Fine-Mapping and Comparative Genomic Analysis Reveal the Gene Composition at the S and Z Self-incompatibility Loci in Grasses.

Self-incompatibility (SI) is a genetic mechanism of hermaphroditic plants to prevent inbreeding after self-pollination. Allogamous Poaceae species exhibit a unique gametophytic SI system controlled by two multi-allelic and independent loci, S and Z. Despite intense research efforts in the last decades, the genes that determine the initial recognition mechanism are yet to be identified. Here, we report the fine-mapping of the Z-locus in perennial ryegrass (Lolium perenne L.) and provide evidence that the pollen and stigma components are determined by two genes encoding DUF247 domain proteins (ZDUF247-I and ZDUF247-II) and the gene sZ, respectively. The pollen and stigma determinants are located side-by-side and were genetically linked in 10,245 individuals of two independent mapping populations segregating for Z. Moreover, they exhibited high allelic diversity as well as tissue-specific gene expression, matching the expected characteristics of SI determinants known from other systems. Revisiting the S-locus using the latest high-quality whole-genome assemblies revealed a similar gene composition and structure as found for Z, supporting the hypothesis of a duplicated origin of the two-locus SI system of grasses. Ultimately, comparative genomic analyses across a wide range of self-compatible and self-incompatible Poaceae species revealed that the absence of a functional copy of at least one of the six putative SI determinants is accompanied by a self-compatible phenotype. Our study provides new insights into the origin and evolution of the unique gametophytic SI system in one of the largest and economically most important plant families.

Half of the 18O enrichment of leaf sucrose is conserved in leaf cellulose of a C3 grass across atmospheric humidity and CO2 levels.

The 18O enrichment (Δ18O) of cellulose (Δ18OCel) is recognized as a unique archive of past climate and plant function. However, there is still uncertainty regarding the proportion of oxygen in cellulose (pex) that exchanges post-photosynthetically with medium water of cellulose synthesis. Particularly, recent research with C3 grasses demonstrated that the Δ18O of leaf sucrose (Δ18OSuc, the parent substrate for cellulose synthesis) can be much higher than predicted from daytime Δ18O of leaf water (Δ18OLW), which could alter conclusions on photosynthetic versus post-photosynthetic effects on Δ18OCel via pex. Here, we assessed pex in leaves of perennial ryegrass (Lolium perenne) grown at different atmospheric relative humidity (RH) and CO2 levels, by determinations of Δ18OCel in leaves, Δ18OLGDZW (the Δ18O of water in the leaf growth-and-differentiation zone) and both Δ18OSuc and Δ18OLW (adjusted for εbio, the biosynthetic fractionation between water and carbohydrates) as alternative proxies for the substrate for cellulose synthesis. Δ18OLGDZW was always close to irrigation water, and pex was similar (0.53 ± 0.02 SE) across environments when determinations were based on Δ18OSuc. Conversely, pex was erroneously and variably underestimated (range 0.02-0.44) when based on Δ18OLW. The photosynthetic signal fraction in Δ18OCel is much more constant than hitherto assumed, encouraging leaf physiological reconstructions.

Nitrogen offset potential in a multiyear farmlet-scale study: Milk and herbage production from grazed perennial ryegrass-white clover swards.

The objective of this study was to quantify the farm gate nitrogen (N) offset potential of perennial ryegrass (Lolium perenne L.; PRG) white clover (Trifolium repens L.; WC) swards by comparing the herbage and milk production from dairy farmlets that were simulations of full farming systems. A study was established where 120 cows were randomly assigned to 4 farmlets of 10.9 ha (stocking rate: 2.75 cow/ha), composed of 20 paddocks each. Cows were fed 526 kg of DM of concentrate on average each year. The 4 grazing treatments were PRG-only at 150 or 250 kg of N/ha and PRG-WC at 150 or 250 kg of N/ha. Cows remained in their treatment group for an entire grazing season and were re-randomized as they calved across treatments each year. As cows calved in the spring as standard practice in Ireland, they were rotationally grazed from early February both day and night (weather permitting) to mid-November, to a target postgrazing sward height of 4.0 cm. Mean sward WC content was 18.1% and 15.4% for the 150 and 250 kg of N/ha PRG-WC treatments, respectively over the 3-yr period. When WC was included, lowering the N rate did not reduce pregrazing yield, pregrazing height, or herbage removed, but those factors decreased significantly when WC was absent. Total annual herbage DM production was 13,771, 15,242, 14,721, and 15,667 kg of DM/ha for PRG-only swards receiving 150 or 250 kg of N/ha and PRG-WC swards receiving 150 or 250 kg of N/ha, respectively. In addition, when WC was present, compressed postgrazing sward heights were lower (4.10 vs. 4.21 cm) and herbage allowance (approximately 17 kg/cow feed allocation per cow per day) higher than the high-N control (+ 0.7 kg of DM/cow per day). There was a significant increase in milk production, both per cow and per hectare, when WC was included in PRG swards. Over the 3-yr study, cows grazing PRG-WC had greater milk (+304 kg) and milk solids (+31 kg of fat + protein) yields than cows grazing PRG-only swards. This significant increase in milk production suggests that the inclusion of WC in grazing systems can be effectively used to increase milk production per cow and per hectare and help offset nitrogen use. This result shows the potential to increase farm gate N use efficiency and reduce the N surplus compared with PRG-dominant sward grazing systems receiving 250 kg of N/ha, without negatively affecting milk solids yield or herbage production, thus increasing farm profit by €478/ha.

