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Background: Reed canary grass has been identified as a suitable species for restoring plateau wetlands and understanding plant adaptation mechanisms in wetland environments. In this study, we subjected a reed canary grass cultivar 'Chuanxi' to waterlogging, salt, and combined stresses to investigate its phenotypic characteristics, physiological indices, and transcriptome changes under these conditions. Results: The results revealed that the growth rate was slower under salt stress than under waterlogging stress. The chlorophyll content and energy capture efficiency of the PS II reaction center decreased with prolonged exposure to each stress. Conversely, while the activities of enzymes associated with respiratory metabolism, as well as MDA, PRO, Na+, and K+-ATPase, increased. The formation of distinct aerenchyma was observed under waterlogging stress and combined stress. Transcriptome sequencing analysis identified 5,379, 4,169, and 14,993 DEGs under CK vs. W, CK vs. S, and CK vs. SW conditions, respectively. The WRKY was found to be the most abundant under waterlogging stress, whereas the MYB predominated under salt stress and combined stress. Glutathione metabolic pathways and Plant hormone signal transduction have also been found to play important roles in stress. Conclusion: By integrating phenotypic, physiological, anatomical, and transcriptomic, this research provides valuable insights into how reed canary grass responds to salt, waterlogging, and combined stresses. These findings may inform the ecological application of reed canary grass in high-altitude wetlands and for breeding purposes. [ABSTRACT FROM AUTHOR]

SUMMARY: Proanthocyanidins (PAs), a group of flavonoids, are found in leaves, flowers, fruits, and seed coats of many plant species. PAs are primarily composed of epicatechin units in the seed coats of the model legume species, Medicago truncatula. It can be synthesized from two separate pathways, the leucoanthocyanidin reductase (MtLAR) pathway and the anthocyanidin synthase (MtANS) pathway, which produce epicatechin through anthocyanidin reductase (MtANR). These pathways are mainly controlled by the MYB–bHLH–WD40 (MBW) ternary complex. Here, we characterize a class IV homeodomain‐leucine zipper (HD‐ZIP IV) transcription factor, GLABRA2 (MtGL2), which contributes to PA biosynthesis in the seed coat of M. truncatula. Null mutation of MtGL2 results in dark brown seed coat, which is accompanied by reduced PAs accumulation and increased anthocyanins content. The MtGL2 gene is predominantly expressed in the seed coat during the early stages of seed development. Genetic and molecular analyses indicate that MtGL2 positively regulates PA biosynthesis by directly activating the expression of MtANR. Additionally, our results show that MtGL2 is strongly induced by the MBW activator complexes that are involved in PA biosynthesis. Taken together, our results suggest that MtGL2 acts as a novel positive regulator in PA biosynthesis, expanding the regulatory network and providing insights for genetic engineering of PA production. [ABSTRACT FROM AUTHOR]

Reed canary grass (Phalaris arundinacea L.) is known for its tolerance to drought, heavy metals, and waterlogging, making it a popular choice for forage production and wetland restoration in the Qinghai-Tibet Plateau (QTP). To accurately assess gene expression in reed canary grass under different abiotic stresses, suitable reference genes need to be identified and validated. Thirteen candidate reference gene sequences were selected and screened using RT-qPCR to detect their expression levels in reed canary grass leaves under drought, salt, cadmium, and waterlogging stresses. Four algorithms were used to assess the stability of the expression levels of the candidate reference genes. The most stably expressed genes were UBC and H3 under drought Cd, ETF and CYT under salt stress, and ETF and TUB under waterlogging stress. GAPDH was found to be less stable under abiotic stresses. PIP-1, PAL, NAC 90, and WRKY 72A were selected as response genes for quantitative expression assessment under drought, salt, Cd, and waterlogging stresses to confirm the accuracy of the selected stable reference genes. These results provide a theoretical reference for assessing gene expression in reed canary grass under abiotic stresses. [ABSTRACT FROM AUTHOR]

