Year One / First Quarter Progress Report

May 31, 2011 “TRPGR IOS-1025974: Comparative genomics of environmental stress responses

in North American hardwoods” Introduction This project is focused on genomic resource building for a range of North American hardwood species. This first progress report summarizes the activities and results of project personnel for the first quarter of this grant period (2/1/2011 – 5/1/2011) as well as progress achieved pre-award. Consequently, the report will reflect progress toward the project objectives by presenting materials on the subjects of 1) population development, 2) EST library construction, 3) BAC library construction, and 4) genetic/QTL map construction, 5) bioinformatics/website development, 6) outreach and 7) project management. Abbreviations: PSU – Pennsylvania State University; UT – University of Tennessee; MU – Missouri University; UWA – University of West Alabama; ND – University of Notre Dame; MTU – Michigan Technological University; CLM – Clemson University

Activities Population Development – Mapping populations

Black Walnut (MU/ND) – Mapping Population: Seed was collected for this species in the fall 2008, from two trees growing in a clonal repository containing 22 nut cultivars at the University of Missouri (MU) Horticulture and Agroforestry Research Center (HARC) in New Franklin, MO. This specific repository is the oldest of four such plantings containing a total of 100 accessions, selected for an array of commercial nut traits, as part of an ongoing breeding program which has been sponsored by the University of Missouri Center for Agroforestry since 2000. Since June 2009, in collaboration with Dr. Romero-Severson at the University of Notre Dame (ND), we have been successful in identifying a total of 320 full sib seedlings representing the cross between ‘Sparrow’ x ‘Schessler’ based on a panel of five DNA markers (using CERVUS 3.0). These 320 full sibs represent 31.7% of the total of 1010 seedlings genotyped so far. To date, 288 of these full sib individuals have been planted at HARC. Prior to establishment at HARC with the rest of the mapping population, 60 ‘Sparrow’ x Schessler’ full sibs were used by Dr. Haiying Liang’s lab at Clemson (summer 2010) for stress treatments and RNA extraction to contribute to transcriptome sequencing. Northern Red Oak (UT) - Mapping Population : Open-pollinated seeds from a select red oak tree growing at Purdue University campus have been collected on several occasions over the last 10 years and subjected to parental analysis using a marker screen to identify full-sib progeny for genetic mapping. Seedlings from multiple cohorts have been raised at the University of Tennessee. This spring, screens protecting the smaller trees in these plantations were removed to stimulate growth. Plantations have been monitored and maintained. Seedlings from this same cross are being used for stress testing, RNA extraction, and EST library construction at Clemson (this quarter). Progeny exclusion using the 5 marker multiplex progeny exclusion kit developed for northern red oak has been used to identify full-sibs from 535 half-sib acorns collected from the SM1 parent’s 2010 progeny. Almost 90% of the samples on the first plate tested (92 samples) were full sibs. This is exceptionally high, but rescoring all chromatograms manually produced the same result. Sweetgum (MU) – Mapping Population: Open pollinated seeds were collected from a single ramet growing in an isolated clonal seed orchard in southern Indiana. This orchard contains a total of 16 clones that were originally selected for their winter hardiness within a provenance/progeny test established by Michigan State University. These selections were made by Dr. Coggeshall in 1996. As of this writing, these seeds are still in stratification, and will be sown in the greenhouse in late May 2011. Seedlings will be subjected to genotyping and paternity analysis to identify a full-sib cohort for mapping. Honeylocust (UT/MTU) – Mapping Population: Seed was collected from a single tree in central Tennessee in autumn 2008, treated with muriatic acid to increase water permeability of the seed

coat and planted in raised nursery beds at UT. Seed germinated over two years (2009 and 2010). Seedlings from the 2009 crop were potted during the dormant season, and leaf samples were taken at the end of the 2010 summer for DNA extraction and genotyping to determine paternal contributions (MTU). A mapping population consisting of 402 full-sibs were identified and established at the East Tennessee State Nursery in February, 2011. The plantation was attacked by voles soon after planting, with a modest loss (mortality - four trees, damaged – unknown). Yellow-Poplar (UT) – Mapping population: Open-pollinated seed was collected from a single clone (#108) in a yellow poplar seed orchard in the 2010 autumn, stratified, and sent to Dr. Liang (Clemson) in January, 2011 for germination on vermiculite. Low germination rates of about 7% were observed. Approximately 20 seedlings have been obtained to date. This material may not serve our needs. But alternatives exist. Controlled crosses between Clone 108 and Clone 23 (male) were made in late April (2011). The parent trees were damaged during a major tornado/hailstorm outbreak and cross success is currently unknown. Clone 108 has been re-grafted to preserve the genotype, as ramets in the Knoxville yellow-poplar orchard are very old (established in 1966) and declining in health. Yellow-Poplar MU – As a backup to the breeding efforts undertaken at UT, open pollinated seeds were collected from a single tree growing at HARC in the fall 2010 and sown at the Vallonia State Nursery in southern Indiana on November 1st. The resulting seedling will be lifted in the spring 2012. It is anticipated that the ability to identify full sibs within this open pollinated family will be facilitated by the fact that yellow-poplar is represented by a total of only four mature trees at this site.

