NEUTRAL AND ADAPTIVE GENETIC VARIATION IN NORTH AMERICAN HARDWOOD TREE SPECIES

\u27Northern Hardwoods,’ are a characteristic composition of multiple tree species and a part of the rich and diverse northeastern forests. Hardwoods e.g. oaks (Quercus: Fagaceae and Sugar maple), serve as foundation species and offer ecological and economical gains to wildlife and humans. Most of the forest trees go through many biotic and abiotic stresses, for example climate change in their long life span. To overcome these threats and to adapt to changing conditions, tree species need to maintain variation especially in adaptive genes. Therefore, it is necessary to analyze genetic diversity within forest tree populations and take appropriate forest management practice decisions. Genetic tools, such as microsatellite markers developed using next generation DNA sequencing technologies, can be used to address these challenges. Here we report the development of nuclear microsatellite markers in one of the most important hardwood tree species, sugar maple (Acer saccharum Marsh.) (Chapter II). We also use genic microsatellite markers to study the introgression patterns of adaptive alleles in two interfertile red oak species, Quercus rubra and Quercus ellipsoidalis (Chapter III). Using the paired-end low coverage Illumina sequencing technology, we developed a set of seven nuclear microsatellite markers (nSSRs) in sugar maple (Acer saccharum Marsh.). Initially, we screened 96 markers in a panel of six unrelated individuals, out of which seven markers produced polymorphic PCR products. These markers were utilized to analyze genetic variation and gene flow in one sugar maple population in an urban setting consisting of 48 individuals. Additionally, 96 seeds from one open pollinated adult tree were used for the gene flow analysis. In addition to these seven markers, we also tested six previously published microsatellites. Paternity analyses displayed effective dispersal of pollen in the sugar maple population with 76% of gene flow from outside the stand. There was no fine-scale genetic structure observed in this population, which also suggested effective dispersal of both seeds and pollen. To check the transferability of these markers to other Acer species, four individuals from each species of Acer rubrum L., Acer saccharinum L., Acer platanoides L., and Acer ginnala Maxim. were tested at these markers. All markers amplified PCR products in these four species. However, only two markers, AS47 and SM37, were locus-specific and polymorphic in one species (A. ginnala). These markers can serve as an important tool to study genetic variation patterns in sugar maple populations in the face of climate change. Quercus rubra L. and Q. ellipsoidalis E. J. Hill are two interfertile red oak species with different adaptations to drought. I have studied the introgression of adaptive microsatellite alleles at a CONSTANS-Like gene (COL) as a response to micro-environment between these two hybridizing species in two sympatric populations. My results indicated that divergent selection in contrasting environments resulted in high interspecific differentiation at COL. However, interspecific differentiation was lower in sympatric stands, where both species occur in similar environments. The introgression of allele 138 from Q. ellipsoidalis into Q. rubra was higher compared to introgression of allele 141 from Q. rubra into Q. ellipsoidalis in contrast to earlier studies in parapatric populations. My results suggest that the introgression of adaptive genes between two red oak species is strongly affected by environmental selection

Leaf morphological and genetic variation between Quercus rubra and Quercus ellipsoidalis: comparison of sympatric and parapatric populations

Species boundaries in oaks are often not clear-cut, which is potentially a result of interspecific hybridization with trait introgression and phenotypic plasticity. Quercus rubra L. and Quercus ellipsoidalis E.J. Hill are two interfertile partially sympatric red oak species (section Lobatae) with different adaptations to drought. Quercus ellipsoidalis is the most drought tolerant of the North American red oak species and is characterized by deep tap roots, a shrubby growth and by deeply dissected leaves. Genetic differentiation between species is low for most molecular markers. However, one genic microsatellite in a CONSTANS-like (COL) gene, FIR013, was previously identified as outlier locus under strong divergent selection between species. In this study, we analyzed leaf morphometric traits in neighboring (parapatric) Q. rubra/Q. ellipsoidalis populations and in one sympatric population from the same region along an environmental gradient. Using multivariate statistics of leaf traits both species showed distinct bimodal frequency distributions for the first canonical discriminant function with some overlap in the phenotypic extremes, especially in the sympatric population. Leaf dissection traits showed strong and consistent differentiation between species in sympatric and parapatric populations, while differentiation for leaf size was lower in the sympatric population under more similar environmental conditions. Leaf phenotypes in F1 hybrids and introgressive forms suggested maternal effects and introgression of leaf traits between species. The association of outlier gene copy number at FIR013 with species-discriminating leaf traits in Quercus rubra can be a reflection of population differences since outlier gene copy number and population membership show significant collinearity. Similar environmental selection pressures on outlier alleles and leaf shape could also have resulted in this association. In future studies, segregating full-sib families could be used to test whether outlier alleles and associated genomic regions are indeed associated with leaf traits or other species-discriminating characters

Preliminary Genomic Characterization of Ten Hardwood Tree Species from Multiplexed Low Coverage Whole Genome Sequencing

