Reducing fecal egg count through selective breeding alters dorper lamb response to Haemonchus contortus in an artificial challenge trial.

Infection by gastrointestinal nematodes (GIN), particularly Haemonchus contortus, can be detrimental to sheep health and performance. Genetic susceptibility to GIN varies between breeds, with those lacking high levels of natural resistance often requiring frequent anthelmintic treatment when facing parasitic challenge. Genetic technology can serve as a tool to decrease GIN susceptibility via selection for sheep with reduced fecal egg count (FEC) estimated breeding values (EBVs). However, the physiological changes that result from implementation of this strategy are not well described. Additionally, there is a need for comparison of animals from recent selective breeding against breeds with inherent GIN resistance. In this study we administered a challenge of H. contortus to Dorper x White Dorper (DWD; n = 92) lambs that have been genetically selected for either low (DWD-) or high (DWD+) FEC EBVs and Barbados Blackbelly x Mouflon (BBM; n = 19) lambs from a genetically resistant breed backgrounds. Lamb FEC, packed-cell volume (PCV) and serum IgG were measured at intermittent levels over 5 weeks. At day 21 and day 35, the selectively bred DWD- had a lower mean FEC compared to DWD+, but were higher than BBM. Reductions in both PCV and serum IgG from initial day 0 levels were observed in DWD lambs, but not in BBM. Furthermore, from a subset of lambs (n = 24) harvested at day 21, DWD- only tended (p = 0.056) to have lower mean worm counts than DWD+, with BBM having the lowest mean worm count. Differentially expressed genes (DEGs) identified via RNA-sequencing of abomasal tissue at day 21 indicate a more pronounced Th2 immune response and more rapid worm expulsion occurred in iBBM than iDWD- and iDWD+ lambs. However, gene expression in DWD- suggests an association between reduced FEC EBV and gastric acid secretion and the ability to limit worm fecundity. Ultimately, selection of Dorper sheep for low FEC EBV can reduce susceptibility to GIN, but it will likely require multiple generations with this trait as a breeding priority before presenting a similar resistance level to Caribbean breeds.

A computational framework for improving genetic variants identification from 5,061 sheep sequencing data.

BACKGROUND: Pan-genomics is a recently emerging strategy that can be utilized to provide a more comprehensive characterization of genetic variation. Joint calling is routinely used to combine identified variants across multiple related samples. However, the improvement of variants identification using the mutual support information from multiple samples remains quite limited for population-scale genotyping. RESULTS: In this study, we developed a computational framework for joint calling genetic variants from 5,061 sheep by incorporating the sequencing error and optimizing mutual support information from multiple samples' data. The variants were accurately identified from multiple samples by using four steps: (1) Probabilities of variants from two widely used algorithms, GATK and Freebayes, were calculated by Poisson model incorporating base sequencing error potential; (2) The variants with high mapping quality or consistently identified from at least two samples by GATK and Freebayes were used to construct the raw high-confidence identification (rHID) variants database; (3) The high confidence variants identified in single sample were ordered by probability value and controlled by false discovery rate (FDR) using rHID database; (4) To avoid the elimination of potentially true variants from rHID database, the variants that failed FDR were reexamined to rescued potential true variants and ensured high accurate identification variants. The results indicated that the percent of concordant SNPs and Indels from Freebayes and GATK after our new method were significantly improved 12%-32% compared with raw variants and advantageously found low frequency variants of individual sheep involved several traits including nipples number (GPC5), scrapie pathology (PAPSS2), seasonal reproduction and litter size (GRM1), coat color (RAB27A), and lentivirus susceptibility (TMEM154). CONCLUSION: The new method used the computational strategy to reduce the number of false positives, and simultaneously improve the identification of genetic variants. This strategy did not incur any extra cost by using any additional samples or sequencing data information and advantageously identified rare variants which can be important for practical applications of animal breeding.

Advanced Skeletal Ossification Is Associated with Genetic Variants in Chronologically Young Beef Heifers.

