Genome assembly and characterization of a complex zfBED-NLR gene-containing disease resistance locus in Carolina Gold Select rice with Nanopore sequencing

Long-read sequencing facilitates assembly of complex genomic regions. In plants, loci containing nucleotide-binding, leucine-rich repeat (NLR) disease resistance genes are an important example of such regions. NLR genes constitute one of the largest gene families in plants and are often clustered, evolving via duplication, contraction, and transposition. We recently mapped the Xo1 locus for resistance to bacterial blight and bacterial leaf streak, found in the American heirloom rice variety Carolina Gold Select, to a region that in the Nipponbare reference genome is NLR gene-rich. Here, toward identification of the Xo1 gene, we combined Nanopore and Illumina reads and generated a high-quality Carolina Gold Select genome assembly. We identified 529 complete or partial NLR genes and discovered, relative to Nipponbare, an expansion of NLR genes at the Xo1 locus. One of these has high sequence similarity to the cloned, functionally similar Xa1 gene. Both harbor an integrated zfBED domain, and the repeats within each protein are nearly perfect. Across diverse Oryzeae, we identified two sub-clades of NLR genes with these features, varying in the presence of the zfBED domain and the number of repeats. The Carolina Gold Select genome assembly also uncovered at the Xo1 locus a rice blast resistance gene and a gene encoding a polyphenol oxidase (PPO). PPO activity has been used as a marker for blast resistance at the locus in some varieties; however, the Carolina Gold Select sequence revealed a loss-of-function mutation in the PPO gene that breaks this association. Our results demonstrate that whole genome sequencing combining Nanopore and Illumina reads effectively resolves NLR gene loci. Our identification of an Xo1 candidate is an important step toward mechanistic characterization, including the role(s) of the zfBED domain. Finally, the Carolina Gold Select genome assembly will facilitate identification of other useful traits in this historically important variety. Author summary Plants lack adaptive immunity, and instead contain repeat-rich, disease resistance genes that evolve rapidly through duplication, recombination, and transposition. The number, variation, and often clustered arrangement of these genes make them challenging to sequence and catalog. The US heirloom rice variety Carolina Gold Select has resistance to two important bacterial diseases. Toward identifying the responsible gene(s), we combined long- and short-read sequencing technologies to assemble the whole genome and identify the resistance gene repertoire. We previously narrowed the location of the gene(s) to a region on chromosome four. The region in Carolina Gold Select is larger than in the rice reference genome (Nipponbare) and contains twice as many resistance genes. One shares unusual features with a known bacterial disease resistance gene, suggesting that it confers the resistance. Across diverse varieties and related species, we identified two widely-distributed groups of such genes. The results are an important step toward mechanistic characterization and deployment of the bacterial disease resistance. The genome assembly also identified a resistance gene for a fungal disease and predicted a marker phenotype used in breeding for resistance. Thus, the Carolina Gold Select genome assembly can be expected to aid in the identification and deployment of other valuable traits

Improving Phrap-Based Assembly of the Rat Using “Reliable” Overlaps

The assembly methods used for whole-genome shotgun (WGS) data have a major impact on the quality of resulting draft genomes. We present a novel algorithm to generate a set of “reliable” overlaps based on identifying repeat k-mers. To demonstrate the benefits of using reliable overlaps, we have created a version of the Phrap assembly program that uses only overlaps from a specific list. We call this version PhrapUMD. Integrating PhrapUMD and our “reliable-overlap” algorithm with the Baylor College of Medicine assembler, Atlas, we assemble the BACs from the Rattus norvegicus genome project. Starting with the same data as the Nov. 2002 Atlas assembly, we compare our results and the Atlas assembly to the 4.3 Mb of rat sequence in the 21 BACs that have been finished. Our version of the draft assembly of the 21 BACs increases the coverage of finished sequence from 93.4% to 96.3%, while simultaneously reducing the base error rate from 4.5 to 1.1 errors per 10,000 bases. There are a number of ways of assessing the relative merits of assemblies when the finished sequence is available. If one views the overall quality of an assembly as proportional to the inverse of the product of the error rate and sequence missed, then the assembly presented here is seven times better. The UMD Overlapper with options for reliable overlaps is available from the authors at http://www.genome.umd.edu. We also provide the changes to the Phrap source code enabling it to use only the reliable overlaps

The Douglas-Fir Genome Sequence Reveals Specialization of the Photosynthetic Apparatus in Pinaceae.