Population Development – The following populations are being created as a resource for the construction of genetic linkage maps in future research. These seedlings will be used for the collection of DNA/RNA for BAC library construction and transcriptome sequencing. The RNA sequence will provide genetic markers for the construction of genetic maps and association studies, The BAC libraries will enable the construction of physical maps in the future. Screening for full-sibs from these populations will progress as funds and time permit. Green Ash (MU) – Open pollinated seeds were collected from a single tree growing on the MU campus in the fall 2010 and sown at the Vallonia State Nursery in southern Indiana on November 1st. The resulting seedling will be lifted in the spring 2012. Sugar Maple (MU) - Open pollinated seeds were collected from a single tree growing on the MU campus in the fall 2010 and sown at the Vallonia State Nursery in southern Indiana on November 1st. The resulting seedling will be lifted in the spring 2012. (Non-project supported activity) Black Gum (MU) - Open pollinated seeds were collected in the fall 2009 from a single tree located in a city park in Columbia, MO (which is outside the native range for this species). A total of five mature black gum trees are growing in this park, which should facilitate the

identification of full sib individuals within this open pollinated family. To date, a total of 140 containerized seedlings have been produced, which are currently growing at HARC. Plans call for an additional seed collection and sowing in the fall 2011.

EST Library / Transciptome Sequencing (Clemson/ND) Black walnut tissue collection (ND).

Tissue was collected from leaves, buds, twigs, cambium, sapwood, nutlets, paper-punch wounded leaves and wounded twigs from one tree of the cultivar Sparrow, the female parent of the mapping population. All tissues were flash frozen in separate tubes and delivered to Haiying Liang at Clemson. This material will be used for RNA extraction and cDNA produced for transcriptome sequencing. Northern red oak tissue collection.

Tissue has been collected from SM1, the female parent of the northern red oak mapping population. Samples were collected from leaves, buds, twigs, cambium, sapwood, paper-punch wounded leaves and wounded twigs. All tissues were flash frozen in separate tubes and are stored in our -80 freezer at Notre Dame for sequencing. In addition to the baseline materials just described, cDNA libraries are being developed from full-sib progeny of the parent trees. These progeny are being subjected to stress treatments to provide contrasting transcriptome profiles. Stress treatments. Stress treatments were completed on full-sib progeny for both black walnut and northern red oak. Treatments included wounding, cold, heat, and drought. RNA extraction from the stressed-treated northern red oak seedlings is in progress. This RNA will contribute to the mRNA extracted from multiple tissue types in the NRO mother tree SM1

For each species, 10 full-sib seedlings were used for wounding, cold, heat, and drought treatments. Another 10 seedlings were included as control. For black walnut, two year-old full-sib seedlings from MU were used, and returned for planting in the mapping trials. For northern red oak, stratified full-sib northern red oak acorns, provided by ND, were grown in the greenhouse at Clemson for two months (see photo) prior to treatment. For each treatment and the control, leaves and fine white roots were collected from all ten seedlings, except for the wounding treatment, in which only leaves were collected. A total of 90 samples were collected for each species. All samples are stored in -80 °C until RNA extraction.

RNA extraction protocols and recovery

Total RNA were extracted from individual samples using the RNAqueous®-Midi kit (Ambion, catalog #1911), while mRNA were isolated with the Poly(A) Purist™ mRNA Purification Kit (Ambion, catalog #1916). Both total RNA and mRNA were checked on an Agilent 2100 Bioanalyzer (Agilent Technologies) for quality and concentration. DNase treatments were conducted before and after mRNA isolation. The amounts of mRNA obtained for each tissue

type in black walnut are listed in Table 1. RNA extractions from the treated and control northern red oak tissues are underway.

