Comparitive Genomics and Analysis of Repetitive Sequence on Rice Chromosome 5

國際合作的水稻基因組定序工作進行多年已臻完成，各項分析工作亦相繼展開，本文採用我國負責的第五條染色體序列，利用生物資訊工具，配合適當的統計分析，探討水稻基因序列的基本特性。 本文主要包含兩個主題，一為秈稻與稉稻基因組序列之比較，以我國完成之稉稻第五條染色體基因組序列比對秈稻品種93-11的全基因組散彈槍法的草圖序列，估計此二品種第五條染色體上序列相似性。以BLAST搜尋結果，估計大約80%的序列可比對，平均相似性97%，若是去除旁系同源的比對，則有60%序列可比對，平均相似性98%。 此外也對第五條染色體基因組序列的重複序列，做一廣泛及深入探討，瞭解各種重複序列在染色體上分布，由本文之結果估計水稻第五條染色體基因組序列中約有20%以上為重複序列，其中最多的一類是轉位因子，另外有2%為短序列重複與低複雜度序列。由中節與端粒區域的分析，搜尋特定重覆序列CentO及Os48的分布範圍，分別為60kb與150kb，以303組CentO及216組Os48重複序列間的親緣分析與位置分布，推估此類連續重複的可能機制均為在附近產生重複。另外，LTR序列的相似性分析亦提供LTR-反轉位子插入基因組的演化參考，由本文結果推估LTR-反轉位子的插入並無位置上的偏好。同時，由中節與端粒重複序列的結構，可推斷第五條染色體基因組序列的完整性，目前已完成中節區域的定序，端粒區域也相當接近邊緣，顯見定序工作已克服高度重複的障礙。 這兩個工作的成果，均可提供設計高密度分子標記之參考，未來可應用於的水稻重要性狀定位與選殖，以期提高水稻品質與產量。The IRGSP (The International Rice Genome Sequencing Program) completed a high quality sequence on schedule in December 2002. This result allows a large scale investigation on rice. We have done some detail analysis on rice chromosome 5, which sequenced by ASPGC（Academia Sinica Plant Genome Center）. This work accomplished basic characteristics of rice genome with some bioinformatics tools and combination with proper statistic approach. This work fell into two parts. The first one was the comparison between genomic sequences of Japonica and Indica. The chromosome 5 sequences of Nipponbare were aligned with whole genome shotgun sequences of 93-11 Indica variety. The similarities between them were estimated by BLAST searching. The result revealed that the average similarity under the aligned region covered 80% of genome was 97%. After those alignments duplicated or over-dispersed were removed, 60% of genome could be aligned and the similarity was up to 98%. The other one was the studying on repetitive sequences on rice chromosome 5. The distributions of different repetitive sequences on chromosome 5 were clarified. And details on centromere and telomere regions were featured. The differences between LTR sequences reflected the histories of insertion of retrotransposons. The completeness of centromere and telomere with highly repetitive sequences has been overcome by map-based approach. These results are important for designing molecular markers. High density of molecular markers is the helpful tool on future breeding and genetic research of rice.圖目錄 IV 表目錄 IX 摘要 1 第一章、前言 4 第二章、前人研究 7 第三章、稉稻第五條染色體序列與秈稻基因組序列之比較 18 一、 目的： 18 二、 材料與方法 19 (一) 資料來源與資料庫之建立 19 (二) 序列相似性分析 21 (三) 模擬試驗驗證BLAST之正確性 24 (四) BLAST結果整理與篩選 24 三、 結果 35 (一) 秈稻品種93-11基因組序列的統計性質 35 (二) BLAST結果的統計分析 39 (三) 模擬試驗的結果 47 (四) 重複比對區域的統計分析 49 四、 討論與結論 53 第四章、水稻第五條染色體基因組重複序列之分布 57 一、 目的： 57 二、 材料與方法 59 (一) 資料來源 59 (二) 重複序列資料庫比對 61 (三) 中節區域序列的重複序列 62 (四) 端粒區域的重複序列 65 (五) 中節區域及端粒區域序列的複雜度估算 66 (六) 長端重複（LTR）的序列差異分析 67 三、 結果 68 (一) 水稻第五條染色體對TIGR水稻重複序列做BLAST比對 68 (二) 水稻第五條染色體的RepeatMasker分析： 74 (三) 中節區域的重複序列分析： 84 (四) 端粒區域的重複序列分析： 90 (五) LTR-retrotransposon序列的相似性 96 (六) CentO與Os48重複序列的親緣分析 103 四、 討論與結論 109 第五章、綜合討論與結論 110 參考文獻： 117 附錄 12

Microarray meta-analysis to explore abiotic stress-specific gene expression patterns in Arabidopsis

