Novel Disease Host Resistances in the World Core Collection of Trifolium subterraneum.

Glasshouse and field investigations of the phenotypic expressions of resistance of a 97-member World Core Collection of subterranean clover (Trifolium subterraneum) collected from its native Mediterranean habitat and representing approximately 80% of the total genetic diversity within the known 10,000 accessions of the species against the most important damping-off and root rot (Phytophthora clandestina, and Pythium irregulare) and foliar (Kabatiella caulivora, Uromyces trifolii-repentis, and Erysiphe trifoliorum) pathogens were performed. An additional 28 diverse cultivars were also included. Associations of these genotypes among 18 disease parameters and 17 morphological traits, and among these disease parameters and 24 climatic and eco-geographic variables from their collection sites, were examined. Many genotypes showed strong phenotypic expression of novel host disease resistance against one or more pathogens, enabling their potential deployment as disease-resistant parents in subterranean clover breeding programs. These new sources of resistance enable future "pyramiding" of different resistance genes to improve resistance against these pathogens. Of particular value were genotypes with multiple disease-resistance across soilborne and/or foliar diseases, because many of these pathogens co-occur. All diseases had some parameters significantly correlated with one or more morphological traits and with one or more sites of origin variables. In particular, there were significant negative correlations between damping-off (i.e., germination) and 8 of the 17 morphological characters. The outcomes of these studies provide crucial information to subterranean clover breeding programs, enabling them to simultaneously select genotypes with multiple resistance to co-occurring soilborne and foliar diseases and desirable traits to offer renewed hope for re-establishing a more productive subterranean clover livestock feedbase despite multiple diseases prevailing widely.

An advanced reference genome of Trifolium subterraneum L. reveals genes related to agronomic performance.

Subterranean clover is an important annual forage legume, whose diploidy and inbreeding nature make it an ideal model for genomic analysis in Trifolium. We reported a draft genome assembly of the subterranean clover TSUd_r1.1. Here we evaluate genome mapping on nanochannel arrays and generation of a transcriptome atlas across tissues to advance the assembly and gene annotation. Using a BioNano-based assembly spanning 512 Mb (93% genome coverage), we validated the draft assembly, anchored unplaced contigs and resolved misassemblies. Multiple contigs (264) from the draft assembly coalesced into 97 super-scaffolds (43% of genome). Sequences longer than >1 Mb increased from 40 to 189 Mb giving 1.4-fold increase in N50 with total genome in pseudomolecules improved from 73 to 80%. The advanced assembly was re-annotated using transcriptome atlas data to contain 31 272 protein-coding genes capturing >96% of the gene content. Functional characterization and GO enrichment confirmed gene expression for response to water deprivation, flavonoid biosynthesis and embryo development ending in seed dormancy, reflecting adaptation to the harsh Mediterranean environment. Comparative analyses across Papilionoideae identified 24 893 Trifolium-specific and 6325 subterranean-clover-specific genes that could be mined further for traits such as geocarpy and grazing tolerance. Eight key traits, including persistence, improved livestock health by isoflavonoid production in addition to important agro-morphological traits, were fine-mapped on the high-density SNP linkage map anchored to the assembly. This new genomic information is crucial to identify loci governing traits allowing marker-assisted breeding, comparative mapping and identification of tissue-specific gene promoters for biotechnological improvement of forage legumes.

ATM deficiency differentially affects expression of proteins related to fatty acid oxidation and oxidative stress in a sex-specific manner in response to Western-type diet prior to and following myocardial infarction.