Table 1. The amounts (µg) of mRNA obtained from each black walnut samples.

Control Wounding Heat Cold Drought

Leaf Root Leaf Leaf Root Leaf Root Leaf Root

mRNA 10,320 2,370 2,520 2,265 3,300 1,300 1,260 1,310 lost

BAC Library Construction

No activity this quarter.

Genetic / QTL Maps (MTU / ND) Honeylocust (Gleditsia triacanthos) – MTU : marker development Leaf samples were collected from the seed parent, six putative pollen parents and 432 progeny by Scott Schlarbaum between August 30 and September 1, 2010 and shipped to Michigan Technological University. All samples were prepared for DNA isolation in a 96-well plate format (DNeasy 96 plant kit, Qiagen) and are stored at -80 °C. DNA was isolated from the seed parent, the putative pollen parent and 16 progeny for primer testing. A total of 14 published EST-SSRs markers have been tested for amplification in the seed parent and putative pollen parents including 8 markers originally developed for Medicago trunculata (Eujayl et al. 2004, Gupta and Prassad 2009) and 6 markers originally developed for Ceratonia siliqua (Caruso et al. 2008), a member of the same subfamily (Caesalpinioideae) as Gleditsia triancanthos (Wojciechowski et al. 2004). In addition, 126 new EST-SSR primer pairs have been derived from the published EST library in Ceratonia siliqua (Caruso et al. 2008) by Meg Staton. Until now, 48 markers have been ordered and 24 of these markers have been tested for amplification and polymorphism in honeylocust after gel electrophoretic separation on the Qiaxcel capillary analyzer (Qiagen) using the high resolution kit. None of the published EST-SSR markers showed amplification in honeylocust. Out of the 24 new EST-SSRs tested, 7 showed amplification in honeylocust and 4 were polymorphic. Northern red oak (Quercus rubra) MTU: marker development A total of 48 EST-SSRs markers (4 markers on each linkage group) originally developed for Q. robur were ordered (Durand et al. 2010). 28 markers have been amplified and tested for polymorphism in the crossing parents of the Q. rubra full-sib family (provided by Jeanne Romero-Severson). Seventeen of the 28 markers tested amplified a product of the expected size, and 10 markers were polymorphic in the Q. rubra crossing parents. 5 markers were heterozygous

for only one parent tree (4 for the male parent, 1 for the female parent) and 5 markers were heterozygous for different alleles in both parents. These markers will enable comparative studies.

Bioinformatics / Website Development (CLM)

Creation of project website The domain name www.hardwoodgenomics.org was purchased for 5 years. A password protected development website was created and shared with the project PIs (dev.hardwoodgenomics.org, username: NSFhardwood, password: tr33s!). This site is based on Drupal, a popular open-source content management system. The software Tripal, initially developed for Fagaceae Genomics Web, has been installed as well. After initial approval of design and content from the project PIs, the site will become public. Results (completed activities) Bioinformatics / Website (CLM) Liriodendron cDNA sequence assembly and marker identification Over 2 million reads were obtained for Liriodendron tulipifera from Genbank and the Ancestral Angiosperm Genome Project. These were trimmed with the software NGS Backbone and assembled with the 454 software Newbler into 33,745 isotigs (putative individual transcripts). A custom SSR script was run to identify short tandem repeats and examine underlying sequence assemblies to detect evidence for polymorphism. Of 9,471 SSRs identified with primers, 3,177 show evidence of polymorphism in the sequence data. The 2 million reads were realigned to the isotigs to detect SNPs with 454 software. After filtering for at least 10 nucleotides at the SNP position and at least 25% of reads containing the minor variant, 27,159 SNPs were identified. Identifying SSRs from a species related to Honeylocust No ESTs are currently available for Gleditsia triacanthos. However Ceratonia siliqua, commonly known as carob, is in the same clade as honeylocust and has 1393 public ESTs in Genbank. The sequences were assembled with cap3 at 95% identity to remove any redundancy in the data set. 126 SSRs were identified in the nonredundant data set of 1,131 sequences. Primers were designed with the software Primer3. These are being used to test for markers in honeylocust.

[Interim results]

Interim results for other activities have been included in the “Activities” section of this report due to their incomplete status.

