Targeted Mutagenesis of the Multicopy Myrosinase Gene Family in Allotetraploid Brassica juncea Reduces Pungency in Fresh Leaves across Environments.

Recent breeding efforts in Brassica have focused on the development of new oilseed feedstock crop for biofuels (e.g., ethanol, biodiesel, bio-jet fuel), bio-industrial uses (e.g., bio-plastics, lubricants), specialty fatty acids (e.g., erucic acid), and producing low glucosinolates levels for oilseed and feed meal production for animal consumption. We identified a novel opportunity to enhance the availability of nutritious, fresh leafy greens for human consumption. Here, we demonstrated the efficacy of disarming the 'mustard bomb' reaction in reducing pungency upon the mastication of fresh tissue-a major source of unpleasant flavor and/or odor in leafy Brassica. Using gene-specific mutagenesis via CRISPR-Cas12a, we created knockouts of all functional copies of the type-I myrosinase multigene family in tetraploid Brassica juncea. Our greenhouse and field trials demonstrate, via sensory and biochemical analyses, a stable reduction in pungency in edited plants across multiple environments. Collectively, these efforts provide a compelling path toward boosting the human consumption of nutrient-dense, fresh, leafy green vegetables.

Construction of a chromosome-scale long-read reference genome assembly for potato.

BACKGROUND: Worldwide, the cultivated potato, Solanum tuberosum L., is the No. 1 vegetable crop and a critical food security crop. The genome sequence of DM1-3 516 R44, a doubled monoploid clone of S. tuberosum Group Phureja, was published in 2011 using a whole-genome shotgun sequencing approach with short-read sequence data. Current advanced sequencing technologies now permit generation of near-complete, high-quality chromosome-scale genome assemblies at minimal cost. FINDINGS: Here, we present an updated version of the DM1-3 516 R44 genome sequence (v6.1) using Oxford Nanopore Technologies long reads coupled with proximity-by-ligation scaffolding (Hi-C), yielding a chromosome-scale assembly. The new (v6.1) assembly represents 741.6 Mb of sequence (87.8%) of the estimated 844 Mb genome, of which 741.5 Mb is non-gapped with 731.2 Mb anchored to the 12 chromosomes. Use of Oxford Nanopore Technologies full-length complementary DNA sequencing enabled annotation of 32,917 high-confidence protein-coding genes encoding 44,851 gene models that had a significantly improved representation of conserved orthologs compared with the previous annotation. The new assembly has improved contiguity with a 595-fold increase in N50 contig size, 99% reduction in the number of contigs, a 44-fold increase in N50 scaffold size, and an LTR Assembly Index score of 13.56, placing it in the category of reference genome quality. The improved assembly also permitted annotation of the centromeres via alignment to sequencing reads derived from CENH3 nucleosomes. CONCLUSIONS: Access to advanced sequencing technologies and improved software permitted generation of a high-quality, long-read, chromosome-scale assembly and improved annotation dataset for the reference genotype of potato that will facilitate research aimed at improving agronomic traits and understanding genome evolution.

Genome-wide Inference of Somatic Translocation Events During Potato Dihaploid Production.

Potato ( L.) breeders often use dihaploids, which are 2× progeny derived from 4× autotetraploid parents. Dihaploids can be used in diploid crosses to introduce new genetic material into breeding germplasm that can be integrated into tetraploid breeding through the use of unreduced gametes in 4× by 2× crosses. Dihaploid potatoes are usually produced via pollination by haploid inducer lines known as in vitro pollinators (IVP). In vitro pollinator chromosomes are selectively degraded from initially full hybrid embryos, resulting in 2× seed. During this process, somatic translocation of IVP DNA may occur. In this study, a genome-wide approach was used to identify such events and other chromosome-scale abnormalities in a population of 95 dihaploids derived from a cross between potato cultivar Superior and the haploid inducing line IVP101. Most Superior dihaploids showed translocation rates of <1% at 16,947,718 assayable sites, yet two dihaploids showed translocation rates of 1.86 and 1.60%. Allelic ratios at translocation sites suggested that most translocations occurred in individual cell lineages and were thus not present in all cells of the adult plants. Translocations were enriched in sites associated with high gene expression and H3K4 dimethylation and H4K5 acetylation, suggesting that they tend to occur in regions of open chromatin. The translocations likely result as a consequence of double-stranded break repair in the dihaploid genomes via homologous recombination during which IVP chromosomes are used as templates. Additionally, primary trisomy was observed in eight individuals. As the trisomic chromosomes were derived from Superior, meiotic nondisjunction may be common in potato.

