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Asset Tag: agriculture

April 21, 2020

Variant Phasing and Haplotypic Expression from Single-molecule Long-read Sequencing in Maize

Haplotype phasing of genetic variants is important for interpretation of the maize genome, population genetic analysis, and functional genomic analysis of allelic activity. Accordingly, accurate methods for phasing full-length isoforms are essential for functional genomics study. In this study, we performed an isoform-level phasing study in maize, using two inbred lines and their reciprocal crosses, based on single-molecule full-length cDNA sequencing. To phase and analyze full-length transcripts between hybrids and parents, we developed a tool called IsoPhase. Using this tool, we validated the majority of SNPs called against matching short read data and identified cases of allele-specific, gene-level, and isoform-level expression. Our results revealed that maize parental and hybrid lines exhibit different splicing activities. After phasing 6,847 genes in two reciprocal hybrids using embryo, endosperm and root tissues, we annotated the SNPs and identified large-effect genes. In addition, based on single-molecule sequencing, we identified parent-of-origin isoforms in maize hybrids, different novel isoforms between maize parent and hybrid lines, and imprinted genes from different tissues. Finally, we characterized variation in cis- and trans-regulatory effects. Our study provides measures of haplotypic expression that could increase power and accuracy in studies of allelic expression.

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April 21, 2020

Genome sequence resource for Ilyonectria mors-panacis, causing rusty root rot of Panax notoginseng.

Ilyonectria mors-panacis is a serious disease hampering the production of Panax notoginseng, an important Chinese medicinal herb, widely used for its anti-inflammatory, anti-fatigue, hepato-protective, and coronary heart disease prevention effects. Here, we report the first Illumina-Pacbio hybrid sequenced draft genome assembly of I. mors-panacis strain G3B and its annotation. The availability of this genome sequence not only represents an important tool toward understanding the genetics behind the infection mechanism of I. mors-panacis strain G3B but also will help illuminate the complexities of the taxonomy of this species.

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April 21, 2020

Genomics-informed molecular detection of Xanthomonas vasicola pv. vasculorum strains causing severe bacterial leaf streak of corn.

Xanthomonas vasicola pv. vasculorum (syn. X. campestris pv. vasculorum) was initially identified as the causal agent of bacterial leaf streak of corn in South Africa. The pathovar vasculorum causes disease on sugarcane and corn, but a subset of these strains was noted for its increased disease severity in corn. This subset was re-classified as Xanthomonas campestris pv. zeae in the early 1990s and was found to have slightly different biochemical and genetic properties than isolates from sugarcane. There has been an emergence of X. campestris pv. zeae-like strains of X. vasicola pv. vasculorum in both the United States and Argentina since 2010. We performed whole genome sequencing on U.S. isolates to confirm their identity. Informed by comparative genomics, we then developed specific TaqMan qPCR and loop-mediated isothermal amplification (LAMP) assays for the detection of this specific subset of X. vasicola pv. vasculorum strains. The qPCR 4909 assay was tested against 27 xanthomonads (diverse representation), 32 DNA extractions from corn leaves confirmed as positive or negative for the bacterium, 41 X. vasicola pv. vasculorum isolates from corn in the United States and Argentina, and 31 additional bacteria associated with corn, sugarcane, or sorghum. In all cases the assay was shown to be specific for the X. vasicola pv. vasculorum isolates that cause more severe disease on corn. We then tested the LAMP 166 assay against the 27 xanthomonads and 32 corn leaf DNA samples, and we found this assay was also specific for this subset of X. vasicola pv. vasculorum isolates. We also developed a live/dead cells distinction protocol using propidium monoazide prior to DNA extraction for analyzing seed washes using these assays. These two detection assays can be useful for both diagnosticians and researchers to specifically identify the X. vasicola pv. vasculorum isolates that cause more severe symptoms on corn.

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April 21, 2020

Decoding and analysis of organelle genomes of Indian tea (Camellia assamica) for phylogenetic confirmation.

