Progress 10/01/15 to 09/30/20
Outputs
Target Audience:All researchers world-wide who compare genomes. Emphasis of my work is on crop and other plant comparative genomics, and to facilitate such work for other plant/crop researchers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Freeling lab publications are multiply advertised on the web, and on the Freeling Lab websitehttp://Freelinglab.berkeley.edu. Freeling's contributions to CoGe "quality control" are crdited in CoGe "about us". Most importantly, all data reported inany Freelibng lab publication are publically available as clearly stated in each publication. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
In the process of pursuing research-- all in collaboration because of the small size of my current lab- I routinely use the tools in the toolbox CoGe. I am familiar with most CoGe functions and report on bugs and comment on possible improvements. The published or submitted manuscripts evidence my research. The continued popularity of CoGeworldwide tells me that my efforts with CoGe are useful. For this last year of this NIFA project, there were 3 published or pending journal papers. I also include here an abstract of research acomplished this last year, it is still in draft form. The qTeller on-line maize gene expression graphic application (J. Schnable, u. Nebraska, developer), has been under repair for the last two years. The vast majority of my lab's research during this period was published as Grover et al, 2020, and is written-up as a rought draft publication. Funding was from NSF Genomes Projectto our a U. Arizona-UCBerkeley collaboration m (Mosher-Freeling). This grant was operating during the last 4 years of this NIFA project. The 2-pager of this unpublished research is as follows: Significant Results: Tentative Title: CLASSY-dependent , maternal smRNAs are abundant in ovule and endosperm from loci (sirens) adjacent to particular families of transposons or gene fragments. Ovule 24-nt siRNAs have a much lower association with transposons and repetitive sequences compared to leaf 24-nt siRNAs. Hot spots for production of 24-nt siRNAs (sirens) also exist in ovule but are rare in leaves. During the past year we continued our characterization of siren loci in B. rapa and A. thaliana. B. rapa ovule 24-nt read counts negatively correlate with TE coverage and positively correlate with gene coverage (Fig. 1). Sixty-seven percent of siren loci overlap an annotated TE. For siren loci TE coverage is highest at the edge of the siren, which is the region with the lowest average read count (Fig. 2). When we looked at which TEs overlap sirens we found two families that are associated with a large number of sirens. A helitron family is associated with 19% of sirens (Fig. 3) and a non-autonomous hAT family is associated with 4.5% of sirens (Fig. 4). To determine whether the hAT family is in general associated with 24-nt hot spots we identified 5 additional family members and found that four of these hAT family members were associated with 24-nt clusters. The hAT family members associated with 24-nt clusters had a 200 bp region in common which was absent from the hAT family member not associated with 24-nt clusters. Similarly, for the helitron family a 74 bp region was present in all siren-associated helitron family members, even those in which most of the reference helitron sequence had been deleted. Three motifs were found to be present in both the 200 bp hAT consensus sequence and the 74 bp helitron consensus sequence (Fig. 5). Whether these motifs are biologically relevant will require further work. How genomic regions are targeted for RNA-directed DNA methylation is not understood. In Arabidopsis the four members of the CLASSY (CLSY) family of SNF2-related chromatin-remodeling factors regulate largely non-overlapping 24 nt-producing loci (1). To determine whether the characteristics that differentiate ovule from leaf 24-nt siRNA clusters could be related to their association with different CLSY family members, we overlapped A. thaliana sirens with flower bud loci dependent on each CLSY family gene (2). All 57 A. thaliana sirens examined overlapped with CLSY3/4-dependent loci. The predominant CLSY gene expressed in ovule in both A. thaliana and B. rapa is CLSY3 whereas CLSY1 is the predominant gene expressed in leaf (Fig. 6). A substantial number of B. rapa and A. thaliana sirens overlap pseudogenes. In A.thaliana 31% of sirens overlap pseudogenes from a list compiled by Zou et al (1). In B. rapa 36% of sirens overlap annotated genes; the majority of these are likely to be pseudogenes since they are unusually small or incomplete relative to best hits from A. thaliana and B. rapa. To determine if sirens overlap additional unannotated gene fragments, we used siren sequences to search for blastn and tblastx hits to A.thaliana and B. rapa genes. 