Project description:Orchids are commercially important flowers, comprising one of the largest families of plants. Model plants such as Arabidopsis do not contain all plant genes. In this study, several molecular biology tools were integrated to isolate the flower-specific gene promoters of Oncidium Gower Ramsey (GR). A cDNA library of reproductive tissues was constructed and a microarray was established and used to compare gene expression in flowers and leaves. Five genes were highly expressed in flower tissues and the subcellular locations of the corresponding proteins were identified using fluorescent protein-fusion protein lip transient transformation, and related to their putative functions. BAC clones of the five genes, together with nine previously published flower and reproductive growth specific genes in Onc. GR, were identified and promoter regions were cloned by screening an Onc. GR BAC library. Interestingly, three of the five novel flower-abundant genes were putative trypsin inhibitor (TI) genes, OnTI1, OnTI2 and OnTI3, which were tandemly duplicated in the same BAC clone. Their promoters were identified using transient GUS reporter gene transformation and stable Arabidopsis transformation analyses. In conclusion, a combination of tools including a cDNA microarray, a BAC library, and a bombardment assay can be successfully employed to identify orchid genes and promoters. A total of 960 cDNAs derived from the Onc. GR flower cDNA library were amplified using PCR incorporating the T3 and M13 reverse universal primers. The PCR products were purified using the QIAquick PCR Purification kit (Qiagen, Germantown, MD) and eluted into 100 M-BM-5l of 0.1M-CM-^W Tris-EDTA buffer, pH 8.0. Purified PCR products were printed on GAPS II-coated slides (Corning, New York, NY) using the OmniGrid 100 microarray (Genomic Solutions, Ann Arbor, MI), and arranged into 1.8 M-CM-^W 1.8-cm arrays (spot size: 100 M-NM-<m) twice. These DNAs were cross-linked to the slide by baking the array at 80M-BM-0C for 2 h. After printing, the slides were left to dry overnight. The total RNA from leaves and flowers (25 M-NM-<g) was used for cDNA synthesis with either Cy3 or Cy5 dye molecules, using the 3DNA Expression Array Detection kit for microarrays (Array 50, version 2, Genisphere, Hatfield, PA). cDNA hybridization and washing procedures were performed according to the manufacturer's instructions. All experiments were carried out in triplicate, with repeats of each dye combination (n = 3).

Project description:The production of heather (Calluna vulgaris) in Germany is highly dependent on cultivars with mutated flower morphology, the so-called diplocalyx bud bloomers. So far, this unique flower type of C. vulgaris has not been reported in any other plant species. The flowers are characterised by an extremely extended flower attractiveness, since the flower buds remain closed throughout the complete flowering season. The flowers of C. vulgaris bud bloomers are male sterile, because the stamens are missing. Furthermore, petals are converted into sepals. Therefore the diplocalyx bud bloomer flowers consist of two whorls of sepals directly followed by the gynoecium. A broad comparison of wild type and bud bloomer’s flowers was undertaken to identify genes differentially expressed in the bud flowering phenotype and in the wild type of C. vulgaris. Transcriptome sequence reads were generated using next generation 454 sequencing of two flower type specific cDNA libraries. In total, 360,000 sequence reads were obtained, assembled to 12,200 contigs, functionally mapped, and annotated. Transcript abundances in wild type and bud bloomer’s libraries were compared and 365 differentially expressed genes detected. Among these differentially genes, CvPI was identified which is the orthologue of the Arabidopsis B gene PISTILLATA (PI) and considered as the most promising candidate gene. Quantitative PCR was performed to analyse the gene expression levels of two C. vulgaris B genes CvPI and CvAP3 in both flower types. CvAP3 which is the orthologue of the Arabidopsis B gene APETALA (AP3) turned out to be ectopically expressed in sepals of wild type and bud bloomer flowers. CvPI expression was proven to be reduced in the flowers of bud blooming cultivars. Differential expression patterns of the B-class genes CvAP3 and CvPI were identified to cause characteristics of flower morphology in C. vulgaris wild type and bud blooming flowers leading to the following hypotheses: ectopic expression of CvAP3 is a convincing explanation for the formation of a completely petaloid perianth in the wild type and the “bud flowering” phenotype. In C. vulgaris, CvPI is essential for determination of petal and stamen identity. The characteristic transition of petals into sepals potentially depends on the observed deficiency of CvPI and CvAP3 expression in bud blooming flowers. However, the complete loss of stamens in bud blooming flowers remains to be explained.

