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An isolate of chili pepper mild mottle virus (CPMMV-Sp; GenBank OQ920979) with a 99% identity to CPMMV (GenBank MN164455.1) was found in symptomatic pepper plants in Spain. RACE analysis, performed using a stem-loop primer developed in this study to prime at the end of the introduced poly(A)/(U) tail, revealed the presence of an extra 22 nt at the 5' end, starting with a cytosine, which were essential to generate infectious clones. However, the 5' terminal cytosine was dispensable for initiating the infection. The design of two specific digoxigenin riboprobes targeting the more divergent area of CPMMV-Sp, compared to the closely related bell pepper mottle virus (BPeMV) (identity percentage of 80.6% and 75.8%, respectively), showed that both probes specifically detected CPMMV-Sp when the hybridization was performed at 68ºC and 60ºC, respectively. However, the BPeMV probe, targeting a region with an 89.4% identity percentage to CPMMV-Sp, showed cross-hybridization at 60ºC but not at 68ºC. The comparison of the detection limits between molecular hybridization and RT-PCR techniques revealed that the former was 125 times less sensitive than RT-PCR. The analysis of the vertical transmission of CPMMV-Sp using seeds from naturally or mechanically infected pepper plants revealed a transmission percentage ranging from 0.9% to 8.5%. Finally, the analysis of the resistance of capsicum species carrying different alleles of the L gene (L1, L2, L3, and L4) revealed that varieties with the L1 gene were infected by CPMMV-Sp (20–40% of inoculated plants), while varieties with the L2, L3, and L4 genes were resistant.

Cathepsin L gene is a member of the cysteine proteinase gene group. In this study Cathepsin L gene was isolated from Kuruma shrimp Marsupenaeus japonicus ( Mj-Cathepsin L ) and the full-length DNA sequence was 1 963 bp. Mj-Cathepsin L protein showed high homologies with other Cathepsin L proteins documented in vertebrates, mollusks and other crustaceans. Expression analysis of Mj-Cathepsin L gene in different tissues revealed that it was predominant in hepatopancreas. During early ontogenetic development stages Mj-Cathepsin L showed a development-regulated expression, and the Mj-Cathepsin L showed a molting stage-regulated expression during the five molting stages, inferring its role in the ontogenic development of M. japonicus. Two kinds of forms of Mj-Cathepsin L protein: pro- Cathepsin L and Cathepsin L were measured in hepatopancreas, stomach and intestine by Western Blotting.

Gene tree discordance in large genomic data sets can be caused by evolutionary processes such as incomplete lineage sorting and hybridization, as well as model violation, and errors in data processing, orthology inference, and gene tree estimation. Species tree methods that identify and accommodate all sources of conflict are not available, but a combination of multiple approaches can help tease apart alternative sources of conflict. Here, using a phylotranscriptomic analysis in combination with reference genomes, we test a hypothesis of ancient hybridization events within the plant family Amaranthaceae s.l. thatwas previously supported bymorphological, ecological, and Sanger-based molecular data. The data set included seven genomes and 88 transcriptomes, 17 generated for this study. We examined gene-tree discordance using coalescent-based species trees and network inference, gene tree discordance analyses, site pattern tests of introgression, topology tests, synteny analyses, and simulations. We found that a combination of processes might have generated the high levels of gene tree discordance in the backbone of Amaranthaceae s.l. Furthermore, we found evidence that three consecutive short internal branches produce anomalous trees contributing to the discordance. Overall, our results suggest that Amaranthaceae s.l. might be a product of an ancient and rapid lineage diversification, and remains, and probably will remain, unresolved. This work highlights the potential problems of identifiability associated with the sources of gene tree discordance including, in particular, phylogenetic network methods. Our results also demonstrate the importance of thoroughly testing for multiple sources of conflict in phylogenomic analyses, especially in the context of ancient, rapid radiations. We provide several recommendations for exploring conflicting signals in such situations.

