Yingjia Shen

Development of Genome-Wide DNA Polymorphism Database for Map-Based Cloning of Rice Genes

DNA polymorphism is the basis to develop molecular markers that are widely used in genetic mapping today. A genome-wide rice (Oryza sativa) DNA polymorphism database has been constructed in this work using the genomes of Nipponbare, a cultivar of japonica, and 93-11, a cultivar of indica. This database contains 1,703,176 single nucleotide polymorphisms (SNPs) and 479,406 Insertion/Deletions (InDels), approximately one SNP every 268 bp and one InDel every 953 bp in rice genome. Both SNPs and InDels in the database were experimentally validated. Of 109 randomly selected SNPs, 107 SNPs (98.2%) are accurate. PCR analysis indicated that 90% (97 of 108) of InDels in the database could be used as molecular markers, and 68% to 89% of the 97 InDel markers have polymorphisms between other indica cultivars (Guang-lu-ai 4 and Long-te-pu B) and japonica cultivars (Zhong-hua 11 and 9522). This suggests that this database can be used not only for Nipponbare and 93-11, but also for other japonica and indica cultivars. While validating InDel polymorphisms in the database, a set of InDel markers with each chromosome 3 to 5 marker was developed. These markers are inexpensive and easy to use, and can be used for any combination of japonica and indica cultivars used in this work. This rice DNA polymorphism database will be a valuable resource and important tool for map-based cloning of rice gene, as well as in other various research on rice (http://shenghuan.shnu.edu.cn/ricemarker).

The genome of the platyfish, Xiphophorus maculatus , provides insights into evolutionary adaptation and several complex traits

Several attributes intuitively considered to be typical mammalian features, such as complex behavior, live birth and malignant disease such as cancer, also appeared several times independently in lower vertebrates. The genetic mechanisms underlying the evolution of these elaborate traits are poorly understood. The platyfish, X. maculatus, offers a unique model to better understand the molecular biology of such traits. We report here the sequencing of the platyfish genome. Integrating genome assembly with extensive genetic maps identified an unexpected evolutionary stability of chromosomes in fish, in contrast to in mammals. Genes associated with viviparity show signatures of positive selection, identifying new putative functional domains and rare cases of parallel evolution. We also find that genes implicated in cognition show an unexpectedly high rate of duplicate gene retention after the teleost genome duplication event, suggesting a hypothesis for the evolution of the behavioral complexity in fish, which exceeds that found in amphibians and reptiles.

Genome level analysis of rice mRNA 3′-end processing signals and alternative polyadenylation

The position of a poly(A) site of eukaryotic mRNA is determined by sequence signals in pre-mRNA and a group of polyadenylation factors. To reveal rice poly(A) signals at a genome level, we constructed a dataset of 55 742 authenticated poly(A) sites and characterized the poly(A) signals. This resulted in identifying the typical tripartite cis-elements, including FUE, NUE and CE, as previously observed in Arabidopsis. The average size of the 3′-UTR was 289 nucleotides. When mapped to the genome, however, 15% of these poly(A) sites were found to be located in the currently annotated intergenic regions. Moreover, an extensive alternative polyadenylation profile was evident where 50% of the genes analyzed had more than one unique poly(A) site (excluding microheterogeneity sites), and 13% had four or more poly(A) sites. About 4% of the analyzed genes possessed alternative poly(A) sites at their introns, 5′-UTRs, or protein coding regions. The authenticity of these alternative poly(A) sites was partially confirmed using MPSS data. Analysis of nucleotide profile and signal patterns indicated that there may be a different set of poly(A) signals for those poly(A) sites found in the coding regions. Based on the features of rice poly(A) signals, an updated algorithm termed PASS-Rice was designed to predict poly(A) sites.

RNA-Seq reveals complex genetic response to deepwater horizon oil release in Fundulus grandis

BackgroundThe release of oil resulting from the blowout of the Deepwater Horizon (DH) drilling platform was one of the largest in history discharging more than 189 million gallons of oil and subject to widespread application of oil dispersants. This event impacted a wide range of ecological habitats with a complex mix of pollutants whose biological impact is still not yet fully understood. To better understand the effects on a vertebrate genome, we studied gene expression in the salt marsh minnow Fundulus grandis, which is local to the northern coast of the Gulf of Mexico and is a sister species of the ecotoxicological model Fundulus heteroclitus. To assess genomic changes, we quantified mRNA expression using high throughput sequencing technologies (RNA-Seq) in F. grandis populations in the marshes and estuaries impacted by DH oil release. This application of RNA-Seq to a non-model, wild, and ecologically significant organism is an important evaluation of the technology to quickly assess similar events in the future.ResultsOur de novo assembly of RNA-Seq data produced a large set of sequences which included many duplicates and fragments. In many cases several of these could be associated with a common reference sequence using blast to query a reference database. This reduced the set of significant genes to 1,070 down-regulated and 1,251 up-regulated genes. These genes indicate a broad and complex genomic response to DH oil exposure including the expected AHR-mediated response and CYP genes. In addition a response to hypoxic conditions and an immune response are also indicated. Several genes in the choriogenin family were down-regulated in the exposed group; a response that is consistent with AH exposure. These analyses are in agreement with oligonucleotide-based microarray analyses, and describe only a subset of significant genes with aberrant regulation in the exposed set.ConclusionRNA-Seq may be successfully applied to feral and extremely polymorphic organisms that do not have an underlying genome sequence assembly to address timely environmental problems. Additionally, the observed changes in a large set of transcript expression levels are indicative of a complex response to the varied petroleum components to which the fish were exposed.

