Recombinant Progeny Research Articles

Engineering a live-attenuated porcine reproductive and respiratory syndrome virus vaccine to prevent RNA recombination by rewiring transcriptional regulatory sequences.

Porcine reproductive and respiratory syndrome viruses (PRRSVs) are genetically diverse, and this is due in part to their extensive recombination. Live vaccines are widely used to prevent and control PRRS in China. However, owing to the wide variety of live vaccines, non-standard use, and the wild viruses prevalent on pig farms, new strains, generated via RNA recombination, are continuously emerging. Vaccine strains are also involved in PRRSV recombination, which leads to the emergence of new variants and alterations in virulence and pathogenesis. A recombination-resistant genome was engineered by rewiring the entire transcriptional regulatory sequence (TRS) circuit of the live PRRSV vaccine strain. Theoretically, after clinical application, once the virus recombines with the genome of the epidemic strain, the base pairing between the two sets of TRS circuits should be disrupted, resulting in a fatal genetic trap for the generation of an RNA recombinant progeny virus. Therefore, the remodeled PRRSV TRS mutant generated in this study can serve as a recombination-resistant platform for the rational design of safe PRRS vaccines in the future.

QTL-Seq: Rapid, Cost-Effective, and Reliable Method for QTL Identification

QTL-seq is a powerful method that integrates whole-genome sequencing (WGS) with bulk-segregant analysis to rapidly and reliably identify quantitative trait loci (QTLs) associated with specific traits. This approach significantly advances traditional QTL mapping by eliminating the need for genome wide DNA markers such as SSR, RFLP, and INDELs, which are typically used in linkage-based QTL mapping. Instead, QTL-seq leverages WGS to detect all genetic variations such as SNPs, Indels, and Structural Variants across the entire genome, providing a comprehensive resource for marker development in marker-assisted selection. The QTL-seq process begins with the creation of genetically diverse mapping populations, such as F2 or RILs, followed by detailed phenotypic characterization. DNA from plants exhibiting similar phenotypes is pooled into bulk groups and sequenced, allowing for cost-effective and efficient QTL identification. Identified QTLs can be further validated through fine mapping using recombinant screenings and progeny testing, leading to the identification of candidate genes associated with traits of interest. In this study, we outline a user-friendly QTL-seq pipeline, from sequencing to data visualization to demonstrate its practical application. While the manuscript primarily focuses on describing the pipeline, we also conducted a case study analysis with real data to showcase its effectiveness. Our work contributes to the broader understanding of QTL-seq applications and offers practical recommendations for optimizing this method in future breeding programs.

Identification of the drug/metabolite transporter 1 as a marker of quinine resistance in a NF54×Cam3.II P. falciparum genetic cross.

The genetic basis of Plasmodium falciparum resistance to quinine (QN), a drug used to treat severe malaria, has long been enigmatic. To gain further insight, we used FRG-NOD human liver-chimeric mice to conduct a P. falciparum genetic cross between QN-sensitive and QN-resistant parasites, which also differ in their susceptibility to chloroquine (CQ). By applying different selective conditions to progeny pools prior to cloning, we recovered 120 unique recombinant progeny. These progeny were subjected to drug profiling and QTL analyses with QN, CQ, and monodesethyl-CQ (md-CQ, the active metabolite of CQ), which revealed predominant peaks on chromosomes 7 and 12, consistent with a multifactorial mechanism of resistance. A shared chromosome 12 region mapped to resistance to all three antimalarials and was preferentially co-inherited with pfcrt. We identified an ATP-dependent zinc metalloprotease (FtsH1) as one of the top candidates and observed using CRISPR/Cas9 SNP-edited lines that ftsh1 is a potential mediator of QN resistance and a modulator of md-CQ resistance. As expected, CQ and md-CQ resistance mapped to a chromosome 7 region harboring pfcrt. However, for QN, high-grade resistance mapped to a chromosome 7 peak centered 295kb downstream of pfcrt. We identified the drug/metabolite transporter 1 (DMT1) as the top candidate due to its structural similarity to PfCRT and proximity to the peak. Deleting DMT1 in QN-resistant Cam3.II parasites significantly sensitized the parasite to QN but not to the other drugs tested, suggesting that DMT1 mediates QN response specifically. We localized DMT1 to structures associated with vesicular trafficking, as well as the parasitophorous vacuolar membrane, lipid bodies, and the digestive vacuole. We also observed that mutant DMT1 transports more QN than the wild-type isoform in vitro. Our study demonstrates that DMT1 is a novel marker of QN resistance and a new chromosome 12 locus associates with CQ and QN response, with ftsh1 is a potential candidate, suggesting these genes should be genotyped in surveillance and clinical settings.