Brazilian Caipira chickens that lay blue eggs are known to possess unique genetic traits. This study investigates the immunogenetic diversity of MHC class II B-Lβ genes (B-LβI and B-LβII) in this population, aiming to assess their potential value in selective breeding programs focused on disease resistance. A total of 100 chickens were analyzed using targeted sequencing of the B-LβI and B-LβII genes. The resulting nucleotide sequences were evaluated for polymorphism and compared with known alleles described in previous studies. Fifteen unique nucleotide sequences were identified, of which five had not been previously reported in the scientific literature. The new alleles exhibited significant polymorphism, confirming high genetic diversity within the population. The genetic variability observed supports earlier findings regarding the diversity of MHC genes in local chicken breeds. These novel alleles may confer advantages in immune responsiveness, reinforcing the importance of preserving local breeds as reservoirs of functional genetic diversity. Brazilian Caipira chickens laying blue eggs display remarkable immunogenetic diversity in MHC class II B-L β genes. This variation includes novel alleles with potential application in future breeding programs. Conservation and utilization of this genetic resource can contribute to the development of healthier and more disease-resistant commercial chicken lines.

We used a novel approach that incorporated chromosome sorting, next-generation sequencing, array hybridization, and systematic exploitation of conserved synteny with model grasses to assign ~86% of the estimated ~32,000 barley [Hordeum vulgare) genes to individual chromosome arms. Using a series of bioinformatically constructed genome zippers that integrate gene indices of rice (Oryza sativa), sorghum [Sorghum bicolor), and Brachypodium distachyon in a conserved synteny model, we were able to assemble 21,766 barley genes in a putative linear order. We show that the barley (H) genome displays a mosaic of structural similarity to hexaploid bread wheat (Triticum aestivum) A, B, and D subgenomes and that orthologous genes in different grasses exhibit signatures of positive selection in different lineages. We present an ordered, information-rich scaffold of the barley genome that provides a valuable and robust framework for the development of novel strategies in cereal breeding.

Lassa virus (LASV) causes a deadly haemorrhagic fever in humans, killing several thousand people in West Africa annually. For 40 years, the Natal multimammate rat, Mastomys natalensis , has been assumed to be the sole host of LASV. We found evidence that LASV is also hosted by other rodent species: the African wood mouse Hylomyscus pamfi in Nigeria, and the Guinea multimammate mouse Mastomys erythroleucus in both Nigeria and Guinea. Virus strains from these animals were isolated in the BSL-4 laboratory and fully sequenced. Phylogenetic analyses of viral genes coding for glycoprotein, nucleoprotein, polymerase and matrix protein show that Lassa strains detected in M. erythroleucus belong to lineages III and IV. The strain from H. pamfi clusters close to lineage I (for S gene) and between II & III (for L gene). Discovery of new rodent hosts has implications for LASV evolution and its spread into new areas within West Africa.

Senna tora is a widely used medicinal plant. Its health benefits have been attributed to the large quantity of anthraquinones, but how they are made in plants remains a mystery. To identify the genes responsible for plant anthraquinone biosynthesis, we reveal the genome sequence of S. tora at the chromosome level with 526 Mb (96%) assembled into 13 chromosomes. Comparison among related plant species shows that a chalcone synthase-like (CHS-L) gene family has lineage-specifically and rapidly expanded in S. tora . Combining genomics, transcriptomics, metabolomics, and biochemistry, we identify a CHS-L gene contributing to the biosynthesis of anthraquinones. The S. tora reference genome will accelerate the discovery of biologically active anthraquinone biosynthesis pathways in medicinal plants. Anthraquinones are aromatic polyketides and have been used for treating various diseases, but the biosynthetic pathway is unclear. Here, the authors assemble the genome of an anthraquinone-producing medicinal plant Senna tora and show the evidences that CHS-like genes may be involved in anthraquinone biosynthesis.

