Analysis Of Sequence Variation Research Articles

Identification and validation of two quantitative trait loci showing pleiotropic effect on multiple grain-related traits in bread wheat (Triticum aestivum L.).

QKl/Tgw/Gns.yaas-2D associates with KL, TGW, and GNS, and QKl/Tgw.yaas-5A associates with KL and TGW. Significantly pleiotropic and additive effects of these two QTL were validated. The YM5 allele both at QKl/Tgw/Gns.yaas-2D and QKl/Tgw.yaas-5A was proved to be the best allelic combination for improving yield potential. Kernel length (KL), kernel width (KW), thousand grain weight (TGW), and grain number per spike (GNS) play important roles in the yield improvement of wheat. In this study, one recombinant inbred line (RIL) derived from a cross between Yangmai 5 (YM5) and Yanzhan 1 (YZ1) was used to identify quantitative trait loci (QTL) associated with KL, KW, TGW, and GNS across three years. Two pleiotropic QTL namely QKl/Tgw/Gns.yaas-2D and QKl/Tgw.yaas-5A were located in two genomic regions on chromosomes 2D and 5A, respectively. Breeder-friendly Kompetitive Allele-Specific PCR (KASP) markers for QKl/Tgw/Gns.yaas-2D and QKl/Tgw.yaas-5A were developed and validated in a set of 246 wheat cultivars/lines. Analysis of allelic combinations indicated that the YM5 allele both at QKl/Tgw/Gns.yaas-2D and QKl/Tgw.yaas-5A is probably the best one to promote TGW, GNS, and grain weight per spike. Based on the analysis of gene annotation, sequence variations, expression patterns, and GO enrichment, twenty-five and twenty-four candidate genes of QKl/Tgw/Gns.yaas-2D and QKl/Tgw.yaas-5A, respectively, were identified. These results provide the basis of fine-mapping the target QTL and marker-assisted selection in wheat yield-breeding programs.

NCBI RefSeq: reference sequence standards through 25years of curation and annotation.

Reference sequences and annotations serve as the foundation for many lines of research today, from organism and sequence identification to providing a core description of the genes, transcripts and proteins found in an organism's genome. Interpretation of data including transcriptomics, proteomics, sequence variation and comparative analyses based on reference gene annotations informs our understanding of gene function and possible disease mechanisms, leading to new biomedical discoveries. The Reference Sequence (RefSeq) resource created at the National Center for Biotechnology Information (NCBI) leverages both automatic processes and expert curation to create a robust set of reference sequences of genomic, transcript and protein data spanning the tree of life. RefSeq continues to refine its annotation and quality control processes and utilize better quality genomes resulting from advances in sequencing technologies as well as RNA-Seq data to produce high-quality annotated genomes, ortholog predictions across more organisms and other products that are easily accessible through multiple NCBI resources. This report summarizes the current status of the eukaryotic, prokaryotic and viral RefSeq resources, with a focus on eukaryotic annotation, the increase in taxonomic representation and the effect it will have on comparative genomics. The RefSeq resource is publicly accessible at https://www.ncbi.nlm.nih.gov/refseq.

Specifications of the ACMG/AMP variant curation guidelines for the analysis of germline ATM sequence variants.

The ClinGen Hereditary Breast, Ovarian, and Pancreatic Cancer (HBOP) Variant Curation Expert Panel (VCEP) is composed of internationally recognized experts in clinical genetics, molecular biology, and variant interpretation. This VCEP made specifications for the American College of Medical Genetics and Association for Molecular Pathology (ACMG/AMP) guidelines for the ataxia telangiectasia mutated (ATM) gene according to the ClinGen protocol. These gene-specific rules for ATM were modified from the ACMG/AMP guidelines and were tested against 33 ATM variants of various types and classifications in a pilot curation phase. The pilot revealed a majority agreement between the HBOP VCEP classifications and the ClinVar-deposited classifications. Six pilot variants had conflicting interpretations in ClinVar, and re-evaluation with the VCEP's ATM-specific rules resulted in four that were classified as benign, one as likely pathogenic, and one as a variant of uncertain significance (VUS) by the VCEP, improving the certainty of interpretations in the public domain. Overall, 28 of the 33 pilot variants were not VUS, leading to an 85% classification rate. The ClinGen-approved, modified rules demonstrated value for improved interpretation of variants in ATM.

