Dietary wheat and reduced methane yield are linked to rumen microbiome changes in dairy cows.

Fermentation of pasture grasses and grains in the rumen of dairy cows and other ruminants produces methane as a by-product, wasting energy and contributing to the atmospheric load of greenhouse gasses. Many feeding trials in farmed ruminants have tested the impact of dietary components on feed efficiency, productivity and methane yield (MeY). Such diets remodel the rumen microbiome, altering bacterial, archaeal, fungal and protozoan populations, with an altered fermentation outcome. In dairy cows, some dietary grains can reduce enteric methane production. This is especially true of wheat, in comparison to corn or barley. Using a feeding trial of cows fed rolled wheat, corn or barley grain, in combination with hay and canola, we identified wheat-associated changes in the ruminal microbiome. Ruminal methane production, pH and VFA concentration data together with 16S rRNA gene amplicon sequences were used to compare ruminal bacterial and archaeal populations across diets. Differential abundance analysis of clustered sequences (OTU) identified members of the bacterial families Lachnospiraceae, Acidaminococcaceae, Eubacteriaceae, Prevotellaceae, Selenomonadaceae, Anaerovoracaceae and Fibrobacteraceae having a strong preference for growth in wheat-fed cows. Within the methanogenic archaea, (at >99% 16S rRNA sequence identity) the growth of Methanobrevibacter millerae was favoured by the non-wheat diets, while Methanobrevibacter olleyae was unaffected. From the wheat-preferring bacteria, correlation analysis found OTU strongly linked to reduced MeY, reduced pH and raised propionic acid levels. OTU from the genera Shuttleworthia and Prevotella_7 and especially Selenomonadaceae had high anti-methane correlations. An OTU likely representing (100% sequence identity) the fumarate-reducing, hydrogen-utilising, rumen bacterium Mitsuokella jalaludinii, had an especially high negative correlation coefficient (-0.83) versus MeY and moderate correlation (-0.6) with rumen pH, strongly suggesting much of the MeY suppression is due to reduced hydrogen availablity. Other OTU, representing as yet unknown species from the Selenomonadaceae family and the genera Prevotella_7, Fibrobacter and Syntrophococcus also had high to moderate negative MeY correlations, but low correlation with pH. These latter likely represent bacterial species able to reduce MeY without causing greater ruminal acidity, making them excellent candidates, provided they can be isolated, for development as anti-methane probiotics.

Genome-wide association studies dissect the G × E interaction for agronomic traits in a worldwide collection of safflowers (Carthamus tinctorius L.).

Genome-wide association studies were conducted using a globally diverse safflower (Carthamus tinctorius L.) Genebank collection for grain yield (YP), days to flowering (DF), plant height (PH), 500 seed weight (SW), seed oil content (OL), and crude protein content (PR) in four environments (sites) that differed in water availability. Phenotypic variation was observed for all traits. YP exhibited low overall genetic correlations (rGoverall) across sites, while SW and OL had high rGoverall and high pairwise genetic correlations (rGij) across all pairwise sites. In total, 92 marker-trait associations (MTAs) were identified using three methods, single locus genome-wide association studies (GWAS) using a mixed linear model (MLM), the Bayesian multi-locus method (BayesR), and meta-GWAS. MTAs with large effects across all sites were detected for OL, SW, and PR, and MTAs specific for the different water stress sites were identified for all traits. Five MTAs were associated with multiple traits; 4 of 5 MTAs were variously associated with the three traits of SW, OL, and PR. This study provided insights into the phenotypic variability and genetic architecture of important safflower agronomic traits under different environments. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01295-8.

Faecalibacterium diversity in dairy cow milk.

