Recombinant Lines Research Articles

Genetic Polymorphisms in SLCO2B1 and ABCC1 Conjointly Modulate Atorvastatin Intracellular Accumulation in HEK293 Recombinant Cell Lines.

Although atorvastatin (ATV) is well-tolerated, patients may report muscle complaints. These are difficult to predict owing to high interindividual variability. Such side effects are linked to intramuscular accumulation of ATV. This study aimed to investigate the relative role of transporters expressed in muscle tissue in promoting or limiting drug access to cells. The impact of common single nucleotide polymorphisms (SNPs) in SLCO2B1 coding for OATP2B1 and ABCC1 coding for MRP1 on ATV transport was also evaluated. HEK293 cells were stably transfected with plasmids containing cDNA encoding wild-type or variant SLCO2B1 and/or ABCC1 to generate single and double stable transfectant HEK293 recombinant models overexpressing variant or wild-type OATP2B1 (influx) and/or MRP1 (efflux) proteins. Variant plasmids were generated by site-directed mutagenesis. Expression analyses were performed to validate recombinant models. Accumulation and efflux experiments were performed at different concentrations. ATV was quantified by LC-MS/MS, and kinetic parameters were compared between single and double HEK transfectants expressing wild-type and variant proteins. The results confirm the involvement of OATP2B1 and MRP1 in ATV cellular transport because it was demonstrated that intracellular accumulation of ATV was boosted by OATP2B1 overexpression, whereas ATV accumulation was decreased by MRP1 overexpression. In double transfectants, it was observed that increased ATV intracellular accumulation driven by OATP2B1 influx was partially counteracted by MRP1 efflux. The c.935G > A SNP in SLCO2B1 was associated with decreased ATV OATP2B1-mediated influx, whereas the c.2012G > T SNP in ABCC1 seemed to increase MRP1 efflux activity against ATV. Intracellular ATV accumulation is regulated by OATP2B1 and MRP1 transporters, whose functionality is modulated by natural genetic variants. This is significant because it may play a role in ATV muscle side-effect susceptibility.

Protein Networks Associated with Native Metabotropic Glutamate 1 Receptors (mGlu1) in the Mouse Cerebellum.

The metabotropic glutamate receptor 1 (mGlu1) plays a pivotal role in synaptic transmission and neuronal plasticity. Despite the fact that several interacting proteins involved in the mGlu1 subcellular trafficking and intracellular transduction mechanisms have been identified, the protein network associated with this receptor in specific brain areas remains largely unknown. To identify novel mGlu1-associated protein complexes in the mouse cerebellum, we used an unbiased tissue-specific proteomic approach, namely co-immunoprecipitation followed by liquid chromatography/tandem mass spectrometry analysis. Many well-known protein complexes as well as novel interactors were identified, including G-proteins, Homer, δ2 glutamate receptor, 14-3-3 proteins, and Na/K-ATPases. A novel putative interactor, KCTD12, was further investigated. Reverse co-immunoprecipitation with anti-KCTD12 antibodies revealed mGlu1 in wild-type but not in KCTD12-knock-out homogenates. Freeze-fracture replica immunogold labeling co-localization experiments showed that KCTD12 and mGlu1 are present in the same nanodomain in Purkinje cell spines, although at a distance that suggests that this interaction is mediated through interposed proteins. Consistently, mGlu1 could not be co-immunoprecipitated with KCTD12 from a recombinant mammalian cell line co-expressing the two proteins. The possibility that this interaction was mediated via GABAB receptors was excluded by showing that mGlu1 and KCTD12 still co-immunoprecipitated from GABAB receptor knock-out tissue. In conclusion, this study identifies tissue-specific mGlu1-associated protein clusters including KCTD12 at Purkinje cell synapses.

