Current Research – Ross Whetten

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Applications of molecular markers in practical tree breeding programs

Summary

Many research studies over the past decade have clearly demonstrated that molecular markers (either protein markers such as isozymes, or DNA markers such as microsatellites, RAPDs, AFLPs, or SNPs) can be used for clonal fingerprinting, parentage analysis, and to identify regions of plant chromosomes that have economically important impacts on traits of commercial interest. Why, then, are these marker systems not being used in forest tree improvement programs?

Simply put, many tree breeders are not yet convinced that molecular markers can be applied in a cost-effective manner, particularly in temperate and boreal ecosystems where rotation times are measured in decades rather than in years. Breeding strategies and propagation systems used to deploy improved genotypes into production planting also have an important role in whether molecular markers can be cost-effective for a particular tree improvement and production program. Within-family selection to identify elite genotypes, coupled with vegetative propagation methods that allow production plantings of thousands of hectares with those elite genotypes, provide a much stronger incentive to use molecular markers than does a population-improvement breeding strategy coupled with open-pollinated seed orchards.

My research interests are aimed at reducing the cost of genetic marker systems suitable for forest tree breeding, and increasing the throughput so that large numbers of individuals can be genotyped. In conjunction with statistical methods for determining which families are the best candidates for marker-assisted breeding, such high-throughput low-cost molecular markers could improve the cost-benefit ratio for applications of markers in temperate and boreal forest tree improvement programs. Next-generation DNA sequencing methods (see next section) have the potential to provide cost-effective markers for both applied breeding programs and for population genetics studies of forest trees.


 

Next-generation DNA Sequencing Technologies

Summary

The development of massively-parallel DNA sequencing technologies has provided new cost-effective tools for discovery and analysis of genetic variation in populations. The yield of DNA sequencing, in terms of bases of sequence per unit cost, has been increasing exponentially since the development of automated DNA sequencing instruments in the mid-1980s, but the doubling time dropped from 19months to 5 months after the introduction of the so-called “next-generation sequencing technologies” in 2005 (Stein, 2010 Genome Biology 11:207). These technologies now provide enormous power to discover genes and genetic variation in populations of species not previously subject to molecular genetic analysis. If the cost continues to drop at the current pace, DNA sequencing will soon become the most cost-effective method of genetic marker analysis.

 

 In collaboration with John Frampton (NC State University Christmas Tree Genetics Program), I have carried out a gene discovery project in Fraser fir (Abies fraseri Pursh. Poir.) using the 454 sequencing platform. Over 900,000 sequence reads (average length about 250 bp) were obtained from root and foliage cDNA libraries from two individual trees. Assembly of these sequence reads into overlapping groups called “contigs” yielded over 30,000 putative cDNA consensus sequences. Searching for possible DNA sequence variation within these sequences identified over 15,000 candidate single-nucleotide polymorphisms (SNPs). About two-thirds of the putative transcript assemblies from one individual tree are similar to putative transcript assemblies from the other individual, suggesting that these sequence collections have identified fragments of many, but not all, genes expressed in the tissue samples used for library preparation and sequencing. Additional DNA sequencing experiments using the Illumina GAIIx platform utilized the reference assembly of contigs from the 454 sequences as a basis for analysis of pooled samples of 10 trees from each of six different provenances of Fraser fir, to discover SNPs and examine the distribution of genetic variation across the disjunct populations of this unique species. About 15,000 putative SNPs were identified, of which about 8,000 were present in all six provenances.


Balsam Woolly Adelgid and Fraser Fir

A collaborative project with:

  • John Frampton and Len van Zyl (Department of Forestry)
  • Fred Hain (Department of Entomology)
  • Ilona Peszlen, Mikhail Balakshin, and Ewellyn Capanema (Department of Wood & Paper Science)


Summary

Fraser fir is important to North Carolina both ecologically and economically. It is a key component of the spruce-fir ecosystem found at the highest elevations of the Appalachian Mountains, including scenic areas along the Blue Ridge Parkway and in Great Smoky Mountains National Park. It is also a major component of the Christmas tree farming industry, which brings over $100 million annually to rural regions of the state. Fraser fir populations in North Carolina have been devastated by an introduced pest, the balsam woolly adelgid (BWA). Death of BWA-infested Fraser fir seems to be due to an over-zealous defense response by the tree, rather than any direct effect of insect feeding. This research will use modern tools of biological research to understand why BWA-infested Fraser firs die, and to look for ways to prevent fir death. Research approaches include metabolic profiling and chemical screening to characterize metabolic changes in both intermediary metabolites and defense-related compounds such as flavonoids and other low-molecular-weight phenolic molecules. Microarray profiling of gene expression in differentiating xylem of non-infested and BWA-infested Fraser fir will be used to characterize changes in gene expression due to BWA attack. All data obtained during this research will be maintained in a Web-accessible database and subjected to multivariate analytical methods to test hypotheses regarding the metabolic and genetic basis for Fraser fir mortality in response to BWA infestation.