The genetic variation in drought resistance in eighteen perennial ryegrass varieties and the underlying adaptation mechanisms.

BACKGROUND: Drought resistance is a complex characteristic closely related to the severity and duration of stress. Perennial ryegrass (Lolium perenne L.) has no distinct drought tolerance but often encounters drought stress seasonally. Although the response of perennial ryegrass to either extreme or moderate drought stress has been investigated, a comprehensive understanding of perennial ryegrass response to both conditions of drought stress is currently lacking. RESULTS: In this study, we investigated the genetic variation in drought resistance in 18 perennial ryegrass varieties under both extreme and moderate drought conditions. The performance of these varieties exhibited obvious diversity, and the survival of perennial ryegrass under severe stress was not equal to good growth under moderate drought stress. 'Sopin', with superior performance under both stress conditions, was the best-performing variety. Transcriptome, physiological, and molecular analyses revealed that 'Sopin' adapted to drought stress through multiple sophisticated mechanisms. Under stress conditions, starch and sugar metabolic enzymes were highly expressed, while CslA was expressed at low levels in 'Sopin', promoting starch degradation and soluble sugar accumulation. The expression and activity of superoxide dismutase were significantly higher in 'Sopin', while the activity of peroxidase was lower, allowing for 'Sopin' to maintain a better balance between maintaining ROS signal transduction and alleviating oxidative damage. Furthermore, drought stress-related transcriptional and posttranscriptional regulatory mechanisms, including the upregulation of transcription factors, kinases, and E3 ubiquitin ligases, facilitate abscisic acid and stress signal transduction. CONCLUSION: Our study provides insights into the resistance of perennial ryegrass to both extreme and moderate droughts and the underlying mechanisms by which perennial ryegrass adapts to drought conditions.

18 O enrichment of sucrose and photosynthetic and nonphotosynthetic leaf water in a C3 grass-atmospheric drivers and physiological relations.

The 18 O enrichment (Δ18 O) of leaf water affects the Δ18 O of photosynthetic products such as sucrose, generating an isotopic archive of plant function and past climate. However, uncertainty remains as to whether leaf water compartmentation between photosynthetic and nonphotosynthetic tissue affects the relationship between Δ18 O of bulk leaf water (Δ18 OLW ) and leaf sucrose (Δ18 OSucrose ). We grew Lolium perenne (a C3 grass) in mesocosm-scale, replicated experiments with daytime relative humidity (50% or 75%) and CO2 level (200, 400 or 800 µmol mol-1 ) as factors, and determined Δ18 OLW , Δ18 OSucrose and morphophysiological leaf parameters, including transpiration (Eleaf ), stomatal conductance (gs ) and mesophyll conductance to CO2 (gm ). The Δ18 O of photosynthetic medium water (Δ18 OSSW ) was estimated from Δ18 OSucrose and the equilibrium fractionation between water and carbonyl groups (εbio ). Δ18 OSSW was well predicted by theoretical estimates of leaf water at the evaporative site (Δ18 Oe ) with adjustments that correlated with gas exchange parameters (gs or total conductance to CO2 ). Isotopic mass balance and published work indicated that nonphotosynthetic tissue water was a large fraction (~0.53) of bulk leaf water. Δ18 OLW was a poor proxy for Δ18 OSucrose , mainly due to opposite Δ18 O responses of nonphotosynthetic tissue water (Δ18 Onon-SSW ) relative to Δ18 OSSW , driven by atmospheric conditions.

Fiber digestibility and protein value of pulp silage for lactating dairy cows: Effects of wet fractionation by screw pressing of perennial ryegrass.

