BIOINFORMATICS TOOL DEVELOPMENT AND SEQUENCE ANALYSIS OF ROSACEAE FAMILY EXPRESSED SEQUENCE TAGS

BACKGROUND: An international community of researchers has generated a significant number of Expressed Sequence Tags (ESTs) for the Rosaceae, an economically important plant family that includes most temperate fruits such as apple, cherry, peach, and strawberry as well as other commercially valuable members. ESTs are fragments of expressed genes that can be used for gene discovery, developing markers for mapping and cultivar improvement via marker assisted selection. Efficient dissemination and integration of this data is best facilitated through a centralized and curated database with associated sequence analysis tools. DESCRIPTION: The Genome Database for Rosaceae (GDR) was initiated to provide a curated and integrated web-based relational database for this family. I developed a key component of GDR to assemble and annotate the publicly available ESTs from the four main genera of the family (Prunus, Malus, Fragaria, Rosa). I created both genera and family level unigenes using the software CAP3 after extensive filtering, trimming and assembly. Further analysis includes marker mining for single nucleotide polymorphisms (SNPs) and simple sequence repeast (SSRs) with putative primer identification, and oligo identification for potential microarray development. Functional genomics efforts are supported with sequence similarity searching against major protein and nucleotide databases, gene product ontology assignment, and protein motif identification. I deployed the entire project on the GDR with all data available for browsing, searching, and downloading. CONCLUSIONS: The GDR and its associated EST unigene project are meeting a major need for timely annotation and curation of sequence data for the Rosaceae community. The results of my analysis highlight major genes and pathways of interest including ripening, disease resistance, and transcription factors. The easily accessible pool of annotated coding sequences should further both functional and structural genomics characterization in Rosaceae. The unigene elucidates the levels of sequence similarity shared across different plant species and the implications for resource sharing across the family. GDR can be accessed at http://www.rosaceae.org/

Developing expressed sequence tag libraries and the discovery of simple sequence repeat markers for two species of raspberry (\u3cem\u3eRubus\u3c/em\u3e L.)

Background Due to a relatively high level of codominant inheritance and transferability within and among taxonomic groups, simple sequence repeat (SSR) markers are important elements in comparative mapping and delineation of genomic regions associated with traits of economic importance. Expressed sequence tags (ESTs) are a source of SSRs that can be used to develop markers to facilitate plant breeding and for more basic research across genera and higher plant orders. Methods Leaf and meristem tissue from ‘Heritage’ red raspberry (Rubus idaeus) and ‘Bristol’ black raspberry (R. occidentalis) were utilized for RNA extraction. After conversion to cDNA and library construction, ESTs were sequenced, quality verified, assembled and scanned for SSRs. Primers flanking the SSRs were designed and a subset tested for amplification, polymorphism and transferability across species. ESTs containing SSRs were functionally annotated using the GenBank non-redundant (nr) database and further classified using the gene ontology database. Results To accelerate development of EST-SSRs in the genus Rubus (Rosaceae), 1149 and 2358 cDNA sequences were generated from red raspberry and black raspberry, respectively. The cDNA sequences were screened using rigorous filtering criteria which resulted in the identification of 121 and 257 SSR loci for red and black raspberry, respectively. Primers were designed from the surrounding sequences resulting in 131 and 288 primer pairs, respectively, as some sequences contained more than one SSR locus. Sequence analysis revealed that the SSR-containing genes span a diversity of functions and share more sequence identity with strawberry genes than with other Rosaceous species. Conclusion This resource of Rubus-specific, gene-derived markers will facilitate the construction of linkage maps composed of transferable markers for studying and manipulating important traits in this economically important genus

Soil indigenous microbiome and plant genotypes cooperatively modify soybean rhizosphere microbiome assembly

