关键词: 陆地棉, DUS测试, SSR标记, 近似品种筛选, 遗传相似度阈值

Abstract:

【Objective】DUS (Distinctness, Uniformity, and Stability) testing provides important technical data for cotton variety approval and intellectual property protection, while the selection of similar varieties is a critical step in the process. Based on the revision of “NY/T 2469-2013, Protocol for the identification of cotton variety-SSR marker method”, this study aims further to construct a DNA fingerprint database for known upland cotton varieties for DUS testing in China and determine a genetic similarity threshold for rigorous and precise similar variety selection. 【Method】 Firstly, preliminary screening and subsequent re-screening were conducted on both the SSR primers from NY/T 2469-2013, and newly collected ones to identify a set of primers with stable PCR amplification, high polymorphism and clear peak patterns, replacing the original SSR primer set in the standard. Secondly, the selected SSR primers were employed to genotype the known cotton varieties to construct a DNA fingerprint database for those varieties. Finally, relationships between phenotypic and genetic differences of cotton varieties were investigated and a genetic similarity threshold for similar variety selection was determined. 【Result】 42 pairs of SSR primers covering all 26 chromosomes of upland cotton were selected, including 12 markers from the original standard. Among the selected 42 pairs of primers, 25 pairs amplified two loci, of which 23 revealed only one polymorphic locus, while the remaining two primer pairs (NAU1167 and HAU1413) had both loci showing polymorphism. A total of 164 alleles were detected from the 44 polymorphic loci, with the number of alleles at each locus ranging from 2 to 7. The polymorphism information content (PIC) of the loci varied from 0.15 to 0.66. 2 100 cotton varieties were genotyped using these primers. A DNA fingerprint database of known cotton varieties was constructed with a data integrity of 98.85%. Pairwise comparisons using the DNA data of 648 protected varieties, 843 approved varieties and 2 100 known upland cotton varieties revealed the distribution patterns of genetic differences between varieties among the three categories. More than 90.00% of comparisons in each category were concentrated in the range of 40.00%-70.00%. The proportion of variety pairs with genetic similarity exceeding 80.00% was 0.28%, 0.31% and 0.31% for the protected, approved and known varieties, respectively. Relationship analysis was conducted between phenotypic and genetic differences for 177 upland cotton varieties and their corresponding similar varieties. It was found that for the 54 cotton applications to which similar varieties with genetic similarity exceeding 90.00% existed, 18 (33.33%) failed to meet the distinctness requirement. In contrast, all 123 cotton applications with genetic similarity to their similar varieties below 90.00% could be clearly distinguished, exhibiting clear trait differences, thus fulfilling the distinctness criteria. The genetic similarity threshold for similar variety selection in upland cotton DUS testing based on these SSR markers could be set at 90.00%. For the variety pairs showing genetic similarity above this threshold, further field-based phenotypic evaluations are required. 【Conclusion】 In summary, NY/T 2469-2013, the standard for identifying upland cotton varieties using SSR markers, was revised. A DNA fingerprint database was constructed to support DUS testing of upland cotton in China. Furthermore, a genetic similarity threshold of 90.00% based on these SSR markers was established for selecting similar varieties, enhancing the precision and reliability of this process within DUS testing.

Key words: Upland cotton, DUS testing, SSR marker, similar variety selection, genetic similarity threshold