Anticipated Activities (Next Quarter) Population Development Black Walnut (MU) – Additional full sibs will be established in the two mapping population outplanting sites in a timely fashion as fingerprinting results are obtained from Dr. Romero-Severson lab at ND. Green Ash (MU) – All potential pollen parents, plus the maternal tree used as the source of the mapping population individuals will be sampled (leaf samples) in the summer 2011 and sent to Dr. Romero-Severson’s lab at ND for genotyping and parental analysis. Honeylocust (UT) - We will sample leaves for Dr. Gailing from an additional 20-25 trees in order to have a population sample from the general area of the mother tree. Also, for construction of the BAC DNA library, tissue will be taken from the mother tree before dawn. Arrangements will be made to take samples in early May for both of these purposes. Population samples will be sent directly to Dr. Gailing. For the cDNA library sample, we will visit the tree prior to dawn and cut branches with leaves. The branches will be placed in a container with ice and sealed with duct tape. It will be transported to Knoxville and given Dr. Liang’s personnel for further transport to Clemson. DNA samples of leaf tissue will be taken from all unsampled honeylocust seedlings growing in Knoxville. The samples will be sent to Dr. Gailing for analysis. The seed crop of the mother tree will be assessed during the summer, and additional seed will be collected in the autumn when mature, if there is a crop. Voles attacking the mapping population will be controlled with wheat seed that has been impregnated with zinc phosphide. Northern Red and White Oak molecular mapping populations (UT) – The plantations will be maintained by mowing and herbicides during the growing season. The 2007 northern red oak seedlings will be rogued per results from Dr. Romero-Severson’s laboratory. The white oak plantation will be rogued per results from Dr. Carlson’s laboratory. Northern red oak full-sibling seedlings from Dr. Liang that have been stress tested at Clemson will be transferred to UT. The seedlings will be grown in the nursery for the 2011 summer, lifted during the dormant season, and added to the existing mapping population at Ames Plantation. Full-sibling northern red oak seedlings used for stress tests by Drs. Shumaker and Carlson will be transferred from Penn State to UT in mid-July and housed for the remainder of the 2011 growing season. The seed crop on the white oak mother trees will be assessed during the summer in case more seedlings are needed for the mapping population or other experiments.

Sugar Maple (MU) – All potential pollen parents, plus the maternal tree used as the source of the mapping population individuals will be sampled (leaf samples) in the summer 2011 and sent to Dr. Romero-Severson’s lab at ND for genotyping. Sweetgum (MU) – The sweetgum seedlot will be sown in late May and the resulting seedlings maintained in containers. It is anticipated that leaf samples will be submitted to PSU in the summer of 2011 to begin the work of identifying full sibs. Leaf samples from all clones in the Indiana seed orchard will also be collected in the summer 2011 for use in the identification of full sibs. Yellow-poplar (UT) – Breeding will continue until flowering has ceased. The resulting seed crop will be assessed in mid-May. Leaf samples for DNA extraction will be taken from Clone 23 and transported to Clemson at the same time of the honeylocust leaf exchange. Black Gum (MU) - All potential pollen parents, plus the maternal tree used as the source of the mapping population individuals will be sampled (leaf samples) in the summer 2011 and sent to Dr. Romero-Severson’s lab at ND. BAC Library Construction BAC library construction will commence for Northern red oak and black walnut during the coming quarter. (CLM/ND/MU) EST Libraries RNA extraction and cDNA synthesis for Northern red oak will be completed (CLM). Research activities at Penn State will commence with the receipt of Black walnut and Northern red oak RNAs from Clemson University and construction of 454 libraries for DNA sequencing. Genetic / QTL Maps (ND, MTU) Black walnut genetic mapping We will screen all of our black walnut and butternut microsatellite markers (40 markers) for informativeness and genotype the informative markers across the mapping population. We will also extract promising markers from the public databases, focusing first on the Julgans regia sequences in GenBank. Northern red oak genetic mapping We will genotype the existing mapping population with all of our informative northern red oak microsatellites (50), then proceed to the EST sequences from Q. robur/Q. petraea which Oliver Gailing sent to us. After that, we will proceed to the EST-SSR markers available from the

Fagaceae Genomics Project, focusing first on the oak EST-SSR. After that, we will include the SNP data from UCLA. Marker development continues at MTU. Honeylocust (Gleditsia triacanthos) 48 additional markers will be tested for polymorphism in the seed parent and in the putative pollen parents. Polymorphic markers will be characterized in a larger population sample (24 individuals). DNA will be isolated from all samples (parent trees and progeny). We will develop a set of markers that will be used to identify full-sib families by paternity exclusion among the progeny of 432 seedlings that were collected from the same mother tree. Bioinformatics / Website (CLM) • PI Staton will attend the ForestTrac workshop on “Interoperability and Integration of