The aim of the study was to investigate the effects of substituting silage of chopped grass with pulp silage of grass fractionated once or twice in a biorefinery using a screw press on fiber kinetics, protein value, and production of CH4 in dairy cows. Six lactating multiparous Holstein cows in mid-lactation (176 ± 93 d in milk), cannulated in the rumen, duodenum, and ileum, were used in an incomplete 6 × 4 Latin square design with a 2 × 3 factorial arrangement of treatments. Perennial ryegrass was harvested in third regrowth from the same field at early and late developmental stage (35 and 44 d of regrowth, respectively) and subjected to 1 of 3 types of processing within each developmental stage. Grass was either harvested for normal silage making (mowed, wilted, chopped, and ensiled), or harvested fresh and fractionated using a screw press. Half of the pulp from the first fractionation was ensiled, whereas the other half of the pulp was rehydrated, fractionated a second time, and pulp hereof was ensiled. The grass and pulp silages were used with concentrates (65:35 forage to concentrate ratio) to make total mixed rations (TMR) based on either silage of chopped grass (GS), pulp silage of grass fractionated once (1×P), or pulp silage of grass fractionated twice (2×P), harvested either at early (E) or late (L) developmental stage resulting in 6 different TMR treatments (EGS, E1×P, E2×P, LGS, L1×P, L2×P). The TMR were fed for ad libitum intake and samples of intestinal digesta and feces were collected for determination of digestibility. The effect of processing on ash-free neutral detergent fiber (aNDFom) concentration in silages depended on developmental stage, but showed that within each developmental stage, pulp silage of grass fractionated twice had higher aNDFom concentration than pulp silage of grass fractionated once and silage of chopped grass. The 2×P resulted in lower (14.9 ± 0.55 vs. 17.5 ± 0.54 kg/d) dry matter intake (DMI) compared with GS. The effects of processing and developmental stage interacted such that apparent total-tract aNDFom digestibility was higher (784 ± 13 vs. 715 ± 13 g/kg) for L2×P compared with LGS, whereas no difference was found between E2×P and EGS. Moreover, the protein value was higher (106 ± 5 vs. 92 ± 5 g AA digested in the small intestine/kg of DMI) for 2×P compared with GS. Unexpectedly, processing had no effect on fractional rate of digestion of digestible aNDFom or CH4 yield (L/kg of DMI), whereas feeding forages harvested at early compared with late developmental stage resulted in lower CH4 yield. Feeding pulp silage of grass fractionated once generally yielded results intermediate to cows fed silage of chopped grass and pulp silage of grass fractionated twice. This study showed that pulp silage of fractionated grass could serve as feed for dairy cows because the fiber digestibility and protein value improved, but further research investigating effects of physical processing of forage on fiber kinetics is required.

Methane emission, nutrient digestibility, and rumen microbiota in Holstein heifers fed 14 different grass or clover silages as the sole feed.

This experiment investigated the variation in enteric methane production and associated gas exchange parameters, nutrient digestibility, rumen fermentation, and rumen microbiome when a range of silages based on different forage types (grass or clover), and different species within the 2 types, were fed as the sole feed to heifers. Three grass species (perennial ryegrass, festulolium, and tall fescue) and 2 clover species (red clover and white clover) were included. Perennial ryegrass was harvested at 2 maturity stages in the primary growth, white clover was harvested once in the primary growth, and 4 cuts of festulolium and tall fescue and 3 cuts of red clover were harvested during the growing season, giving 14 different silage batches in total. Sixteen Holstein heifers 16 to 21 mo old and 2 to 5 mo in pregnancy were fed the silages ad libitum as the sole feed in an incomplete crossover design. Each silage was fed to 4 heifers, except for the 2 perennial ryegrass silages, which were fed to 8 heifers; in total 64 observations. The CH4 production was measured for 3 d in respiration chambers. Heifers fed clover silages had higher dry matter intake (DMI) compared with heifers fed grass silages, and heifers fed tall fescue silages had the numerically the lowest DMI. Compared with grass silages, feeding clover silages led to higher crude protein digestibility but lower neutral detergent fiber (NDF) digestibility. Rumen pH was higher in heifers fed clover silages compared with those fed grass silages. Based on composition analysis, the rumen microbiota of the heifers clustered clearly according to forage type and species. More specifically, 7 of the 34 dominating rumen bacterial genus-level groups showed higher relative abundances for the clover silages, whereas 7 genus-level groups showed higher abundances for the grass silages. Methane yield was higher for heifers fed grass silages than for those fed clover silages when methane production was related to dry matter and digestible organic matter intake, whereas the opposite was seen when related to NDF digestion. The gross energy lost as methane (CH4 conversion factor, %) reduced from 7.5% to 6.7%, equivalent to an 11% reduction. The present study gives the outlines for choosing the optimal forage type and forage species with respect to nutrient digestibility and enteric methane emission in ruminants.

Chromosome-scale assembly and annotation of the perennial ryegrass genome.