Immunodiagnostic detection of banana bract mosaic virus (BBrMV) infecting abaca through the recombinant coat protein and its sequence diversity analysis in the Philippines

Abaca is an indigenous fiber crop in the Philippines and is an important source of raw fiber worldwide. Banana bract mosaic virus (BBrMV) causes the abaca bract mosaic disease which results in decreased fiber yield and quality. Out of the 261 abaca samples tested, 175 tested positive via ELISA using raw antiserum against the recombinant BBrMV coat protein (CP). To further validate the antisera produced, a total of 48 samples were screened for BBrMV CP fragment via RT-PCR. Out of the 26 positives for RT-PCR, 22 were cloned and sequenced to validate the RT-PCR amplified fragments. Nucleotide analysis showed a high percentage identity (97–98%) towards BBrMV CP Pacific and Indian isolates while phylogenetic analysis revealed clustering of BBrMV CP Philippine abaca isolates with the Pacific group. The results validate the application of polyclonal antibodies against the recombinant coat protein of BBrMV (rCP) for its serological detection in infected abaca.

DNA Barcoding Using 18S rRNA Gene Fragments for Identification of Tick-Borne Protists in Ticks in the Republic of Korea.

The objective of this study was to evaluate the diversity and prevalence of tick-borne protists in the Republic of Korea via DNA barcoding using 18S rRNA gene fragments and PCR. Between 2021 and 2022, questing ticks were collected using the flagging method, with a total of 13,375 ticks collected and pooled into 1003 samples. Of these, 50 tick pools were selected for DNA barcoding targeting the V4 and V9 regions of 18S rRNA using the MiSeq platform. A taxonomic analysis of the amplicon sequence variants identified three genera of protozoa, namely Hepatozoon canis, Theileria luwenshuni, and Gregarine sp. However, the number and abundance of protists detected were different depending on the primer sets, and T. gondii was not identified in DNA barcoding. Furthermore, conventional PCR confirmed the presence of H. canis, Toxoplasma gondii, T. luwenshuni, and Theileria sp. in the collected ticks. This study identified H. canis and T. gondii in Ixodes nipponensis for the first time. It demonstrated that the results of DNA barcoding using 18S rRNA gene fragments can vary depending on the primer sets and further optimization is required for library construction to identify tick-borne protists in ticks. Despite these limitations, the findings highlight the potential of DNA barcoding using 18S rRNA gene fragments for screening the diversity of tick-borne protists in ticks.

A point mutation in the zinc-finger transcription factor CqLOL1 controls the green flesh color in chieh-qua (Benincasa hispida Cogn. var. Chieh-qua How).

Flesh color is an essential trait in chieh-qua (Benincasa hispida Cogn. var. Chieh-qua How); however, the inheritance and molecular basis of green flesh trait remain unclear. In the present study, two F2 populations, derived from 1742 (white flesh) × FJ3211 (green flesh) and J16 (white flesh) × FJ5 (green flesh), were used to identify the green flesh (Cqgf) locus. Genetic analysis revealed that the presence of green flesh was a quantitative trait that closely followed a normal distribution. Combining the results from QTL mapping and BSA-seq analysis, the Cqgf locus was preliminarily determined to be located on chromosome 05 and was narrowed down to a 2.55-Mb interval by linkage analysis. A large J16 × FJ5 F2 population comprising 3,180 individuals was subsequently used to screen the recombinants, and the Cqgf locus was fine-mapped to a region of 329.70 kb that harbors six genes. One of the candidate genes, Bch05G003700, the zinc-finger transcription factor LOL1 (lsd one like 1 protein; CqLOL1), was the strongest candidate gene for the Cqgf locus according to sequence variation and expression analysis. Additionally, a point mutation (A > C) in CqLOL1 resulted in the substitution of threonine (T) with proline (P) in the amino acid sequence, showing a complete relationship linked with flesh color in a panel of 45 germplasms. The study suggests that CqLOL1 promotes the accumulation of chlorophyll content in chieh-qua and lead to green flesh. Our findings establish a theoretical and technical foundation for breeding different flesh color lines and elucidating the underlying mechanisms of flesh color in chieh-qua.

