Forest genetics in the genomics era
David B. Neale 1, 2

1 Dept. Plant Sciences, University of California, Davis

2 Whitebark Pine Ecosystem Foundation, Missoula, Montana


In this presentation I will chronicle the experimental approaches and progress over the last 50 years toward discovering the individual genes underlying complex traits and determining adaptation to the environment in forest trees. Historically, forest geneticists used the common garden to infer the genetic component to phenotypic variation. Beginning in the 1970s, the first of the individual gene marker systems were developed (allozymes, RFLPs, AFLPs, SSRs), however these markers generally coded for neutral genetic variation and were thus quite useful for understanding demographic processes in forest trees (gene flow, genetic drift, mating systems) but did not contribute to an understanding of non-neutral processes (natural and artificial selection). It was not until the human genome was sequenced in the year 2000 that modern genomic technologies were developed that it became possible to discover individual genes underlying complex traits and adaptation to the environment. I will describe research done by my research group and many collaborators in a few conifer species (loblolly pine, sugar pine, Douglas-fir, coast redwood, giant sequoia, whitebark park and bristlecone pine) to sequence genomes and transcriptomes and subsequently perform genome wide association studies and environmental association studies.


Evolutionary and conservation genomics of Tertiary relict tree genus Cercidiphyllum
Shanshan Zhu 3, Yingxiong Qiu 4

3 Systematic & Evolutionary Botany and Biodiversity group, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China

4 Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan, 430074, Hubei, China


Considerable resilience in the face of cyclic climatic changes has been regarded as a necessary common feature for Tertiary relict trees. At the same time, demographic processes associated with Pleistocene climatic oscillations and human-driven habitat loss have led to their highly fragmented ranges and/or low population sizes. However, it is not yet well understood how demographic history, selection and introgression jointly determine their genomic differentiation, contemporary genetic diversity and mutational load, and thus impinge on their survival and population viability. Cercidiphyllum (Cercidiphyllaceae) is a Tertiary relict tree genus, containing two extant species: C. japonicum and C. magnificum. C. japonicum is one of the most widely distributed forest species in the East Asia Tertiary deciduous forest with distribution across subtropical China all the way to North Japan; by contrast, C. magnificum, a small tree or shrub, is restricted to the cool-temperate/subalpine forests of Central Honshu/Japan. In this study, we assembled two whole genomes of both species using a combination of PacBio long reads and Hi-C sequencing technologies, and performed whole genome resequencing of total 135 Cercidiphyllum individuals (C. japonicum: 86; C. magnificum: 49). Population genomic analyses revealed high genomic differentiation between C. japonicum and C. magnificum. Based on coalescent-based demographic analyses, we dated the divergence of these two species to the mid-Miocene, and the intraspecific lineage divergence of C. japonicum (China vs. Japan) to the Early Pliocene. Population bottlenecks owing to climatic upheavals of the Late Tertiary/Quaternary greatly reduced the genetic diversity of C. japonicum and C. manificum. For the widely distributed C. japonicum, long-term balancing selection may have maintained genome-wide variation at multiple chromosomal and heterozygous gene regions, while selective sweeps at stress response and growth-related genes are likely involved in local adaptation. In terms of inbreeding, C. magnificum genomes were found to have a greater number of runs of homozygosity, when compared to C. japonicum. At the species level, C. magnificum genome accumulated more mildly and highly deleterious mutations than C. japonicum, indicating a lack of genetic purging of these mutations in the narrow endemic C. magnificum. We also detected several blocks containing introgression signal between these two genomes in which possible adaptive genes were involved. Introgression signals observed between these two species were more random and less frequent, and therefore probably due to secondary contact. Our results have broader implications for management of genetic variation related to conservation of Tertiary relict trees at the species and population levels.