Assessment of Multiple Tolerance Indices to Identify Rice Lines Suitable for the Aerobic System of Cultivation

Climate resilience is the most concentrated subject in the current scenario for rice improvement. The aerobic system of rice cultivation involving direct seeding with need-based irrigation in non-puddled soil is gaining ground with respect to a current scenario of water scarcity. The selection of lines suitable and stable under aerobic along with irrigated conditions without any yield penalty is one of the focus areas of the breeding programme for resource use efficiency. In the present study, we have screened a panel of 118 rice lines under aerobic i.e. limited water conditions and irrigated conditions at ARS Dhadesugur Karnataka to identify ideal selection indices viz. STI, TOL, SSI, YSI, YR, YI, PYR, MP and GMP for selecting the best high-yielding and stable lines under both rice cultivation methods. The deployment of selection indices here only pertains to finding the differences in yield per plant under aerobic and irrigated conditions. According to the results of multivariate analysis (correlation and PCA), STI, YI, MP and GMP exhibited a strong correlation with YP and YS. Therefore, they appear to be the most effective stress indices for the selection of lines with good yield potential under water-limited and irrigated conditions. These indices serve as valuable selection criteria for the identification of aerobic-tolerant cultivars from both water-limited and normal conditions. These indices identified lines, DB 5 (Swarna × Oryza nivara (IRGC 81848)) and NPK-40 (Swarna × Oryza nivara (IRGC81832)) wild introgression lines. GNV-14-96-1 (BPT-5204 × Nerica line) Advanced breeding line. JBB 631-1 ((Swarna*2/ IRGC 4105) (RP 5405-JBB-631-1-1-1-1-1-1)) Tropical japonica × indica introgression line. KR-209 (Wazuhophek × ISM) and KR-262 (Wazuhophek × ISM) recombinant introgression lines. TI-36 and TI-124 Ethyl Methane Sulfonate (EMS) mutants of BPT-5204. WB-10 (Langphou) and WB-16 (Phouoibi) North-Eastern Landraces were promising for both environments. These lines are suitable because of low grain yield loss under aerobic conditions and can be further considered for cultivation.

Long term culture promotes changes to growth, gene expression, and metabolism in CHO cells that are independent of production stability.

Phenotypic stability of Chinese hamster ovary (CHO) cells over long term culture (LTC) presents one of the most pressing challenges in the development of therapeutic protein manufacturing processess. However, our current understanding of the consequences of LTC on recombinant (r-) CHO cell lines is still limited, particularly as clonally-derived cell lines present distinct production stability phenotypes. This study evaluated changes of culture performance, global gene expression, and cell metabolism of two clonally-derived CHO cell lines with a stable or unstable phenotype during the LTC (early [EP] vs. late [LP] culture passages). Our findings indicated that LTC altered the behavior of CHO cells in culture, in terms of growth, overall gene expression, and cell metabolism. Regardless whether cells were categorized as stable or unstable in terms of r-protein production, CHO cells at LP presented an earlier decline in cell viability and loss of any observable stationary phase. These changes were parallelled by the upregulation of genes involved in cell proliferation and survival pathways (i.e., MAPK/ERK, PI3K-Akt). Stable and unstable CHO cell lines both showed increased consumption of glucose and amino acids at LP, with a parallel accumulation of greater amounts of lactate and TCA cycle intermediates. In terms of production stability, we found that decreased r-protein production in the unstable cell line directly correlated to the loss in r-gene copy number and r-mRNA expression. Our data revealed that LTC produced ubiquitious effects on CHO cell phenotypes, changes that were rooted in alterations in cell transcriptome and metabolome. Overall, we found that CHO cells adapted their cellular function to proliferation and survival during the LTC, some of these changes may well have limited effects on overall yield or specific productivity of the desired r-product, but they may be critical towardthe capacity of cells to handle r-proteins with specific molecular features.

Nanopore Cas9-targeted sequencing enables accurate and simultaneous identification of transgene integration sites, their structure and epigenetic status in recombinant Chinese hamster ovary cells.