The bacterial species, Faecalibacterium prausnitzii, beneficial to humans and animals and found in mammalian and avian gut, is also occasionally found in dairy cow milk. It is one of the butyrate-producing bacteria of the colon, has anti-inflammatory properties and its abundance in the gut is negatively correlated with obesity in humans. Several strains differing in their functional capability, have been identified. It is important therefore, milk being a potential source of F. prausnitzii as a novel probiotic, to investigate the diversity of this species in bovine milk. Using 16s rRNA gene amplicons we find 292 different dereplicated Faecalibacterium-related amplicons in a herd of 21 dairy cows. The distribution of the 20 most abundant amplicons with >97% identity to a Greengenes OTU varies from cow to cow. Clustering of the 292 pooled sequences from all cows at 99.6% identity finds 4 likely Faecalibacterium phylotypes with >98.5% identity to an F. prausnitzii reference sequence. Sequence alignment and phylogenetic analysis shows these phylotypes are distinct from 34 other species from the Ruminococcaceae family and displaying the sequence clusters as a network illustrates how each cluster is composed of sequences from multiple cows. We conclude there are several phylotypes of Faecalibacterium prausnitzii (the only species so far defined for the genus) in this dairy herd with cows being inoculated with a mixture of several strains from a common source. We conclude that not only can Faecalibacterium be detected in dairy cow milk (as noted by others) but that there exist multiple different strains in the milk of a dairy herd. Therefore milk, as an alternative to faeces, offers the opportunity of discovering new strains with potential probiotic application.

Metabolomics approaches for the discrimination of disease suppressive soils for Rhizoctonia solani AG8 in cereal crops using 1H NMR and LC-MS.

The suppression of soilborne crop pathogens such as Rhizoctonia solani AG8 may offer a sustainable and enduring method for disease control, though soils with these properties are difficult to identify. In this study, we analysed the soil metabolic profiles of suppressive and non-suppressive soils over 2 years of cereal production. We collected bulk and rhizosphere soil at different cropping stages and subjected soil extracts to liquid chromatography-mass spectrometry (LC-MS) and proton nuclear magnetic resonance spectroscopy (1H NMR) analyses. Community analyses of suppressive and non-suppressive soils using principal component analyses and predictive modelling of LC-MS and NMR datasets respectively, revealed distinct biochemical profiles for the two soil types with clustering based on suppressiveness and cropping stage. NMR spectra revealed the suppressive soils to be more abundant in sugar molecules than non-suppressive soils, which were more abundant in lipids and terpenes. LC-MS features that were significantly more abundant in the suppressive soil were identified and assessed as potential biomarkers for disease suppression. The structures of a potential class of LC-MS biomarkers were elucidated using accurate mass data and MS fragmentation spectrum information. The most abundant compound found in association with suppressive soils was confirmed to be a macrocarpal, which is an antimicrobial secondary metabolite. Our study has demonstrated the utility of environmental metabolomics for the study of disease suppressive soils, resulting in the discovery of a macrocarpal biomarker for R. solani AG8 suppressive soil which can be further studied functionally in association with suppression pot trials and microbial isolation studies.

Comparative Metatranscriptomics of Wheat Rhizosphere Microbiomes in Disease Suppressive and Non-suppressive Soils for Rhizoctonia solani AG8.

The soilborne fungus Rhizoctonia solani anastomosis group (AG) 8 is a major pathogen of grain crops resulting in substantial production losses. In the absence of resistant cultivars of wheat or barley, a sustainable and enduring method for disease control may lie in the enhancement of biological disease suppression. Evidence of effective biological control of R. solani AG8 through disease suppression has been well documented at our study site in Avon, South Australia. A comparative metatranscriptomic approach was applied to assess the taxonomic and functional characteristics of the rhizosphere microbiome of wheat plants grown in adjacent fields which are suppressive and non-suppressive to the plant pathogen R. solani AG8. Analysis of 12 rhizosphere metatranscriptomes (six per field) was undertaken using two bioinformatic approaches involving unassembled and assembled reads. Differential expression analysis showed the dominant taxa in the rhizosphere based on mRNA annotation were Arthrobacter spp. and Pseudomonas spp. for non-suppressive samples and Stenotrophomonas spp. and Buttiauxella spp. for the suppressive samples. The assembled metatranscriptome analysis identified more differentially expressed genes than the unassembled analysis in the comparison of suppressive and non-suppressive samples. Suppressive samples showed greater expression of a polyketide cyclase, a terpenoid biosynthesis backbone gene (dxs) and many cold shock proteins (csp). Non-suppressive samples were characterised by greater expression of antibiotic genes such as non-heme chloroperoxidase (cpo) which is involved in pyrrolnitrin synthesis, and phenazine biosynthesis family protein F (phzF) and its transcriptional activator protein (phzR). A large number of genes involved in detoxifying reactive oxygen species (ROS) and superoxide radicals (sod, cat, ahp, bcp, gpx1, trx) were also expressed in the non-suppressive rhizosphere samples most likely in response to the infection of wheat roots by R. solani AG8. Together these results provide new insight into microbial gene expression in the rhizosphere of wheat in soils suppressive and non-suppressive to R. solani AG8. The approach taken and the genes involved in these functions provide direction for future studies to determine more precisely the molecular interplay of plant-microbe-pathogen interactions with the ultimate goal of the development of management options that promote beneficial rhizosphere microflora to reduce R. solani AG8 infection of crops.

