A cluster of putative resistance genes is associated with a dominant resistance to sunflower broomrape.

KEY MESSAGE: The HaOr5 resistance gene is located in a large genomic insertion containing putative resistance genes and provides resistance to O. cumana, preventing successful connection to the sunflower root vascular system. Orobanche cumana (sunflower broomrape) is a parasitic plant that is part of the Orobanchaceae family and specifically infests sunflower crops. This weed is an obligate parasitic plant that does not carry out photosynthetic activity or develop roots and is fully dependent on its host for its development. It produces thousands of dust-like seeds per plant. It possesses a high spreading ability and has been shown to quickly overcome resistance genes successively introduced by selection in cultivated sunflower varieties. The first part of its life cycle occurs underground. The connection to the sunflower vascular system is essential for parasitic plant survival and development. The HaOr5 gene provides resistance to sunflower broomrape race E by preventing the connection of O. cumana to the root vascular system. We mapped a single position of the HaOr5 gene by quantitative trait locus mapping using two segregating populations. The same location of the HaOr5 gene was identified by genome-wide association. Using a large population of thousands of F2 plants, we restricted the location of the HaOr5 gene to a genomic region of 193 kb. By sequencing the whole genome of the resistant line harboring the major resistance gene HaOr5, we identified a large insertion of a complex genomic region containing a cluster of putative resistance genes.

Direct Comparative Analysis of a Pharmacogenomics Panel with PacBio Hifi® Long-Read and Illumina Short-Read Sequencing.

BACKGROUND: Pharmacogenetics (PGx) aims to determine genetic signatures that can be used in clinical settings to individualize treatment for each patient, including anti-cancer drugs, anti-psychotics, and painkillers. Taken together, a better understanding of the impacts of genetic variants on the corresponding protein function or expression permits the prediction of the pharmacological response: responders, non-responders, and those with adverse drug reactions (ADRs). OBJECTIVE: This work provides a comparison between innovative long-read sequencing (LRS) and short-read sequencing (SRS) techniques. METHODS AND MATERIALS: The gene panel captured using PacBio HiFi® sequencing was tested on thirteen clinical samples on GENTYANE's platform. SRS, using a comprehensive pharmacogenetics panel, was performed in routine settings at the Civil Hospitals of Lyon. We focused on complex regions analysis, including copy number variations (CNVs), structural variants, repeated regions, and phasing-haplotyping for three key pharmacogenes: CYP2D6, UGT1A1, and NAT2. RESULTS: Variants and the corresponding expected star (*) alleles were reported. Although only 38.4% concordance was found for haplotype determination and 61.5% for diplotype, this did not affect the metabolism scoring. A better accuracy of LRS was obtained for the detection of the CYP2D6*5 haplotype in the presence of the duplicated wild-type CYP2D6*2 form. A total concordance was performed for UGT1A1 TA repeat detection. Direct phasing using the LRS approach allowed us to correct certain NAT2 profiles. CONCLUSIONS: Combining an optimized variant-calling pipeline and with direct phasing analysis, LRS is a robust technique for PGx analysis that can minimize the risk of mis-haplotyping.

Complete genome sequences of two Bacillus thuringiensis serovar kurstaki strains isolated from Lebanon and Tunisia, highly toxic against lepidopteran larvae.

Bacillus thuringiensis-based products are key in the biopesticides market. Bacillus thuringiensis kurstaki strains Lip and BLB1 were isolated from Lebanese and Tunisian soils, respectively. These strains are highly toxic against lepidopteran larvae, Ephestia kuehniella. Here, we report Lip and BLB1 complete genomes, including their plasmid and toxin contents.

The genomics of linkage drag in inbred lines of sunflower.