Impact of the Transboundary Interference Inhibitor on RNAi and the Baculovirus Expression System in Insect Cells.

RNA interference inhibitors were initially discovered in plant viruses, representing a unique mechanism employed by these viruses to counteract host RNA interference. This mechanism has found extensive applications in plant disease resistance breeding and other fields; however, the impact of such interference inhibitors on insect cell RNA interference remains largely unknown. In this study, we screened three distinct interference inhibitors from plant and mammal viruses that act through different mechanisms and systematically investigated their effects on the insect cell cycle and baculovirus infection period at various time intervals. Our findings demonstrated that the viral suppressors of RNA silencing (VSRs) derived from plant and mammal viruses significantly attenuated the RNA interference effect in insect cells, as evidenced by reduced apoptosis rates, altered gene regulation patterns in cells, enhanced expression of exogenous proteins, and improved production efficiency of recombinant virus progeny. Further investigations revealed that the early expression of VSRs yielded superior results compared with late expression during RNA interference processes. Additionally, our results indicated that dsRNA-binding inhibition exhibited more pronounced effects than other modes of action employed by these interference inhibitors. The outcomes presented herein provide novel insights into enhancing defense mechanisms within insect cells using plant and mammal single-stranded RNA virus-derived interference inhibitors and have potential implications for expanding the scope of transformation within insect cell expression systems.

The effect of UV radiation on the frequency of crossing over in Drosophila melanogaster

The increasing ozone layer depletion due to exposure to high environmental pollutants may contribute to the high probability of cells being exposed to UV radiation, causing various cellular responses, such as genomic instability. This study aimed to determine the effect of UV on the frequency of crossing over in a cross between the wild-type and the black vestigial strain of Drosophila melanogaster, using four variations of duration UV exposure as follows: 0’, 3’, 6’, and 9’. Recombinant progeny were calculated and analyzed using single-factor ANOVA. The results showed that: (1) The longer the duration of the UV exposure, the higher the percentage of recombinant progeny. (2) The body size of the D. melanogaster progeny exposed to UV light was more petite, and their movements were slower. (3) The single factor ANOVA showed that the length of UV had not affected the frequency of crossing over; but there was a phenomenon of crossing over, which has been showed that UV exposure made higher the percentage of recombinant progeny and affected to the body size of D. melanogaster. Therefore, further research is needed to examine the effect of UV radiation with longer periods of crossing over frequency and molecular analysis needs to be considered.

Genetic crosses within and between species of Cryptosporidium.

Parasites and their hosts are engaged in reciprocal coevolution that balances competing mechanisms of virulence, resistance, and evasion. This often leads to host specificity, but genomic reassortment between different strains can enable parasites to jump host barriers and conquer new niches. In the apicomplexan parasite Cryptosporidium, genetic exchange has been hypothesized to play a prominent role in adaptation to humans. The sexual lifecycle of the parasite provides a potential mechanism for such exchange; however, the boundaries of Cryptosporidium sex are currently undefined. To explore this experimentally, we established a model for genetic crosses. Drug resistance was engineered using a mutated phenylalanyl tRNA synthetase gene and marking strains with this and the previously used Neo transgene enabled selection of recombinant progeny. This is highly efficient, and genomic recombination is evident and can be continuously monitored in real time by drug resistance, flow cytometry, and PCR mapping. Using this approach, multiple loci can now be modified with ease. We demonstrate that essential genes can be ablated by crossing a Cre recombinase driver strain with floxed strains. We further find that genetic crosses are also feasible between species. Crossing Cryptosporidium parvum, a parasite of cattle and humans, and Cryptosporidium tyzzeri a mouse parasite resulted in progeny with a recombinant genome derived from both species that continues to vigorously replicate sexually. These experiments have important fundamental and translational implications for the evolution of Cryptosporidium and open the door to reverse- and forward-genetic analysis of parasite biology and host specificity.

