*2.4. Primer Selection*

After Tandem repeat detection, a total of 13,264 SSRs were obtained. Then, after lobSTR processing, a total of 145 high quality SSRs and ranges were finally selected. Based on the predicted results of the SSRs, 12 pairs of specific primers were selected from the developed primer sequences, namely primer pairs Cun463, Cun378, Cun500, Cun626, Cun672, Cun586, Cun148, Cun752, Cun230, Cun27, Cun485, and Cun484 following the principle of high polymorphism at the loci. Each primer pair consisted of one forward primer and one reverse primer. The dosage ratio of the forward and reverse primers was 1:4 [27]. Furthermore, two fluorescently labeled universal primers M13 and PQE-F were included: the fluorescent marker that is compatible with the universal primer M13 is 5-FAM, and the fluorescent marker that is compatible with the universal primer PQE-F

is 5-HEX [28]. The 12 primer pairs were divided into two groups: G1 group primers, including Cun463, Cun378, Cun500, Cun626, Cun672, Cun586, and Cun148, and G2 group primers, including primer pairs Cun752, Cun230, Cun27, Cun485, and Cun484. Primers were synthesized by Beijing Rui Bo Xing Ke Biotechnology Co. Site names and primer sequences are listed, and the generic primer names and primer sequence information are shown in Table 1.

**Table 1.** Information about site combination and primer concentration of Multiplex PCR1 and Multiplex PCR2.


#### *2.5. Establishment of a Microsatellite Multiplex PCR Amplification System*

The reaction volume for multiplex PCR in both G1 and G2 groups was 25 μL. Specific information is shown in Table 2. For PCR amplification, the amplification procedure used was as follows: 98 ◦C for 10 s, 57 ◦C for 40 s, 72 ◦C for 60 s, 35 cycles; 98 ◦C for 10 s, 53 ◦C for 40 s, 72 ◦C for 60 s, 15 cycles; and 72 ◦C for 30 min for extension [26]. After PCR, 5 μL was taken on an agarose gel to detect bright bands of the desired size. Gel electrophoresis was used to confirm that the primers amplified the target bands. The remaining samples were sent to a commercial company (Beijing Ruibio BioTech Co., Ltd., Beijing, China) for genotyping using an ABI 3730XL sequencer.

**Table 2.** Group G1 and Group G2 multiplex PCR reaction system.



**Table 2.** *Cont.*

#### *2.6. Polymorphism Analysis*

The amplification products were subjected to capillary electrophoresis usingABI3730XL. Peak types were converted to alleles using GeneMarker (version 2.2.2.0, SoftGenetics, Pennsylvania, USA) [29]. Genotype data were counted using GenAlex (version 6.5, ANU, Canberra, Australia) [30] and population genetics parameters were calculated including the number of individuals (N), number of different alleles (Na), number of effective alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), unbiased expected heterozygosity(uHe), fixation index (F) and Hardy–Weinberg equilibrium (HW). The null allele frequency (F(null)) and polymorphism information content (PIC) were calculated using the software Cervus (version 3.0.7, Field Genetics, London, UK).
