Progress 05/01/07 to 04/30/13
Outputs
OUTPUTS: Activities: Multiple sequencing experiments have been conducted for 5 years in four main closely related Southern pines, P. taeda, P. elliottii, P. echinata, and P. palustris (subsection Australes) to study nucleotide variation of the following 14 genes that can be considered as adaptive trait and wood quality related candidate genes: arabinogalactan-like protein 1519 (agpl-like or agp1519), arabinogalactan 4 (agp-4), arabinogalactan 6 (agp-6), aquaporin membrane intrinsic protein (aqua-MIP), coumarate 3-hydroxylase (c3h), cinnamoyl alcohol dehydrogenase (cad), cinnamoyl CoA reductase (ccr), caffeate O-methyltransferase 2 (comt-2), calcium-dependent protein kinase (cpk3), dehydrin 2 (dhn-2), loblolly pine water-stress inducible protein 3 (lp3-3), putative wall-associated protein kinase (ppap12), LIM domain protein 1 (ptlim-1), and s-adenosyl methionine synthetase 2 (sams-2). The signatures of selection in these genes were studied using neutrality and Fst-outlier tests, association with environmental variables and logistic regression analysis of clinal variation. Association mapping was used also to study if allelic variation of these candidate genes was associated with phenotypic variation of breeding traits. The sequence data have been used also to study phylogenetic relationships between species. The obtained data facilitated mentoring two PhD students and teaching two graduate courses "Population Genetics" and "Molecular Ecology". Events: The obtained results were presented at the numerous conferences and meetings, and were used as training material in several workshops. Services: The obtained results were used for consulting, counseling, and tutoring the Western Gulf Forest Tree Improvement Program (WGFTIP) hosted by the Texas A&M Forest Service and its coop members. Products: sequence data deposited in Genbank database and single nucleotide polymorphism (SNP) genotyping data deposited in the TreeGenes Data Repository (TreeGenes Accession is TGDR006 (https://dendrome.ucdavis.edu/tgdr/index.phpacc=TGDR006); the USDA-NIFA the Conifer Translational Genomics Network (CTGN) and US FS collaborations fostered by the project; computer program (python script) StrAuto v0.3.1 to automate the entire population structure analysis using the STRUCTURE program. This script and its documentation are available for download from http://www.crypticlineage.net/pages/software.html. Dissemination: The obtained results were disseminated for practical tree improvement and breeding by the Western Gulf Forest Tree Improvement Program (WGFTIP) and its coop members. PARTICIPANTS: (1) Dr. Judy Brooks (jebrooks@tamu.edu), Research Assistant, 0.5 FTE; (2) Tomasz Koralewski (tkoral@tamu.edu), PhD student (successfully graduated in August 2010), now postdoc; 3) Vikram Chhatre (crypticlineage@gmail.com), PhD student (successfully defended his thesis on February 27th 2013, projected graduation in May 2013). Education is embedded throughout the research process. Educational activities do not only include a Ph.D. graduate student but also a wide range of research-sharing emanate from this important aspect of the project. While virtually all steps of the study involve tasks to be performed by the graduate student, which includes DNA isolation, PCR amplification, design of primers, sequencing reaction and analysis, SNP genotyping, gel electrophoresis, statistical analysis of data, and writing reports and papers, the graduate student takes leadership in the development and execution of complementary education activities. Existing programs for graduate students at Texas A&M University benefits from the current research, as well. Both Genetics and MEPS (Molecular and Environmental Plant Sciences) major students going through rotation during their first year of graduate studies will engage in the research and receive additional options from which to choose areas of specialization and research. Our project will also serve as a platform to train college students, including community college students. Our expectation is to enrich the biology program of Blinn College, a local community college, through an existing agreement between Texas A&M University and the College. TARGET AUDIENCES: Forest geneticists and breeders community, such as, for instance, the Western Gulf Forest Tree Improvement Program and the Gene Conservation Program (Texas A&M Forest Service, Forest Science Laboratory, Texas A&M University, College Station, TX 77845-2585; http://www.ars-grin.gov/misc/wgftip) PROJECT MODIFICATIONS: The research is more, if not primarily focused now on loblolly pine taking advantage of having high-throughput SNP genotyping assay developed for loblolly pine.
Impacts
Change in knowledge: New sequence and SNP data for 14 adaptive trait and wood quality related candidate genes have been obtained in P. taeda, P. elliottii, P. echinata, and P. palustris, and deposited in the NCBI Genbank database, which is publicly available to the scientific community. The multiple associations were obtained between SNPs representing more than 4000 genes and adaptive and breeding traits in loblolly pine that represent new fundamental and applied knowledge significant enough to be included in publications, as well as in breeding methods and techniques, and should help tree breeders in their decision-making. Obtained data on association of SNPs with environmental variables can help researcher make predictions on how global climate change may affect loblolly pine populations. Change in actions: Newly obtained genotypic and association data for more than 1700 trees and 4000 genes and multiple breeding traits in the first and second generations of breeding populations in the WGFTIP will be directly applied in tree improvement and selection for fast growing, drought resistant and more resilient trees with better wood quality.