Drought is one of the most significant limiting factors affecting plant growth and development on the Qinghai–Tibet Plateau (QTP). Mining the drought-tolerant genes of the endemic perennial grass of the QTP, Siberian wildrye (Elymus sibiricus), is of great significance to creating new drought-resistant varieties which can be used in the development of grassland livestock and restoring natural grassland projects in the QTP. To investigate the transcriptomic responsiveness of E. sibiricus to drought stress, PEG-induced short- and long-term drought stress was applied to two Siberian wildrye genotypes (drought-tolerant and drought-sensitive accessions), followed by third- and second-generation transcriptome sequencing analysis. A total of 40,708 isoforms were detected, of which 10,659 differentially expressed genes (DEGs) were common to both genotypes. There were 2107 and 2498 unique DEGs in the drought-tolerant and drought-sensitive genotypes, respectively. Additionally, 2798 and 1850 DEGs were identified in the drought-tolerant genotype only under short- and long-term conditions, respectively. DEGs numbering 1641 and 1330 were identified in the drought-sensitive genotype only under short- and long-term conditions, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that all the DEGs responding to drought stress in E. sibiricus were mainly associated with the mitogen-activated protein kinase (MAKP) signaling pathway, plant hormone signal transduction, the linoleic acid metabolism pathway, the ribosome pathway, and plant circadian rhythms. In addition, Nitrate transporter 1/Peptide transporter family protein 3.1 (NPF3.1) and Auxin/Indole-3-Acetic Acid (Aux/IAA) family protein 31(IAA31) also played an important role in helping E. sibiricus resist drought. This study used transcriptomics to investigate how E. sibiricus responds to drought stress, and may provide genetic resources and references for research into the molecular mechanisms of drought resistance in native perennial grasses and for breeding drought-tolerant varieties. [ABSTRACT FROM AUTHOR]

Low temperature is an important limiting factor in the environment that affects the distribution, growth and development of warm-season grasses. Transcriptome sequencing has been widely used to mine candidate genes under low-temperature stress and other abiotic stresses. However, the molecular mechanism of centipedegrass in response to low-temperature stress was rarely reported. To understand the molecular mechanism of centipedegrass in response to low-temperature stress, we measured physiological indicators and sequenced the transcriptome of centipedegrass under different stress durations. Under cold stress, the SS content and APX activity of centipedegrass increased while the SOD activity decreased; the CAT activity, POD activity and flavonoid content first increased and then decreased; and the GSH-Px activity first decreased and then increased. Using full-length transcriptome and second-generation sequencing, we obtained 38.76 G subreads. These reads were integrated into 177,178 isoforms, and 885 differentially expressed transcripts were obtained. The expression of AUX_IAA and WRKY transcription factors and HSF transcription-influencing factors increased during cold stress. Through KEGG enrichment analysis, we determined that arginine and proline metabolism, plant circadian rhythm, plant hormone signal transduction and the flavonoid biosynthesis pathways played important roles in the cold stress resistance of centipedegrass. In addition, by using weighted gene coexpression network analysis (WGCNA), we determined that the turquoise module was significantly correlated with SS content and APX activity, while the blue module was significantly negatively correlated with POD and CAT activity. This paper is the first to report the response of centipedegrass to cold stress at the transcriptome level. Our results help to clarify the molecular mechanisms underlying the cold tolerance of warm-season grasses. [ABSTRACT FROM AUTHOR]

Kengyilia melanthera is one of the most important forages, and has received significant attention as a desirable ecological pioneer grass for conserving grasslands and mitigating degradation pressure in a region. It is widely distributed in the alpine sandy meadow zone of the Eastern Qinghai–Tibet Plateau (QTP). Therefore, determining the optimal harvest time of this species is critical. A two-year field experiment (2016–2017) was utilized in this study to evaluate the impact of eight harvest times on the seed yield and quality of K. melanthera 'Aba'. The results show that the fresh weight (FW), dry weight (DW), seed yield (SY), thousand-grain weight (TGW), accelerated aging germination percentage (AAGP), and dehydrogenase activity (DA) of seeds increased with the extension of harvest time in two trial years, while the water content (WC) of seeds decreased. In both trial years, the germination percentage (GEP), germination potential (GP), and acid phosphatase activity (APA) of seeds increased initially and then decreased with the extension of harvest time. The comprehensive evaluation of the membership function and hierarchical clustering revealed that the 30th day after peak anthesis was the optimal harvest time for both 2016 and 2017. During this time, FW, DW, SY, TGW, AAGP, DA, WC, GEP, GP, and APA reached their optimal value, with 0.815 g, 0.518 g, 1773.67 kg·hm−2, 143.81%, 66%, 16.23 µg·mL−1, 36.01%, 87%, 85%, and 2.50 nmol·min−1 in 2016 and 0.805 g, 0.515 g, 1847.42 kg·hm−2, 133.96%, 65%, 16.52 µg·mL−1, 36.04%, 86.5%, 83.5%, and 2.55 nmol·min−1 in 2017, respectively. This study uncovers several high-efficiency and long-term technological solutions for cultivating high yields and quality production of K. melanthera in East QTP. [ABSTRACT FROM AUTHOR]