Transcriptome sequencing of field pea and faba bean for discovery and validation of SSR genetic markers.

BACKGROUND: Field pea (Pisum sativum L.) and faba bean (Vicia faba L.) are cool-season grain legume species that provide rich sources of food for humans and fodder for livestock. To date, both species have been relative 'genomic orphans' due to limited availability of genetic and genomic information. A significant enrichment of genomic resources is consequently required in order to understand the genetic architecture of important agronomic traits, and to support germplasm enhancement, genetic diversity, population structure and demographic studies. RESULTS: cDNA samples obtained from various tissue types of specific field pea and faba bean genotypes were sequenced using 454 Roche GS FLX Titanium technology. A total of 720,324 and 304,680 reads for field pea and faba bean, respectively, were de novo assembled to generate sets of 70,682 and 60,440 unigenes. Consensus sequences were compared against the genome of the model legume species Medicago truncatula Gaertn., as well as that of the more distantly related, but better-characterised genome of Arabidopsis thaliana L.. In comparison to M. truncatula coding sequences, 11,737 and 10,179 unique hits were obtained from field pea and faba bean. Totals of 22,057 field pea and 18,052 faba bean unigenes were subsequently annotated from GenBank. Comparison to the genome of soybean (Glycine max L.) resulted in 19,451 unique hits for field pea and 16,497 unique hits for faba bean, corresponding to c. 35% and 30% of the known gene space, respectively. Simple sequence repeat (SSR)-containing expressed sequence tags (ESTs) were identified from consensus sequences, and totals of 2,397 and 802 primer pairs were designed for field pea and faba bean. Subsets of 96 EST-SSR markers were screened for validation across modest panels of field pea and faba bean cultivars, as well as related non-domesticated species. For field pea, 86 primer pairs successfully obtained amplification products from one or more template genotypes, of which 59% revealed polymorphism between 6 genotypes. In the case of faba bean, 81 primer pairs displayed successful amplification, of which 48% detected polymorphism. CONCLUSIONS: The generation of EST datasets for field pea and faba bean has permitted effective unigene identification and functional sequence annotation. EST-SSR loci were detected at incidences of 14-17%, permitting design of comprehensive sets of primer pairs. The subsets from these primer pairs proved highly useful for polymorphism detection within Pisum and Vicia germplasm.

Transcriptome sequencing of lentil based on second-generation technology permits large-scale unigene assembly and SSR marker discovery.

BACKGROUND: Lentil (Lens culinaris Medik.) is a cool-season grain legume which provides a rich source of protein for human consumption. In terms of genomic resources, lentil is relatively underdeveloped, in comparison to other Fabaceae species, with limited available data. There is hence a significant need to enhance such resources in order to identify novel genes and alleles for molecular breeding to increase crop productivity and quality. RESULTS: Tissue-specific cDNA samples from six distinct lentil genotypes were sequenced using Roche 454 GS-FLX Titanium technology, generating c. 1.38 × 106 expressed sequence tags (ESTs). De novo assembly generated a total of 15,354 contigs and 68,715 singletons. The complete unigene set was sequence-analysed against genome drafts of the model legume species Medicago truncatula and Arabidopsis thaliana to identify 12,639, and 7,476 unique matches, respectively. When compared to the genome of Glycine max, a total of 20,419 unique hits were observed corresponding to c. 31% of the known gene space. A total of 25,592 lentil unigenes were subsequently annoated from GenBank. Simple sequence repeat (SSR)-containing ESTs were identified from consensus sequences and a total of 2,393 primer pairs were designed. A subset of 192 EST-SSR markers was screened for validation across a panel 12 cultivated lentil genotypes and one wild relative species. A total of 166 primer pairs obtained successful amplification, of which 47.5% detected genetic polymorphism. CONCLUSIONS: A substantial collection of ESTs has been developed from sequence analysis of lentil genotypes using second-generation technology, permitting unigene definition across a broad range of functional categories. As well as providing resources for functional genomics studies, the unigene set has permitted significant enhancement of the number of publicly-available molecular genetic markers as tools for improvement of this species.