The NCBI database has >15 chloroplast (cp) genome sequences available for different Camellia species but none for C. assamica. There is no report of any mitochondrial (mt) genome in the Camellia genus or Theaceae family. With the strong believes that these organelle genomes can play a great tool for taxonomic and phylogenetic analysis, we successfully assembled and analyzed cp and mt genome of C. assamica. We assembled the complete mt genome of C. assamica in a single circular contig of 707,441?bp length comprising of a total of 66 annotated genes, including 35 protein-coding genes, 29 tRNAs and two rRNAs. The first ever cp genome of C. assamica resulted in a circular contig of 157,353?bp length with a typical quadripartite structure. Phylogenetic analysis based on these organelle genomes showed that C. assamica was closely related to C. sinensis and C. leptophylla. It also supports Caryophyllales as Superasterids. Copyright © 2019. Published by Elsevier Inc.

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April 21, 2020

Insect genomes: progress and challenges.

In the wake of constant improvements in sequencing technologies, numerous insect genomes have been sequenced. Currently, 1219 insect genome-sequencing projects have been registered with the National Center for Biotechnology Information, including 401 that have genome assemblies and 155 with an official gene set of annotated protein-coding genes. Comparative genomics analysis showed that the expansion or contraction of gene families was associated with well-studied physiological traits such as immune system, metabolic detoxification, parasitism and polyphagy in insects. Here, we summarize the progress of insect genome sequencing, with an emphasis on how this impacts research on pest control. We begin with a brief introduction to the basic concepts of genome assembly, annotation and metrics for evaluating the quality of draft assemblies. We then provide an overview of genome information for numerous insect species, highlighting examples from prominent model organisms, agricultural pests and disease vectors. We also introduce the major insect genome databases. The increasing availability of insect genomic resources is beneficial for developing alternative pest control methods. However, many opportunities remain for developing data-mining tools that make maximal use of the available insect genome resources. Although rapid progress has been achieved, many challenges remain in the field of insect genomics. © 2019 The Royal Entomological Society.

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April 21, 2020

Phenomics and genomics of finger millet: current status and future prospects.

Diverse gene pool, advanced plant phenomics and genomics methods enhanced genetic gain and understanding of important agronomic, adaptation and nutritional traits in finger millet. Finger millet (Eleusine coracana L. Gaertn) is an important minor millet for food and nutritional security in semi-arid regions of the world. The crop has wide adaptability and can be grown right from high hills in Himalayan region to coastal plains. It provides food grain as well as palatable straw for cattle, and is fairly climate resilient. The crop has large gene pool with distinct features of both Indian and African germplasm types. Interspecific hybridization between Indian and African germplasm has resulted in greater yield enhancement and disease resistance. The crop has shown numerous advantages over major cereals in terms of stress adaptation, nutritional quality and health benefits. It has indispensable repository of novel genes for the benefits of mankind. Although rapid strides have been made in allele mining in model crops and major cereals, the progress in finger millet genomics is lacking. Comparative genomics have paved the way for the marker-assisted selection, where resistance gene homologues of rice for blast and sequence variants for nutritional traits from other cereals have been invariably used. Transcriptomics studies have provided preliminary understanding of the nutritional variation, drought and salinity tolerance. However, the genetics of many important traits in finger millet is poorly understood and need systematic efforts from biologists across disciplines. Recently, deciphered finger millet genome will enable identification of candidate genes for agronomically and nutritionally important traits. Further, improvement in genome assembly and application of genomic selection as well as genome editing in near future will provide plethora of information and opportunity to understand the genetics of complex traits.

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April 21, 2020

Extended haplotype phasing of de novo genome assemblies with FALCON-Phase

Haplotype-resolved genome assemblies are important for understanding how combinations of variants impact phenotypes. These assemblies can be created in various ways, such as use of tissues that contain single-haplotype (haploid) genomes, or by co-sequencing of parental genomes, but these approaches can be impractical in many situations. We present FALCON-Phase, which integrates long-read sequencing data and ultra-long-range Hi-C chromatin interaction data of a diploid individual to create high-quality, phased diploid genome assemblies. The method was evaluated by application to three datasets, including human, cattle, and zebra finch, for which high-quality, fully haplotype resolved assemblies were available for benchmarking. Phasing algorithm accuracy was affected by heterozygosity of the individual sequenced, with higher accuracy for cattle and zebra finch (>97%) compared to human (82%). In addition, scaffolding with the same Hi-C chromatin contact data resulted in phased chromosome-scale scaffolds.