Project description:To facilitate the functional annotation of the pepper genome, we generated 90.84 Gb of RNA-Seq data from 33 libraries representing all major tissue types and developmental stages of Zunla 1, as well as fruits from other accessions with significant phenotypic differences. Pepper ‘Zunla 1’ and other inbred lines were grown in a greenhouse as described in Table S1, with their different developmental stages Plants at full-bloom stage were harvested for roots, stems, and leaves as the same as the samples for phased small RNAs (see text S3.4.2 for details). Mature plants were harvested for unopened flower buds (buds) and fully open flowers (flowers). Additional flowers were allowed to self-pollinate and fruit was harvested at four pre-breaker stages (1-3cm, 3-4cm, 4-5cm fruit length, and mature green), the breaker stage (when the fruit was turning red) and three post-breaker stages (3, 5, and 7 days after breaker). These samples will respectively be referred to as Root, Stem, Leaf, Bud, Flower, F-Dev-1, F-Dev-2, F-Dev-3, F-Dev-4, F-Dev-5, F-Dev-6, F-Dev-7, F-Dev-8, and F-Dev-9. Similar roots, stems, leaves, immature fruit and red fruit were harvested from other inbred lines from domesticated Capsicum species. Meanwhile, chiltepin plants were grown under long days at controlled temperature and RNA was extracted from a mix of leaves from four stages (seedling, early blooming, full bloom, and fruit breaker phases), a mix of flowers from unopened flower buds (buds) and fully open flowers (flowers), and fruit at breaker and breaker plus five days respectively. All tissues were frozen in liquid nitrogen and then stored at -80℃. Total RNA was isolated from different samples by using the Trizol Reagent (Invitrogen) according to manufacturer’s instructions. Strand-specific RNA-Seq library preparations were performed as previously described (39) with 12 independently bar-coded samples sequenced on one lane of an Illumina HiSeq2000 system. The 200 bp paired-end libraries were sequenced using Illumina HiSeq 2000 (90 bp PE).

Project description:The production of heather (Calluna vulgaris) in Germany is highly dependent on cultivars with mutated flower morphology, the so-called diplocalyx bud bloomers. So far, this unique flower type of C. vulgaris has not been reported in any other plant species. The flowers are characterised by an extremely extended flower attractiveness, since the flower buds remain closed throughout the complete flowering season. The flowers of C. vulgaris bud bloomers are male sterile, because the stamens are missing. Furthermore, petals are converted into sepals. Therefore the diplocalyx bud bloomer flowers consist of two whorls of sepals directly followed by the gynoecium. A broad comparison of wild type and bud bloomer’s flowers was undertaken to identify genes differentially expressed in the bud flowering phenotype and in the wild type of C. vulgaris. Transcriptome sequence reads were generated using next generation 454 sequencing of two flower type specific cDNA libraries. In total, 360,000 sequence reads were obtained, assembled to 12,200 contigs, functionally mapped, and annotated. Transcript abundances in wild type and bud bloomer’s libraries were compared and 365 differentially expressed genes detected. Among these differentially genes, CvPI was identified which is the orthologue of the Arabidopsis B gene PISTILLATA (PI) and considered as the most promising candidate gene. Quantitative PCR was performed to analyse the gene expression levels of two C. vulgaris B genes CvPI and CvAP3 in both flower types. CvAP3 which is the orthologue of the Arabidopsis B gene APETALA (AP3) turned out to be ectopically expressed in sepals of wild type and bud bloomer flowers. CvPI expression was proven to be reduced in the flowers of bud blooming cultivars. Differential expression patterns of the B-class genes CvAP3 and CvPI were identified to cause characteristics of flower morphology in C. vulgaris wild type and bud blooming flowers leading to the following hypotheses: ectopic expression of CvAP3 is a convincing explanation for the formation of a completely petaloid perianth in the wild type and the “bud flowering” phenotype. In C. vulgaris, CvPI is essential for determination of petal and stamen identity. The characteristic transition of petals into sepals potentially depends on the observed deficiency of CvPI and CvAP3 expression in bud blooming flowers. However, the complete loss of stamens in bud blooming flowers remains to be explained. two samples were analysed, each representing a flower type

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Integration of molecular biology tools to obtain information about abundantly expressed genes in flowers of Oncidium Gower Ramsey

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