Genome-Wide Divergence and Linkage Disequilibrium Analyses for Capsicum baccatum Revealed by Genome-Anchored Single Nucleotide Polymorphisms

Principal component analysis (PCA) with 36,621 polymorphic genome-anchored single nucleotide polymorphisms (SNPs) identified collectively for Capsicum annuum and Capsicum baccatum was used to show the distribution of these 2 important incompatible cultivated pepper species. Estimated mean nucleotide diversity (π) and Tajima’s D across various chromosomes revealed biased distribution toward negative values on all chromosomes (except for chromosome 4) in cultivated C. baccatum, indicating a population bottleneck during domestication of C. baccatum. In contrast, C. annuum chromosomes showed positive π and Tajima’s D on all chromosomes except chromosome 8, which may be because of domestication at multiple sites contributing to wider genetic diversity. For C. baccatum, 13,129 SNPs were available, with minor allele frequency (MAF) ≥0.05; PCA of the SNPs revealed 283 C. baccatum accessions grouped into 3 distinct clusters, for strong population structure. The fixation index (FST) between domesticated C. annuum and C. baccatum was 0.78, which indicates genome-wide divergence. We conducted extensive linkage disequilibrium (LD) analysis of C. baccatum var. pendulum cultivars on all adjacent SNP pairs within a chromosome to identify regions of high and low LD interspersed with a genome-wide average LD block size of 99.1 kb. We characterized 1742 haplotypes containing 4420 SNPs (range 9–2 SNPs per haplotype). Genome-wide association study of peduncle length, a trait that differentiates wild and domesticated C. baccatum types, revealed 36 genome-wide SNPs significantly associated. Population structure, identity by state (IBS) and LD patterns across the genome will be of potential use for future genome-wide association study of economically important traits in C. baccatum peppers

cavin-2の発現低下は、口腔癌の成長に寄与する

研究科: 千葉大学大学院医学薬学府学位:千大院医薬博甲第医1120号要約博士(医学)千葉大

Genome-Wide Differentiation of Various Melon Horticultural Groups for Use in GWAS for Fruit Firmness and Construction of a High Resolution Genetic Map

Ajuts: Funding support is provided by Gus R. Douglass Institute (Evans Allen Project to Nimmakayala) and USDA-NIFA (2010-02247 and 2012-02511).Melon (Cucumis melo L.) is a phenotypically diverse eudicot diploid (2n = 2x = 24) has climacteric and non-climacteric morphotypes and show wide variation for fruit firmness, an important trait for transportation and shelf life. We generated 13,789 SNP markers using genotyping-by-sequencing (GBS) and anchored them to chromosomes to understand genome-wide fixation indices (Fst) between various melon morphotypes and genomewide linkage disequilibrium (LD) decay. The FST between accessions of cantalupensis and inodorus was 0.23. The FST between cantalupensis and various agrestis accessions was in a range of 0.19-0.53 and between inodorus and agrestis accessions was in a range of 0.21-0.59 indicating sporadic to wide ranging introgression. The EM (Expectation Maximization) algorithm was used for estimation of 1436 haplotypes. Average genome-wide LD decay for the melon genome was noted to be 9.27 Kb. In the current research, we focused on the genome-wide divergence underlying diverse melon horticultural groups. A high-resolution genetic map with 7153 loci was constructed. Genome-wide segregation distortion and recombination rate across various chromosomes were characterized. Melon has climacteric and non-climacteric morphotypes and wide variation for fruit firmness, a very important trait for transportation and shelf life. Various levels of QTLs were identified with high to moderate stringency and linked to fruit firmness using both genome-wide association study (GWAS) and biparental mapping. Gene annotation revealed some of the SNPs are located in β-D-xylosidase, glyoxysomal malate synthase, chloroplastic anthranilate phosphoribosyltransferase, and histidine kinase, the genes that were previously characterized for fruit ripening and softening in other crops