Leishmania genetic exchange is mediated by IgM natural antibodies.

Host factors that mediate Leishmania genetic exchange are not well defined. Here we demonstrate that natural IgM (IgMn)1-4 antibodies mediate parasite genetic exchange by inducing the transient formation of a spherical parasite clump that promotes parasite fusion and hybrid formation. We establish that IgMn from Leishmania-free animals binds to the surface of Leishmania parasites to induce significant changes in the expression of parasite transcripts and proteins. Leishmania binding to IgMn is partially lost after glycosidase treatment, although parasite surface phosphoglycans, including lipophosphoglycan, are not required for IgMn-induced parasite clumping. Notably, the transient formation of parasite clumps is essential for Leishmania hybridization in vitro. In vivo, we observed a 12-fold increase in hybrid formation in sand flies provided a second blood meal containing IgMn compared with controls. Furthermore, the generation of recombinant progeny from mating hybrids and parental lines were only observed in sand flies provided with IgMn. Both in vitro and in vivo IgM-induced Leishmania crosses resulted in full genome hybrids that show equal patterns of biparental contribution. Leishmania co-option of a host natural antibody to facilitate mating in the insect vector establishes a new paradigm of parasite-host-vector interdependence that contributes to parasite diversity and fitness by promoting genetic exchange.

Pigment composition analysis of fruit pulp in the recombinant progenies reveals the polygenic nature of pulp color inheritance in guava (Psidium guajava L.)

Pigment composition analysis of fruit pulp in the recombinant progenies reveals the polygenic nature of pulp color inheritance in guava (Psidium guajava L.)

Genetic Determination of Regressive Pattern of Walker 256 Carcinosarcoma in Rats with Hypothalamic Diabetes Insipidus.

We studied the growth dynamics of Walker 256 carcinosarcoma in recombinant progeny of dihybrid crosses of Brattleboro and WAG rats. A mutation in the vasopressin gene determining hypothalamic diabetes insipidus was detected in Brattleboro rats. WAG rats are carriers of normal vasopressin gene. Another interlinear difference was linked to tyrosinase gene controlling melanin synthesis. WAG rats express mutant allele determining albino phenotype. Brattleboro rats had normal working tyrosinase gene. F2 segregation yielded phenotypic classes with two patterns of tumor growth: linear growth or regression. Tumors regression was not linked to tyrosinase activity and was observed only in rats with diabetes insipidus. Analysis of mutants in next generations F3 and F4 confirmed this regularity in the Walker 256 carcinosarcoma growth pattern.

A method for quantifying sporulation efficiency and isolating meiotic progeny in non-GMO strains of Saccharomyces cerevisiae.

Meiotic mapping, a linkage-based method for analyzing the recombinant progeny of a cross, has long been a cornerstone of genetic research. The yeast Saccharomyces cerevisiae is a powerful system because it is possible to isolate and cultivate the four products (spores) of a single meiotic event. However, the throughput of this process has historically been limited by the process of identifying tetrads in a heterogeneous population of vegetative cells, tetrads, and dyads followed by manual separation (dissection) of the spores contained in a tetrad. To date, methods that facilitate high throughput characterization and isolation of meiotic progeny have relied on genetic engineering. Here, we characterize the ability of the fluorescent dye DiBAC4 (5) to stain yeast tetrads and dyads as well as to adhere to spores following bulk tetrad disruption. Applications include quantitative assays of sporulation rates and efficiency by flow cytometry as well as enrichment of intact tetrads, dyads, or disrupted spores by fluorescence-activated cell sorting in strains that have not been genetically modified.

Generated Randomly and Selected Functionally? The Nature of Enterovirus Recombination.