The integration of a transgene expression construct into the host genome is the initial step for the generation of recombinant cell lines used for biopharmaceutical production. The stability and level of recombinant gene expression in Chinese hamster ovary (CHO) can be correlated to the copy number, its integration site as well as the epigenetic context of the transgene vector. Also, undesired integration events, such as concatemers, truncated, and inverted vector repeats, are impacting the stability of recombinant cell lines. Thus, to characterize cell clones and to isolate the most promising candidates, it is crucial to obtain information on the site of integration, the structure of integrated sequence and the epigenetic status. Current sequencing techniques allow to gather this information separately but do not offer a comprehensive and simultaneous resolution.In this study, we present a fast and robust nanopore Cas9-targeted sequencing (nCats) pipeline to identify integration sites, the composition of the integrated sequence as well as its DNA methylation status in CHO cells that can be obtained simultaneously from the same sequencing run. A Cas9-enrichment step during library preparation enables targeted and directional nanopore sequencing with up to 724× median on-target coverage and up to 153 kb long reads. The data generated by nCats provides sensitive, detailed, and correct information on the transgene integration sites and the expression vector structure, which could only be partly produced by traditional Targeted Locus Amplification-seq data. Moreover, with nCats the DNA methylation status can be analyzed from the same raw data without prior DNA amplification.

Fine mapping of KLW1 that conditions kernel weight mainly through regulating kernel length in wheat (Triticum aestivum L.).

KLW1 was localized to a 0.6 cM interval near the centromere of chromosome 4B and found to be dominant in conditioning longer kernels and higher kernel weight. Kernel weight is a major wheat yield component and affected by kernel dimensions, filling process and kernel density. Because of this complexity, the mechanism underlying kernel weight is still far from clear. Qtgw.nau-4B or KLW1 was a major kernel weight QTL identified in the Nanda2419 × Wangshuibai population. We showed that introduction of the Nanda2419 allele into elite cultivar Wenmai6 resulted in longer kernels as well as higher kernel weight, without affecting other traits such as spike number per plant, plant height, spike length, spikelet number per spike, and kernel number per spike. KLW1 was dominant in conditioning higher kernel weight and functioned mainly through affecting kernel length. Using F2 plants derived from KLW1 NIL, a high-density genetic map covering the QTL was constructed. KLW1 was consequently confined to the 0.6cM Xwgrc4219-Xwgrc4067 interval by evaluating the recombinant lines in three field trials. KLW1 is complementary to KT1, the QTL on chromosome 5A of Nanda2419 for thicker and heavier kernels, in producing larger kernels with higher commercial value, augmenting its usefulness in wheat breeding.

Variability of photosynthesis parameters and yield in recombinant lines of bread wheat with introgressions from Triticum timopheevii into 2A chromosome under different water supply conditions

Variability of photosynthesis parameters and yield in recombinant lines of bread wheat with introgressions from Triticum timopheevii into 2A chromosome under different water supply conditions

Untargeted Metabolomics Reveals a Multi-Faceted Resistance Response to Fusarium Head Blight Mediated by the Thinopyrum elongatum Fhb7E Locus Transferred via Chromosome Engineering into Wheat.

The Thinopyrum elongatum Fhb7E locus has been proven to confer outstanding resistance to Fusarium Head Blight (FHB) when transferred into wheat, minimizing yield loss and mycotoxin accumulation in grains. Despite their biological relevance and breeding implications, the molecular mechanisms underlying the resistant phenotype associated with Fhb7E have not been fully uncovered. To gain a broader understanding of processes involved in this complex plant-pathogen interaction, we analysed via untargeted metabolomics durum wheat (DW) rachises and grains upon spike inoculation with Fusarium graminearum (Fg) and water. The employment of DW near-isogenic recombinant lines carrying or lacking the Th. elongatum chromosome 7E region including Fhb7E on their 7AL arm, allowed clear-cut distinction between differentially accumulated disease-related metabolites. Besides confirming the rachis as key site of the main metabolic shift in plant response to FHB, and the upregulation of defence pathways (aromatic amino acid, phenylpropanoid, terpenoid) leading to antioxidants and lignin accumulation, novel insights were revealed. Fhb7E conferred constitutive and early-induced defence response, in which specific importance of polyamine biosynthesis, glutathione and vitamin B6 metabolisms, along with presence of multiple routes for deoxynivalenol detoxification, was highlighted. The results suggested Fhb7E to correspond to a compound locus, triggering a multi-faceted plant response to Fg, effectively limiting Fg growth and mycotoxin production.