Morphometric trait normalization, cluster analysis and molecular phylogenetic analysis in delimiting sympatric Neolissochilus hexastichus and N. hexagonolepis in Umngi River, Meghalaya (India)

Aim: To assess the ability of an integrative approach comprising multivariate morphometric data analysis and mitochondrial cytochrome oxidase subunit I (CO I) sequence divergence analysis in delimiting sympatric Neolissochilus hexastichus (McClleland, 1839) and N. hexagonolepis (McClleland, 1839) mahseer populations in River Umngi, Meghalaya. Methodology: Authoritative descriptions of mahseers were referred during the sampling of 40 individuals from River Umngi, Meghalaya. Multivariate morphometric data were normalized with respect to standard length. The normalization procedure maintained the particular shape factor for each sample. Normalised data were subjected to hierarchical clustering on principal components Analysis (HCPC) for grouping samples according to morphometric features. Genetic affinities based on CO I sequence divergence were assessed by Bayesian Inference (BI) and Automatic Barcode Gap Discovery (ABGD) protocols. Results: The normalization protocols removed the variations in traits due to body size effects and facilitated the identification of allometric traits. HCPC analysis revealed morphometric features that had a significant impact on clustering. Nine N. hexagonolepis and 13 N. hexastichus individuals were identified by integrative morphological and molecular phylogenetic approach. The remaining 18 individuals had incongruence in their memberships in the morphometric and phylogenetic clusters. Interpretation: The integrative approach, apart from delineating N. hexagonolepis and N. hexastichus congeners, highlighted the need to include nuclear genetic markers in future studies for confirming the presence of natural hybrids in sympatric populations of Neolissochilus mahseers. Key words: Cluster, Morphometric, Neolissochilus, Normalization, Phylogenetics

Characterization of Nuclear Mitochondrial Insertions in Canine Genome Assemblies

Background: The presence of mitochondrial sequences in the nuclear genome (Numts) confounds analyses of mitochondrial sequence variation, and is a potential source of false positives in disease studies. To improve the analysis of mitochondrial variation in canines, we completed a systematic assessment of Numt content across genome assemblies, canine populations and the carnivore lineage. Results: Centering our analysis on the UU_Cfam_GSD_1.0/canFam4/Mischka assembly, a commonly used reference in dog genetic variation studies, we found a total of 321 Numts located throughout the nuclear genome and encompassing the entire sequence of the mitochondria. A comparison with 14 canine genome assemblies identified 63 Numts with presence–absence dimorphism among dogs, wolves, and a coyote. Furthermore, a subset of Numts were maintained across carnivore evolutionary time (arctic fox, polar bear, cat), with eight sequences likely more than 10 million years old, and shared with the domestic cat. On a population level, using structural variant data from the Dog10K Consortium for 1879 dogs and wolves, we identified 11 Numts that are absent in at least one sample, as well as 53 Numts that are absent from the Mischka assembly. Conclusions: We highlight scenarios where the presence of Numts is a potentially confounding factor and provide an annotation of these sequences in canine genome assemblies. This resource will aid the identification and interpretation of polymorphisms in both somatic and germline mitochondrial studies in canines.

Mitochondrial DNA sequence-based identification of two subterranean termite species, from Riyadh Province, Kingdom of Saudi Arabia.