Crop wild relatives represent valuable sources of alleles for crop improvement, including adaptation to climate change and emerging diseases. However, introgressions from wild relatives might have deleterious effects on desirable traits, including yield, due to linkage drag. Here, we analyzed the genomic and phenotypic impacts of wild introgressions in inbred lines of cultivated sunflower to estimate the impacts of linkage drag. First, we generated reference sequences for seven cultivated and one wild sunflower genotype, as well as improved assemblies for two additional cultivars. Next, relying on previously generated sequences from wild donor species, we identified introgressions in the cultivated reference sequences, as well as the sequence and structural variants they contain. We then used a ridge-regression best linear unbiased prediction (BLUP) model to test the effects of the introgressions on phenotypic traits in the cultivated sunflower association mapping population. We found that introgression has introduced substantial sequence and structural variation into the cultivated sunflower gene pool, including >3,000 new genes. While introgressions reduced genetic load at protein-coding sequences, they mostly had negative impacts on yield and quality traits. Introgressions found at high frequency in the cultivated gene pool had larger effects than low-frequency introgressions, suggesting that the former likely were targeted by artificial selection. Also, introgressions from more distantly related species were more likely to be maladaptive than those from the wild progenitor of cultivated sunflower. Thus, breeding efforts should focus, as far as possible, on closely related and fully compatible wild relatives.

An ancient truncated duplication of the anti-Müllerian hormone receptor type 2 gene is a potential conserved master sex determinant in the Pangasiidae catfish family.

The evolution of sex determination (SD) in teleosts is amazingly dynamic, as reflected by the variety of different master sex-determining genes identified. Pangasiids are economically important catfishes in South Asian countries, but little is known about their SD system. Here, we generated novel genomic resources for 12 Pangasiids and characterized their SD system. Based on a Pangasianodon hypophthalmus chromosome-scale genome assembly, we identified an anti-Müllerian hormone receptor type Ⅱ gene (amhr2) duplication, which was further characterized as being sex-linked in males and expressed only in testes. These results point to a Y chromosome male-specific duplication (amhr2by) of the autosomal amhr2a. Sequence annotation revealed that the P. hypophthalmus Amhr2by is truncated in its N-terminal domain, lacking the cysteine-rich extracellular part of the receptor that is crucial for ligand binding, suggesting a potential route for its neofunctionalization. Reference-guided assembly of 11 additional Pangasiids, along with sex-linkage studies, revealed that this truncated amhr2by duplication is a male-specific conserved gene in Pangasiids. Reconstructions of the amhr2 phylogeny suggested that amhr2by arose from an ancient duplication/insertion event at the root of the Siluroidei radiation that is dated to ~100 million years ago. Together these results bring multiple lines of evidence supporting that amhr2by is an ancient and conserved master sex-determining gene in Pangasiids, a finding that highlights the recurrent use of the transforming growth factor ß pathway, which is often used for the recruitment of teleost master SD genes, and provides another empirical case towards firther understanding of dynamics of SD systems.

Cantaloupe melon genome reveals 3D chromatin features and structural relationship with the ancestral cucurbitaceae karyotype.

Cucumis melo displays a large diversity of horticultural groups with cantaloupe melon the most cultivated type. Using a combination of single-molecule sequencing, 10X Genomics link-reads, high-density optical and genetic maps, and chromosome conformation capture (Hi-C), we assembled a chromosome scale C. melo var. cantalupensis Charentais mono genome. Integration of RNA-seq, MeDip-seq, ChIP-seq, and Hi-C data revealed a widespread compartmentalization of the melon genome, segregating constitutive heterochromatin and euchromatin. Genome-wide comparative and evolutionary analysis between melon botanical groups identified Charentais mono genome increasingly more divergent from Harukei-3 (reticulatus), Payzawat (inodorus), and HS (ssp. agrestis) genomes. To assess the paleohistory of the Cucurbitaceae, we reconstructed the ancestral Cucurbitaceae karyotype and compared it to sequenced cucurbit genomes. In contrast to other species that experienced massive chromosome shuffling, melon has retained the ancestral genome structure. We provide comprehensive genomic resources and new insights in the diversity of melon horticultural groups and evolution of cucurbits.

Building a cluster of NLR genes conferring resistance to pests and pathogens: the story of the Vat gene cluster in cucurbits.

