南京林业大学学报(自然科学版) ›› 2024, Vol. 48 ›› Issue (4): 12-24.doi: 10.12302/j.issn.1000-2006.202405026

所属专题: 专题报道Ⅰ:郑万钧先生诞辰120周年纪念专题Ⅱ

• 专题报道Ⅰ:郑万钧先生诞辰120周年纪念专题Ⅱ(执行主编 曹福亮、尹佟明、李维林、方升佐) • 上一篇    下一篇

基于表型性状和SNP标记构建桂花主要品种资源的分子身份证

王一涵1(), 刘姣姣1, 金沛权1, 李书情1, 魏建芬2, 郭朋1, 尚富德1,*()   

  1. 1.河南农业大学生命科学学院,河南省桂花种质创新与资源利用工程研究中心, 河南 郑州 450046
    2.杭州桂花国家种质资源库, 浙江 杭州 310020
  • 收稿日期:2024-05-15 修回日期:2024-06-07 出版日期:2024-07-30 发布日期:2024-08-05
  • 通讯作者: *尚富德(shangfude@henau.edu.cn),教授。
  • 作者简介:

    王一涵(yihanwang@vip.163.com),副教授。

  • 基金资助:
    国家自然科学基金项目(32371911);河南省重大公益专项(201300110900)

Construction of molecular ID for Osmanthus fragrans cultivars based on phenotypic traits and single nucleotide polymorphisms (SNPs)

WANG Yihan1(), LIU Jiaojiao1, JIN Peiquan1, LI Shuqing1, WEI Jianfen2, GUO Peng1, SHANG Fude1,*()   

  1. 1. College of Life Sciences, Henan Agricultural University, Henan Engineering Research Center for Osmanthus Germplasm Innovation and Resource Utilization, Zhengzhou 450046, China
    2. National Osmanthus Germplasm Resource Repository, Hangzhou 310020, China
  • Received:2024-05-15 Revised:2024-06-07 Online:2024-07-30 Published:2024-08-05

摘要:

【目的】筛选核心单核苷酸多态性(single nucleotide polymorphism,SNP)位点,建立基于KASP平台的桂花(Osmanthus fragrans)品种基因型快速检测方法,构建品种特异性分子身份证,为桂花品种鉴定、溯源、知识产权保护等提供理论基础。【方法】实地调查主流桂花栽培品种的重要表型特征。通过两轮严格筛选,从基因组SNP中保留一组能够完全鉴别测序品种的最优SNP标记,计算SNP 位点的多态信息含量(PIC)、期望杂合度(He)等信息。以‘日香桂’(‘Rixianggui’)基因组为参考,设计桂花特异性KASP引物并进行批量扩增,根据基因分型结果建立品种指纹图谱,评估核心SNP标记的品种鉴别力。结合品种表型信息码和品种分子指纹码构建桂花品种资源的分子身份证。【结果】筛选出14个能够完全鉴别测序品种的核心SNP位点。各位点PIC值的变化范围为0.246~0.375,平均值为0.335;He的变化范围为 0.288~0.500,平均值为0.431。针对核心位点设计的KASP引物基因分型准确。根据扩增结果构建DNA指纹图谱,可区分全部包括未测序品种在内的90个参试品种。对品种表型特征赋值,结合指纹码构建由34位数字组成的桂花品种资源分子身份证。【结论】确定了SNP1—SNP14共14个核心SNP位点,能够实现至少90个桂花品种的有效鉴别。结合品种群类型、表型特征和分子指纹码构建了90个桂花品种的唯一分子身份证,并生成对应的条形码和二维码。

关键词: 桂花, 品种鉴别, 单核苷酸多态性, 表型性状, 分子身份证

Abstract:

【Objective】This study selected core genomic single-nucleotide polymorphism (SNP) loci to establish a rapid SNP genotyping method on the KASP platform, and to construct molecular IDs for Osmanthus fragrans cultivars. This study provides a theoretical foundation for identifying, tracing and protecting the intellectual property of O. fragrans cultivars.【Method】Field surveys were conducted to investigate key phenotypic characteristics of O. fragrans cultivars. Following two rounds of rigorous screening, we identified a set of core SNP markers capable of completely distinguishing previously sequenced cultivars. Subsequently, we analyzed the polymorphic information content (PIC) and expected heterozygosity (He) of each SNP locus. Using the genome sequences of ‘Rixianggui’ as a reference, species-specific KASP primers were designed for PCR amplification. Based on the genotyping results, we constructed cultivar DNA fingerprints and assessed the efficiency of core SNP markers for cultivar identification. Molecular IDs for O. fragrans cultivars were established by integrating phenotypic information codes with molecular fingerprint codes.【Result】We retained a total of 14 core SNP loci from genomic SNPs that fully discriminated the sequenced cultivars. The PIC values of these loci ranged from 0.246 to 0.375, with an average of 0.335, and the He indices ranged from 0.288 to 0.500, averaging 0.431. The KASP primers designed for these core SNP loci produced accurate genotyping results, enabling us to construct DNA fingerprints capable of distinguishing all 90 tested cultivars, including those not previously sequenced. Each cultivar was assigned a molecular ID composed of 34 digits.【Conclusion】In conclusion, 14 core SNP loci (SNP1 to SNP14) were identified that effectively discriminate among at least 90 O. fragrans cultivars. Unique molecular ID codes were constructed using DNA fingerprint codes along with serial codes derived from cultivar group types and phenotypic characteristics. Finally, barcode and quick response (QR) codes were generated for each cultivar.

Key words: Osmanthus fragrans, cultivar identification, single nucleotide polymorphism (SNP), phenotype, molecular ID

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