J Integr Plant Biol ›› 2023, Vol. 65 ›› Issue (3): 633-645.DOI: 10.1111/jipb.13395

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  • 收稿日期:2022-08-02 接受日期:2022-10-21 出版日期:2023-03-01 发布日期:2023-03-15

A prolific and robust whole-genome genotyping method using PCR amplification via primer-template mismatched annealing

Sheng Zhao1,2†, Cuicui Zhang2†, Liqun Wang1†, Minxuan Luo1,2, Peng Zhang2, Yue Wang2, Waqar Afzal Malik2, Yue Wang2, Peng Chen2, Xianjin Qiu3, Chongrong Wang4, Hong Lu2, Yong Xiang2, Yuwen Liu2, Jue Ruan2, Qian Qian2*, Haijian Zhi1* and Yuxiao Chang2*   

  1. 1. National Key Laboratory for Crop Genetics and Germplasm Enhancement, National Center for Soybean Improvement, Nanjing Agricultural University, Nanjing 210095, China;
    2. Guangdong Laboratory of Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518120, China;
    3. College of Agriculture, Yangtze University, Jingzhou 434023, China;
    4. Guangdong Provincial Key Laboratory of New Technology in Rice Breeding, Rice Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
    †These authors contributed equally to this work.
    *Correspondences: Haijian Zhi (zhj@njau.edu.cn); Yuxiao Chang (changyuxiao@caas.cn, Dr. Chang is fully responsible for the distribution of the materials associated with this article); Qian Qian (qianqian188@hotmail.com)
  • Received:2022-08-02 Accepted:2022-10-21 Online:2023-03-01 Published:2023-03-15

Abstract: Whole-genome genotyping methods are important for breeding. However, it has been challenging to develop a robust method for simultaneous foreground and background genotyping that can easily be adapted to different genes and species. In our study, we accidently discovered that in adapter ligation-mediated PCR, the amplification by primer-template mismatched annealing (PTMA) along the genome could generate thousands of stable PCR products. Based on this observation, we consequently developed a novel method for simultaneous foreground and background integrated genotyping by sequencing (FBI-seq) using one specific primer, in which foreground genotyping is performed by primer-template perfect annealing (PTPA), while background genotyping employs PTMA. Unlike DNA arrays, multiple PCR, or genome target enrichments, FBI-seq requires little preliminary work for primer design and synthesis, and it is easily adaptable to different foreground genes and species. FBI-seq therefore provides a prolific, robust, and accurate method for simultaneous foreground and background genotyping to facilitate breeding in the post-genomics era.

Key words: background selection, foreground genotyping, primer-template mismatched annealing, marker-assisted breeding, whole-genome genotyping

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