Osteogenesis is a developmental process critical for structural support and the establishment of a dynamic reservoir for calcium and phosphorus. Changes in livestock breeding over the past 100 years have resulted in earlier bone development and increased physical size of cattle. Advanced skeletal maturity is now commonly observed at harvest, with heifers displaying more mature bone than is expected at 30 months of age (MOA). We surmise that selection for growth traits and earlier reproductive maturity resulted in co-selection for accelerated skeletal ossification. This study examines the relationship of single nucleotide polymorphisms (SNPs) in 793 beef heifers under 30 MOA with USDA-graded skeletal maturity phenotypes (A-, B-, C- skeletal maturity). Further, the estrogen content of FDA-approved hormonal implants provided to heifers prior to harvest was evaluated in association with the identified SNPs and maturities. Association tests were performed, and the impact of the implants were evaluated as covariates against genotypes using a logistic regression model. SNPs from the ESR1, ALPL, PPARGC1B, SORCS1 genes, and SNPs near KLF14, ANKRD61, USP42, H1C1, OVCA2, microRNA mir-29a were determined to be associated with the advanced skeletal ossification phenotype in heifers. Higher dosage estrogen implants increased skeletal maturity in heifers with certain SNP genotypes.

Transcriptome analysis of flower colour reveals the correlation between SNP and differential expression genes in Phalaenopsis.

BACKGROUND: Phalaenopsis is an important ornamental plant that has great economic value in the world flower market as one of the most popular flower resources. OBJECTIVE: To investigate the flower colour formation of Phalaenopsis at the transcription level, the genes involved in flower color formation were identified from RNA-seq in this study. METHODS: In this study, white and purple petals of Phalaenopsis were collected and analyzed to obtained (1) differential expression genes (DEGs) between white and purple flower color and (2) the association between single nucleotide polymorphisms (SNP) mutations and DEGs at the transcriptome level. RESULTS: The results indicated that a total of 1,175 DEGs were identified, and 718 and 457 of them were up- and down-regulated genes, respectively. Gene Ontology and pathway enrichment showed that the biosynthesis of the secondary metabolites pathway was key to color formation, and the expression of 12 crucial genes (C4H, CCoAOMT, F3'H, UA3'5'GT, PAL, 4CL, CCR, CAD, CALDH, bglx, SGTase, and E1.11.17) that are involved in the regulation of flower color in Phalaenopsis. CONCLUSION: This study reported the association between the SNP mutations and DEGs for color formation at RNA level, and provides a new insight to further investigate the gene expression and its relationship with genetic variants from RNA-seq data in other species.

Comparative Analysis of Codon Usage Patterns in Nuclear and Chloroplast Genome of Dalbergia (Fabaceae).

The Dalbergia plants are widely distributed across more than 130 tropical and subtropical countries and have significant economic and medicinal value. Codon usage bias (CUB) is a critical feature for studying gene function and evolution, which can provide a better understanding of biological gene regulation. In this study, we comprehensively analyzed the CUB patterns of the nuclear genome, chloroplast genome, and gene expression, as well as systematic evolution of Dalbergia species. Our results showed that the synonymous and optimal codons in the coding regions of both nuclear and chloroplast genome of Dalbergia preferred ending with A/U at the third codon base. Natural selection was the primary factor affecting the CUB features. Furthermore, in highly expressed genes of Dalbergia odorifera, we found that genes with stronger CUB exhibited higher expression levels, and these highly expressed genes tended to favor the use of G/C-ending codons. In addition, the branching patterns of the protein-coding sequences and the chloroplast genome sequences were very similar in the systematic tree, and different with the cluster from the CUB of the chloroplast genome. This study highlights the CUB patterns and features of Dalbergia species in different genomes, explores the correlation between CUB preferences and gene expression, and further investigates the systematic evolution of Dalbergia, providing new insights into codon biology and the evolution of Dalbergia plants.

High-throughput and high-accuracy single-cell RNA isoform analysis using PacBio circular consensus sequencing.

