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...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.展开更多
Whole-genome genotyping(WGG)stands as a pivotal element in genomic-assisted plant breeding.Nevertheless,sequencing-based approaches for WGG continue to be costly,primarily owing to the high expenses associated with li...Whole-genome genotyping(WGG)stands as a pivotal element in genomic-assisted plant breeding.Nevertheless,sequencing-based approaches for WGG continue to be costly,primarily owing to the high expenses associated with library preparation and the laborious protocol.During prior development of foreground and background integrated genotyping by sequencing(FBI-seq),we discovered that any sequence-specific primer(SP)inherently possesses the capability to amplify a massive array of stable and reproducible non-specific PCR products across the genome.Here,we further improved FBI-seq by replacing the adapter ligated by Tn5 transposase with an arbitrary degenerate(AD)primer.The protocol for the enhanced FBI-seq unexpectedly mirrors a simplified thermal asymmetric interlaced(TAIL)-PCR,a technique that is widely used for isolation of flanking sequences.However,the improved TAIL-PCR maximizes the primer-template mismatched annealing capabilities of both SP and AD primers.In addition,leveraging of next-generation sequencing enhances the ability of this technique to assay tens of thousands of genome-wide loci for any species.This cost-effective,user-friendly,and powerful WGG tool,which we have named TAIL-PCR by sequencing(TAIL-peq),holds great potential for widespread application in breeding programs,thereby facilitating genome-assisted crop improvement.展开更多
基金supported by the National Natural Science Foundation of China(31970379 and 32172086)the Jiangsu Collaborative Innovation Center for Modern Crop Production (JCIC-MCP)+3 种基金the National Key R&D Program of China (ZZ202001)the R&D program of Shenzhen (KCXFZ20211020164207012)the R&D program in key areas of Guangdong Province (2021B0707010006)the Science and Technology Planning Project of Guangdong Province (2022B0202060002)。
文摘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.
基金supported by the Science and Technology Planning Project of Guangdong Province(2022B0202060002)the National Natural Science Foundation of China(32300340,32172086)+2 种基金the R&D program of Shenzhen(KCXFZ20211020164207012)the R&D Program in Key Areas of Guangdong Province(2021B0707010006)ACKNOWLEDGMENTS We thank Lili Dong(China National Center for Bioinformation/Beijing Institute of Genomics,Chinese Academy of Sciences)for assistance with uploading the raw sequencing data.Y.C.,S.Z.,P.C.,et al.are listed as co-inventors on a patent application(CN202211418)。
文摘Whole-genome genotyping(WGG)stands as a pivotal element in genomic-assisted plant breeding.Nevertheless,sequencing-based approaches for WGG continue to be costly,primarily owing to the high expenses associated with library preparation and the laborious protocol.During prior development of foreground and background integrated genotyping by sequencing(FBI-seq),we discovered that any sequence-specific primer(SP)inherently possesses the capability to amplify a massive array of stable and reproducible non-specific PCR products across the genome.Here,we further improved FBI-seq by replacing the adapter ligated by Tn5 transposase with an arbitrary degenerate(AD)primer.The protocol for the enhanced FBI-seq unexpectedly mirrors a simplified thermal asymmetric interlaced(TAIL)-PCR,a technique that is widely used for isolation of flanking sequences.However,the improved TAIL-PCR maximizes the primer-template mismatched annealing capabilities of both SP and AD primers.In addition,leveraging of next-generation sequencing enhances the ability of this technique to assay tens of thousands of genome-wide loci for any species.This cost-effective,user-friendly,and powerful WGG tool,which we have named TAIL-PCR by sequencing(TAIL-peq),holds great potential for widespread application in breeding programs,thereby facilitating genome-assisted crop improvement.