Databases in Europe and North America” from May 23-24th in Baden, Austria. • Launch of the Project (Abbreviated here as the Hardwood Genomics Project (HGP)) website

to the public. • Expansion of the HGP website to include the Liriodendron transcriptome sequences,

assembly, functional annotation and mined markers. • Inclusion of the data from the Fagaceae Genomics Web into HGP website. • Developing use case scenarios for the new mapping, marker and phenotype section of the

website. This will include answering the following questions: o What types of data do the mapping PIs currently store? What format is it in? o How can this data be appropriately stored in the chado database schema? o What types of data would breeders (traditional and marker-assisted) like to access?

What display and download formats would be the most useful? o What types of searches would users perform to access this data?

Outreach UT / ND – Genomic / Genetic / Forestry Training for Cherokee Tribal Youth As part of a comprehensive outreach plan to develop field and classroom activities that can be delivered to eastern band of the Cherokee tribes in North Carolina, PI’s Schlarbaum and Romero-Severson have hosted multiple conference calls and attended one site visit with Carmaleta Monteith, curriculum designer for the new Ravensford School (K-12 grades) in Cherokee, NC. PI Schlarbaum presented an invited seminar on exotic forest pests and related research to an audience that included tribal members, Cherokee artisans, BIA forestry personnel, the Tribal (Eastern Band of Cherokee Indians) Forester (Mr. Tommy Cabe), and the Ravenford School’s forestry class and their instructor. He was invited by the Revitalization of Traditional Cherokee Artisans Resources program (RTCAR), which is funded by the Cherokee Preservation Foundation. During the seminar, he talked about our outreach effort in respect to butternut activities at the Ravensford School, ate lunch with the Ravensford High School forestry class students, and discussed the forthcoming outreach effort. The PI’s will continue discussions and planning for the outreach effort over the next quarter by traveling to Cherokee (SS) to meet with Dr. Monteith, the Ravensford School’s forestry instructor, and representatives from the USDI Bureau of Indian Affairs forestry and Eastern Band of Cherokee Indians Natural Resources office. The purpose of the visit will be to develop a draft of age-appropriate classroom exercises, inventory available equipment, and strengthen external support. The outreach effort will attempt to engage the BIA and Tribe foresters in certain field activities. The RTCAR program will be approached to fund any minor equipment needed by the Ravensford School in conjunction with this outreach effort. PI Romero-Severson has spoken to two Notre Dame teaching faculty (Michelle Whaley and Kristen Lewis) who do science lab outreach for middle and high school students in underserved schools in South Bend. They have agreed to serve as advisors to us if needed as we develop labs and outdoor activities. PSU / UWA: Summer Internship for Students at PSU

PSU: Planning was completed for the summer research visit by Dr. Ketia Shumaker and two undergraduate students from the University of West Alabama. They will conduct research on the response of Northern red oak to ozone stress, under the supervision of the PI and his post-doctoral fellow Theodora Best.

UWA: As part of the comparative hardwood genomics project’s outreach and education component, The University of West Alabama’s (UWA) Associate Professor, Dr. Ketia Shumaker, is serving as coordinator, researcher, and student mentor for the summer research internship program at Pennsylvania State University (PSU). In the fall of 2010, Shumaker developed an internship application (refer to the last page of this report) and coordinated a faculty review committee composed of UWA faculty members from the biology and chemistry departments. Starting in late November, emails were sent to all of our science majoring students about the summer internship at PSU. Verbal announcements were also made in lecture classes and biology club meetings. African American students were especially targeted in these announcements. The review committee met to discuss the applicants and during the first week of March students were contacted of their acceptance status. Selected students had two days to

acceptance the offer and sign a commitment contract. In April, Shumaker had an information dinner meeting with the students’ parents on UWA’s campus. Shumaker will travel with undergraduate students Dantria Grace and Christen Nelms on June 1st to start their 10-week summer research experience at PSU.

