Phenotypic changes and DNA methylation status in cryopreserved seeds of cereal rye (Secale cereale L.)

Authors: LU, JIA - Colorado State University; Greene, Stephanie; REID, SCOTT - Colorado State University; CRUZ, VON MARK - Bridgestone Americas Tire Operations; DIERIG, DAVID - Bridgestone Americas Tire Operations

Submitted to: Cryobiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/26/2018
Publication Date: 4/30/2018
Citation: Lu, J., Greene, S.L., Reid, S., Cruz, V.V., Dierig, D. 2018. Phenotypic changes and DNA methylation status in cryopreserved seeds of cereal rye (Secale cereale L.). Cryobiology. 82:8-14. https://doi.org/10.1016/j.cryobiol.2018.04.015.
DOI: https://doi.org/10.1016/j.cryobiol.2018.04.015

Interpretive Summary: Conserving genetic diversity is a major priority of the National Laboratory for Genetic Resources Preservation (NLGRP), operated by the U.S. Department of Agriculture, Agricultural Research Service. There are two long-term preservation methods employed in the NLGRP:storage in a -18 °C freezer (conventional storage) and storage in liquid nitrogen vapor phase at -135 to -180 oC (cryopreservation). To test the phenotypic and epigenetic effects of long-term cryopreservation of orthodox seeds, we evaluated 40 cereal rye accessions (20 spring habit and 20 winter habit) stored for 25 years under both conventional storage and cryogenic conditions. In field evaluations we found that spikes of plants grown from conventionally stored seeds were slightly longer than those from cryopreserved seeds. In laboratory evaluations, seeds from cryopreserved samples had significantly higher normal germination percentage. No differences in methylation were detected between storage treatments on an individual locus basis. Our study indicated that cryopreservation slowed seed deterioration as evidenced by higher germination rates compared to conventional storage, had only minimal effects on other phenotypic traits, and had no significant effects on DNA methylation status.

Technical Abstract: Conserving genetic diversity is a major priority of the National Laboratory for Genetic Resources Preservation (NLGRP), operated by the U.S. Department of Agriculture, Agricultural Research Service. There are two long-term preservation methods employed in the NLGRP:storage in a -18 °C freezer (conventional storage) and storage in liquid nitrogen vapor phase at -135 to -180 oC (cryopreservation). To test the phenotypic and epigenetic effects of long-term cryopreservation of orthodox seeds, we evaluated 40 cereal rye accessions (20 spring habit and 20 winter habit) stored for 25 years under both conventional storage and cryogenic conditions. In field evaluations conducted in Fort Collins, Colorado in 2014-15, spikes of plants grown from conventionally stored seeds of the winter accessions were slightly longer than those from cryopreserved seeds (P=0.045). In laboratory evaluations, seeds from cryopreserved samples had significantly higher normal germination percentage (P<0.01) and lower abnormal germination percentage (P<0.01) than those stored under conventional conditions for winter habit rye and higher normal germination percentage (P<0.01) for spring habit rye. In addition, seedlings from cryopreserved seeds had longer roots and smaller root diameter (P<0.05) than seedlings from conventionally stored seeds in winter habit rye. To detect DNA methylation changes, a methylation-sensitive amplified fragment length polymorphism (metAFLP) technique was applied to two accessions. After false discovery rate adjustment, no differences in methylation were detected between storage treatments on an individual locus basis. Our study indicated that cryopreservation slowed seed deterioration as evidenced by higher germination rates compared to conventional storage, had only minimal effects on other phenotypic traits, and had no significant effects on DNA methylation status.