Functional analysis of SIZ1 and LGD1 involved in rice growth and development

稻米是世界上近半數人口的主要糧食作物來源。而水稻植株的形態對於稻米的產量有著關鍵性影響，其形態結構是由株植的高低、分蘗的多寡、角度以及花序的形態所決定。此外，有性生殖的健全發育及授粉作用的成功與否也顯著地影響稻米產量，例如，花藥的開裂異常都可能造成雄蕊的不稔性，而雄不稔會顯著的降低作物的產量。因此，如何有效地利用功能基因組的研究策略對水稻重要性狀例如雄不稔、分蘗的多寡與角度、開花時期和花序形態等性狀的研究，可增進我們對於這些性狀如何受到基因調控機制的認知及未來的應用性。而T-DNA插入突變體是各種生物之功能基因組研究的重點之一，我們利用台灣水稻功能基因組研究之突變族資料庫中（TRIM database) 搜尋近20株與上述性狀相關的T-DNA插入突變體，本論文即針對其中兩個基因進行功能性分析與研究。 本論文的第一個部分，我們以siz1 T-DNA突變株和SIZ1-RNAi轉殖株兩種不同的遺傳學方法來証明了水稻SIZ1是一具有生物功能的SUMO E3黏接酶並參與調控水稻花藥開裂的過程。遺傳分析的結果顯示siz1 T-DNA突變株為單一T-DNA插入而導致了一個隱性的突變，此突變株的外表型會和T-DNA插入基因型共同分離至子代中。siz1 T-DNA突變株和SIZ1-RNAi轉殖株除了在植株的高度、分蘗的多寡及種子數目方面較野生型水稻有顯著降低的外表型，它們同時也造成花藥的開裂異常，但對花粉的活性卻無任何不良的影響；近一步分析發現siz1突變株花藥無法正常開裂可能肇因於開花期前花藥的內皮層發育異常所致。特別值得一提的是阿拉伯芥AtIRX和玉米ZmMADS2基因被發現是花藥開裂時花藥內皮層的發育所必需，而水稻這兩個基因的同源基因在siz1突變株中的表現量有明顯降低的現象。和野生型水稻相較，在siz1 T-DNA突變株和SIZ1-RNAi轉殖株的成熟小穗中高分子量蛋白質被sumoylation的樣式有隨著SIZ1基因表現量下降而減少的情形。在以基因槍轟擊洋葱表皮細胞的實驗中，我們發現位於SIZ1蛋白質羧基端的細胞核定位序列 (NLS) 已足夠將其本身帶入細胞核中。綜合所有的結果顯示SIZ1為一具功能的SUMO E3黏合酶參與調控水稻花藥的開裂過程。 論文第二部分是著重於研究一未知功能的基因LGD1是如何調控水稻分蘗和花序的發育。在這部分的研究中亦是利用lgd1 T-DNA突變株和LGD1-RNAi轉殖株來分析水稻LGD1的功能為何。我們發現由於T-DNA的插入而導致一隱性的突變，使得突變株呈現生長遲緩、分蘗數目減少、植株高度降低、花序的結構改變以及稻米的產量縮減等性狀，因此將此基因命名為lagging growth and development1 (lgd1)。LGD1-RNAi轉殖株和lgd1突變株有著相似的外表型。lgd1突變使得未延長的節間 (internode) 數目下降進而造成了分蘗數的減少；植株高度的降低是由於其細胞數目減少所致。水稻LGD1基因為單一基因，可轉譯出一具有vWA domain的蛋白質，在高梁中也有此基因的同源基因存在。從5’-RACE、GUS分析、RT-PCR和北方墨漬的結果中發現LGD1基因的各個多重轉錄之間的表現是具有時間及空間上的特異性。我們利用5’-RACE聚合酶連鎖反應技術獲得LGD1的多重轉錄片段，再利用螢光酵素 (Luciferase) 證實LGD1不同長度的起動子 (promoter) 區域均具有轉錄活性。LGD1-GFP融合蛋白質同時存在於細胞質及細胞核中。綜合以上結果我們推測LGD1基因藉由不同的起動子片段和不同的轉錄起始位置 (Transcription Start Sites) 來產生多重轉錄進而調控著營養生長及生殖生長，但其詳細的分子細胞調控機制則有待深入的探討。未來將近一步設法找出直接參與SIZ1及LGD1 作用的下游分子和其詳細的分子細胞調控機制，期待相關研究將會在經水稻生物科技在榖類的改良過程中能有其重要應用價值。Rice is a major staple food for nearly half of the world's population and rice plant architecture including plant height, tiller number and angle, and panicle morphology is crucial for grain yield. In addition to these characteristics, the higher grain production of rice depends on successful sexual reproduction. Male sterility, caused by various defects including anther dehiscence, has adverse effects on agricultural productivity and significantly reduces the crop yield. The phenomenon of male sterility is highly exploited to produce successful hybrids with higher yield. By taking the advantage of available resources of functional genomics and T-DNA insertional mutants, functional studies of the tagged genes with important characters such as male sterility, tillering, early flowering and panicle morphology are the main foci of this dissertation. In the first part, I provided the experimental evidence for the biological function of the SUMO E3 ligase, SIZ1, in rice for anther dehiscence. In this study, we used two genetic approaches, the siz1 T-DNA mutant and SIZ1-RNAi transgenic plants, to characterize the function of rice SIZ1. Genetic results revealed co-segregation of single T-DNA insertional recessive mutation with the observed phenotypes in siz1. In