BACKGROUND: The availability of chromosome-scale genome assemblies is fundamentally important to advance genetics and breeding in crops, as well as for evolutionary and comparative genomics. The improvement of long-read sequencing technologies and the advent of optical mapping and chromosome conformation capture technologies in the last few years, significantly promoted the development of chromosome-scale genome assemblies of model plants and crop species. In grasses, chromosome-scale genome assemblies recently became available for cultivated and wild species of the Triticeae subfamily. Development of state-of-the-art genomic resources in species of the Poeae subfamily, which includes important crops like fescues and ryegrasses, is lagging behind the progress in the cereal species. RESULTS: Here, we report a new chromosome-scale genome sequence assembly for perennial ryegrass, obtained by combining PacBio long-read sequencing, Illumina short-read polishing, BioNano optical mapping and Hi-C scaffolding. More than 90% of the total genome size of perennial ryegrass (approximately 2.55 Gb) is covered by seven pseudo-chromosomes that show high levels of collinearity to the orthologous chromosomes of Triticeae species. The transposon fraction of perennial ryegrass was found to be relatively low, approximately 35% of the total genome content, which is less than half of the genome repeat content of cultivated cereal species. We predicted 54,629 high-confidence gene models, 10,287 long non-coding RNAs and a total of 8,393 short non-coding RNAs in the perennial ryegrass genome. CONCLUSIONS: The new reference genome sequence and annotation presented here are valuable resources for comparative genomic studies in grasses, as well as for breeding applications and will expedite the development of productive varieties in perennial ryegrass and related species.

Responses of antioxidant enzymes and key resistant substances in perennial ryegrass (Lolium perenne L.) to cadmium and arsenic stresses.

Cadmium (Cd) and arsenic (As) exist simultaneously in soil environment, which poses a serious threat to the safety of agricultural products and forage production. Four Perennial Ryegrass (Lolium perenne L.) cultivars with different accumulation characteristics ('Nicaragua', 'Venus', 'Excellent' and 'Monro') were selected as the material for pot experiment. The coupled responses of key components and related enzyme activities under combined stresses of Cd and As were investigated. key components contents include Non protein sulfhydryl (NPT), glutathione (GSH) and phytochelatins (PCs). The related enzyme includes (superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), γ-glutamylcysteine synthetase (γ-ECS), glutathione synthetase (GSS), phytochelatin synthetases (PCSase) and arsenate reductase (AR). The results showed that Cd contents of perennial ryegrass were higher than those of As contents with TFCd/As < 1. Cd and As contents in roots were in the higher proportion than those in shoots. Compared to control, POD activities increased by 2.72 folds under 120 mg kg-1 As treatment. The contents of PCs increased by 5.68 folds under 120 mg kg-1 As treatment. Under combined Cd and As stress, the MDA contents and antioxidant enzyme activities of 'Venus' were higher than those of 'Nicaragua'. 'Nicaragua', a high accumulation cultivar. Under the combined stresses of Cd and As, the enzyme activities and the key components were significantly correlated (P < 0.05) with the contents of Cd and As. The tolerance to Cd and As was improved with increase in GSH and PCs contents and γ-ECS, GSS, PCSase and AR activities. In conclusion, the antioxidant enzyme system and key resistant substances of perennial ryegrass have important and antagonistic effects on Cd and As stresses.

Effects on feed intake, milk production, and methane emission in dairy cows fed silage or fresh grass with concentrate or fresh grass harvested at early or late maturity stage without concentrate.