Termites are economically important wood-destroying and agricultural pests. The termite fauna almost consists of 2900 described species in 286 genera worldwide. In the present study, hundreds of termite samples from 42 different locations in the Riyadh province were collected. These samples were previously used for morphometric identification and reported two subterranean termite species, Coptotermes heimi and Psammotermes hypostoma, in the family Rhinotermitidae. In the present study, these samples were analysed using DNA barcoding with the mitochondrial cytochrome c oxidase subunit 1 gene to confirm the conventional taxonomical identification on a molecular basis. The obtained COI gene sequences of all 42 termite specimens were submitted to GenBank (accession numbers: ON529959-ON529969, OP825131-OP825132, and OP890882-OP890910). Eleven of the 42 samples were thus identified as C. heimi and the remaining 31 samples as P. hypostoma, which were phylogenetically analysed. All the 11 C. heimi sequences were grouped in a single clade, indicating close relatedness. While 31 sequences of P. hypostoma constituted two clades in the phylogenetic tree. Pairwise nucleotide sequence identity and divergence analysis showed that C. heimi sequences showed high nucleotide identities of 87.6-99.5% and less divergence ranging from 0.5% to 13.6%. Similarly, sequences of P. hypostoma also showed high nucleotide identity of 78.6-100% and low divergence among them ranging from 0-10.7%. A further application, significance, and shortcomings of COI-based DNA barcoding have been discussed. DNA barcoding using the COI gene is a reliable tool to distinguish C. heimi and P. hypostoma genotypes.

Comparative analysis based on shared amplicon sequence variants reveals that cohabitation influences gut microbiota sharing between humans and dogs.

The One Health concept is a comprehensive understanding of the interaction between humans, animals, and the environment. The cohabitation of humans and pets positively affects their physical, mental, and social well-being. It is recognized as an essential factor from the One Health perspective. Furthermore, a healthy balance in the gut microbiome is essential for good health, and the changes in the gut microbiome associated with cohabitation between humans and pets could potentially affect various aspects of the health of both hosts. Therefore, elucidating the sharing of gut bacteria between humans and pets associated with cohabitation is important for understanding One Health. However, most studies have examined sharing at the taxonomic level, and it remains unclear whether the same bacteria are transferred between humans and pets, and whether they mutually influence each other. Here, microbiome analysis and shared 16S rRNA gene amplicon sequence variant (ASV) analysis were conducted before the start of cohabitation between humans and dogs, as well as at 2 weeks, 1 month, and 3 months after cohabitation. 16S rRNA gene ASVs analysis indicated that gut microbes have been transferred between humans and dogs. The overall structure of the gut microbiota within human-dog pairs remained unchanged after 3 months of adaptation. However, 11ASVs were shared within human-dog pairs. Many shared ASVs were highly abundant within each host, and this high abundance may be considered a factor that influences bacterial transfer between hosts. Our results provide important insights into the potential for the transfer of gut bacteria between humans and dogs. These findings are considered crucial for understanding the impact of human-dog cohabitation on various aspects of health.

Integration Linkage Mapping and Comparative Transcriptome Analysis to Dissect the Genetic Basis of Rice Salt Tolerance Associated with the Germination Stage.

Soil salinity poses a serious threat to rice production. The salt tolerance of rice at the germination stage is one of the major determinants of stable stand establishment, which is very important for direct seeding in saline soil. The complexity and polygenic nature of salt tolerance have limited the efficiency of discovering and cloning key genes in rice. In this study, an RIL population with an ultra-high-density genetic map was employed to investigate the salt-tolerant genetic basis in rice, and a total of 20 QTLs were detected, including a major and stable QTL (qRCL3-1). Subsequently, salt-specific DEGs from a comparative transcriptome analysis were overlaid onto annotated genes located on a stable QTL interval, and eight putative candidate genes were further identified. Finally, from the sequence alignment and variant analysis, OsCam1-1 was confirmed to be the most promising candidate gene for regulating salinity tolerance in rice. This study provides important information for elucidating the genetic and molecular basis of rice salt tolerance at the germination stage, and the genes detected here will be useful for improvements in rice salt tolerance.

Characterisation of Ovine KRTAP19-3 and Its Impact on Wool Traits in Chinese Tan Sheep.

