A Bioinformatic Workflow for InDel Analysis in the Wheat Multi-Copy α-Gliadin Gene Family Engineered with CRISPR/Cas9

Abstract

The α-gliadins of wheat, along with other gluten components, are responsible for bread viscoelastic properties. However, they are also related to human pathologies as celiac disease or non-celiac wheat sensitivity. CRISPR/Cas was successfully used to knockout α-gliadin genes in bread and durum wheat, therefore, obtaining low gluten wheat lines. Nevertheless, the mutation analysis of these genes is complex as they present multiple and high homology copies arranged in tandem in A, B, and D subgenomes. In this work, we present a bioinformatic pipeline based on NGS amplicon sequencing for the analysis of insertions and deletions (InDels) in α-gliadin genes targeted with two single guides RNA (sgRNA). This approach allows the identification of mutated amplicons and the analysis of InDels through comparison to the most similar wild type parental sequence. TMM normalization was performed for inter-sample comparisons; being able to study the abundance of each InDel throughout generations and observe the effects of the segregation of Cas9 coding sequence in different lines. The usefulness of the workflow is relevant to identify possible genomic rearrangements such as large deletions due to Cas9 cleavage activity. This pipeline enables a fast characterization of mutations in multiple samples for a multi-copy gene family.

Reduced Glyphosate Movement and Mutation of the EPSPS Gene (Pro106Ser) Endow Resistance in Conyza canadensis Harvested in Mexico

“…Copy number was calculated based on the ratio of EPSPS to ALS characterized as a single copy gene. Copy number was analyzed following the methodology described by Guzmán-López et al eq was used to obtain the efficiency for each target gene analyzed E = E normalC normalF normalX 100 + 1 where E CFX is the efficiency calculated by the CFX manager software ( E EPSPS = 2.016; E ALS = 2.089). Following eq , the ratios between the EPSPS and ALS gene were obtained normalr normala normalt normali normalo = N × false( E false( normalA normalL normalS false) false) normalM normalC normalq false( A L S false) E false( normalE normalP normalS normalP normalS false) normalM normalC normalq false( normalE normalP normalS normalP normalS false) where N is the number of copies per haploid genome for the ALS gene ( N = 1 ), E is the efficiency of the two genes analyzed, and MCq is the quantification of the average cycle.…”

“…Copy number was calculated based on the ratio of EPSPS to ALS characterized as a single copy gene. Copy number was analyzed following the methodology described by Guzmán-López et al eq was used to obtain the efficiency for each target gene analyzed where E CFX is the efficiency calculated by the CFX manager software ( E EPSPS = 2.016; E ALS = 2.089). Following eq , the ratios between the EPSPS and ALS gene were obtained where N is the number of copies per haploid genome for the ALS gene ( N = 1 ), E is the efficiency of the two genes analyzed, and MCq is the quantification of the average cycle.…”