Publications
- Krutovsky, K.V., T.E. Koralewski, 2012. Evolution of exon-intron gene structure and alternative splicing: what we can learn from completely sequenced genomes and predict for non-model species. The 8th International Conference on the Bioinformatics of Genome Regulation and Structure\Systems Biology, June 25-29, 2012, Novosibirsk, Russia, p. 171 (http://conf.nsc.ru/BGRSSB2012/en/scientific_program)
- Krutovsky, K.V., 2012. Forest Genomics for Mitigating Climate Change and Breeding Resilient Trees. In: Plant & Animal Genome XX. The International Conference on the Status of Plant and Animal Genome Research, Final Program and Abstracts Guide, W166, p. 144, January 14-18, 2012. San Diego, CA, USA (http://pag.confex.com/pag/xx/webprogram/Paper1651.html)
- Chhatre, V., T. Byram, D.B. Neale, J.L. Wegrzyn, and K.V. Krutovsky, 2012. Genome Wide Analysis of Genetic Associations with Environmental Variables in East Texas Loblolly Pine (Pinus taeda L.). In: Plant & Animal Genome XX. The International Conference on the Status of Plant and Animal Genome Research, Final Program and Abstracts Guide, P0513, p. 233, January 14-18, 2012. San Diego, CA, USA (http://pag.confex.com/pag/xx/webprogram/Paper3066.html)
- Chhatre, V., T. Byram, D.B. Neale, J.L. Wegrzyn, and K.V. Krutovsky, 2013 Genetic structure and association mapping of adaptive and selective traits in the East Texas loblolly pine (Pinus taeda L.) breeding populations. Tree Genetics and Genomes DOI 10.1007/s11295-013-0624-x
- Krutovsky, K.V., J. Burczyk, I. Chybicki, R. Finkeldey, T. Pyhajarvi, J.J. Robledo-Arnuncio, 2012 Gene flow, spatial structure, local adaptation and assisted migration in trees, pp. 71-116, Ch. 4 in Genomics of Tree Crops, edited by R.J. Schnell and P.M. Priyadarshan. Springer Science, Inc.
- Chhatre, V.E., M. Resende Jr., P.R. Munoz, G.F. Peter, J.M Davis, M. Kirst, C.S. Echt, K.V. Krutovsky, C. Dana Nelson, 2013. An Integrated Linkage Map of Loblolly Pine and its Application in QTL Mapping in a Multi-Parent, Pedigree-Structured Population. In: Plant & Animal Genome XXI. The International Conference on the Status of Plant and Animal Genome Research, Final Program and Abstracts Guide, W289, p. , January 12-16, 2013. San Diego, CA, USA (http://pag13.mapyourshow.com/5_0/sessions/sessiondetails.cfmSchedul edSessionID=1DAF)
- Chhatre, V.E., T.D. Byram, D.B. Neale, J.L. Wegrzyn, and K.V. Krutovsky, 2013. Landscape Genomics of East Texas Loblolly Pine (Pinus taeda L.). In: Plant & Animal Genome XXI. The International Conference on the Status of Plant and Animal Genome Research, Final Program and Abstracts Guide, P0759, p. , January 12-16, 2013. San Diego, CA, USA (http://pag13.mapyourshow.com/5_0/sessions/sessiondetails.cfmSchedul edSessionID=18A9CFC6)
- Krutovsky, K.V. 2012. Forest ecogenomics and genomic selection for mitigating climate change and breeding resilient trees. Final Conference and Workshops of Noveltree Project. Tree Breeding, Genomics and Evolutionary Biology: New synergies to tackle the impact of climate change in the 21st century, 16-17 October 2012, Helsinki, Finland. Abstracts of conference, p. 66 (http://www.metla.fi/tapahtumat/2012/Noveltree/Book%20of%20Abstracts% 20-%20Final%20Conference%20Noveltree.pdf)
- Krutovsky, K.V. 2012. Forest Genomics for Mitigating Climate Change and Breeding Resilient Trees. Keynote presentation at the AdapCAR and IUFRO (WP 2.02.00) meeting 3-5 October 2012 in Riga, Latvia: Genetic aspects of adaptation and Mitigation: forest health, wood quality and biomass production (http://www.nordicforestresearch.org/adapcar/files/2012/10/Program-Ad apCAR-IUFRO-Meeting.pdf).
- Krutovsky, K.V., V. Chhatre, and T. Byram, 2012 How genomics can help restore the Lost Pines. Western Gulf Forest Tree Improvement Program (WGFTIP) Contact Representatives Meeting, May 22-23, 2012, Bastrop, TX, USA.
- Chhatre, V., T. Byram, and K.V. Krutovsky, 2012 Evolutionary & population genetics of the Lost Pines: Lessons from history. Western Gulf Forest Tree Improvement Program (WGFTIP) Contact Representatives Meeting, May 22-23, 2012, Bastrop, TX, USA.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Single nucleotide polymorphisms (SNPs) in the following 10 wood quality and drought resistance related genes were genotyped in 1706 loblolly pine trees representing the first and second generation selection trees in the Western Gulf Forest Tree Improvement Program (WGFTIP), one of the largest tree improvement programs in the US, in collaboration with Dr. Thomas Byram (Director, WGFTIP): Ccoaomt (CL544Contig1-03-112), Ccr1 (CL594Contig1-06-236), Comt2 (0-10914-02-55 and 0-10914-02-331), Cpk-3 (CL2332Contig1-01-175 and CL2332Contig1-01-314), Lp3-3 (CL1740Contig1-03-78), Pal1 (CL863Contig1-03-164), Ppap12 (CL3898Contig1-04-256), Ptlim1 (CL1905Contig1-03-377 and CL1905Contig1-06-353), Ptlim2 (CL711Contig1-04-212), 4CL (0-7767-01-191 and UMN-CL379Contig1-12-117). Association between allelic variation in these 14 SNPs and variation in (a) adaptive and breeding traits (such as growth rate, DBH, mortality, drought resistance, etc.), (b) eco-physical or geographic variables (latitude and longitude), and (c) environmental or climatic variables (temperature, precipitation, aridity, etc.) was studied using TASSEL (Bradbury et al. 2007) and logistic regression (Krutovsky et al. 2009), and a few significant associations were found. PARTICIPANTS: (1) Dr. Judy Brooks (jebrooks@neo.tamu.edu), Research Assistant, 0.5 FTE; (2) Tomasz Koralewski (tkoral@tamu.edu), PhD student (successfully graduated in August 2010); 3) Vikram Chhatre (crypticlineage@gmail.com), PhD student. Education is embedded throughout the research process. Educational activities do not only include a Ph.D. graduate student but also a wide range of research-sharing emanate from this important aspect of the project. While virtually all steps of the study involve tasks to be performed by the graduate student, which includes DNA isolation, PCR amplification, design of primers, sequencing reaction and analysis, SNP genotyping, gel electrophoresis, statistical analysis of data, and writing reports and papers, the graduate student takes leadership in the development and execution of complementary education activities. Existing programs for graduate students at Texas A&M University benefits from the current research, as well. Both Genetics and MEPS (Molecular and Environmental Plant Sciences) major students going through rotation during their first year of graduate studies will engage in the research and receive additional options from which to choose areas of specialization and research. Our project will also serve as a platform to train college students, including community college students. Our expectation is to enrich the biology program of Blinn College, a local community college, through an existing agreement between Texas A&M University and the College. TARGET AUDIENCES: Forest geneticists and breeders community, such as, for instance, the Western Gulf Forest Tree Improvement Program and the Gene Conservation Program (Texas Forest Service, Forest Science Laboratory, Texas A&M University, College Station, TX 77845-2585; http://www.ars-grin.gov/misc/wgftip) PROJECT MODIFICATIONS: The research is more, if not primarily focused now on loblolly pine taking advantage of having high-throughput SNP genotyping assay developed for loblolly pine.
