Dr. Kirst will give an invited presentation on Tuesday, August 26, for the IUFRO-CTIA Main Conference.  The abstract of his presentation is provided below.

Bridging Genomics and Breeding in Hardwood Tree Models

Matias KIRST [1], Derek DROST [2] and Evandro NOVAES [3]

Forest ecosystems cover approximately 3.9 million hectares or 30% of the world’s land surface (FAO, 2001), where trees are the dominant species. Tree species are essential for the survival of these ecosystems and the stability of their composition and diversity. Trees also provide a source of materials for bioenergy and wood, one of the world’s most important renewable materials, and contribute an estimated 354 billion US dollars to the world’s economy (FAO, 2001). Wood consumption per capita has increased only slightly in the past decades, but world population growth has lead to a significant expansion in demand. In the meantime, wood production and the area dedicated to plantation forestry have remained relatively stable or even declined in parts of Europe and North America. The consequent loss in wood production has been partially offset by a rapid increase in the area of planted forests in Asia and South America and gains in productivity supported by aggressive tree breeding programs and biotechnology.

This presentation will focus on Eucalyptus and Populus, two woody angiosperms of great economic importance for the forest industry worldwide. Populus trichocarpa was the first woody plant genome to be sequenced (Tuskan et al., 2006), and the genome of the Eucalyptus grandis genotype Brasuz1 will soon be unveiled by JGI/DOE. Both species are differentiated from conifers in wood morphology, chemistry, and several reproductive, developmental and adaptive traits. As will be discussed below, translating genomics to breeding in these species will present its own challenges. On the other hand, the relative simplicity of their genomes and the availability of a reference sequence may lead to the rapid development and adoption of genomics tools in traditional genetic improvement programs.

Genomics offers a platform to learn about the relationships of genes and phenotypes. For genomic breeders the long term goal is to develop predictive models that will permit estimation of performance and adaptability of genotypes across sites/ecosystems based on genetic data alone. In that scenario, knowledge of the optimal allele combinations necessary for biosynthesis of an ideal product will be essential. The development of genotype-based predictive models is still a long way from reality. However, the first step to develop these models – the discovery of allelic variants that contribute to adaptation and phenotypic diversity – is well under way. Here I will discuss the approaches that we and others are applying in Populus and Eucalyptus genomic research, aimed at the identification of genes and alleles of value. Current limitations and pitfalls of these methods will also be discussed. Finally, rapidly changing technologies of genomic analyses and their benefits to molecular breeding will also be discussed.

The development of the first genetics maps in the early 1990’s created the foundation for quantitative trait loci (QTL) analysis and the characterization of the genetic architecture of wood quality and growth traits in Eucalyptus, Populus and other tree species. However, major barriers to the effective application of QTL analysis to breeding rapidly became apparent. High levels of genetic heterogeneity and linkage equilibrium of breeding populations meant that marker/trait linkages were of limited application to different families. QTL studies were also limited in resolution and in the genetic variation captured.

Despite these limitations, a QTL approach combined with genome information can be particularly powerful for detection of genes and alleles that confer unique/superior properties to a species. Tree breeders have long recognized that some hardwood species that have strikingly distinct wood quality, growth and adaptive properties can be intercrossed, producing hybrid genotypes that capture the superior characteristics of each species in one individual. This genetic variation has been and will increasingly be a powerful source for detection of major effect genes in experimental hybrid populations. We recently characterized a broad spectrum of phenotypes in a hybrid cross of P. trichocarpa and P. deltoides, including leaf shape, vessel development and other developmental traits. Analysis of QTLs identified significant genomic regions of interest that, in combination with the Populus reference genome sequence and transcriptome information defined genes that are strong candidates to regulate these traits. These alleles of large effect may be relatively easy to detect in a combined QTL/genomic analysis of hybrid segregating populations. For many hardwood species of commercial value, highly significant gains may be achievable through the introgression of these valuable species-specific alleles into the breeding populations through marker-assisted selection or transgenesis.

Some of the limitations of QTL analysis have favored the use of another forward genetic approach, linkage disequilibrium mapping or association genetics, for discovery of genes that regulate quantitative phenotypic variation. Association studies typically rely on the genetic analysis of populations with unknown ancestry and from a diverse genetic background. Success of association mapping depends on the level of nucleotide diversity and linkage disequilibrium in the surveyed population. In the few genes that we and others have analyzed in Populus and Eucalyptus, significant levels of linkage disequilibrium (LD) only extends to a few hundred base pairs and nucleotide diversity is among the highest recorded in any species. Therefore significant association may readily identify genes that contribute to phenotypes. The limited genetic structure generally observed in these natural populations also minimizes the detection of spurious associations. In fact, the first successful identification of a significant association between a polymorphism and a phenotype, microfibril angle, was reported for Eucalyptus globulus. While it demonstrated the feasibility of association genetic studies in long-lived, perennial woody species, several limitations and pitfalls associated with the biology and breeding methods in hardwoods must be recognized.