The objective of the study was to quantify the effects on dry matter intake (DMI), nutrient digestibility, gas exchange, milk production, and milk quality in dairy cows fed fresh grass harvested at different maturity stages. Sixteen Danish Holstein cows in mid-lactation were divided into 4 blocks and used in 4 incomplete 4 × 2 Latin squares with 2 periods of 21 d. The cows received 1 of 4 treatments in each period, resulting in 8 cows per treatment, as follows: grass-clover silage supplemented with 6 kg/d concentrate pellets (SILc), fresh grass harvested at late maturity stage supplemented with 6 kg/d concentrate pellets (LATc), fresh grass harvested at late maturity stage (LAT), and fresh grass harvested at early maturity stage (ERL). The cows were housed in tiestalls and milked twice daily. The cows had ad libitum access to the forage, and concentrate pellets were divided into equal amounts and fed separately in the morning and afternoon. Fecal samples were collected to determine apparent total-tract digestibility, and samples of rumen fluid were collected for determination of short chain fatty acid composition. Halters were used for measuring eating and rumination time. Gas exchange was measured in open-circuit respiration chambers. Total DMI was higher in LATc and ERL (16.9 ± 0.45 and 15.5 ± 0.39 kg/d, respectively) compared with LAT (14.1 ± 0.42 kg/d). Relative to SILc, cows fed fresh grass experienced a convex pattern in DMI during the experiment. The changes in DMI were related to changes in leaf to stem ratio, fiber concentration, and organic matter digestibility determined in vitro in samples of the fresh grass harvested throughout the experiment. The apparent total-tract digestibility of organic matter was higher in SILc and LAT compared with LATc. Methane yield was lower for LATc compared with LAT (19.5 ± 0.61 vs. 22.6 ± 0.55 g of CH4/kg of DMI), and was not different between LAT and ERL. Compared with LAT, milk yield was higher for ERL (21.1 ± 1.14 vs. 23.4 ± 1.11 kg/d) and energy-corrected milk (ECM) yield was higher for LATc (21.5 ± 0.99 vs. 25.3 ± 1.03 kg/d). We detected no differences in milk or ECM yield between SILc and LATc. Milk protein yield was higher and milk fat concentration was lower in LATc compared with LAT. The fatty acid percentages of ∑C4-C14:1 and ∑C16 in milk were higher for SILc compared with LATc, signifying pronounced de novo synthesis. The n-6:n-3 ratio in milk fatty acids was lower for SILc and LAT compared with LATc, indicating improved nutritional quality for SILc and LAT. However, retinol concentration in milk was lower in SILc compared with all other treatments. The study implies that feeding silage instead of fresh grass has no effect on DMI, ECM yield, or CH4 yield, and that concentrate supplementation can increase milk production, affects milk quality, and reduces the effect on climate, whereas feeding less mature grass increases DMI and milk yield, but has no effect on CH4 yield.

Near-Infrared Hyperspectral Imaging Pipelines for Pasture Seed Quality Evaluation: An Overview.

Near-infrared (800-2500 nm; NIR) spectroscopy coupled to hyperspectral imaging (NIR-HSI) has greatly enhanced its capability and thus widened its application and use across various industries. This non-destructive technique that is sensitive to both physical and chemical attributes of virtually any material can be used for both qualitative and quantitative analyses. This review describes the advancement of NIR to NIR-HSI in agricultural applications with a focus on seed quality features for agronomically important seeds. NIR-HSI seed phenotyping, describing sample sizes used for building high-accuracy calibration and prediction models for full or selected wavelengths of the NIR region, is explored. The molecular interpretation of absorbance bands in the NIR region is difficult; hence, this review offers important NIR absorbance band assignments that have been reported in literature. Opportunities for NIR-HSI seed phenotyping in forage grass seed are described and a step-by-step data-acquisition and analysis pipeline for the determination of seed quality in perennial ryegrass seeds is also presented.

Temperature-sensitive biochemical 18 O-fractionation and humidity-dependent attenuation factor are needed to predict δ18 O of cellulose from leaf water in a grassland ecosystem.

We explore here our mechanistic understanding of the environmental and physiological processes that determine the oxygen isotope composition of leaf cellulose (δ18 Ocellulose ) in a drought-prone, temperate grassland ecosystem. A new allocation-and-growth model was designed and added to an 18 O-enabled soil-vegetation-atmosphere transfer model (MuSICA) to predict seasonal (April-October) and multi-annual (2007-2012) variation of δ18 Ocellulose and 18 O-enrichment of leaf cellulose (Δ18 Ocellulose ) based on the Barbour-Farquhar model. Modelled δ18 Ocellulose agreed best with observations when integrated over c. 400 growing-degree-days, similar to the average leaf lifespan observed at the site. Over the integration time, air temperature ranged from 7 to 22°C and midday relative humidity from 47 to 73%. Model agreement with observations of δ18 Ocellulose (R2  = 0.57) and Δ18 Ocellulose (R2  = 0.74), and their negative relationship with canopy conductance, was improved significantly when both the biochemical 18 O-fractionation between water and substrate for cellulose synthesis (εbio , range 26-30‰) was temperature-sensitive, as previously reported for aquatic plants and heterotrophically grown wheat seedlings, and the proportion of oxygen in cellulose reflecting leaf water 18 O-enrichment (1 - pex px , range 0.23-0.63) was dependent on air relative humidity, as observed in independent controlled experiments with grasses. Understanding physiological information in δ18 Ocellulose requires quantitative knowledge of climatic effects on pex px and εbio .

Inter-annual and spatial climatic variability have led to a balance between local fluctuating selection and wide-range directional selection in a perennial grass species.