Impacts
Several SNPs were Fst outliers with unusually high or low differentiation above or below thresholds for neutral markers that could be signatures of selection. The genetic differentiation was above the threshold expected for neutral markers in Ccoaomt (CL544Contig1-03-112) = 0.024 (P < 0.03) and Cpk-3 (CL2332Contig1-01-314) = 0.022 (P < 0.03), and below threshold in Ptlim2 (CL711Contig1-04-212) = 0.000 (P < 0.007) despite the relatively high heterozygosity for these markers, which could be signatures of positive and balancing selections, respectively. A few SNPs were significantly associated with important adaptive and breeding traits but none of them passed the false positive rate test. For instance, Comt2 (0-10914-02-55) was associated with 1) bole length at age 20, 2) diameter at breast height age 7, 3) height at age 20, and 4) max branch diameter at age 7; Ptlim2 (CL711Contig1-04-212) was associated with 1) clear bole length at age 12, and 2) fusiform rust resistance (measured as the total number of fusiform rust stem galls at age 3); 4CL (UMN-CL379Contig1-12-117) was associated with 1) crown width at age 7; 2) fusiform rust resistance, and 3) height at age 3; Cpk-3 (CL2332Contig1-01-314) was associated with 1) clear bole length at age 12. The SNPs in Lp3_3 (CL1740Contig1-03-78) were associated with mortality rate. Several SNPs were associated with geographic gradients and demonstrated clinal allelic variation that is usually a result of interaction between gene flow and local adaptation to environmental variables, such as temperature and precipitation that also usually have gradual distribution. SNPs in Lp3_3 (CL1740Contig1-03-78), Ppap12 (CL3898Contig1-04-256) and 4CL (0-7767-01-191) were associated with latitude gradient, and SNPs in Ppap12 (CL3898Contig1-04-256) were associated also with longitude gradient. CONCLUSIONS: 1. Several SNPs demonstrated extremely high or extremely low Fst values that could be signatures of positive and balancing selections, respectively. 2. Several significant associations were detected between SNPs and geographic and demonstrated clinal allelic variation that could be also signatures of selection and local adaptation to environmental variables.
Publications
- Chhatre, V., T. Byram, D.B. Neale, J.L. Wegrzyn and K.V. Krutovsky 2011 High-Density SNP Genotyping Of East Texas Loblolly Pine (Pinus taeda L.). In: Plant & Animal Genome XIX. The International Conference on the Status of Plant and Animal Genome Research, Final Program and Abstracts Guide, W238, p. 139, January 15-19, 2011. San Diego, CA, USA (http://www.intl-pag.org/19/abstracts/W35_PAGXIX_238.html)
- Krutovsky, K.V., J. Burczyk, I. Chybicki, R. Finkeldey, T. Pyhajarvi, J.J. Robledo-Arnuncio, 2011 Gene flow, spatial structure, local adaptation and assisted migration in trees, pp. , Ch. in Genomics of Tree Crops, edited by R.J. Schnell and P.M. Priyadarshan. Springer Science, Inc.
- Koralewski, T.E., and K.V. Krutovsky, 2011 Evolution of exon-intron structure and alternative splicing. PLoS One 6(3): e18055. doi:10.1371/journal.pone.0018055.
- Chhatre, V.E., T.D. Byram, D.B. Neale, J.L. Wegrzyn, and K.V. Krutovsky, 2011. Association mapping of adaptive and breeding traits in East Texas Loblolly Pine (Pinus taeda L.) breeding populations using high-density SNP genotyping. In: Proceedings 31st Southern Forest Tree Improvement Conference, June 13-16, 2011. Biloxi, MS, USA (http://www.rngr.net/publications/tree-improvement-proceedings/sftic)
- Chhartre, V.E., T.D. Byram, D.B. Neale, J.L. Wegrzyn, and K.V. Krutovsky, 2011. Association mapping of adaptive and breeding traits in East Texas Loblolly Pine (Pinus taeda L.) breeding populations using high-density SNP genotyping. In: Proceedings 31st Southern Forest Tree Improvement Conference, June 13-16, 2011. Biloxi, MS, USA (http://www.rngr.net/publications/tree-improvement-proceedings/sftic)
- Chhatre, V.E., T.D. Byram, D.B. Neale, J.L. Wegrzyn, and K. V. Krutovsky, 2011. Linkage Disequilibrium and Association Mapping of Adaptive and Selected Traits in Breeding Populations of East Texas Loblolly Pine. International Symposium: Genomics-based breeding in forestry. June 22-24, 2011, Davis, CA, USA.
- Chhatre, V., T. Byram, D.B. Neale, J.L. Wegrzyn and K.V. Krutovsky 2011 Genetic structure and association mapping of adaptive and selected traits in East Texas loblolly pine breeding populations. National Association of Plant Breeding Annual Meeting, 5th Annual Plant Breeding Coordinating Committee Meeting, 1st Annual National Association of Plant Breeders Meeting, Texas A&M University, College Station, Texas, May 23-25, 2011, Abstracts Guide, p. 11 (http://www.plantbreeding.org/napb/Meetings/2011/Abstracts11e05dsSJ.p df)
- Chhatre, V., T. Byram, D.B. Neale, J.L. Wegrzyn and K.V. Krutovsky 2011 Association Mapping Analysis of Economic Traits in East Texas Loblolly Pine (Pinus taeda L.). Western Gulf Forest Tree Improvement Program (WGFTIP) Contact Representatives Meeting, May 10-11, 2011, Jasper, TX, USA.