Drought is one of the most important factors affecting plant growth and production due to ongoing global climate change. Elymus sibiricus has been widely applied for ecological restoration and reseeding of degraded grassland in the Qinghai–Tibetan Plateau (QTP) because of its strong adaptability to barren, salted, and drought soils. To explore the mechanism of drought resistance in E. sibiricus, drought-tolerant and drought-sensitive genotypes of E. sibiricus were used in metabolomic studies under simulated long-term and short-term drought stress. A total of 1091 metabolites were detected, among which, 27 DMs were considered to be the key metabolites for drought resistance of E. sibiricus in weighted gene co-expression network analysis (WGCNA). Ten metabolites, including 3-amino-2-methylpropanoic acid, coniferin, R-aminobutyrate, and so on, and 12 metabolites, including L-Proline, L-histidine, N-acetylglycine, and so on, showed differential accumulation patterns under short-term and long-term drought stress, respectively, and thus, could be used as biomarkers for drought-tolerant and drought-sensitive E. sibiricus. In addition, different metabolic accumulation patterns and different drought response mechanisms were also found in drought-tolerant and drought-sensitive genotypes of E. sibiricus. Finally, we constructed metabolic pathways and metabolic patterns for the two genotypes. This metabolomic study on the drought stress response of E. sibiricus can provide resources and a reference for the breeding of new drought-tolerant cultivars of E. sibiricus. [ABSTRACT FROM AUTHOR]

Summary: Strigolactones (SLs) play a critical role in regulating plant tiller number. LATERAL BRANCHING OXIDOREDUCTASE (LBO) encodes an important late‐acting enzyme for SL biosynthesis and regulates shoot branching in Arabidopsis. However, little is known about the function of LBO in monocots including switchgrass (Panicum virgatum L.), a dual‐purpose fodder and biofuel crop.We studied the function of PvLBO via the genetic manipulation of its expression levels in both the wild‐type and miR156 overexpressing (miR156OE) switchgrass. Co‐expression analysis, quantitative real‐time polymerase chain reaction (qRT‐PCR), transient dual luciferase assay, and chromatin immunoprecipitation‐qPCR were all used to determine the activation of PvLBO by miR156‐targeted Squamosa Promoter Binding Protein‐like 2 (PvSPL2) in regulating tillering of switchgrass.PvLBOtranscripts dramatically declined in miR156OE transgenic switchgrass, and the overexpression of PvLBO in the miR156OE transgenic line produce fewer tillers than the control. Furthermore, we found that PvSPL2 can directly bind to the promoter of PvLBO and activate its transcription, suggesting that PvLBO is a novel downstream gene of PvSPL2.We propose that PvLBO functions as an SL biosynthetic gene to mediate tillering and acts as an important downstream factor in the crosstalk between the SL biosynthetic pathway and the miR156‐SPL module in switchgrass. [ABSTRACT FROM AUTHOR]

