关键词: 多花黑麦草, EST-SSR分子标记, 指纹图谱, 荧光检测, 毛细管电泳

Abstract: 【Objective】In this study, a Italian ryegrass (Lolium multiflorum Lam.) variety identification system based on fluorescently labeled ETS-SSR markers was developed to provide a high-throughput DNA profiling means for identification of Italian ryegrass varieties, which can provide valuable information for the use of Italian ryegrass production and an effective method of protecting farmers’ benefits and breeders’ rights.【Method】Using three Italian ryegrass varieties (Tetragold, ChangjiangNo.2 and Aubade) with high difference of phenotypic traits, 30 primers were screened from the original 200 EST-SSR primers by polyacrylamide gel electrophoresis, which had clear amplification bands, rich polymorphism and stable amplification. Markers selected were labeled at the 5′ end of forward primer using fluorescent tags FAM, DNA analyzer was employed to detect different allelic variations of 200 individual PCR-amplified fragments by capillary electrophoresis. After further screening, 25 out of 30 fluorescent markers were chosen based on stable amplification to construct a high throughput identification system for Lolium multiflorum L..【Result】DNA fingerprint of ten Italian ryegrass materials were constructed using 25 EST-SSR primers for variety identification. A total of 127 alleles were amplified by 25 primer pairs, and the amplification fragment length ranged from 51 to 249. The effective number of alleles ranged from 2 to 11 on each pair of primers among them, primer N101 with 11 specific alleles was the most of all primers and each pair of primers had 4.00 specific alleles on average. The ratio of polymorphism sites ranged from 33.33% to 100.00%. The average value of polymorphic information content (PIC) was 0.702, the largest Shannon’s value (I) was 3.322 (N101) and the average value was 1.929, the Nei’s genetic diversity (H) ranged from 0.159 to 0.500 and the average value was 0.318. The amount of identified materials was from 0 to 10, and 14 of 25 primer pairs had 25 specific amplification sites among ten varieties (strains). Taken together, the results showed that the N101 fluorescence primer had the highest identification capability, which can identify these varieties (strains) directly. In addition, specific alleles were detected in primer ‘N101’ in three varieties such as Changjiang No.2, Ganxuan No.1 and Chuannong No.2. But due to high variation exists in same variety and different varieties of Lolium multiflorum L., in order to identify other materials, 6 EST-SSR primers (N54, N101, N146, N151, N154, N156) with good amplification and detection effect were chosen from 25 primers, the numbers of stable alleles were no less than 19, and the varieties “Double Barrel” and “Abundant” with 22 alleles were the most of all materials. Six high-efficiency primers were used for construction of DNA fingerprint spectrum including the standard model, fingerprint code and QR encodes of fingerprint spectrum. In this study, the fingerprint code and the unique QR encode for ten varieties of Italian ryegrass were firstly obtained using EST-SSR molecular marker with the aid of capillary electrophoresis and fluorescence labeling technology.【Conclusion】In this study, a SSR high-throughput identification system was constructed according to six pairs high efficiency primers, in which the ‘N101’ fluorescence primer having the most polymorphism can directly apply to identification of 10 Italian ryegrass varieties (strains).

Key words: Italian ryegrass (Lolium multiflorum Lam.), EST-SSR marker, fingerprinting, fluorescence detection, capillary electrophoresis