*Chamaecyparis* *obtusa* (Sieb. & Zucc.) Endl. (Cupressaceae), an economically important tree species frequently used for construction and native to Japan, was first introduced to Korea in 1904 and mainly planted in the southern provinces ([@bib12]). Recently, this species has received attention in Korea for its ability to adapt to ongoing climate change and to be used for outdoor recreation as a "healing forest" ([@bib1]). Therefore, the demand for seeds of *C. obtusa* has increased. With this increasing demand, there has been a growing interest in breeding research on *C. obtusa* using molecular markers. Microsatellite markers, with codominance and high polymorphism, are preferred for molecular breeding programs because they allow high genetic resolution ([@bib9]; [@bib2]). Previous studies have developed microsatellite markers for *C. obtusa* and conducted investigations of its genetic variation and population structure. [@bib7] and [@bib5] developed nine and 15 polymorphic markers, respectively. These developed markers were employed in further genetic studies on genetic diversity and structure in a natural fragmented population of *C. obtusa* ([@bib6]). However, microsatellite markers have some shortcomings, such as genotyping error caused by null alleles, mutation, or mistyping, which can generate false exclusion or produce ghost individuals. To overcome such problems, likelihood-based approaches are preferred. The accuracy of estimates resulting from these analyses is dependent on (i.e., increased with) the number of loci ([@bib11]). Accordingly, previously developed markers might be insufficient to employ various methodologies of genetic analysis used in molecular breeding and breeding population management. Therefore, to secure enough polymorphic loci, we developed and evaluated additional microsatellite markers for further genetic studies for *C. obtusa*.

METHODS AND RESULTS
===================

A microsatellite-enrichment library was constructed on *C. obtusa* plants collected from seed production stands throughout South Korea. Genomic DNA was extracted from fresh leaves with a QIAamp 96 DNA QIAcube HT Kit (QIAGEN, Hilden, Germany) using QIAxtractor (QIAGEN). The library was constructed according to the method of [@bib3]. Briefly, genomic DNA from the leaves of *C. obtusa* was digested with a restriction enzyme, *Rsa*I, to obtain DNA fragments approximately ranging from 300 to 1000 bp and then ligated with a modified SNX linker incorporated with a GTTT PIG-tail. This method should facilitate that the PCR product can be cloned directly without obtaining a large proportion of small DNA fragments and have efficient A-tailing to yield good results from TA cloning. The ligation products were subjected to double enrichment steps by hybridization with 3′-biotinylated microsatellite probes. The DNA fragments containing microsatellites were ligated to pGEM-T vectors (Promega Corporation, Madison, Wisconsin, USA), and the ligation mixture was transformed into competent *Escherichia coli* DH5α cells. The resulting colonies were subjected to colony PCR to identify recombinant clones using M13 forward and reverse primer sets. The PCR products were purified and directly used for sequencing by the ABI 3730 DNA Analyzer (Applied Biosystems, Waltham, Massachusetts, USA). After the trimming of vector and linker sequences, the nucleotide sequences were assembled to generate nonredundant contigs using Lasergene SeqMan (ver. 7.0.0; DNASTAR, Madison, Wisconsin, USA). Putative SSRs were identified by MISA software ([@bib10]) with the following criteria: a minimum of three repeats for dinucleotides to hexanucleotides and a gap within 100 bp for composite class. Criteria for primer design were as follows: amplicon size of 85--350 bp and annealing temperature of 57--60°C. Twenty-one primers used in this study were synthesized by Biomedic Co. Ltd. (Bucheon, Korea; [www.ibiomedic.co.kr](http://www.ibiomedic.co.kr)). The primer specificity was validated by routine PCR using genomic DNA as templates. For the preliminary screening of markers to amplify putative single loci, routine genomic PCR was performed using genomic DNA from five samples of *C. obtusa* as templates. The PCR products were separated on a 2% agarose gel. Twenty-one candidate microsatellite primers, which amplified putative single loci, were selected to screen polymorphic markers and labeled with fluorescent dye (FAM).

