BackgroundSheepgrass [Leymus chinensis (Trin.) Tzvel.] is an important perennial forage grass across the Eurasian Steppe and is known for its adaptability to various environmental conditions. However, insufficient data resources in public databases for sheepgrass limited our understanding of the mechanism of environmental adaptations, gene discovery and molecular marker development.ResultsThe transcriptome of sheepgrass was sequenced using Roche 454 pyrosequencing technology. We assembled 952,328 high-quality reads into 87,214 unigenes, including 32,416 contigs and 54,798 singletons. There were 15,450 contigs over 500 bp in length. BLAST searches of our database against Swiss-Prot and NCBI non-redundant protein sequences (nr) databases resulted in the annotation of 54,584 (62.6%) of the unigenes. Gene Ontology (GO) analysis assigned 89,129 GO term annotations for 17,463 unigenes. We identified 11,675 core Poaceae-specific and 12,811 putative sheepgrass-specific unigenes by BLAST searches against all plant genome and transcriptome databases. A total of 2,979 specific freezing-responsive unigenes were found from this RNAseq dataset. We identified 3,818 EST-SSRs in 3,597 unigenes, and some SSRs contained unigenes that were also candidates for freezing-response genes. Characterizations of nucleotide repeats and dominant motifs of SSRs in sheepgrass were also performed. Similarity and phylogenetic analysis indicated that sheepgrass is closely related to barley and wheat.ConclusionsThis research has greatly enriched sheepgrass transcriptome resources. The identified stress-related genes will help us to decipher the genetic basis of the environmental and ecological adaptations of this species and will be used to improve wheat and barley crops through hybridization or genetic transformation. The EST-SSRs reported here will be a valuable resource for future gene-phenotype studies and for the molecular breeding of sheepgrass and other Poaceae species.

Lotus root is a popular wetland vegetable which produces edible rhizome. At the molecular level, the regulation of rhizome formation is very complex, which has not been sufficiently addressed in research. In this study, to identify differentially expressed genes (DEGs) in lotus root, four libraries (L1 library: stolon stage, L2 library: initial swelling stage, L3 library: middle swelling stage, L4: later swelling stage) were constructed from the rhizome development stages. High-throughput tag-sequencing technique was used which is based on Solexa Genome Analyzer Platform. Approximately 5.0 million tags were sequenced, and 4542104, 4474755, 4777919, and 4750348 clean tags including 151282, 137476, 215872, and 166005 distinct tags were obtained after removal of low quality tags from each library respectively. More than 43% distinct tags were unambiguous tags mapping to the reference genes, and 40% were unambiguous tag-mapped genes. From L1, L2, L3, and L4, total 20471, 18785, 23448, and 21778 genes were annotated, after mapping their functions in existing databases. Profiling of gene expression in L1/L2, L2/L3, and L3/L4 libraries were different among most of the selected 20 DEGs. Most of the DEGs in L1/L2 libraries were relevant to fiber development and stress response, while in L2/L3 and L3/L4 libraries, major of the DEGs were involved in metabolism of energy and storage. All up-regulated transcriptional factors in four libraries and 14 important rhizome formation-related genes in four libraries were also identified. In addition, the expression of 9 genes from identified DEGs was performed by qRT-PCR method. In a summary, this study provides a comprehensive understanding of gene expression during the rhizome formation in lotus root.