Elymus sibiricus L. is a perennial allotetraploid belonging to Triticeae of Poaceae, Elymus L., as the type species of genus Elymus L. The existing geographical distribution pattern and genetic spatial structure of E. sibiricus on Qinghai–Tibetan Plateau (QTP) are not yet clear. In this study, population genetic structure and demography history of 216 individuals from 44 E. sibiricus populations on QTP were studied used specific-locus amplified fragment sequencing (SLAF-seq). The result of genetic diversity showed that there was no single genetic diversity center was observed across all E. sibiricus populations. The results of genetic variation showed that 44 populations were clearly divided into the following three groups: Qinghai Plateau (Group I), South Tibet (Group II), and Hengduan Mountains (Group III). From the three analyses of AMOVA, Mantel test and Treemix, strong genetic differentiation across all populations and low genetic differentiation among populations within three groups. Molecular dating indicated that E. sibiricus diverged at 16.08 Ma (during the early Miocene) can be linked to the Himalayan Motion stage of QTP uplift. It is speculated that the reasons affecting the current phylogeographical pattern are as follows: (1) The environmental changes due to the uplift of the QTP; (2) The geographic distance between the populations (Groups I and III are close in geographic distance, and gene flow are frequent); (3) Geographical barriers (the Tanggula and Bayangela Mountains between Groups I and II). This study provides new evidence and historical perspective to the future exploration of the evolution and geographic distribution pattern of Elymus L. [ABSTRACT FROM AUTHOR]

S -adenosyl- l -methionine (SAM) is the methyl donor involved in the biosynthesis of guaiacyl (G) and syringyl (S) lignins in vascular plants. SAM is synthesized from methionine through the catalysis of the enzyme S -adenosylmethionine synthase (SAMS). However, the detailed function of SAMS in lignin biosynthesis has not been widely investigated in plants, particularly in monocot species. In this study, we identified PvSAMS genes from switchgrass (Panicum virgatum L.), an important dual-purpose fodder and biofuel crop, and generated numerous transgenic switchgrass lines through PvSAMS RNA interference technology. Down-regulation of PvSAMS reduced the contents of SAM, G-lignins, and S-lignins in the transgenic switchgrass. The methionine and glucoside derivatives of caffeoyl alcohol were found to accumulate in the transgenic plants. Moreover, down-regulation of PvSAMS in switchgrass resulted in brownish stems associated with reduced lignin content and improved cell wall digestibility. Furthermore, transcriptomic analysis revealed that most sulfur deficiency-responsive genes were differentially expressed in the transgenic switchgrass, leading to a significant increase in total sulfur content; thus implying an important role of SAMS in the methionine cycle, lignin biosynthesis, and sulfur assimilation. Taken together, our results suggest that SAMS is a valuable target in lignin manipulation, and that manipulation of PvSAMS can simultaneously regulate the biosynthesis of SAM and methylated monolignols in switchgrass. [ABSTRACT FROM AUTHOR]

The genetic adaptations to harsh climatic conditions in high altitudes and genetic basis of important agronomic traits are poorly understood in Elymus sibiricus L. In this study, an association population of 210 genotypes was used for population structure, selective sweep analysis, and genome-wide association study (GWAS) based on 88,506 single nucleotide polymorphisms (SNPs). We found 965 alleles under the natural selection of high altitude, which included 7 hub genes involved in the response to UV, and flavonoid and anthocyanin biosynthetic process based on the protein–protein interaction (PPI) analysis. Using a mixed linear model (MLM), the GWAS test identified a total of 1,825 significant loci associated with 12 agronomic traits. Based on the gene expression data of two wheat cultivars and the PPI analysis, we finally identified 12 hub genes. Especially, in plant height traits, the top hub gene (TOPLESS protein) encoding auxins and jasmonic acid signaling pathway, shoot apical meristem specification, and xylem and phloem pattern formation was highly overexpressed. These genes might play essential roles in controlling the growth and development of E. sibiricus. Therefore, this study provides fundamental insights relevant to hub genes and will benefit molecular breeding and improvement in E. sibiricus and other Elymus species. [ABSTRACT FROM AUTHOR]