Background: Plants have evolved intimate interactions with soil microbes for a range of beneficial functions including nutrient acquisition, pathogen resistance and stress tolerance. Further understanding of this system is a promising way to advance sustainable agriculture by exploiting the versatile benefits offered by the plant microbiome. The rhizosphere is the interface between plant and soil, and functions as the first step of plant defense and root microbiome recruitment. It features a specialized microbial community, intensive microbe-plant and microbe-microbe interactions, and complex signal communication. To decipher the rhizosphere microbiome assembly of soybean (Glycine max), we comprehensively characterized the soybean rhizosphere microbial community using 16S rRNA gene sequencing and evaluated the structuring influence from both host genotype and soil source. Results: Comparison of the soybean rhizosphere to bulk soil revealed significantly different microbiome composition, microbe-microbe interactions and metabolic capacity. Soil type and soybean genotype cooperatively modulated microbiome assembly with soil type predominantly shaping rhizosphere microbiome assembly while host genotype slightly tuned this recruitment process. The undomesticated progenitor species, Glycine soja, had higher rhizosphere diversity in both soil types tested in comparison to the domesticated soybean genotypes. Rhizobium, Novosphingobium, Phenylobacterium, Streptomyces, Nocardioides, etc. were robustly enriched in soybean rhizosphere irrespective of the soil tested. Co-occurrence network analysis revealed dominant soil type effects and genotype specific preferences for key microbe-microbe interactions. Functional prediction results demonstrated converged metabolic capacity in the soybean rhizosphere between soil types and among genotypes, with pathways related to xenobiotic degradation, plant-microbe interactions and nutrient transport being greatly enriched in the rhizosphere. Conclusion: This comprehensive comparison of the soybean microbiome between soil types and genotypes expands our understanding of rhizosphere microbe assembly in general and provides foundational information for soybean as a legume crop for this assembly process. The cooperative modulating role of the soil type and host genotype emphasizes the importance of integrated consideration of soil condition and plant genetic variability for future development and application of synthetic microbiomes. Additionally, the detection of the tuning role by soybean genotype in rhizosphere microbiome assembly provides a promising way for future breeding programs to integrate host traits participating in beneficial microbiota assembly

Soil indigenous microbiome and plant genotypes cooperatively modify soybean rhizosphere microbiome assembly

Background Plants have evolved intimate interactions with soil microbes for a range of beneficial functions including nutrient acquisition, pathogen resistance and stress tolerance. Further understanding of this system is a promising way to advance sustainable agriculture by exploiting the versatile benefits offered by the plant microbiome. The rhizosphere is the interface between plant and soil, and functions as the first step of plant defense and root microbiome recruitment. It features a specialized microbial community, intensive microbe-plant and microbe-microbe interactions, and complex signal communication. To decipher the rhizosphere microbiome assembly of soybean (Glycine max), we comprehensively characterized the soybean rhizosphere microbial community using 16S rRNA gene sequencing and evaluated the structuring influence from both host genotype and soil source. Results Comparison of the soybean rhizosphere to bulk soil revealed significantly different microbiome composition, microbe-microbe interactions and metabolic capacity. Soil type and soybean genotype cooperatively modulated microbiome assembly with soil type predominantly shaping rhizosphere microbiome assembly while host genotype slightly tuned this recruitment process. The undomesticated progenitor species, Glycine soja, had higher rhizosphere diversity in both soil types tested in comparison to the domesticated soybean genotypes. Rhizobium, Novosphingobium, Phenylobacterium, Streptomyces, Nocardioides,etc. were robustly enriched in soybean rhizosphere irrespective of the soil tested. Co-occurrence network analysis revealed dominant soil type effects and genotype specific preferences for key microbe-microbe interactions. Functional prediction results demonstrated converged metabolic capacity in the soybean rhizosphere between soil types and among genotypes, with pathways related to xenobiotic degradation, plant-microbe interactions and nutrient transport being greatly enriched in the rhizosphere. Conclusion This comprehensive comparison of the soybean microbiome between soil types and genotypes expands our understanding of rhizosphere microbe assembly in general and provides foundational information for soybean as a legume crop for this assembly process. The cooperative modulating role of the soil type and host genotype emphasizes the importance of integrated consideration of soil condition and plant genetic variability for future development and application of synthetic microbiomes. Additionally, the detection of the tuning role by soybean genotype in rhizosphere microbiome assembly provides a promising way for future breeding programs to integrate host traits participating in beneficial microbiota assembly