Gustatory polymorphism mediates a new adaptive courtship strategy.

Human-imposed selection can lead to adaptive changes in sensory traits. However, rapid evolution of the sensory system can interfere with other behaviours, and animals must overcome such sensory conflicts. In response to intense selection by insecticide baits that contain glucose, German cockroaches evolved glucose-aversion (GA), which confers behavioural resistance against baits. During courtship the male offers the female a nuptial gift that contains maltose, which expediates copulation. However, the female's saliva rapidly hydrolyses maltose into glucose, which causes GA females to dismount the courting male, thus reducing their mating success. Comparative analysis revealed two adaptive traits in GA males. They produce more maltotriose, which is more resilient to salivary glucosidases, and they initiate copulation faster than wild-type males, before GA females interrupt their nuptial feeding and dismount the male. Recombinant lines of the two strains showed that the two emergent traits of GA males were not genetically associated with the GA trait. Results suggest that the two courtship traits emerged in response to the altered sexual behaviour of GA females and independently of the male's GA trait. Although rapid adaptive evolution generates sexual mismatches that lower fitness, compensatory behavioural evolution can correct these sensory discrepancies.

Implementation of ubiquitous chromatin opening elements as artificial integration sites for CRISPR/Cas9-mediated knock-in in mammalian cells.

CRISPR/Cas9-mediated targeted gene integration (TI) has been used to generate recombinant mammalian cell lines with predictable transgene expression. Identifying genomic hot spots that render high and stable transgene expression and knock-in (KI) efficiency is critical for fully implementing TI-mediated cell line development (CLD); however, such identification is cumbersome. In this study, we developed an artificial KI construct that can be used as a hot spot at different genomic loci. The ubiquitous chromatin opening element (UCOE) was employed because of its ability to open chromatin and enable stable and site-independent transgene expression. UCOE KI cassettes were randomly integrated into CHO-K1 and HEK293T cells, followed by TI of enhanced green fluorescent protein (EGFP) onto the artificial UCOE KI site. The CHO-K1 random pool harboring 5'2.2A2UCOE-CMV displayed a significant increase in EGFP expression level and KI efficiency compared with that of the control without UCOE. In addition, 5'2.2A2UCOE-CMV showed improved Cas9 accessibility in the HEK293T genome, leading to an increase in indel frequency and homology-independent KI. Overall, this assessment revealed the potential of UCOE KI constructs as artificial integration sites in streamlining the screening of high-production targeted integrants by mitigating the selection of genomic hot spots.

Influence of wheat-rye translocations on grain quality indices during bread winter wheat breeding in the South of Ukraine

Topicality. The use of introgressive material developed due to the involvement of wheat-rye translocations 1AL.1RS, 1BL.1RS in the modern gene pool of bread winter wheat is quite relevant at this stage of breeding development. This makes it possible to expand the genetic diversity of breeding material to identify new genetic sources of valuable traits and obtain genotypes based on introgressive material with wheat-rye translocations 1AL.1RS, 1BL.1RS with a sufficiently high grain quality in breeding programs in the southern Ukraine. Purpose. To establish the role of genetic environment as a factor of directional influence of wheat-rye translocations on baking properties, and to develop breeding techniques for neutralizing the negative effects of translocations to produce genotypes with high grain quality indices of valuable and strong wheat. Materials and Methods. Field experiments were carried out in the fields of the Plant Breeding and Genetic Institute - National Center for Seed Breeding and Variety Research in the 2010–2020. Meteorological conditions over the years of research were generally typical arid for the Steppe zone. The lines developed by crossing local varieties-carriers of translocations and the best varieties of the Plant Breeding and Genetic Institute breeding were studied that to identify the influence of wheat-rye translocations 1AL.1RS, 1BL.1RS on the quality of winter wheat grain in the process of breeding. Results. It was established that the winter wheat grain quality indices were changed (protein content increased, rheological properties decreased) by the introduction of wheat-rye translocations 1AL.1RS, 1BL.1RS into the local gene pool. These changes depend on the genetic environment, hybrid combination and the influence of genetic factors that reduce the negative effect of wheat-rye translocations on baking properties of the lines. It was proved that development of recombinant lines combining high yield and increased baking properties increases when using introgressive lines with wheat-rye translocations 1AL.1RS. Conclusions. It is possible to reduce the negative impact of wheat-rye translocations on the bread winter wheat grain quality and develop varieties with quality parameters of valuable and strong wheat by combining wheat-rye translocations with alleles in genotype positively influencing baking properties, and also by creating heterogeneity in genotypes with and without wheat-rye translocations in a certain ratio. An example is the highly heat-resistant variety of strong wheat Oktava Odeska, which is included in the State Register for dissemination in all agroclimatic zones of Ukraine. Keywords: bread winter wheat, recombinant lines, baking quality, wheat-rye translocations 1AL.1RS and 1BL.1RS

