Project description:Nucellar embryony is a form of apomixis found in citrus where somatic nucellar cells differentiate into embryos and are included in the seed resulting from the normal sexual process. The nucellar cells giving rise to adventive embryo start proliferating prior to anthesis and fully differentiate obtaining nourishment from sexually derived endosperm. To identify transcripts differentially expressed during nucellar embryo initiation we have taken RNA samplesfrom different developing stages of ovules from polyembryonic (cv. Vaniglia Sanguigno) and monoembryonic (cv. Temple) cultivars. We used microarray for a detailed analysis of global gene expression during nucellar embryony initiation and development. We have further validated the differentially expressed genes using qRT-PCR.
Project description:Nucellar embryony is a form of apomixis found in citrus where somatic nucellar cells differentiate into embryos and are included in the seed resulting from the normal sexual process. The nucellar cells giving rise to adventive embryo start proliferating prior to anthesis and fully differentiate obtaining nourishment from sexually derived endosperm. To identify transcripts differentially expressed during nucellar embryo initiation we have taken RNA samplesfrom different developing stages of ovules from polyembryonic (cv. Vaniglia Sanguigno) and monoembryonic (cv. Temple) cultivars. We used microarray for a detailed analysis of global gene expression during nucellar embryony initiation and development. We have further validated the differentially expressed genes using qRT-PCR. Citrus ovules were harvested from the excised ovaries of different flower developmental stages. These developing stages were decided on the basis of morphological and histological characteristics of flower and female gametophyte, respectively. We selected, Pre anthesis, post anthesis, and initial fruit set stage of flower to excise ovules from Polyembryonic cultivar Vaniglia Sanguigno and Monoembryonic cultivar Temple. Leaf tissue of Vaniglia Sanguigno was used to check the overall transcriptome level differences between leaf and ovule tissues. For each sample, three technical replicates were used.
Project description:We compared gene expression profiles of SFZ and deep AC of articular cartilage through laser microdissection (LMD) using adhesive tape, linear amplification of mRNA, and mRNA-seq analysis.
Project description:Seed developmental arrest is one of the early phenotypes of seed abortion. However, the molecular mechanism underlying seed developmental arrest of citrus is still unclear. In this study, laser capture microdissection (LCM) was used to accurately divide the seeds of seedless Ponkan ‘Huagan No.4’ (Citrus reticulata) (HG) and seeded Ponkan ‘Egan No.1’ (Citrus reticulata) (EG) into nucellus and integument/seed coat tissues. The captured tissues were used for subsequent RNA-seq. Moreover, single-molecule real-time (SMRT) sequencing was used to generate full-length transcripts of EG, which were used as reference transcripts for RNA-seq. These data can be utilized to analyse the causes of citrus seedlessness formation and the molecular regulatory network in the process of seed abortion.
Project description:We compared gene expression profiles of hypertrophic zone of WT and Adamts17-/- growth plates through laser microdissection (LMD) using adhesive tape, linear amplification of mRNA, and mRNA-seq analysis.
Project description:Somatic embryogenesis in nucellar tissues is widely recognized to induce polyembryony in major citrus varieties such as sweet oranges, satsuma mandarins and lemons. This capability for apomixis is attractive in agricultural production systems using hybrid seeds, and many studies have been performed to elucidate the molecular mechanisms of various types of apomixis. To identify the gene responsible for somatic embryogenesis in citrus, a custom oligo-DNA microarray including predicted genes in the citrus polyembryonic locus was used to compare the expression profiles in reproductive tissues between monoembryonic and polyembryonic varieties. The full length of CitRKD1, which was identified as a candidate gene responsible for citrus somatic embryogenesis, was isolated from satsuma mandarin and its molecular function was investigated using transgenic ‘Hamlin’ sweet orange by antisense-overexpression. The candidate gene CitRKD1, predominantly transcribed in reproductive tissues of polyembryonic varieties, is a member of the plant RWP-RK domain proteins. CitRKD1 of satsuma mandarin comprised two alleles (CitRKD1-mg1 and CitRKD1-mg2) at the polyembryonic locus controlling embryony type (mono/polyembryony) that were structurally divided into two types with or without a miniature inverted-repeat transposable element (MITE)-like insertion in the upstream region. CitRKD1-mg2 with the MITE insertion was the predominant transcript in flowers and young fruits where somatic embryogenesis of nucellar cells occurred. Loss of CitRKD1 function by antisense-overexpression abolished somatic embryogenesis in transgenic sweet orange and the transgenic T1 plants were confirmed to derive from zygotic embryos produced by self-pollination by DNA diagnosis. Genotyping PCR analysis of 95 citrus traditional and breeding varieties revealed that the CitRKD1 allele with the MITE insertion (polyembryonic allele) was dominant and major citrus varieties with the polyembryonic allele produced polyembryonic seeds.
Project description:E12.5 mouse lens epithelium and fiber cells were collected using Leica LMD 6000 Laser microdissection system. Total RNA was isolated from pepitheliam and fiber cell samples using total RNA was extracted from the tissue using the RNeasy Microkit.
Project description:Apomixis differs from sexual reproduction only in three major aspects: While the sexual megaspore mother cell undergoes meiosis, the apomictic initial cell omits or aborts meiosis (apomeiosis); the unreduced egg cell of apomicts forms an embryo without fertilization (parthenogenesis), and formation of functional endosperm requires specific developmental adaptations. Currently, our knowledge about the gene regulatory programs underlying apomixis is scarce. We used the apomict Boechera gunnisoniana, a close relative of Arabidopsis thaliana, to investigate the transcriptional basis underlying apomeiosis and parthenogenesis. Here, we present the first comprehensive reference transcriptome for reproductive development in an apomictic species. To compare sexual and apomictic development at the cellular level, we then used a combination of laser-assisted microdissection with microarray and RNA-Seq analysis. Our study yields important new insights into the transcriptional basis underlying apomixis.