Although long-read single-cell RNA isoform sequencing (scISO-Seq) can reveal alternative RNA splicing in individual cells, it suffers from a low read throughput. Here, we introduce HIT-scISOseq, a method that removes most artifact cDNAs and concatenates multiple cDNAs for PacBio circular consensus sequencing (CCS) to achieve high-throughput and high-accuracy single-cell RNA isoform sequencing. HIT-scISOseq can yield >10 million high-accuracy long-reads in a single PacBio Sequel II SMRT Cell 8M. We also report the development of scISA-Tools that demultiplex HIT-scISOseq concatenated reads into single-cell cDNA reads with >99.99% accuracy and specificity. We apply HIT-scISOseq to characterize the transcriptomes of 3375 corneal limbus cells and reveal cell-type-specific isoform expression in them. HIT-scISOseq is a high-throughput, high-accuracy, technically accessible method and it can accelerate the burgeoning field of long-read single-cell transcriptomics.

Genome-wide characterization of R2R3-MYB gene family in Santalum album and their expression analysis under cold stress.

Sandalwood (Santalum album) is a high-value multifunctional tree species that is rich in aromatic substances and is used in medicine and global cosmetics. Due to the scarcity of land resources in tropical and subtropical regions, land in temperate regions is a potential resource for the development of S. album plantations in order to meet the needs of S. album production and medicine. The R2R3-MYB transcription factor family is one of the largest in plants and plays an important role in the response to various abiotic stresses. However, the R2R3-MYB gene family of S. album has not been studied. In this study, 144 R2R3-MYB genes were successfully identified in the assembly genome sequence, and their characteristics and expression patterns were investigated under various durations of low temperature stress. According to the findings, 31 of the 114 R2R3-MYB genes showed significant differences in expression after cold treatment. Combining transcriptome and weighted gene co-expression network analysis (WGCNA) revealed three key candidate genes (SaMYB098, SaMYB015, and SaMYB068) to be significantly involved in the regulation of cold resistance in S. album. The structural characteristics, evolution, and expression pattern of the R2R3-MYB gene in S. album were systematically examined at the whole genome level for the first time in this study. It will provide important information for future research into the function of the R2R3-MYB genes and the mechanism of cold stress response in S. album.

An eco-evo-devo genetic network model of stress response.

The capacity of plants to resist abiotic stresses is of great importance to agricultural, ecological and environmental sustainability, but little is known about its genetic underpinnings. Existing genetic tools can identify individual genetic variants mediating biochemical, physiological, and cellular defenses, but fail to chart an overall genetic atlas behind stress resistance. We view stress response as an eco-evo-devo process by which plants adaptively respond to stress through complex interactions of developmental canalization, phenotypic plasticity, and phenotypic integration. As such, we define and quantify stress response as the developmental change of adaptive traits from stress-free to stress-exposed environments. We integrate composite functional mapping and evolutionary game theory to reconstruct omnigenic, information-flow interaction networks for stress response. Using desert-adapted Euphrates poplar as an example, we infer salt resistance-related genome-wide interactome networks and trace the roadmap of how each SNP acts and interacts with any other possible SNPs to mediate salt resistance. We characterize the previously unknown regulatory mechanisms driving trait variation; i.e. the significance of a SNP may be due to the promotion of positive regulators, whereas the insignificance of a SNP may result from the inhibition of negative regulators. The regulator-regulatee interactions detected are not only experimentally validated by two complementary experiments, but also biologically interpreted by their encoded protein-protein interactions. Our eco-evo-devo model of genetic interactome networks provides an approach to interrogate the genetic architecture of stress response and informs precise gene editing for improving plants' capacity to live in stress environments.

Evaluation on the phenotypic diversity of Calamansi (Citrus microcarpa) germplasm in Hainan island.