Wool, a natural fibre derived from sheep, can present a challenge to wool processing and manufacturing industries because of the variation in fibre traits. Genetic improvement offers one solution to this challenge, and having a better understanding of the genes that affect wool fibre traits is therefore important. Here, we describe ovine KRTAP19-3, a new member of the KAP19 gene family. Phylogenetic analysis revealed its relationship to other known KRTAP19 gene sequences, and an analysis of the nucleotide sequence variation in KRTAP19-3 from 288 sheep of a variety of breeds revealed six unique variant sequences. Among these variants, eleven single nucleotide polymorphisms (SNPs) were detected, with six located in the coding region. Three of these coding region SNPs were non-synonymous and would result in amino acid changes. Associations were observed between the presence of specific sequence variants in Chinese Tan sheep and wool trait variation, particularly an increase in fibre diameter variability in the heterotypic hair fibres. These findings enhance our understanding of the genes that encode sheep wool proteins.

Species limits and hybridization in Andean leaf-eared mice (Phyllotis).

Leaf-eared mice (genus Phyllotis) are among the most widespread and abundant small mammals in the Andean Altiplano, but species boundaries and distributional limits are often poorly delineated due to sparse survey data from remote mountains and high-elevation deserts. Here we report a combined analysis of mitochondrial DNA variation and whole-genome sequence (WGS) variation in Phyllotis mice to delimit species boundaries, to assess the timescale of diversification of the group, and to examine evidence for interspecific hybridization. Estimates of divergence dates suggest that most diversification of Phyllotis occurred during the past 3 million years. Consistent with the Pleistocene Aridification hypothesis, our results suggest that diversification of Phyllotis largely coincided with climatically induced environmental changes in the mid- to late Pleistocene. Contrary to the Montane Uplift hypothesis, most diversification in the group occurred well after the major phase of uplift of the Central Andean Plateau. Species delimitation analyses revealed surprising patterns of cryptic diversity within several nominal forms, suggesting the presence of much undescribed alpha diversity in the genus. Results of genomic analyses revealed evidence of ongoing hybridization between the sister species Phyllotis limatus and P. vaccarum and suggest that the contemporary zone of range overlap between the two species represents an active hybrid zone.

Yeasts of Pichia (Pichiaceae) dominate the mycobiome of Hermetia illucens (Diptera: Stratiomyidae) larvae during urban composting

Introduction: Hermetia illucens is a fly found worldwide in tropical and temperate regions that feeds on decaying organic matter in its larval stage, which makes them useful to accelerate composing processes. Bacterial component on the larval guts that helps to degrade organic matter is well studied, however fungal communities information is more scarse, specially in tropical regions. Objective: To determine fungal communities in the gut of H. illucens larvae naturally occurring during urban composting processes in a tropical region. Methods: ITS sequencing was employed to characterize fungal communities present in H. illucens larval gut. Results: The analysis of amplicon sequence variants (ASVs) unveiled a notable dominance of Saccharomycetales, being yeasts of the genus Pichia the most abundant. Other relatively abundant yeasts were Candida and Galactomyces and the genus Archaeospora from Archaeosporales. The last two groups not being reported in H. illucens before. Conclusions: Yeasts of the genus Pichia are the most abundant group, this result is in concordance with previous studies which suggest a stable insect-yeast relation. The description of previously unreported fungal groups highlights the importance of continuing to explore the mycobiome dynamics of this larva. This study offers insight into the mycobiome of naturally occurring H. illucens larvae in a tropical region.

Chloroplast Genome Variation and Phylogenetic Relationships of Autochthonous Varieties of Vitis vinifera from the Don Valley.

