Jae-Pil Choi

Small RNA and transcriptome deep sequencing proffers insight into floral gene regulation in Rosa cultivars

BackgroundRoses (Rosa sp.), which belong to the family Rosaceae, are the most economically important ornamental plants—making up 30% of the floriculture market. However, given high demand for roses, rose breeding programs are limited in molecular resources which can greatly enhance and speed breeding efforts. A better understanding of important genes that contribute to important floral development and desired phenotypes will lead to improved rose cultivars. For this study, we analyzed rose miRNAs and the rose flower transcriptome in order to generate a database to expound upon current knowledge regarding regulation of important floral characteristics. A rose genetic database will enable comprehensive analysis of gene expression and regulation via miRNA among different Rosa cultivars.ResultsWe produced more than 0.5 million reads from expressed sequences, totalling more than 110 million bp. From these, we generated 35,657, 31,434, 34,725, and 39,722 flower unigenes from Rosa hybrid: ‘Vital’, ‘Maroussia’, and ‘Sympathy’ and Rosa rugosa Thunb. , respectively. The unigenes were assigned functional annotations, domains, metabolic pathways, Gene Ontology (GO) terms, Plant Ontology (PO) terms, and MIPS Functional Catalogue (FunCat) terms. Rose flower transcripts were compared with genes from whole genome sequences of Rosaceae members (apple, strawberry, and peach) and grape. We also produced approximately 40 million small RNA reads from flower tissue for Rosa, representing 267 unique miRNA tags. Among identified miRNAs, 25 of them were novel and 242 of them were conserved miRNAs. Statistical analyses of miRNA profiles revealed both shared and species-specific miRNAs, which presumably effect flower development and phenotypes.ConclusionsIn this study, we constructed a Rose miRNA and transcriptome database, and we analyzed the miRNAs and transcriptome generated from the flower tissues of four Rosa cultivars. The database provides a comprehensive genetic resource which can be used to better understand rose flower development and to identify candidate genes for important phenotypes.

A Genome-Wide Comparison of NB-LRR Type of Resistance Gene Analogs (RGA) in the Plant Kingdom

Plants express resistance (R) genes to recognize invaders and prevent the spread of pathogens. To analyze nucleotide binding site, leucine-rich repeat (NB-LRR) genes, we constructed a fast pipeline to predict and classify the R gene analogs (RGAs) by applying in-house matrices. With predicted ∼37,000 RGAs, we can directly compare RGA contents across entire plant lineages, from green algae to flowering plants. We focused on the highly divergent NBLRRs in land plants following the emergence of mosses. We identified entire loss of Toll/Interleukin-1 receptor, NBLRR (TNL) in Poaceae family of monocots and interestingly from Mimulus guttatus (a dicot), which leads to the possibility of species-specific TNL loss in other sequenced flowering plants. Using RGA maps, we have elucidated a positive correlation between the cluster sizes of NB-LRRs and their numbers. The cluster members were observed to consist of the same class of NB-LRRs or their variants, which were probably generated from a single locus for an R gene. Our website (http://sol.kribb.re.kr/PRGA/), called plant resistance gene analog (PRGA), provides useful information, such as RGA annotations, tools for predicting RGAs, and analyzing domain profiles. Therefore, PRGA provides new insights into R-gene evolution and is useful in applying RGA as markers in breeding and or systematic studies.

Functional Domain Marker (FDM): an In Silico Demonstration in Solanaceae Using Simple Sequence Repeats (SSRs)

A simple sequence repeat–functional domain marker (SSR-FDM) relies on development of molecular markers for putative functional domains using simple sequence repeats and in silico annotated information of those sequences using biological databases. A total of 148,921 tomato ESTs and 115,598 pepper ESTs were analyzed, resulting in the identification of 439 tomato SSR-FDMs and 489 pepper SSR-FDMs. Among them, 54 pepper SSR-FDMs were tested on pepper. Several genomic databases were used for the in silico annotation of the SSR-FDM sequences that revealed a wide range of candidate genes. This study demonstrates that SSR-FDMs provide information regarding transcribed genetic markers and putative function as a genomic resource database for Solanaceae. This system could be applied to the development of a functional marker database for any crop species.

Functional innovations of three chronological mesohexaploid Brassica rapa genomes

BackgroundThe Brassicaceae family is an exemplary model for studying plant polyploidy. The Brassicaceae knowledge-base includes the well-annotated Arabidopsis thaliana reference sequence; well-established evidence for three rounds of whole genome duplication (WGD); and the conservation of genomic structure, with 24 conserved genomic blocks (GBs). The recently released Brassica rapa draft genome provides an ideal opportunity to update our knowledge of the conserved genomic structures in Brassica, and to study evolutionary innovations of the mesohexaploid plant, B. rapa.ResultsThree chronological B. rapa genomes (recent, young, and old) were reconstructed with sequence divergences, revealing a trace of recursive WGD events. A total of 636 fast evolving genes were unevenly distributed throughout the recent and young genomes. The representative Gene Ontology (GO) terms for these genes were ‘stress response’ and ‘development’ both through a change in protein modification or signaling, rather than by enhancing signal recognition. In retention patterns analysis, 98% of B. rapa genes were retained as collinear gene pairs; 77% of those were singly-retained in recent or young genomes resulting from death of the ancestral copies, while others were multi-retained as long retention genes. GO enrichments indicated that single retention genes mainly function in the interpretation of genetic information, whereas, multi-retention genes were biased toward signal response, especially regarding development and defense. In the recent genome, 13,302, 5,790, and 20 gene pairs were multi-retained following Brassica whole genome triplication (WGT) events with 2, 3, and 4 homoeologous copies, respectively. Enriched GO-slim terms from B. rapa homomoelogues imply that a major effect of the B. rapa WGT may have been to acquire environmental adaptability or to change the course of development. These homoeologues seem to more frequently undergo subfunctionalization with spatial expression patterns compared with other possible events including nonfunctionalization and neofunctionalization.ConclusionWe refined Brassicaceae GB information using the latest genomic resources, and distinguished three chronologically ordered B. rapa genomes. B. rapa genes were categorized into fast evolving, single- and multi-retention genes, and long retention genes by their substitution rates and retention patterns. Representative functions of the categorized genes were elucidated, providing better understanding of B. rapa evolution and the Brassica genus.