Calamansi or Philippine lime (Citrofortunella macrocarpa) is an important crop for local economic in Hainan Island. There is no study about Calamansi germplasm evaluation and cultivar development. In this study, Calamansi data were collected from 151 of Calamansi seedling trees, and 37 phenotypic traits were analyzed to investigate their genetic diversities. The cluster analysis and principal component analysis were conducted aiming to provide a theoretical basis for the Calamansi genetic improvement. The results of the diversity analysis revealed: (1) the diversity indexes for qualitative traits were ranged from 0.46-1.39, and the traits with the highest genetic diversity level were fruit shaped and pulp colored (H' > 1.20); and the diversity indexes for quantitative traits ranged from 0.67-2.10, with the exception of a lower in fruit juice rate (1.08) and lower in number of petals (0.67). (2) The clustering analysis of phenotypic traits have arranged the samples into 4 categories: the first group characterized by fewer flesh Segment number per fruit (SNF) and more Oil cell number (OCN); the second group had 7 samples, all characterized with larger Crown breadth (CB), higher Yield per tree (YPT), the lager leaf, the higher Ascorbic acid (AA), and less Seed number per fruit (SNPF); the third group had 25 samples characterized by smaller Tree foot diameter (TFD),smaller Fruit shape index (FSI) and higher Total soluble solids (TSS) contain; the fourth group had 87 samples, they were characterized by shorter Petiole length (PEL), larger fruit, higher Juice ratio (JR), multiple Stamen number (SN) and longer Pistil length (PIL). (3) The principal component analysis showed the values of the first 9 major components characteristic vectors were all greater than 3, the cumulative contribution rate reach 72.20%, including the traits of single fruit weight, fruit diameter, tree height, tree canopy width etc. Finally, based on the comprehensive main component value of all samples, the Calamansi individuals with higher testing scores were selected for further observation. This study concludes that Calamansi seedling populations in the Hainan Island holds great genetic diversity in varies traits, and can be useful for the Calamansi variety improvements.

Applications and potentials of nanopore sequencing in the (epi)genome and (epi)transcriptome era.

The Human Genome Project opened an era of (epi)genomic research, and also provided a platform for the development of new sequencing technologies. During and after the project, several sequencing technologies continue to dominate nucleic acid sequencing markets. Currently, Illumina (short-read), PacBio (long-read), and Oxford Nanopore (long-read) are the most popular sequencing technologies. Unlike PacBio or the popular short-read sequencers before it, which, as examples of the second or so-called Next-Generation Sequencing platforms, need to synthesize when sequencing, nanopore technology directly sequences native DNA and RNA molecules. Nanopore sequencing, therefore, avoids converting mRNA into cDNA molecules, which not only allows for the sequencing of extremely long native DNA and full-length RNA molecules but also document modifications that have been made to those native DNA or RNA bases. In this review on direct DNA sequencing and direct RNA sequencing using Oxford Nanopore technology, we focus on their development and application achievements, discussing their challenges and future perspective. We also address the problems researchers may encounter applying these approaches in their research topics, and how to resolve them.

SCSit: A high-efficiency preprocessing tool for single-cell sequencing data from SPLiT-seq.

SPLiT-seq provides a low-cost platform to generate single-cell data by labeling the cellular origin of RNA through four rounds of combinatorial barcoding. However, an automatic and rapid method for preprocessing and classifying single-cell sequencing (SCS) data from SPLiT-seq, which directly identified and labeled combinatorial barcoding reads and distinguished special cell sequencing data, is currently lacking. Here, we develop a high-efficiency preprocessing tool for single-cell sequencing data from SPLiT-seq (SCSit), which can directly identify combinatorial barcodes and UMI of cell types and obtain more labeled reads, and remarkably enhance the retained data from SCS due to the exact alignment of insertion and deletion. Compared with the original method used in SPLiT-seq, the consistency of identified reads from SCSit increases to 97%, and mapped reads are twice than the original. Furthermore, the runtime of SCSit is less than 10% of the original. It can accurately and rapidly analyze SPLiT-seq raw data and obtain labeled reads, as well as effectively improve the single-cell data from SPLiT-seq platform. The data and source of SCSit are available on the GitHub website https://github.com/shang-qian/SCSit.

The complete chloroplast genome sequence of Oxalis corniculata (L.).

Oxalis corniculata L. is a perennial herb with a world-wide distribution. In this study, we sequenced the complete chloroplast genome of O. corniculata, which exhibited a circular genome of 155,182 bp in length with 37.5% GC content. The chloroplast genome contained a canonical quadripartite structure with a large single copy (LSC) region of 83,936 bp, a small single copy (SSC) region of 17,048 bp and a pair of 25,581 bp inverted repeats (IRs). A total of 108 unique genes, including 76 protein-coding genes (PCGs), 28 tRNA genes and four rRNA genes were found in this chloroplast genome. The phylogenetic tree was constructed based on O. corniculata and other 11 chloroplast genome sequences, which showed that O. corniculata was closely grouped with of O. corymbosa and O. drummondii.