addition to showing reduced plant height, tiller number, and seed set percentage, both siz1 mutant and SIZ1-RNAi transgenic plants showed obvious defects in anther dehiscence but not in pollen viability. The anther indehiscence in siz1 was due to defects in endothecium development before anthesis. Interestingly, the rice orthologs of AtIRX and ZmMADS2, which are essential for endothecium development during anther dehiscence, were significantly downregulated in siz1. Compared to the wild-type, the sumoylation profile of high molecular weight proteins in mature spikelet was significantly reduced in siz1 and SIZ1-RNAi line that showed notable reduction in SIZ1 expression. The NLS located in SIZ1 C-terminus was sufficient for its nuclear targeting in bombarded onion epidermis. All results suggest the functional role of SIZ1, a SUMO E3 ligase, in regulating rice anther dehiscence. The second part of this dissertation mainly addresses the functional characterization of an unknown gene regulating tillering and panicle development. Two genetic approaches, T-DNA insertional mutant and RNAi transgenic lines, were used to characterize its functions. Later T-DNA insertion recessive mutant was named as lagging growth and development1 (lgd1), which exhibited slow growth, reduced tillers and plant height, altered panicle architecture and grain yield. LGD1-RNAi plants showed similar phenotypes found in the lgd1. Reduced number of unelongated internodes caused few tiller numbers and semi-dwarf was due to reduced number of cells in the lgd1 mutant. LGD1, encoding a vWA domain-containing protein(s), is a single gene in rice and has ortholog in sorghum. Interestingly, 5'-RACE, GUS assay, RT-PCR and RNA blot revealed the existence of multiple transcripts and distinctive spatiotemporal expression of LGD1 transcripts. Additionally, 5'-RACE-PCR and luciferase promoter assay were used to clone multiple transcripts and to analyze different promoters of LGD1, respectively. The LGD1-GFP fusion protein is located both in the nucleus and cytoplasm. We propose that LGD1 regulates both vegetative and reproductive growth through multiple transcripts from different promoters and TSSs and the regulatory mechanism is yet to be studied. Notably, the direct downstream targets of these genes SIZ1 and LGD1 with known mechanisms will shed light on multiple phenotypes towards crop improvement through rice biotechnology.中文摘要 i Abstract iii List of tables vii Lists of figures viii Lists of appendices x Chapter 1. Rice Functional Genomics for Vegetative and Reproductive Traits 1 Chapter 2. Rice SIZ1, a Sumo E3 Ligase, Controls Spikelet Fertility through Regulation of Anther Dehiscence 5 Abstract 6 Introduction 7 Materials and Methods 11 Results 15 Discussion 21 Chapter 3. Multiple Transcripts of OsLGD1 Pleiotropically Regulate Rice Growth and Development 38 Abstract 39 Introduction 40 Materials and Methods 44 Results 48 Discussion 54 Chapter 4. Conclusion and Future Prospects 80 References 83 Appendices 9

