Aspergillus flavus pangenome (AflaPan) uncovers novel aflatoxin and secondary metabolite associated gene clusters.

BACKGROUND: Aspergillus flavus is an important agricultural and food safety threat due to its production of carcinogenic aflatoxins. It has high level of genetic diversity that is adapted to various environments. Recently, we reported two reference genomes of A. flavus isolates, AF13 (MAT1-2 and highly aflatoxigenic isolate) and NRRL3357 (MAT1-1 and moderate aflatoxin producer). Where, an insertion of 310 kb in AF13 included an aflatoxin producing gene bZIP transcription factor, named atfC. Observations of significant genomic variants between these isolates of contrasting phenotypes prompted an investigation into variation among other agricultural isolates of A. flavus with the goal of discovering novel genes potentially associated with aflatoxin production regulation. Present study was designed with three main objectives: (1) collection of large number of A. flavus isolates from diverse sources including maize plants and field soils; (2) whole genome sequencing of collected isolates and development of a pangenome; and (3) pangenome-wide association study (Pan-GWAS) to identify novel secondary metabolite cluster genes. RESULTS: Pangenome analysis of 346 A. flavus isolates identified a total of 17,855 unique orthologous gene clusters, with mere 41% (7,315) core genes and 59% (10,540) accessory genes indicating accumulation of high genomic diversity during domestication. 5,994 orthologous gene clusters in accessory genome not annotated in either the A. flavus AF13 or NRRL3357 reference genomes. Pan-genome wide association analysis of the genomic variations identified 391 significant associated pan-genes associated with aflatoxin production. Interestingly, most of the significantly associated pan-genes (94%; 369 associations) belonged to accessory genome indicating that genome expansion has resulted in the incorporation of new genes associated with aflatoxin and other secondary metabolites. CONCLUSION: In summary, this study provides complete pangenome framework for the species of Aspergillus flavus along with associated genes for pathogen survival and aflatoxin production. The large accessory genome indicated large genome diversity in the species A. flavus, however AflaPan is a closed pangenome represents optimum diversity of species A. flavus. Most importantly, the newly identified aflatoxin producing gene clusters will be a new source for seeking aflatoxin mitigation strategies and needs new attention in research.

Transcriptome Profile Reveals Drought-Induced Genes Preferentially Expressed in Response to Water Deficit in Cultivated Peanut (Arachis hypogaea L.).

Cultivated peanut (Arachis hypogaea) is one of the most widely grown food legumes in the world, being valued for its high protein and unsaturated oil contents. Drought stress is one of the major constraints that limit peanut production. This study's objective was to identify the drought-responsive genes preferentially expressed under drought stress in different peanut genotypes. To accomplish this, four genotypes (drought tolerant: C76-16 and 587; drought susceptible: Tifrunner and 506) subjected to drought stress in a rainout shelter experiment were examined. Transcriptome sequencing analysis identified that all four genotypes shared a total of 2,457 differentially expressed genes (DEGs). A total of 139 enriched gene ontology terms consisting of 86 biological processes and 53 molecular functions, with defense response, reproductive process, and signaling pathways, were significantly enriched in the common DEGs. In addition, 3,576 DEGs were identified only in drought-tolerant lines in which a total of 74 gene ontology terms were identified, including 55 biological processes and 19 molecular functions, mainly related to protein modification process, pollination, and metabolic process. These terms were also found in shared genes in four genotypes, indicating that tolerant lines adjusted more related genes to respond to drought. Forty-three significantly enriched Kyoto Encyclopedia of Genes and Genomes pathways were also identified, and the most enriched pathways were those processes involved in metabolic pathways, biosynthesis of secondary metabolites, plant circadian rhythm, phenylpropanoid biosynthesis, and starch and sucrose metabolism. This research expands our current understanding of the mechanisms that facilitate peanut drought tolerance and shed light on breeding advanced peanut lines to combat drought stress.

Draft Genome Sequences of One Aspergillus parasiticus Isolate and Nine Aspergillus flavus Isolates with Varying Stress Tolerance and Aflatoxin Production.

Aspergillus flavus and Aspergillus parasiticus produce carcinogenic aflatoxins during crop infection, with extensive variations in production among isolates, ranging from atoxigenic to highly toxigenic. Here, we report draft genome sequences of one A. parasiticus isolate and nine A. flavus isolates from field environments for use in comparative, functional, and phylogenetic studies.

Two New Aspergillus flavus Reference Genomes Reveal a Large Insertion Potentially Contributing to Isolate Stress Tolerance and Aflatoxin Production.