To validate the applicability of the 21 candidate primers to genetic studies, PCR amplifications were conducted for 90 samples from three seed production stands of *C. obtusa* located in Jeonbuk, Jeonnam, and Gyeongnam provinces, Korea. To test the transferability of the markers to the other related species, 12 samples of *C. pisifera* (Siebold & Zucc.) Endl. were collected in Chungbuk Province and used for further analysis. All the DNA samples used in this study were deposited at the Gene Bank of the National Forest Seed and Variety Center (NFSV, Korea); accession numbers and locality information are provided in [Appendix 1](#app1){ref-type="app"}. PCR was performed in 11-μL reactions containing 9 ng of template DNA, 1.5 or 2.5 mM MgCl~2~, 200 μM of each dNTP, 0.2 μM of 6-FAM fluorescent dye--labeled forward primer and reverse primer, 0.75 units of NeoTherm *Taq* DNA polymerase (GeneCraft, Hulme, United Kingdom), and 1× reaction buffer (GeneCraft). The PCR cycling was conducted as follows: 5 min at 94°C for predenaturation; 34 cycles of 30 s at 94°C, 1 min at 54--62°C for each primer, and 1 min at 72°C; and a final extension for 10 min at 72°C. The fluorescent PCR products were mixed with Hi-Di formamide and GeneScan 500 ROX Size Standard (Applied Biosystems). Those were visualized and scored using an ABI 3730 Genetic Analyzer (Applied Biosystems) and GeneMapper 4.1 software (Applied Biosystems).

Fifteen (71.4%) of the 21 candidate primers were successfully amplified for *C. obtusa*. Ten of these produced polymorphic DNA fragments, and the remaining five primers produced monomorphic amplicons ([Table 1](#tbl1){ref-type="table"}). The percentage of amplification for *C. pisifera* was 33.3% (5/15). Genetic properties of the 10 polymorphic primers for *C. obtusa* were evaluated ([Table 2](#tbl2){ref-type="table"}). The number of alleles (*A*), number of effective alleles (*A*~e~), and observed (*H*~o~) and expected heterozygosity (*H*~e~) were obtained by GenAlEx version 6.41 ([@bib8]), and polymorphic information content (PIC) was calculated with CERVUS version 3.0.3 ([@bib4]). In the total samples (90), *A* ranged from four to 24 per locus, *A*~e~ ranged from 1.2 to 14.2, and PIC values ranged from 0.160 to 0.927. *H*~o~ and *H*~e~ varied from 0.000 to 0.988 and from 0.165 to 0.929, respectively. The Co2047 marker, which had a high *A*~e~ value, could be a most efficient marker to infer current pollen flow using an indirect method such as TwoGener or a direct method such as parentage analysis, because high PIC values can be the result of relatively even allele frequency.

###### 

Repeat motif, primer sequence, and size range for amplified microsatellite loci in *Chamaecyparis* species.

