2012 Annual Report
1a.Objectives (from AD-416):
Develop a detailed understanding of the seasonal changes of gene expression coincident with major fluxes of C and N into and out of the crown and rhizomes.
(1) Use 454 sequencing technology to profile barcoded cDNA libraries derived from crowns, rhizomes and dormant buds during critical phases of transition.
(2) Employ stable 13C and 15N isotope enrichment for C and N utilization, sequestration, and cycling analysis.
(3) Perform metabolic profiling of sugars, starch, phenolics, protein, amino acids and lipids in genotypes and cultivars with known differences in winter survival and pre-frost maturity and senescence differences.
(4) We have developed unique Upland switchgrass populations by recurrent breeding for forage digestibility which is strongly associated with low lignin concentration in which winter survival and plant fitness has been significantly decreased.
We will utilize the 454 sequence analysis platform on populations and genotypes with known differences in winter survival to uncover marker-trait associations that can be used to reduce the generations, and within generation time and expense of phenotyping in the breeding process by use of marker assisted selection.
1b.Approach (from AD-416):
C and N Partitioning and recycling in switchgrass over two growing seasons using stableisotope methods, transcript profiling and metabolomics of crown and rhizome tissues:
Plants from two contrasting tetraploid backgrounds: cv Kanlow (Lowland, high yield, not well adapted to Upper Midwest); and cv Summer, (Upland, lower yield and in a different heterotic group than Kanlow) and Kanlow N1, which is a Lowland cold-adapted, high yield population selected from a Kanlow base population with significantly improved winter survival, will be planted in a field nursery as well as in large pots maintained under ambient atmospheric conditions. For C-cycling, replicated rows of plants in the field will be pulsed labeled with 13CO2 during the growing season (mid vegetative approximately mid/late May, three labeling/week for 1 week to obtain well labeled plants. Above ground plant parts will be harvested at specific times (see below) after labeling, separated into leaves, sheaths and stems, dried, ground and analyzed for 13C- enrichment using stable-isotope ratio mass spectrometry. For N-recycling, 15N will be supplied as ammonium nitrate just prior to green-up (mid April) and above ground plant materials will be harvested, separated and analyzed as described above.
Gene Profiling During Regreening and Dormancy of Bulked Segregants:
The populations selected for this objective have been growing in replicated field nurseries in NE, and select genotypes with low winter survival have been clonally maintained in greenhouses. RNA will be isolated in early spring at the first sign of regreening, just post anthesis, and following first killing frost. RNA will be pooled and bulked by genotype. These RNAs will be analyzed as above by the creation of barcoded cDNA libraries and 454 sequencing.
Creation and Analysis of QTL and Association Mapping Panel in Breeding Stock Developed for the Northern Plains Climate that Show Significant Heterosis:
There are 148 single-seed descent tetraploid plants in a marker population derived from a cross between an upland cv Summer and an adapted lowland cv Kanlow plant. This population will be expanded and replicated, along with 500 tetraploid upland and lowland lines derived from a foundation nursery. Material from this association panel will be evaluated for biomass yield, rapid spring regrowth, and heading date. Using available mapped EST-microsatellites will enable evaluation of population structure, and based on sequence profiling, we will resequence up to 10 kb from selected candidate loci in the 500 individual association panel.
This project is a collaboration between ARS scientists at the University of Nebraska, Lincoln, NE, the Western Regional Research Center (WRRC), and scientists at the University of Nebraska, Lincoln, NE. Work at the WRRC was initiated on field material from 5 different switchgrass populations differing in yield and maturity. The fields have been clonally replicated and have all been sampled this year. Transcriptional profiling of several lines has been conducted on crown tissue to better understand the transcriptome in this tissue that is responsible for winter hardiness and spring regrowth. Reduced representation genotyping by sequencing approaches has been attempted on a pilot scale to determine which barcoding system is best for simultaneous sequencing of large numbers of samples.
This project is relevant to the development of switchgrass genetic resources in support of bioenergy feedstock improvement that is an objective of the parent project.