Efforts in genome sequencing in the Aspergillus genus have led to the development of quality reference genomes for several important species including A. nidulans, A. fumigatus, and A. oryzae However, less progress has been made for A. flavus As part of the effort of the USDA-ARS Annual Aflatoxin Workshop Fungal Genome Project, the isolate NRRL3357 was sequenced and resulted in a scaffold-level genome released in 2005. Our goal has been biologically driven, focusing on two areas: isolate variation in aflatoxin production and drought stress exacerbating aflatoxin production by A. flavus Therefore, we developed two reference pseudomolecule genome assemblies derived from chromosome arms for two isolates: AF13, a MAT1-2, highly stress tolerant, and highly aflatoxigenic isolate; and NRRL3357, a MAT1-1, less stress tolerant, and moderate aflatoxin producer in comparison to AF13. Here, we report these two reference-grade assemblies for these isolates through a combination of PacBio long-read sequencing and optical mapping, and coupled them with comparative, functional, and phylogenetic analyses. This analysis resulted in the identification of 153 and 45 unique genes in AF13 and NRRL3357, respectively. We also confirmed the presence of a unique 310 Kb insertion in AF13 containing 60 genes. Analysis of this insertion revealed the presence of a bZIP transcription factor, named atfC, which may contribute to isolate pathogenicity and stress tolerance. Phylogenomic analyses comparing these and other available assemblies also suggest that the species complex of A. flavus is polyphyletic.

Machine Learning as an Effective Method for Identifying True Single Nucleotide Polymorphisms in Polyploid Plants.

Single nucleotide polymorphisms (SNPs) have many advantages as molecular markers since they are ubiquitous and codominant. However, the discovery of true SNPs in polyploid species is difficult. Peanut ( L.) is an allopolyploid, which has a very low rate of true SNP calling. A large set of true and false SNPs identified from the Axiom_ 58k array was leveraged to train machine-learning models to enable identification of true SNPs directly from sequence data to reduce ascertainment bias. These models achieved accuracy rates above 80% using real peanut RNA sequencing (RNA-seq) and whole-genome shotgun (WGS) resequencing data, which is higher than previously reported for polyploids and at least a twofold improvement for peanut. A 48K SNP array, Axiom_2, was designed using this approach resulting in 75% accuracy of calling SNPs from different tetraploid peanut genotypes. Using the method to simulate SNP variation in several polyploids, models achieved >98% accuracy in selecting true SNPs. Additionally, models built with simulated genotypes were able to select true SNPs at >80% accuracy using real peanut data. This work accomplished the objective to create an effective approach for calling highly reliable SNPs from polyploids using machine learning. A novel tool was developed for predicting true SNPs from sequence data, designated as SNP machine learning (SNP-ML), using the described models. The SNP-ML additionally provides functionality to train new models not included in this study for customized use, designated SNP machine learner (SNP-MLer). The SNP-ML is publicly available.

A Comparison of sun, ovate, fs8.1 and Auxin Application on Tomato Fruit Shape and Gene Expression.

Elongated tomato fruit shape is the result of the action of the fruit shape genes possibly in coordination with the phytohormone auxin. To investigate the possible link between auxin and the fruit shape genes, a series of auxin (2,4-D) treatments were performed on the wild-type and the fruit shape near-isogenic lines (NILs) in Solanum pimpinellifolium accession LA1589 background. Morphological and histological analyses indicated that auxin application approximately 3 weeks before anthesis led to elongated pear-shaped ovaries and fruits, which was mainly attributed to the increase of ovary/fruit proximal end caused by the increase of both cell number and cell size. Fruit shape changes caused by SUN, OVATE and fs8.1 were primarily due to the alterations of cell number along different growth axes. Particularly, SUN caused elongation by extending cell number along the entire proximal-distal axis, whereas OVATE caused fruit elongation in the proximal area, which was most similar to the effect of auxin on ovary shape. Expression analysis of flower buds at different stages in fruit shape NILs indicated that SUN had a stronger impact on the transcriptome than OVATE and fs8.1. The sun NIL differentially expressed genes were enriched in several biological processes, such as lipid metabolism, ion transmembrane and actin cytoskeleton organization. Additionally, SUN also shifted the expression of the auxin-related genes, including those involved in auxin biosynthesis, homeostasis, signal transduction and polar transport, indicating that SUN may regulate ovary/fruit shape through modifying the expression of auxin-related genes very early during the formation of the ovary in the developing flower.