Breeding of woody angiosperms such as Eucalyptus and Populus generally explores the vigor of hybrids for creating superior genotypes. While overdominance and epistasis appear to be a significant driving force behind heterosis in these species, it is unclear if association genetics will be able to identify the interspecific allelic combinations that produce these superior phenotypes. It is also unclear if high levels of polymorphism and low LD will be repeated in hardwood species of commercial value but with a narrow natural distribution, or in provenances that may have originated from relatively recent migration events. Selective sweeps around loci that are essential to adaptation will also lead to high levels of LD. Although typically localized, these regions of high LD will in some cases be specifically the ones targeted by breeders. Finally, many of these outcrossing tree species are likely to contain an abundance of high-effect, low-frequency alleles that will be difficult to identify in traditional association studies. Some of these rare polymorphisms may contribute significantly to diversity in the phenotypes most sought by tree breeders. In a recent survey we identified almost 300 rare large-effect SNPs – i.e. polymorphism that lead to the introduction or removal of a STOP codon – in transcribed sequences of a pool of E. grandis individuals. The probability of maintaining rare alleles in outcrossing, long-lived perennial species with large effective population sizes is high. It may be possible that the majority of the variance that contributes to superior phenotype detected in some individuals may be due to such uncommon, high effect allelic variants. Therefore, the largest gains from molecular breeding may be achieved not through the continuous incorporation of frequent alleles of small positive effect, but rather through the identification of rare, high-value alleles.

Identifying these rare alleles and increasing their frequency in breeding populations will be challenging. Furthermore, in many hardwood species that utilize a hybrid breeding/clonal deployment approach the challenge will go beyond detecting rare allele of large effects, but rather rare allelic epistatic combinations, which will require analysis of much larger populations. Uncovering them will be unlikely using the current approaches but we can foresee that emerging high-throughput sequencing and genotyping technologies may rapidly change this scenario. While the initial sequencing of the model plant Arabidopsis thaliana was a multi-year, multi-million dollar project, several hundred new genotypes have since been sequenced and characterized in a fraction of that cost and time. Similarly, sequencing of Populus or Eucalyptus genotypes, when supported by the availability of a reference genome, may be achievable now for a cost 2-3 orders of magnitude smaller than that of a decade ago. The consequence of the rapid improvements of genotyping and sequencing methods and major advances in bioinformatics are that it will soon be possible to identify all the sequence variants in very large breeding populations. Already in human genetics, genotyping of several million nucleotide polymorphisms in thousands of individuals is commonly carried out. Unraveling the alleles and interactions of value will provide the foundation for an understanding of all the genetic factors that contribute to the immense phenotypic diversity of tree species, and will be the first step of effectively applying this knowledge to breeding.

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[1] School of Forest Resources and Conservation, University of Florida; University of Florida Genetics Institute, Gainesville, FL 32611

[2] Graduate Program in Plant Molecular and Cellular Biology, University of Florida, Gainesville, FL 32611

[3] Graduate Program in Forestry, School of Forest Resources and Conservation, University of Florida, Gainesville, FL 32611

Assistant Professor in Quantitative Genetics and Genomics

School of Forest Resources and Conservation, University of Florida Genetics Institute, Plant Molecular and Cellular Biology Program, University of Florida

Dr. Matias Kirst is an Assistant Professor in Quantitative Genetics and Genomics at the School of Forest Resources and Conservation at the University of Florida. He has a B.S. in Forestry Engineering, a M.Sc. in Genetics and Improvement (University of Vicosa, Brazil) and Ph.D. in Genetics and Genomics Sciences (North Carolina State University, USA). Dr. Kirst is the lead investigator of the Quantitative Genomics Laboratory at the University of Florida Genetics Institute. Research efforts are focused on the genetic regulation of gene expression and gene expression networks; and discovery of genes, metabolic and regulatory networks that control variation in wood quality, growth and other important traits for the forestry and agronomic industry. These studies are supported by the implementation and development of new genomic tools for polymorphism discovery and genotyping.

Link to web page: http://forestgenomics.ifas.ufl.edu/kirstlab.shtml