BacPhase: Long-insert paired-end sequencing for bin marker construction and genome phasing.

Area of Science:

• Genomics
• Plant genetics
• Bioinformatics

Background:

• Phasing, determining genetic variants on the same chromosome, is crucial for genome assembly but challenging in autopolyploid plants like potato due to high chromosome similarity.
• Traditional bacterial artificial chromosome (BAC)-based phasing methods are labor-intensive and require single colony selection, limiting their efficiency.
• Existing methods like Hi-C rely on chromatin interactions, which may not provide precise haplotype resolution.

Purpose of the Study:

• To develop an innovative, sequence-based method for efficient and high-resolution phasing in autopolyploid plants.
• To overcome the limitations of traditional BAC-based methods and improve genome assembly contiguity.
• To generate a large number of high-confidence bin markers for applications in plant genetics and breeding.

Main Methods:

• Developed BacPhase, a novel sequence-based approach involving BAC digestion, self-ligation, PCR amplification, and sequencing to generate bin markers.
• Evaluated 14 restriction enzymes to identify optimal ones for marker evenness and spacing across multiple crop species.
• Utilized PacBio HiFi long-read sequencing to span repetitive genomic regions and generate high-resolution bin markers.

Main Results:

• Generated 39,484 high-confidence, high-resolution bin markers in potato using the BacPhase method.
• BacPhase directly uses sequence polymorphisms for precise haplotype resolution, distinguishing it from chromatin interaction-based methods.
• Successfully anchored 59.58% of scaffolds to chromosomes in the autotetraploid potato cultivar C88, significantly improving genome assembly contiguity.

Conclusions:

• The BacPhase method offers a powerful, efficient, and precise approach for phasing and generating bin markers in complex polyploid genomes.
• This method substantially improves genome assembly contiguity without the need for physical maps or Hi-C data.
• BacPhase has broad applicability for trait mapping, genomic selection, and accelerating breeding programs in polyploid crops such as potato, sugarcane, and alfalfa.