Plants have evolved sophisticated mechanisms to adapt to fluctuating environmental nitrogen availability. However, more underlying genes regulating the response to nitrate have yet to be characterized. We report here the identification of a nitrate regulatory mutant whose mutation mapped to the Cleavage and Polyadenylation Specificity Factor 30 gene (CPSF30-L). In the mutant, induction of nitrate-responsive genes was inhibited independent of the ammonium conditions and was restored by expression of the wild-type 65 kDa encoded by CPSF30-L. Molecular and genetic evidence suggests that CPSF30-L works upstream of NRT1.1 and independently of NLP7 in response to nitrate. Analysis of the 3′-UTR of NRT1.1 showed that the pattern of polyadenylation sites was altered in the cpsf30 mutant. Transcriptome analysis revealed that four nitrogen-related clusters were enriched in the differentially expressed genes of the cpsf30 mutant. Nitrate uptake was decreased in the mutant along with reduced expression of the nitrate transporter/sensor gene NRT1.1, while nitrate reduction and amino acid content were enhanced in roots along with increased expression of several nitrate assimilatory genes. These findings indicate that the 65 kDa protein encoded by CPSF30-L mediates nitrate signaling in part by regulating NRT1.1 expression, thus adding an important component to the nitrate signaling network.

Recent reverse genetics technologies have enabled genetic manipulation of plant negative‐strand RNA virus (NSR) genomes. Here, we report construction of an infectious clone for the maize‐infecting Alphanucleorhabdovirus maydis, the first efficient NSR vector for maize. The full‐length infectious clone was established using agrobacterium‐mediated delivery of full‐length maize mosaic virus (MMV) antigenomic RNA and the viral core proteins (nucleoprotein N, phosphoprotein P, and RNA‐directed RNA polymerase L) required for viral transcription and replication into Nicotiana benthamiana. Insertion of intron 2 ST‐LS1 into the viral L gene increased stability of the infectious clone in Escherichia coli and Agrobacterium tumefaciens. To monitor virus infection in vivo, a green fluorescent protein (GFP) gene was inserted in between the N and P gene junctions to generate recombinant MMV‐GFP. Complementary DNA (cDNA) clones of MMV‐wild type (WT) and MMV‐GFP replicated in single cells of agroinfiltrated N. benthamiana. Uniform systemic infection and high GFP expression were observed in maize inoculated with extracts of the infiltrated N. benthamiana leaves. Insect vectors supported virus infection when inoculated via feeding on infected maize or microinjection. Both MMV‐WT and MMV‐GFP were efficiently transmitted to maize by planthopper vectors. The GFP reporter gene was stable in the virus genome and expression remained high over three cycles of transmission in plants and insects. The MMV infectious clone will be a versatile tool for expression of proteins of interest in maize and cross‐kingdom studies of virus replication in plant and insect hosts. The maize mosaic virus infectious clone enables stable and robust expression of heterologous genes in maize and planthoppers, and is a versatile tool for cross‐kingdom studies of virus replication in plant and insect hosts.

Rice black-streaked dwarf virus (RBSDV) causes maize rough dwarf disease (MRDD), which is a viral disease that significantly affects maize yields worldwide. Plants tolerate stress through transcriptional reprogramming at the alternative splicing (AS), transcriptional, and fusion gene (FG) levels. However, it is unclear whether and how AS and FG interfere with transcriptional reprogramming in MRDD. In this study, we performed global profiling of AS and FG on maize response to RBSDV and compared it with transcriptional changes. There are approximately 1.43 to 2.25 AS events per gene in maize infected with RBSDV. GRMZM2G438622 was only detected in four AS modes (A3SS, A5SS, RI, and SE), whereas GRMZM2G059392 showed downregulated expression and four AS events. A total of 106 and 176 FGs were detected at two time points, respectively, including six differentially expressed genes and five differentially spliced genes. The gene GRMZM2G076798 was the only FG that occurred at two time points and was involved in two FG events. Among these, 104 GOs were enriched, indicating that nodulin-, disease resistance-, and chloroplastic-related genes respond to RBSDV stress in maize. These results provide new insights into the mechanisms underlying post-transcriptional and transcriptional regulation of maize response to RBSDV stress.