Substantial genome synteny preservation among woody angiosperm species: comparative genomics of Chinese chestnut (\u3cem\u3eCastanea mollissima\u3c/em\u3e) and plant reference genomes

Background Chinese chestnut (Castanea mollissima) has emerged as a model species for the Fagaceae family with extensive genomic resources including a physical map, a dense genetic map and quantitative trait loci (QTLs) for chestnut blight resistance. These resources enable comparative genomics analyses relative to model plants. We assessed the degree of conservation between the chestnut genome and other well annotated and assembled plant genomic sequences, focusing on the QTL regions of most interest to the chestnut breeding community. Results The integrated physical and genetic map of Chinese chestnut has been improved to now include 858 shared sequence-based markers. The utility of the integrated map has also been improved through the addition of 42,970 BAC (bacterial artificial chromosome) end sequences spanning over 26 million bases of the estimated 800 Mb chestnut genome. Synteny between chestnut and ten model plant species was conducted on a macro-syntenic scale using sequences from both individual probes and BAC end sequences across the chestnut physical map. Blocks of synteny with chestnut were found in all ten reference species, with the percent of the chestnut physical map that could be aligned ranging from 10 to 39 %. The integrated genetic and physical map was utilized to identify BACs that spanned the three previously identified QTL regions conferring blight resistance. The clones were pooled and sequenced, yielding 396 sequence scaffolds covering 13.9 Mbp. Comparative genomic analysis on a microsytenic scale, using the QTL-associated genomic sequence, identified synteny from chestnut to other plant genomes ranging from 5.4 to 12.9 % of the genome sequences aligning. Conclusions On both the macro- and micro-synteny levels, the peach, grape and poplar genomes were found to be the most structurally conserved with chestnut. Interestingly, these results did not strictly follow the expectation that decreased phylogenetic distance would correspond to increased levels of genome preservation, but rather suggest the additional influence of life-history traits on preservation of synteny. The regions of synteny that were detected provide an important tool for defining and cataloging genes in the QTL regions for advancing chestnut blight resistance research

Overexpression of Strigolactone-Associated Genes Exerts Fine-Tuning Selection on Soybean Rhizosphere Bacterial and Fungal Microbiome

Strigolactones are a recently discovered class of carotenoid-derived plant hormones with a wide variety of functions, including acting as signaling molecules in the rhizosphere to promote arbuscular mycorrhizal fungi colonization and parasitic seed germination. To determine whether strigolactones influence the recruitment of microbes to the rhizosphere, we characterized both bacterial and fungal communities in response to the overexpression of genes involved in strigolactone biosynthesis (MAX1d) and signaling perception (D14 and MAX2a) in soybean (Glycine max). The amplicon sequencing-based results suggest that strigolactone overexpression lines had altered soybean rhizosphere bacteria composition at both the community level and individual taxa level with genera including Shinella and Bdellovibrio consistently more abundant across all three overexpression constructs. In addition, the responses of the soybean rhizosphere bacteria community differed significantly across the genes, with lines overexpressing genes involved in strigolactone biosynthesis (MAX1d) yielding a divergent bacterial community in comparison with those with altered expression of strigolactone perception genes (D14 and MAX2a). The overexpressed genes did not significantly impact the overall fungal community distribution; however, some individual taxa were altered in abundance. MAX1d and D14 overexpression lines had significantly enriched abundance of Fusarium solani. The mediating role of strigolactone biosynthesis and signaling genes on soybean rhizosphere bacterial and fungal communities confirmed strigolactone’s importance in the rhizosphere host–microbe communication and microbial community structure

The methylome of soybean roots during the compatible interaction with the soybean cyst nematode, Heterodera glycines