Current status and future directions of forest tree breeding in Japan
Makoto Takahashi 5

5 Forest Tree Breeding Center, Forestry and Forest Products Research Institute


Nationwide forest breeding project has started in 1950s in Japan, since then more than sixty years has passed. Approximately nine thousand plus-trees were selected as breeding materials for major forestry species such as Cryptomeria japonica, Chamaecyparis obtusa, Larix kaempferi and Abies sachalinensis from national, public or private stands throughout Japan, and they have been tested in clonal or progeny tests. The plus-tree clones have been evaluated principally in traits of growth performance and wood properties. As of the end of March 2022, 627 C. japonica, 301 C. obtusa, 122 L. kaempferi, and 50 A. sachalinensis second generation plus-trees have been selected. Although the progress of the selection somewhat varies among regions or tree species, the selection and dissemination of the second generation plus trees and the establishment of breeding populations for the third generation selection are currently ongoing. Other than the growth performance and wood properties, tree breeding activities were extended to improve tolerances against diseases and extreme meteorological conditions such as cold or freezing weather and snowfall. The most characteristic target trait in the tree breeding in Japan over the last couple of decades has been male fecundity or sterility in C. japonica and C. obtusa, which are directly connected to the pollen allergy, one of the serious social problem in Japanese society. More than ten million seedlings with less male fecundity or male sterility, that account for approximately 50% of planting seedlings, are planted throughout Japan in recent years.

Shortening the breeding period has been a longstanding challenge for forest tree breeding. In order to achieve this, it is important to enlarge knowledges about the genomes of targeted species and develop accelerated breeding techniques that utilize the genome information. As the pioneer case, we have focused on C. japonica and have been comprehensively collecting expressed sequence data. Some DNA markers tightly linked to the male sterilities were developed using those sequence data, and phenotypically-hidden heterozygotes have been successfully screened from breeding materials. Those markers enabled MAS in the male sterility breeding. Those markers enlarged the male sterility genetic resources and can shorten the breeding period for selecting new male sterile individuals. We also examined the potential of GWAS approach in C. japonica. We are going to continue to make efforts to develop technologies for accelerated forest tree breeding and to extend applications that utilize genome information.


Forest genetic studies using genome-wide SNP data by MIG-seq analysis
Yoshihisa Suyama 6

6 Graduate School of Agricultural Science, Tohoku University


The development of high-throughput DNA sequencing technology provides drastic progress in genetic studies. Genome-wide single-nucleotide polymorphism (SNP) genotyping is an effective application of high-throughput DNA sequencing and is widely used in marker-assisted genetic studies such as molecular phylogeny, phylogeography, population genetics, and so on. We developed a novel genome-wide SNP genotyping method, which is named “multiplexed inter-simple sequence repeat (ISSR) genotyping by sequencing (MIG-seq)”, and applied it to a variety of forest genetic studies. One of the advantages of this method is the ability to apply to a wide range of biological hierarchies from individuals to populations within and among species, and even among genus levels, without optimization for each target taxon. Here I will present a brief introduction of the MIG-seq method and examples of applied studies in forest genetics starting from the individual to genus or family levels.

MIG-seq is a PCR-based next-generation sequencing (NGS) method capable of constructing highly reduced representation libraries for genome-wide SNP genotyping. In the MIG-seq method, numerous genome-wide regions can be amplified from a wide variety of DNA samples using the universal multiplexed ISSR primers. Then the library can be simply sequenced using NGS, and normally more than thousands of genome-wide regions can be detected as comparable genomic information with other samples. This method provides a quick, simple, and economical approach for genome-wide SNP genotyping. This approach is effectively and reliably applicable for a wide variety of species, including forest plants, animals, and fungi, using the same protocol without any prior genetic information and protocol optimization. Using this method, a wide variety of data sets have been already obtained for tens of population genetic, molecular phylogeographic, and systematic studies, including quick surveys of genetic differentiation among individuals, populations, hybrids, and species, as follows.

Clone identification analysis is a good example of an individual-level study by MIG-seq. It works very well in clone identification of clonal plants such as bamboos, for instance. In addition, the DNA identification of closely related cultivars or breeding varieties is also an effective application example of this method. Population genetic and molecular phylogeographic studies are one of the most suitable targets for this method. An advantage of using thousands of SNP markers detected by this method is not only to reveal basic population genetic information such as genetic diversity but also to reconstruct population demographic histories such as a change in effective population size and diversification process. Molecular phylogenetic taxonomy is another ‘hot’ target of this method, including its application to museum specimens and for species identification and discovery. For example, we have applied the MIG-seq approach to international projects of species discovery at global biodiversity hotspots, such as Southeast Asian tropical forests and New Caledonia, as well as a domestic project to investigate more than 70% of the Japanese vascular plants. These example studies will be presented in my talk.

I hope that the MIG-seq method will contribute to various researches in forest genetics and the progress of related research fields.