Genetic recombination in RNA viruses is an important evolutionary mechanism. It contributes to population diversity, host/tissue adaptation, and compromises vaccine efficacy. Both the molecular mechanism and initial products of recombination are relatively poorly understood. We used an established poliovirus-based in vitro recombination assay to investigate the roles of sequence identity and RNA structure, implicated or inferred from an analysis of circulating recombinant viruses, in the process. In addition, we used next-generation sequencing to investigate the early products of recombination after cellular coinfection with different poliovirus serotypes. In independent studies, we find no evidence for a role for RNA identity or structure in determining recombination junctions location. Instead, genome function and fitness are of greater importance in determining the identity of recombinant progeny. These studies provide further insights into this important evolutionary mechanism and emphasize the critical nature of the selection process on a mixed virus population.

Identification of QTLs Controlling Resistance to Anthracnose Disease in Water Yam (Dioscorea alata).

Anthracnose disease caused by a fungus Colletotrichum gloeosporioides is the primary cause of yield loss in water yam (Dioscorea alata), the widely cultivated species of yam. Resistance to yam anthracnose disease (YAD) is a prime target in breeding initiatives to develop durable-resistant cultivars for sustainable management of the disease in water yam cultivation. This study aimed at tagging quantitative trait loci (QTL) for anthracnose disease resistance in a bi-parental mapping population of D. alata. Parent genotypes and their recombinant progenies were genotyped using the Genotyping by Sequencing (GBS) platform and phenotyped in two crop cycles for two years. A high-density genetic linkage map was built with 3184 polymorphic Single Nucleotide Polymorphism (NSP) markers well distributed across the genome, covering 1460.94 cM total length. On average, 163 SNP markers were mapped per chromosome with 0.58 genetic distances between SNPs. Four QTL regions related to yam anthracnose disease resistance were identified on three chromosomes. The proportion of phenotypic variance explained by these QTLs ranged from 29.54 to 39.40%. The QTL regions identified showed genes that code for known plant defense responses such as GDSL-like Lipase/Acylhydrolase, Protein kinase domain, and F-box protein. The results from the present study provide valuable insight into the genetic architecture of anthracnose resistance in water yam. The candidate markers identified herewith form a relevant resource to apply marker-assisted selection as an alternative to a conventional labor-intensive screening for anthracnose resistance in water yam.

Interbacterial Transfer of Carbapenem Resistance and Large Antibiotic Resistance Islands by Natural Transformation in Pathogenic Acinetobacter.

ABSTRACTAcinetobacter baumannii infection poses a major health threat, with recurrent treatment failure due to antibiotic resistance, notably to carbapenems. While genomic analyses of clinical strains indicate that homologous recombination plays a major role in the acquisition of antibiotic resistance genes, the underlying mechanisms of horizontal gene transfer often remain speculative. Our understanding of the acquisition of antibiotic resistance is hampered by the lack of experimental systems able to reproduce genomic observations. We here report the detection of recombination events occurring spontaneously in mixed bacterial populations and which can result in the acquisition of resistance to carbapenems. We show that natural transformation is the main driver of intrastrain but also interstrain recombination events between A. baumannii clinical isolates and pathogenic species of Acinetobacter. We observed that interbacterial natural transformation in mixed populations is more efficient at promoting the acquisition of large resistance islands (AbaR4 and AbaR1) than when the same bacteria are supplied with large amounts of purified genomic DNA. Importantly, analysis of the genomes of the recombinant progeny revealed large recombination tracts (from 13 to 123 kb) similar to those observed in the genomes of clinical isolates. Moreover, we highlight that transforming DNA availability is a key determinant of the rate of recombinants and results from both spontaneous release and interbacterial predatory behavior. In the light of our results, natural transformation should be considered a leading mechanism of genome recombination and horizontal gene transfer of antibiotic resistance genes in Acinetobacter baumannii.

Isolation and Identification of Inter-Species Enterovirus Recombinant Genomes.