The Response of Chromosomally Engineered Durum Wheat-Thinopyrum ponticum Recombinant Lines to the Application of Heat and Water-Deficit Stresses: Effects on Physiological, Biochemical and Yield-Related Traits.

Abiotic stress occurrence and magnitude are alarmingly intensifying worldwide. In the Mediterranean basin, heat waves and precipitation scarcity heavily affect major crops such as durum wheat (DW). In the search for tolerant genotypes, the identification of genes/QTL in wild wheat relatives, naturally adapted to harsh environments, represents a useful strategy. We tested three DW-Thinopyrum ponticum recombinant lines (R5+, R112+, R23+), their control sibs lacking any alien introgression, and the heat-tolerant cv. Margherita for their physiological, biochemical and yield response to heat stress (HS) application at anthesis, also in combination with water-deficit stress applied from booting until maturity. Under HS, R5+ and R112+ (23%- and 28%-long 7el1L Th. ponticum chromosome segment distally inserted on DW 7AL, respectively) showed remarkable stability of the yield-related traits; in turn, R23+ (40%-long 7el1L segment), despite a decreased grain yield, exhibited a greater spike fertility index and proline content in spike than its control sib. Under water-deficit + HS, R5+ showed the highest increment in water use efficiency and in flag leaf proline content, accompanied by the lowest yield penalty even vs. Margherita. This research confirms the value of harnessing wild gene pools to enhance DW stress tolerance and represents a starting point for elucidating the mechanisms of Thinopyrum spp. contribution to this relevant breeding target.

Overexpression of geraniol-10-hydroxylase improves valtrate accumulation in Valeriana jatamansi

AbstractValtrate is the major active component of iridoids in Valeriana jatamansi plants, and has been widely used for the treatment of various diseases, giving rise to rapidly increasing market demands. The gene VjG10H from V. jatamansi encoding geraniol 10-hydroxylase (G10H) was introduced into V. jatamansi hairy roots and a transgenic hairy root culture system of V. jatamansi with good valtrate production ability was successfully established for the first time. The valtrate content of VjG10H-transformed lines ranged from 8.12 to 10.77 mg/g, with an average valtrate content (9.52 mg/g) of all the four G lines being higher than the non-transgenic hairy root line. Overexpression of VjG10H significantly improved valtrate production, indicating that G10H played an important role in stimulating valtrate accumulation. MeJA treatment stimulated valtrate accumulation in all of the VjG10H overexpression recombinant lines compared to untreated cultures, indicating that methyl jasmonate treatment was another effective regulation target for metabolic engineering of valtrate biosynthesis in V. jatamansi. Thus, transgenic hairy root technology coupled with elicitor treatments may be a promising strategy to increase valtrate yield in V. jatamansi.