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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 mRNA, we identified 4114 and 5880 pepper genes forming natural antisense transcript (NAT) genes in-cis and in-trans, respectively. Most of these genes accumulate small RNAs in their overlapping regions. By analyzing these NAT gene expression patterns in our transcriptome data, we identified many NAT pairs responsive to a variety of biological processes in pepper. Pepper formate dehydrogenase 1 (FDH1), which is required for R-gene-mediated disease resistance, may be regulated by nat-siRNAs and participate in a positive feedback loop in salicylic acid biosynthesis during resistance responses. Several cis-NAT pairs and subgroups of trans-NAT genes were responsive to pepper pericarp and placenta development, which may play roles in capsanthin and capsaicin biosynthesis. Using a comparative genomics approach, the evolutionary mechanisms of cis-NATs were investigated, and we found that an increase in intergenic sequences accounted for the loss of most cis-NATs, while transposon insertion contributed to the formation of most new cis-NATs. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.

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April 21, 2020

Draft Genome Assembly and Annotation of Red Raspberry Rubus Idaeus

The red raspberry, Rubus idaeus, is widely distributed in all temperate regions of Europe, Asia, and North America and is a major commercial fruit valued for its taste, high antioxidant and vitamin content. However, Rubus breeding is a long and slow process hampered by limited genomic and molecular resources. Genomic resources such as a complete genome sequencing and transcriptome will be of exceptional value to improve research and breeding of this high value crop. Using a hybrid sequence assembly approach including data from both long and short sequence reads, we present the first assembly of the Rubus idaeus genome (Joan J. variety). The de novo assembled genome consists of 2,145 scaffolds with a genome completeness of 95.3% and an N50 score of 638 KB. Leveraging a linkage map, we anchored 80.1% of the genome onto seven chromosomes. Using over 1 billion paired-end RNAseq reads, we annotated 35,566 protein coding genes with a transcriptome completeness score of 97.2%. The Rubus idaeus genome provides an important new resource for researchers and breeders.

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April 21, 2020

The Ptr1 locus of Solanum lycopersicoides confers resistance to race 1 strains of Pseudomonas syringae pv. tomato and to Ralstonia pseudosolanacearum by recognizing the type III effectors AvrRpt2/RipBN.

Race 1 strains of Pseudomonas syringae pv. tomato, which cause bacterial speck disease of tomato, are becoming increasingly common and no simply-inherited genetic resistance to such strains is known. We discovered that a locus in Solanum lycopersicoides, termed Pseudomonas tomato race 1 (Ptr1), confers resistance to race 1 Pst strains by detecting the activity of type III effector AvrRpt2. In Arabidopsis, AvrRpt2 degrades the RIN4 protein thereby activating RPS2-mediated immunity. Using site-directed mutagenesis of AvrRpt2 we found that, like RPS2, activation of Ptr1 requires AvrRpt2 proteolytic activity. Ptr1 also detected the activity of AvrRpt2 homologs from diverse bacteria including one in Ralstonia pseudosolanacearum. The genome sequence of S. lycopersicoides revealed no RPS2 homolog in the Ptr1 region. Ptr1 could play an important role in controlling bacterial speck disease and its future cloning may shed light on an example of convergent evolution for recognition of a widespread type III effector.

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April 21, 2020

The radish genome database (RadishGD): an integrated information resource for radish genomics.

Radish (Raphanus sativus L.) is an important root vegetable crop in the family Brassicaceae, which provides diverse nutrients for human health and is closely related to the Brassica crop species. Recently, we sequenced and assembled the radish genome into nine chromosome pseudomolecules. In addition, we developed diverse genomic resources, including genetic maps, molecular markers, transcriptome, genome-wide methylation and variome data. In this study, we describe the radish genome database (RadishGD), including details of data sets that we generated and the web interface that allows access to these data. RadishGD comprises six major units that enable researchers and general users to search, browse and analyze the radish genomic data in an integrated manner. The Search unit provides gene structures and sequences for gene models through keyword or BLAST searches. The Genome browser displays graphic representations of gene models, mRNAs, repetitive sequences, genome-wide methylation and variomes among various genotypes. The Functional annotation unit offers gene ontology, plant ontology, pathway and gene family information for gene models. The Genetic map unit provides information about markers and their genetic locations using two types of genetic maps. The Expression unit presents transcriptional characteristics and methylation levels for each gene in 18 tissues. All sequence data incorporated into RadishGD can be downloaded from the Data resources unit. RadishGD will be continually updated to serve as a community resource for radish genomics and breeding research.

