Manyuan Long - US grants

The questions to be asked center on the molecular evolutionary mechanisms that create initial gene structures and control subsequent evolution. The objectives of this research are: (1) to understand molecular evolutionary relationships among the new genes in the jgw (jinwei)-ynd(yande) gene system by characterizing their gene structures and flanking sequences in D. teisseiri, D, yakuba, and D melanogaster, using various methods of molecular analysis (2) to determine when the two mosiac genes, ynd and ymp(yellow- emperor) originated,by determining their phylogenetic distribution using molecular phylogenetic techniques (3) to determine the roles of various evolutionary forces governing the early stages of new genes, by analyzing molecular evolution and population genetics of jgw, ynd, and ymp (4) in order to look for the correspondence between the newly evolved function and the fast sequence evolution of jgw as revealed by previous analysis of molecular population genetics, the biochemical functions of the jgw proteins at three stages: the ancestral jgw when first created, the evolved ancestral jgw before speciation, and the modern jgw genes will be investigated. The two ancestral jgw sequences are inferred by evolutionary analysis and they will be made by site-direced mutagenesis.

The long-term goal of this research is to elucidate how new genes with novel functions originate, a fundamentally important problem of gene evolution. Studying the origin of a gene in detail requires the discovery of a young gene, since the early evolution of old genes has often been obscured by later evolutionary processes. The recently identified jingwei-yande gene system in Drosophila contains several genes only 2.5 to 30 million years old jingwei (jgw) originated as an Adh retrosequence that recruited several additional exons, while yande (ynd) evolved through exon recombination with yellow-emperor (ymp). Therefore, these young genes, with other related genes in the gene system that evolved recently in gene structure and copy number, provide an excellent opportunity to examine early processes in the origin of new genes.

In this project, Dr. Long investigates the rates and patterns of new gene origination in the model organism Drosophila (the fruit fly) by using experimental and computational genomic analysis in research and teaching. Previous investigations have revealed a number of young genes with peculiar evolutionary features, which include the rapid emergence of novel expression patterns, accelerated rates of evolution, and an asymmetrical distribution of new gene donor sequences within the Drosophila genome. This project initiates a large-scale effort to explore gene originations via recent (within 10 million years) retroposition events in Drosophila genomes. (1) The investigator will measure the rate of new gene origination via retroposition by undertaking a genomic screen of recently evolved genes in the D. melanogaster subgroup species using an approach integrating genomic analysis and molecular evolution. He will identify recently retroposed genes in the D. melanogaster genome by means of genomic hybridization experiments and computational analysis of D. melanogaster genome sequences. (2) He recently observed in his laboratory the first pattern of new gene origination, asymmetric retroposition. New autosomal genes that retroposed from X-linked parental genes are more common than are new X-linked genes that retroposed from autosomal parental genes. (3) He will investigate divergence in function of new genes by examining their tissue-specific expression patterns using DNA microarrays and other RNA analysis techniques. (4) He will train young researchers at different levels, including undergraduates, graduates, and postdoctoral fellows, to meet the urgent needs in the field. He will enhance the education of undergraduates at selected small midwestern colleges by giving lectures on evolutionary genomics at the colleges and providing summer research projects in his laboratory to two students from these schools each year.

These studies will add significantly to the understanding of one of the most basic phenomena of biology: how do new genes and coding portions of genomes evolve? Not only does this work promise to enhance our knowledge base, the large number of new genes identified will also provide a valuable resource for the further study of evolutionary genomics. Furthermore, this project will train young scientists, including undergraduates, in the broad issues of evolutionary genomics

DESCRIPTION (provided by applicant): The objective of this research is to study evolutionary forces that govern unusual variation of the D. melanogaster fourth chromosome uncovered recently by us. For a long time, this chromosome has been viewed as a canonical textbook example of how natural selection can result in low within-species variation in a genome region of low recombination. Our preliminary work has indicated that the previous conclusion that the entire chromosome 4 harbors low variation is incorrect and should not be taken as a paradigm any more. We have found a high level of nucleotide variation in large blocks of this chromosome in D. melanogaster and simulans. The following objectives will be pursued. (1) Using Monte Carlo simulations based on the coalescent process with selection and recombination, we will investigate a theoretical population genetic process associated with a low average recombination rate in the whole chromosome but with sporadic recombination hot spots. We expect that this theoretical analysis will shed new light on the relationship between recombination and variation. (2) To determine the length of the high variation region in the chromosome, we will characterize the polymorphism distribution in natural populations in whole euchromatin regions of the D. melanogaster fourth chromosome. (3) To compare the high variation region of D. melanogaster with the corresponding region in other sibling species, we will investigate sequence variation in D. simulans and D. yakuba. Finally, using these theoretical results and variation data, we will test several hypotheses that might explain the variation and evolution of the chromosome: selective sweep, background selection, balancing selection, and neutrality. We will also characterize the variation and evolution of the sphinx gene, a gene that recently originated on the D. melanogaster fourth chromosome, with structural and expression analysis, and investigate its potential role in the evolution of the chromosome. These studies should add significantly to the understanding of the evolutionary genetics of the Drosophila fourth chromosome.