Most molecularly characterized plant resistance genes (R genes) belong to the nucleotide-binding-site-leucine-rich-repeat (NLR) receptor family and are prone to duplication and transposition with high sequence diversity. In this family, the Vat gene in melon is one of the few R genes known for conferring resistance to insect, i.e., Aphis gossypii, but it has been misassembled and/or mispredicted in the whole genomes of Cucurbits. We examined 14 genomic regions (about 400 kb) derived from long-read assemblies spanning Vat-related genes in Cucumis melo, Cucumis sativus, Citrullus lanatus, Benincasa hispida, Cucurbita argyrosperma, and Momordica charantia. We built the phylogeny of those genes. Investigating the paleohistory of the Vat gene cluster, we revealed a step by step process beginning from a common ancestry in cucurbits older than 50 my. We highlighted Vat exclusively in the Cucumis genera, which diverged about 20 my ago. We then focused on melon, evaluating a minimum duplication rate of Vat in 80 wild and cultivated melon lines using generalist primers; our results suggested that duplication started before melon domestication. The phylogeny of 44 Vat-CDS obtained from 21 melon lines revealed gain and loss of leucine-rich-repeat domains along diversification. Altogether, we revealed the high putative recognition scale offered in melon based on a combination of SNPs, number of leucine-rich-repeat domains within each homolog and number of homologs within each cluster that might jointly confer resistance to a large pest and pathogen spectrum. Based on our findings, we propose possible avenues for breeding programs.

Harnessing the power of next-generation sequencing technologies to the purpose of high-throughput pesticide resistance diagnosis.

BACKGROUND: Next Generation Sequencing (NGS) technologies offer tremendous possibilities for high-throughput pesticide resistance diagnosis via massive genotyping-by-sequencing. Herein, we used Illumina sequencing combined with a simple, non-commercial bioinformatics pipe-line to seek mutations involved in herbicide resistance in two weeds. RESULTS: DNA was extracted from 96 pools of 50 plants for each species. Three amplicons encompassing 15 ALS (acetolactate-synthase) codons crucial for herbicide resistance were amplified from each DNA extract. Above 18 and 20 million quality 250-nucleotide sequence reads were obtained for groundsel (Senecio vulgaris, tetraploid) and ragweed (Ambrosia artemisiifolia, diploid), respectively. Herbicide resistance-endowing mutations were identified in 45 groundsel and in eight ragweed field populations. The mutations detected and their frequencies assessed by NGS were checked by individual plant genotyping or Sanger sequencing. NGS results were fully confirmed, except in three instances out of 12 where mutations present at a frequency of 1% were detected below the threshold set for reliable mutation detection. CONCLUSION: Analyzing 9600 plants requested 192 DNA extractions followed by 1728 PCRs and two Illumina runs. Equivalent results obtained by individual analysis would have necessitated 9600 individual DNA extractions followed by 216 000 genotyping PCRs, or by 121 500 PCRs and 40 500 Sanger sequence runs. This clearly demonstrates the interest and power of NGS-based detection of pesticide resistance from pools of individuals for diagnosing resistance in massive numbers of individuals. © 2019 Society of Chemical Industry.

Discovery of High Abundances of Aster-Like Nanoparticles in Pelagic Environments: Characterization and Dynamics.

This study reports the discovery of Aster-Like Nanoparticles (ALNs) in pelagic environments. ALNs are pleomorphic, with three dominant morphotypes which do not fit into any previously defined environmental entities [i.e., ultramicro-prokaryotes, controversed nanobes, and non-living particles (biomimetic mineralo-organic particles, natural nanoparticles or viruses)] of similar size. Elemental composition and selected-area electron diffraction patterns suggested that the organic nature of ALNs may prevail over the possibility of crystal structures. Likewise, recorded changes in ALN numbers in the absence of cells are at odds with an affiliation to until now described viral particles. ALN abundances showed marked seasonal dynamics in the lakewater, with maximal values (up to 9.0 ± 0.5 × 107 particles·mL-1) reaching eight times those obtained for prokaryotes, and representing up to about 40% of the abundances of virus-like particles. We conclude that (i) aquatic ecosystems are reservoirs of novel, abundant, and dynamic aster-like nanoparticles, (ii) not all virus-like particles observed in aquatic systems are necessarily viruses, and (iii) there may be several types of other ultra-small particles in natural waters that are currently unknown but potentially ecologically important.

Allele-specific expression and genetic determinants of transcriptomic variations in response to mild water deficit in tomato.