- Chhatre, V., T. Byram, D.B. Neale, J.L. Wegrzyn and K.V. Krutovsky 2011 Population Structure And Association Mapping Analysis Of Adaptive Traits In East Texas Loblolly Pine (Pinus taeda L.). In: Plant & Animal Genome XIX. The International Conference on the Status of Plant and Animal Genome Research, Final Program and Abstracts Guide, P189, p. 193, January 15-19, 2011. San Diego, CA, USA (http://www.intl-pag.org/19/abstracts/P03e_PAGXIX_189.html)
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Single nucleotide polymorphisms (SNPs) in the following 10 wood quality and drought resistance related genes were genotyped in 1706 loblolly pine trees representing the first and second generation selection trees in the Western Gulf Forest Tree Improvement Program (WGFTIP), one of the largest tree improvement programs in the US, in collaboration with Dr. Thomas Byram (Director, WGFTIP): Ccoaomt (CL544Contig1-03-112), Ccr1 (CL594Contig1-06-236), Comt2 (0-10914-02-55 and 0-10914-02-331), Cpk-3 (CL2332Contig1-01-175 and CL2332Contig1-01-314), Lp3-3 (CL1740Contig1-03-78), Pal1 (CL863Contig1-03-164), Ppap12 (CL3898Contig1-04-256), Ptlim1 (CL1905Contig1-03-377 and CL1905Contig1-06-353), Ptlim2 (CL711Contig1-04-212), 4CL (0-7767-01-191 and UMN-CL379Contig1-12-117). These 14 SNPs are based on amplicons representing partial sequences of ~3000 expressed genes including these 10 genes (one or two SNPs per gene) and were originally discovered in a small rangewide population set in the NSF funded ADEPT2 resequencing project. The nucleotide sequences for all genes were deposited into GenBank (loblolly pine PopGene sequence data). The trees were genotyped using Illumina Infinium SNP genotyping assay developed through the USDA Conifer Translational Genomics Network (CTGN) project in collaboration with Dr. Davis Neale (UC in Davis). The tree samples were subdivided into 14 (first generation) and 8 (second generation) populations based on their geographical origin and 30 breeding groups based on their pedigree and breeding strategy. The first generation selection trees were selected from natural and old plantations in East Texas that represents the westernmost area of the natural loblolly pine distribution. The first generation selection trees were subsequently partitioned into sublines and subjected to breeding and controlled pollination. Their progeny established the second generation selection trees. Genetic variation, population substructure and adaptive trait associations were investigated for 10 genes in both first and second generation populations. The SNPs were polymorphic in Ccoaomt (CL544Contig1-03-112), Ccr1 (CL594Contig1-06-236), Cpk-3 (CL2332Contig1-01-175 and CL2332Contig1-01-314), Lp3-3 (CL1740Contig1-03-78), Ptlim2 (CL711Contig1-04-212), but weakly polymorphic in Comt2 (0-10914-02-55 and 0-10914-02-331), Pal1 (CL863Contig1-03-164), Ppap12 (CL3898Contig1-04-256), and almost monomorphic in 4CL (0-7767-01-191 and UMN-CL379Contig1-12-117), and Ptlim1 (CL1905Contig1-03-377 and CL1905Contig1-06-353). Family pedigrees were verified using kinship coefficients derived from SNPs. Population structure was analyzed using the Bayesian analysis as implemented in software STRUCTURE (Pritchard et al, 2000) and also using the ΔK parameter of Evanno et al (2005). FST outlier method was used to detect candidate markers for natural selection. The blast homology search was done to assess their functional significance. Significant associations between markers and adaptive traits were also studied using TASSEL (Bradbury et al. 2007). PARTICIPANTS: (1) Dr. Judy Brooks (jebrooks@neo.tamu.edu), Research Assistant, 0.5 FTE; (2) Tomasz Koralewski (tkoral@tamu.edu), PhD student (successfully graduated in August 2010); 3) Vikram Chhatre (crypticlineage@gmail.com), PhD student. Education is embedded throughout the research process. Educational activities do not only include a Ph.D. graduate student but also a wide range of research-sharing emanate from this important aspect of the project. While virtually all steps of the study involve tasks to be performed by the graduate student, which includes DNA isolation, PCR amplification, design of primers, sequencing reaction and analysis, SNP genotyping, gel electrophoresis, statistical analysis of data, and writing reports and papers, the graduate student will take leadership in the development and execution of complementary education activities. Existing programs for graduate students at Texas A&M University will benefit from the current research, as well. Both Genetics and MEPS (Molecular and Environmental Plant Sciences) major students going through rotation during their first year of graduate studies will engage in the research and receive additional options from which to choose areas of specialization and research. Our project will also serve as a platform to train college students, including community college students. Our expectation is to enrich the biology program of Blinn College, a local community college, through an existing agreement between Texas A&M University and the College. TARGET AUDIENCES: Forest geneticists and breeders community, such as, for instance, the Western Gulf Forest Tree Improvement Program and the Gene Conservation Program (Texas Forest Service, Forest Science Laboratory, Texas A&M University, College Station, TX 77845-2585; http://www.ars-grin.gov/misc/wgftip) PROJECT MODIFICATIONS: The research is more, if not primarily focused now on loblolly pine taking advantage of having high-throughput SNP genotyping assay developed for loblolly pine.