Photo Key : The University of West Alabama faculty Dr. Ketia Shumaker (center) and undergraduate students Dantria Grace (left) and Christen Nelms (right). Grace is a junior majoring in biology and

Nelms is a first semester senior majoring in chemistry. Newsletter The project manager and PI’s developed and distributed the first project newsletter describing the project goals and personnel within a month of project initiation. It was distributed to over 200 recipients by email, and will be permanently hosted on the public website. Presentations/Posters/Publications related to project activities Schlarbaum, S. E. 2011. Treats from Exotic Pests to Appalachian Forests. Gathering Places Seminar Series, Revitalization of Traditional Cherokee Artisan Resources program, Cherokee Preservation Foundation. 1 April 2011. (approximately 80 attendees representing the Eastern Band of Cherokee Indians, USDI Bureau of Indian Affairs, North Carolina State Extension Service, Ravensford School, and private individuals). Coggeshall, M.V. 2011. TCD screening strategies: The search for resistance in black walnut. Thousand Cankers Disease Workshop at Purdue University. 31 March 2011. Sponsored by USDA Forest Service, Northern Research Station, Hardwood Tree Improvement and Regeneration Center and Walnut Council. (80 attendees representing 18 states). Coggeshall, M.V. 2011. The black walnut breeding program at the University of Missouri Center for Agroforestry. 9 April 2011. Michigan Nut Growers Association Spring Meeting in East Lansing, MI. (60 attendees). Gailing, O. 2011. Development of genomic resources in oaks to study local adaptation to drought. (8th Annual Cottonwood Symposium, February 23-25. Flagstaff. USA. Attendance: ~ 50 scientists including graduate students from the US and Canada. (In Press) H. Liang, S. Ayyampalayam, N. Wickett, A. Barakat, Y. Xu*, L Landherr, P. Ralph, T. Xu*, S.E. Schlarbaum, J. H. Leebens-Mack, C.W. dePamphilis. Generation of a large-scale genomic resource for functional and comparative genomics in Liriodendron. Tree Genetics and Genome. DOI: 10.1007/s11295-011-0386-2

(In Press) Y. Jiao, N. Wickett, S. Ayyampalayam, A. Chanderbali, L. Landherr, P.E. Ralph, L.P. Tomsho, H. Liang, P.S. Soltis, D.E. Soltis, S.E. Clifton, S.E. Schlarblum, S.C. Shuster, H. Ma, J. Leebens-Mack, C.W. dePamphilis. Phylogenomic detection of ancient polyploidy in seed plants and angiosperms. Nature. (http://www.nature.com/nature/journal/vaop/ncurrent/pdf/nature09916.pdf) PI Carlson and co-PI Jeanne Romero-Severson presented the project and discussed international collaborations at the FoResTTraC Workshop on Genomics of Forest and Ecosystem Health in the Fagaceae held February 23-25, 2011 at INRA Headquarters in Paris, France.

Project Management PI Carlson (PSU) PI Carlson’s activities in Quarter 1 focused on working with Sponsored Programs at Penn State to secure transfer of the funding for year one and setting up the main research account, the sub-awards to the 6 partner institutions, and the contract to support the project manager, Dr. Nicholas Wheeler.

A one day meeting of the project participants was convened on Jan 15, 2010, prior to the Plant and Animal Genome Conference XIX. At the organizational meeting revised project objectives, responsibilities, budgets, and time frames as accepted by NSF were reviewed, along with pre-award research results by the project participants. The team set priorities and milestones for year one of the project. We started planning our outreach and education activities and covered publication and data release policies. Finally plans were laid for the project’s web site content. Subsequent to the organizational meeting in January, a steady stream of emails and calls have ensued among project members to discuss questions and opportunities arising at this early stage in the project.

Project Manager – (Wheeler, PSU)

A project management website (Basecamp) was established within weeks of the project obtaining approved funding. This site has been used extensively by project personnel to post and review documents, conduct and archive discussions about on-going activities, and communicate milestones and to-do lists.

The projects first Newsletter was drafted and released. These will follow on an every other quarter basis.

Planning for the first annual project meeting was also conducted during this quarter. The meeting will be held July 10, 2011, at the Mont Alto campus of Penn State University, in conjunction with the Schatz Genetic Colloquium that will follow. This first meeting is being held at the mid-point of year one so that we can benefit from feedback by the SAB on our research plans and milestones early enough for modifications to be made as needed.

Project progress is being maintained on a quarterly basis to insure all project personnel are aware of activities across the group, and so that we can respond in a timely manner to potential issues or pitfalls. Reports will be archived on our Basecamp site and executive summaries made available to NSF personnel.