Abstract Background Abiotic stresses are the major limiting factors that affect plant growth, development, yield and final quality. Deciphering the underlying mechanisms of plants’ adaptations to stresses using few datasets might overlook the different aspects of stress tolerance in plants, which might be simultaneously and consequently operated in the system. Fortunately, the accumulated microarray expression data offer an opportunity to infer abiotic stress-specific gene expression patterns through meta-analysis. In this study, we propose to combine microarray gene expression data under control, cold, drought, heat, and salt conditions and determined modules (gene sets) of genes highly associated with each other according to the observed expression data. Results By analyzing the expression variations of the Eigen genes from different conditions, we had identified two, three, and five gene modules as cold-, heat-, and salt-specific modules, respectively. Most of the cold- or heat-specific modules were differentially expressed to a particular degree in shoot samples, while most of the salt-specific modules were differentially expressed to a particular degree in root samples. A gene ontology (GO) analysis on the stress-specific modules suggested that the gene modules exclusively enriched stress-related GO terms and that different genes under the same GO terms may be alternatively disturbed in different conditions. The gene regulatory events for two genes, DREB1A and DEAR1, in the cold-specific gene module had also been validated, as evidenced through the literature search. Conclusions Our protocols study the specificity of the gene modules that were specifically activated under a particular type of abiotic stress. The biplot can also assist to visualize the stress-specific gene modules. In conclusion, our approach has the potential to further elucidate mechanisms in plants and beneficial for future experiments design under different abiotic stresses

MOESM4 of Microarray meta-analysis to explore abiotic stress-specific gene expression patterns in Arabidopsis

Additional file 5: Table S3. Lists of enriched GO terms of each abiotic stress-specific gene module. These tables are shown the enriched GO terms in the (A) cold-specific, (B) heat-specific, (C) salt-specific, and (D) all condition-specific gene modules

Genetic and Evolutionary Analysis of Purple Leaf Sheath in Rice

Background: Anthocyanin accumulates in many plant tissues or organs, in rice for example leading to red, purple red and purple phenotypes for protection from damage by biotic and abiotic stresses and for reproduction. Purple leaf, leaf sheath, stigma, pericarp, and apiculus are common in wild rice and landraces and occasionally found in modern cultivars. No gene directly conferring anthocyanin deposited in a purple leaf sheath has yet been isolated by using natural variants. An F-2 population derived from ssp. japonica cv. Tainung 72 (TNG72) with purple leaf sheath (PSH) crossed with ssp. indica cv. Taichung Sen 17 (TCS17) with green leaf sheath (GSH) was utilized to isolate a gene conferring leaf sheath color. Results: By positional cloning, 10-and 3-bp deletions in the R2R3 Myb domain of OsC1 were uncovered in GSH varieties TCS17 and Nipponbare, respectively. Allelic diversity, rather than gene expression levels of OsC1, might be responsible for anthocyanin accumulation. Parsimony-based analysis of genetic diversity in 50 accessions, including cultivars, landraces, and A-genome wild rice, suggests that independent mutation occurred in Asian, African, South American, and Australian species, while O. meridionalis had a divergent sequence. OsC1 was thought of as a domestication related gene, with up to 90 % reduction of genetic diversity in GSH; however, no values from three tests showed significant differences from neutral expectations, implying that OsC1 had not been subjected to recent selection. Haplotype network analysis revealed that species from different continents formed unique haplotypes with no gene flow. Two major groups of haplotypes corresponding to 10-bp deletion and other sequences were formed in Asian rice, including O. rufipogon, O. nivara and O. sativa. Introgressions of OsC1 between subspecies through natural and artificial hybridization were not rare. Because artificial and natural selection imposed admixture on rice germplasm in Taiwan, the genealogy of OsC1 might not be congruent with the current distribution of alleles through lineage diversification. Conclusion: OsC1 is responsible for purple leaf sheath, and much new information about OsC1 is provided e.g., new alleles, non-domestication syndrome, and incongruence of genealogy with geographic distribution

Five different piscidins from Nile tilapia, Oreochromis niloticus: analysis of their expressions and biological functions.

Piscidins are antimicrobial peptides (AMPs) that play important roles in helping fish resist pathogenic infections. Through comparisons of tilapia EST clones, the coding sequences of five piscidin-like AMPs (named TP1∼5) of Nile tilapia, Oreochromis niloticus, were determined. The complete piscidin coding sequences of TP1, -2, -3, -4, and -5 were respectively composed of 207, 234, 231, 270, and 195 bases, and each contained a translated region of 68, 77, 76, 89, and 64 amino acids. The tissue-specific, Vibrio vulnificus stimulation-specific, and Streptococcus agalactiae stimulation-specific expressions of TP2, -3, and -4 mRNA were determined by a comparative RT-PCR. Results of the tissue distribution analysis revealed high expression levels of TP2 mRNA in the skin, head kidneys, liver, and spleen. To study bacterial stimulation, S. agalactiae (SA47) was injected, and the TP4 transcript was upregulated by >13-fold (compared to the wild-type (WT) control, without injection) and was 60-fold upregulated (compared to the WT control, without injection) 24 h after the S. agalactiae (SA47) injection in the spleen and gills. Synthesized TP3 and TP4 peptides showed antimicrobial activities against several bacteria in this study, while the synthesized TP1, -2, and -5 peptides did not. The synthesized TP2, -3, and -4 peptides showed hemolytic activities and synthesized TP3 and TP4 peptides inhibited tilapia ovary cell proliferation with a dose-dependent effect. In summary, the amphiphilic α-helical cationic peptides of TP3 and TP4 may represent novel and potential antimicrobial agents for further peptide drug development