Impacts
The average heterozygosity was relatively high in the following SNPs: Ccoaomt (CL544Contig1-03-112) = 0.489, Ccr1 (CL594Contig1-06-236) = 0.457, Cpk-3 (CL2332Contig1-01-175 = 0.155 and CL2332Contig1-01-314 = 0.365), Lp3-3 (CL1740Contig1-03-78) = 0.293, Ptlim2 (CL711Contig1-04-212) = 0.154, but low in Comt2 (0-10914-02-55 = 0.087 and 0-10914-02-331 = 0.080), Pal1 (CL863Contig1-03-164) = 0.028, Ppap12 (CL3898Contig1-04-256) = 0.044, and very low in 4CL (0-7767-01-191 = 0.001 and UMN-CL379Contig1-12-117 = 0.001) and Ptlim1 (CL1905Contig1-03-377 = 0.015 and CL1905Contig1-06-353 = 0.015). The genetic differentiation was relatively low in general (Fst = 0.005), but it was above the threshold expected for neutral markers in Ccoaomt (CL544Contig1-03-112) = 0.024 (P < 0.03) and Cpk-3 (CL2332Contig1-01-314) = 0.022 (P < 0.03), and below threshold in Ptlim2 (CL711Contig1-04-212) = 0.000 (P < 0.007) despite the relatively high heterozygosity for these markers, which could be signatures of positive and balancing selections, respectively. A few SNPs were significantly associated with important adaptive and breeding traits but none of them passed the false positive rate test. For instance, Comt2 (0-10914-02-55) was associated with 1) bole length at age 20, 2) diameter at breast height age 7, 3) height at age 20, and 4) max branch diameter at age 7; Ptlim2 (CL711Contig1-04-212) was associated with 1) clear bole length at age 12, and 2) fusiform rust resistance (measured as the total number of fusiform rust stem galls at age 3); 4CL (UMN-CL379Contig1-12-117) was associated with 1) crown width at age 7; 2) fusiform rust resistance, and 3) height at age 3; Cpk-3 (CL2332Contig1-01-314) was associated with 1) clear bole length at age 12. CONCLUSIONS: 1. The SNP polymorphism was relatively high in 10 wood quality and drought resistance related genes genotyped in 1706 loblolly pine trees in the studied populations. 2. Population differentiation was relatively low, but two SNPs demonstrated extremely high and one SNP extremely low FST values that could be signatures of positive and balancing selections, respectively. 3. Several significant associations were detected between SNPs and adaptive and/or breeding phenotypic traits but, none of them passed the false positive rate test.
Publications
- Krutovsky, K.V., 2010 What can we learn about evolutionary responses to environmental change from ecological population genomics The keynote speaker at the conference "Forest Ecosystem Genomics and Adaptation", June 9-11, 2010, Spain, Madrid (http://www.ecosystemgenomics2010.fgua.es//invi/invi.cfm). A joint event co-organized by the research partners in the Network of Excellence EVOLTREE (www.evoltree.eu), funded by the EC 6th Framework Programme for Research.
- Krutovsky, K.V., V. Chhatre and T. Byram, 2010 Genetic Structure in Loblolly Pine Breeding Populations. Western Gulf Forest Tree Improvement Program (WGFTIP) Contact Representatives Meeting, May 18-19, 2010, Lake De Gray State Park Lodge, Arkadelphia, AR, USA.
- Koralewski, T.E., and K.V. Krutovsky, 2010 Evolution of intron-exon structure and alternative splicing: what did we learn from genomes of completely sequenced species and what can we predict for insufficiently studied species 11th Annual Ecological Integration Symposium: Understanding Patterns and Processes Across Scales at Texas A&M University, March 26-27, 2010, College Station, TX, USA (http://wfsc.tamu.edu/eis/EIS_Student_Symposium_Schedule.pdf)
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: (1) The study of molecular evolution of wood quality and drought resistance related genes in four Southern pines from subsection Australes, Pinus taeda, P. elliottii, P. echinata, and P. palustris was continued using available nucleotide sequences and newly sequenced homologous and orthologous adaptive trait related genes, including drought tolerance or drought response related genes. Multiple nucleotide sequence alignments were generated for 34 genes in loblolly pine and most of other Southern pine species: 4cl, agpl-like or agp1519, agp-4, agp-6, alpha-tubulin, ccoaomt-1, c3h, c4h-1, c4h-2, cad, ccr, cesA3, comt-2, glyhmt, ptlim-1, ptlim-2, pal-1, sam-1, sam-2, aqua-MIP, cpk3, dhn-1, dhn-2, erd3, ferritin, lp3-1, lp3-3, lp5-like, mt-like, pp2c, ppap12, rd21A-like, sod-chl, and ug-2_498. Neutrality tests, such as Tajima's D, HKA, MK and others, were performed using intra- and interspecific approaches to find signatures of selection in genes of interest. (2) The phylogenetic study to define the phylogenetic relationships of the four Southern pine species within the subsection Australes, and other pine species in the family Pinaceae was enhanced using nucleotide sequences of 3 nuclear protein coding genes sequenced in 58 pine and 2 outgroup species (Picea sitchensis and Pseudotsuga menziesii): ifg8612 (lea), 4cl, and agp-6. (3) The study of proteomic complexity expected from exon-intron gene structure due to alternative splicing was continued using a computer program (Perl script) developed in the previous year that could parse genomic data of completely sequenced and thoroughly annotated model species downloaded from NCBI GenBank and infer alternative splicing data for exon-intron structure and alternative splicing analysis. Alternative splicing was categorized using binary approach (Nagasaki et al. 2006). We developed and applied an approach to test our hypothesis that alternative splicing contributes greatly to proteomic and metabolomic complexity of more evolutionary advanced organisms. We studied the frequency of alternative splicing in evolutionary different completely sequenced and fully annotated species. One of our objectives was to use these data for developing a statistical model that would allow us to predict metabolomic and proteomic complexity in the non-model organisms, including forest trees. Number of exons, alternative splicing rate and diversity and, therefore, proteomic complexity are significantly increased in more evolutionary advanced complex organisms. (4) Single nucleotide polymorphisms (SNPs) in the following 11 wood quality and drought resistance related genes were genotyped in a large range wide population sample of more than 2,000 loblolly pine trees from East Texas in collaboration with Dr. Thomas Byram (Western Gulf Tree Improvement Program): ccoaomt-1, ccr-1, comt-2, pal1, ptlim2, cpk3, lp3-3, ppap12, 4cl, comt2, and ptlim1. Polymorphic SNPs were selected from the GenBank loblolly pine PopGene sequence data and genotyped using Illumina Infinium SNP genotyping assay in collaboration with Dr. Davis Neale (UC in Davis). PARTICIPANTS: (1) Dr. Judy Brooks (jebrooks@neo.tamu.edu), Research