关键词: 望春玉兰, SSR, 引物开发, 玉兰商业品种, DNA指纹图

Abstract:

Objective: Genome-wide microsatellite sequence and distribution characteristics of Magnolia biondii were analyzed, and highly polymorphic and stable simple sequence repeat (SSR) primer pairs were developed. Furthermore, DNA fingerprinting profiles were constructed for important commercial Magnolia cultivars. Results of this study would provide molecular marker resources for population genetic structure and diversity analyses, and also provide technological support and scientific basis for identification of elite Magnolia cultivars, and also for the protection of intellectual property rights of breeders. Method: Based on the published whole genome sequence of M. biondii, the MISA program was used to characterize the microsatellites. The Primer Premier 5.0 software was used to design SSR primers, and 203 pairs of primers were randomly synthesized and screened using six M. biondii individual plants. The selected polymorphic primers were further used to construct the DNA fingerprinting profiles of 26 commercial Magnolia cultivars. Result: A total of 2 820 303 microsatellites were identified in the genome of M. biondii, among which the hexanucleotide repeats were most abundant (62.13%), followed by dinucleotide repeats (12.02%) and mononucleotide repeats (9.98%). Sequence analysis revealed 501 different repeat motifs, among which AAACCT/AGGTTT (16.46%) repeats were the most frequent, followed by A/T (8.63%) and AG/CT (5.9%). Among the 203 randomly synthesized primers, 94 (46.31%) primers were able to effectively amplify the genomic DNA of six M. biondii individuals, and 25 (12.32%) primers could generate a clear, easily scored and highly polymorphic banding pattern, with the polymorphism information content (PIC) values ranging from 0.24 to 0.72. The top 10 primers ranked by the PIC value were used to construct DNA fingerprinting profiles of 26 commercial varieties, and a total of 85 genotypes were obtained. The number of genotypes produced by each primer varied from 4 to 18. The discrimination power of each primer ranged from 0 to 13. The 26 commercial Magnolia cultivars could be distinguished by a subset of three primers. Conclusion: On the basis of microsatellite sequence statistics and analysis of the entire genome of M. biondii, in this study 10 SSR primer pairs have been developed with clear bands, high polymorphism and good reproducibility. These primers have been used to construct DNA fingerprint profiles for 26 commercial Magnolia cultivars. Comparison of the fingerprint profiles has revealed that there are no synonyms or homonyms within the 26 commercial Magnolia cultivars.

Key words: Magnolia biondii, SSR, primer development, Magnolia commercial cultivar, DNA fingerprint