Gene Discovery in Gelsemium Highlights Conserved Gene Clusters in Monoterpene Indole Alkaloid Biosynthesis.

Genome mining is a routine technique in microbes for discovering biosynthetic pathways. In plants, however, genomic information is not commonly used to identify novel biosynthesis genes. Here, we present the genome of the medicinal plant and oxindole monoterpene indole alkaloid (MIA) producer Gelsemium sempervirens (Gelsemiaceae). A gene cluster from Catharanthus roseus, which is utilized at least six enzymatic steps downstream from the last common intermediate shared between the two plant alkaloid types, is found in G. sempervirens, although the corresponding enzymes act on entirely different substrates. This study provides insights into the common genomic context of MIA pathways and is an important milestone in the further elucidation of the Gelsemium oxindole alkaloid pathway.

Genome Reduction in Tetraploid Potato Reveals Genetic Load, Haplotype Variation, and Loci Associated With Agronomic Traits.

The cultivated potato (Solanum tuberosum) has a complex genetic structure due to its autotetraploidy and vegetative propagation which leads to accumulation of mutations and a highly heterozygous genome. A high degree of heterozygosity has been considered to be the main driver of fitness and agronomic trait performance in potato improvement efforts, which is negatively impacted by genetic load. To understand the genetic landscape of cultivated potato, we constructed a gynogenic dihaploid (2n = 2x = 24) population from cv. Superior, prior to development of a high-density genetic map containing 12,753 single nucleotide polymorphisms (SNPs). Common quantitative trait loci (QTL) were identified for tuber traits, vigor and height on chromosomes 2, 4, 7, and 10, while specific QTL for number of inflorescences per plant, and tuber shape were present on chromosomes 4, 6, 10, and 11. Simplex rather than duplex loci were mainly associated with traits. In general, the Q allele (main effect) detected in one or two homologous chromosomes was associated with lower mean trait values suggesting the importance of dosage allelic effects, and the presence of up to two undesired alleles in the QTL region. Loss of heterozygosity has been associated with a lower rate of fitness, yet no correlation between the percent heterozygosity and increased fitness or agronomic performance was observed. Based upon linkage phase, we reconstructed the four homologous chromosome haplotypes of cv. Superior. revealing heterogeneity throughout the genome yet nearly duplicate haplotypes occurring among the homologs of particular chromosomes. These results suggest that the potentially deleterious mutations associated with genetic load in tetraploid potato could be mitigated by multiple loci which is consistent with the theory that epistasis complicates the identification of associations between markers and phenotypic performance.

Genome sequence of M6, a diploid inbred clone of the high-glycoalkaloid-producing tuber-bearing potato species Solanum chacoense, reveals residual heterozygosity.

Cultivated potato (Solanum tuberosum L.) is a highly heterozygous autotetraploid that presents challenges in genome analyses and breeding. Wild potato species serve as a resource for the introgression of important agronomic traits into cultivated potato. One key species is Solanum chacoense and the diploid, inbred clone M6, which is self-compatible and has desirable tuber market quality and disease resistance traits. Sequencing and assembly of the genome of the M6 clone of S. chacoense generated an assembly of 825 767 562 bp in 8260 scaffolds with an N50 scaffold size of 713 602 bp. Pseudomolecule construction anchored 508 Mb of the genome assembly into 12 chromosomes. Genome annotation yielded 49 124 high-confidence gene models representing 37 740 genes. Comparative analyses of the M6 genome with six other Solanaceae species revealed a core set of 158 367 Solanaceae genes and 1897 genes unique to three potato species. Analysis of single nucleotide polymorphisms across the M6 genome revealed enhanced residual heterozygosity on chromosomes 4, 8 and 9 relative to the other chromosomes. Access to the M6 genome provides a resource for identification of key genes for important agronomic traits and aids in genome-enabled development of inbred diploid potatoes with the potential to accelerate potato breeding.