Assistant, 0.5 FTE; (2) Tomasz Koralewski (tkoral@tamu.edu), PhD student. Education is embedded throughout the research process. Educational activities do not only include a Ph.D. graduate student but also a wide range of research-sharing emanate from this important aspect of the project. While virtually all steps of the study involve tasks to be performed by the graduate student, which includes DNA isolation, PCR amplification, design of primers, sequencing reaction and analysis, SNP genotyping, gel electrophoresis, statistical analysis of data, and writing reports and papers, the graduate student will take leadership in the development and execution of complementary education activities. Existing programs for graduate students at Texas A&M University will benefit from the current research, as well. Both Genetics and MEPS (Molecular and Environmental Plant Sciences) major students going through rotation during their first year of graduate studies will engage in the research and receive additional options from which to choose areas of specialization and research. Our project will also serve as a platform to train college students, including community college students. Our expectation is to enrich the biology program of Blinn College, a local community college, through an existing agreement between Texas A&M University and the College. TARGET AUDIENCES: Forest geneticists and breeders community, such as, for instance, the Western Gulf Forest Tree Improvement Program and the Gene Conservation Program (Texas Forest Service, Forest Science Laboratory, Texas A&M University, College Station, TX 77845-2585; http://www.ars-grin.gov/misc/wgftip) PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
(1) For interspecific neutrality test Tajima's D, the loblolly pine population set included our data and those downloaded from the PopSet database at GenBank. HKA and MK tests were run to compare the loblolly pine set to the three remaining species. More than 20 genes were completely resequenced in all four species. (2) Phylogenetic analysis demonstrated very close phylogenetic relationships between the Southern pines. Both neighbor-joining (NJ) and maximum parsimony (MP) trees give very similar pattern and are in a good consensus with the existing pine phylogenies. (3) Examined exon-intron structure and alternative splicing in 36 most studied model species was used to generate regression models for pines and other species. The obtained results were compared with available exon-intron data in loblolly pine and were used to predict proteomic complexity in pines. It was found that number of gene products and exons in the genome, exon/gene ratio and AS ratio increase as species become more evolutionarily advanced. Statistical models were developed to reveal relationships between these characteristics. The models and parameters estimated from limited data sets (e.g. exon length or exon/gene ratio in loblolly pine) can be used as good predictors of characteristics that are difficult to assess (e.g. total number of genes, gene products and exons). Based on the developed models ~20,000 protein coding genes and ~21,000 total number of genes are expected in loblolly pine. Obtained results confirmed our hypothesis that regulatory and other post-transcriptional processes such as alternative splicing might play an increasingly more important role in maintaining complexity of more evolved organisms. (4) Analysis of SNP variation in 11 wood quality and drought resistance related genes genotyped in more than 2,000 loblolly pine trees from East Texas is in progress and will include estimates of allele frequencies, expected and observed heterozygosity, genetic differentiation, geographic distribution (spatial and clinal variation analysis and subpopulation clustering), testing for outliers (possible indication of selection), and association with environmental factors. CONCLUSIONS: (1) Neutrality tests demonstrated significant deviation from neutrality in cinnamoyl alcohol dehydrogenase (cad) and coumarate 3-hydroxylase (c3h) genes. The HKA test demonstrated signatures of selection in the LIM domain protein 1 (ptlim-1) gene. (2) The phylogenetic position of the four Southern pine species among other pines was refined with inclusion of additional genes. Nucleotide-based phylogenetic and haplotype network analysis confirmed very close relationships between four Southern pine species. (3) Our study demonstrated a great diversity of alternative splicing types. The most common alternative splicing types were alternative 3' splice site in plants and exon skipping in animals. The models and parameters estimated from limited data sets (e.g. exon length or exon/gene ratio in loblolly pine) can be used as good predictors of total number of genes, gene products and exons.
Publications
- Krutovsky, K.V., 2009 Population genomics and forest genetic conservation. In: Forest Genetics Workshop "Opportunities, challenges and limitations of genomics-based technologies in forest tree breeding and forest genetics", Program and Abstracts Guide, October 7-9, 2009, Freiburg, Germany (http://www.efi.int/portal/news___events/events/extra/2009/workshop_f orest_genetics/programme/)
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: New data were obtained in 2008 on comparative genomic study in which we studied nucleotide variation in adaptive trait related orthologous genes, including drought tolerance or drought response related ones in the four Southern pine species, P. taeda, P. elliottii, P. echinata, and P. palustris. The water management and drought resistance-related genes have been sampled and resequenced in these species using primers developed earlier for loblolly pine by Brown et al. (2004) and Gonzalez-Martinez et al. (2006). Neutrality tests (Tajima's D, HKA and MK) were performed using DnaSP. In case of Tajima's D, the loblolly pine population set included our data and those downloaded from the PopSet database at GenBank. HKA and MK tests were run to compare the loblolly pine set to the three remaining species. Phylogenetic analysis was done using MEGA4 and based on the AGP6 gene sequenced in 21 pine species and Picea sitchensis that was used as an out-group species. Single nucleotide polymorphisms (SNPs) in expressed sequence tags (ESTs) representing 34 loblolly pine genes were studied and compared with SNPs that were independently discovered via direct resequencing of megagametophytes from population samples representing 32 individual trees in earlier studies. This is the first study that compared ESTs and genomic sequences from a population sample for a relatively large set of the same genes. A high correspondence