Rise of the clones: apomixis in plant breeding

artículo -- Universidad de Costa Rica. escuela de Agronomía, 2011. Ese documento es privado debido a limitaciones de derechos de autor.In flowering plants the transfer of traits from one generation to the other involves fertilization of female gametophyte with sperm cells delivered by pollen tube, and the subsequent reassortment of traits (alleles) in the developing progeny. In nature DNA recombination and segregation of traits to the progeny prevents the accumulation of deleterious genes and loss of fitness; however for breeding purposes it is advantageous to fix superior trait combinations (genotypes) and by-pass sexual reproduction. The formation of sexual seed without fertilization of the egg is called apomixis and is considered the holy grail of plant breeding because top-performing varieties can be reproduced indefinitely without changes in the genotypes themselves or in their expression patterns. Apomixis is a dominant trait and consists on several processes working in tandem, separately each process is detrimental for plants, however, as a single unit they allow development of embryo and endosperm from unfertilized eggs. The three processes that constitute apomixis are: 1) apomeiosis, or cell division without DNA recombination in pollen and eggs, 2) parthenogenesis, autonomous development of eggs into fully formed embryos, and 3) stable development of the endosperm, the part of the seed, needed for the embryo to grow. Unfortunately full expression and transmission of apomixis is affected by DNA recombination, therefore introgression of the trait from wild relatives into commercial varieties is extremely difficult. In this review we present genes that have been identified to regulate each step of apomixis and discuss strategies to allow transmission of the trait in full using tools from molecular biology.Academia Sinica (Taiwan). National Science Council (Taiwan). Universidad de Costa RicaUCR::Vicerrectoría de Docencia::Ciencias Agroalimentarias::Facultad de Ciencias Agroalimentarias::Escuela de Agronomí

Rice LGD1 containing RNA binding activity affects growth and development through alternative promoters

Tiller initiation and panicle development are important agronomical traits for grain production in Oryza sativa L. (rice), but their regulatory mechanisms are not yet fully understood. In this study, T-DNA mutant and RNAi transgenic approaches were used to functionally characterize a unique rice gene, LAGGING GROWTH AND DEVELOPMENT 1 (LGD1). The lgd1 mutant showed slow growth, reduced tiller number and plant height, altered panicle architecture and reduced grain yield. The fewer unelongated internodes and cells in lgd1 led to respective reductions in tiller number and to semi-dwarfism. Several independent LGD1-RNAi lines exhibited defective phenotypes similar to those observed in lgd1. Interestingly, LGD1 encodes multiple transcripts with different transcription start sites (TSSs), which were validated by RNA ligase-mediated rapid amplification of 5� and 3� cDNA ends (RLM-RACE). Additionally, GUS assays and a luciferase promoter assay confirmed the promoter activities of LGD1.1 and LGD1.5. LGD1 encoding a von Willebrand factor type A (vWA) domain containing protein is a single gene in rice that is seemingly specific to grasses. GFP-tagged LGD1 isoforms were predominantly detected in the nucleus, and weakly in the cytoplasm. In vitro northwestern analysis showed the RNA-binding activity of the recombinant C-terminal LGD1 protein. Our results demonstrated that LGD1 pleiotropically regulated rice vegetative growth and development through both the distinct spatiotemporal expression patterns of its multiple transcripts and RNA binding activity. Hence, the study of LGD1 will strengthen our understanding of the molecular basis of the multiple transcripts, and their corresponding polypeptides with RNA binding activity, that regulate pleiotropic effects in rice

