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

Cross amplification of 15 EST-SSR markers in the genus Fraxinus

Abstract Ash (Fraxinus, Oleaceae) species occur on most continents, within a wide range of forest tree communities. Emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), introduced into the U.S. from Asia in the late twentieth century, has caused widespread mortality, primarily in green ash, Fraxinus pennsylvanica Marsh. (Section: Melioides) and now impacts other North American ash species. The development and successful reintroduction of resistant trees requires genetic tools to evaluate population dynamics and aid in species identification. Here, we report 15 novel EST-SSR markers developed in green ash, most of which amplify and are polymorphic across 18 species of Fraxinus, including six species native to North America. The high average polymorphism information content (0.741) and allelic richness (15.3) revealed in six disparate populations of green ash indicate that these markers also have utility for investigating population dynamics in this species

Development of Genomic Microsatellites in <i>Gleditsia triacanthos</i> (Fabaceae) Using Illumina Sequencing

Premise of the study: Fourteen genomic microsatellite markers were developed and characterized in honey locust, Gleditsia triacanthos, using Illumina sequencing. Due to their high variability, these markers can be applied in analyses of genetic diversity and structure, and in mating system and gene flow studies. Methods and Results: Thirty-six individuals from across the species range were included in a genetic diversity analysis and yielded three to 20 alleles per locus. Observed heterozygosity and expected heterozygosity ranged from 0.214 to 0.944 and from 0.400 to 0.934, respectively, with minimal occurrence of null alleles. Regular segregation of maternal alleles was observed at seven loci and moderate segregation distortion at four of 11 loci that were heterozygous in the seed parent. Conclusions: Honey locust is an important agroforestry tree capable of very fast growth and tolerance of poor site conditions. This is the first report of genomic microsatellites for this species

High-quality genetic mapping with ddRADseq in the non-model tree Quercus rubra

Abstract Background Restriction site associated DNA sequencing (RADseq) has the potential to be a broadly applicable, low-cost approach for high-quality genetic linkage mapping in forest trees lacking a reference genome. The statistical inference of linear order must be as accurate as possible for the correct ordering of sequence scaffolds and contigs to chromosomal locations. Accurate maps also facilitate the discovery of chromosome segments containing allelic variants conferring resistance to the biotic and abiotic stresses that threaten forest trees worldwide. We used ddRADseq for genetic mapping in the tree Quercus rubra, with an approach optimized to produce a high-quality map. Our study design also enabled us to model the results we would have obtained with less depth of coverage. Results Our sequencing design produced a high sequencing depth in the parents (248×) and a moderate sequencing depth (15×) in the progeny. The digital normalization method of generating a de novo reference and the SAMtools SNP variant caller yielded the most SNP calls (78,725). The major drivers of map inflation were multiple SNPs located within the same sequence (77% of SNPs called). The highest quality map was generated with a low level of missing data (5%) and a genome-wide threshold of 0.025 for deviation from Mendelian expectation. The final map included 849 SNP markers (1.8% of the 78,725 SNPs called). Downsampling the individual FASTQ files to model lower depth of coverage revealed that sequencing the progeny using 96 samples per lane would have yielded too few SNP markers to generate a map, even if we had sequenced the parents at depth 248×. Conclusions The ddRADseq technology produced enough high-quality SNP markers to make a moderately dense, high-quality map. The success of this project was due to high depth of coverage of the parents, moderate depth of coverage of the progeny, a good framework map, an optimized bioinformatics pipeline, and rigorous premapping filters. The ddRADseq approach is useful for the construction of high-quality genetic maps in organisms lacking a reference genome if the parents and progeny are sequenced at sufficient depth. Technical improvements in reduced representation sequencing (RRS) approaches are needed to reduce the amount of missing data

Development of genic and genomic microsatellites in Gleditsia triacanthos L. (Fabaceae) using illumina sequencing

© 2017, Editura Silvica. All rights reserved. Twenty new polymorphic genic SSRs (EST-SSRs) and 13 genomic SSRs were developed in honeylocust (Gleditsia triacanthos) using Illumina transcriptome and low-coverage genome sequencing. A diversity panel of 40 honeylocust samples covering large parts of the species distribution range was characterized. As expected the level of genetic variation was lower in EST-SSRs than for non-genic genomic SSRs. This is the first report of EST-SSRs for honeylocust. All markers are polymorphic and produce clear single locus amplification products and can be used for genetic diversity and gene flow analyses. The transcriptome sequencing data provide a rich resource for new marker development

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

A high‐quality reference genome for Fraxinus pennsylvanica for ash species restoration and research.

Green ash (Fraxinus pennsylvanica) is the most widely distributed ash tree in North America. Once common, it has experienced high mortality from the non‐native invasive emerald ash borer (EAB; Agrilus planipennis). A small percentage of native green ash trees that remain healthy in long‐infested areas, termed “lingering ash,” display partial resistance to the insect, indicating that breeding and propagating populations with higher resistance to EAB may be possible. To assist in ash breeding, ecology and evolution studies, we report the first chromosome‐level assembly from the genus Fraxinus for F. pennsylvanica with over 99% of bases anchored to 23 haploid chromosomes, spanning 757 Mb in total, composed of 49.43% repetitive DNA, and containing 35,470 high‐confidence gene models assigned to 22,976 Asterid orthogroups. We also present results of range‐wide genetic variation studies, the identification of candidate genes for important traits including potential EAB‐resistance genes, and an investigation of comparative genome organization among Asterids based on this reference genome platform. Residual duplicated regions within the genome probably resulting from a recent whole genome duplication event in Oleaceae were visualized in relation to wild olive (Olea europaea var. sylvestris). We used our F. pennsylvanica chromosome assembly to construct reference‐guided assemblies of 27 previously sequenced Fraxinus taxa, including F. excelsior. Thus, we present a significant step forward in genomic resources for research and protection of Fraxinus species