Plant Gene and Trait 2026, Vol.17, No.1, 1-11 http://genbreedpublisher.com/index.php/pgt 8 No. Group Region Locality Longitude Latitude Elevation (m) Aspect 66 a Shanting District Northwest Mountain 117°36′29″ 35°03′40″ 280 Southeast 68 a Shanting District Northwest Mountain 117°36′31″ 35°03′43″ 280 East 70 a Shanting District Northwest Mountain 117°36′38″ 35°03′22″ 280 South 72 a Shanting District Northwest Mountain 117°36′29″ 35°03′40″ 290 Southeast 74 a Shanting District Northwest Mountain 117°36′28″ 35°03′40″ 240 South 76 a Shanting District Northwest Mountain 117°36′27″ 35°03′39″ 280 Southwest 78 a Shanting District Northwest Mountain 117°36′40″ 35°03′09″ 280 South 80 a Shanting District Northwest Mountain 117°36′25″ 35°03′38″ 270 South 81 a Shanting District Yanggang Mountain 117°36′30″ 35°02′55″ 230 East 83 a Shanting District Yanggang Mountain 117°36′28″ 35°02′57″ 280 Southeast 85 a Shanting District Yanggang Mountain 117°36′10″ 35°03′21″ 300 Southwest 89 a Shanting District Yanggang Mountain 117°36′29″ 35°02′56″ 290 Southeast 96 a Shanting District Shengshan’an Pass 117°34′08″ 35°00′19″ 180 North 98 a Shanting District Shengshan’an Pass 117°34′10″ 34°59′38″ 290 Northwest 99 a Shanting District Yanggang Mountain 117°36′00″ 35°03′22″ 280 South 100 a Shanting District Shengshan’an Pass 117°34′10″ 34°59′38″ 216 Southwest 102 a Shanting District Shengshan’an Pass 117°34′10″ 34°59′39″ 213 Southwest 103 a Shanting District Jiguan Gu 117°36′57″ 34°58′15″ 270 Southwest 107 a Shanting District Jiguan Gu 117°37′09″ 34°58′09″ 260 Southwest 109 a Shanting District Jiguan Gu 117°36′46″ 34°58′37″ 300 Southwest 113 a Shanting District Baodu Gu 117°42′54″ 34°59′11″ 330 Northwest 115 a Shanting District Baodu Gu 117°42′55″ 34°59′08″ 340 Southwest 118 b Yicheng District Qingtan Temple 117°34′23″ 34°57′39″ 410 East 135 c Shizhong District Guishan Forest Farm 117°40′51″ 34°46′43″ 200 Northeast 151 d Tengzhou City East Mountain 117°23′13″ 34°55′51″ 120 East 155 d Tengzhou City Hutou Mountain 117°16′44″ 34°53′28″ 120 Northwest 166 d Tengzhou City Mushi Forest Farm 117°16′59″ 34°58′08″ 100 South 183 d Tengzhou City Hulutao 117°16′34″ 34°52′58″ 80 Northeast 200 a Shanting District Beiyu 117°25′53″ 35°08′46″ 360 Northeast 3.2 Experimental methods 3.2.1 Extraction and quality assessment of total plant DNA Genomic DNA was extracted from the leaves of 100 Platycladus orientalis samples using a modified cetyltrimethylammonium bromide (CTAB) method. After the extracted DNA was completely dissolved, its quality was assessed by electrophoresis on 2% agarose gels. 3.2.2 Primer screening In this study, SSR primers of Platycladus orientalis were developed based on the identification of SSR loci and primer screening from P. orientalis transcriptome sequences, and a total of 45 pairs of SSR primers were initially selected. Prior to large-scale PCR amplification and sequencing of all individuals, eight individuals were randomly selected for preliminary screening of the 45 microsatellite primers (Figure 5). Using the annealing temperature of 58 °C reported in the literature as a reference, PCR products were examined by 3% agarose gel electrophoresis and capillary sequencing. Primers with poor amplification efficiency or unclear banding patterns were discarded. Ultimately, seven pairs of SSR primers that produced clear gel electrophoresis profiles, could be stably amplified in all populations, showed satisfactory performance, and were easy to score and statistically analyze were selected for subsequent analyses. 3.2.3 SSR analysis In this study, seven pairs of polymorphic primers with clear and well-resolved DNA bands were selected (Table 6). Detailed information on the PCR reaction system (Table 7) and amplification program is provided below.
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