Using CRISPR as a Gene Editing Tool for Validating Adaptive Gene Function in Tree Landscape Genomics

Anthropogenic activities have substantially modified natural forested ecosystems around the world through species exploitation, land-use changes, soil degradation, pollution and introduction of exotic species. The impacts of these activities are being exacerbated today by climate change that is expected to become more severe over the coming decades. Modern landscape genomics has made advances in identifying genes that are associated with phenotypic expression, but they have been unable to prove that the associations are more than correlative. The threats to biological diversity raised by climate change, underscore the need to have an improved understanding of the genetic basis of phenotypic traits. In sedentary, long-lived tree species this becomes of utmost importance, as the success of populations is likely to depend, in large part, on existing standing genetic variation. The most recent technologies of gene editing (CRISPR/Cas9) promise to be an elegant approach that will move forest tree genomics to the next level, by allowing the rigorous testing of gene function and its role in the adaptation of trees to their environment. This perspectives paper looks at how genome editing technologies can be used to advance our understanding of the role genes play in adaptation to climate change in woody plants. We discuss the different CRISPR modes than can be used in studies of adaptive traits in perennial species

Using CRISPR as a Gene Editing Tool for Validating Adaptive Gene Function in Tree Landscape Genomics

Anthropogenic activities have substantially modified natural forested ecosystems around the world through species exploitation, land-use changes, soil degradation, pollution and introduction of exotic species. The impacts of these activities are being exacerbated today by climate change that is expected to become more severe over the coming decades. Modern landscape genomics has made advances in identifying genes that are associated with phenotypic expression, but they have been unable to prove that the associations are more than correlative. The threats to biological diversity raised by climate change, underscore the need to have an improved understanding of the genetic basis of phenotypic traits. In sedentary, long-lived tree species this becomes of utmost importance, as the success of populations is likely to depend, in large part, on existing standing genetic variation. The most recent technologies of gene editing (CRISPR/Cas9) promise to be an elegant approach that will move forest tree genomics to the next level, by allowing the rigorous testing of gene function and its role in the adaptation of trees to their environment. This perspectives paper looks at how genome editing technologies can be used to advance our understanding of the role genes play in adaptation to climate change in woody plants. We discuss the different CRISPR modes than can be used in studies of adaptive traits in perennial species

Molecular Breeding and Genomics

Molecular breeding is the use of genetic manipulation at the DNA molecular level to improve traits of interest in plants and to shorten the time for developing new crop varieties to be brought to the market. In traditional plant breeding, genetic variation and characterization are usually identified by visual selection of morphological traits. However, with the development of molecular biology, the problems related to environmental factors and, in some cases, the slow development process, might be solved by the use of molecular markers. 'DNA marker' is a term used to refer to a specific DNA variation between individuals that has been found to be associated with a certain characteristic. These different DNA or genetic variants are known as alleles. Thus, the use of DNA markers to define the genotype and predict the performance of a plant is a powerful aid to plant breeding. Molecular breeding implies molecular marker-assisted selection (MAS), which is a strategy to facilitate the exploitation of existing genetic diversity in breeding populations and therefore, in combination with linkage maps and genomics, can be used to improve a whole range of desirable traits

Population Genetic Diversity of Quercus ilex subsp. ballota (Desf.) Samp. Reveals Divergence in Recent and Evolutionary Migration Rates in the Spanish Dehesas