Soybean cyst nematode (SCN, Heterodera glycines) induces the formation of a multinucleated feeding site, or syncytium, whose etiology includes massive gene expression changes. Nevertheless, the genetic networks underlying gene expression control in the syncytium are poorly understood. DNA methylation is a critical epigenetic mark that plays a key role in regulating gene expression. To determine the extent to which DNA methylation is altered in soybean roots during the susceptible interaction with SCN, we generated whole-genome cytosine methylation maps at single nucleotide resolution. The methylome analysis revealed that SCN induces hypo-methylation to a much higher extent than hyper-methylation. We identified 2,465 differentially hyper-methylated regions and 4,692 hypo-methylated regions in the infected roots compared with the non-infected control. In addition, a total number of 703 and 1346 unique genes were identified as overlapping with hyper- or hypo-methylated regions, respectively. The differential methylation in genes apparently occurs independently of gene size and GC content but exhibits strong preference for recently duplicated paralogs. Furthermore, a set of 278 genes was identified as specifically syncytium differentially methylated genes. Of these, we found genes associated with epigenetic regulation, phytohormone signaling, cell wall architecture, signal transduction and ubiquitination. This study provides new evidence that differential methylation is part of the regulatory mechanisms controlling gene expression changes in the nematode-induced syncytium, which seems to be heavily influenced by the traditional well-known transcription factor-based regulatory mechanisms

An Extension of the Plant Ontology Project Supporting Wood Anatomy and Development Research

A wealth of information on plant anatomy and morphology is available in the current and historical literature, and molecular biologists are producing massive amounts of transcriptome and genome data that can be used to gain better insights into the development, evolution, ecology, and physiological function of plant anatomical attributes. Integrating anatomical and molecular data sets is of major importance to the field of wood science, but this is often hampered by the lack of a standardized, controlled vocabulary that allows for cross-referencing among disparate data types. One approach to overcome this obstacle is through the annotation of data using a common controlled vocabulary or “ontology” (Ashburner et al. 2000; Smith et al. 2007). An ontology is a way of representing knowledge in a given domain that includes a set of terms to describe the classes in that domain, as well as the relationships among terms. Each term can be associated with an array of data such as names, definitions, identification numbers, and genes involved. Ontologies are fundamental for unifying diverse terminologies and are increasingly used by scientists, philosophers, the military and online web search engines. In an ontology, terms are carefully defined, allowing a wide array of researchers to (1) use terms consistently in scientific publications or standardized handbooks on quality/trait evaluations, and (2) search for and integrate data linked to these terms in anatomical, genetic, genomic, and other types of biological databases.This is the publisher’s final pdf. The published article is copyrighted by the International Association of Wood Anatomists and can be found at: http://science.naturalis.nl/media/337871/lens%20et%20al-2012%20iawa%20j%20plant%20ontology.pdf)

Preliminary Genomic Characterization of Ten Hardwood Tree Species from Multiplexed Low Coverage Whole Genome Sequencing

Forest health issues are on the rise in the United States, resulting from introduction of alien pests and diseases, coupled with abiotic stresses related to climate change. Increasingly, forest scientists are finding genetic/genomic resources valuable in addressing forest health issues. For a set of ten ecologically and economically important native hardwood tree species representing a broad phylogenetic spectrum, we used low coverage whole genome sequencing from multiplex Illumina paired ends to economically profile their genomic content. For six species, the genome content was further analyzed by flow cytometry in order to determine the nuclear genome size. Sequencing yielded a depth of 0.8X to 7.5X, from which in silico analysis yielded preliminary estimates of gene and repetitive sequence content in the genome for each species. Thousands of genomic SSRs were identified, with a clear predisposition toward dinucleotide repeats and AT-rich repeat motifs. Flanking primers were designed for SSR loci for all ten species, ranging from 891 loci in sugar maple to 18,167 in redbay. In summary, we have demonstrated that useful preliminary genome information including repeat content, gene content and useful SSR markers can be obtained at low cost and time input from a single lane of Illumina multiplex sequence.All raw sequence data are available through NCBI SRA under project number SRP021923. Sequences containing SSRs and designed primers are attached as supplementary material