Single-cell RNA sequencing reveals homogeneous transcriptome patterns and low variance in a suspension CHO-K1 and an adherent HEK293FT cell line in culture conditions

Recombinant mammalian host cell lines, in particular CHO and HEK293 cells, are used for the industrial production of therapeutic proteins. Despite their well-known genomic instability, the control mechanisms that enable cells to respond to changes in the environmental conditions are not yet fully understood, nor do we have a good understanding of the factors that lead to phenotypic shifts in long-term cultures. A contributing factor could be inherent diversity in transcriptomes within a population. In this study, we used a full-length coverage single-cell RNA sequencing (scRNA-seq) approach to investigate and compare cell-to-cell variability and the impact of standardized and homogenous culture conditions on the diversity of individual cell transcriptomes, comparing suspension CHO-K1 and adherent HEK293FT cells. Our data showed a critical batch effect from the sequencing of four 96-well plates of CHO-K1 single cells stored for different periods of time, which was and may be therefore identified as a technical variable to consider in experimental planning. Besides, in an artificial and controlled culture environment such as used in routine cell culture technology, the gene expression pattern of a given population does not reveal any marker gene capable to disclose relevant cell population substructures, both for CHO-K1 cells and for HEK293FT cells. The variation observed is primarily driven by the cell cycle.

An automated, low volume, and high-throughput analytical platform for aggregate quantitation from cell culture media

High throughput screening methods have driven a paradigm shift in biopharmaceutical development by reducing the costs of good manufactured (COGM) and accelerate the launch to market of novel drug products. Scale-down cell culture systems such as shaken 24- and 96-deep-well plates (DWPs) are used for initial screening of hundreds of recombinant mammalian clonal cell lines to quickly and efficiently select the best producing strains expressing product quality attributes that fit to industry platform. A common modification monitored from early-stage product development is protein aggregation due to its impact on safety and efficacy. This study aims to integrate high-throughput analysis of aggregation-prone therapeutic proteins with 96-deep well plate screening to rank clones based on the aggregation levels of the expressed proteins. Here we present an automated, small-scale analytical platform workflow combining the purification and subsequent aggregation analysis of protein biopharmaceuticals expressed in 96-DWP cell cultures. Product purification was achieved by small-scale solid-phase extraction using dual flow chromatography (DFC) automated on a robotic liquid handler for the parallel processing of up to 96 samples at a time. At-line coupling of size-exclusion chromatography (SEC) using a 2.1 mm ID column enabled the detection of aggregates with sub-2 µg sensitivity and a 3.5 min run time. The entire workflow was designed as an application to aggregation-prone mAbs and “mAb-like” next generation biopharmaceuticals, such as bispecific antibodies (BsAbs). Application of the high-throughput analytical workflow to a shake plate overgrow (SPOG) screen, enabled the screening of 384 different clonal cell lines in 32 h, requiring < 2 μg of protein per sample. Aggregation levels expressed by the clones varied between 9 and 76%. This high-throughput analytical workflow allowed for the early elimination of clonal cell lines with high aggregation, demonstrating the advantage of integrating analytical testing for critical quality attributes (CQAs) earlier in product development to drive better decision making.

Combination of untargeted and targeted proteomics for secretome analysis of L-WRN cells.

Organoid culture is a promising biomedical technology that requires specialized growth factors. Recently, a recombinant L-WRN cell line has been extensively used to generate conditioned medium (L-CM) for organoid culture. Nevertheless, methods for evaluating the stability of the L-WRN cells have been limited. In this study, a novel proteomics-based approach was developed to analyze the secretome of the cells. Serum-free L-CM was lyophilized, precipitated by trichloroacetic acid, and desalted prior to analysis by liquid chromatography-tandem mass spectrometry. Data-dependent acquisition (DDA) was conducted for the untargeted secretome profiling of the cells, and parallel reaction monitoring (PRM) was applied for the targeted quantification of the Wnt3A, R-spondin3, and noggin proteins (WRNs). This study also compared the performance of two types of PRM methods, namely MS1-independent PRM and MS1-dependent PRM, that can be executed on an Orbitrap instrument. The results showed that the growth of mouse intestinal organoids was closely related to the use of L-CM. The composition of L-CM could be markedly affected by the medium collection scheme. A total of 1725, 2302, and 2681 proteins were identified from the L-CM collected on day 5, day 9, and day 13, respectively. The MS1-independent PRM outperformed the MS1-dependent PRM and effectively quantified the WRNs with high repeatability and specificity. In conclusion, by integrating untargeted and targeted proteomics, this study develops a mass spectrometry-based method for the secretome analysis and quality control of the L-WRN cells. The methodology and findings of the present work will benefit future studies on organoids and secretomes.