he Spanish dehesas have been severely affected by human activities that date to the prehistoric period and have suffered accelerated decline since the 1980s. Holm oak (Quercus ilex subsp. ballota (Desf.) Samp.) is a key component of this system, and its acorns provide an important food source for wildlife and domesticated livestock. Our earlier work showed structured variation in acorn morphology and biochemistry. Here, we used chloroplast and nuclear microsatellites to detect genetic structure among populations of Q. ilex from the major biogeographic regions of Andalusia. We found high levels of spatial differentiation with chloroplast DNA indicating little seed dispersal among populations. Spatial differentiation was weaker for nuclear DNA, presumably as a result of more widespread pollen dispersal and its larger effective population size. The Baetic Cordillera (Cádiz) population consistently appeared well separated from populations of the northern Sierra Morena, suggesting that the Guadalquivir Valley has played an important role in determining population divergence. This may be, in part, evolutionary, as suggested by chloroplast DNA, and, in part, a result of human-induced population isolation, as Q. ilex has been removed from the Guadalquivir Valley. Evolutionary gene flow rates were greater than contemporary rates, which were limited to unidirectional gene flow from Córdoba to other populations in the Sierra Morena and, surprisingly, to the southern population at Almería. The inconsistency between evolutionary and recent migration rates suggests an effect of anthropogenic activity over the last few generations of Q. ilex

Population Genetic Diversity of Quercus ilex subsp. ballota (Desf.) Samp. Reveals Divergence in Recent and Evolutionary Migration Rates in the Spanish Dehesas

he Spanish dehesas have been severely affected by human activities that date to the prehistoric period and have suffered accelerated decline since the 1980s. Holm oak (Quercus ilex subsp. ballota (Desf.) Samp.) is a key component of this system, and its acorns provide an important food source for wildlife and domesticated livestock. Our earlier work showed structured variation in acorn morphology and biochemistry. Here, we used chloroplast and nuclear microsatellites to detect genetic structure among populations of Q. ilex from the major biogeographic regions of Andalusia. We found high levels of spatial differentiation with chloroplast DNA indicating little seed dispersal among populations. Spatial differentiation was weaker for nuclear DNA, presumably as a result of more widespread pollen dispersal and its larger effective population size. The Baetic Cordillera (Cádiz) population consistently appeared well separated from populations of the northern Sierra Morena, suggesting that the Guadalquivir Valley has played an important role in determining population divergence. This may be, in part, evolutionary, as suggested by chloroplast DNA, and, in part, a result of human-induced population isolation, as Q. ilex has been removed from the Guadalquivir Valley. Evolutionary gene flow rates were greater than contemporary rates, which were limited to unidirectional gene flow from Córdoba to other populations in the Sierra Morena and, surprisingly, to the southern population at Almería. The inconsistency between evolutionary and recent migration rates suggests an effect of anthropogenic activity over the last few generations of Q. ilex

Genomic Sequencing of Japanese Plum (Prunus salicina Lindl.) Mutants Provides a New Model for Rosaceae Fruit Ripening Studies

It has recently been described that the Japanese plum “Santa Rosa” bud sport series contains variations in ripening pattern: climacteric, suppressed-climacteric and non-climacteric types. This provides an interesting model to study the role of ethylene and other key mechanisms governing fruit ripening, softening and senescence. The aim of the current study was to investigate such differences at the genomic level, using this series of plum bud sports, with special reference to genes involved in ethylene biosynthesis, signal transduction, and sugar metabolism. Genomic DNA, isolated from leaf samples of six Japanese plum cultivars (“Santa Rosa”, “July Santa Rosa”, “Late Santa Rosa”, “Sweet Miriam”, “Roysum”, and “Casselman”), was used to construct paired-end standard Illumina libraries. Sequences were aligned to the Prunus persica genome, and genomic variations (SNPs, INDELS, and CNV's) were investigated. Results determined 12 potential candidate genes with significant copy number variation (CNV), being associated with ethylene perception and signal transduction components. Additionally, the Maximum Likelihood (ML) phylogenetic tree showed two sorbitol dehydrogenase genes grouping into a distinct clade, indicating that this natural group is well-defined and presents high sequence identity among its members. In contrast, the ethylene group, which includes ACO1, ACS1, ACS4, ACS5, CTR1, ERF1, ERF3, and ethylene-receptor genes, was widely distributed and clustered into 10 different groups. Thus, ACS, ERF, and sorbitol dehydrogenase proteins potentially share a common ancestor for different plant genomes, while the expansion rate may be related to ancestral expansion rather than species-specific events. Based on the distribution of the clades, we suggest that gene function diversification for the ripening pathway occurred prior to family extension. We herein report all the frameshift mutations in genes involved in sugar transport and ethylene biosynthesis detected as well as the gene CNV implicated in ripening differences