Studies of new gene origination provide insight into the mechanisms by which biological diversity is created. Dr. Manyuan Long and James Emerson will employ microarray technology to identify genes in the fruit fly that have originated so recently that they have yet to spread through the entire population. Subsequent analysis using comparative genomics will allow us to determine how new genes come to be preserved. They will estimate the fitness effects of these new genes by comparing data within one species (Drosophila melanogaster) to data between species (D. melanogaster vs D. pseudoobscura). They will make the most accurate estimate of the gene duplication rate to date, which will be the first such study of new gene acquisition in any organism.

These studies will influence human welfare: for example, duplications of resistance genes frequently confer pesticide resistance, and gene amplifications frequently lead to tumorogenesis. For this project we plan to train young scientists from institutions that do not have access to facilities that allow such research to be conducted.

[unreadable] DESCRIPTION (provided by applicant): The objective of this research is to systematically study new genes in the Drosophila melanogaster subgroup and to investigate the tempo and mode of new gene originations. We will focus on the new chimerical genes created by RNA-involved retroposition that often evolve novel functions associated with the mosaic gene structures. To meet these objectives, the following four-prong integrated approaches will be pursued using the D. melanogaster subgroup as our model taxa. (1) Detection of New Genes by Genomic Analyses: Taking advantage of the known divergence times within the subgroup (0.5-12 million years (mys)), new gene detection will be based upon an interdisciplinary approach that integrates computational and experimental genomic analyses. Our computational analyses will utilize available genome sequences and work in conjunction with Fluorescence In Situ Hybridization with multiple species genomes (MFISH) and Comparative Genomic Hybridization (CGH) using DMA microarrays. These analyses will detect and provide new gene candidates on the genome-wide scale. (2) Inference of the Functionality of the New Genes: To infer functionality for these new genes we will examine the molecular evolutionary and transcriptional constraints of each new gene and their temporal-spatial expression patterns. Molecular evolution and population genetic analyses will be also conducted to detect adaptive evolution of new functional genes. (3) Analysis of phylogenetic Distribution of New Gene Originations: We will map all detected origination events of new genes on the phylogenetic tree of the melanogaster subgroup species. We will estimate the rate of new gene origination and its variation along branches of the phylogeny, using related statistical analyses including maximum likelihood approach. (4) Sex Chromosome-dependence of the Origination of New Genes: Taking advantage of the young ages of the identified new genes (between 0.5 and 12 mys), we will investigate whether or not the male gene traffic that we observed in ancient retrogene samples is an on- going process by analyzing the chromosomal location distribution of new genes and their parental genes. The results from these four categories of studies will generate the first phylogenetic distribution of new genes and provide the fresh-insight into the rates and patterns of new gene originations in genomes. [unreadable] [unreadable] [unreadable]

PI: Rod A. Wing (University of Arizona)

CoPIs: Scott A. Jackson (Purdue University), Manyuan Long (University of Chicago), Carlos A. Machado (University of Maryland), and Michael J. Sanderson (University of Arizona)

Collaborators: O. Panaud (University of Perpignan, France) and D. Weigel (Max Planck Institute for Developmental Biology, Germany), Doreen Ware (Cold Spring Harbor Laboratory), Qifa Zhang and Sibin Yu (Huazhong Agricultural University, China), Bin Han (National Center for Gene Research, China), Marco Wopereis (Africa Rice Center (WARDA), Benin), Mathias Lorieux (International Center for Tropical Agriculture (CIAT), Columbia, and Georgia Eizenga (DB NRRC, Germany.