Nine multiparous Holstein-Friesian cows (initially 97 d in milk), were used in a 3×3 lattice square design experiment with 4-wk periods. All cows received 4 kg/d concentrates and dietary treatments were based on silages offered ad libitum: perennial ryegrass (PRG); timothy (TIM); tall fescue (TF); red clover (RC); red clover/corn silage mixture [40/60 on a dry matter (DM) basis; RCC]; red clover/whole-crop oat silage mixture (40/60 on a DM basis; RCO); or red clover/whole-crop oat silage mixture (25/75 on a DM basis; ORC). The remaining treatments were based on RCO with feed intake restricted to the level of PRG (RCOr) or with a low protein concentrate (50/50 mixture of barley and molassed sugar beet pulp; RCOlp). Experiment objectives were to evaluate diet effects on N partitioning and N isotopic fractionation. Yields of milk and milk protein were consistently high for diets RC, RCC, and RCO and low for the diets based on poorly ensiled grass silages. Restriction of intake (RCOr) and inclusion of a higher proportion of whole-crop oat silage (ORC) and the low-protein concentrate (RCOlp) led to some loss of production. Diet had little effect on milk fat, protein, and lactose concentrations: low concentrations of milk protein and lactose reflect the restricted energy intakes for all treatments. The highest diet digestibilities were measured for RC and PRG, whereas increasing inclusion of the whole-crop oat silage (0, 60, and 75% of forage DM) led to a marked decrease in diet digestibility (0.717, 0.624, and 0.574 g/g, respectively). Urinary excretion of purine derivatives, an indicator for rumen microbial protein synthesis, was significantly higher for RCC than for TIM and TF. Nitrogen intake ranged between 359 and 626 g/d (treatment means). Partitioning of N intake to feces and urine was closely related to N intake, although urinary N losses were less than predicted from N intake for the 60/40 mixtures of cereal silage and red clover silage. The 15N content of milk, urine, and feces were all influenced by diet 15N content. Isotopic fractionation meant that feces and milk were enriched and urine was depleted in 15N relative to the diet. Significant relationships were observed between the extent of enrichment of urine, feces, and milk, suggesting some commonality in fractionation pathways. The trend for the lowest 15N enrichment in milk protein occurring in diets with low N-use efficiency (milk N/feed N) was contrary to expectations, possibly because of endogenous contributions to milk protein or fractionation when dietary ammonia was incorporated into microbial protein.

a b s t r a c tCombining sweet sorghum (SS) with alfalfa (AF) for ensiling has the potential to improve the nutritive value and fermentation characteristics of resultant silages. However, the optimal combination and the associative effects of SS and AF for ensilage have not been studied. Therefore, the aim of this study was to determine the fermentation characteristic and nutritive value of silage mixtures with six different SS to AF ratios. The two forages were ensiled in air free silos for 150 days at room temperature as mixtures containing 0:100, 20:80, 40:60, 60:40, 80:20, and 100:0 of SS:AF on a fresh weight basis. As the proportion of SS increased in silage, the content of ash, crude protein, saponins, ammonia, acetic acid, propionic acid and pH decreased, while neutral detergent fiber, acid detergent fiber in organic matter, acid detergent lignin, water-soluble carbohydrate, starch, total phenolics and condensed tannins content increased. The silages were evaluated in 24-h incubations with rumen liquor. The in vitro rumen degradability of dry matter and organic matter as well as gas production, pH, ammonia, total volatile fatty acids and methane decreased as the proportion of SS increased in the silage mixtures. This study suggests that high quality silages can be made with SS:AF ratios of 20:80 and 40:60. These silage mixtures offer an opportunity to optimize the nutrient supply for ruminant production.

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Water is a critical environmental factor that restricts the geographic distribution of plants. Sheepgrass [Leymus chinensis, (Trin.) Tzvel] is an important forage grass in the Eurasia Steppe and a close germplasm for wheat and barley. This native grass adapts well to adverse environments such as cold, salinity, alkalinity and drought, and it can survive when the soil moisture may be less than 6% in dry seasons. However, little is known about how sheepgrass tolerates water stress at the molecular level. Here, drought stress experiment and RNA-sequencing (RNA-seq) was performed in three pools of RNA samples (control, drought stress, and rewatering). We found that sheepgrass seedlings could still survive when the soil water content (SWC) was reduced to 14.09%. Differentially expressed genes (DEGs) analysis showed that 7320 genes exhibited significant responses to drought stress. Of these DEGs, 2671 presented opposite expression trends before and after rewatering. Furthermore, ~680 putative sheepgrass-specific water responsive genes were revealed that can be studied deeply. Gene ontology (GO) annotation revealed that stress-associated genes were activated extensively by drought treatment. Interestingly, cold stress-related genes were up-regulated greatly after drought stress. The DEGs of MAPK and calcium signal pathways, plant hormone ABA, jasmonate, ethylene, brassinosteroid signal pathways, cold response CBF pathway participated coordinatively in sheepgrass drought stress response. In addition, we identified 288 putative transcription factors (TFs) involved in drought response, among them, the WRKY, NAC, AP2/ERF, bHLH, bZIP, and MYB families were enriched, and might play crucial and significant roles in drought stress response of sheepgrass. Our research provided new and valuable information for understanding the mechanism of drought tolerance in sheepgrass. Moreover, the identification of genes involved in drought response can facilitate the genetic improvement of crops by molecular breeding.