Table2.xlsx

<p>It has recently been described that the Japanese plum “Santa Rosa” bud sport series contains variations in ripening pattern: climacteric, suppressed-climacteric and non-climacteric types. This provides an interesting model to study the role of ethylene and other key mechanisms governing fruit ripening, softening and senescence. The aim of the current study was to investigate such differences at the genomic level, using this series of plum bud sports, with special reference to genes involved in ethylene biosynthesis, signal transduction, and sugar metabolism. Genomic DNA, isolated from leaf samples of six Japanese plum cultivars (“Santa Rosa”, “July Santa Rosa”, “Late Santa Rosa”, “Sweet Miriam”, “Roysum”, and “Casselman”), was used to construct paired-end standard Illumina libraries. Sequences were aligned to the Prunus persica genome, and genomic variations (SNPs, INDELS, and CNV's) were investigated. Results determined 12 potential candidate genes with significant copy number variation (CNV), being associated with ethylene perception and signal transduction components. Additionally, the Maximum Likelihood (ML) phylogenetic tree showed two sorbitol dehydrogenase genes grouping into a distinct clade, indicating that this natural group is well-defined and presents high sequence identity among its members. In contrast, the ethylene group, which includes ACO1, ACS1, ACS4, ACS5, CTR1, ERF1, ERF3, and ethylene-receptor genes, was widely distributed and clustered into 10 different groups. Thus, ACS, ERF, and sorbitol dehydrogenase proteins potentially share a common ancestor for different plant genomes, while the expansion rate may be related to ancestral expansion rather than species-specific events. Based on the distribution of the clades, we suggest that gene function diversification for the ripening pathway occurred prior to family extension. We herein report all the frameshift mutations in genes involved in sugar transport and ethylene biosynthesis detected as well as the gene CNV implicated in ripening differences.</p

Image1.PNG

<p>It has recently been described that the Japanese plum “Santa Rosa” bud sport series contains variations in ripening pattern: climacteric, suppressed-climacteric and non-climacteric types. This provides an interesting model to study the role of ethylene and other key mechanisms governing fruit ripening, softening and senescence. The aim of the current study was to investigate such differences at the genomic level, using this series of plum bud sports, with special reference to genes involved in ethylene biosynthesis, signal transduction, and sugar metabolism. Genomic DNA, isolated from leaf samples of six Japanese plum cultivars (“Santa Rosa”, “July Santa Rosa”, “Late Santa Rosa”, “Sweet Miriam”, “Roysum”, and “Casselman”), was used to construct paired-end standard Illumina libraries. Sequences were aligned to the Prunus persica genome, and genomic variations (SNPs, INDELS, and CNV's) were investigated. Results determined 12 potential candidate genes with significant copy number variation (CNV), being associated with ethylene perception and signal transduction components. Additionally, the Maximum Likelihood (ML) phylogenetic tree showed two sorbitol dehydrogenase genes grouping into a distinct clade, indicating that this natural group is well-defined and presents high sequence identity among its members. In contrast, the ethylene group, which includes ACO1, ACS1, ACS4, ACS5, CTR1, ERF1, ERF3, and ethylene-receptor genes, was widely distributed and clustered into 10 different groups. Thus, ACS, ERF, and sorbitol dehydrogenase proteins potentially share a common ancestor for different plant genomes, while the expansion rate may be related to ancestral expansion rather than species-specific events. Based on the distribution of the clades, we suggest that gene function diversification for the ripening pathway occurred prior to family extension. We herein report all the frameshift mutations in genes involved in sugar transport and ethylene biosynthesis detected as well as the gene CNV implicated in ripening differences.</p