AIMS: Published work has shown that ataxia-telangiectasia mutated kinase (ATM) deficiency is associated with cardioprotective effects in Western-type diet (WD)-fed female mice. This study assessed the expression of proteins related to fatty acid oxidation (FAO) and oxidative stress in WD-fed male and female mouse hearts, and investigated if sex-specific cardioprotective effects in WD-fed female ATM-deficient mice are maintained following myocardial infarction (MI). MAIN METHODS: Wild-type (WT) and ATM-deficient (hKO) mice (both sexes) were placed on WD for 14 weeks. Myocardial tissue from a subset of mice was used for western blot analyses, while another subset of WD-fed mice underwent MI. Heart function was analyzed by echocardiography prior to and 1 day post-MI. KEY FINDINGS: CPT1B (mitochondrial FAO enzyme) expression was lower in male hKO-WD, while it was higher in female hKO-WD vs WT-WD. WD-mediated decrease in ACOX1 (peroxisomal FAO enzyme) expression was only observed in male WT-WD. PMP70 (transports fatty acyl-CoA across peroxisomal membrane) expression was lower in male hKO-WD vs WT-WD. Catalase (antioxidant enzyme) expression was higher, while Nox4 (pro-oxidant enzyme) expression was lower in female hKO-WD vs WT-WD. Heart function was better in female hKO-WD vs WT-WD. However, post-MI heart function was not significantly different among all MI groups. Post-MI, CPT1B and catalase expression was higher in male hKO-WD-MI vs WT-WD-MI, while Nox4 expression was higher in female hKO-WD-MI vs WT-WD-MI. SIGNIFICANCE: Increased mitochondrial FAO and decreased oxidative stress contribute towards ATM deficiency-mediated cardioprotective effects in WD-fed female mice which are abolished post-MI with increased Nox4 expression.

Leaf formononetin content of Trifolium subterraneum increases in response to waterlogging but its proportion of total isoflavones is little changed.

The isoflavone formononetin (F) impacts livestock fertility and cultivars of the pasture legume Trifolium subterraneum L. (subclover) have been selected for F levels ≤0.2% of leaf dry weight. However, the impact of waterlogging (WL) on isoflavones is little studied. We investigated the response of isoflavones, biochanin A (BA), genistein (G) and F, to WL for: (1) Yarloop (high F) and eight low F cultivars each from subspecies subterraneum , brachycalycinum and yanninicum (Experiment 1); and (2) four cultivars and 12 ecotypes of ssp. yanninicum (Experiment 2). WL impacted F: estimated means increased from 0.19% (control) to 0.31% (WL) in Experiment 1 and from 0.61% to 0.97% in Experiment 2. Isoflavones under WL were highly heritable, particularly F (H 2 =95%). The proportions of BA, G and F were little changed by WL, with strong positive correlations between free-drained and waterlogged treatments. Isoflavone contents were not related to WL tolerance, as assessed by shoot relative growth rate. In conclusion, isoflavones varied among genotypes and increased with WL, but the proportion of individual isoflavones in each genotype was stable. High F under WL was unrelated with genotype tolerance to WL. Instead, it was a consequence of inherently high F for that particular genotype.

Morphological diversity within a core collection of subterranean clover (Trifolium subterraneum L.): Lessons in pasture adaptation from the wild.

Subterranean clover (Trifolium subterraneum L.) is a diploid self-pollinated annual pasture legume native to the Mediterranean region and widely sown in southern Australia and other countries with Mediterranean-type climates. This study utilised a core collection of 97 lines, representing around 80% of the genetic diversity of the species, to examine morphological diversity within subterranean clover. A total of 23 quantitative agro-morphological and 13 semi-quantitative morphological marker traits were assayed on the core collection and 28 diverse Australian cultivars as spaced plants in a replicated common garden experiment. Relationships between these traits and 24 climatic and edaphic parameters at their sites of origin were also examined within the core collection. Significant diversity was present for all traits. The Australian cultivars had similar levels of diversity to the core collection for several traits. Among the agro-morphological traits, time to flowering, leaf size and petiole diameter in mid-winter, plant area in late winter, maximum stem length, content of the oestogenic isoflavone biochanin A and total isoflavone content, were correlated with seven or more environmental variables. These can be considered highly adaptive, being the result of strong environmental selection pressure over time. For the first time in a clover species, morphological markers, including leaf mark, anthocyanin pigmentation and pubescence traits, have been associated with rainfall and soil parameters. This suggests they either have an adaptive role or the genes controlling them may be linked to other genes controlling adaptive traits. This study demonstrated the value of core collections to examine diversity within much larger global collections. It also identified adaptive traits from wild plants that can be utilised to develop more productive and persistent subterranean clover cultivars. The high heritability of these traits indicates that selection gains can be readily made.

