Construction And Application Of Accurate Identification System Of Osmanthus Fragrans Varieties Based On Whole-Genome SNP

Osmanthus fragrans is an economically and ornamentally important tree species in the Oleaceae family.The long history of artificial selection and domestication in O.fragrans resulted in abundant intraspecific variation and cultivated accessions.The technical and scientific basis for variety protection is the testing of new plant varieties’ specificity,consistency and stability.DUS testing based on morphological characteristics is time-consuming,laborious and susceptible to environmental influences.Single nucleotide polymorphisms(SNPs)have been widely used as a molecular marker in crop species with advances in next-generation sequencing technology.The use of SNP markers for variety identification is a cost-effective strategy to protect breeder’s intellectual property rights.However,until now,the application of SNP markers and high-throughput genotyping techniques in the identification and evaluation of O.fragrans varieties is still lack.In this study,based on the extensive collection of mainstream O.fragrans varieties in the Chinese market,genetic variation identification and population genetic analysis were conducted using resequencing technology and whole-genome SNP analysis to explore the origin and evolution of cultivated varieties.Uniformly distributed and highly polymorphic loci were selected for conversion into KASP(Kompetitive Allele Specific PCR)markers.Core SNP loci were screened,and high-throughput genotyping of 85 O.fragrans varieties was performed using the KASP technology platform to construct a high-resolution DNA fingerprint pattern and molecular ID for these varieties.The main results of this study are as follows:1)Based on a wide sampling of commercial O.fragrans varieties in China,we re-sequenced the whole genomes of 121 cultivars and 3 related species in Osmanthus,and obtained 1,599,694 high-quality SNPS,of which 22.89% were located in the gene coding region,including 184,998 non-synonymous mutations and 178,946 synonymous mutations.Based on genome-wide SNPs,we performed population genetic structure and LD decay analysis,which indicated that ‘Yingui group’ and Jingui group’ is the more original than ‘Dangui group’ and ‘Sijigui group’;‘Sijigui group’ are probably artificially domesticated species rather than wild species.In the process of variety breeding,flower color,flowering time and leaf color were subjected to strong artificial selection pressure.2)Then,585 SNPS were selected from the genome wide SNPs for KASP maker development,and a subset of 143 markers with clear and stable clustering patterns were retained as core SNP markers for variety identification.The core markers are highly polymorphic(91.61% with MAF > 0.20 and 86.71% with PIC > 0.30),and were effective in differentiating all the 145 varieties we genotyped.Finally,we constructed DNA fingerprints for O.fragrans varieties based on the 143 core SNP markers.The fingerprint patterns were validated using a mixed pool population consisting of 15 known varieties and 20 new varieties,with three replicates each.The genotyping results of three samples from the same variety were identical,indicating consistent results within the same variety.The genotyping results of the 15 known varieties matched perfectly with the corresponding varieties in the fingerprint database.Among the 20 new varieties,19 had no corresponding variety in the fingerprint library,except for ’Huihuang’,which had the same genotype as ’Bo Ye Jin Gui’ in the fingerprint library.The identification system achieved a discrimination efficiency of 95% for new varieties,demonstrating good repeatability and stability.The genetic similarity coefficients ranged from 0.441 to 1.000,with an average of0.616.A genetic similarity threshold of > 0.993 was proposed,indicating that varieties with a difference in loci <2 can be considered similar.This threshold can be used to assist in the screening of similar O.fragrans varieties and the identification of new varieties in conjunction with DUS testing.Furthermore,when the number of core markers was reduced to 71 and 36,the identification efficiencies were 97.58% and93.33%,respectively,indicating a high level of identification.3)Based on the principle of using the fewest markers to differentiate the most varieties,14 SNPs(PIC: 0.246~0.375;MAF: 0.174~0.489;HE: 0.288~0.500)were selected from the 143 core SNP markers,enabling 100% identification of 89 O.fragrans varieties.The genotype data of the 89 varieties at the 14 selected SNPs were transformed into numerical codes,resulting in the construction of fingerprint codes for the 89 O.fragrans varieties.Four morphological traits of industrial significance(flower color,fragrance,flower yield,and fruit set)were investigated to construct commodity codes.By combining the fingerprint codes with the commodity codes,cultivar IDs for the 89 O.fragrans varieties were generated using barcode generation software.This provides an effective approach for the simplified management and protection of germplasm resources and promotes the rapid and intelligent querying of varieties.