The Pyramiding of Elite Allelic Genes Related to Grain Number Increases Grain Number per Panicle Using the Recombinant Lines Derived from Indica-japonica Cross in Rice.

Indica(xian)-japonica(geng) hybrid rice has many heterosis traits that can improve rice yield. However, the traditional hybrid technology will struggle to meet future needs for the development of higher-yield rice. Available genomics resources can be used to efficiently understand the gene-trait association trait for rice breeding. Based on the previously constructed high-density genetic map of 272 high-generation recombinant inbred lines (RILs) originating from the cross of Luohui 9 (indica, as female) and RPY geng (japonica, as male) and high-quality genomes of parents, here, we further explore the genetic basis for an important complex trait: possible causes of grain number per panicle (GNPP). A total of 20 genes related to grains number per panicle (GNPP) with the differences of protein amino acid between LH9 and RPY were used to analyze genotype combinations, and PCA results showed a combination of PLY1, LAX1, DTH8 and OSH1 from the RPY geng with PYL4, SP1, DST and GNP1 from Luohui 9 increases GNPP. In addition, we also found that the combination of LAX1-T2 and GNP1-T3 had the most significant increase in GNPP. Notably, Molecular Breeding Knowledgebase (MBK) showed a few aggregated rice cultivars, LAX1-T2 and GNP1-T3, which may be a result of the natural geographic isolation between the two gene haplotypes. Therefore, we speculate that the pyramiding of japonica-type LAX-T2 with indica-type GNP1-T3 via hybridization can significantly improve rice yield by increasing GNPP.

Isolation of a Novel QTL, qSCM4, Associated with Strong Culm Affects Lodging Resistance and Panicle Branch Number in Rice.

Rice breeders are now developing new varieties with semi-high or even high plant height to further increase the grain yield, and the problem of lodging has re-appeared. We identified a major quantitative trait locus (QTL), qSCM4, for resistance to lodging by using an F2 segregant population and a recombinant self-incompatible line population from the cross between Shennong265 (SN265) and Lijiangxintuanheigu (LTH) after multiple years and multiple environments. Then, the residual heterozygous derived segregant population which consisted of 1781 individual plants, and the BC3F2 segregant population which consisted of 3216 individual plants, were used to shorten the physical interval of qSCM4 to 58.5 kb including 11 genes. DNA sequencing revealed the most likely candidate gene for qSCM4 was Os04g0615000, which encoded a functional protein with structural domains of serine and cysteine. There were 13 DNA sequence changes in LTH compared to SN265 in this gene, including a fragment deletion, two base changes in the 3' UTR region, six base changes in the exons, and four base changes in the introns. A near-isogenic line carrying qSCM4 showed that it improved the lodging resistance through increasing stem thickness by 25.3% and increasing stem folding resistance by 20.3%. Furthermore, it was also discovered that qSCM4 enhanced the primary branch per panicle by 16.7%, secondary branch by per panicle 9.9%, and grain number per panicle by 14.7%. All the above results will give us a valuable genetic resource for concurrently boosting culm strength and lodging resistance, and they will also provide a basis for further research on the lodging resistance mechanism of rice.