Investigation of the Correlation between Graves’ Ophthalmopathy and CTLA4 Gene Polymorphism

Graves’ disease (GD) is an autoimmune inflammatory disease, and Graves’ ophthalmopathy (GO) occurs in 25–50% of patients with GD. Several susceptible genes were identified to be associated with GO in some genetic analysis studies, including the immune regulatory gene CTLA4. We aimed to find out the correlation of CTLA4 gene polymorphism and GO. A total of 42 participants were enrolled in this study, consisting of 22 patients with GO and 20 healthy controls. Chi-square or Fisher’s exact test were used to appraise the association between Graves’ ophthalmopathy and CTLA4 single nucleotide polymorphisms (SNPs). All regions of CTLA4 including promoter, exon and 3’UTR were investigated. There was no nucleotide substitution in exon 2 and exon 3 of CTLA4 region, and the allele frequencies of CTLA4 polymorphisms had no significant difference between patients with GO and controls. However, the genotype frequency of “TT” genotype in rs733618 significantly differed between patients with GO and healthy controls (OR = 0.421, 95%CI: 0.290–0.611, p = 0.043), and the “CC” and “CT” genotype in rs16840252 were nearly significantly differed in genotype frequency (p = 0.052). Haplotype analysis showed that CTLA4 Crs733618Crs16840252 might increase the risk of GO (OR = 2.375, 95%CI: 1.636–3.448, p = 0.043). In conclusion, CTLA4 Crs733618Crs16840252 was found to be a potential marker for GO, and these haplotypes would be ethnicity-specific. Clinical application of CTLA4 Crs733618Crs16840252 in predicting GO in GD patients may be beneficial

Additional file 1: Table S1. of Genetic and Evolutionary Analysis of Purple Leaf Sheath in Rice

Newly designed markers used in coarse mapping. (DOCX 17 kb

Comparative genomics revealed the gene evolution and functional divergence of magnesium transporter families in Saccharum

Abstract Background Sugarcane served as the model plant for discovery of the C4 photosynthetic pathway. Magnesium is the central atom of chlorophyll, and thus is considered as a critical nutrient for plant development and photosynthesis. In plants, the magnesium transporter (MGT) family is composed of a number of membrane proteins, which play crucial roles in maintaining Mg homeostasis. However, to date there is no information available on the genomics of MGTs in sugarcane due to the complexity of the Saccharum genome. Results Here, we identified 10 MGTs from the Saccharum spontaneum genome. Phylogenetic analysis of MGTs suggested that the MGTs contained at least 5 last common ancestors before the origin of angiosperms. Gene structure analysis suggested that MGTs family of dicotyledon may be accompanied by intron loss and pseudoexon phenomena during evolution. The pairwise synonymous substitution rates corresponding to a divergence time ranged from 142.3 to 236.6 Mya, demonstrating that the MGTs are an ancient gene family in plants. Both the phylogeny and Ks analyses indicated that SsMGT1/SsMGT2 originated from the recent ρWGD, and SsMGT7/SsMGT8 originated from the recent σ WGD. These 4 recently duplicated genes were shown low expression levels and assumed to be functionally redundant. MGT6, MGT9 and MGT10 weredominant genes in the MGT family and werepredicted to be located inthe chloroplast. Of the 3 dominant MGTs, SsMGT6 expression levels were found to be induced in the light period, while SsMGT9 and SsMTG10 displayed high expression levels in the dark period. These results suggested that SsMGT6 may have a function complementary to SsMGT9 and SsMTG10 that follows thecircadian clock for MGT in the leaf tissues of S. spontaneum. MGT3, MGT7 and MGT10 had higher expression levels Insaccharum officinarum than in S. spontaneum, suggesting their functional divergence after the split of S. spontaneum and S. officinarum. Conclusions This study of gene evolution and expression of MGTs in S. spontaneum provided basis for the comprehensive genomic study of the entire MGT genes family in Saccharum. The results are valuable for further functional analyses of MGT genes and utilization of the MGTs for Saccharum genetic improvement