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ADEPT2 PhenotypingGene Expression Phenotyping and Association Genetics
Nearly 1500 rooted cuttings were received from. North Carolina State University in spring 2006. That fall, they were
harvested and roots, stems and needles were stored for RNA isolations.
RNA was isolated from stems of two ramets per clone.
Over 200 genes with potential roles in wood development or responses to biotic and abiotic stresses were chosen based on previous research in our laboratories and literature reviews. Real-time qPCR has been used to analyze 182 genes to date. The last 23 genes are currently being analyzed. We have discovered high levels of variation in gene expression between genotypes. The differences between high-expressing and low-expressing genotypes usually ranges between three and seven (rarely to 14) PCR cycles (usually 8 to 128 fold). In many cases we observe
strong correlations between the expression levels of various genes.
In some cases, this suggests coordinate regulation. The DDCT values
for phenylalanine ammonia lyase, a gene involved in lignin biosynthesis,
and a myb transcription factor are compared below.
Figure 1. A loblolly pine myb promoter contains a SNP in a putative myb-binding site. CTAACCA - putative myb binding site TTAACCA - not predicted to be a myb binding site. Such correlations caused us to clone promoters
in search of SNPs in putative binding sites of trans-acting elements.
Promoters from 13 transcription factors and three other genes induced
by drought stress were cloned using a Genome Walker kit and sequenced.
SNP discovery in the promoters is planned for this year in collaboration
with UC-Davis. Four of the promoters were sequenced in a small number
of individuals and an interesting SNP was found in the promoter of a
myb gene induced by drought stress. One allele results in a putative
binding site for another MYB with a known role in response to drought
stress. The other allele does not have a putative binding site. In a
preliminary experiment, four individuals from The Lost Pines, a drought-resistant
population, contain one copy of the binding site while four individuals
from Louisiana do not. This SNP was included on the Illumina chip. Experiments in Progress In addition to completing
the remaining gene expression analyses and the promoter SNP genotyping,
association analyses are just starting. One of the graduate students
on the project, Sreenath Palle, went to UC-Davis in April 2008 to be
trained in the use of the program “Tassel?  With the exception of
some LEA genes, most of the genes with potential roles in drought stress
did not show much variation between clones. The plants had not been
drought stressed before harvesting. The disease genes did not produce
good quality data with differences between ramets, possibly due to a
fungal infection in the greenhouse. Therefore, a previously unplanned,
small experiment was initiated. 24 clones were selected for inclusion
based on pitch canker data from the University of Florida and water
use efficiency data from North Carolina State. The plants are currently
ready to start a factorial experiment involving drought
stress and inoculation with pitch canker. Three ramets per clone exposed
to each of the four treatments (drought ?no disease; no drought ?
no disease; drought ?pitch canker inoculation; no drought ?pitch
canker inoculation) will have physiological traits measured and RNA
isolated from the stems. qPCR will be performed to determine the expression
of approximately 100 genes in the different clones and in response to
the treatments. Training ?Two Ph.D.
students are being trained in the laboratory as part of this project.
Both have completed all their coursework and have passed their preliminary
examinations. Both are involved in the Graduate Student Teaching Academy
to help prepare them to be the educators of the future. Sreenath Palle
will graduate in August 2009 and Candace Seeve in August 2010. One visiting
scientist from Nicaragua has been in the laboratory this semester and
has received training in real-time qPCR, sequencing, and genotyping.
Seven high school and middle school teachers from Texas were recruited
and sent to the genomics workshop help at Clemson University.
TASKS: Measure the wood mechanical, chemical and anatomical properties in a clonally propagated population. This structured CCLONES population contains 31 parents mated in a circular dialell. Approximately 15 progeny per cross were used to create hedges from which rooted cuttings were taken and planted in 2003. In all, 999 genotypes from 62 families are represented in the population that is being sampled. Four clonal replications per genotype (2 replications from each of 2 sites) have been cored (total of 3888 cores) and the wood properties are being measured from rings 3 and/or 4, depending on the property. Two significant challenges/set backs for the wood property phenotyping needed to be overcome. First, the closure of the wood x-ray micro ct instrument at Oakridge National Laboratory was over come with new funding to G. Peter from DOE which was used together with funds from this award to purchase an x-ray micro ct that is now up and running at UF. Second, the dramatic increase in cost by Silviscan for cellulose microfibril angle (MFA) measurement was overcome by moving to acoustic based approaches for measuring velocity wood stiffness as a surrogate for MFA, but very highly correlated trait.
MECHANICAL PROPERTIES
CHEMICAL PROPERTIES
ANATOMICAL PROPERTIES
Summary of wood property phenotyping
in CCLONES.
TASKS: Measure
the disease resistance of the unstructured NSF association population.
This population contains 498 randomly selected trees from across the
range of loblolly pine. Clonally propagated ramets were inoculated
with spores from Fusarium circinatum
and Cronartium quercuum f.sp. fusiforme
and scored for lesion length and gall characteristics, respectively,
as measures of resistance. DISEASE PHENOTYPING FOR PITCH CANKER RESISTANCE A population of 498 clonally
propagated, unrelated genotypes (NSF association population) was screened
for resistance to pitch canker under greenhouse conditions. Lesion
length measurements were taken at 4, 8 and 12 weeks after inoculation. Figure below shows
that resistance is a complex trait, which validates previous screens
(Kayihan et al. 2005) implying that resistance is inherited quantitatively. 