33% of sirens were found to overlap unannotated gene fragments. One siren, for instance, had adjacent gene fragments related to a protein kinase that phosphorylates an auxin efflux carrier and to a pectinesterase expressed during early silique development. The juxtaposition of unrelated gene fragments is commonly associated with helitrons and this siren overlaps a truncated member of the helitron family discussed above. The majority of 24-nt siRNAs overlap the pectinesterase gene fragment rather than the helitron fragment. Transcript accumulation of the best hit B. rapa pectinesterase gene is greatly impacted by nrpd1a in both ovule and 10 dpf seed, increasing from 0.18 to 13.1 FPKM in ovule (48-fold) and from 0.017 to 47.7 FPKM in 10dpf seed (2806-fold), suggesting the possibility that siRNAs produced by the siren-associated gene fragment or DNA methylation of this gene fragment could affect expression of the pectinesterase gene in trans. The best hit B. rapa pectinesterase gene also shows CHH- and CHG-methylation in ovule that is specific to the exon corresponding to the siren-associated gene fragment. This methylation is RDR2-dependent and absent in leaves. Pectinesterase fragments are found in 9 other sirens. Sixteen sirens include DUF239-related gene fragments; in some cases these correspond to different exons of the same best hit B. rapa gene, with RDR2-dependent CHH- methylation confined to the corresponding exons. Ten sirens have gene fragments from TMK receptor-like kinases, which are involved in regulating cell proliferation. In a number of cases a siren overlaps an exon that has been duplicated from an adjacent gene, and expression of the adjacent gene is in some cases increased in a nrpd1 background. These observations suggest the possibility that sirens may in some cases regulate the expression of genes in trans, but further work is required to test this hypothesis. 1. C. Zou et al., Evolutionary and expression signatures of pseudogenes in Arabidopsis and rice. Plant Physiol 151, 3-15 (2009). 2. M. Zhou, A. M. S. Palanca, J. A. Law, Locus-specific control of the de novo DNA methylation pathway in Arabidopsis by the CLASSY family. Nat Genet 50, 865-873 (2018).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Grover, JW et al., 2019. Abundant expression of maternal siRNAs is a conserved feature of seed development. PNAS USA 117: 15305-15315.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2020
Citation:
Yocca et al.,m 2020. Evolution of conserved noncoding sequences in Arabidopsis thaliana. revision under review in Molecular biology and evolution.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2020
Citation:
Hao, Y. et al. 2020. Submitted and bioRxiv2020.08 10.245258. The contributions from the progenitor genomes of the mesopolyploid Brassiceae are evolutionarily distinct but functionally compatible. bioRxiv, 2020.08. 10.245258
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Progress 10/01/18 to 09/30/19
Outputs
Target Audience:My lab's target audiences for this past year. 1) Published papers derived from research accomplished on the CoGe public comparative genomics platform aimed at the world-wide research community, especially those interested in plants. 2) All researchers world-wide who use, or are tempted to use, the online CoGe comparative genomic platform within CyVerse are my intended audience. My lab's research is designed to continuously test and proof CoGe's capabilities and my lab's input helps CoGe remain useful and bug-free as a public resource for plant and agricultural research. Changes/Problems: This year, the public web tool "qTeller", a graphic display of all RNAseq data for the B73 inbred line of maize was off line for construction. This, my development testing role was not needed. This is probably temporary. According to the CoGe online portal, Freeling lis listed as a "development tester". Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Our papers are published and our data is disseminated. CoGe is available free to all with access to the web, world-wide. What do you plan to do during the next reporting period to accomplish the goals?Next (this) year's research will be driven by the datasets available from the ovules of B. rapa r-o-18 as a part of the Mosher-Freeling collaboration. We now have small RNA and mRNA data on mutant ovules where the mutant's action was either in the male or female parent, and all from reciprocal crosses. These data will generate genes where the mode of transmission matters to expression or silencing. The description of these loci is now under submission and is "published" in BioRxiv : https://www.biorxiv.org/content/10.1101/866806v1.abstract, where there is a PDF to download/preview. While this paper is next year's produce, the research therein was this year's progress. In this paper--a paper in which CoGe results are featured--we show that the silencing RNA ovule loci we are studying are expressed from maternal alleles, in the ovule, and also "end up" in the endosperm. We will report our results during the next reporting period. We are also now writing up an additional paper wherein we use CoGe extensively to discover the origin of our ovule-specific vast overexpressing siRNA targets. It turns out that the origin involves small sequences within transposons and pseudogenes that exist outside of the siRNA target loci themselves. That result is being written-up now. My lab will study these loci. Our ongoing work as CoGe development testers should help CoGe hold its value as a platform for on-the-fly comparative genomics research. We are proud to play a small part in the health of this well-utilized public resource.