Extensive genome heterogeneity leads to preferential allele expression and copy number-dependent expression in cultivated potato.

Relative to homozygous diploids, the presence of multiple homologs or homeologs in polyploids affords greater tolerance to mutations that can impact genome evolution. In this study, we describe sequence and structural variation in the genomes of six accessions of cultivated potato (Solanum tuberosum L.), a vegetatively propagated autotetraploid and their impact on the transcriptome. Sequence diversity was high with a mean single nucleotide polymorphisms (SNP) rate of approximately 1 per 50 bases suggestive of high levels of allelic diversity. Additive gene expression was observed in leaves (3605 genes) and tubers (6156 genes) that contrasted the preferential allele expression of between 2180 and 3502 and 3367 and 5270 genes in the leaf and tuber transcriptome, respectively. Preferential allele expression was significantly associated with evolutionarily conserved genes suggesting selection of specific alleles of genes responsible for biological processes common to angiosperms during the breeding selection process. Copy number variation was rampant with between 16 098 and 18 921 genes in each cultivar exhibiting duplication or deletion. Copy number variable genes tended to be evolutionarily recent, lowly expressed, and enriched in genes that show increased expression in response to biotic and abiotic stress treatments suggestive of a role in adaptation. Gene copy number impacts on gene expression were detected with 528 genes having correlations between copy number and gene expression. Collectively, these data suggest that in addition to allelic variation of coding sequence, the heterogenous nature of the tetraploid potato genome contributes to a highly dynamic transcriptome impacted by allele preferential and copy number-dependent expression effects.

De novo genome assembly of Camptotheca acuminata, a natural source of the anti-cancer compound camptothecin.

Camptotheca acuminata is 1 of a limited number of species that produce camptothecin, a pentacyclic quinoline alkaloid with anti-cancer activity due to its ability to inhibit DNA topoisomerase. While transcriptome studies have been performed previously with various camptothecin-producing species, no genome sequence for a camptothecin-producing species is available to date. We generated a high-quality de novo genome assembly for C. acuminata representing 403 174 860 bp on 1394 scaffolds with an N50 scaffold size of 1752 kbp. Quality assessments of the assembly revealed robust representation of the genome sequence including genic regions. Using a novel genome annotation method, we annotated 31 825 genes encoding 40 332 gene models. Based on sequence identity and orthology with validated genes from Catharanthus roseus as well as Pfam searches, we identified candidate orthologs for genes potentially involved in camptothecin biosynthesis. Extensive gene duplication including tandem duplication was widespread in the C. acuminata genome, with 2571 genes belonging to 997 tandem duplicated gene clusters. To our knowledge, this is the first genome sequence for a camptothecin-producing species, and access to the C. acuminata genome will permit not only discovery of genes encoding the camptothecin biosynthetic pathway but also reagents that can be used for heterologous expression of camptothecin and camptothecin analogs with novel pharmaceutical applications.

EGRINs (Environmental Gene Regulatory Influence Networks) in Rice That Function in the Response to Water Deficit, High Temperature, and Agricultural Environments.

Environmental gene regulatory influence networks (EGRINs) coordinate the timing and rate of gene expression in response to environmental signals. EGRINs encompass many layers of regulation, which culminate in changes in accumulated transcript levels. Here, we inferred EGRINs for the response of five tropical Asian rice (Oryza sativa) cultivars to high temperatures, water deficit, and agricultural field conditions by systematically integrating time-series transcriptome data, patterns of nucleosome-free chromatin, and the occurrence of known cis-regulatory elements. First, we identified 5447 putative target genes for 445 transcription factors (TFs) by connecting TFs with genes harboring known cis-regulatory motifs in nucleosome-free regions proximal to their transcriptional start sites. We then used network component analysis to estimate the regulatory activity for each TF based on the expression of its putative target genes. Finally, we inferred an EGRIN using the estimated transcription factor activity (TFA) as the regulator. The EGRINs include regulatory interactions between 4052 target genes regulated by 113 TFs. We resolved distinct regulatory roles for members of the heat shock factor family, including a putative regulatory connection between abiotic stress and the circadian clock. TFA estimation using network component analysis is an effective way of incorporating multiple genome-scale measurements into network inference.