Characterizing the natural diversity of gene expression across environments is an important step in understanding how genotype-by-environment interactions shape phenotypes. Here, we analyzed the impact of water deficit onto gene expression levels in tomato at the genome-wide scale. We sequenced the transcriptome of growing leaves and fruit pericarps at cell expansion stage in a cherry and a large fruited accession and their F1 hybrid grown under two watering regimes. Gene expression levels were steadily affected by the genotype and the watering regime. Whereas phenotypes showed mostly additive inheritance, ~80% of the genes displayed non-additive inheritance. By comparing allele-specific expression (ASE) in the F1 hybrid to the allelic expression in both parental lines, respectively, 3005 genes in leaf and 2857 genes in fruit deviated from 1:1 ratio independently of the watering regime. Among these genes, ~55% were controlled by cis factors, ~25% by trans factors and ~20% by a combination of both types of factors. A total of 328 genes in leaf and 113 in fruit exhibited significant ASE-by-watering regime interaction, among which ~80% presented trans-by-watering regime interaction, suggesting a response to water deficit mediated through a majority of trans-acting loci in tomato. We cross-validated the expression levels of 274 transcripts in fruit and leaves of 124 recombinant inbred lines (RILs) and identified 163 expression quantitative trait loci (eQTLs) mostly confirming the divergences identified by ASE. Combining phenotypic and expression data, we observed a complex network of variation between genes encoding enzymes involved in the sugar metabolism.

FRIZZY PANICLE drives supernumerary spikelets in bread wheat.

Bread wheat (Triticum aestivum) inflorescences, or spikes, are characteristically unbranched and normally bear one spikelet per rachis node. Wheat mutants on which supernumerary spikelets (SSs) develop are particularly useful resources for work towards understanding the genetic mechanisms underlying wheat inflorescence architecture and, ultimately, yield components. Here, we report the characterization of genetically unrelated mutants leading to the identification of the wheat FRIZZY PANICLE (FZP) gene, encoding a member of the APETALA2/Ethylene Response Factor transcription factor family, which drives the SS trait in bread wheat. Structural and functional characterization of the three wheat FZP homoeologous genes (WFZP) revealed that coding mutations of WFZP-D cause the SS phenotype, with the most severe effect when WFZP-D lesions are combined with a frameshift mutation in WFZP-A. We provide WFZP-based resources that may be useful for genetic manipulations with the aim of improving bread wheat yield by increasing grain number.

Using next-generation sequencing to detect mutations endowing resistance to pesticides: application to acetolactate-synthase (ALS)-based resistance in barnyard grass, a polyploid grass weed.

BACKGROUND: Next-generation sequencing (NGS) technologies offer tremendous possibilities for accurate detection of mutations endowing pesticide resistance, yet their use for this purpose has not emerged in crop protection. This study aims at promoting NGS use for pesticide resistance diagnosis. It describes a simple procedure accessible to virtually any scientist and implementing freely accessible programs for the analysis of NGS data. RESULTS: Three PCR amplicons encompassing seven codons of the acetolactate-synthase gene crucial for herbicide resistance were sequenced using non-quantified pools of crude DNA extracts from 40 plants in each of 28 field populations of barnyard grass, a polyploid weed. A total of 63,959 quality NGS sequence runs were obtained using the 454 technology. Three herbicide-resistance-endowing mutations (Pro-197-Ser, Pro-197-Leu and/or Trp-574-Leu) were identified in seven populations. The NGS results were confirmed by individual plant Sanger sequencing. CONCLUSION: This work demonstrated the feasibility of NGS-based detection of pesticide resistance, and the advantages of NGS compared with other molecular biology techniques for analysing large numbers of individuals. NGS-based resistance diagnosis has the potential to play a substantial role in monitoring resistance, maintaining pesticide efficacy and optimising pesticide applications.

Deciphering the genomic structure, function and evolution of carotenogenesis related phytoene synthases in grasses.