Impacts
What was accomplished under these goals?
Published accomplishments. Please see publication and the BioRx prepublication linked in "plan for next year". The core of my lab's-- Dian Burgess' and my-- accomplishments were to continuously developmental-test the CoGe applications for real time research capabilities and durabilities. We are pleased that CoGe continues to be a "go to" comparative genomics research tool world-wide. That sucess derives from the efforts of the Lyons team at the University of Arizona. We play a small part in this sucess.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Edger PP1,2, Poorten TJ3, VanBuren R4,5, Hardigan MA3, Colle M4, McKain MR6, Smith RD7, Teresi SJ, Nelson ADL, Wai CM, Alger EI, Bird K, Yocca AE, Pumplin N, Ou S, Ben-Zvi G, Brodt A, Baruch K, Swale T, Shiue L, Acharya CB, Cole GS, Mower JP, Childs KL, Jiang N, Lyons E, Freeling M, Puzey JR, Knapp SJ, 2019. Origin and evolution of the octoploid strawberry genome. Nat Genet. 2019:541-547. doi: 10.1038/s41588-019-0356-4.
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:My lab's target audience is two-fold during this last year. 1) Published papers derived from research accomplished on the CoGe public comparitive genomics platformare aimed at the world-wide research community, especially those interested in plants. 2) All researchers world-wide who use, or are tempted to use, the CoGe comparative genomic platform are my intended audience. My lab's research is designed to continuously test and proof CoGe's capabilities and my lab's input helps CoGe remain useful and bug-free as a public resource for plant and agricultural research. Changes/Problems:This year, the public web tool "qTeller", a graphic display of all RNAseq data for the B73 inbred line of maize, was taken off line for reconstruction. This, my development testing role was not needed. This is probably temporary. My original grant application's goals included qTeller work. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?By publication, by listing on my lab's website, often listing on MaizeGDB, and CoGe is a toolbox available on the web:https://genomevolution.org/coge/. Since my lab at Berkeley has been around for over 45 years, my lab's produce is not under-exposed. Occasionally a "splsh" article advertizes my lab's results. This year McCormick (2018, The Plant Journal 94: 573-574.) said nice things about my lab's contributions to the Grover et al., 2018 paper that is part of this progress. What do you plan to do during the next reporting period to accomplish the goals?Next year's research will be driven by the datasets avaialble from the ovules of B. rapa r-o-18. For example: We expect some small and mRNA data on mutant ovules where the mutant's action was either in the male or female parent from reciprocal crosses. These data will generate genes where the mode of trassmission matters to expression or silencing. My lab will study these loci. Our ongoing work as CoGe development testers should help CoGe hold its value as a supplement to comparative genomics research. We are proud to play a small part in the health of this well-utilized public resource.
Impacts
What was accomplished under these goals?