Intellectual merit. Asian cultivated rice (Oryza sativa) feeds more people than any other crop. As the rice-dependent population is expected to double in about 25 years, breeders are faced with the enormous task of doubling rice yields with less land, water, and fertilizers, and on poorer soils. It is therefore critical that the scientific community unites to provide the tools and biological/evolutionary insights required to meet future needs. The project's long-term goal is to exploit 15 million years of evolutionary diversification and adaptation that have been locked in the genomes of wild and domesticated Oryza species, to both improve cultivated rice and address fundamental questions in evolutionary and comparative genomics. To accomplish this goal the project will first generate a set of publicly available genomics resources for the genus Oryza including: i) physical maps for three wild relatives of cultivated rice and the closely related outgoup species Leersia perrieri; ii) complete sequences for the short arms of chromosome 3 of these species; iii) complete reference genome sequences for O. barthii (the wild progenitor of West African cultivated rice), and O. punctata (a wild species that will serve as the evolutionary outgroup the eight AA genome species--the species group containing O. sativa); iv) gene and small RNA expression data sets for 11 Oryza species and L. perrieri; and v) DNA polymorphism data sets across the AA genome phylogeny. The project will integrate these new resources with 5 to 8 forthcoming new Oryza reference genome sequences to create the most comprehensive within-genus comparative genomics platform for any plant system. The project will interrogate this comparative genomics platform to discover key evolutionary events and characterize the forces that led to the formation of 24 Oryza species with 10 different genome types. Specific research areas that will be addressed include: analyses of structural variation, phylogenomics, population genomics, genome evolution and the origin of new genes, and the role of transposable elements in genome evolution. Libraries, genome sequences and their annotations will be accessible via public databases and repositories, such as the AGI BAC/TEST Resource Center (http://www.genome.arizona.edu/orders/), NCBI (http://www.ncbi.nlm.nih.gov/), and Gramene (www.gramene.org).

Broader impacts. This project will have broad impacts at several levels, including basic and applied research communities, postdoctoral/graduate/undergraduate and high school student training and mentoring, community outreach, and K-5 children and their families. For basic/applied research communities, the project will enhance and develop a genus-level resource as a platform for accessing new traits and alleles that is unparalleled in the plant world. This transformative platform can be used to address fundamental hypothesis-driven questions as well as provide essential tools to improve the world's most important food crop. The project will provide training and mentoring to postdoctoral scientists, graduate/undergraduate students and high school students interested in genome evolution, plant breeding, and careers in academic and corporate science. Finally, the project will develop a biannual "Plant Science Family Night" program, targeting K-5 students and families, to get children and their families excited about plants and the role plant science plays in ensuring a safe, sustainable, and secure food supply for our planet.

To understand the evolutionary diversification of animals, the applicants will investigate the variation of phenotypes and their genetic basis in multiple species of fruitflies in the genus Drosophila. The connection to diverse phenotypic features has been mapped to protein-coding changes, cis-regulatory changes and lineage-specific genes. The applicants' recent discoveries showed that many young genes played essential roles in organism development. This research will (1) investigate the reproductive contribution of young genes using the evolutionary genetic analysis and gene silencing techniques, (2) detect their interactions with other genes in the genome using the gene-chip based techniques of experimental genomics, and (3) study their roles in the species divergence by comparative analysis of related species. These efforts will advance the understanding of fundamental problems of gene evolution and phenotypic evolution.

These studies will bring conceptual innovations in the understanding of biological systems. The applicants will also create new data and share with the public. Furthermore, the applicants will train undergraduate students, visiting students from other schools and different disciplines, to encourage more talented undergraduates to enter graduate schools and nurture cross-disciplinary investigation. The applicants will purposely initiate more collaborate with multiple research groups in various institutions both nationally and internationally. The knowledge on gene origination is important to the understanding of human evolution. The proposed project will be of general interest to the broad audience including scientists and non-scientists and the discoveries from this project will play a role in scientific education for the general public.

Intellectual Merit: Gene duplication is a mechanism to generate new genes, an important genetic novelty for evolution of new functions associated with phenotypic variation. How a gene duplicate arises and changes in abundance over time in a species population is unclear. This research aims to understand the genetic mechanisms and evolutionary forces that lead to new gene functions in some individuals in a population and not in others. Using fruit flies as a model, this study applies an integrated approach to address key questions related to the evolution of new gene functions on a population level. First, to determine the frequency of tandem versus dispersed duplications, hundreds of polymorphic gene duplicates, known to be present at intermediate or high frequencies, will assayed for their chromosomal locations and sequences, using florescence labeling and other techniques. Second, to test the importance of evolutionary constraints related to functionality of the gene duplicates, sequence variation and expression patterns of the new gene duplicates will be examined and compared among functional and non-functional sites. Third, the influence of evolutionary forces governing survival and natural selection will be investigated by comparing within- and between-species variation. Together, the results of these experiments will provide important insights into the evolution of new gene functions.

Broader Impacts: This project will generate a large well-characterized dataset of polymorphic duplicates that will be made publicly available. This project will be closely tied to the education of students and the public and will promote education of under-represented ethnic groups in the communities neighboring the University of Chicago. In collaboration with Chicago State University (CSU), this project will initiate a new direction of teaching and research training in molecular evolution for CSU biology students, including opportunities to participate in summer research, to take undergraduate courses about genome evolution, and to engage in related educational activities. In addition, this project will train graduate students and postdoctoral students, thus contributing to their career development as independent scientists.