was observed between both datasets. However, out of 243 SNPs found in population studies only 113 (46.5%) were also found among the ESTs. The discrepancies concerned mainly rare alleles: only 17 out of 79 unique SNPs were confirmed (21.5%) while 96 out of 164 polymorphic SNPs were confirmed (58.5%). Our results demonstrate a good potential of in silico SNP discovery in loblolly pine. Diversity, complexity and difference in anatomy, physiology and behavior are greatly increasing from relatively simple, less evolved organisms toward more evolutionary advanced species. However, this does not affect significantly the number of genes that constitute the structural and functional basis for every living organism. For instance, such evolutionary distant species as Arabidopsis and human have a similar number of genes. This suggests that regulatory and other post-transcriptional processes such as alternative splicing might play an increasingly more important role in maintaining complexity of more evolved organisms. Therefore, we developed an approach to test our hypothesis that alternative splicing contributes greatly to proteomic and metabolomic complexity of more evolutionary advanced organisms. We studied the frequency of alternative splicing in evolutionary different completely sequenced and fully annotated species. We developed a computer program that allowed us to infer the types and frequency of alternative splicing from the annotation provided in the GenBank files. One of our objectives was to use these data for developing a computer model that would allow us to predict metabolomic and proteomic complexity in the non-model organisms, including forest trees. Here we present only the first steps in this direction. PARTICIPANTS: (1) Dr. Judy Brooks (jebrooks@neo.tamu.edu), Research Assistant, 0.5 FTE; (2) Tomasz Koralewski (tkoral@tamu.edu), PhD student. Education is embedded throughout the research process. Educational activities do not only include a Ph.D. graduate student but also a wide range of research-sharing emanate from this important aspect of the project. While virtually all steps of the study involve tasks to be performed by the graduate student, which includes DNA isolation, PCR amplification, design of primers, sequencing reaction and analysis, SNP genotyping, gel electrophoresis, statistical analysis of data, and writing reports and papers, the graduate student will take leadership in the development and execution of complementary education activities. Existing programs for graduate students at Texas A&M University will benefit from the current research, as well. Both Genetics and MEPS (Molecular and Environmental Plant Sciences) major students going through rotation during their first year of graduate studies will engage in the research and receive additional options from which to choose areas of specialization and research. Our project will also serve as a platform to train college students, including community college students. Our expectation is to enrich the biology program of Blinn College, a local community college, through an existing agreement between Texas A&M University and the College. TARGET AUDIENCES: Forest geneticists and breeders community, such as, for instance, the Western Gulf Forest Tree Improvement Program and the Gene Conservation Program (Texas Forest Service, Forest Science Laboratory, Texas A&M University, College Station, TX 77845-2585; http://www.ars-grin.gov/misc/wgftip) PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
(1) More than 20 genes were completely resequenced in all four species. Neutrality tests demonstrated significant deviation from neutrality in cinnamoyl alcohol dehydrogenase (cad) and coumarate 3-hydroxylase (c3h) genes. The HKA test demonstrated signatures of selection in the LIM domain protein 1 (ptlim-1) gene. (2) Phylogenetic analysis demonstrated very close phylogenetic relationships between the Southern pines. Both neighbor-joining (NJ) and maximum parsimony (MP) trees give very similar pattern and are in a good consensus with the existing pine phylogenies. There was practically no difference between trees based on all nucleotide positions vs. those based only on the 3rd position in codons. (3) We compared nucleotide sequences of 34 resequenced genes from the PopSets (504,224 bp) and the ESTs that represent them (243,518 bp). The number of segregating sites was higher among the ESTs than in the PopSets, 1 SNP per every 28 bp among ESTs vs. 1 SNP per every 60 bp in the PopSets, respectively. For polymorphic SNPs, the ratios were less different, 1 SNP per every 55 bp among ESTs vs. 1 SNP per every 88 bp in the PopSets, respectively. This can be explained by relatively more sequencing errors among ESTs. The confirmation rate varied widely from 0% to 100%, depending on gene and EST sequencing intensity. It is interesting to note that while the confirmation rate significantly correlated with the number of ESTs for polymorphic SNPs (r = 0.30, P < 0.05), the correlation was insignificant for unique ESTs. This cannot be explained by more sequencing errors among ESTs. It is likely that a significant portion of unique SNPs really represent very rare mutations. (4) The information was gathered about exon/intron structure and number and type of alternative protein products that are encoded by each locus in model species. Based on these data, a decision was made on the type of alternative splicing, if any. Our data demonstrate a great diversity of alternative splicing types. The most common 10 types represent only ~12% of the total number of alternative splicing events. The most common alternative splicing types are due to alternative 3' splice site and alternative promoters. Finally, we summarized data on alternative splicing types and rates in the studied model species. CONCLUSIONS: (1) There are signatures of selection for cinnamoyl alcohol dehydrogenase (cad), coumarate 3-hydroxylase (c3h) and LIM domain protein 1 (ptlim-1) genes. (2) Nucleotide-based phylogenetic and haplotype network analysis demonstrated very close relationships between four Southern pine species. (3) ESTs are a good and reliable source of SNPs; the confirmation rate was relatively high for polymorphic SNPs (58.5%) but low for unique SNPs (21.5%); a significant portion of unique SNPs likely represent really rare mutations; SNPs discovered in silico will have numerous important applications in association genetics studies, marker-aided breeding, genome mapping and assembling a physical map. (4) Number of exons, alternative splicing rate and diversity and, therefore, proteomic complexity are significantly increased in more evolutionary advanced complex organisms.
