Progress 08/15/08 to 09/30/10
Outputs
OUTPUTS: This seed project has begun to develop a strong foundation of genomic resources for the development of high-resolution linkage maps of pedigrees developed in North America that will enable the selection and improvement of shrub willow varieties to move forward using genomic selection and association mapping. The experimental objectives of this project included: 1) use of flow cytometry to determine ploidy in key parents and pedigrees; 2) cloning and sequencing of cDNAs; and 3) discovery of new markers and adaptation of existing markers to North American pedigrees. Leaf samples were analyzed by the Flow Cytometry Center at Benaroya Research Institute of Virginia Mason. Nuclear DNA content was estimated using an internal control of chicken erythrocyte nuclei. The following species were analyzed and determined to be diploid (2n=2X): Salix purpurea, S. viminalis and hybrids, S. nigra, S. integra, and SV1. Based on these data, it is unlikely that SV1 is a S. dasyclados, which is typically described as 6X, but SV1 is more likely a S. viminalis x S. cinerea hybrid. The following were determined to be tetraploid (2n=4X): S. miyabeana, S. sachalinensis, and their hybrids. Logically, the following hybrids were determined to be triploid (3X): S. viminalis x S. miyabeana and S. purpurea x S. miyabeana. In collaboration with Dr. Gerald Tuskan and Dr. Chris Town a project to sequence the genome of Salix purpurea L. clone 94006 was initiated by the US DOE Joint Genome Institute. To complement the next-generation sequencing of the genome, we have developed BAC libraries that can be used to create a physical map and for improved scaffolding of the sequence. A random shear BAC library consisting of 25,344 clones and representing 5x coverage of the Salix purpurea 94006 genome was constructed by Lucigen Corp. In addition, enzyme digested BAC libraries were also constructed at the Clemson University Genomics Institute (CUGI) using funds provided by Dr. Haiying Liang. We cloned fragments of 20 genes from young stem cDNA of S. sachalinensis and S. miyabeana. Expression profiling in willow stem apical tissue and developing stem tissue was performed for each isolated gene using probe-based qRT-PCR. Two willow parental genotypes and six progeny within a hybrid family were selected for analysis and significant differences in expression among the individuals and between tissue types were observed for most of the genes. To characterize the willow transcriptome, RNA from 8 different tissue samples of S. purpurea was sequenced at JGI using RNA-Seq. Preliminary analysis of selected willow varieties using AFLP and microsatellite marker technologies was performed to begin to identify usable polymorphic markers. Small scale preliminary studies have identified over 100 polymorphic bands in 16 willow clones using 3 AFLP primer combinations, and a panel of 7 SSRs produced polymorphic bands in 10 different cultivars of willow. We performed phylogentic analyses on important willow varieties using matK and ITS sequences. Sequences have been completed for over 35 individuals revealing phylogenetic relationships. PARTICIPANTS: Dr. Lawrence Smart (PI) - Associate Professor, SUNY-ESF Michelle Serapiglia - Graduate student - SUNY-ESF Partnering organizations: US DOE Joint Genome Institute, Oak Ridge National Laboratory, J. Craig Venter Institute, Cornell University, Clemson University TARGET AUDIENCES: The target audience of this project includes the academic research community involved in the development of perennial feedstock crops. The knowledge gained through this project provides tangible resources for the research community to further develop shrub willow crops. This project also impacts growers, landowners, entrepreneurs, and businesses involved in the cultivation, management and production of biomass feedstocks, since these results have direct impact on the development of cultivars producing higher and more sustainable yields across a broader range of marginal sites. These project is also targeted at the biofuels and biopower industry, which is in need of cheaper feedstock biomass for production of carbon-neutral renewable energy. PROJECT MODIFICATIONS: With the advance of next-generation sequencing technology, some technical approaches based on conventional library construction and cloning were replaced with high-throughput sequencing strategies. These aspects of the project gained significant financial support through partnerships with the US Department of Energy Oak Ridge National Lab and Joint Genome Institute, the J. Craig Venter Institute, and Clemson University.