Production and characterisation of a SARS-CoV-2 S-protein RBD homodimer with increased avidity for specific antibodies

Monitoring of the proportion of immune individuals and the effectiveness of vaccination in a population involves evaluation of several important parameters, including the level of virus-neutralising antibodies. In order to combat the COVID-19 pandemic, it is essential to develop approaches to detecting SARS-CoV-2 neutralising antibodies by safe, simple and rapid methods that do not require live viruses. To develop a test system for enzyme-linked immunosorbent assay (ELISA) that detects potential neutralising antibodies, it is necessary to obtain a highly purified recombinant receptor-binding domain (RBD) of the spike (S) protein with high avidity for specific antibodies.The aim of the study was to obtain and characterise a SARS-CoV-2 S-protein RBD homodimer and a recombinant RBD-expressing cell line, as well as to create an ELISA system for detecting potential neutralising antibodies.Materials and methods: the genetic construct was designed in silico. To generate a stable producer cell line, the authors transfected CHO-S cells, subjected them to antibiotic pressure, and selected the optimal clone. To isolate monomeric and homodimeric RBD forms, the authors purified the recombinant RBD by chromatographic methods. Further, they analysed the activity of the RBD forms by Western blotting, bio-layer interferometry, and indirect ELISA. The analysis involved mono clonal antibodies GamXRH19, GamP2C5, and h6g3, as well as serum samples from volunteers vaccinated with Gam-COVID-Vac (Sputnik V) and unvaccinated ones.Results: the authors produced the CHO-S cell line for stable expression of the recombinant SARS-CoV-2 S-protein RBD. The study demonstrated the recombinant RBD’s ability to homodimerise after fed-batch cultivation of the cell line for more than 7 days due to the presence of unpaired cysteines. The purified recombinant RBD yield from culture broth was 30–50 mg/L. Monomeric and homodimeric RBD forms were separated using gel-filtration chromatography and characterised by their ability to interact with specific monoclonal antibodies, as well as with serum samples from vaccinated volunteers. The homodimeric recombinant RBD showed increased avidity for both monoclonal and immune sera antibodies.Conclusions: the homodimeric recombinant RBD may be more preferable for the analysis of levels of antibodies to the receptor-binding domain of the SARS-CoV-2 S protein.

TRIPLE TEST CROSS ANALYSIS FOR DETERMINING GENE ACTION, GENETIC CORRELATIONS AND PREDICTION FOR YIELD AND YIELD COMPONENTS IN BREAD WHEAT

In two crosses of bread wheat (Gemmeiza11 x Misr1 and Gemmeiza12 x Misr2) to investigate gene action, genetic correlations and predicating new recombinant lines for days to heading, plant height, flag leaf area, number of spikes/plant, number of grains/spike, 1000-grain weight, spike grain weight and grain yield /plant, triple test cross was used. The starting materials for triple test cross was the F1 and their grand parents (P1 and P2) for the two studied wheat crosses. The first cross Gemmeiza 11 x Misr1 and the second cross was Gemmeiza 12x Misr2. The F1’s were selfed to produce F2’s grains. In order to create F1 and F2 plants, the obtained materials (F1 and F2 grain genotypes) and the parental genotypes were seeded together. Thirty individual F2 plants were randomly labeled from each cross and crossed back to their grand parents (P1 and P2) and F1 between then to produce three types of families L1 (F2i x P1i), L2 (F2i x P2i) and L3 (F2i x F1i) in each cross. On November 10, 2018, during the winter growing season, the triple test cross families (L1, L2 and L3), along with the parents, F1 and F2 in each cross, were seeded. Results showed that in the genetic system for the investigated features for two crosses, epistatic gene effects were significant. The types of epistasis (additive x additive, additive x dominance and dominance x dominance) were significant for all studied characters. For the majority of traits in both crossings, both additive and dominant genetic components were significant and involved in the genetic system. The average level of dominance for spikes/plant in cross 1 and spike grain weight in cross -2 was overdominance, while for the remaining characters, additive gene effects were more pronounced. The F value was positive and significant for 1000-grain weight in two crosses, suggesting that dominant genes controlling these characters were unidirectional. The highest proportion of inbreds excepted to outperform parental rang in cross 1 for days to heading and flag leaf area. In both crosses, grain yield per plant was positively and significantly correlated with number of spikes per plant, number of grains per spike, and 1000-grain weight, according to epistasis, additive, and dominance genetic correlations.