Mailloux, K. | Search bioRxiv

bioRxiv2024

Targeted genotyping-by-sequencing of potato and software for imputation

Mid-density targeted genotyping-by-sequencing (GBS) combines trait-specific markers with thousands of genomic markers at an attractive price for linkage mapping and genomic selection. A 2.5K targeted GBS assay for potato was developed using the DArTagTM technology and later expanded to 4K targets. Genomic markers were selected from the potato InfiniumTM SNP array to maximize genome coverage and polymorphism rates. The DArTag and SNP array platforms produced equivalent dendrograms in a test set of 298 tetraploid samples, and 83% of the common markers showed good quantitative agreement, with RMSE (root-mean-squared-error) less than 0.5. DArTag is suited for genomic selection candidates in the clonal evaluation trial, coupled with imputation to a higher density platform for the training population. Using the software polyBreedR, an R package for the manipulation and analysis of polyploid marker data, the RMSE for imputation by linkage analysis was 0.15 in a small half-diallel population (N=85), which was significantly lower than the RMSE of 0.42 with the Random Forest method. Regarding high-value traits, the DArTag markers for resistance to potato virus Y, golden cyst nematode, and potato wart appeared to track their targets successfully, as did multi-allelic markers for maturity and tuber shape. In summary, the potato DArTag assay is a transformative and publicly available technology for potato breeding and genetics. Core IdeasO_LIA mid-density, targeted genotyping-by-sequencing (GBS) assay was developed for potato. C_LIO_LIThe GBS assay includes markers for resistance to potato virus Y, golden cyst nematode, and potato wart. C_LIO_LIThe GBS assay includes multi-allelic markers for potato maturity and tuber shape. C_LIO_LIThe polyBreedR software has functions for manipulating and imputing polyploid marker data in Variant Call Format. C_LIO_LILinkage Analysis was more accurate than the Random Forest method when imputing from 2K to 10K markers. C_LI

genomics↗

bioRxiv2022

Nuclei isolation protocol from diverse angiosperm species

The ability to generate intact nuclei is crucial to the success of a variety of genomics experiments, such as Assay for Transposase-Accessible Chromatin using sequencing (ATAC- seq), Cleavage Under Targets and Tagmentation (CUT&Tag), and nuclei-based single cell sequencing (e.g., single nuclei ATAC-seq and single nuclei RNA-seq). For plants, the presence of the cell wall presents significant challenges in the isolation of nuclei from tissues. Here, we report an optimized nuclei isolation protocol that can be adapted for diverse angiosperm species, including maize, soybean, tomato, potato, and wheat, starting from fresh or frozen tissues. Nuclei release is achieved through chopping tissue on ice, where a key parameter affecting nuclei integrity is the concentration of detergent TritonX-100 in the nuclei isolation buffer. The method is simple, quick, and largely centrifugation-free, in which debris is removed by serial filtration. Initial nuclei release and filtration can be performed within 20 min. Fluorescence activated nuclei sorting is then used for final nuclei purification to remove other organelles such as plastids. The protocol uses 500 mg or less plant tissue as input and typically yields at least 100,000 - 200,000 purified nuclei per sample, a common input amount for downstream experiments. Throughout the protocol, we provide guidelines for optimization if performing nuclei isolation from a given species and tissue for the first time.

plant biology↗