A SNP-Based Linkage Map Revealed QTLs for Resistance to Early and Late Leaf Spot Diseases in Peanut (Arachis hypogaea L.).

Cultivated peanut (Arachis hypogaea L.) is an important oilseed crop that is grown extensively in Africa, Asia and America. The diseases early and late leaf spot severely constrains peanut production worldwide. Because multiple genes control resistance to leaf spot diseases, conventional breeding is a time-consuming approach for pyramiding resistance genes into a single genotype. Marker-assisted selection (MAS) would complement and accelerate conventional breeding once molecular markers tightly associated with the resistance genes are identified. In this study, we have generated a large number of SNPs through genotyping by sequencing (GBS) and constructed a high-resolution map with an average distance of 1.34 cM among 2,753 SNP markers distributed on 20 linkage groups. QTL mapping has revealed that major QTL within a confidence interval could provide an efficient way to detect putative resistance genes. Analysis of the interval sequences has indicated that a major QTL for resistance to late leaf spot anchored by two NBS-LRR resistance genes on chromosome B05. Two major QTLs located on chromosomes A03 and B04 were associated with resistance genes for early leaf spot. Sequences within the confidence interval would facilitate identifying resistance genes and applying marker-assisted selection for resistance.

Haplotype-Based Genotyping in Polyploids.

Accurate identification of polymorphisms from sequence data is crucial to unlocking the potential of high throughput sequencing for genomics. Single nucleotide polymorphisms (SNPs) are difficult to accurately identify in polyploid crops due to the duplicative nature of polyploid genomes leading to low confidence in the true alignment of short reads. Implementing a haplotype-based method in contrasting subgenome-specific sequences leads to higher accuracy of SNP identification in polyploids. To test this method, a large-scale 48K SNP array (Axiom Arachis2) was developed for Arachis hypogaea (peanut), an allotetraploid, in which 1,674 haplotype-based SNPs were included. Results of the array show that 74% of the haplotype-based SNP markers could be validated, which is considerably higher than previous methods used for peanut. The haplotype method has been implemented in a standalone program, HAPLOSWEEP, which takes as input bam files and a vcf file and identifies haplotype-based markers. Haplotype discovery can be made within single reads or span paired reads, and can leverage long read technology by targeting any length of haplotype. Haplotype-based genotyping is applicable in all allopolyploid genomes and provides confidence in marker identification and in silico-based genotyping for polyploid genomics.

The control of tomato fruit elongation orchestrated by sun, ovate and fs8.1 in a wild relative of tomato.

Within the cultivated tomato germplasm, sun, ovate and fs8.1 are the three predominant QTLs controlling fruit elongation. Although SUN and OVATE have been cloned, their role in plant growth and development are not well understood. To compare and contrast the effects of the three QTLs in a homogeneous background, we developed near isogenic lines (NILs) in the wild species Solanum pimpinellifolium LA1589 background. We carried out detailed morphological characterization of reproductive and vegetative organs in the single, double and triple NILs and determined the epistatic interactions of the three loci affecting fruit shape. The phenotypic evaluations demonstrated that the three loci regulate unique aspects of ovary and fruit elongation and in different temporal manners. The strongest effect on organ shape was caused by sun. In addition to fruit shape, sun also affected leaf and sepal elongation and stem thickness. The synergistic interaction between sun and ovate or fs8.1 suggested that the pathways involving SUN, OVATE and the gene(s) underlying fs8.1 may converge at a common node. The results of an extensive profiling analysis suggested that the degree of fruit elongation was not related to the accumulation of any of the classical hormones.

SWEEP: A Tool for Filtering High-Quality SNPs in Polyploid Crops.

High-throughput next-generation sequence-based genotyping and single nucleotide polymorphism (SNP) detection opens the door for emerging genomics-based breeding strategies such as genome-wide association analysis and genomic selection. In polyploids, SNP detection is confounded by a highly similar homeologous sequence where a polymorphism between subgenomes must be differentiated from a SNP. We have developed and implemented a novel tool called SWEEP: Sliding Window Extraction of Explicit Polymorphisms. SWEEP uses subgenome polymorphism haplotypes as contrast to identify true SNPs between genotypes. The tool is a single command script that calls a series of modules based on user-defined options and takes sorted/indexed bam files or vcf files as input. Filtering options are highly flexible and include filtering based on sequence depth, alternate allele ratio, and SNP quality on top of the SWEEP filtering procedure. Using real and simulated data we show that SWEEP outperforms current SNP filtering methods for polyploids. SWEEP can be used for high-quality SNP discovery in polyploid crops.

