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

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 primertemplate 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 postgenomics era.

Genetic Diversity and Population Structure Analysis of Hollyhock (Alcea rosea Cavan) Using High-Throughput Sequencing

“…The extensive whole-genome SNP dataset coupled with the elucidation of distinct genetic structures holds immense value for the advancement of hollyhock breeding research. Furthermore, the advent of advanced whole-genome genotyping techniques, such as all-in-one sequencing [56] and simultaneous foreground and background integrated genotyping by sequencing [57], will further enhance the significance of our current study, marking a pivotal stride toward molecular marker-assisted breeding in hollyhock.…”

MultiPrime: A reliable and efficient tool for targeted next‐generation sequencing

“…This mechanism gives rise to primer‐template mismatch annealing—a dynamic that, although it may affect PCR efficiency, does not hinder the amplification process outright. Empirical support from a plethora of studies underscores the robustness of this approach, placing it on par with the outcomes attained through perfect primer‐template annealing [ 50 , 53 , 54 , 55 , 56 , 57 , 58 ], such as established amplification refractory mutation system PCR (ARMS‐PCR) [ 59 ]. In the dynamic landscape of nucleotide sequence analysis, multiPrime aims to strike a delicate balance between the specificity and efficiency of primers [ 60 ].…”