The project manager will work with PI Romero-Severson to set up and conduct frequent conference calls among all PIs working on genetic/QTL mapping projects. Project wide conference calls have and will continue to be called as needed.

Project Personnel – Students, Post-Doctoral Fellows, Staff Clemson Lanae Neild is the new systems administrator at Clemson University Genomics Institute. She earned her MS degree in Computer Science at Clemson University in 2004. She is responsible for maintaining the web server and other computational equipment. Her expertise in Drupal website development will be instrumental in building the project website and expanding the Tripal software package. A Ph.D. student was recruited for the fall of 2011 to work with Dr. Liang. Michigan Technological University The following students are working directly on the project or related projects at MTU.

o Jennifer Lind (PhD student, funded by MTU): Local genetic adaptation in Quercus rubra (anticipated date of graduation: fall 2013)

o Alexis Sullivan (Master student, funded by the Atlantis program, Forest service,

Hanes Fund), Hybridization and local adaptation in Quercus rubra and Q. ellipsoidalis (anticipated date of graduation: spring 2013)

o Jessica Goodin (Master student) Adaptation of Ceratonia silqua EST-SSRs for Gleditsia triacanthos (anticipated date of graduation: spring 2012)

o Erin Hickey (undergraduate), Adaptation of Querus robur EST-SSRs for Quercus rubra

Table 1. Time table for project activities: (values in boxes are % work completed; √, finishing points)

-1 Year 1 Year 2 Year 3 Year 4 RESEARCH ACTIVITIES Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Northern red oak

Treatments; RNA isol’n 50 EST sequencing √ √ High Density Mapping √ BAC library construct √

Black walnut Treatments; RNA isol’n 100 EST sequencing √ √ High Density Mapping √ BAC library construct √

Sweetgum Treatments; RNA isol’n EST sequencing √ √ Framework genetic map √ BAC library construct √

Honey Locust Treatments; RNA isol’n EST sequencing √ √ Framework genetic map √ BAC library construct √

Sugar maple Treatments; RNA isol’n EST sequencing √ √ BAC library construct √

Green Ash Treatments; RNA isol’n EST sequencing √ √ BAC library construct √

Tulip poplar Framework Map √ √

Year 1 Year 2 Year 3 Year 4 OUTREACH PROJECTS Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4

Cherokee Central Schools Planning with Monteith Preparation of materials Summer project with students Evaluation and redesign Integration into curriculum Summer project with students Evaluation, integration, redesign Integrated projects with students Evaluation, long term planning

University of West Alabama Training workshop at PSU Summer research at PSU Attend ASPB Conference

Table 2. Yearly goals with participant roles (blackened boxes)

Participants: Goals:

JEC SES JRS MVC OG KLS HL MS

Year 1 Oak and walnut seedling treatments and RNA isolations

Finish Oak EST sequencing Start oak and walnut genetic maps Start oak and walnut BAC libraries Website development Reference population development Cherokee Central Schools outreach Student summer research at PSU Year 2 Sweetgum and honeylocust seedling treatments and RNA isolations

Finish walnut, sweetgum, and honeylocust EST sequencing

Finish oak, walnut BAC libraries Continue oak, walnut genetic maps Start yellow-poplar genetic map Start sweetgum, honeylocust BACs Start maple and green ash seedling treatments and RNA isolations

Reference population development Website development; EST analysis Cherokee Central Schools outreach Student summer research at PSU Year 3 Finish maple and green ash seedling treatments and RNA isolations

Finish maple EST sequencing Finish oak, walnut genetic maps Finish yellow-poplar genetic map Finish sweetgum, honeylocust BAC libraries Start maple and green ash ESTs Start maple, green ash BAC libraries Start sweetgum, honeylocust maps Website development; EST analysis Cherokee Central Schools outreach Student summer research at PSU Year 4 Finish green ash EST sequencing Finish sweetgum, honeylocust genetic maps Finish maple, green ash BAC libraries Website development; EST analysis Cherokee Central Schools outreach Student summer research at PSU

Abbreviations: JEC, John E. Carlson; SES, Scott E. Schlarbaum; JRS, Jeanne Romero-Severson; MVC, Mark V Coggeshall; HL, Haiying Liang; OG, Oliver Gailing; KLS, Ketia L. Shumaker; MS,Meg Staton; UWA, University West Alabama; PSU, Penn State University