Unique Features of Nuclear mRNA Poly(A) Signals and Alternative Polyadenylation in Chlamydomonas reinhardtii

To understand nuclear mRNA polyadenylation mechanisms in the model alga Chlamydomonas reinhardtii, we generated a data set of 16,952 in silico-verified poly(A) sites from EST sequencing traces based on Chlamydomonas Genome Assembly v.3.1. Analysis of this data set revealed a unique and complex polyadenylation signal profile that is setting Chlamydomonas apart from other organisms. In contrast to the high-AU content in the 3′-UTRs of other organisms, Chlamydomonas shows a high-guanylate content that transits to high-cytidylate around the poly(A) site. The average length of the 3′-UTR is 595 nucleotides (nt), significantly longer than that of Arabidopsis and rice. The dominant poly(A) signal, UGUAA, was found in 52% of the near-upstream elements, and its occurrence may be positively correlated with higher gene expression levels. The UGUAA signal also exists in Arabidopsis and in some mammalian genes but mainly in the far-upstream elements, suggesting a shift in function. The C-rich region after poly(A) sites with unique signal elements is a characteristic downstream element that is lacking in higher plants. We also found a high level of alternative polyadenylation in the Chlamydomonas genome, with a range of up to 33% of the 4057 genes analyzed having at least two unique poly(A) sites and ∼1% of these genes having poly(A) sites residing in predicted coding sequences, introns, and 5′-UTRs. These potentially contribute to transcriptome diversity and gene expression regulation.

Identification of transcriptome SNPs between Xiphophorus lines and species for assessing allele specific gene expression within F1 interspecies hybrids

Variations in gene expression are essential for the evolution of novel phenotypes and for speciation. Studying allelic specific gene expression (ASGE) within interspecies hybrids provides a unique opportunity to reveal underlying mechanisms of genetic variation. Using Xiphophorus interspecies hybrid fishes and high-throughput next generation sequencing technology, we were able to assess variations between two closely related vertebrate species, Xiphophorus maculatus and Xiphophorus couchianus, and their F(1) interspecies hybrids. We constructed transcriptome-wide SNP polymorphism sets between two highly inbred X. maculatus lines (JP 163 A and B), and between X. maculatus and a second species, X. couchianus. The X. maculatus JP 163 A and B parental lines have been separated in the laboratory for ≈70 years and we were able to identify SNPs at a resolution of 1 SNP per 49 kb of transcriptome. In contrast, SNP polymorphisms between X. couchianus and X. maculatus species, which diverged ≈5-10 million years ago, were identified about every 700 bp. Using 6524 transcripts with identified SNPs between the two parental species (X. maculatus and X. couchianus), we mapped RNA-seq reads to determine ASGE within F(1) interspecies hybrids. We developed an in silico X. couchianus transcriptome by replacing 90,788 SNP bases for X. maculatus transcriptome with the consensus X. couchianus SNP bases and provide evidence that this procedure overcomes read mapping biases. Employment of the in silico reference transcriptome and tolerating 5 mismatches during read mapping allow direct assessment of ASGE in the F(1) interspecies hybrids. Overall, these results show that Xiphophorus is a tractable vertebrate experimental model to investigate how genetic variations that occur during speciation may affect gene interactions and the regulation of gene expression.

A classification-based prediction model of messenger RNA polyadenylation sites

Messenger RNA polyadenylation is one of the essential processing steps during eukaryotic gene expression. The site of polyadenylation [(poly(A) site] marks the end of a transcript, which is also the end of a gene. A computation program that is able to recognize poly(A) sites would not only prove useful for genome annotation in finding genes ends, but also for predicting alternative poly(A) sites. Features that define the poly(A) sites can now be extracted from the poly(A) site datasets to build such predictive models. Using methods, including K-gram pattern, Z-curve, position-specific scoring matrix and first-order inhomogeneous Markov sub-model, numerous features were generated and placed in an original feature space. To select the most useful features, attribute selection algorithms, such as information gain and entropy, were employed. A training model was then built based on the Bayesian network to determine a subset of the optimal features. Test models corresponding to the training models were built to predict poly(A) sites in Arabidopsis and rice. Thus, a prediction model, termed Poly(A) site classifier, or PAC, was constructed. The uniqueness of the model lies in its structure in that each sub-model can be replaced or expanded, while feature generation, selection and classification are all independent processes. Its modular design makes it easily adaptable to different species or datasets. The algorithms high specificity and sensitivity were demonstrated by testing several datasets and, at the best combinations, they both reached 95%. The software package may be used for genome annotation and optimizing transgene structure.