BACKGROUND AND AIMS: The persistence of a plant population under a specific local climatic regime requires phenotypic adaptation with underlying particular combinations of alleles at adaptive loci. The level of allele diversity at adaptive loci within a natural plant population conditions its potential to evolve, notably towards adaptation to a change in climate. Investigating the environmental factors that contribute to the maintenance of adaptive diversity in populations is thus worthwhile. Within-population allele diversity at adaptive loci can be partly driven by the mean climate at the population site but also by its temporal variability. METHODS: The effects of climate temporal mean and variability on within-population allele diversity at putatively adaptive quantitative trait loci (QTLs) were evaluated using 385 natural populations of Lolium perenne (perennial ryegrass) collected right across Europe. For seven adaptive traits related to reproductive phenology and vegetative potential growth seasonality, the average within-population allele diversity at major QTLs (HeA) was computed. KEY RESULTS: Significant relationships were found between HeA of these traits and the temporal mean and variability of the local climate. These relationships were consistent with functional ecology theory. CONCLUSIONS: Results indicated that temporal variability of local climate has likely led to fluctuating directional selection, which has contributed to the maintenance of allele diversity at adaptive loci and thus potential for further adaptation.

The effect of concentrate supplement type on milk production, nutrient intake, and total-tract nutrient digestion in mid-lactation, spring-calving dairy cows grazing perennial ryegrass (Lolium perenne L.) pasture.

The objective of this study was to evaluate the effect of concentrate supplement type on milk production, nutrient intake, and total-tract nutrient digestion in lactating dairy cows grazing mid-season perennial ryegrass (Lolium perenne L.; PRG) pasture. Twelve primiparous (mean ± standard deviation; 95 ± 30 d in milk and 470 ± 43 kg of body weight) and 68 multiparous (99 ± 24 d in milk and 527 ± 64 kg of body weight) lactating dairy cows were blocked based on pre-study milk yield and parity and randomly assigned to 1 of 4 dietary treatments. The 4 dietary treatments were a non-supplemented PRG control (PRG); PRG supplemented with 4.4 kg of dry matter (DM) per cow per day of citrus pulp and 0.067 kg of DM/cow per day of urea (PRG+C); PRG supplemented with 0.8 kg of DM/cow per day of heat-treated soybean meal (PRG+PP); and PRG supplemented with 3.1 kg of DM/cow per day of a combination of heat-treated soybean meal and citrus pulp (PRG+C+PP). The study consisted of a 2-wk adaptation period and a 10-wk period of data collection. Weekly measurements of milk yield, body weight, body condition score, and feeding and rumination time were made. Nutrient intake and total-tract digestibility were measured during wk 6 of the study. A large soil moisture deficit was experienced during the study that probably reduced herbage growth rate and likely altered the chemical composition of the PRG offered when compared with typical mid-season PRG. Total dry matter intake was increased in cows fed PRG+C compared with cows fed PRG and PRG+PP and was similar to cows fed PRG+C+PP (18.0, 15.9, 16.4, and 17.2 ± 0.41 kg of DM/d, respectively). The apparent total-tract neutral detergent fiber digestibility of cows fed the PRG+C diet was lower compared with the PRG and PRG+PP diets and was similar to the PRG+C+PP diet (0.67, 0.70, 0.70, and 0.69 ± 0.01 g/g, respectively). The energy-corrected milk (ECM) yield of cows fed PRG+C+PP was highest (23.7 kg/d), PRG+C was intermediate (22.2 kg/d), and PRG was lowest (20.8 kg/d). Cows fed PRG+PP produced more ECM (22.9 kg/d) compared with cows fed PRG and produced similar ECM compared with cows fed PRG+C and PRG+C+PP diets. The PRG+PP diet increased milk protein yield compared with the PRG diet, tended to increase milk protein yield compared with the PRG+C diet, and was similar to the PRG+C+PP diet. Milk fat concentration and the composition of milk fat were not influenced by treatment. The results demonstrated that, for cows consuming pasture-based diets, increasing metabolizable protein supply allowed higher milk yield as metabolizable protein was more limiting than metabolizable energy. However, due to the large soil moisture deficit experienced during this experiment, caution is recommended when extrapolating these results to cows consuming typical mid-season PRG herbage.

Using estimated nutrient intake from pasture to formulate supplementary concentrate mixes for grazing dairy cows.