My lab's research is funded by NSF to Freeling as a Co-PI via a subaward from the U. Arizona: PI R. Mosher. The grant is to study imprining in Brassica rapa. My lab's research has almost entirely relied on multiple sequence comparisons between regions within the B. rapa genome as imformed by the nature of the "big data" collected by the project: small RNAs and RNA-seq for wild-type and a number of mutants in the ovules of B. rapa. Interesting loci are compared. This process provides my lab with a continuing opportunity to proof and test CoGe tools. When we find problems, we report them. CoGe tools are coninuously being developed; "development tester", my lab's job, is continuously needed. The Grover et al. publication of this period included my lab's entire reannotation of the transposon content of the r-o-18 line of B. rapa, the used by the Mosher project. Most notably, we found many unannotated helitrons. Current research by my lab has found that helitrons are associated with mysterious loci that generate 24nt RNAs, but only in the ovules, and, furthermore, these are the most silenced loci in the entire genome. We have no idea what is causing these loci to be silenced, but there is a significant association of these loci with helitrons. These mysterious loci occupy positions in the genome; they are flanked by particular ancestral genes. Sometimes these positions are silenced in homologous chromosomes even though there is no sequence similarity to the original locus silenced. The mystery deepens. Solving this mystery, one we didn;t anticipate when our research began, is now a priority. All of our research uses the CoGe toolbox. The Feng et al. paper includes a few pages and a Figure that illustratesour explanation for heterosis in plant and animals. Heterosis is the founding discovery on which the Big Seed Industry rests. We think that there are several interactions with possible unanticipated grow consequences happebning when two independently-evolved genotypes make a hybrid. Vigor, or heterosis, is one such consequence. However, hybrid dysgenesis or nothing-at-all are other possible consequences. Denome dominance-- one of the discoveries from my lab, historically-- is one such interaction that should inhibit heterosis. Our solution tothe heterosis mystery, perhaps best described as "a solution of no solution", is that there are too many possible interactions to make useful predictions at this time, and the epigenetic interactions are the most unpredictable of all. Time will tell if the "solution of no solution" is actually true.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Gorman et al. (including D. Burgess and M. Freeling), 2017. Extensive gene content variation in the Brachypodium distachyon pan-genome correlates with population structure. Nature Communications 8: 2184
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Grover et al. (incl. D. Burgess and M. Freeling). Maternal components of RNA-directed DNA methylation are required for seed development in Brassica rapa. The Plant Journal 94: 575-582.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Cheng, F. et al. (including M. Freeling), 2018. Gene retention, fractionation and subgenome differences in polyploid plants. Nature Plants 4: 258-268.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Reiser, L. et al. (including M. Freeling), 2018. FAIR: A call to make published data more findable, accessible, interoperable and reusable. Molecular Plant 11: 1105-1108.
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:My lab's audience is as it has been for my lab's 44.5 years: the world-wide community of biologists with an emphasis on those working in the area of plant genetics and genomics. Occasionally I write a report in layman's language, so then my audience is the general English-reading public, but I do not keep a record of such communication. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two of my lab's collaborations,reported above, are with PRC (Chinese) labs. There is probably a bit of "training young professionals" involved, but that is probably a part of all of my lab's collaborations. As a professor and instructor in comparative genomics to graduate students and molecular genetics for upper division undergrads, and everyday activities on student committees, Freeling still has some "training and professional development" activities even though the Freeling lab is closed to all students that are not primarily mentored by someone else. How have the results been disseminated to communities of interest?My lab did not produce primary data during this period. We produced ideas and analyses. We publish in open-aqccess journals where all data is available. While our data did not require deposits into the Small Reads Archive, CoGe or NCBI, these deposits were made by the "contributing author" of collaborative publications as is expected for all genomicpublished work. What do you plan to do during the next reporting period to accomplish the goals?Please refer to our project's description. My lab's primary effort, beyond CoGe and qTeller product testing, will be to work on primary research on B. rapa r-o-18 as part of the Mosher team's NSF project. We hope to understand the relationship between subgenome location, promoter complexity, transposon involvement and epigenetic marks on those genes that are and are not imprinted as assayed in the organs of developing B. rapa seeds.
Impacts
What was accomplished under these goals?