Publications
- Koralewski, T.E., J.E. Brooks and K.V. Krutovsky, 2008 Molecular ecology and evolution of drought resistance related genes in four Southern pines from subsection Australes. In: Metacommunities: Connectivity, Dispersal and Invasion. 9th Ecological Integration Symposium, Final Program and Abstracts Guide, p. 54, March 28-29, 2008. Texas A&M University, College Station, TX, USA (http://wfscnet.tamu.edu/eis/EIS%202008.pdf)
- Krutovsky, K.V., and J.E. Brooks, 2008 Efficiency of in silico SNP discovery in Loblolly Pine. In: Plant & Animal Genome XVI. The International Conference on the Status of Plant and Animal Genome Research, Final Program and Abstracts Guide, p. 245, January 12-16, 2008. San Diego, CA, USA (http://www.intl-pag.org/16/abstracts/PAG16_P05j_507.html)
- Koralewski, T.E., J.E. Brooks and K.V. Krutovsky, 2008 Comparative nucleotide sequence analysis in four Southern pines from Subsection Australes. In: Plant & Animal Genome XVI. The International Conference on the Status of Plant and Animal Genome Research, Final Program and Abstracts Guide, p. 247, January 12-16, 2008. San Diego, CA, USA (http://www.intl-pag.org/16/abstracts/PAG16_P05j_514.html)
- Koralewski, T.E., L.A. Zhivotovsky, K.V. Krutovsky, 2008 Proteomic complexity expected from intron-exon gene structure due to alternative splicing. Texas A&M AgriLife Conference, Texas A&M University, January 7-11, 2008 (http://agnews.tamu.edu/showstory.php?id=285)
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Progress 05/01/07 to 12/31/07
Outputs
OUTPUTS: The main Southern pines, P. taeda, P. elliottii, P. echinata, and P. palustris, are closely related members of the subsection Australes. The current natural range of these Southern pines includes the warm-temperate to subtropical climate of 13 southeastern United States. Moreover, they are planted as important exotics in many other parts of the world. They are well-adapted to drought conditions. Their populations grow in different environments and probably accumulated significant adaptive genetic variation. They are distributed in large, out-crossing communities with high gene flow and little population structure at neutral markers. Since the natural range covers large variation in temperature and rainfall from the coastal plains to the interior southern uplands, very interesting variation in adaptive trait related genes can be expected All together, current data suggest that Southern pines are evolutionarily very young species. These data provide us with a unique
opportunity to study adaptation and evolution "in progress". Here we present our preliminary data obtained in 2007 on comparative genomic study in which we studied nucleotide variation in adaptive trait related orthologous genes, including drought tolerance or drought response related ones. The available genomic resources of one of the most-studied pine species, loblolly pine, greatly helped us sequence and study other Southern pine species. The examined water use efficiency and drought resistance-related genes have been sampled and resequenced in the four abovementioned pine species, using primers developed earlier for loblolly pine by Brown et al. (2004) and Gonzalez-Martinez et al. (2006). Several hundred single nucleotide polymorphisms (SNPs) were found in these studies. At this stage of our research, only a few individuals from each of the other species were sequenced. Both forward and reverse strands were sequenced, and consensus was generated using the Sequencher (v. 4.2)
computer program. This step helped us maintain high quality of the data. Multiple alignment was performed using BioEdit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) software; neutrality tests (Tajima's D, HKA and MK) were run using DnaSP (http://www.ub.edu/dnasp/). In the case of Tajima's D, the loblolly pine population set included our data and those downloaded from the PopSet database at GenBank (http://www.ncbi.nlm.nih.gov). HKA and MK tests were run to compare the loblolly pine set to the three remaining species.
PARTICIPANTS: (1) Dr. Judy Brooks (jebrooks@neo.tamu.edu), Research Assistant, 0.5 FTE; (2) Tomasz Koralewski (tkoral@tamu.edu), PhD student. Education is embedded throughout the research process. Educational activities do not only include a Ph.D. graduate student but also a wide range of research-sharing emanate from this important aspect of the project. While virtually all steps of the study involve tasks to be performed by the graduate student, which includes DNA isolation, PCR amplification, design of primers, sequencing reaction and analysis, SNP genotyping, gel electrophoresis, statistical analysis of data, and writing reports and papers, the graduate student will take leadership in the development and execution of complementary education activities. Existing programs for graduate students at Texas A&M University will benefit from the current research, as well. Both Genetics and MEPS (Molecular and Environmental Plant Sciences) major students going through rotation during their
first year of graduate studies will engage in the research and receive additional options from which to choose areas of specialization and research. Our project will also serve as a platform to train college students, including community college students. Our expectation is to enrich the biology program of Blinn College, a local community college, through an existing agreement between Texas A&M University and the College.
TARGET AUDIENCES: Forest geneticists and breeders community.
Impacts
The following 14 genes were completely resequenced in all four species: arabinogalactan-like protein 1519 (agpl-like or agp1519), arabinogalactan 4 (agp-4), arabinogalactan 6 (agp-6), aquaporin membrane intrinsic protein (aqua-MIP), coumarate 3-hydroxylase (c3h), cinnamoyl alcohol dehydrogenase (cad), cinnamoyl CoA reductase (ccr), caffeate O-methyltransferase 2 (comt-2), calcium-dependent protein kinase (cpk3), dehydrin 2 (dhn-2), loblolly pine water-stress inducible protein 3 (lp3-3), putative wall-associated protein kinase (ppap12), LIM domain protein 1 (ptlim-1), and s-adenosyl methionine synthetase 2 (sams-2). At 0.95 confidence level Tajima's D test demonstrated significant deviation from neutrality in cinnamoyl alcohol dehydrogenase (cad) alone. Coumarate 3-hydroxylase's (c3h) neutrality can be rejected at 0.9 confidence level (TABLE 2). The MK test failed to reject neutrality for observed polymorphism in any of the 14 genes (TABLE 3). The HKA test demonstrated
signatures of selection in the LIM domain protein 1 (ptlim-1) gene (TABLE 4). One of the possible causes could be the small sample size for P. elliottii, P. echinata and P. palustris. Results of both tests would be more representative if more individuals were sequenced from these species; this work is currently in progress. Preliminary phylogenetic and haplotype network analysis demonstrated very close relationships between these four species. However, this analysis requires an appropriate outgroup species to delineate relationships between these species. We are in the process of defining and sequencing the orthologous genes in a few outgroup pine species. CONCLUSIONS: (1) There are signatures of selection for cinnamoyl alcohol dehydrogenase (cad), coumarate 3-hydroxylase (c3h) and LIM domain protein 1 (ptlim-1) genes. (2) The sample size should be extended to carry out more powerful and informative neutrality tests. (3) Nucleotide-based phylogenetic and haplotype network analysis
demonstrated very close relationships between these four species, but additional analysis involving outgroup species is required to delineate relationships between these species.
Publications
- No publications reported this period
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