Identification of cassava alternative splicing-related genes and functional characterization of MeSCL30 involvement in drought stress.

Alternative splicing (AS) is an important post-transcriptional regulation strategy that can increase the proteome diversity and regulate mRNA level in eukaryote. Multi-exon genes can be alternative spliced to generate two or more transcripts, thereby increasing the adaptation to the external stress conditions in planta. However, AS-related proteins were less explored in cassava which is an important staple crop in the tropical area. A total of 365 genes encoding AS-related proteins were identified and renamed in the cassava genome, and the transcriptional and splicing changes of 15 randomly selected genes were systematically investigated in the tissues under diverse abiotic stress conditions. 13 out of 15 genes undergo AS in the tissues and under diverse environmental stress condition. Importantly, the greatest changes of splicing patterns were found in the leaf or in response to temperature stress, indicating that AS-related proteins had their tissue-specific regulation patterns and might be participated in the plant adaptation to temperature stress. We then found that overexpression of MeSCL30 in Arabidopsis enhanced the tolerance to drought stress through maintaining reactive oxygen species (ROS) homeostasis and increasing the expression of drought-responsive genes. Therefore, these findings refined the AS-related protein-coding genes and provided novel insights for manipulation of AS-related genes in order to enhance the resistance to environmental stress in plant.

Compacting a synthetic yeast chromosome arm.

BACKGROUND: Redundancy is a common feature of genomes, presumably to ensure robust growth under different and changing conditions. Genome compaction, removing sequences nonessential for given conditions, provides a novel way to understand the core principles of life. The synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) system is a unique feature implanted in the synthetic yeast genome (Sc2.0), which is proposed as an effective tool for genome minimization. As the Sc2.0 project is nearing its completion, we have begun to explore the application of the SCRaMbLE system in genome compaction. RESULTS: We develop a method termed SCRaMbLE-based genome compaction (SGC) and demonstrate that a synthetic chromosome arm (synXIIL) can be efficiently reduced. The pre-introduced episomal essential gene array significantly enhances the compacting ability of SGC, not only by enabling the deletion of nonessential genes located in essential gene containing loxPsym units but also by allowing more chromosomal sequences to be removed in a single SGC process. Further compaction is achieved through iterative SGC, revealing that at least 39 out of 65 nonessential genes in synXIIL can be removed collectively without affecting cell viability at 30 °C in rich medium. Approximately 40% of the synthetic sequence, encoding 28 genes, is found to be dispensable for cell growth at 30 °C in rich medium and several genes whose functions are needed under specified conditions are identified. CONCLUSIONS: We develop iterative SGC with the aid of eArray as a generic yet effective tool to compact the synthetic yeast genome.

Efficient assembly of nanopore reads via highly accurate and intact error correction.

Long nanopore reads are advantageous in de novo genome assembly. However, nanopore reads usually have broad error distribution and high-error-rate subsequences. Existing error correction tools cannot correct nanopore reads efficiently and effectively. Most methods trim high-error-rate subsequences during error correction, which reduces both the length of the reads and contiguity of the final assembly. Here, we develop an error correction, and de novo assembly tool designed to overcome complex errors in nanopore reads. We propose an adaptive read selection and two-step progressive method to quickly correct nanopore reads to high accuracy. We introduce a two-stage assembler to utilize the full length of nanopore reads. Our tool achieves superior performance in both error correction and de novo assembling nanopore reads. It requires only 8122 hours to assemble a 35X coverage human genome and achieves a 2.47-fold improvement in NG50. Furthermore, our assembly of the human WERI cell line shows an NG50 of 22 Mbp. The high-quality assembly of nanopore reads can significantly reduce false positives in structure variation detection.

Comparative Genomics Analysis Provides New Strategies for Bacteriostatic Ability of Bacillus velezensis HAB-2.