My lab's major accomplishments this year, in additionto primary published or to be published research mostly as part of collaborative efforts, is to product-test the updates and innovations added to the two on-line comparative genomics toolboxes identified in this proposal: CoGe (genomevolution.com,) and qTeller. These public comparative genomics websites are administered and developed by the teams lead by Eric Lyons (University of Arizona and CyVerse) and James Schnable (U. Nebraska), respectively. My lab's (myself and Diane Burgess) job is to use these webtools regularly and to report bugs and malfunctions to their programming team. My lab has been active this year. CoGe tools were used in our research published this year, and have and are being used in pursuit of our various collaborative research projects. These collaborations are: 1) Studies of the Brachypodium genus' pan genome centered in the lab of John Vogel, JGI, LBL; produce is now published and will be reported nextyear since its opublication date was after the deadline for this report. 2) We are funded by a sub-grant on a collaborative NSF Plant Genome Research Project researchingoRNA directed DNA Methylation (RdDM) and imprinting in Brassica rapa centered in the lab of R. Mosher, University of Arizona. We have a collaborative paper submitted, to be reported next, have created an athoritative list of (almost) all transposons in the genome of B. rapa line r-o-18, and have ongoing but unpublished research on small RNA biology. This Mosher-team work isour major research effort, and our sub-project progress is dependent on using CoGe web tools. 3) We are exploring how we can contiribute to the meaning within the sugarcane genome sequence as itis being assembled by the team headed by R. Ming at the U. of Illinois. 4) We are engaged with collaborative projects with two Chinese (PRC) labs and collaborative papers have been (reported) or will be (submitted) published. The lab of Xiaowu Wang (with Feng Cheng), CAAS, Beijing, Chinaand the lab of Jie Xu, p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 10.0px Helvetica} Maize Research Institute, Sichuan Agricultural University, Wenjiang 611130, Sichuan, China. Most or all of Freeling lab research depends on using the webtools we are dedicated to product-test.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Freeling, M. 2017. Picking up the ball at the K/Pg boundary: the distribution of ancient polyploidies in the plant phylogenetic tree as a spandrel of asexuality with accasional sex. The Plant Cell 29: 202-206
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Xu, J, Q. Wang M. Freeling et al., 2017. Nucleic Acids Research 45: 5126-5141
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:My lab's audience is as it has been for my lab's 43.5 years: the world-wide community of biologists with an emphasis on those working in the area of plant genetics and genomics. Occasionally I write a report in layman's language, so then my audience is the general public, but I do not keep a record of such communication. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The Freeling lab's last student and postdoc left the lab before this project began. The Freeling lab now participates in student lab work teaching informally, through collabortions, or through having Departmental graduate students rotate in my lab, during which time we teach them mostly about how to work won CyVerse platforms, and how to use Coge and qTeller, the two web-based toolboxes (applications) identified in this proposal. How have the results been disseminated to communities of interest?My labs work is published in a way that insures open accesss. Even when my funding agencies hae no money for open access, the University of California provides funds that may be used for this essential function. We did not publish any large datasets during this project year, so we did not need to use any of the "permanent data storage" alternatives. What do you plan to do during the next reporting period to accomplish the goals?My lab has already found numerous bugs and irregularities while working with Coge applications. These have all beeen corrected by the responsible programmers on Eric Lyon's CoGe team at the University of Arizona. During the current project period (next), both Coge and qTeller tools are being and will be put to use on three different research projects. One of these is funded and two are not. The funded project is NSG PGRP to Rosher and team, U. Arizona on RNA-dependent DNA Methylation in Brassica rapa; my lab's sub-=project is to study subgenomes and imprinting. The not-funded projects are to complete work started on a NIFA Plant Biology research project that inded in 2015 on maize mutational mechanisms, and to wotk with R. Ming and team to find meaning in the single-molecule-sequenced (PABbio) sugarcane genome.
Impacts
What was accomplished under these goals?
All of the publications from this product period included reserch accomplished using the Coge toolbox of compartive genomic applications. CoGe is a part of CyVerse. In the process of accomplishing this research, my two-person lab proofed the following applications: GEvo, CoGeBlast, SynMap, Feat View and Epic CoGe. At the same time, these publications advertize the CoGe toolbox.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Epigenetic regulation of subgenome dominance following whole genome tripliction in Brassica rapa. New Phytologist 211: 288-299.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Freeling, M. A solution to the C-Value Paradox and the Function of Junk DNA. Plant and Animal Genome Conference XXIV: Published Abstract of talk delivered
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Freeling M. et al. Fractionation and subfunctionalization following genome duplications: mechanisms that drive gene content and their consequences. Current Opinions in Genetics and Development 35: 110-118
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Ming, R and coworkers (incl. M. Freeling). The pineapple genome and the evolution of CAM photosynthesis. Nature Genetics 47:1435-1442
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
VanBuren, R et al. incl. M. Freeling Single molecule sequencing of the dessication-tolerant grass Oropetium thomaeum. Nature 527: 508-511.
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