A detailed understanding of the distribution and degree of genetic variation within a species is important for determining their evolutionary potential, which in return facilitates the development of efficient conservation strategies aimed at preserving adaptive genetic variation. As an important perennial, cool-season grass in temperate Eurasia, increasing attention has been paid to Siberian wildrye (Elymus sibiricus) due to its excellent ecological utilization value and forage production potential in China, particularly in the Qinghai–Tibet Plateau (QTP) regions. In this study, we applied two chloroplast (cp) genes (matK and rbcL), three cp spacer regions (trnY - GUA ∼ trnD - GUC , atpH ∼ atpF , and rps4 ∼ trnT - UGU), and six cpSSR markers to the genetic and phylogenetic analysis of 137 wild E. sibiricus accessions from 23 natural populations that represent the main distribution regions in China. The results show the highest genetic diversity (h = 0.913) and haplotype richness (10 haplotypes) for the QTP population, which indicates QTP as the probable diversity center and geographic origin of E. sibiricus in China. Population divergence was high, indicating a significant phylogeographic structure together with a significantly higher Nst value (Nst > Gst, P < 0.05) at the species level, QTP+XJ (combined populations from QTP and Xinjiang), QTP+NC (combined populations from QTP and North China), and XJ+NC (combined populations from Xinjiang and North China) group levels, respectively. An expansion was revealed in the distributional range of E. sibiricus in China from paleo times up to the recent past, while a dramatic range of contraction was predicted for the near future. The predicted main limiting factor for the further spread of E. sibiricus is an increasing global mean temperature. We recommend that the combination of Es-cpDNA1 and Es-cpDNA3+4+5 can be used as effective markers for phylogenetic analysis and phylogeographical history analysis of E. sibiricus. These findings shed new light on the historical population dynamics of cold-season herbs in the QTP region and the north of China and are of great significance for the future establishment of protection and collection strategies for wild E. sibiricus germplasm. [ABSTRACT FROM AUTHOR]

A detailed understanding of the distribution and degree of genetic variation within a species is important for determining their evolutionary potential, which in return facilitates the development of efficient conservation strategies aimed at preserving adaptive genetic variation. As an important perennial, cool-season grass in temperate Eurasia, increasing attention has been paid to Siberian wildrye (Elymus sibiricus) due to its excellent ecological utilization value and forage production potential in China, particularly in the Qinghai–Tibet Plateau (QTP) regions. In this study, we applied two chloroplast (cp) genes (matK and rbcL), three cp spacer regions (trnY - GUA ∼ trnD - GUC , atpH ∼ atpF , and rps4 ∼ trnT - UGU), and six cpSSR markers to the genetic and phylogenetic analysis of 137 wild E. sibiricus accessions from 23 natural populations that represent the main distribution regions in China. The results show the highest genetic diversity (h = 0.913) and haplotype richness (10 haplotypes) for the QTP population, which indicates QTP as the probable diversity center and geographic origin of E. sibiricus in China. Population divergence was high, indicating a significant phylogeographic structure together with a significantly higher Nst value (Nst > Gst, P < 0.05) at the species level, QTP+XJ (combined populations from QTP and Xinjiang), QTP+NC (combined populations from QTP and North China), and XJ+NC (combined populations from Xinjiang and North China) group levels, respectively. An expansion was revealed in the distributional range of E. sibiricus in China from paleo times up to the recent past, while a dramatic range of contraction was predicted for the near future. The predicted main limiting factor for the further spread of E. sibiricus is an increasing global mean temperature. We recommend that the combination of Es-cpDNA1 and Es-cpDNA3+4+5 can be used as effective markers for phylogenetic analysis and phylogeographical history analysis of E. sibiricus. These findings shed new light on the historical population dynamics of cold-season herbs in the QTP region and the north of China and are of great significance for the future establishment of protection and collection strategies for wild E. sibiricus germplasm. [ABSTRACT FROM AUTHOR]