                                                                                                     Allele size range (bp)                  
  ------------------------------------------ ------------------------------- ----------------------- ------------------------ --------- ---- ----------
  Co2011                                     F: TAAAAATCGCAAGGCCAACT         (GAA)~3~                244                      238/241   54   KF689697
                                             R: TCGACCCAATTTGGAGTTACTT                                                                       
  Co2018[\*](#tblfn1){ref-type="table-fn"}   F: GCCCTAGAAACTCGGGGTAA         (CA)~16~.(CA)~11~       240--270                 ---       62   KF689702
                                             R: GTTTGGGGCAATACACAGCGTAATA                                                                    
  Co2023[\*](#tblfn1){ref-type="table-fn"}   F: CACTATCGGGATGCTTGTGA         (CT)~28~                210--310                 ---       55   KF689706
                                             R: GTTTAGAGAATGGACTCGATGCAAA                                                                    
  Co2033                                     F: CCATTGGGCAAGGTTTCTTA         (AC)~9~                 145                      145       55   KF689713
                                             R: GATGCTTCAGATGGGACCTC                                                                         
  Co2043[\*](#tblfn1){ref-type="table-fn"}   F: GGAATTAATTACCACGCTGAAA       (CT)~15~TTCTC(CA)~6~    160--210                 ---       60   KF689719
                                             R: CCCCAAAAGCCTATGAGACA                                                                         
  Co2047[\*](#tblfn1){ref-type="table-fn"}   F: ATGCCCAAAACACACAAACA         (CT)~21~                200--280                 ---       56   KF689720
                                             R: GTTTCTTCCCTTTGTCTCTTTGTCA                                                                    
  Co2050[\*](#tblfn1){ref-type="table-fn"}   F: GCCCTCAACCTCAAACCATA         (GA)~31~                110--180                 224/228   56   KF689723
                                             R: GTTTCACACTCCCTTATCTCCCTCA                                                                    
  Co3008                                     F: GAGAATGCACCAGGCAATAAA        (AG)~4~                 152                      ---       55   KF689734
                                             R: GTTTGATCTTCAATTCCCTCCCATA                                                                    
  Co3016                                     F: GGAAAAAGTTTGGCATCGAG         (ACAG)~5~               157                      157       55   KF689739
                                             R: GTTTCTCCTTGTGCCAACACAGAA                                                                     
  Co3026[\*](#tblfn1){ref-type="table-fn"}   F: CGAAACGAAACCAATTCCTC         (CTAT)~19~              210--290                 ---       60   KF689746
                                             R: GTTTACCTCCTCCCACAATTCG                                                                       
  Co3033[\*](#tblfn1){ref-type="table-fn"}   F: TGTCTCACCAATGCCAAAAG         (ATAG)~8~               220--250                 ---       60   KF689750
                                             R: CCACTCCCCCTTTTCAAATC                                                                         
  Co3038                                     F: TTTCTCCCTTATCCACCTTCC        (TCTG)~5~               242                      242       55   KF689753
                                             R: TTCAACTCATCACATCATCCA                                                                        
  Co4012[\*](#tblfn1){ref-type="table-fn"}   F: CTTGCATCCCTACCTTGCAT         (CATC)~8~...(CTAC)~7~   140--190                 ---       55   KF689767
                                             R: TTCCACTTCCATGTCAACCA                                                                         
  Co4014\*                                   F: TTCCACTTCCATGTCAACCA         (TAGG)~7~               150--200                 ---       62   KF689769
