DNA markers based on retrotransposon insertion polymorphisms can detect short DNA fragments for strawberry cultivar identification.

In this study, DNA markers were developed for discrimination of strawberry (Fragaria × ananassa L.) cultivars based on retrotransposon insertion polymorphisms. We performed a comprehensive genomic search to identify retrotransposon insertion sites and subsequently selected one retrotransposon family, designated CL3, which provided reliable discrimination among strawberry cultivars. Through analyses of 75 strawberry cultivars, we developed eight cultivar-specific markers based on CL3 retrotransposon insertion sites. Used in combination with 10 additional polymorphic markers, we differentiated 35 strawberry cultivars commonly cultivated in Japan. In addition, we demonstrated that the retrotransposon-based markers were effective for PCR detection of DNA extracted from processed food materials, whereas a SSR marker was ineffective. These results indicated that the retrotransposon-based markers are useful for cultivar discrimination for processed food products, such as jams, in which DNA may be fragmented or degraded.

Efficient screening of long terminal repeat retrotransposons that show high insertion polymorphism via high-throughput sequencing of the primer binding site.

Retrotransposons have been used frequently for the development of molecular markers by using their insertion polymorphisms among cultivars, because multiple copies of these elements are dispersed throughout the genome and inserted copies are inherited genetically. Although a large number of long terminal repeat (LTR) retrotransposon families exist in the higher eukaryotic genomes, the identification of families that show high insertion polymorphism has been challenging. Here, we performed an efficient screening of these retrotransposon families using an Illumina HiSeq2000 sequencing platform with comprehensive LTR library construction based on the primer binding site (PBS), which is located adjacent to the 5' LTR and has a motif that is universal and conserved among LTR retrotransposon families. The paired-end sequencing library of the fragments containing a large number of LTR sequences and their insertion sites was sequenced for seven strawberry (Fragaria × ananassa Duchesne) cultivars and one diploid wild species (Fragaria vesca L.). Among them, we screened 24 families with a "unique" insertion site that appeared only in one cultivar and not in any others, assuming that this type of insertion should have occurred quite recently. Finally, we confirmed experimentally the selected LTR families showed high insertion polymorphisms among closely related cultivars.

A GAL4-based targeted activation tagging system in Arabidopsis thaliana.

Activation tagging is a powerful tool for discovering novel genes that are not easily identified by loss-of-function (lof) screening due to genetic redundancy or lethality. Although the current activation tagging system, which involves a viral enhancer sequence, has been used for a decade, alternative methods that allow organ- or tissue-specific activation are required to identify genes whose strong activation leads to loss of fertility or viability. Here, we established a GAL4/UAS activation-tagging system in Arabidopsis thaliana. Host plants that express a synthetic transcription activator GAL4:VP16 (GV) in an organ- or tissue-specific manner were transformed with a T-DNA harboring tandem copies of UAS, a GAL4-binding sequence. Using a post-embryonic and root-specific GV-expressing line as the host plant, we isolated several dominant mutants with abnormal root tissue patterns, designated as uas-tagged root patterning (urp) mutants, and identified their causal genes. Notably, most URP genes encoded putative transcription factors, indicating that the GAL4/UAS activation tagging system effectively identifies genes with regulatory functions. lof phenotypes of most URP genes were either local patterning defects or visible only if homologous genes were disrupted simultaneously or independently. Systemic overexpression of some URP genes resulted in seedling lethality. These results indicate that GAL4/UAS activation tagging is a powerful method for identifying genes with biological functions that are not readily identified by conventional screening methods.

The Arabidopsis RWP-RK protein RKD4 triggers gene expression and pattern formation in early embryogenesis.

Morphogenesis of seed plants commences with highly stereotypical cell division sequences in early embryogenesis [1, 2]. Although a small number of transcription factors and a mitogen-activated protein (MAP) kinase cascade have been implicated in this process [3-8], pattern formation in early embryogenesis remains poorly understood. We show here that the Arabidopsis RKD4, a member of the RWP-RK motif-containing putative transcription factors [9], is required for this process. Loss-of-function rkd4 mutants were defective in zygotic cell elongation, as well as subsequent cell division patterns. As expected from this mutant phenotype, RKD4 was transcribed preferentially in early embryos. RKD4 possessed functional characteristics of transcription factors and was able to ectopically induce early embryo-specific genes when overexpressed in seedlings. Strikingly, induced overexpression of RKD4 primed somatic cells for embryogenesis independently of external growth regulators. These results reveal that RKD4 is a novel key regulator of the earliest stage of plant development.