Climate Clever Clovers: New Paradigm to Reduce the Environmental Footprint of Ruminants by Breeding Low Methanogenic Forages Utilizing Haplotype Variation.

Mitigating methane production by ruminants is a significant challenge to global livestock production. This research offers a new paradigm to reduce methane emissions from ruminants by breeding climate-clever clovers. We demonstrate wide genetic diversity for the trait methanogenic potential in Australia's key pasture legume, subterranean clover (Trifolium subterraneum L.). In a bi-parental population the broadsense heritability in methanogenic potential was moderate (H2 = 0.4) and allelic variation in a region of Chr 8 accounted for 7.8% of phenotypic variation. In a genome-wide association study we identified four loci controlling methanogenic potential assessed by an in vitro fermentation system. Significantly, the discovery of a single nucleotide polymorphism (SNP) on Chr 5 in a defined haplotype block with an upstream putative candidate gene from a plant peroxidase-like superfamily (TSub_g18548) and a downstream lectin receptor protein kinase (TSub_g18549) provides valuable candidates for an assay for this complex trait. In this way haplotype variation can be tracked to breed pastures with reduced methanogenic potential. Of the quantitative trait loci candidates, the DNA-damage-repair/toleration DRT100-like protein (TSub_g26967), linked to avoid the severity of DNA damage induced by secondary metabolites, is considered central to enteric methane production, as are disease resistance (TSub_g26971, TSub_g26972, and TSub_g18549) and ribonuclease proteins (TSub_g26974, TSub_g26975). These proteins are good pointers to elucidate the genetic basis of in vitro microbial fermentability and enteric methanogenic potential in subterranean clover. The genes identified allow the design of a suite of markers for marker-assisted selection to reduce rumen methane emission in selected pasture legumes. We demonstrate the feasibility of a plant breeding approach without compromising animal productivity to mitigate enteric methane emissions, which is one of the most significant challenges to global livestock production.

Draft genome sequence of subterranean clover, a reference for genus Trifolium.

Clovers (genus Trifolium) are widely cultivated across the world as forage legumes and make a large contribution to livestock feed production and soil improvement. Subterranean clover (T. subterraneum L.) is well suited for genomic and genetic studies as a reference species in the Trifolium genus, because it is an annual with a simple genome structure (autogamous and diploid), unlike the other economically important perennial forage clovers, red clover (T. pratense) and white clover (T. repens). This report represents the first draft genome sequence of subterranean clover. The 471.8 Mb assembled sequence covers 85.4% of the subterranean clover genome and contains 42,706 genes. Eight pseudomolecules of 401.1 Mb in length were constructed, based on a linkage map consisting of 35,341 SNPs. The comparative genomic analysis revealed that different clover chromosomes showed different degrees of conservation with other Papilionoideae species. These results provide a reference for genetic and genomic analyses in the genus Trifolium and new insights into evolutionary divergence in Papilionoideae species.

Knowledge, attitudes, and behaviors regarding hydration and fluid replacement of collegiate athletes.

The purpose of this study was to determine collegiate athletes' knowledge, attitudes, and behaviors concerning hydration and fluid replacement. A survey containing questions pertaining to demographics and knowledge, attitude, and behavior on hydration and fluid replacement was distributed to the athletes during team meetings and practices. A total of 139 out of 171 (81.3%) athletes participated in the study. The mean age of the athletes was 19.8 y. The mean score for knowledge, attitude, and behavior was 13.9 +/-1.8, 9.8 +/- 2.2, and 12.4 +/- 2.5, respectively, with higher scores indicating positive hydration knowledge, attitudes, and behaviors. Significant positive correlation was observed between knowledge, attitude, and behavior scores (P < 0.05). Significant difference (P < 0.05) was observed in the reported hydration behaviors between skilled (11.79 +/- 2.08) and endurance (12.71 +/- 2.63) athletes. Most athletes correctly answered the general hydration questions on the survey, but the majority did not correctly answer statements in regards to National Athletic Trainers' Association (NATA) and the American College of Sports Medicine (ACSM) position stands and lacked knowledge regarding appropriate use of sports drink. The results of this study identify specific areas of education for athletes with regards to hydration.