Switchgrass (Panicum virgatum L.) is an important perennial C4 species due to its large potential for cellulosic bioenergy feedstock production. Identification of quantitative trait loci (QTL) controlling important developmental traits is valuable to understanding the genetic basis and using marker-assisted selection (MAS) in switchgrass breeding. One F1 hybrid population derived from NL94 (♀) × SL93 (♂) and one S1 (first-generation selfed) population from NL94 were used in this study. Both the populations showed significant variations for genotype and genotype by environment interactions for three traits studied: plant vigor, spring green-up, and plant biomass. Plant vigor had strong and positive correlations with plant biomass in both populations. Broad-sense heritability estimates for plant vigor ranged from 0.46 to 0.74 and 0.45 to 0.74 in the hybrid and selfed population, respectively. Spring green-up had similar heritability estimates, 0.42–0.78 in the hybrid population, and 0.47–0.82 in the selfed population. Heritability of plant biomass was 0.54–0.64 in the hybrid population and 0.64–0.74 in the selfed population. Fifteen QTLs for spring green-up, 6 QTLs for plant vigor, and 3 QTLs for biomass yield were detected in the hybrid population, whereas 4 QTLs for spring green-up, 4 QTLs for plant vigor, and 1 QTL for biomass yield were detected in the selfed population. Markers associated with these QTLs can be used in MAS to accelerate switchgrass breeding program. This study provided new information in understanding the genetic control of biomass components and demonstrated substantial heterotic vigor that could be explored for breeding hybrid cultivars in switchgrass. [ABSTRACT FROM AUTHOR]

Background: This study aimed to investigate the effect of phenyllactic acid as an additive on silage fermentation and bacterial community of reed canary grass (RCG, Phalaris arundinacea L.) on the Qinghai Tibetan Plateau. At the heading stage, RCG was harvested, chopped and ensiled in small bag silos. The silage was treated without (control, 1.0 g/mL sterile water, on a fresh matter basis (FM)) or with phenyllactic acid (PLA, 3 mg/mL, FM), antimicrobial additive (PSB, a mixture of potassium sorbate and sodium benzoate, 2%, FM), lactic acid bacteria inoculant (LABi, L. plantarum + L. curvatus, 1 × 106 cfu/g, FM) and PLA + LABi, and then stored in a dark room at the ambient temperature (5 ~ 15 °C) for 60 days. Results: Compared with control, PLA decreased lactic acid, acetic acid and ammonia-N contents, and subsequently increased CP content of RCG silage. PLA enhanced the growth of lactic acid bacteria and reduced the count of yeasts (P < 0.05) in RCG silage, with reduced bacterial richness index (Chao1), observed operational taxonomic units and diversity index (Simpson). In relative to control, moreover, PLA and PLA + LABi increased the relative abundance of Lactococcus in RCG silage by 27.73 and 16.93%, respectively. Conclusions: Therefore, phenyllactic acid at ensiling improved nutritional quality of RCG silage by advancing the disappearance of yeasts and the dominance of Lactococcus. [ABSTRACT FROM AUTHOR]

The most intriguing characteristics of plant mitochondrial genomes (mitogenomes) include their high variation in both sequence and structure, the extensive horizontal gene transfer (HGT), and the important role they play in hypoxic adaptation. However, the investigation of the mechanisms of hypoxic adaptation and HGT in plant mitochondria remains challenging due to the limited number of sequenced mitogenomes and non-coding nature of the transferred DNA. In this study, the mitogenome of Elymus sibiricus (Gramineae, Triticeae), a perennial grass species native to the Qinghai-Tibet plateau (QTP), was de novo assembled and compared with the mitogenomes of eight Gramineae species. The unique haplotype composition and higher TE content compared to three other Triticeae species may be attributed to the long-term high-altitude plateau adaptability of E. sibiricus. We aimed to discover the connection between mitogenome simple sequence repeats (SSRs) (mt-SSRs) and HGT. Therefore, we predicted and annotated the mt-SSRs of E. sibiricus along with the sequencing of 87 seed plants. The clustering result based on all of the predicted compound mitogenome SSRs (mt-c-SSRs) revealed an expected synteny within systematic taxa and also inter-taxa. The mt-c-SSRs were annotated to 11 genes, among which "(ATA)3agtcaagtcaag (AAT)3" occurred in the nad5 gene of 8 species. The above-mentioned results further confirmed the HGT of mitogenomes sequences even among distant species from the aspect of mt-c-SSRs. Two genes, nad4 and nad7 , possessed a vast number of SSRs in their intron regions across the seed plant mitogenomes. Furthermore, five pairs of SSRs developed from the mitogenome of E. sibiricus could be considered as potential markers to distinguish between the species E. sibiricus and its related sympatric species E. nutans. [ABSTRACT FROM AUTHOR]