In pasture-based dairy systems, feeding a complex concentrate mix in the parlor during milking that contains cereal grains and protein supplements has been shown to have milk production advantages over feeding straight cereal grain. This experiment had the aim of testing whether further milk production advantages could be elicited by adjusting the composition of the concentrate mix in an attempt to match the expected nutrient intake from pasture during late spring. The experiment used 96 lactating dairy cows, grazing perennial ryegrass pasture offered at a target allowance of 30 kg of dry matter/cow per day (to ground level) during late spring (mid October to November) in southeastern Australia. Cows were allocated into 3 replicates of 4 treatment groups, with 24 cows in each treatment. Each treatment group was offered 1 of 4 dietary treatments in the parlor at milking: control consisting of crushed wheat and barley grains; formulated grain mix (FGM) consisting of crushed wheat, barley, and corn grains and canola meal; designer grain mix 1 (DGM1) consisting of the same ingredients as the FGM grain mix but formulated using the CPM Dairy nutrition model to take into account the expected nutrient intake from pasture; and designer grain mix 2 (DGM2) consisting of the same ingredients as DGM1 but with canola meal replaced by urea and a fat supplement (Megalac, Volac Wilmar, Gresik, Indonesia). Concentrate mixes were offered at 8.0 kg of dry matter/cow per day, except for DGM2 cows, which were offered 7.5 kg of dry matter/cow per day. The experiment ran for a total of 28 d; after a 14-d adaptation period, nutrient intake, milk production, and body weight were measured over a 14-d measurement period. Milk yield (kg) of cows fed the FGM diet was greater than that of the control cows but was not different from that of the DGM1 and DGM2 cows. However, milk fat and protein yields (kg) were greater for cows fed the FGM diet than for all other diets. There was no difference in estimated daily pasture or total dry matter intakes between the 4 treatment groups, despite cows fed the DGM2 treatment consuming less of the concentrate mix (average 6.5 kg of dry matter/cow per day when offered 7.5 kg of dry matter/cow per day). This research has demonstrated the potential for using a nutrition model to take into account the expected nutrient intake from pasture to formulate a concentrate mix (DGM1) to achieve similar milk yields, but also highlighted the need for near real-time analyses of the pasture to be grazed so as to also capture benefits in terms of milk fat and protein yield.

The simulated environmental impact of incorporating white clover into pasture-based dairy production systems.

White clover (WC) offers an alternative source of nitrogen (N) for pasture-based systems. Substituting energy- and carbon-intensive synthetic N fertilizers with N derived from biological fixation by WC has been highlighted as a promising environmental mitigation strategy through the omission of emissions, pollutants, and energy usage during the production and application of synthetic fertilizer. Therefore, the objective was to investigate the effect of the inclusion of WC in perennial ryegrass (PRG) swards on the environmental impact of pasture-based dairy systems. Cradle-to-farm gate life cycle assessment of 3 pasture-based dairy systems were conducted: (1) a PRG-WC sward receiving 150 kg of N/ha per year (CL150), (2) a PRG-WC sward receiving 250 kg of N/ha per year (CL250), and (3) a PRG-only sward receiving 250 kg of N/ha per year (GR250). A dairy environmental model was updated with country-specific N excretion equations and recently developed N2O, NH3, and NO3- emission factors. The environmental impact categories assessed were global warming potential, nonrenewable energy, acidification potential, and eutrophication potential (marine and freshwater). Impact categories were expressed using 2 functional units: per hectare and per metric tonne of fat- and protein-corrected milk. The GR250 system had the lowest milk production and highest global warming potential, nonrenewable energy, and acidification potential per tonne of fat- and protein-corrected milk for all systems. The CL250 system produced the most milk and had the highest environmental impact across all categories when expressed on an area basis. It also had the highest marine eutrophication potential for both functional units. The impact category freshwater eutrophication potential did not differ across the 3 systems. The CL150 system had the lowest environmental impact across all categories and functional units. This life cycle assessment study demonstrates that the substitution of synthetic N fertilizer with atmospheric N fixed by WC has potential to reduce the environmental impact of intensive pasture-based dairy systems in temperate regions, not only through improvement in animal performance but also through the reduction in total emissions and pollutants contributing to the environmental indicators assessed.

Incorporation of the grazing utilization subindex and new updates to the Pasture Profit Index.

Grazing efficiency has been shown to differ between perennial ryegrass varieties. Such differences affect the utilization of grass within grazing systems, influencing the profitability of grass-based ruminant production systems. The Pasture Profit Index (PPI) is an economic merit grass variety selection tool developed to identify varieties with the greatest economic potential for grass-based dairy production systems. A new grass utilization subindex was developed and incorporated into the PPI to identify varieties with superior grazing efficiency. The subindex rewards varieties with superior grazing efficiency, measured as Residual grazed height, as these varieties allow increased amounts of herbage dry matter to be used by grazing animals. The economic values of all other traits within the PPI were reviewed and updated to ensure that the index was reflective of the current economic scenarios with appropriate assumptions included in the models, thus ensuring that varieties excelling in the agronomic traits with the greatest effect on profitability were recognized. The difference between the highest and lowest performing varieties for the grass utilization trait ranged from €23 to -€24. A range of €211 to €43 was recorded between the highest and lowest ranked varieties within the updated PPI. Spearman's rank correlation between the updated and original PPI lists was 0.96. The introduction of the utilization subindex will allow farmers to make informed variety selection decisions when reseeding pasture, particularly on their grazing platforms and it will allow a demand-based communication process between the farmer and the grass merchant or breeder, ultimately affecting trait selection for future breeding strategies.

