Measuring the change in deleterious mutation burden of barley germplasm conserved over one regeneration cycle.

Deleterious mutations will occur and accumulate in germplasm conserved under long-term cold storage. However, little is known about the extent of deleterious mutation accumulation in conserved germplasm. This study represents an attempt to measure the change in deleterious mutation burden of barley germplasm conserved over 1 regeneration cycle. Specifically, seed-based RNA-Seq analysis was performed on paired samples of 90 barley accessions representing germplasm conserved under cold storage since its original acquisition (G0) and since the first regeneration (G1). The analysis identified 1,642 deleterious variants by 2 SNP calling methods. These deleterious variants were widely distributed across 7 chromosomes, were present in only 8 or fewer samples, occurred predominantly in heterozygotes, were associated with diverse biological processes, and were largely predicted to be weakly detrimental. Based on BCFtools-based SNP calls, the change in total mutation burden per deleterious locus per conservation year was estimated at 0.00123, with a standard deviation of 0.00084, for an accession conserved through the first regeneration. This estimate suggests that a conserved barley accession accumulated 28 to 37 deleterious variants over the G0 to G1 period. More deleterious variants were found in G1 than in G0 samples; variants unique to each sample type were also observed; and a high proportion of variants occurred in heterozygotes. These findings clearly demonstrated the technical and practical feasibility of measuring mutation accumulation in barley germplasm conserved in a genebank.