ASPMF : A new approach for identifying alternative splicing isoforms using peptide mass fingerprinting

Alternative splicing is generally accepted as a mechanism that explains the discrepancy between the number of genes and proteins. We used peptide mass fingerprinting with a theoretical database and scoring method to discover and identify alternative splicing isoforms. Our theoretical database was built using published alternative splicing databases such as ECgene, H-DBAS, and TISA. According to our theoretical database of 190,529 isoforms, 37% of human genes have multiple isoforms. The isoforms produced from a gene partially share common peptide fragments because they have common exons, making it difficult to distinguish isoforms. Therefore, we developed a new method that effectively distinguishes a true isoform among multiple isoforms in a gene. In order to evaluate our algorithm, we made test sets for 4226 protein isoforms extracted from our theoretical database randomly. Consequently, 94% of true isoforms were identified by our scoring algorithm.

RISA: a new web-tool for Rapid Identification of SSRs and Analysis of primers

The simple sequence repeats (SSRs) are short tandem arrayed sequence motifs consisting of 2–6 bp, which are not only involved in causing human fatal diseases but also have various applications in plant genetic studies. Thanks to the advancements made in sequencing technology, now we can easily generate genomic/transcriptomic sequences in a shorter period of time. Therefore, trend to identify SSR markers needs up gradation to handle these high-throughput data. Unfortunately, existing web programs for identifying SSR markers are useful but they are unable to process high-throughput data. To overcome this disadvantage, we have constructed a web-based tool, RISA (http://sol.kribb.re.kr/RISA/), with a goal of one-click service to identify SSR markers from high-throughput data (up to 200 Mbp). RISA controls automatic input and output pipeline by demon which combines the SSR classification and investigation by Robert Kofler (SciRoKo) to search SSRs and Primer3 to identify primers specific to SSRs simultaneously. In our test, 45,495 qualified primer sets specific to 47,070 SSRs were identified by RISA from whole Arabidopsis lyrata genome (about 207 Mbp) in 15 minutes. In results, it includes SSR statistics generated from user’s queries and SSR markers information along with primers suitable for their amplification. To support handling of large amount of results, RISA provides various filtering options such as motif length, repeat units, total length and PCR product size. Therefore, we propose that RISA minimizes labour-intensive works or any other considerations which can be required during the development of SSR markers without having deep understanding of computer system and/or algorithms.

Intracellular Ca 2+ and K + concentration in Brassica oleracea leaf induces differential expression of transporter and stress-related genes

BackgroundOne of the most important members of the genus Brassica, cabbage, requires a relatively high level of calcium for normal growth (Plant Cell Environ 7: 397–405, 1984; Plant Physiol 60: 854–856, 1977). Localized Ca2+ deficiency in cabbage leaves causes tip-burn, bringing about serious economic losses (Euphytica 9:203–208, 1960; Ann Bot 43:363–372, 1979; Sci Hortic 14:131–138, 1981). Although it has been known that the occurrence of tip-burn is related to Ca2+ deficiency, there is limited information on the underlying mechanisms of tip-burn or the relationship between Ca2+ and tip-burn incidence. To obtain more information on the genetic control of tip-burn symptoms, we focused on the identification of genes differentially expressed in response to increasing intracellular Ca2+ and K+ concentrations in B. oleracea lines derived from tip-burn susceptible, tip-burn resistant cabbages (B. oleracea var. capitata), and kale (B. oleracea var. acephala).ResultsWe compared the levels of major macronutrient cations, including Ca2+ and K+, in three leaf segments, the leaf apex (LA), middle of leaf (LM), and leaf base (LB), of tip-burn susceptible, tip-burn resistant cabbages, and kale. Ca2+ and K+ concentrations were highest in kale, followed by tip-burn resistant and then tip-burn susceptible cabbages. These cations generally accumulated to a greater extent in the LB than in the LA. Transcriptome analysis identified 58,096 loci as putative non-redundant genes in the three leaf segments of the three B. oleracea lines and showed significant changes in expression of 27,876 loci based on Ca2+ and K+ levels. Among these, 1844 loci were identified as tip-burn related phenotype-specific genes. Tip-burn resistant cabbage and kale-specific genes were largely related to stress and transport activity based on GO annotation. Tip-burn resistant cabbage and kale plants showed phenotypes clearly indicative of heat-shock, freezing, and drought stress tolerance compared to tip-burn susceptible cabbages, demonstrating a correlation between intracellular Ca2+ and K+ concentrations and tolerance of abiotic stress with differential gene expression. We selected 165 genes that were up- or down-regulated in response to increasing Ca2+ and K+ concentrations in the three leaf segments of the three plant lines. Gene ontology enrichment analysis indicated that these genes participated in regulatory metabolic processes or stress responses.ConclusionsOur results indicate that the genes involved in regulatory metabolic processes or stress responses were differentially expressed in response to increasing Ca2+ and K+ concentrations in the B. oleracea leaf. Our transcriptome data and the genes identified may serve as a starting point for understanding the mechanisms underlying essential macronutrient deficiencies in plants, as well as the features of tip-burn in cabbage and other Brassica species.