The effect of Lolium perenne L. ploidy and Trifolium repens L. inclusion on dry matter intake and production efficiencies of spring-calving grazing dairy cows.

The objective of this study was to investigate the effect of perennial ryegrass (Lolium perenne L.; PRG) ploidy and white clover (Trifolium repens L.) inclusion on milk production, dry matter intake (DMI), and milk production efficiencies. Four separate grazing treatments were evaluated: tetraploid PRG only, diploid PRG only, tetraploid PRG with white clover, and diploid PRG with white clover. Individual DMI was estimated 8 times during the study (3 times in 2015, 2 times in 2016, and 3 times in 2017) using the n-alkane technique. Cows were, on average, 64, 110, and 189 d in milk during the DMI measurement period, corresponding to spring, summer, and autumn, respectively. Measures of milk production efficiency were total DMI/100 kg of body weight (BW), milk solids (kg of fat + protein; MSo)/100 kg of BW, solids-corrected milk/100 kg of BW, and MSo/kg of total DMI. Perennial ryegrass ploidy had no effect on DMI; however, a significant increase in DMI (+0.5 kg/cow per day) was observed from cows grazing PRG-white clover swards compared with PRG-only swards. Sward white clover content influenced DMI as there was no increase in DMI in spring (9% sward white cover content), whereas DMI was greater in summer and autumn for cows grazing PRG-white clover swards (+0.8 kg/cow per day) compared with PRG-only swards (14 and 23% sward white clover content, respectively). The greater DMI of cows grazing PRG-white clover swards led to increased milk (+1.3 kg/cow per day) and MSo (+0.10 kg/cow per day) yields. Cows grazing PRG-white clover swards were also more efficient for total DMI/100 kg of BW, solids-corrected milk/100 kg of BW, and MSo/100 kg of BW compared with cows grazing PRG-only swards due to their similar BW but higher milk and MSo yields. The results highlight the potential of PRG-white clover swards to increase DMI at grazing and to improve milk production efficiency in pasture-based systems.

Cadmium stress suppresses the tillering of perennial ryegrass and is associated with the transcriptional regulation of genes controlling axillary bud outgrowth.

Perennial ryegrass (Lolium perenne L.), a grass species with superior tillering capacity, plays a potential role in the phytoremediation of cadmium (Cd)-contaminated soils. Tiller production is inhibited in response to serious Cd stress. However, the regulatory mechanism of Cd stress-induced inhibition of tiller development is not well documented. To address this issue, we investigated the phenotype, the expression levels of genes involved in axillary bud initiation and bud outgrowth, and endogenous hormone biosynthesis and signaling pathways in seedlings of perennial ryegrass under Cd stress. The results showed that the number of tillers and axillary buds in the Cd-treated seedlings decreased by 67% and 21%, respectively. The suppression of tiller production in the Cd-treated seedlings was more closely associated with the inhibition of axillary bud outgrowth than with bud initiation. Cd stress upregulated the expression level of genes related to axillary bud dormancy and downregulated bud activity genes. Additionally, genes involved in strigolactone biosynthesis and signaling, auxin transport and signaling, and cytokinin degradation were upregulated in Cd-treated seedlings, and cytokinin biosynthesis gene expression were decreased by Cd stress. The content of zeatin in the Cd-treated pants was significantly reduced by 69~85% compared to the control plants. The content of indole-3-acetic acid (IAA) remains constant under Cd stress. Overall, Cd stress induced axillary bud dormancy and subsequently inhibited axillary bud outgrowth. The decrease of zeatin content and upregulation of genes involved in strigolactone signaling and bud dormancy might be responsible for the inhibition of axillary bud outgrowth.

Genome-wide investigation of the NAC transcript factor family in perennial ryegrass (Lolium perenne L.) and expression analysis under various abiotic stressor.

NAC is one of the largest family of plant-specific transcription factors, and it plays important roles in plant development and stress responses. The study identified 72 LpNACs genes from the perennial ryegrass genome database. Gene length, MW and pI of NAC family transcription factors varied, but the gene structure and motifs were relatively conserved in bioinformatics analysis. Phylogenetic analyses of perennial ryegrass, rice and Arabidopsis were performed to study the evolutionary and functional relationships in various species. The expression of LpNAC genes that respond to various abiotic stresses including high salinity, ABA, high temperature, polyethylene glycol (PEG) and heavy metal was comprehensively analyzed. The present study provides a basic understanding of the NAC gene family in perennial ryegrass for further abiotic stress studies and improvements in breeding.