Positive-strand RNA virus evolution is partly attributed to the process of recombination. Although common between closely genetically related viruses, such as within species of the Enterovirus genus of the Picornaviridae family, inter-species recombination is rarely observed in nature. Recent studies have shown recombination is a ubiquitous process, resulting in a wide range of recombinant genomes and progeny viruses. While not all recombinant genomes yield infectious progeny virus, their existence and continued evolution during replication have critical implications for the evolution of the virus population. In this study, we utilised an in vitro recombination assay to demonstrate inter-species recombination events between viruses from four enterovirus species, A-D. We show that inter-species recombinant genomes are generated in vitro with polymerase template-switching events occurring within the virus polyprotein coding region. However, these genomes did not yield infectious progeny virus. Analysis and attempted recovery of a constructed recombinant cDNA revealed a restriction in positive-strand but not negative-strand RNA synthesis, indicating a significant block in replication. This study demonstrates the propensity for inter-species recombination at the genome level but suggests that significant sequence plasticity would be required in order to overcome blocks in the virus life cycle and allow for the production of infectious viruses.

A quadruple recombination event discovered in hepatitis E virus.

Hepatitis E virus (HEV) can infect humans, pigs, and many other animals, but recombination in HEV has rarely been reported. In the present study, phylogenetic and recombination analysis was performed on 557 complete HEV genome sequences from the GenBank database. A potentially significant quadruple recombination event was identified by recombination detection analysis. The recombinant progeny virus, HEV_32_Manchester_301214, was produced by inter-genotype recombination between the major parent HEPAC-44 and the minor parent HE-JA15-1335. HEV_32_Manchester_301214 and HEPAC-44 belong to genotype 3, while HE-JA15-1335 belongs to genotype 1, and these three strains were all isolated from humans. Three breakpoints of the four recombination events occurred in the ORF2 region, while another occurred in the ORF1 region. This quadruple recombination event was confirmed by phylogenetic analysis. The genotype, host, and recombination regions of the three strains were analyzed, and the analysis results provide valuable information for future research on HEV diversity.

The power and promise of genetic mapping from Plasmodium falciparum crosses utilizing human liver-chimeric mice

Genetic crosses are most powerful for linkage analysis when progeny numbers are high, parental alleles segregate evenly and numbers of inbred progeny are minimized. We previously developed a novel genetic crossing platform for the human malaria parasite Plasmodium falciparum, an obligately sexual, hermaphroditic protozoan, using mice carrying human hepatocytes (the human liver-chimeric FRG NOD huHep mouse) as the vertebrate host. We report on two genetic crosses—(1) an allopatric cross between a laboratory-adapted parasite (NF54) of African origin and a recently patient-derived Asian parasite, and (2) a sympatric cross between two recently patient-derived Asian parasites. We generated 144 unique recombinant clones from the two crosses, doubling the number of unique recombinant progeny generated in the previous 30 years. The allopatric African/Asian cross has minimal levels of inbreeding and extreme segregation distortion, while in the sympatric Asian cross, inbred progeny predominate and parental alleles segregate evenly. Using simulations, we demonstrate that these progeny provide the power to map small-effect mutations and epistatic interactions. The segregation distortion in the allopatric cross slightly erodes power to detect linkage in several genome regions. We greatly increase the power and the precision to map biomedically important traits with these new large progeny panels.

Identification of superior progenies between common bean gene groups for root system obtained by recurrent selection

Genetic variability is essential for gains in breeding programs. The cyclic process of progeny recombination is a strategy to raise the chances of selecting better genotype combinations. The objective of this study was to identify superior progenies between common bean genes groups in first recurrent selection cycle for root system traits. Parents of the Andean and Middle American gene groups were hybridized in a complete diallel scheme. Thereafter, the parents and F1 and F2 populations were planted in the field. To establish the base population of recurrent selection (C0), seven segregating populations (F2) with superior performance for root system were selected and intercrossed, resulting in recombinant progenies (C1). To estimate the selection gain, the parents and C0 and C1 genotypes were compared with regard to the following variables: total root length (TRL, cm), projected root area (PRA, cm2), root volume (VOL, cm3) and number of root tips (RT). The difference between genotype combinations indicates the presence of genetic variability and effectiveness of recurrent selection. The mean genetic progress for root system-related traits was 12.9% (TRL), 12.6% (PRA), 12% (VOL) and 11.5% (RT) in the first recombination cycle. The mean phenotypic performance of seven of the C1 progenies exceeded that of their parents for all root system traits. These progenies are promising as base populations of the next selection cycle.