Genome Reduction Uncovers a Large Dispensable Genome and Adaptive Role for Copy Number Variation in Asexually Propagated Solanum tuberosum.

Clonally reproducing plants have the potential to bear a significantly greater mutational load than sexually reproducing species. To investigate this possibility, we examined the breadth of genome-wide structural variation in a panel of monoploid/doubled monoploid clones generated from native populations of diploid potato (Solanum tuberosum), a highly heterozygous asexually propagated plant. As rare instances of purely homozygous clones, they provided an ideal set for determining the degree of structural variation tolerated by this species and deriving its minimal gene complement. Extensive copy number variation (CNV) was uncovered, impacting 219.8 Mb (30.2%) of the potato genome with nearly 30% of genes subject to at least partial duplication or deletion, revealing the highly heterogeneous nature of the potato genome. Dispensable genes (>7000) were associated with limited transcription and/or a recent evolutionary history, with lower deletion frequency observed in genes conserved across angiosperms. Association of CNV with plant adaptation was highlighted by enrichment in gene clusters encoding functions for environmental stress response, with gene duplication playing a part in species-specific expansions of stress-related gene families. This study revealed unique impacts of CNV in a species with asexual reproductive habits and how CNV may drive adaption through evolution of key stress pathways.

Whole genome re-sequencing of date palms yields insights into diversification of a fruit tree crop.

Date palms (Phoenix dactylifera) are the most significant perennial crop in arid regions of the Middle East and North Africa. Here, we present a comprehensive catalogue of approximately seven million single nucleotide polymorphisms in date palms based on whole genome re-sequencing of a collection of 62 cultivars. Population structure analysis indicates a major genetic divide between North Africa and the Middle East/South Asian date palms, with evidence of admixture in cultivars from Egypt and Sudan. Genome-wide scans for selection suggest at least 56 genomic regions associated with selective sweeps that may underlie geographic adaptation. We report candidate mutations for trait variation, including nonsense polymorphisms and presence/absence variation in gene content in pathways for key agronomic traits. We also identify a copia-like retrotransposon insertion polymorphism in the R2R3 myb-like orthologue of the oil palm virescens gene associated with fruit colour variation. This analysis documents patterns of post-domestication diversification and provides a genomic resource for this economically important perennial tree crop.

Whole-Genome Resequencing Reveals Extensive Natural Variation in the Model Green Alga Chlamydomonas reinhardtii.

We performed whole-genome resequencing of 12 field isolates and eight commonly studied laboratory strains of the model organism Chlamydomonas reinhardtii to characterize genomic diversity and provide a resource for studies of natural variation. Our data support previous observations that Chlamydomonas is among the most diverse eukaryotic species. Nucleotide diversity is ∼3% and is geographically structured in North America with some evidence of admixture among sampling locales. Examination of predicted loss-of-function mutations in field isolates indicates conservation of genes associated with core cellular functions, while genes in large gene families and poorly characterized genes show a greater incidence of major effect mutations. De novo assembly of unmapped reads recovered genes in the field isolates that are absent from the CC-503 assembly. The laboratory reference strains show a genomic pattern of polymorphism consistent with their origin as the recombinant progeny of a diploid zygospore. Large duplications or amplifications are a prominent feature of laboratory strains and appear to have originated under laboratory culture. Extensive natural variation offers a new source of genetic diversity for studies of Chlamydomonas, including naturally occurring alleles that may prove useful in studies of gene function and the dissection of quantitative genetic traits.