Impacts
Genetic improvement of fast-growing shrub willow (Salix spp.), a proven perennial bioenergy crop for temperate climates, will be expedited by a thorough understanding of the molecular basis for cell wall composition and by molecular tools for early selection in breeding programs. By identifying the genes that are critical for regulating variation in biomass composition, we may be able to develop molecular markers for the early selection of genotypes with varied and improved biomass properties. Patterns of gene expression were correlated with variation in the biomass composition of those genotypes to provide insights into the genetic regulation of lignocellulosic deposition in this important bioenergy crop. These candidate genes will prove useful in the screening of germplasm for variations in gene expression that may be an early indicator of harvest-age biomass compositional characteristics. Future breeding of shrub willow feedstock crops will include selection based on biomass composition, so that the feedstock is optimized for conversion efficiency. Future selection for yield improvements will be greatly improved by using the genomic resources developed in this project to accomplish genomic selection.
Publications
- No publications reported this period
|
Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: This seed project is establishing a strong foundation of genomic resources for the development of high-resolution linkage maps of pedigrees developed in New York that will enable mapping of quantitative trait loci and marker-assisted selection. These marker resources will also be used for association mapping of a diverse population of natural accessions. The experimental objectives include: 1) use of flow cytometry to determine ploidy in key parents and pedigrees; 2) cloning and sequencing of cDNAs; and 3) discovery of new markers and adaptation of existing markers to New York pedigrees. Leaf samples were collected from greenhouse or field-grown samples and were sent to the Flow Cytometry Center at Benaroya Research Institute of Virginia Mason. Nuclear DNA content was estimated using an internal control of chicken erythrocyte nuclei. The following species and varieties were analyzed and determined to be diploid (2n): Salix purpurea 9882-34 (Fish Creek), 9882-41 (Wolcott), 94006 (4 times); S. viminalis Jorr; S. viminalis x S. schwerinii, Bjorn, S. viminalis x (S. schwerinii x S. viminalis) Olof and Sven; S. nigra clone IDs 95311, 01-01-063, 01-03-161, 01-04-142, 01-06-218, 01-07-243, 01-13-013, 01-18-140; S. integra P63 and 05X-278-005; S. dasyclados SV1. The following were determined to be tetraploid (4n): S. miyabeana SX64 (twice), SX67, and 05X-287-059; S. sachalinensis SX61. The following species hybrid varieties were determined to be triploid (3n): (S. schwerinii x S. viminalis) x S. linderstipularis Nimrod; (S. triandra x S. viminalis) x S. linderstipularis Terra Nova; S. viminalis x S. miyabeana 99202-001 and 99202-011 (Tully Champion); S. sachalinensis x S. miyabeana 9970-036 (Canastota) ; S. purpurea x S. miyabeana 9980-005 (Oneida), 99217-004, and 05X-295-011. To gain an understanding of the molecular basis for difference in biomass composition, we are studying genes encoding enzymes involved in lignin biosynthesis and a set of carbohydrate active enzymes selected based on their functional characterization and conservation in Populus trichocarpa and Arabidopsis thaliana. Fragments of nearly 20 genes have been cloned from cDNA made from young stems of Salix sachalinensis and S. miyabeana. Expression profiling in willow stem apical tissue and developing stem tissue was performed for each isolated gene using probe-based quantitative real-time PCR. Two willow parental genotypes and six progeny within a hybrid family were selected for analysis and significant differences in expression among the individuals and between tissue types were observed for most of the genes. Funds were used to train one Ph.D. student and to support the training of a summer undergraduate student. PARTICIPANTS: Dr. Lawrence B. Smart, PI Michelle J. Serapiglia, Ph.D. student Amy Fox, undergraduate Dr. Kimberly D. Cameron, collaborator TARGET AUDIENCES: Willow breeding professionals Bioenergy companies Plant biologists Plant geneticists PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Genetic improvement of fast-growing shrub willow (Salix spp.), a proven perennial bioenergy crop for temperate climates, will be expedited by a thorough understanding of the molecular basis for cell wall composition and by molecular tools for early selection in breeding programs. By identifying the genes that are critical for regulating variation in biomass composition, we may be able to develop molecular markers for the early selection of genotypes with varied and improved biomass properties. Patterns of gene expression were correlated with variation in the biomass composition of those genotypes to provide insights into the genetic regulation of lignocellulosic deposition in this important bioenergy crop. These candidate genes will prove useful in the screening of germplasm for variations in gene expression that may be an early indicator of harvest-age biomass compositional characteristics.
Publications
- No publications reported this period
|
|