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April 21, 2020

BjuWRR1, a CC-NB-LRR gene identified in Brassica juncea, confers resistance to white rust caused by Albugo candida.

BjuWRR1, a CNL-type R gene, was identified from an east European gene pool line of Brassica juncea and validated for conferring resistance to white rust by genetic transformation. White rust caused by the oomycete pathogen Albugo candida is a significant disease of crucifer crops including Brassica juncea (mustard), a major oilseed crop of the Indian subcontinent. Earlier, a resistance-conferring locus named AcB1-A5.1 was mapped in an east European gene pool line of B. juncea-Donskaja-IV. This line was tested along with some other lines of B. juncea (AABB), B. rapa (AA) and B. nigra (BB) for resistance to six isolates of A. candida collected from different mustard growing regions of India. Donskaja-IV was found to be completely resistant to all the tested isolates. Sequencing of a BAC spanning the locus AcB1-A5.1 showed the presence of a single CC-NB-LRR protein encoding R gene. The genomic sequence of the putative R gene with its native promoter and terminator was used for the genetic transformation of a susceptible Indian gene pool line Varuna and was found to confer complete resistance to all the isolates. This is the first white rust resistance-conferring gene described from Brassica species and has been named BjuWRR1. Allelic variants of the gene in B. juncea germplasm and orthologues in the Brassicaceae genomes were studied to understand the evolutionary dynamics of the BjuWRR1 gene.

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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 significantly diverged, with rapid genome differentiation after hybridization and intensive local selective breeding. The first historical introgression of B. rapa dramatically broadened the allelic pool but decreased the deleterious variations of Asian rapeseed. The second historical introgression of the double-low traits of European rapeseed (canola) has reshaped Asian rapeseed into two groups (double-low and double-high), accompanied by an increase in genetic load in the double-low group. This study demonstrates distinctive genomic footprints and deleterious SNP (single nucleotide polymorphism) variants for local adaptation by recent intra- and interspecies introgression events and provides novel insights for understanding the rapid genome evolution of a young allopolyploid crop. © 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

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April 21, 2020

Optimized Cas9 expression systems for highly efficient Arabidopsis genome editing facilitate isolation of complex alleles in a single generation.

Genetic resources for the model plant Arabidopsis comprise mutant lines defective in almost any single gene in reference accession Columbia. However, gene redundancy and/or close linkage often render it extremely laborious or even impossible to isolate a desired line lacking a specific function or set of genes from segregating populations. Therefore, we here evaluated strategies and efficiencies for the inactivation of multiple genes by Cas9-based nucleases and multiplexing. In first attempts, we succeeded in isolating a mutant line carrying a 70 kb deletion, which occurred at a frequency of ~?1.6% in the T2 generation, through PCR-based screening of numerous individuals. However, we failed to isolate a line lacking Lhcb1 genes, which are present in five copies organized at two loci in the Arabidopsis genome. To improve efficiency of our Cas9-based nuclease system, regulatory sequences controlling Cas9 expression levels and timing were systematically compared. Indeed, use of DD45 and RPS5a promoters improved efficiency of our genome editing system by approximately 25-30-fold in comparison to the previous ubiquitin promoter. Using an optimized genome editing system with RPS5a promoter-driven Cas9, putatively quintuple mutant lines lacking detectable amounts of Lhcb1 protein represented approximately 30% of T1 transformants. These results show how improved genome editing systems facilitate the isolation of complex mutant alleles, previously considered impossible to generate, at high frequency even in a single (T1) generation.

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April 21, 2020

Complete Genome Sequence of Agrobacterium fabrum Strain 1D159.

This work reports the draft genome sequence of Agrobacterium fabrum strain 1D159 (also known as ATCC strain 27912). The assembled genome is composed of a 2,861,352-bp circular chromosome, a 2,058,040-bp linear chromosome, a 519,735-bp AT plasmid, and the 223,394-bp Ti virulence plasmid. The wild nondisarmed strain produces small gall-like structures in citrus.

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