                                             R: GTTTCTTGCATCCCTACCTTGCAT                                                                     
  Co4049[\*](#tblfn1){ref-type="table-fn"}   F: TTTGTCTGTTTATCTGCCTTTCTC     (TCTA)~7~               340--390                 ---       56   KF689781
                                             R: GTTTGCTCCTATTGTGATTTTGAGTG                                                                   

*Note*: --- = information not available; *T*~a~ = annealing temperature.

Polymorphic microsatellite loci.

###### 

Genetic properties of 10 polymorphic microsatellite loci of *Chamaecyparis obtusa*.

           Jeonbuk (*N* = 30)   Jeonnam (*N* = 45)   Gyeongnam (*N* = 15)   Total (*N* = 90)                                                                                                                                                                                                        
  -------- -------------------- -------------------- ---------------------- ----------------------------------------- ------- ---- ---- ------ ----------------------------------------- ------- ---- ---- ----- ----------------------------------------- ------- ---- ---- ------ ------- ------- -------
  Co2023   29                   16                   5.8                    0.759[\*](#tblfn2){ref-type="table-fn"}   0.827   42   17   10.9   0.881[\*](#tblfn2){ref-type="table-fn"}   0.908   14   9    4.8   0.857[\*](#tblfn2){ref-type="table-fn"}   0.791   85   24   9.9    0.835   0.899   0.889
  Co2043   30                   10                   2.7                    0.533[\*](#tblfn2){ref-type="table-fn"}   0.635   42   7    3.6    0.690[\*](#tblfn2){ref-type="table-fn"}   0.720   15   10   6.5   0.933                                     0.847   87   13   3.8    0.678   0.734   0.704
  Co2047   27                   12                   8.1                    1.000[\*](#tblfn2){ref-type="table-fn"}   0.876   43   20   12.3   1.000[\*](#tblfn2){ref-type="table-fn"}   0.919   15   12   9.2   0.933[\*](#tblfn2){ref-type="table-fn"}   0.891   85   22   14.2   0.988   0.929   0.927
  Co2050   29                   14                   9.7                    1.000[\*](#tblfn2){ref-type="table-fn"}   0.897   44   17   9.2    0.773[\*](#tblfn2){ref-type="table-fn"}   0.891   15   10   7.8   0.867[\*](#tblfn2){ref-type="table-fn"}   0.871   88   22   11.3   0.864   0.911   0.904
  Co3026   27                   4                    1.8                    0.000[\*](#tblfn2){ref-type="table-fn"}   0.453   41   5    1.8    0.000[\*](#tblfn2){ref-type="table-fn"}   0.458   13   3    2.6   0.000[\*](#tblfn2){ref-type="table-fn"}   0.615   81   5    2.0    0.000   0.496   0.439
  Co3033   26                   2                    1.8                    0.385                                     0.453   44   4    2.2    0.500                                     0.537   15   2    1.8   0.667[\*](#tblfn2){ref-type="table-fn"}   0.444   85   4    2.0    0.494   0.497   0.413
  Co4012   24                   4                    1.7                    0.500                                     0.416   44   5    1.4    0.341                                     0.301   15   4    1.6   0.467                                     0.389   83   5    1.6    0.410   0.355   0.336
  Co4014   29                   3                    1.2                    0.172                                     0.160   43   3    1.1    0.116                                     0.111   14   3    1.4   0.071[\*](#tblfn2){ref-type="table-fn"}   0.304   86   5    1.2    0.128   0.165   0.160
  Co4049   30                   6                    3.6                    0.867[\*](#tblfn2){ref-type="table-fn"}   0.722   43   9    5.8    0.953                                     0.826   15   6    4.8   0.733[\*](#tblfn2){ref-type="table-fn"}   0.791   88   9    5.1    0.886   0.802   0.775
  Co2018   18                   6                    4.2                    0.556                                     0.761   29   9    4.5    0.690[\*](#tblfn2){ref-type="table-fn"}   0.780   12   5    3.3   0.583                                     0.694   59   9    4.6    0.627   0.781   0.750