High temperature (HT) is an important factor for limiting global plant distribution and agricultural production. As the global temperature continues to rise, it is essential to clarify the physiological and molecular mechanisms of alfalfa responding the high temperature, which will contribute to the improvement of heat resistance in leguminous crops. In this study, the physiological and proteomic responses of two alfalfa (Medicago sativa L.) varieties contrasting in heat tolerance, MS30 (heat-tolerant) and MS37 (heat-sensitive), were comparatively analyzed under the treatments of continuously rising temperatures for 42 days. The results showed that under the HT stress, the chlorophyll content and the chlorophyll fluorescence parameter (Fv/Fm) of alfalfa were significant reduced and some key photosynthesis-related proteins showed a down-regulated trend. Moreover, the content of Malondialdehyde (MDA) and the electrolyte leakage (EL) of alfalfa showed an upward trend, which indicates both alfalfa varieties were damaged under HT stress. However, because the antioxidation-reduction and osmotic adjustment ability of MS30 were significantly stronger than MS37, the damage degree of the photosynthetic system and membrane system of MS30 is significantly lower than that of MS37. On this basis, the global proteomics analysis was undertaken by tandem mass tags (TMT) technique, a total of 6,704 proteins were identified and quantified. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that a series of key pathways including photosynthesis, metabolism, adjustment and repair were affected by HT stress. Through analyzing Venn diagrams of two alfalfa varieties, 160 and 213 differentially expressed proteins (DEPs) that had dynamic changes under HT stress were identified from MS30 and MS37, respectively. Among these DEPs, we screened out some key DEPs, such as ATP-dependent zinc metalloprotease FTSH protein, vitamin K epoxide reductase family protein, ClpB3, etc., which plays important functions in response to HT stress. In conclusion, the stronger heat-tolerance of MS30 was attributed to its higher adjustment and repair ability, which could cause the metabolic process of MS30 is more conducive to maintaining its survival and growth than MS37, especially at the later period of HT stress. This study provides a useful catalog of the Medicago sativa L. proteomes with the insight into its future genetic improvement of heat-resistance. [ABSTRACT FROM AUTHOR]

Background: Siberian wildrye (Elymus sibiricus L.) attracts considerable interest for grassland establishment and pasture recovery in the Qinghai-Tibet Plateau (QTP) due to its excellence in strong stress tolerance, high nutritional value and ease to cultivate. However, the lack of genomic information of E. sibiricus hampers its genetics study and breeding process. Results: In this study, we performed a genome survey and developed a set of SSR markers for E. sibiricus based on Next-generation sequencing (NGS). We generated 469.17 Gb clean sequence which is 58.64× of the 6.86 Gb estimated genome size. We assembled a draft genome of 4.34 Gb which has 73.23% repetitive elements, a heterozygosity ratio of 0.01% and GC content of 45.68%. Based on the gnomic sequences we identified 67,833 SSR loci and from which four hundred were randomly selected to develop markers. Finally, 30 markers exhibited polymorphism between accessions and ten were identified as single-locus SSR. These newly developed markers along with previously reported 30 ones were applied to analyze genetic polymorphism among 27 wild E. sibiricus accessions. We found that single-locus SSRs are superior to multi-loci SSRs in effectiveness. Conclusions: This study provided insights into further whole genome sequencing of E. sibiricus in strategy selection. The novel developed SSR markers will facilitate genetics study and breeding for Elymus species. [ABSTRACT FROM AUTHOR]