Candidate gene selection and detailed morphological evaluations of fs8.1, a quantitative trait locus controlling tomato fruit shape.

fs8.1 is a major quantitative trait locus (QTL) that controls the elongated shape of tomato (Solanum lycopersicum) fruit. In this study, we fine-mapped the locus from a 47Mb to a 3.03Mb interval on the long arm of chromosome 8. Of the 122 annotated genes found in the fs8.1 region, 51 were expressed during floral development and six were differentially expressed in anthesis-stage ovaries in fs8.1 and wild-type (WT) lines. To identify possible nucleotide polymorphisms that may underlie the fruit shape phenotype, genome sequence analyses between tomato cultivars carrying the mutant and WT allele were conducted. This led to the identification of 158 single-nucleotide polymorphisms (SNPs) and five small indels in the fs8.1 interval, including 31 that could be associated with changes in gene expression or function. Morphological and histological analyses showed that the effects of fs8.1 were mainly on reproductive organ elongation by increasing cell number in the proximal-distal direction. Fruit weight was also increased in fs8.1 compared with WT, which was predominantly attributed to the increased fruit length. By combining the findings from the different analyses, we consider 12 likely candidate genes to underlie fs8.1, including Solyc08g062580 encoding a pentatricopeptide repeat protein, Solyc08g061560 encoding a putative orthologue of ERECTA, which is known to control fruit morphology and inflorescence architecture in Arabidopsis, Solyc08g061910 encoding a GTL2-like trihelix transcription factor, Solyc08g061930 encoding a protein that regulates cytokinin degradation, and two genes, Solyc08g062340 and Solyc08g062450, encoding 17.6kDa class II small heat-shock proteins.

Network Analyses Reveal Shifts in Transcript Profiles and Metabolites That Accompany the Expression of SUN and an Elongated Tomato Fruit.

SUN controls elongated tomato (Solanum lycopersicum) shape early in fruit development through changes in cell number along the different axes of growth. The gene encodes a member of the IQ domain family characterized by a calmodulin binding motif. To gain insights into the role of SUN in regulating organ shape, we characterized genome-wide transcriptional changes and metabolite and hormone accumulation after pollination and fertilization in wild-type and SUN fruit tissues. Pericarp, seed/placenta, and columella tissues were collected at 4, 7, and 10 d post anthesis. Pairwise comparisons between SUN and the wild type identified 3,154 significant differentially expressed genes that cluster in distinct gene regulatory networks. Gene regulatory networks that were enriched for cell division, calcium/transport, lipid/hormone, cell wall, secondary metabolism, and patterning processes contributed to profound shifts in gene expression in the different fruit tissues as a consequence of high expression of SUN. Promoter motif searches identified putative cis-elements recognized by known transcription factors and motifs related to mitotic-specific activator sequences. Hormone levels did not change dramatically, but some metabolite levels were significantly altered, namely participants in glycolysis and the tricarboxylic acid cycle. Also, hormone and primary metabolite networks shifted in SUN compared with wild-type fruit. Our findings imply that SUN indirectly leads to changes in gene expression, most strongly those involved in cell division, cell wall, and patterning-related processes. When evaluating global coregulation in SUN fruit, the main node represented genes involved in calcium-regulated processes, suggesting that SUN and its calmodulin binding domain impact fruit shape through calcium signaling.

What lies beyond the eye: the molecular mechanisms regulating tomato fruit weight and shape.

Domestication of fruit and vegetables resulted in a huge diversity of shapes and sizes of the produce. Selections that took place over thousands of years of alleles that increased fruit weight and altered shape for specific culinary uses provide a wealth of resources to study the molecular bases of this diversity. Tomato (Solanum lycopersicum) evolved from a wild ancestor (S. pimpinellifolium) bearing small and round edible fruit. Molecular genetic studies led to the identification of two genes selected for fruit weight: FW2.2 encoding a member of the Cell Number Regulator family; and FW3.2 encoding a P450 enzyme and the ortholog of KLUH. Four genes were identified that were selected for fruit shape: SUN encoding a member of the IQD family of calmodulin-binding proteins leading to fruit elongation; OVATE encoding a member of the OVATE family proteins involved in transcriptional repression leading to fruit elongation; LC encoding most likely the ortholog of WUSCHEL controlling meristem size and locule number; FAS encoding a member in the YABBY family controlling locule number leading to flat or oxheart shape. For this article, we will provide an overview of the putative function of the known genes, when during floral and fruit development they are hypothesized to act and their potential importance in regulating morphological diversity in other fruit and vegetable crops.