A reference genome sequence for Pseudotsuga menziesii var. menziesii (Mirb.) Franco (Coastal Douglas-fir) is reported, thus providing a reference sequence for a third genus of the family Pinaceae. The contiguity and quality of the genome assembly far exceeds that of other conifer reference genome sequences (contig N50 = 44,136 bp and scaffold N50 = 340,704 bp). Incremental improvements in sequencing and assembly technologies are in part responsible for the higher quality reference genome, but it may also be due to a slightly lower exact repeat content in Douglas-fir vs. pine and spruce. Comparative genome annotation with angiosperm species reveals gene-family expansion and contraction in Douglas-fir and other conifers which may account for some of the major morphological and physiological differences between the two major plant groups. Notable differences in the size of the NDH-complex gene family and genes underlying the functional basis of shade tolerance/intolerance were observed. This reference genome sequence not only provides an important resource for Douglas-fir breeders and geneticists but also sheds additional light on the evolutionary processes that have led to the divergence of modern angiosperms from the more ancient gymnosperms

Transposable element islands facilitate adaptation to novel environments in an invasive species

Peer reviewe

The evolution of synaptic and cognitive capacity: insights from the nervous system transcriptome of Aplysia

© The Author(s), 2022. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Orvis, J., Albertin, C., Shrestha, P., Chen, S., Zheng, M., Rodriguez, C., Tallon, L., Mahurkar, A., Zimin, A., Kim, M., Liu, K., Kandel, E., Fraser, C., Sossin, W., & Abrams, T. The evolution of synaptic and cognitive capacity: insights from the nervous system transcriptome of Aplysia. Proceedings of the National Academy of Sciences of the United States of America, 119(28), (2022): e2122301119, https://doi.org/10.1073/pnas.2122301119.The gastropod mollusk Aplysia is an important model for cellular and molecular neurobiological studies, particularly for investigations of molecular mechanisms of learning and memory. We developed an optimized assembly pipeline to generate an improved Aplysia nervous system transcriptome. This improved transcriptome enabled us to explore the evolution of cognitive capacity at the molecular level. Were there evolutionary expansions of neuronal genes between this relatively simple gastropod Aplysia (20,000 neurons) and Octopus (500 million neurons), the invertebrate with the most elaborate neuronal circuitry and greatest behavioral complexity? Are the tremendous advances in cognitive power in vertebrates explained by expansion of the synaptic proteome that resulted from multiple rounds of whole genome duplication in this clade? Overall, the complement of genes linked to neuronal function is similar between Octopus and Aplysia. As expected, a number of synaptic scaffold proteins have more isoforms in humans than in Aplysia or Octopus. However, several scaffold families present in mollusks and other protostomes are absent in vertebrates, including the Fifes, Lev10s, SOLs, and a NETO family. Thus, whereas vertebrates have more scaffold isoforms from select families, invertebrates have additional scaffold protein families not found in vertebrates. This analysis provides insights into the evolution of the synaptic proteome. Both synaptic proteins and synaptic plasticity evolved gradually, yet the last deuterostome-protostome common ancestor already possessed an elaborate suite of genes associated with synaptic function, and critical for synaptic plasticity.This work was supported by NSF EAGER Award IOS-1255695 and NIH grant R01 MH 55880 grant to T.W.A.; by a Natural Sciences and Engineering Research Council of Canada Discovery grant and Canadian Institutes of Health Research project grant 340328 to W.S.; by funding from the HHMI to E.R.K.; and by a Hibbitt Early Career Fellowship to C.A. W.S. is James McGill Professor at McGill University

A pseudomolecule assembly of the Rocky Mountain elk genome

Rocky Mountain elk (Cervus canadensis) populations have significant economic implications to the cattle industry, as they are a major reservoir for Brucella abortus in the Greater Yellowstone area. Vaccination attempts against intracellular bacterial diseases in elk populations have not been successful due to a negligible adaptive cellular immune response. A lack of genomic resources has impeded attempts to better understand why vaccination does not induce protective immunity. To overcome this limitation, PacBio, Illumina, and Hi-C sequencing with a total of 686-fold coverage was used to assemble the elk genome into 35 pseudomolecules. A robust gene annotation was generated resulting in 18,013 gene models and 33,422 mRNAs. The accuracy of the assembly was assessed using synteny to the red deer and cattle genomes identifying several chromosomal rearrangements, fusions and fissions. Because this genome assembly and annotation provide a foundation for genome-enabled exploration of Cervus species, we demonstrate its utility by exploring the conservation of immune system-related genes. We conclude by comparing cattle immune system-related genes to the elk genome, revealing eight putative gene losses in elk

De novo assembly of the cattle reference genome with single-molecule sequencing.

BackgroundMajor advances in selection progress for cattle have been made following the introduction of genomic tools over the past 10-12 years. These tools depend upon the Bos taurus reference genome (UMD3.1.1), which was created using now-outdated technologies and is hindered by a variety of deficiencies and inaccuracies.ResultsWe present the new reference genome for cattle, ARS-UCD1.2, based on the same animal as the original to facilitate transfer and interpretation of results obtained from the earlier version, but applying a combination of modern technologies in a de novo assembly to increase continuity, accuracy, and completeness. The assembly includes 2.7 Gb and is >250× more continuous than the original assembly, with contig N50 >25 Mb and L50 of 32. We also greatly expanded supporting RNA-based data for annotation that identifies 30,396 total genes (21,039 protein coding). The new reference assembly is accessible in annotated form for public use.ConclusionsWe demonstrate that improved continuity of assembled sequence warrants the adoption of ARS-UCD1.2 as the new cattle reference genome and that increased assembly accuracy will benefit future research on this species