Rice LGD1 containing RNA binding activity affects growth and development through alternative promoters

Tiller initiation and panicle development are important agronomical traits for grain production in Oryza sativa L. (rice), but their regulatory mechanisms are not yet fully understood. In this study, T-DNA mutant and RNAi transgenic approaches were used to functionally characterize a unique rice gene, LAGGING GROWTH AND DEVELOPMENT 1 (LGD1). The lgd1 mutant showed slow growth, reduced tiller number and plant height, altered panicle architecture and reduced grain yield. The fewer unelongated internodes and cells in lgd1 led to respective reductions in tiller number and to semi-dwarfism. Several independent LGD1-RNAi lines exhibited defective phenotypes similar to those observed in lgd1. Interestingly, LGD1 encodes multiple transcripts with different transcription start sites (TSSs), which were validated by RNA ligase-mediated rapid amplification of 5′ and 3′ cDNA ends (RLM-RACE). Additionally, GUS assays and a luciferase promoter assay confirmed the promoter activities of LGD1.1 and LGD1.5. LGD1 encoding a von Willebrand factor type A (vWA) domain containing protein is a single gene in rice that is seemingly specific to grasses. GFP-tagged LGD1 isoforms were predominantly detected in the nucleus, and weakly in the cytoplasm. In vitro northwestern analysis showed the RNA-binding activity of the recombinant C-terminal LGD1 protein. Our results demonstrated that LGD1 pleiotropically regulated rice vegetative growth and development through both the distinct spatiotemporal expression patterns of its multiple transcripts and RNA binding activity. Hence, the study of LGD1 will strengthen our understanding of the molecular basis of the multiple transcripts, and their corresponding polypeptides with RNA binding activity, that regulate pleiotropic effects in rice

Transcriptome Analysis of Cambium Tissue of Paulownia Collected during Winter and Spring

Paulownia (Paulownia elongata) is a fast-growing, multipurpose deciduous hardwood species that grows in a wide range of temperatures from –30 °C to 45 °C. Seasonal cues influence the secondary growth of tree stems, including cambial activity, wood chemistry, and transition to latewood formation. In this study, a de novo transcriptome approach was conducted to identify the transcripts expressed in vascular cambial tissue from senescent winter and actively growing spring seasons. An Illumina paired-end sequenced cambial transcriptome generated 297,049,842 clean reads, which finally yielded 61,639 annotated unigenes. Based on non-redundant protein database analyses, Paulownia cambial unigenes shared the highest homology (64.8%) with Erythranthe guttata. KEGG annotation of 35,471 unigenes identified pathways enriched in metabolic activities. Transcriptome-wide DEG analysis showed that 2688 and 7411 genes were upregulated and downregulated, respectively, in spring tissues compared to winter. Interestingly, several transcripts encoding heat shock proteins were upregulated in the spring season. RT-qPCR expression results of fifteen wood-forming candidate genes involved in hemicellulose, cellulose, lignin, auxin, and cytokinin pathways showed that the hemicellulose genes (CSLC4, FUT1, AXY4, GATL1, and IRX19) were significantly upregulated in spring season tissues when compared to winter tissues. In contrast, lignin pathway genes CCR1 and CAD1 were upregulated in winter cambium. Finally, a transcriptome-wide marker analysis identified 11,338 Simple Sequence Repeat (SSRs). The AG/CT dinucleotide repeat predominately represented all SSRs. Altogether, the cambial transcriptomic analysis reported here highlights the molecular events of wood formation during winter and spring. The identification of candidate genes involved in the cambial growth provides a roadmap of wood formation in Paulownia and other trees for the seasonal growth variation