The autochthonous grape varieties of the Don Valley, situated in southern Russia, constitute a distinctive element of regional cultural heritage. These varieties have been adapted over centuries to the region's specific local climatic and soil conditions. For the most part, these varieties are not imported from other countries. They are closely related to varieties found in Crimea and the North Caucasus. In this study, we obtained the first complete, unfragmented sequences of the chloroplast genomes of eight autochthonous varieties from the Don Valley and one from Crimea. We also performed a comparative analysis of their genomic features. The size of Vitis vinifera chloroplast genome sequences varied from 160,925 to 160,991 bp, depending on the cultivar, with a uniform GC ratio of 37.38%. Each genome consists of four subregions: a single copy region (LSC) ranging from 89,158 to 89,336 bp, a small single copy region (SSC) ranging from 19,070 to 19,073 bp, and a pair of inverted repeat regions (IRa and IRb) in the range of 26,292 to 26,353 bp. The chloroplast genomes of the studied V. vinifera varieties contained 130 genes, including 85 protein-coding genes, 8 rRNA genes, and 37 tRNA genes. The sequence divergence analysis has enabled the identification of four highly variable regions, which may be utilized as potential markers for phylogenetic analysis. The analysis revealed the presence of 58 to 61 SSRs and multiple long repeated sequences in the chloroplast genomes of these varieties. The phylogenetic analyses of the sequences obtained and complete chloroplast genomes available from public databases indicated that the majority of autochthonous V. vinifera varieties do not have a direct origin from any European variety.

Phylodynamic and Epistatic Analysis of Coxsackievirus A24 and Its Variant.

Coxsackievirus A24 (CV-A24) is a human enterovirus that causes acute flaccid paralysis. However, a Coxsackievirus A24 variant (CV-A24v) is the most common cause of eye infections. The causes of these variable pathogenicity and tissue tropism remain unclear. To elucidate the phylodynamics of CV-A24 and CV-A24v, we analyzed a dataset of 66 strains using Bayesian phylodynamic approach, along with detailed sequence variation and epistatic analyses. Six CV-A24 strains available in GenBank and 60 CV-A24v strains, including 11 Taiwanese strains, were included in this study. The results revealed striking differences between CV-A24 and CV-A24v exhibiting long terminal branches in the phylogenetic tree, respectively. CV-A24v presented distinct ladder-like clustering, indicating immune escape mechanisms. Notably, 10 genetic recombination events in the 3D regions were identified. Furthermore, 11 missense mutation signatures were detected to differentiate CV-A24 and CV-A24v; among these mutations, the F810Y substitution may significantly affect the secondary structure of the GH loop of VP1 and subsequently affect the epitopes of the capsid proteins. In conclusion, this study provides critical insights into the evolutionary dynamics and epidemiological characteristics of CV-A24 and CV-A24v, and highlights the differences in viral evolution and tissue tropism.

Transcriptome analysis reveals genes associated with late blight resistance in potato

Late blight is a serious disease of potato worldwide. Our study aimed to unveil genes involved in late blight resistance in potato by RNA-seq analysis after artificial inoculation under controlled conditions. In this study, two potato somatic hybrids (P7 and Crd6) and three varieties such as Kufri Girdhari, Kufri Jyoti and Kufri Bahar (control) were used. Transcriptiome analysis revealed statistically significant (p < 0.05) differentially expressed genes (DEGs), which were analysed into up-regulated and down-regulated genes. Further, DEGs were functionally characterized by the Gene Ontology annotations and the Kyoto Encyclopedia of Genes and Genomes pathways. Overall, some of the up-regulated genes in resistant genotypes were disease resistance proteins such as CC-NBS-LRR resistance protein, ankyrin repeat family protein, cytochrome P450, leucine-rich repeat family protein/protein kinase family, and MYB transcription factor. Sequence diversity analysis based on 38 peptide sequences representing 18 genes showed distinct variation and the presence of three motifs in 15 amino acid sequences. Selected genes were also validated by real-time quantitative polymerase chain reaction analysis. Interestingly, gene expression markers were developed for late blight resistant genotypes. Our study elucidates genes involved in imparting late blight resistance in potato, which will be beneficial for its management strategies in the future.

