The giant sequoia genome and proliferation of disease resistance genes

Abstract

The giant sequoia ( Sequoiadendron giganteum ) of California are massive, long-lived trees that grow along the U.S. Sierra Nevada mountains. As they grow primarily in isolated groves within a narrow range, conservation of existing trees has been a national goal for over 150 years.Genomic data are limited in giant sequoia, and the assembly and annotation of the first giant sequoia genome has been an important goal to allow marker development for restoration and management. Using Illumina and Oxford Nanopore sequencing combined with Dovetail chromosome conformation capture libraries, 8.125 Gbp of sequence was assembled into eleven chromosome-scale scaffolds. This giant sequoia assembly represents the first genome sequenced in the Cupressaceae family, and lays a foundation for using genomic tools to aid in giant sequoia conservation and management. Beyond conservation and management applications, the giant sequoia assembly is a resource for answering questions about the life history of this enigmatic and robust species. Here we provide an example by taking an inventory of the large and complex family of NLR type disease resistance genes.assembly and annotation presented here is an unprecedented resource in conifer genomics, both for the quality of the assembly and because it represents an understudied branch of the gymnosperm tree of life. MATERIALS AND METHODS Sequencing and assembly Megagametophyte DNA extraction and sequencingCones were collected from a 1,360-year-old giant sequoia (SEGI21, Sillett et al., 2015) in Sequoia/Kings Canyon National Park in 2012. As in previous conifer genome sequencing projects (e.g. Zimin et al., 2014), the megagametophyte from a single fertilized seed was dissected out and its haploid DNA extracted with a Qiagen DNeasy Plant Kit (Hilden, Germany), followed by library preparation with an Illumina TruSeq Nano kit using the low throughput protocol. This megagametophyte library was then sequenced on 10 lanes of an Illumina HiSeq 4000 with 150 bp paired-end reads at the UC Davis Genome Center DNA Technologies Core facility. Foliage DNA extraction and Nanopore sequencingIn 2017 foliage was collected from the upper canopy of the same giant sequoia tree (SEGI21).From this foliage, high molecular weight DNA was extracted following the protocol described here (dx.doi.org/10.17504/protocols.io.4vbgw2n) . Briefly, purified genomic DNA was isolated through a nuclei extraction and lysis protocol. First, mature leaf tissue was homogenized in liquid nitrogen until well-ground, then added to a gentle lysis buffer (after Zhang et al.,

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“…Long reads can indeed be used alone at a high depth of coverage permitting autocorrection (Koren et al, 2017; Shafin et al, 2020) or in combination with short reads for (1) scaffolding short-read contigs (Armstrong et al, 2020; Kwan et al, 2019), (2) using short reads to polish long-read contigs (Batra et al, 2019b; Datema et al, 2016; Jansen et al, 2017; Michael et al, 2018), or (3) optimizing the assembly process by using information from both long and short reads (Díaz-Viraqué et al, 2019; Gan et al, 2019; Jiang et al, 2019; Kadobianskyi et al, 2019; Tan et al, 2018; Wang et al, 2020; Zimin et al, 2017). Given the previously demonstrated efficiency of the MaSuRCA tool for the assembly of large genomes (Scott et al, 2020; Wang et al, 2020; Zimin et al, 2017), we decided to rely on hybrid sequencing data combining the advantages of Illumina short-read and Nanopore long-read sequencing technologies.…”

“…Furthermore, this method is designed to tolerate a significant level of sequencing error. Initially developed to address short reads from Sanger sequencing and longer reads from 454 Life Sciences instruments, this method has already shown promising results for combining Illumina and ONT/PacBio sequencing data in several taxonomic groups, such as plants (Scott et al, 2020; Wang et al, 2020; Zimin et al, 2017), birds (Gan et al, 2019), and fishes (Jiang et al, 2019; Kadobianskyi et al, 2019; Tan et al, 2018) but not yet in mammals.…”

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“…These genomes were 8.2 and 26.5 Gbp, respectively, demonstrating an ability for the MinION to be used for very large genomes [69]. The giant sequoia's reference genome also features the longest scaffolds assembled in any organism to date with the largest being 985 Mbp and the scaffold N50 being 690.55 Mb [69]. ONT's ability to characterise complex genomic regions has the potential to increase the accuracy of reference genome assemblies in livestock.…”

“…This decreases the cost of sequencing and increases the individual base accuracy of genome assemblies. Reference genomes for the endangered giant redwood ( Sequoia sempervirens ) and giant sequoia ( Sequoiadendron giganteum ), both trees native to California, were also created using Illumina and Nanopore data [ 69 ]. These genomes were 8.2 and 26.5 Gbp, respectively, demonstrating an ability for the MinION to be used for very large genomes [ 69 ].…”

“…Reference genomes for the endangered giant redwood ( Sequoia sempervirens ) and giant sequoia ( Sequoiadendron giganteum ), both trees native to California, were also created using Illumina and Nanopore data [ 69 ]. These genomes were 8.2 and 26.5 Gbp, respectively, demonstrating an ability for the MinION to be used for very large genomes [ 69 ]. The giant sequoia’s reference genome also features the longest scaffolds assembled in any organism to date with the largest being 985 Mbp and the scaffold N50 being 690.55 Mb [ 69 ].…”