A simple and efficient method to extract polar metabolites from guar leaves (Cyamopsis tetragonoloba (L.) Taub.) for GC-MS metabolome analysis

Guar (Cyamopsis tetragonoloba (L.) Taub.) is an agricultural crop species new to Russia and is in demand by the gas, oil and food industries. Due to the progress of “omics” technologies and the marker-assisted selection, there is a huge interest in the studies that compare the metabolites of various guar varieties, employing metabolomics as a method of functional genomics. For a large-scale screening of guar germplasm from the VIR collection, it is important to choose an efficient method to extract metabolites from samples. The accuracy of the assessment of the content of metabolites in samples is crucial for distinguishing varieties within the crop, since the metabolome profiles of plants within the same species differ mainly in the quantitative ratio of metabolites, and not in their qualitative composition. In metabolome practice, two methods of extracting polar compounds are usually employed in the preparation of samples for GC-MS analysis. One of the widely used methods of sample preparation is the long-term extraction of metabolites from whole tissues with the aid of a methanol solvent. Another method of sample preparation is based on the short-term methanol extraction of metabolites from frozen and homogenized material. The advantages and disadvantages of these two methods revealed in the course of our work have prompted us to develop a new approach that avoids some difficulties in analyzing the metabolic profiles of leaves of various guar varieties. The method we suggested combines the advantages of the two above-mentioned approaches of sample preparation, namely eliminates the loss of metabolites due to centrifugation and ensures the complete destruction of all cell walls, ensuring the maximum extraction level of polar metabolites. The essence of the new method is that the leaf is rapidly frozen in liquid nitrogen with subsequent thawing in cold methanol. Thus, leaf tissues retain morphological integrity, and subsequent centrifugation, necessary for homogenization, is skipped. We have checked the effectiveness of this improved method by experiments with leaf samples of three guar genotypes. It has been shown that the amount of extracted metabolites increases more than 5-fold compared to extraction with methanol from fresh unfrozen leaf tissues and more than 2-fold compared to extraction with methanol after freezing and homogenization. Extraction of metabolites using the new method allows the GC-MS analysis of guar samples to be conducted with the least loss and high accuracy required to distinguish varieties

Genome assembly of Vitis rotundifolia Michx. using third-generation sequencing (Oxford Nanopore Technologies)

The immune North American grapevine species Vitis rotundifolia Michaux (subgen. Muscadinia Planch.) is regarded as a potential donor of disease resistance genes, withstanding such dangerous diseases of grapes as powdery and downy mildews. The cultivar ‘Dixie’ is the only representative of this species preserved ex situ in Russia: it is maintained by the N.I. Vavilov All-Russian Institute of Plant Genetic Resources (VIR) in the orchards of its branch, Krymsk Experiment Breeding Station. Third-generation sequencing on the MinION platform was performed to obtain information on the primary structure of the cultivar’s genomic DNA, employing also the results of Illumina sequencing available in databases. A detailed description of the technique with modifications at various stages is presented, as it was used for grapevine genome sequencing and whole-genome sequence assembly. The modified technique included the main stages of the original protocol recommended by the MinION producer: 1) DNA extraction; 2) preparation of libraries for sequencing; 3) MinION sequencing and bioinformatic data processing; 4) de novo whole-genome sequence assembly using only MinION data or hybrid assembly (MinION+Illumina data); and 5) functional annotation of the whole-genome assembly. Stage 4 included not only de novo sequencing, but also the analysis of the available bioinformatic data, thus minimizing errors and increasing precision during the assembly of the studied genome. The DNA isolated from the leaves of cv. ‘Dixie’ was sequenced using two MinION flow cells (R9.4.1)

Impact of growing conditions on the gum properties of different genotypes of guar (Cyamopsis tetragonoloba (L.) Taub.)

