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.

• Metabolite Phenotyping and Association Genetics

• Wood Quality Phenotyping and Association Genetics

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

• For the 3888 trees in the study, in-tree velocity stiffness has been measured. As expected, wood stiffness shows a continuous distribution (Figure 1), indicating that it is inherited as a complex trait. The clonal repeatability, a measure of broad sense heritability for clones, for in-tree wood stiffness is 0.42, similar to other published results with slash and radiata pine families (Kumar et al., 2002; Kumar et al., 2006; Li et al., 2007), and has one of highest levels of genetic control of all wood properties. 65% of the genetic variation is additive demonstrating that breeding for improved juvenile wood stiffness will be quite successful. For a small sample of 60 trees, cellulose microfibril angle of the two outermost rings and in-tree velocity stiffness showed positive, genetic correlation (0.84).
  
  

  Figure 2. Distribution of LS means for In-tree velocity stiffness from 999 genotypes from two sites.

• In the lab, the velocity stiffness of the earlywood and latewood of rings 3 and 4 is being measured with a newly purchased and installed ultrasonic tester. Lab measurements of velocity from 5mm wood cores are being measured to confirm the rankings from the in-tree results and to relate stiffness to cellulose microfibril angle (MFA). Velocity measurements from the new ultrasonic tester and MFA measured with x-ray diffraction are highly, linearly correlated (r²=0.88) for both earlywood and latewood, demonstrating that velocity stiffness can be used to precisely estimate MFA. Velocity stiffness is a much more cost effective and higher throughput method compared with x-ray diffraction.

CHEMICAL PROPERTIES

• For all 3888 wood cores, wood chemical composition of rings 3 plus 4 has been measured by pyrolysis molecular beam mass spectrometry at the National Renewable Energy Lab, a method used previously for mapping QTL for these traits in loblolly pine (Sewell et al., 2002). Lignin, C5 and C6 sugar contents were inherited quantitatively with across site clonal repeatabilities of C5 – 0.11+0.03, C6- 0.14+0.03, and lignin – 0.13+0.02. Overall these show that wood chemical contents are under low genetic control in this population. The heritabilities found for CCLONES are slightly lower than the only published study for the genetic control of wood chemical content in pine; however the published study sampled substantially fewer trees and hence had large standard errors of the estimates (Sykes et al., 2006). C5 and C6 contents are strongly negatively genetically correlated with lignin content, -0.87 and -0.83, respectively. Interestingly, lignin content was positively correlated genetically with height and stem diameter growth, 0.67 and 0.57, respectively. Lignin content showed substantial genetic by environmental (GxE) interaction, while C6 showed little indication of GxE.
  

  Figure 3. Cross sectional plane of wood core scanned with x-ray micro ct at 10 uM

ANATOMICAL PROPERTIES

• Wood density, tracheid cell dimensions, and lumen diameters in rings 3 and 4 are being measured using x-ray micro ct. An x-ray micro ct was purchased and recently installed. X-ray micro ct allows for low and high resolution scans and 3-dimensional tomographic reconstruction of the density profiles of wood. Wood density profiles will be measured at 74 uM resolution. From these profiles the average density and the percentage of both earlywood and latewood will be quantified for rings 3 and 4. Tracheid cell dimensions and lumen diameters will be quantified from 10 uM scans of ring 4.

Summary of wood property phenotyping in CCLONES.


Wood Property	Status
Density	rings 3-4 in progress, expected 2/09
% Latewood	rings 3-4 in progress, expected 2/09
Tracheid dimensions	ring 4, expected 6/09
Cellulose microfibril angle/velocity stiffness	rings 3-4 in progress, expected 1/09
In-tree velocity stiffness	rings 3-4 – complete
Lignin content	rings 3+4 – complete
C6 content	rings 3+4 – complete
C5 content	rings 3+4 – complete