BACKGROUND: Carotenoids are isoprenoid pigments, essential for photosynthesis and photoprotection in plants. The enzyme phytoene synthase (PSY) plays an essential role in mediating condensation of two geranylgeranyl diphosphate molecules, the first committed step in carotenogenesis. PSY are nuclear enzymes encoded by a small gene family consisting of three paralogous genes (PSY1-3) that have been widely characterized in rice, maize and sorghum. RESULTS: In wheat, for which yellow pigment content is extremely important for flour colour, only PSY1 has been extensively studied because of its association with QTLs reported for yellow pigment whereas PSY2 has been partially characterized. Here, we report the isolation of bread wheat PSY3 genes from a Renan BAC library using Brachypodium as a model genome for the Triticeae to develop Conserved Orthologous Set markers prior to gene cloning and sequencing. Wheat PSY3 homoeologous genes were sequenced and annotated, unravelling their novel structure associated with intron-loss events and consequent exonic fusions. A wheat PSY3 promoter region was also investigated for the presence of cis-acting elements involved in the response to abscisic acid (ABA), since carotenoids also play an important role as precursors of signalling molecules devoted to plant development and biotic/abiotic stress responses. Expression of wheat PSYs in leaves and roots was investigated during ABA treatment to confirm the up-regulation of PSY3 during abiotic stress. CONCLUSIONS: We investigated the structural and functional determinisms of PSY genes in wheat. More generally, among eudicots and monocots, the PSY gene family was found to be associated with differences in gene copy numbers, allowing us to propose an evolutionary model for the entire PSY gene family in Grasses.

A detailed survey of seed coat flavonoids in developing seeds of Brassica napus L.

Proanthocyanidins (PAs) are seed coat flavonoids that impair the digestibility of Brassica napus meal. Development of low-PA lines is associated with a high-quality meal and with increased contents in oil and proteins, but requires better knowledge of seed flavonoids. Flavonoids in Brassica mature seed are mostly insoluble so that very few qualitative and quantitative data are available yet. In the present study, the profiling of seed coat flavonoids was established in eight black-seeded B. napus genotypes, during seed development when soluble flavonoids were present and predominated over the insoluble forms. Thirteen different flavonoids including (-)-epicatechin, five procyanidins (PCs which are PAs composed of epicatechin oligomers only) and seven flavonols (quercetin-3-O-glucoside, quercetin-dihexoside, isorhamnetin-3-O-glucoside, isorhamnetin-hexoside-sulfate, isorhamnetin-dihexoside, isorhamnetin-sinapoyl-trihexoside and kaempferol-sinapoyl-trihexoside) were identified and quantified using liquid chromatography coupled to electrospray ionization-mass spectrometry (LC-ESI-MS(n)). These flavonol derivatives were characterized for the first time in the seed coat of B. napus, and isorhamnetin-hexoside-sulfate and isorhamnetin-sinapoyl-trihexoside were newly identified in Brassica spp. High amounts of PCs accumulated in the seed coat, with solvent-soluble polymers of (-)-epicatechin reaching up to 10% of the seed coat weight during seed maturation. In addition, variability for both PC and flavonol contents was observed within the panel of eight black-seeded genotypes. Our results provide new insights into breeding for low-PC B. napus genotypes.

1-O-alkylglycerols improve boar sperm motility and fertility.

1-O-alkylglycerols are naturally occurring ether lipids with potent biological activities. They may interfere with lipidic signaling, and they amplify platelet-activating factor (PAF) biosynthesis in a monocyte cell line. The PAF is produced by mammalian sperm and is an important activator of sperm motility. The aim of this study was to evaluate the effect of in vitro treatment of boar spermatozoa with natural 1-O-alkylglycerols (10 microM) on 1) boar sperm motility; 2) production of PAF and its metabolite, lyso-PAF, by spermatozoa; and 3) fertility in artificial inseminations of breeding sows. Using a computer-assisted spermatozoa analyzer, we found that 1-O-alkylglycerols increased percentage motility as well as velocity parameters after 24 h. These effects were partially or totally reversed by the PAF receptor-antagonist SR 27417. After [3H]-1-O-alkylglycerol incubation with boar spermatozoa, we identified [3H]lyso-PAF by high-performance liquid chromatography. Production of PAF and lyso-PAF was measured with a biological assay using [3H]serotonin release from rabbit platelets. 1-O-alkylglycerols significantly increased lyso-PAF production but had no effect on PAF production. The effect of 1-O-alkylglycerols on fertilization was also evaluated in industrial breedings: 1-O-alkylglycerol-treated or untreated semen dilutions were alternately used for artificial inseminations of sows on 12 farms. 1-O-alkylglycerol treatment increased the number of farrows but had no effect on the mean size of the litters. This study demonstrates that 1-O-alkylglycerol treatment of boar spermatozoa in vitro improves their motility and fertility, and it suggests that this effect is related to PAF metabolism and function in boar spermatozoa.