*2.4. High-Throughput Genotyping of SNPs in Intra-Specific Mapping Individuals*

In order to validate the *BoPur* QTL, obtained by QTL-seq, classical QTL mapping was performed, via the selection of the SNP, with a polymorphism between the genotypes of the parents (BT 126 and SN 60). Through the Hi-SNP method, a total of 33 SNPs, differentiating BT 126 and SN60, were utilized for validation and high-throughput genotyping in the F2 segregating population with 280 individuals and parents. The HI-SNP technology was developed by Shanghai BioWing Applied Biotechnology Company (http://www.biowing.com.cn/) (accessed on 9 June 2021) the genotyping of 33 SNPs was carried out by multiplex PCR with NGS on Illumina X-10 (Illumina, CA, USA) [41]. The genotyping primers were shown in the Supplementary Table S1. Based on the phenotypic and genotypic data, linkage maps were constructed by QTL IciMapMaker (Chinese Academy of Agricultural Sciences, Beijing, China) with a logarithm of the odds (LOD) value [42]. The LOD score is a measure of the strength of the evidence for the presence of a QTL at a particular location (the LOD score = log10 likelihood ratio, comparing single-QTL model to the "no QTL anywhere" mode).