Galactomannan (gum), a water-soluble polysaccharide, is widely used as a gelling agent in liquids, including in the oil and gas industry for hydraulic fracturing. The most effective source of this valuable plant material is seeds of guar (Cyamopsis tetragonoloba (L.) Taub.), a legume crop new for Russia. Although in recent years progress has been made in the selection of guar varieties adapted to the conditions of the Russian Federation, the question of the most appropriate region for the cultivation of this crop remains open. The purpose of the study was to investigate how a region and technology of guar cultivation can affect the main indicators of the final target product: the content and viscosity of guar gum extracted from the seeds of various guar genotypes. To understand this, ecogeographical tests of 13 guar accessions from the VIR collection were conducted at the experimental stations of the Vavilov Institute (VIR), where climatic conditions correspond to the temperature requirements of the crop. To compare the properties of gum extracted from the seeds of various genotypes, a fast-tracked laboratory method was suggested allowing gum extracts to be obtained for assessing their viscosity. The method allows fast screening of the breeding material and selecting guar genotypes with beneficial properties of guar gum which are in demand by the oil industry. Applying the fast laboratory method for assessing the properties of gum in seeds of 13 guar varieties showed that the content and viscosity of gum of the same variety vary greatly depending on growing conditions. The same set of 13 guar accessions was grown in 2018 at the Volgograd, Astrakhan, Dagestan and Kuban VIR experimental stations. As a result, the maximum viscosity values were obtained for the seeds reproduced at the Astrakhan region, where the guar was grown on irrigated lands. On the other hand, the maximum gum content in the seeds of all accessions was recorded when they were grown in the Volgograd region. The results showed that the guar gum extracted from seeds of guar plants grown in the Russian Federation can be used as a gelling agent in the processes of intensification of oil production by the method of hydraulic fracturing. This experience is new to the Russian Federation

Introgressions of Vitis rotundifolia Michx. to obtain grapevine genotypes with complex resistance to biotic and abiotic stresses

Vitis rotundifolia Michx. is one of the species of the family Vitaceae, with resistance to both biotic and abiotic stresses. The present study reports new scientific knowledge about the inheritance of resistance to downy mildew, powdery mildew and frost by V. vinifera varieties from V. rotundifolia. Recombinant lines of three hybrid populations from the crossing of the maternal genotype ♀M. 31-77-10 with V. rotundifolia hybrids were used as the object of the study. As a result of laboratory screening, more than 40 % of recombinants of the ♀M. 31-77-10× ×[DRX-M5-734+DRX-M5-753+DRX-M5-790] population showed a high degree of frost resistance (–24 °C), while 6 % of transgressive recombinants were characterized by a very high degree of resistance (–27 °С). The maternal genotype ♀M. 31-77-10 does not carry alleles of resistance to powdery mildew at the Run1 locus and in the field suffers from powdery mildew much more than the paternal genotypes. The prevalence of powdery mildew on vegetative organs in the three recombinant populations over the years varies on average between 3.2–17.1, 0.3–17.7 and 0.6–5.2 %, respectively. As a result, almost all recombinant genotypes that received a resistant allele from the paternal genome are highly resistant to powdery mildew

Alteration of the embryo transcriptome of hexaploid winter wheat (Triticum aestivum cv. Mercia) during maturation and germination

Grain dormancy and germination are areas of biology that are of considerable interest to the cereal community. We have used a 9,155-feature wheat unigene cDNA microarray resource to investigate changes in the wheat embryo transcriptome during late grain development and maturation and during the first 48 h of postimbibition germination. In the embryo 392 mRNAs accumulated by twofold or greater over the time course from 21 days postanthesis (dpa) to 40 dpa and on through 1 and 2 days postgermination. These included mRNAs encoding proteins involved in amino acid biosynthesis and metabolism, cell division and subsequent cell development, signal transduction, lipid metabolism, energy production, protein turnover, respiration, initiation of transcription, initiation of translation and ribosomal composition. A number of mRNAs encoding proteins of unknown function also accumulated over the time course. Conversely 163 sequences showed decreases of twofold or greater over the time course. A small number of mRNAs also showed rapid accumulation specifically during the first 48 h of germination. We also examined alterations in the accumulation of transcripts encoding proteins involved in abscisic acid signalling. Thus, we describe changes in the level of transcripts encoding wheat Viviparous 1 (Vp1) and other interacting proteins. Interestingly, the transcript encoding wheat Viviparous-interacting protein 1 showed a pattern of accumulation that correlates inversely with germination. Our data suggests that the majority of the transcripts required for germination accumulate in the embryo prior to germination and we discuss the implications of these findings with regard to manipulation of germination in wheat

Differential gene expression in nearly isogenic lines with QTL for partial resistance to Puccinia hordei in barley