*Note*: *A* = number of alleles per locus; *A*~e~ = number of effective alleles per locus; *H*~e~ = expected heterozygosity; *H*~o~ = observed heterozygosity; *N* = number of individuals sampled; *n* = number of individuals genotyped; PIC = polymorphism information content.

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

CONCLUSIONS
===========

Ten polymorphic microsatellite markers developed for *C. obtusa* are available for genetic studies, such as analyses of current pollen flow, seed flow, mating system, and population genetic structure. Of these microsatellite markers developed in *C. obtusa*, 33.3% were successfully amplified in the related species *C. pisifera*. The genetic information gathered by these markers could be useful for breeding program management of *C. obtusa*.

###### 

Accession numbers and locality information of *Chamaecyparis* samples used in this study. All DNA samples are deposited at the Gene Bank of the National Forest Seed and Variety Center (NFSV), Chungju, Korea.

  Species                                            Accession no.                        Collection locality[^a^](#tblfn3){ref-type="table-fn"}   Geographic coordinates
  -------------------------------------------------- ------------------------------------ -------------------------------------------------------- -------------------------------
  *Chamaecyparis obtusa* (Siebold & Zucc.) Endl.     0300-03-00041555                     Jeonbuk                                                  35°43′01.83″N, 127°13′29.72″E
                                                     0300-03-00041556                     Jeonbuk                                                  35°43′01.67″N, 127°13′29.56″E
                                                     0300-03-00041557                     Jeonbuk                                                  35°43′01.74″N, 127°13′29.91″E
                                                     0300-03-00041558                     Jeonbuk                                                  35°43′01.83″N, 127°13′29.87″E
                                                     0300-03-00041559                     Jeonbuk                                                  35°43′01.61″N, 127°13′29.67″E
                                                     0300-03-00041560                     Jeonbuk                                                  35°43′01.64″N, 127°13′29.48″E
                                                     0300-03-00041561                     Jeonbuk                                                  35°43′01.48″N, 127°13′29.04″E
                                                     0300-03-00041562                     Jeonbuk                                                  35°43′01.41″N, 127°13′29.32″E
                                                     0300-03-00041567                     Jeonbuk                                                  35°43′01.54″N, 127°13′29.75″E
                                                     0300-03-00041568                     Jeonbuk                                                  35°43′01.48″N, 127°13′29.75″E
                                                     0300-03-00041569                     Jeonbuk                                                  35°43′01.48″N, 127°13′30.03″E
                                                     0300-03-00041637                     Jeonbuk                                                  35°43′00.02″N, 127°13′30.51″E
                                                     0300-03-00041661                     Jeonbuk                                                  35°42′59.56″N, 127°13′30.31″E
                                                     0300-03-00041693                     Jeonbuk                                                  35°42′59.14″N, 127°13′31.26″E
                                                     0300-03-00041725                     Jeonbuk                                                  35°42′58.39″N, 127°13′32.17″E
                                                     0300-03-00039814                     Jeonbuk                                                  35°24′14.47″N, 126°43′23.35″E
                                                     0300-03-00039838                     Jeonbuk                                                  35°24′14.96″N, 126°43′24.42″E
                                                     0300-03-00039870                     Jeonbuk                                                  35°24′14.64″N, 126°43′26.56″E
                                                     0300-03-00039902                     Jeonbuk                                                  35°24′14.05″N, 126°43′24.58″E
                                                     0300-03-00039916                     Jeonbuk                                                  35°24′14.38″N, 126°43′25.84″E
                                                     0300-03-00039917                     Jeonbuk                                                  35°24′14.38″N, 126°43′26.24″E
                                                     0300-03-00039919                     Jeonbuk                                                  35°24′14.25″N, 126°43′26.04″E
                                                     0300-03-00039922                     Jeonbuk                                                  35°24′14.38″N, 126°43′26.76″E
                                                     0300-03-00039923                     Jeonbuk                                                  35°24′14.29″N, 126°43′26.88″E
                                                     0300-03-00039924                     Jeonbuk                                                  35°24′14.22″N, 126°43′26.68″E
                                                     0300-03-00039925                     Jeonbuk                                                  35°24′14.16″N, 126°43′26.84″E
                                                     0300-03-00039928                     Jeonbuk                                                  35°24′14.26″N, 126°43′27.23″E
                                                     0300-03-00039929                     Jeonbuk                                                  35°24′14.19″N, 126°43′27.11″E
                                                     0300-03-00039930                     Jeonbuk                                                  35°24′14.09″N, 126°43′27.15″E
                                                     0300-03-00039931                     Jeonbuk                                                  35°24′13.96″N, 126°43′27.03″E
                                                     0300-03-00040129                     Jeonnam                                                  35°21′22.27″N, 126°53′33.46″E
                                                     0300-03-00040161                     Jeonnam                                                  35°21′22.85″N, 126°53′31.88″E
                                                     0300-03-00040177                     Jeonnam                                                  35°21′23.28″N, 126°53′32.43″E
                                                     0300-03-00040185                     Jeonnam                                                  35°21′22.92″N, 126°53′32.87″E
                                                     0300-03-00040495                     Jeonnam                                                  35°21′20.72″N, 126°53′35.68″E
                                                     0300-03-00040498                     Jeonnam                                                  35°21′20.88″N, 126°53′35.96″E
                                                     0300-03-00040502                     Jeonnam                                                  35°21′20.59″N, 126°53′35.76″E
                                                     0300-03-00040505                     Jeonnam                                                  35°21′20.68″N, 126°53′36.00″E
                                                     0300-03-00042405                     Jeonnam                                                  35°00′45.27″N, 127°10′51.45″E
                                                     0300-03-00042429                     Jeonnam                                                  35°00′45.17″N, 127°10′52.08″E
                                                     0300-03-00042461                     Jeonnam                                                  35°00′45.66″N, 127°10′52.32″E
                                                     0300-03-00042485                     Jeonnam                                                  35°00′46.11″N, 127°10′51.03″E
