Identification of Seed Storage Protein Genes Repressors in Arabidopsis Seedlings Using Next-Generation Mapping

Seed maturation is a highly regulated developmental phase when the embryos accumulate high quantities of storage compounds, including seed storage proteins (SSPs). Genes encoding SSPs are specifically and highly expressed in the seed during maturation. However, the mechanisms that repress the expression of these genes in vegetative tissues are not well understood. To identify the repressors of SSP in vegetative organs, transgenic Arabidopsis plants expressing the GUS reporter gene under the control of a SSP gene promoter were generated. Homozygous transgenic plants were mutagenized and the progeny of mutated plants were screened for individuals displaying ectopic expression of seed storage protein genes (essp). Two mutant lines, essp7 and essp8, were used in this study. Genetic analysis revealed that essp7 and essp8 mutations are both recessive. Bulked-segregant analysis with simple sequence length polymorphism (SSLP) markers, located the mutations on chromosome 1 and chromosome 3 in essp7, and on chromosome 2 in essp8. Next-generation mapping (NGM) was used to detect the genes responsible for the observed mutant phenotypes in the mapped regions. essp7 and essp8 plants were sequenced using 100nt paired-end sequencing on the Illumina HiSeq and 300nt paired-end sequencing on the Illumina MiSeq, respectively. The NGM results were consistent with prior mapping results for both mutants. All non-synonymous mutations identified within the candidate regions were detected and used for further validation. NGM could provide high-resolution genotypic information that can be used as a complement for traditional map-based cloning by direct identification of mutations in a fast and cost-effective way.