UVB-induced gene expression in the skin of Xiphophorus maculatus Jp 163 B

Xiphophorus fish and interspecies hybrids represent long-standing models to study the genetics underlying spontaneous and induced tumorigenesis. The recent release of the Xiphophorus maculatus genome sequence will allow global genetic regulation studies of genes involved in the inherited susceptibility to UVB-induced melanoma within select backcross hybrids. As a first step toward this goal, we report results of an RNA-Seq approach to identify genes and pathways showing modulated transcription within the skin of X. maculatus Jp 163 B upon UVB exposure. X. maculatus Jp 163 B were exposed to various doses of UVB followed by RNA-Seq analysis at each dose to investigate overall gene expression in each sample. A total of 357 genes with a minimum expression change of 4-fold (p-adj<0.05) were identified as responsive to UVB. The molecular genetic response of Xiphophorus skin to UVB exposure permitted assessment of; (1) the basal expression level of each transcript for each skin sample, (2) the changes in expression levels for each gene in the transcriptome upon exposure to increasing doses of UVB, and (3) clusters of genes that exhibit similar patterns of change in expression upon UVB exposure. These data provide a foundation for understanding the molecular genetic response of fish skin to UVB exposure.

Characterization of telomeres and telomerase expression in Xiphophorus

Research investigating telomere lengths and telomerase expression in vertebrates has progressively become important due to the association of these two biological endpoints with cellular aging and cancer in humans. Studies that rely upon the traditional use of laboratory mice have been faced with limitations largely due to inbred mice possessing large telomeres and ubiquitous expression of telomerase. Recently, a number of small fish species have been shown to provide potentially informative models for examining the role of telomeres and telomerase within intact vertebrate animals. Xiphophorus fishes represent a new world live-bearing genus that has not previously been assessed for telomere length or telomerase expression. To add to the knowledge base of telomere and telomerase biology in vertebrates we assessed telomere length and telomerase expression among several species of Xiphophorus. The telomere lengths in several organs (gill, brain, eyes, testis, ovary and liver) in three species (Xiphophorus hellerii, Xiphophorus maculatus, Xiphophorus couchianus) and also in F(1) interspecies hybrids were approximately 2-6 kb. This size was consistent within the same organs of the same species, as well as between species and F(1) hybrids. Despite possessing relatively short telomere lengths compared to humans, the consistency of size among Xiphophorus species and organs may allow experimental detection of telomere shortening. The relative expression of telomerase reverse transcriptase (TERT) was determined by quantitative real-time PCR. Expression levels of TERT was measured in seven organs (ovary, testis, liver, gill, brain, heart, skin) from X. maculatus, X. hellerii and in control and ultraviolet light (UVB) exposed skin samples from X. maculatus, X. hellerii, and F(1) interspecies hybrids. TERT gene expression was significantly higher in ovary and testis, while all other organs showed low relative TERT expression. Detectable increases in TERT expression were found in skin samples upon UVB exposure. Our findings suggest that Xiphophorus may serve as a suitable model for future studies investigating the association of telomere length and telomerase expression in regard to aging and disease.

Novel Method for Analysis of Allele Specific Expression in Triploid Oryzias latipes Reveals Consistent Pattern of Allele Exclusion

Assessing allele-specific gene expression (ASE) on a large scale continues to be a technically challenging problem. Certain biological phenomena, such as X chromosome inactivation and parental imprinting, affect ASE most drastically by completely shutting down the expression of a whole set of alleles. Other more subtle effects on ASE are likely to be much more complex and dependent on the genetic environment and are perhaps more important to understand since they may be responsible for a significant amount of biological diversity. Tools to assess ASE in a diploid biological system are becoming more reliable. Non-diploid systems are, however, not uncommon. In humans full or partial polyploid states are regularly found in both healthy (meiotic cells, polynucleated cell types) and diseased tissues (trisomies, non-disjunction events, cancerous tissues). In this work we have studied ASE in the medaka fish model system. We have developed a method for determining ASE in polyploid organisms from RNAseq data and we have implemented this method in a software tool set. As a biological model system we have used nuclear transplantation to experimentally produce artificial triploid medaka composed of three different haplomes. We measured ASE in RNA isolated from the livers of two adult, triploid medaka fish that showed a high degree of similarity. The majority of genes examined (82%) shared expression more or less evenly among the three alleles in both triploids. The rest of the genes (18%) displayed a wide range of ASE levels. Interestingly the majority of genes (78%) displayed generally consistent ASE levels in both triploid individuals. A large contingent of these genes had the same allele entirely suppressed in both triploids. When viewed in a chromosomal context, it is revealed that these genes are from large sections of 4 chromosomes and may be indicative of some broad scale suppression of gene expression.