Biocontrol formulations prepared from biocontrol bacteria are increasingly applied in sustainable agriculture. Notably, inoculants prepared from Bacillus strains have been proven efficient and environmentally friendly alternatives to chemical bactericides. The bacterium Bacillus velezensis HAB-2 (formerly classified as B. amyloliquefaciens HAB-2) is used as a biological control agent in agricultural fields. In this study, we reported a high-quality genome sequence of HAB-2 using third-generation sequencing technology (PacBio RS II). The 3.89 Mb genome encoded 3,820 predicted genes. Comparative analysis among the genome sequences of reference strains B. velezensis FZB42, B. amyloliquefaciens DSM7 and B. subtilis 168 with the HAB-2 genome revealed obvious differences in the variable part of the genomes, while the core genome shared by FZB42 and HAB-2 was similar (96.14%). However, there were differences in the prophage region among the four strains. The numbers of prophage regions and coding genes in HAB-2 and FZB42 were smaller than the other two strains. The HAB-2 genome showed superior ability to produce secondary metabolites and harbored 13 gene clusters involved in synthesis of antifungal and antibacterial acting secondary metabolites. Furthermore, there were two unique clusters: one cluster which encoded lanthipeptide was involved in mersacidin synthesis and another cluster which encoded ladderane was shown to direct an unknown compound. Multidomain enzymes, such as non-ribosomal peptide synthetase and polyketide synthase, control the biosynthesis of secondary metabolites and rely on 4'-phosphopantetheinyl transferases (PPTases). Key genes lpaH2 and a encoded PPTases in HAB-2 encoded 224 and 120 amino acids, respectively. The genomic features revealed that HAB-2 possesses immense potential to synthesize antimicrobial acting secondary metabolites by regulating and controlling gene clusters. The prophage regions and genes encoding PPTases may provide novel insight for the bacteriostatic mechanism of Bacillus in the biological control of plant diseases.

Corrigendum: Distribution Patterns of DNA N6-Methyladenosine Modification in Non-coding RNA Genes.

[This corrects the article DOI: 10.3389/fgene.2020.00268.].

DNA N6-Methyladenine Modification in Wild and Cultivated Soybeans Reveals Different Patterns in Nucleus and Cytoplasm.

DNA 6mA modification, an important newly discovered epigenetic mark, plays a crucial role in organisms and has been attracting more and more attention in recent years. The soybean is economically the most important bean in the world, providing vegetable protein for millions of people. However, the distribution pattern and function of 6mA in soybean are still unknown. In this study, we decoded 6mA modification to single-nucleotide resolution in wild and cultivated soybeans, and compared the 6mA differences between cytoplasmic and nuclear genomes and between wild and cultivated soybeans. The motif of 6mA in the nuclear genome was conserved across the two kinds of soybeans, and ANHGA was the most dominant motif in wild and cultivated soybeans. Genes with 6mA modification in the nucleus had higher expression than those without modification. Interestingly, 6mA distribution patterns in cytoplasm for each soybean were significantly different from those in nucleus, which was reported for the first time in soybean. Our research provides a new insight in the deep analysis of cytoplasmic genomic DNA modification in plants.

Distribution Patterns of DNA N6-Methyladenosine Modification in Non-coding RNA Genes.

N6-methyladenosine (6mA) DNA modification played an important role in epigenetic regulation of gene expression. And the aberrational expression of non-coding genes, as important regular elements of gene expression, was related to many diseases. However, the distribution and potential functions of 6mA modification in non-coding RNA (ncRNA) genes are still unknown. In this study, we analyzed the 6mA distribution of ncRNA genes and compared them with protein-coding genes in four species (Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, and Homo sapiens) using single-molecule real-time (SMRT) sequencing data. The results indicated that the consensus motifs of short nucleotides at 6mA location were highly conserved in four species, and the non-coding gene was less likely to be methylated compared with protein-coding gene. Especially, the 6mA-methylated lncRNA genes were expressed significant lower than genes without methylation in A. thaliana (p = 3.295e-4), D. melanogaster (p = 3.439e-11), and H. sapiens (p = 9.087e-3).. The detection and distribution profiling of 6mA modification in ncRNA regions from four species reveal that 6mA modifications may have effects on their expression level.