| To establish a molecular identification system and DNA fingerprint database for hulless barley (Hordeum vulgare L. var. nudum Hook. f.) varieties in China, this study aimed to screen a set of SSR core primers suitable for national variety identification standards, thereby providing technical support for variety authentication, management, and new plant variety protection. Based on previously reported SSR markers of hulless barley, PCR amplification, marker screening, and validation were performed across diverse varieties, followed by analyses of genetic diversity and discrimination efficiency. Using 155 hulless barley accessions, 200 SSR primer pairs were subjected to polymorphism screening. Twenty-one core primers were selected based on criteria including clear and easily interpretable peak patterns, high polymorphism (mean PIC = 0.68), amplification stability, and even chromosomal distribution. Reference varieties (e.g., Yunqing4hao, Diqing4hao, and Kunlun8hao) were employed to validate the stability and reliability of these primers. The constructed DNA fingerprint database for 155 accessions detected 136 alleles, ranging from 2 to 10 per locus (mean = 6.48). The polymorphism information content (PIC) values varied between 0.39 and 0.85 (mean = 0.68), with 95.24% of loci classified as highly polymorphic (PIC > 0.5). These 21 primers achieved a discrimination efficiency of 99.991%, distinguishing 153 out of 155 accessions. Cluster analysis, population structure analysis, and principal component analysis classified the 155 accessions into three distinct groups, revealing rich genetic diversity and simple genetic backgrounds in 45% of the accessions. The SSR-based identification system developed in this study demonstrated high accuracy and practical applicability, enabling authenticity verification, auxiliary screening of approximate varieties in DUS testing, and intellectual property protection. Furthermore, the DNA fingerprint database and genetic diversity analysis provide valuable molecular-level insights for future hulless barley breeding programs. |
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