DESCRIPTION (provided by applicant): Although energy homeostasis is central in feeding behavior, the decision to eat is not merely a question of hunger. In a natural environment in which food is often scarce, evaluating the benefit of gaining food over the energy cost or risk in seeking and acquiring food, and the decision to either seek food or stay put and conserve energy is essential for survival. However, the genetic and molecular basis of food-seeking decision-making behaviors, and their functional significance in fitness are poorly understood. We propose a bottom-up approach: starting from well-defined behaviors that are essential for food-seeking decision-making in Drosophila, taking advantage of its rich genetic tools and genomic resources. Moreover, flies are ideal for viability studies to examine the relations between genetic variations, differences in decision- making and differences in fitness under different environmental conditions. We have already taken the critical step and designed novel assays. We will optimize the following assays for quantitative analysis in this application. Decision-making in choosing between 1) small free reward or large reward that requires work; 2) small reward or large reward with a relatively low probability in its availability; 3) small reward or large reward that is paired with potential risks of getting noxious food; and 4) immediate small reward or delayed large reward. We will take advantage of natural variation in genetics and behaviors between different D. melanogaster populations living in African. We will establish 200 recombinant inbred strains derived from original African strains with extreme behavioral phenotypes for each decision-making behavior. We will perform QTL mapping and identify QTLs that contribute to the variations in food-seeking decision-making and fitness. We will then use RNAi transgenic lines to identify specific genes and variants responsible for the above behavioral variations. Although the most likely immediate clinical relevance of our study is obesity, it will have a much broader impact on neuroeconomics, the genetic basis of behavior in general, evolution and ecology.

Project Summary Several compelling questions in genome sequence analysis have been compromised by errors and gaps in the available genome assemblies. A telomere-to-telomere platinum-quality genome sequence of a human would open doors to investigating many problems associated with genetic disease, and development of platinum-quality model organism genomes will allow early exploration of the most efficient ways to pursue these questions. To demonstrate the utility of multiple reference-quality genomes, we have formulated several questions about genome evolution that make use of the Drosophila model system. These questions include (1) identifying new genes that originated within the Drosophila-specific clade, (2) estimating the rates of new gene evolution and examine the variation and constancy of those rates among Drosophila lineages, (3) quantifying rates and patterns of divergence of piRNA clusters, critical to host regulation of transposable elements, (4) analysis of sequence divergence in heterochromatic repeats, known to play key roles in centromere and telomere function as well as modulating chromatin states, and (5) analysis of Y chromosome gene and loss across the pan-Y chromosome. By obtaining and annotating reference-quality genome sequences of 19 Drosophila species spanning 40-60 MY of evolutionary history, using an efficient scheme that combines deep long-read (PacBio) assembly coupled with targeted sequencing of bacterial artificial chromosomes, we will produce a resource that will pave the way for the Drosophila community to tackle pressing hypothesis-driven questions in the field, including embryonic development, neurobiology, and aging ? all within a phylogenomics perspective.

Sexual selection is a major driver of evolution, often resulting in drastically different characteristics between males and females. Recently it has become clear that gene duplication is also a powerful contributor to evolutionary diversification. This research seeks to understand how young gene duplicates evolve sex-specific essential functions under sexually antagonistic selection. Males and females, while sharing most of their DNA, satisfy different functional requirements for reproduction and survival. Sexual selection on traits with a shared genetic basis can have opposing effects in the two sexes, causing sexual conflict (SC) that may reduce fitness until the conflict is resolved by new genetic changes. This project addresses the hypothesis that gene duplication is an effective mechanism to resolve sexual conflict, using the fruit fly as a convenient model organism. In pursuing these aims, postdocs, graduates, undergraduates and high school students will be trained in molecular and evolutionary biology. The project will also partner with the Illinois Mathematics and Science Academy to involve diverse high school students in the research through lectures and summer internships.

This project focuses on a pair of recently duplicated genes (Apl and Arts) in Drosophila that have opposing fitness effects in the two sexes. The functional evolution in the protein sequences will be characterized to understand the role of protein divergence in mitigation of sexual conflict. The ancestral gene from which Apl and Arts were derived will then be computationally reconstructed and experimentally synthesized. A series of engineered Drosophila strains will then be created using gene editing technology, with different versions of the synthesized ancestral gene replacing the wildtype gene locus which harbors Apl and Arts. Divergence in expression of constructs with different regulatory regions will reveal their role in mitigating sexual conflict. The generality of the duplication-mediated SC resolution model will be tested by examining a score of recent gene duplicates with sex complementary expression patterns using gene knockout and rescue analyses.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.