Figure 4. Distribution of BLUP clonal estimates for pitch canker lesion length (log-transformed), highlighting the 50 most resistant and susceptible clones with >0.3 cuttings. Inserts show examples of resistant and susceptible genotypes. Genotypes with extreme resistance (45) and susceptibility (47) to pitch canker were selected based on their ranks from best linear unbiased predictions (BLUP) of clonal performance. Clonally propagated ramets were inoculated and used to confirm their rank positions in the primary screen. Clonal values and variation
for lesion length increased as disease progressed in all measurements,
particularly in the susceptible tails. Significant differences
between tails were observed (p<0.0001).
Clonal repeatability, a measure of broad-sense heritability, was estimated for both the overall population as well as for the extreme genotypes (tails). These results compared well with previous genetic data obtained from the structured CCLONES population with known pedigree (Kayihan et al., 2005). Clonal repeatability ranged between 0.21 and 0.28 in the first inoculation and between 0.35 and 0.39 in the second inoculation. Repeatabilities increased throughout measurements in both inoculation experiments. These results are comparable to the broad-sense heritability estimates reported by Kayihan et al. (2005) in a population with known pedigree (H2 = 0.37 ?0.43).
DISEASE PHENOTYPING
FOR FUSIFORM RUST RESISTANCE A total of 497 genotypes
were inoculated with three fungal cultures (SC 20-21, NC 2-36 or the
P2 hybrid culture of the previous two) at the Resistance Screening Center
in Asheville, NC in June, 2008. Four replicates were used for
each inoculation, totaling 5,760 cuttings. Most genotypes (386) had
enough cuttings (12) for inoculations with all 3 fungal cultures and
4 replicates. Clones that had between 3 and 11 cuttings were randomly
assigned, such that at least one cutting of each clone was inoculated
with each one of the 3 fungal cultures. The plants were transported
to the University of Florida greenhouse facilities and were set in a
random incomplete block design with four replicates. They are
currently being scored for presence of galls. A collection of
pycnial drops from the galls is also being established to obtain genotypic
data of the fungus. Summary of fungal disease
phenotyping in NSF Association Population.
IDENTIFICATION OF POTENTIALLY SIGNIFICANT ASSOCIATIONS IN NSF ASSOCIATION POPULATION WITH PITCH CANKER RESISTANCE A total of 3,938 SNP
markers for 404 of the 498 clones in the NCSU association population
were analyzed using the Bayesian method. Potentially significant
associations with pitch canker resistance have been identified for 24
SNP markers in 24 genes. The EST sequences from the putative significant
SNPs were obtained from the UGA and UMN databases, as well as from the
Dendrome Plone (http://dendrome.ucdavis.edu/ A BLASTx search was performed
against the entire NCBI protein database and the best hits were obtained.
In cases where the best hit sequence from the database was an unknown
protein, the next available sequence was considered until a best hit
with known function was identified. This provided an idea of a
possible protein function that may be influenced by SNP polymorphisms.
The potential effects of SNP polymorphisms (changes in amino acids,
premature termination codons, etc) are currently being assessed. TASKS: Develop
robust methods for detecting significant associations between SNP genotypes
and phenotypes. The models must be capable of controlling for
both population structures at a coarse level, such as those that arise
from geographic location, and for fine population structures which arises
from controlled mating designs. Construct a pipeline for handling
data and running association analyses. MODEL DEVELOPMENT
AND VALIDATION WITH SIMULATIONS A new Bayesian association
framework has been developed and validated. The Bayesian framework
accommodates analyses of unstructured and structured populations by
implementing population structure and relationship matrices. Distinct
advantages of this framework are that 1) simultaneous analysis of the
set of SNPs is less biased and more likely to identify multiple causal
SNPs, 2) informative priors can be assigned with the relationships are
known. In addition, a Gibbs sampler has been implemented that
imputes missing SNP data from all available relationship and phenotypic
data, permitting the retention of more SNPs compared with mixed linear
model methods. Sparse matrix technology together with improved
programming methods has permitted the development of an R-package that
is straightforward to use. The approach and program have been
validated using simulated data. Using data simulated based on
the CCLONES design; SNPs with small phenotypic effects were readily
identified, even with 10% missing SNP data. COMPARISON OF QTdT
AND BAYESIAN MODEL WITH CCLONES SNP GENOTYPIC AND
D13C PHENOTYPIC DATA From previously funded ADEPT1research, 46 SNPs in candidate drought tolerance and disease resistance genes were genotyped in the CCLONES population and d13C carbon isotope discrimination data were collected from two sites (Gonzales-Martinez, et al., 2008). Using the quantitative transmission disequilibrium test (QTdT), 6 potential SNPs were identified as candidate genes controlling carbon isotope discrimination. With the Bayesian method with informative priors 4 of the 6 SNPs were identified. |
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