                                                     0300-03-00042299                     Jeonnam                                                  35°00′44.72″N, 127°10′48.85″E
                                                     0300-03-00042300                     Jeonnam                                                  35°00′44.75″N, 127°10′48.97″E
                                                     0300-03-00042303                     Jeonnam                                                  35°00′44.88″N, 127°10′49.08″E
                                                     0300-03-00041744                     Jeonnam                                                  35°00′45.82″N, 127°10′48.18″E
                                                     0300-03-00039118                     Jeonnam                                                  34°45′37.04″N, 126°44′05.36″E
                                                     0300-03-00039174                     Jeonnam                                                  34°45′36.27″N, 126°44′07.17″E
                                                     0300-03-00039200                     Jeonnam                                                  34°45′37.96″N, 126°44′08.66″E
                                                     0300-03-00039201                     Jeonnam                                                  34°45′37.89″N, 126°44′08.81″E
                                                     0300-03-00039206                     Jeonnam                                                  34°45′36.56″N, 126°44′08.05″E
                                                     0300-03-00039210                     Jeonnam                                                  34°45′37.31″N, 126°44′08.42″E
                                                     0300-03-00039211                     Jeonnam                                                  34°45′37.47″N, 126°44′08.05″E
                                                     0300-03-00042486                     Jeonnam                                                  34°40′08.12″N, 126°56′06.41″E
                                                     0300-03-00042487                     Jeonnam                                                  34°40′07.99″N, 126°56′06.37″E
                                                     0300-03-00042488                     Jeonnam                                                  34°40′08.02″N, 126°56′06.29″E
                                                     0300-03-00042489                     Jeonnam                                                  34°40′08.06″N, 126°56′06.13″E
                                                     0300-03-00042501                     Jeonnam                                                  34°40′08.09″N, 126°56′05.43″E
                                                     0300-03-00042525                     Jeonnam                                                  34°40′07.54″N, 126°56′05.07″E
                                                     0300-03-00042557                     Jeonnam                                                  34°40′07.05″N, 126°56′05.27″E
                                                     0300-03-00042589                     Jeonnam                                                  34°40′06.03″N, 126°56′03.15″E
                                                     0300-03-00045093                     Jeonnam                                                  34°54′29.34″N, 126°49′36.74″E
                                                     0300-03-00045101                     Jeonnam                                                  34°54′30.38″N, 126°49′37.96″E
                                                     0300-03-00045102                     Jeonnam                                                  34°54′30.12″N, 126°49′37.92″E
                                                     0300-03-00045104                     Jeonnam                                                  34°54′30.06″N, 126°49′38.28″E
                                                     0300-03-00045117                     Jeonnam                                                  34°54′29.67″N, 126°49′38.95″E
                                                     0300-03-00045149                     Jeonnam                                                  34°54′30.81″N, 126°49′40.09″E
                                                     0300-03-00045155                     Jeonnam                                                  34°54′30.61″N, 126°49′40.44″E
                                                     0300-03-00045157                     Jeonnam                                                  34°54′30.52″N, 126°49′40.76″E
                                                     0300-03-00045163                     Jeonnam                                                  34°54′29.64″N, 126°49′39.07″E
                                                     0300-03-00045164                     Jeonnam                                                  34°54′29.57″N, 126°49′39.58″E
                                                     0300-03-00045170                     Jeonnam                                                  34°54′29.74″N, 126°49′40.96″E
                                                     0300-03-00045171                     Jeonnam                                                  34°54′29.74″N, 126°49′40.08″E
                                                     0300-03-00045172                     Jeonnam                                                  34°54′29.08″N, 126°49′41.35″E
                                                     0300-03-00045173                     Jeonnam                                                  34°54′29.93″N, 126°49′40.99″E
                                                     0300-03-00047493                     Gyeongnam                                                34°46′58.18″N, 127°58′19.49″E
                                                     0300-03-00047517                     Gyeongnam                                                34°46′57.89″N, 127°58′20.39″E
                                                     0300-03-00047549                     Gyeongnam                                                34°46′57.17″N, 127°58′20.07″E
                                                     0300-03-00047581                     Gyeongnam                                                34°46′57.07″N, 127°58′20.31″E
                                                     0300-03-00046437                     Gyeongnam                                                34°46′59.12″N, 127°58′04.09″E
                                                     0300-03-00046438                     Gyeongnam                                                34°46′59.12″N, 127°58′04.28″E
                                                     0300-03-00046439                     Gyeongnam                                                34°46′58.99″N, 127°58′04.16″E
                                                     0300-03-00046440                     Gyeongnam                                                34°46′58.86″N, 127°58′04.12″E
                                                     0300-03-00046441                     Gyeongnam                                                34°46′58.83″N, 127°58′04.28″E
                                                     0300-03-00046442                     Gyeongnam                                                34°46′58.73″N, 127°58′04.16″E
                                                     0300-03-00046443                     Gyeongnam                                                34°46′58.73″N, 127°58′04.28″E
                                                     0300-03-00046444                     Gyeongnam                                                34°46′58.79″N, 127°58′04.36″E
                                                     0300-03-00046445                     Gyeongnam                                                34°46′58.92″N, 127°58′04.04″E
                                                     0300-03-00046446                     Gyeongnam                                                34°46′58.95″N, 127°58′04.56″E
                                                     0300-03-00046447                     Gyeongnam                                                34°46′58.82″N, 127°58′04.56″E
  *Chamaecyparis pisifera* (Siebold & Zucc.) Endl.   0300-03-00085617--0300-03-00085628   Chungbuk                                                 36°52′37.02″N, 127°58′25.80″E

Province in Korea.

[^1]: This work was supported by the Research Project of the National Forest Seed and Variety Center, Korea (NFSV).
