \+                                          123--132                                      100
  JS11    ---          \+                 129--149           97    \+                                          137--145                                      100   \+                                          137--149                                      100
  JS12    ---          \+                 121--139           100   \+                                          121--124                                      100   ---                                         121                                           100
  JS13    ---          \+                 129--149           97    ---                                         137                                           67    \+                                          137--143                                      100
  JS14    \+           \+                 138--155           100   \+                                          128--143                                      100   \+                                          128--140                                      100
  JS15    \+           \+                 138--150           100   \+                                          135--138                                      100   ---                                         135                                           100
  JS16    \+           \+                 196--223           97    \+                                          193--205                                      100   ---                                         196                                           100
  JS17    \+           \+                 199--211           100   ---                                         202                                           67    ---                                         202                                           100
  JS18    **+**        **+**              197--221           97    ---                                         209                                           33    [\*](#aps311328-note-0007){ref-type="fn"}   [\*\*](#aps311328-note-0008){ref-type="fn"}   0
  JS19    ---          ---                228--234           97    \+                                          219--237                                      100   \+                                          216--219                                      100
  JS20    ---          \+                 234--254           100   \+                                          239--254                                      67    ---                                         239                                           100
  JS21    ---          \+                 248--251           100   \+                                          245--248                                      100   \+                                          248--251                                      100
  JS22    ---          \+                 272--282           100   \+                                          266--281                                      100   \+                                          266--275                                      100
  JS23    \+           \+                 292--304           100   \+                                          289--298                                      33    ---                                         298                                           67
  JS24    \+           \+                 281--305           97    \+                                          293--299                                      67    ---                                         299                                           67
  JS25    \+           \+                 320--328           86    \+                                          316                                           67    ---                                         316                                           100
  JS26    \+           \+                 310--334           100   \+                                          310--319                                      100   \+                                          322--328                                      100
  JS27    \+           ---                312--316           86    \+                                          312--316                                      67    ---                                         316                                           100
  JS28    ---          ---                324                93    \+                                          312--324                                      33    \+                                          312--324                                      100
  JS29    \+           ---                318--330           93    ---                                         330                                           100   ---                                         330                                           100
  JS30    ---          \+                 319--323           93    ---                                         323                                           67    \+                                          319--327                                      100
  JS31    \+           \+                 321--345           31    \+                                          330--336                                      67    \+                                          336--339                                      67
  JS32    ---          ---                418                100   ---                                         418                                           67    ---                                         418                                           100

--- = monomorphic (only one allele was detected); + = polymorphic (two or more alleles were observed).

Locality and voucher information are provided in Appendix [1](#aps311328-app-0001){ref-type="app"}.

Unsuccessful PCR amplification.

No result.

John Wiley & Sons, Ltd

CONCLUSIONS {#aps311328-sec-0011}
===========

We developed 32 polymorphic microsatellite loci in *J. sigillata*. These primers can be used to analyze the genetic diversity and structure of *Juglans* populations, not only in *J. sigillata* but also in closely related species. Moreover, these primers can also be used to examine species boundaries and infraspecific taxonomy within the genus, thus allowing for the development of a DNA fingerprinting system. Fundamentally, these markers offer a new opportunity to solve and understand the origin and domestication history of walnut tree species.

AUTHOR CONTRIBUTIONS {#aps311328-sec-0013}
====================

J.L., D.‐Z.L., and L.‐M.G. conceived and designed the experiments. J.L. collected the samples. H.‐T.Q. and L.‐J.Y. designed the primers. Z.‐C.X. and Y.‐C.J. performed the molecular laboratory work. Z.‐Y.X. and Z.‐C.X. analyzed the data. Z.‐C.X., J.L., and R.I.M. drafted the manuscript, and all authors contributed to the manuscript revision.

This research was funded by the National Natural Science Foundation of China (grant no. 31770367, 41571059), the Science and Technology Innovation Program of the Forestry Department of Yunnan Province (\[2016\]cx03), the Natural Science Foundation of Yunnan (2017FB027), and the Top‐notch Young Talents Project of Yunnan Provincial "Ten Thousand Talents Program" (YNWR‐QNBJ‐2018‐146). Molecular experiments were performed at the Laboratory of Molecular Biology at the Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences.

DATA AVAILABILITY {#aps311328-sec-0014}
=================

Genomic data for the two *Juglans regia* specimens were downloaded from the National Center for Biotechnology Information (NCBI) database (PRJNA291087 \[Martínez‐García et al., [2016](#aps311328-bib-0009){ref-type="ref"}\] and PRJNA356989 \[Bai et al., [2018](#aps311328-bib-0001){ref-type="ref"}\]). Primer sequences for the newly developed primers have been deposited to NCBI\'s GenBank database; accession numbers are listed in Table [1](#aps311328-tbl-0001){ref-type="table"}.

 {#aps311328-sec-0015}

SpeciesPopulation codeLocation (County, province, country)Latitude (°N)Longitude (°E)Elevation (m)*N*Voucher specimen[a](#aps311328-note-0009){ref-type="fn"}*J. sigillata* DodeGDGongshan, Yunnan, China27.91660898.339602210020LiuJ9781--LiuJ9800*J. sigillata*TQTTengchong, Yunnan, China25.25506998.700549220020LiuJ9761--LiuJ9780*J. sigillata*LBTLongyangqu, Yunnan, China25.30298498.791248195020LiuJ9809--LiuJ9828*J. regia* L.GLRGongliu, Xinjiang, China43.35333382.83541713222016CS12110*J. regia*XYRXinyuan, Xinjiang, China43.52133383.93966612509LiuJ167917--LiuJ167925*J. cathayensis* DodeMGYMeigu, Sichuan, China29.352999103.5323321453LiuJ9844--LiuJ9846*J. mandshurica* Maxim.DTLTonghua, Jilin, China42.511167125.914675133LiuJ167742--LiuJ167744[^1]

[^1]: Voucher specimens have been deposited at the herbarium of Kunming Institute of Botany, Chinese Academy of Sciences (KUN).