FlyORF-TaDa allows rapid generation of new lines for in vivo cell-type-specific profiling of protein-DNA interactions in Drosophila melanogaster.

Targeted DamID (TaDa) is an increasingly popular method of generating cell-type-specific DNA-binding profiles in vivo. Although sensitive and versatile, TaDa requires the generation of new transgenic fly lines for every protein that is profiled, which is both time-consuming and costly. Here, we describe the FlyORF-TaDa system for converting an existing FlyORF library of inducible open reading frames (ORFs) to TaDa lines via a genetic cross, with recombinant progeny easily identifiable by eye color. Profiling the binding of the H3K36me3-associated chromatin protein MRG15 in larval neural stem cells using both FlyORF-TaDa and conventional TaDa demonstrates that new lines generated using this system provide accurate and highly reproducible DamID-binding profiles. Our data further show that MRG15 binds to a subset of active chromatin domains in vivo. Courtesy of the large coverage of the FlyORF library, the FlyORF-TaDa system enables the easy creation of TaDa lines for 74% of all transcription factors and chromatin-modifying proteins within the Drosophila genome.

Superinfection and recombination of infectious laryngotracheitis virus vaccines in the natural host

Infectious laryngotracheitis virus (ILTV, Gallid alphaherpesvirus 1) causes severe respiratory disease in chickens and has a major impact on the poultry industry worldwide. Live attenuated vaccines are widely available and are administered early in the life of commercial birds, often followed by one or more rounds of revaccination, generating conditions that can favour recombination between vaccines. Better understanding of the factors that contribute to the generation of recombinant ILTVs will inform the safer use of live attenuated herpesvirus vaccines. This study aimed to examine the parameters of infection that allow superinfection and may enable the generation of recombinant progeny in the natural host. In this study, 120 specific-pathogen free (SPF) chickens in 8 groups were inoculated with two genetically distinct live-attenuated ILTV vaccine strains with 1–4 days interval between the first and second vaccinations. After inoculation, viral genomes were detected in tracheal swabs in all groups, with lowest copies detected in swabs collected from the groups where the interval between inoculations was 4 days. Superinfection of the host was defined as the detection of the virus that was inoculated last, and this was detected in tracheal swabs from all groups. Virus could be isolated from swabs at a limited number of timepoints, and these further illustrated superinfection of the birds as recombinant viruses were detected among the progeny. This study has demonstrated superinfection at host level and shows recombination events occur under a very broad range of infection conditions. The occurrence of superinfection after unsynchronised infection with multiple viruses, and subsequent genomic recombination, highlight the importance of using only one type of vaccine per flock as the most effective way to limit recombination.

Deciphering the Impact of a Bacterial Infection on Meiotic Recombination in Arabidopsis with Fluorescence Tagged Lines

Plants are under strong evolutionary pressure to maintain surveillance against pathogens. One major disease resistance mechanism is based on NB-LRR (NLR) proteins that specifically recognize pathogen effectors. The cluster organization of the NLR gene family could favor sequence exchange between NLR genes via recombination, favoring their evolutionary dynamics. Increasing data, based on progeny analysis, suggest the existence of a link between the perception of biotic stress and the production of genetic diversity in the offspring. This could be driven by an increased rate of meiotic recombination in infected plants, but this has never been strictly demonstrated. In order to test if pathogen infection can increase DNA recombination in pollen meiotic cells, we infected Arabidopsis Fluorescent Tagged Lines (FTL) with the virulent bacteria Pseudomonas syringae. We measured the meiotic recombination rate in two regions of chromosome 5, containing or not an NLR gene cluster. In all tested intervals, no significant difference in genetic recombination frequency between infected and control plants was observed. Although it has been reported that pathogen exposure can sometimes increase the frequency of recombinant progeny in plants, our findings suggest that meiotic recombination rate in Arabidopsis may be resilient to at least some pathogen attack. Alternative mechanisms are discussed.