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Wednesday, January 6, 2021

PAG Conference: Update on sequencing of the Cabernet sauvignon genome

Grant Cramer from the University of Nevada, Reno, and Dario Cantu from the Univeristy of Callifornia, Davis, discuss past challenges with sequencing Clone 8 of Cabernet Sauvignon (Vitis vinifera). An assembly of the genome was attempted with approximately 110x Illumina reads and 5x PacBio reads. The PacBio SMRT Sequencing read made major improvements in the assembly compared with the results of Illumina reads only. However, the assembly results were still unsatisfactory, so an additional 100-fold SMRT Sequencing coverage had been generated. An update on the current sequencing results and status of the assembly are presented.

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Tuesday, December 22, 2020

Structural variant detection in crops using low-fold coverage long-read sequencing

Genomics studies have shown that the insertions, deletions, duplications, translocations, inversions, and tandem repeat expansions in the structural variant (SV) size range (>50 bp) contribute to the evolution of traits and often have significant associations with agronomically important phenotypes. However, most SVs are too small to detect with array comparative genomic hybridization and too large to reliably discover with short-read DNA sequencing. While de novo assembly is the most comprehensive way to identify variants in a genome, recent studies in human genomes show that PacBio SMRT Sequencing sensitively detects structural variants at low coverage. Here we present SV characterization in…

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Tuesday, December 22, 2020

Rapid full-length Iso-Seq cDNA sequencing of rice mRNA to facilitate annotation and identify splice-site variation.

PacBio’s new Iso-Seq technology allows for rapid generation of full-length cDNA sequences without the need for assembly steps. The technology was tested on leaf mRNA from two model O. sativa ssp. indica cultivars – Minghui 63 and Zhenshan 97. Even though each transcriptome was not exhaustively sequenced, several thousand isoforms described genes over a wide size range, most of which are not present in any currently available FL cDNA collection. In addition, the lack of an assembly requirement provides direct and immediate access to complete mRNA sequences and rapid unraveling of biological novelties.

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Tuesday, December 22, 2020

Targeted sequencing of genes from soybean using NimbleGen SeqCap EZ and PacBio SMRT Sequencing

Full-length gene capture solutions offer opportunities to screen and characterize structural variations and genetic diversity to understand key traits in plants and animals. Through a combined Roche NimbleGen probe capture and SMRT Sequencing strategy, we demonstrate the capability to resolve complex gene structures often observed in plant defense and developmental genes spanning multiple kilobases. The custom panel includes members of the WRKY plant-defense-signaling family, members of the NB-LRR disease-resistance family, and developmental genes important for flowering. The presence of repetitive structures and low-complexity regions makes short-read sequencing of these genes difficult, yet this approach allows researchers to obtain complete sequences…

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Tuesday, April 21, 2020

The bracteatus pineapple genome and domestication of clonally propagated crops.

Domestication of clonally propagated crops such as pineapple from South America was hypothesized to be a ‘one-step operation’. We sequenced the genome of Ananas comosus var. bracteatus CB5 and assembled 513?Mb into 25 chromosomes with 29,412 genes. Comparison of the genomes of CB5, F153 and MD2 elucidated the genomic basis of fiber production, color formation, sugar accumulation and fruit maturation. We also resequenced 89 Ananas genomes. Cultivars ‘Smooth Cayenne’ and ‘Queen’ exhibited ancient and recent admixture, while ‘Singapore Spanish’ supported a one-step operation of domestication. We identified 25 selective sweeps, including a strong sweep containing a pair of tandemly duplicated…

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Tuesday, April 21, 2020

The Chinese chestnut genome: a reference for species restoration

Forest tree species are increasingly subject to severe mortalities from exotic pests, diseases, and invasive organisms, accelerated by climate change. Forest health issues are threatening multiple species and ecosystem sustainability globally. While sources of resistance may be available in related species, or among surviving trees, introgression of resistance genes into threatened tree species in reasonable time frames requires genome-wide breeding tools. Asian species of chestnut (Castanea spp.) are being employed as donors of disease resistance genes to restore native chestnut species in North America and Europe. To aid in the restoration of threatened chestnut species, we present the assembly of…

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Tuesday, April 21, 2020

Genome data of Fusarium oxysporum f. sp. cubense race 1 and tropical race 4 isolates using long-read sequencing.

Fusarium wilt of banana is caused by the soil-borne fungal pathogen Fusarium oxysporum f. sp. cubense (Foc). We generated two chromosome-level assemblies of Foc race 1 and tropical race 4 strains using single-molecule real-time sequencing. The Foc1 and FocTR4 assemblies had 35 and 29 contigs with contig N50 lengths of 2.08 Mb and 4.28 Mb, respectively. These two new references genomes represent a greater than 100-fold improvement over the contig N50 statistics of the previous short read-based Foc assemblies. The two high-quality assemblies reported here will be a valuable resource for the comparative analysis of Foc races at the pathogenic…

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Tuesday, April 21, 2020

Full-length mRNA sequencing and gene expression profiling reveal broad involvement of natural antisense transcript gene pairs in pepper development and response to stresses.

