A prolific and robust whole‐genome genotyping method using PCR amplification via primer‐template mismatched annealing. Issue 3 (13th January 2023)

• Record Type: Journal Article
• Authors: Zhao, Sheng; Zhang, Cuicui; Wang, Liqun; Luo, Minxuan; Zhang, Peng; Wang, Yue; Malik, Waqar Afzal; Wang, Yue; Chen, Peng; Qiu, Xianjin; Wang, Chongrong; Lu, Hong; Xiang, Yong; Liu, Yuwen; Ruan, Jue; Qian, Qian; Zhi, Haijian; Chang, Yuxiao
• Is Part Of: Journal of integrative plant biology. Volume 65:Issue 3(2023)
• Page Start: 633
• Page End: 645
• Publication Date: 2023-01-13
• Subjects: background selection -- foreground genotyping -- primer‐template mismatched annealing -- marker‐assisted breeding -- whole‐genome genotyping; Plants -- Periodicals; Plants -- China -- Periodicals; Electronic journals; 580.5
• DOI: 10.1111/jipb.13395
• Languages: English
• ISSNs: 1672-9072

Abstract

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. Abstract : An accidental discovery during PCR generated abundant stable PCR products through non‐specific amplification, which resulted in the development of foreground and background integrated genotyping by sequencing (FBI‐seq) using a single specific primer, wherein the foreground is genotyped by primer's specific amplification, while background by non‐specific.