Effect of dynamic exclusion and the use of FAIMS, DIA and MALDI-mass spectrometry imaging with ion mobility on amyloid protein identification

Amyloidosis is a disease characterized by local and systemic extracellular deposition of amyloid protein fibrils where its excessive accumulation in tissues and resistance to degradation can lead to organ failure. Diagnosis is challenging because of approximately 36 different amyloid protein subtypes. Imaging methods like immunohistochemistry and the use of Congo red staining of amyloid proteins for laser capture microdissection combined with liquid chromatography tandem mass spectrometry (LMD/LC–MS/MS) are two diagnostic methods currently used depending on the expertise of the pathology laboratory. Here, we demonstrate a streamlined in situ amyloid peptide spatial mapping by Matrix Assisted Laser Desorption Ionization–Mass Spectrometry Imaging (MALDI-MSI) combined with Trapped Ion Mobility Spectrometry for potential transthyretin (ATTR) amyloidosis subtyping. While we utilized the standard LMD/LC–MS/MS workflow for amyloid subtyping of 31 specimens from different organs, we also evaluated the potential introduction in the MS workflow variations in data acquisition parameters like dynamic exclusion, or testing Data Dependent Acquisition combined with High-Field Asymmetric Waveform Ion Mobility Spectrometry (DDA FAIMS) versus Data Independent Acquisition (DIA) for enhanced amyloid protein identification at shorter acquisition times. We also demonstrate the use of Mascot’s Error Tolerant Search and PEAKS de novo sequencing for the sequence variant analysis of amyloidosis specimens.

The microbiota of ensiled forages and of bulk tank milk on dairy cattle farms in northern Sweden

Factors contributing to variations in the quality and microbiota of ensiled forages and in bulk tank microbiota in milk from cows fed different forages were investigated. Nutritional quality, fermentation parameters and hygiene quality of forage samples and corresponding bulk tank milk samples collected in 3 periods from 18 commercial farms located in northern Sweden were compared. Principal coordinates analysis revealed that the microbiota in forage and bulk milk, analyzed using 16S rRNA gene-based amplicon sequencing, were significantly different. The genera Lactobacillus, Weissella, and Leuconostoc dominated in forage samples, whereas Pseudomonas, Staphylococcus, and Streptococcus dominated in bulk milk samples. Forage quality and forage-associated microbiota were affected by ensiling method and by use of silage additive. Forages stored in bunker and tower silos (confounded with use of additive) were associated with higher levels of acetic and lactic acid and Lactobacillus. Forage ensiled as bales (confounded with no use of additive) was associated with higher DM content, water-soluble carbohydrate content, pH, yeast count, and the genera Weissella, Leuconostoc, and Enterococcus. For bulk tank milk samples, milking system was identified as the major factor affecting the microbiota and type of forage preservation had little effect. Analysis of common amplicon sequence variants (ASV) suggested that forage was not the major source of Lactobacillus found in bulk tank milk.

Viral Envelope Evolution in Simian-HIV-Infected Neonate and Adult-Dam Pairs of Rhesus Macaques.

We recently demonstrated that Simian-HIV (SHIV)-infected neonate rhesus macaques (RMs) generated heterologous HIV-1 neutralizing antibodies (NAbs) with broadly-NAb (bNAb) characteristics at a higher frequency compared with their corresponding dam. Here, we characterized genetic diversity in Env sequences from four neonate or adult/dam RM pairs: in two pairs, neonate and dam RMs made heterologous HIV-1 NAbs; in one pair, neither the neonate nor the dam made heterologous HIV-1 NAbs; and in another pair, only the neonate made heterologous HIV-1 NAbs. Phylogenetic and sequence diversity analyses of longitudinal Envs revealed that a higher genetic diversity, within the host and away from the infecting SHIV strain, was correlated with heterologous HIV-1 NAb development. We identified 22 Env variable sites, of which 9 were associated with heterologous HIV-1 NAb development; 3/9 sites had mutations previously linked to HIV-1 Env bNAb development. These data suggested that viral diversity drives heterologous HIV-1 NAb development, and the faster accumulation of viral diversity in neonate RMs may be a potential mechanism underlying bNAb induction in pediatric populations. Moreover, these data may inform candidate Env immunogens to guide precursor B cells to bNAb status via vaccination by the Env-based selection of bNAb lineage members with the appropriate mutations associated with neutralization breadth.