Pepper is an important vegetable with great economic value and unique biological features. In the past few years, significant development has been made towards understanding the huge complex pepper genome; however, pepper functional genomics has not been well studied. To better understand the pepper gene structure and pepper gene regulation, we conducted full-length mRNA sequencing by PacBio sequencing and obtained 57862 high-quality full-length mRNA sequences derived from 18362 previously annotated and 5769 newly detected genes. New gene models were built that combined the full-length mRNA sequences and corrected approximately 500 fragmented gene models from previous annotations. Based on the full-length…

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Tuesday, April 21, 2020

Genome-wide selection footprints and deleterious variations in young Asian allotetraploid rapeseed.

Brassica napus (AACC, 2n = 38) is an important oilseed crop grown worldwide. However, little is known about the population evolution of this species, the genomic difference between its major genetic groups, such as European and Asian rapeseed, and the impacts of historical large-scale introgression events on this young tetraploid. In this study, we reported the de novo assembly of the genome sequences of an Asian rapeseed (B. napus), Ningyou 7, and its four progenitors and compared these genomes with other available genomic data from diverse European and Asian cultivars. Our results showed that Asian rapeseed originally derived from European rapeseed but subsequently…

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Tuesday, April 21, 2020

Relative Performance of MinION (Oxford Nanopore Technologies) versus Sequel (Pacific Biosciences) Third-Generation Sequencing Instruments in Identification of Agricultural and Forest Fungal Pathogens.

Culture-based molecular identification methods have revolutionized detection of pathogens, yet these methods are slow and may yield inconclusive results from environmental materials. The second-generation sequencing tools have much-improved precision and sensitivity of detection, but these analyses are costly and may take several days to months. Of the third-generation sequencing techniques, the portable MinION device (Oxford Nanopore Technologies) has received much attention because of its small size and possibility of rapid analysis at reasonable cost. Here, we compare the relative performances of two third-generation sequencing instruments, MinION and Sequel (Pacific Biosciences), in identification and diagnostics of fungal and oomycete pathogens from…

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Tuesday, April 21, 2020

The genomes of pecan and Chinese hickory provide insights into Carya evolution and nut nutrition.

Pecan (Carya illinoinensis) and Chinese hickory (C. cathayensis) are important commercially cultivated nut trees in the genus Carya (Juglandaceae), with high nutritional value and substantial health benefits.We obtained >187.22 and 178.87 gigabases of sequence, and ~288× and 248× genome coverage, to a pecan cultivar (“Pawnee”) and a domesticated Chinese hickory landrace (ZAFU-1), respectively. The total assembly size is 651.31 megabases (Mb) for pecan and 706.43 Mb for Chinese hickory. Two genome duplication events before the divergence from walnut were found in these species. Gene family analysis highlighted key genes in biotic and abiotic tolerance, oil, polyphenols, essential amino acids, and…

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Tuesday, April 21, 2020

Chromosome-scale genome assembly of kiwifruit Actinidia eriantha with single-molecule sequencing and chromatin interaction mapping.

Kiwifruit (Actinidia spp.) is a dioecious plant with fruits containing abundant vitamin C and minerals. A handful of kiwifruit species have been domesticated, among which Actinidiaeriantha is increasingly favored in breeding owing to its superior commercial traits. Recently, elite cultivars from A. eriantha have been successfully selected and further studies on their biology and breeding potential require genomic information, which is currently unavailable.We assembled a chromosome-scale genome sequence of A. eriantha cultivar White using single-molecular sequencing and chromatin interaction map-based scaffolding. The assembly has a total size of 690.6 megabases and an N50 of 21.7 megabases. Approximately 99% of the…

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Tuesday, April 21, 2020

Effector gene reshuffling involves dispensable mini-chromosomes in the wheat blast fungus.

Newly emerged wheat blast disease is a serious threat to global wheat production. Wheat blast is caused by a distinct, exceptionally diverse lineage of the fungus causing rice blast disease. Through sequencing a recent field isolate, we report a reference genome that includes seven core chromosomes and mini-chromosome sequences that harbor effector genes normally found on ends of core chromosomes in other strains. No mini-chromosomes were observed in an early field strain, and at least two from another isolate each contain different effector genes and core chromosome end sequences. The mini-chromosome is enriched in transposons occurring most frequently at core…

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Tuesday, April 21, 2020

A whole genome scan of SNP data suggests a lack of abundant hard selective sweeps in the genome of the broad host range plant pathogenic fungus Sclerotinia sclerotiorum.

The pathogenic fungus Sclerotinia sclerotiorum infects over 600 species of plant. It is present in numerous environments throughout the world and causes significant damage to many agricultural crops. Fragmentation and lack of gene flow between populations may lead to population sub-structure. Within discrete recombining populations, positive selection may lead to a ‘selective sweep’. This is characterised by an increase in frequency of a favourable allele leading to reduction in genotypic diversity in a localised genomic region due to the phenomenon of genetic hitchhiking. We aimed to assess whether isolates of S. sclerotiorum from around the world formed genotypic clusters associated…

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