title: Rapid identification of lettuce seed germination mutants by bulked segregant analysis and whole genome sequencing.
abstract: Lettuce (Lactuca sativa) seeds exhibit thermoinhibition, or failure to complete germination when imbibed at warm temperatures. Chemical mutagenesis was employed to develop lettuce lines that exhibit germination thermotolerance. Two independent thermotolerant lettuce seed mutant lines, TG01 and TG10, were generated through ethyl methanesulfonate mutagenesis. Genetic and physiological analyses indicated that these two mutations were allelic and recessive. To identify the causal gene(s), we applied bulked segregant analysis by whole genome sequencing. For each mutant, bulked DNA samples of segregating thermotolerant (mutant) seeds were sequenced and analyzed for homozygous single-nucleotide polymorphisms. Two independent candidate mutations were identified at different physical positions in the zeaxanthin epoxidase gene (ABSCISIC ACID DEFICIENT 1/ZEAXANTHIN EPOXIDASE, or ABA1/ZEP) in TG01 and TG10. The mutation in TG01 caused an amino acid replacement, whereas the mutation in TG10 resulted in alternative mRNA splicing. Endogenous abscisic acid contents were reduced in both mutants, and expression of the ABA1 gene from wild-type lettuce under its own promoter fully complemented the TG01 mutant. Conventional genetic mapping confirmed that the causal mutations were located near the ZEP/ABA1 gene, but the bulked segregant whole genome sequencing approach more efficiently identified the specific gene responsible for the phenotype.
authors.affiliation: Seed Biotechnology Center, Department of Plant Sciences, University of California, Davis, CA, 95616, USA.
authors.forename: Heqiang
authors.initials: H
authors.lastname: Huo
authors.affiliation: Department of Plant Biology and Genome Center, University of California, Davis, CA, 95616, USA.
authors.forename: Isabelle M
authors.initials: IM
authors.lastname: Henry
authors.affiliation: Rijk Zwaan Breeding B.V., 2678, ZG De Lier, The Netherlands.
authors.forename: Eric R
authors.initials: ER
authors.lastname: Coppoolse
authors.affiliation: Rijk Zwaan Breeding B.V., 2678, ZG De Lier, The Netherlands.
authors.forename: Miriam
authors.initials: M
authors.lastname: Verhoef-Post
authors.affiliation: Rijk Zwaan Breeding B.V., 2678, ZG De Lier, The Netherlands.
authors.forename: Johan W
authors.initials: JW
authors.lastname: Schut
authors.affiliation: Rijk Zwaan Breeding B.V., 2678, ZG De Lier, The Netherlands.
authors.forename: Han
authors.initials: H
authors.lastname: de Rooij
authors.affiliation: Rijk Zwaan Breeding B.V., 2678, ZG De Lier, The Netherlands.
authors.forename: Aat
authors.initials: A
authors.lastname: Vogelaar
authors.affiliation: Rijk Zwaan Breeding B.V., 2678, ZG De Lier, The Netherlands.
authors.forename: Ronny V L
authors.initials: RV
authors.lastname: Joosen
authors.affiliation: Rijk Zwaan Breeding B.V., 2678, ZG De Lier, The Netherlands.
authors.forename: Leo
authors.initials: L
authors.lastname: Woudenberg
authors.affiliation: Department of Plant Biology and Genome Center, University of California, Davis, CA, 95616, USA.
authors.forename: Luca
authors.initials: L
authors.lastname: Comai
authors.affiliation: Seed Biotechnology Center, Department of Plant Sciences, University of California, Davis, CA, 95616, USA.
authors.forename: Kent J
authors.initials: KJ
authors.lastname: Bradford
mesh_terms: D000040:Abscisic Acid / Q000378:metabolism; D018745:Genome, Plant / Q000235:genetics; D018525:Germination / Q000235:genetics / Q000502:physiology*; D018545:Lactuca / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D012639:Seeds / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*
chemical_list: D000040:Abscisic Acid
keywords: bulked segregant analysis; ethyl methanesulfonate mutation; lettuce (Lactuca sativa); seed; thermoinhibition; whole genome sequencing