<p>Abstract</p> <p>Background</p> <p>The barley-<it>Puccinia hordei </it>(barley leaf rust) pathosystem is a model for investigating partial disease resistance in crop plants and genetic mapping of phenotypic resistance has identified several quantitative trait loci (QTL) for partial resistance. Reciprocal QTL-specific near-isogenic lines (QTL-NILs) have been developed that combine two QTL, <it>Rphq</it>2 and <it>Rphq</it>3, the largest effects detected in a recombinant-inbred-line (RIL) population derived from a cross between the super-susceptible line L94 and partially-resistant line Vada. The molecular mechanism underpinning partial resistance in these QTL-NILs is unknown.</p> <p>Results</p> <p>An Agilent custom microarray consisting of 15,000 probes derived from barley consensus EST sequences was used to investigate genome-wide and QTL-specific differential expression of genes 18 hours post-inoculation (hpi) with <it>Puccinia hordei</it>. A total of 1,410 genes were identified as being significantly differentially expressed across the genome, of which 55 were accounted for by the genetic differences defined by QTL-NILs at <it>Rphq</it>2 and <it>Rphq</it>3. These genes were predominantly located at the QTL regions and are, therefore, positional candidates. One gene, encoding the transcriptional repressor Ethylene-Responsive Element Binding Factor 4 (<it>HvERF4</it>) was located outside the QTL at 71 cM on chromosome 1H, within a previously detected eQTL hotspot for defence response. The results indicate that <it>Rphq</it>2 or <it>Rphq</it>3 contains a <it>trans</it>-eQTL that modulates expression of <it>HvERF4</it>. We speculate that HvERF4 functions as an intermediate that conveys the response signal from a gene(s) contained within <it>Rphq</it>2 or <it>Rphq</it>3 to a host of down-stream defense responsive genes. Our results also reveal that barley lines with extreme or intermediate partial resistance phenotypes exhibit a profound similarity in their spectrum of <it>Ph</it>-responsive genes and that hormone-related signalling pathways are actively involved in response to <it>Puccinia hordei</it>.</p> <p>Conclusions</p> <p>Differential gene expression between QTL-NILs identifies genes predominantly located within the target region(s) providing both transcriptional and positional candidate genes for the QTL. Genetically mapping the differentially expressed genes relative to the QTL has the potential to discover <it>trans</it>-eQTL mediated regulatory relays initiated from genes within the QTL regions.</p

Quantitative and Qualitative Stem Rust Resistance Factors in Barley Are Associated with Transcriptional Suppression of Defense Regulons

Stem rust (Puccinia graminis f. sp. tritici; Pgt) is a devastating fungal disease of wheat and barley. Pgt race TTKSK (isolate Ug99) is a serious threat to these Triticeae grain crops because resistance is rare. In barley, the complex Rpg-TTKSK locus on chromosome 5H is presently the only known source of qualitative resistance to this aggressive Pgt race. Segregation for resistance observed on seedlings of the Q21861 × SM89010 (QSM) doubled-haploid (DH) population was found to be predominantly qualitative, with little of the remaining variance explained by loci other than Rpg-TTKSK. In contrast, analysis of adult QSM DH plants infected by field inoculum of Pgt race TTKSK in Njoro, Kenya, revealed several additional quantitative trait loci that contribute to resistance. To molecularly characterize these loci, Barley1 GeneChips were used to measure the expression of 22,792 genes in the QSM population after inoculation with Pgt race TTKSK or mock-inoculation. Comparison of expression Quantitative Trait Loci (eQTL) between treatments revealed an inoculation-dependent expression polymorphism implicating Actin depolymerizing factor3 (within the Rpg-TTKSK locus) as a candidate susceptibility gene. In parallel, we identified a chromosome 2H trans-eQTL hotspot that co-segregates with an enhancer of Rpg-TTKSK-mediated, adult plant resistance discovered through the Njoro field trials. Our genome-wide eQTL studies demonstrate that transcript accumulation of 25% of barley genes is altered following challenge by Pgt race TTKSK, but that few of these genes are regulated by the qualitative Rpg-TTKSK on chromosome 5H. It is instead the chromosome 2H trans-eQTL hotspot that orchestrates the largest inoculation-specific responses, where enhanced resistance is associated with transcriptional suppression of hundreds of genes scattered throughout the genome. Hence, the present study associates the early suppression of genes expressed in this host–pathogen interaction with enhancement of R-gene mediated resistance

Complex nature of SNP genotype effects on gene expression in primary human leucocytes