Forest health issues are on the rise in the United States, resulting from introduction of alien pests and diseases, coupled with abiotic stresses related to climate change. Increasingly, forest scientists are finding genetic/genomic resources valuable in addressing forest health issues. For a set of ten ecologically and economically important native hardwood tree species representing a broad phylogenetic spectrum, we used low coverage whole genome sequencing from multiplex Illumina paired ends to economically profile their genomic content. For six species, the genome content was further analyzed by flow cytometry in order to determine the nuclear genome size. Sequencing yielded a depth of 0.8X to 7.5X, from which in silico analysis yielded preliminary estimates of gene and repetitive sequence content in the genome for each species. Thousands of genomic SSRs were identified, with a clear predisposition toward dinucleotide repeats and AT-rich repeat motifs. Flanking primers were designed for SSR loci for all ten species, ranging from 891 loci in sugar maple to 18,167 in redbay. In summary, we have demonstrated that useful preliminary genome information including repeat content, gene content and useful SSR markers can be obtained at low cost and time input from a single lane of Illumina multiplex sequence.All raw sequence data are available through NCBI SRA under project number SRP021923. Sequences containing SSRs and designed primers are attached as supplementary material

Evidence for environment-dependent introgression of adaptive genes between two red oak species with different drought adaptations

© 2017 Botanical Society of America. PREMISE OF THE STUDY: Introgression of adaptive alleles between hybridizing species is likely an important mechanism to generate new genetic variation for adaptation to rapidly changing environmental conditions. Oaks provide a model for the study of adaptive gene introgression because environmental selection maintains high interspecific differentiation at a few outlier loci and species-specific adaptations despite recurrent interspecific gene flow. Previously, we identified a CONSTANS-like gene under strong divergent selection between drought-tolerant Quercus ellipsoidalis and drought-averse Quercus rubra. Neighboring (parapatric) populations of both species were fixed for alternate alleles, Q. ellipsoidalis for allele 138 and Q. rubra for allele 141. METHODS: In this study, we analyzed introgression of these outlier alleles in sympatric and in parapatric Q. rubra/Q. ellipsoidalis populations. KEY RESULTS: We found evidence that environmental selection affected the level and direction of outlier allele introgression. Thus, in the face of symmetric interspecific gene flow, outlier allele introgression was asymmetric and introgression of allele 138 into Q. rubra was consistently higher than introgression of allele 141 into Q. ellipsoidalis in sympatric populations, but the opposite pattern was found in parapatric populations. Furthermore, the rate of introgression of outlier alleles between species in sympatric stands was related to soil quality (i.e., soil water holding capacity, nutrient availability). CONCLUSIONS: This pattern suggests that introgression of adaptive genes between these two red oak species is strongly affected by environmental selection and is an important mechanism for species’ adaptation to changing environmental conditions in a changing climate

Nuclear microsatellite markers for population genetic studies in sugar maple (Acer saccharum Marsh.)

© 2015, Editura Silvica. All rights reserved. A set of seven new nuclear microsatellite markers (nSSRs) was developed for sugar maple (Acer saccharum Marsh.) using paired-end Illumina sequencing. Out of 96 primers screened in a panel of six unrelated individuals, seven markers amplified polymorphic products. The utility of these markers, in addition to six already published microsatellites, for genetic variation and gene flow studies was assessed. Out of the seven newly developed markers, three amplified multiple fragments and were interpreted as dominant (absence/presence) markers, while four markers amplified a maximum of two amplification products per sample. The six published microsatellites and three of the four newly developed markers showed regular segregation in an open-pollinated single tree progeny. Observed heterozygosity (Ho) and expected heterozygosity (He) in 48 individuals from one population ranged from 0.436 to 0.917 and from 0.726 to 0.894, respectively. Dominant markers revealed 64 variable positions and moderate genetic variation with- in the population (He = 0.102, Shannon’s I = 0.193). Paternity analyses in the program CERVUS at co-dominant markers showed effective dispersal of pollen in the sugar maple population both at 95% and 80% confidence levels. Dependent on the confidence level, the mean pollen dispersal distance within the population ranged from 33.25 m to 38.75 m and gene flow from outside the stand from 78% to 82%. The absence of fine-scale Spatial Genetic Structure (SGS) suggested effective dispersal of both seeds and pollen

Preliminary Genomic Characterization of Ten Hardwood Tree Species from Multiplexed Low Coverage Whole Genome Sequencing

© 2015, Public Library of Science. All rights reserved. This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Forest health issues are on the rise in the United States, resulting from introduction of alien pests and diseases, coupled with abiotic stresses related to climate change. Increasingly, forest scientists are finding genetic/genomic resources valuable in addressing forest health issues. For a set of ten ecologically and economically important native hardwood tree species representing a broad phylogenetic spectrum, we used low coverage whole genome sequencing from multiplex Illumina paired ends to economically profile their genomic content. For six species, the genome content was further analyzed by flow cytometry in order to determine the nuclear genome size. Sequencing yielded a depth of 0.8X to 7.5X, from which in silico analysis yielded preliminary estimates of gene and repetitive sequence content in the genome for each species. Thousands of genomic SSRs were identified, with a clear predisposition toward dinucleotide repeats and AT-rich repeat motifs. Flanking primers were designed for SSR loci for all ten species, ranging from 891 loci in sugar maple to 18,167 in redbay. In summary, we have demonstrated that useful preliminary genome information including repeat content, gene content and useful SSR markers can be obtained at low cost and time input from a single lane of Illumina multiplex sequence

Number of PALs (potentially amplifiable loci) for each of ten hardwood tree species.

<p>Hundreds to thousands of PALs were identified for each species sequenced. For all species the most commonly identified repeat motif was 2 bases, followed by 3 base motifs. Reptitive motifs of 4 bases were found the least often.</p

Identified repetitive elements and genes in genomic reads.

<p>The percent of reconstructed fragments with sequence similarity to known plant repetitive elements and gene sequences vary across species. The majority of identified repetitive elements originate from the retrotransposon classes of Gypsy and Copia.</p