A neurotropic herpesvirus infecting the gastropod, abalone, shares ancestry with oyster herpesvirus and a herpesvirus associated with the amphioxus genome.

BACKGROUND: With the exception of the oyster herpesvirus OsHV-1, all herpesviruses characterized thus far infect only vertebrates. Some cause neurological disease in their hosts, while others replicate or become latent in neurological tissues. Recently a new herpesvirus causing ganglioneuritis in abalone, a gastropod, was discovered. Molecular analysis of new herpesviruses, such as this one and others, still to be discovered in invertebrates, will provide insight into the evolution of herpesviruses. RESULTS: We sequenced the genome of a neurotropic virus linked to a fatal ganglioneuritis devastating parts of a valuable wild abalone fishery in Australia. We show that the newly identified virus forms part of an ancient clade with its nearest relatives being a herpesvirus infecting bivalves (oyster) and, unexpectedly, one we identified, from published data, apparently integrated within the genome of amphioxus, an invertebrate chordate. Predicted protein sequences from the abalone virus genome have significant similarity to several herpesvirus proteins including the DNA packaging ATPase subunit of (putative) terminase and DNA polymerase. Conservation of amino acid sequences in the terminase across all herpesviruses and phylogenetic analysis using the DNA polymerase and terminase proteins demonstrate that the herpesviruses infecting the molluscs, oyster and abalone, are distantly related. The terminase and polymerase protein sequences from the putative amphioxus herpesvirus share more sequence similarity with those of the mollusc viruses than with sequences from any of the vertebrate herpesviruses analysed. CONCLUSIONS: A family of mollusc herpesviruses, Malacoherpesviridae, that was based on a single virus infecting oyster can now be further established by including a distantly related herpesvirus infecting abalone, which, like many vertebrate viruses is neurotropic. The genome of Branchiostoma floridae (amphioxus) provides evidence for the existence of a herpesvirus associated with this invertebrate chordate. The virus which likely infected amphioxus is, by molecular phylogenetic analysis, more closely related to the other 2 invertebrate viruses than to herpesviruses infecting vertebrates (ie chordates).

Haemonchus contortus: molecular characterisation of a small heat shock protein.

A cDNA encoding a predicted small heat shock protein, HSP20, was isolated from the parasitic nematode Haemonchus contortus. This cDNA encoded a predicted protein of 156 amino acids, which had high sequence identity with other nematode small heat shock proteins. Southern blot analysis of genomic DNA suggested that in H. contortus HSP20 is encoded by a single copy gene. The HSP20 transcript and protein were expressed in the infective larvae (L3), early L4 and adult stages, but expression was not increased by heat shock treatment. In situ hybridisation analysis was used to localise expression of HSP20 mRNA in the adult parasite. Similar HSPs (heat shock protein) were detected by Western blotting in Ancylostoma caninum, Dictyocaulus viviparus, and Toxocara canis, but not in Trichostronglyus colubriformis. The conservation of HSP20 in several different nematode species may reflect its importance to parasites that require mammalian hosts as a part of their development. Index Descriptors and Abbreviations: Haemonchus contortus; nematode; small heat shock protein; L3, infective larvae; xL3, exsheathed L3; eL4, early L4; EST, expressed sequence tag; HSP20, heat shock protein 20; sHSP, small heat shock protein