<p>Abstract</p> <p>Background</p> <p>Genome wide association studies have been hugely successful in identifying disease risk variants, yet most variants do not lead to coding changes and how variants influence biological function is usually unknown.</p> <p>Methods</p> <p>We correlated gene expression and genetic variation in untouched primary leucocytes (n = 110) from individuals with celiac disease – a common condition with multiple risk variants identified. We compared our observations with an EBV-transformed HapMap B cell line dataset (n = 90), and performed a meta-analysis to increase power to detect non-tissue specific effects.</p> <p>Results</p> <p>In celiac peripheral blood, 2,315 SNP variants influenced gene expression at 765 different transcripts (< 250 kb from SNP, at FDR = 0.05, <it>cis </it>expression quantitative trait loci, eQTLs). 135 of the detected SNP-probe effects (reflecting 51 unique probes) were also detected in a HapMap B cell line published dataset, all with effects in the same allelic direction. Overall gene expression differences within the two datasets predominantly explain the limited overlap in observed <it>cis</it>-eQTLs. Celiac associated risk variants from two regions, containing genes <it>IL18RAP </it>and <it>CCR3</it>, showed significant <it>cis </it>genotype-expression correlations in the peripheral blood but not in the B cell line datasets. We identified 14 genes where a SNP affected the expression of different probes within the same gene, but in opposite allelic directions. By incorporating genetic variation in co-expression analyses, functional relationships between genes can be more significantly detected.</p> <p>Conclusion</p> <p>In conclusion, the complex nature of genotypic effects in human populations makes the use of a relevant tissue, large datasets, and analysis of different exons essential to enable the identification of the function for many genetic risk variants in common diseases.</p

Gains in QTL Detection Using an Ultra-High Density SNP Map Based on Population Sequencing Relative to Traditional RFLP/SSR Markers

Huge efforts have been invested in the last two decades to dissect the genetic bases of complex traits including yields of many crop plants, through quantitative trait locus (QTL) analyses. However, almost all the studies were based on linkage maps constructed using low-throughput molecular markers, e.g. restriction fragment length polymorphisms (RFLPs) and simple sequence repeats (SSRs), thus are mostly of low density and not able to provide precise and complete information about the numbers and locations of the genes or QTLs controlling the traits. In this study, we constructed an ultra-high density genetic map based on high quality single nucleotide polymorphisms (SNPs) from low-coverage sequences of a recombinant inbred line (RIL) population of rice, generated using new sequencing technology. The quality of the map was assessed by validating the positions of several cloned genes including GS3 and GW5/qSW5, two major QTLs for grain length and grain width respectively, and OsC1, a qualitative trait locus for pigmentation. In all the cases the loci could be precisely resolved to the bins where the genes are located, indicating high quality and accuracy of the map. The SNP map was used to perform QTL analysis for yield and three yield-component traits, number of tillers per plant, number of grains per panicle and grain weight, using data from field trials conducted over years, in comparison to QTL mapping based on RFLPs/SSRs. The SNP map detected more QTLs especially for grain weight, with precise map locations, demonstrating advantages in detecting power and resolution relative to the RFLP/SSR map. Thus this study provided an example for ultra-high density map construction using sequencing technology. Moreover, the results obtained are helpful for understanding the genetic bases of the yield traits and for fine mapping and cloning of QTLs

An eQTL Analysis of Partial Resistance to Puccinia hordei in Barley

Background - Genetic resistance to barley leaf rust caused by Puccinia hordei involves both R genes and quantitative trait loci. The R genes provide higher but less durable resistance than the quantitative trait loci. Consequently, exploring quantitative or partial resistance has become a favorable alternative for controlling disease. Four quantitative trait loci for partial resistance to leaf rust have been identified in the doubled haploid Steptoe (St)/Morex (Mx) mapping population. Further investigations are required to study the molecular mechanisms underpinning partial resistance and ultimately identify the causal genes.Methodology/Principal Findings - We explored partial resistance to barley leaf rust using a genetical genomics approach. We recorded RNA transcript abundance corresponding to each probe on a 15K Agilent custom barley microarray in seedlings from St and Mx and 144 doubled haploid lines of the St/Mx population. A total of 1154 and 1037 genes were, respectively, identified as being P. hordei-responsive among the St and Mx and differentially expressed between P. hordei-infected St and Mx. Normalized ratios from 72 distant-pair hybridisations were used to map the genetic determinants of variation in transcript abundance by expression quantitative trait locus (eQTL) mapping generating 15685 eQTL from 9557 genes. Correlation analysis identified 128 genes that were correlated with resistance, of which 89 had eQTL co-locating with the phenotypic quantitative trait loci (pQTL). Transcript abundance in the parents and conservation of synteny with rice allowed us to prioritise six genes as candidates for Rphq11, the pQTL of largest effect, and highlight one, a phospholipid hydroperoxide glutathione peroxidase (HvPHGPx) for detailed analysis.Conclusions/Significance - The eQTL approach yielded information that led to the identification of strong candidate genes underlying pQTL for resistance to leaf rust in barley and on the general pathogen response pathway. The dataset will facilitate a systems appraisal of this host-pathogen interaction and, potentially, for other traits measured in this populatio