Comparison of the two up-to-date sequencing technologies for genome assembly: HiFi reads of Pacific Biosciences Sequel II system and ultralong reads of Oxford Nanopore.

BACKGROUND: The availability of reference genomes has revolutionized the study of biology. Multiple competing technologies have been developed to improve the quality and robustness of genome assemblies during the past decade. The 2 widely used long-read sequencing providers-Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT)-have recently updated their platforms: PacBio enables high-throughput HiFi reads with base-level resolution of >99%, and ONT generated reads as long as 2 Mb. We applied the 2 up-to-date platforms to a single rice individual and then compared the 2 assemblies to investigate the advantages and limitations of each. RESULTS: The results showed that ONT ultralong reads delivered higher contiguity, producing a total of 18 contigs of which 10 were assembled into a single chromosome compared to 394 contigs and 3 chromosome-level contigs for the PacBio assembly. The ONT ultralong reads also prevented assembly errors caused by long repetitive regions, for which we observed a total of 44 genes of false redundancies and 10 genes of false losses in the PacBio assembly, leading to over- or underestimation of the gene families in those long repetitive regions. We also noted that the PacBio HiFi reads generated assemblies with considerably fewer errors at the level of single nucleotides and small insertions and deletions than those of the ONT assembly, which generated an average 1.06 errors per kb and finally engendered 1,475 incorrect gene annotations via altered or truncated protein predictions. CONCLUSIONS: It shows that both PacBio HiFi reads and ONT ultralong reads had their own merits. Further genome reference constructions could leverage both techniques to lessen the impact of assembly errors and subsequent annotation mistakes rooted in each.

Comparative Genomics of Pathogens Causing Brown Spot Disease of Tobacco: Alternaria longipes and Alternaria alternata.

The genus Alternaria is a group of infectious/contagious pathogenic fungi that not only invade a wide range of crops but also induce severe allergic reactions in a part of the human population. In this study, two strains Alternaria longipes cx1 and Alternaria alternata cx2 were isolated from different brown spot lesions on infected tobacco leaves. Their complete genomes were sequenced, de novo assembled, and comparatively analyzed. Phylogenetic analysis revealed that A. longipes cx1 and A. alternata cx2 diverged 3.3 million years ago, indicating a recent event of speciation. Seventeen non-ribosomal peptide synthetase (NRPS) genes and 13 polyketide synthase (PKS) genes in A. longipes cx1 and 13 NRPS genes and 12 PKS genes in A. alternata cx2 were identified in these two strains. Some of these genes were predicted to participate in the synthesis of non-host specific toxins (non-HSTs), such as tenuazonic acid (TeA), alternariol (AOH) and alternariol monomethyl ether (AME). By comparative genome analysis, we uncovered that A. longipes cx1 had more genes putatively involved in pathogen-plant interaction, more carbohydrate-degrading enzymes and more secreted proteins than A. alternata cx2. In summary, our results demonstrate the genomic distinction between A. longipes cx1 and A. altenata cx2. They will not only improve the understanding of the phylogenetic relationship among genus Alternaria, but more importantly provide valuable genomic resources for the investigation of plant-pathogen interaction.

Genome of Plant Maca (Lepidium meyenii) Illuminates Genomic Basis for High-Altitude Adaptation in the Central Andes.

Maca (Lepidium meyenii Walp, 2n = 8x = 64), belonging to the Brassicaceae family, is an economic plant cultivated in the central Andes sierra in Peru (4000-4500 m). Considering that the rapid uplift of the central Andes occurred 5-10 million years ago (Ma), an evolutionary question arises regarding how plants such as maca acquire high-altitude adaptation within a short geological period. Here, we report the high-quality genome assembly of maca, in which two closely spaced maca-specific whole-genome duplications (WGDs; ∼6.7 Ma) were identified. Comparative genomic analysis between maca and closely related Brassicaceae species revealed expansions of maca genes and gene families involved in abiotic stress response, hormone signaling pathway, and secondary metabolite biosynthesis via WGDs. The retention and subsequent functional divergence of many duplicated genes may account for the morphological and physiological changes (i.e., small leaf shape and self-fertility) in maca in a high-altitude environment. In addition, some duplicated maca genes were identified with functions in morphological adaptation (i.e., LEAF CURLING RESPONSIVENESS) and abiotic stress response (i.e., GLYCINE-RICH RNA-BINDING PROTEINS and DNA-DAMAGE-REPAIR/TOLERATION 2) under positive selection. Collectively, the maca genome provides useful information to understand the important roles of WGDs in the high-altitude adaptation of plants in the Andes.

Transcriptomic profile of tobacco in response to Alternaria longipes and Alternaria alternata infections.

Tobacco brown spot caused by Alternaria fungal species is one of the most damaging diseases, and results in significant yield losses. However, little is known about the systematic response of tobacco to this fungal infection. To fill this knowledge gap, de novo assemblies of tobacco leaf transcriptomes were obtained in cultivars V2 and NC89 after the inoculation of either Alternaria longipes (AL) or Alternaria alternata (AA) at three different time points. We studied the gene expression profile of each cultivar-pathogen combination, and identified eight differentially expressed genes shared among all combinations. Gene ontology enrichment analysis of the differentially expressed genes revealed key components during the fungal infection, which included regulation of gene expression (GO:0010468), regulation of RNA metabolic process (GO:0051252), tetrapyrrole binding (GO:0046906), and external encapsulating structure (GO:0030312). Further analyses of the continuously upregulated/downregulated genes and the resistance genes demonstrated that the gene expression profile upon fungal infection was contingent on the specific cultivar and pathogen. In conclusion, this study provides a solid foundation for the investigation of plant-pathogen interaction, and is of great importance for disease prevention and molecular breeding.

SNP-based genetic linkage map of tobacco (Nicotiana tabacum L.) using next-generation RAD sequencing.

BACKGROUND: Tobacco (Nicotiana tabacum L.) is an important model system, which has been widely used in plant physiological studies and it is particularly useful as a bioreactor. Despite its importance, only limited molecular marker resources are available for genome analysis, genetic mapping and breeding. Restriction-site associated DNA sequencing (RAD-seq) is a powerful new method for targeted sequencing across the genomes of many individuals. This approach has broad potential for genetic analysis through linkage mapping. RESULTS: We constructed a RAD library using genomic DNA from a BC1 backcross population. Sequencing of 196 individuals was performed on an Illumina HiSeq 2500. Two linkage maps were constructed, one with a reference genome and another, termed as de novo identification of single nucleotide polymorphism (SNP) by RAD-seq, without a reference genome. Overall, 4138 and 2162 SNP markers with a total length of 1944.74 and 2000.9 cM were mapped to 24 linkage groups in the genetic maps based on reference genome and without reference, respectively. CONCLUSIONS: Using two different SNP discovery methods based on next generation RAD sequencing technology, we have respectively mapped 2162 and 4318 SNPs in our backcross population. This study gives an excellent example for high density linkage map construction, irrespective of genome sequence availability, and provides saturated information for downstream genetic investigations such as quantitative trait locus analyses or genomic selection (e.g. bioreactor suitable cultivars).