Two experiments were conducted to investigate the bacterial community of fresh and ensiled paper mulberry prepared with or without lactic acid bacteria (LAB) inoculants in South China. In Experiment 1, the bacterial community, chemical composition, and fermentation products of paper mulberry were analyzed. The results showed that fresh paper mulberry had high crude protein content, buffering capacity value, and amounts of uncultured bacteria. Ensiled paper mulberry showed poor fermentation with high pH value, ammonia–N content, and butyric acid content. In addition, Enterobacter was the dominant genus in silage, followed by Lactobacillus and Enterococcus. Water‐soluble carbohydrates, ammonia–N, propionic acid, pH, and lactic acid (LA) were the main factors affecting bacterial community of silage. In Experiment 2, the BP17 (Lactobacillus plantarum) from natural fermented paper mulberry silage and two commercial inoculants (Silage‐help [SH] and Chikuso‐1 [CH]) were used as additives. Compared with other treatments, BP17 inoculant decreased (p < 0.05) pH and ammonia–N content and increased (p < 0.05) LA content of silage. Inoculation of BP17 also increased the dominance of desirable Lactobacillus and inhibited the growth of harmful bacteria in silage. These results confirmed that paper mulberry could be ensiled and epiphytic LAB inoculant can improve its fermentation quality. [ABSTRACT FROM AUTHOR]

The objective of the present study was to evaluate the silage quality of forage soybean‐corn stover silage in the south of China. Forage soybean were combined manually with corn stover by ratios of R1 (25:75), R2 (50:50) and R3 (75:25) in fresh weight (FM) and ensiled in bag silos for 60 days at room temperature (20–25°C). All silages were treated without (control) or with lactic acid bacteria (LAB) inoculant (G, Lactobacillus plantarum and Lactobacillus buchneri, 105 cfu/g, of FM), cellulase (C, 0.02% of FM) and C + G. R1 silage had appropriate V‐score value with high lactic acid content and low pH value and ammonia‐N (NH3‐N) content, undetectable levels of propionic acid (PA) and butyric acid (BA). C and C + G reduced undesirable microorganisms of aerobic bacteria, yeasts and molds, resulting in improved nutritive value of R2 silage, as indicated by lower neutral detergent fiber and acid detergent fiber contents, and higher crude protein content, in vitro ruminal dry matter digestibility, gross energy and metabolizable energy values as compared to the control. Additives did not improve the quality of R3 silage with high pH value and NH3‐N content, BA and PA contents and undesirable microorganism counts. These results confirmed that high‐quality silage produced from mixtures (25:75) of forage soybean and corn stover in the south of China; additives (especially for C + G) could enhance the silage quality of forage soybean‐corn stover (50:50); and high proportion (>75%) of forage soybean was difficult to ensile. [ABSTRACT FROM AUTHOR]

Background: Elymus sibiricus is an ecologically and economically important perennial, self-pollinated, and allotetraploid (StStHH) grass, widely used for forage production and animal husbandry in Western and Northern China. However, it has low seed yield mainly caused by seed shattering, which makes seed production difficult for this species. The goals of this study were to construct the high-density genetic linkage map, and to identify QTLs and candidate genes for seed-yield related traits. Results: An F2 mapping population of 200 individuals was developed from a cross between single genotype from "Y1005" and "ZhN06". Specific-locus amplified fragment sequencing (SLAF-seq) was applied to construct the first genetic linkage map. The final genetic map included 1971 markers on the 14 linkage groups (LGs) and was 1866.35 cM in total. The length of each linkage group varied from 87.67 cM (LG7) to 183.45 cM (LG1), with an average distance of 1.66 cM between adjacent markers. The marker sequences of E. sibiricus were compared to two grass genomes and showed 1556 (79%) markers mapped to wheat, 1380 (70%) to barley. Phenotypic data of eight seed-related traits (2016–2018) were used for QTL identification. A total of 29 QTLs were detected for eight seed-related traits on 14 linkage groups, of which 16 QTLs could be consistently detected for two or three years. A total of 6 QTLs were associated with seed shattering. Based on annotation with wheat and barley genome and transcriptome data of abscission zone in E. sibiricus, we identified 30 candidate genes for seed shattering, of which 15, 7, 6 and 2 genes were involved in plant hormone signal transcription, transcription factor, hydrolase activity and lignin biosynthetic pathway, respectively. Conclusion: This study constructed the first high-density genetic linkage map and identified QTLs and candidate genes for seed-related traits in E. sibiricus. Results of this study will not only serve as genome-wide resources for gene/QTL fine mapping, but also provide a genetic framework for anchoring sequence scaffolds on chromosomes in future genome sequence assembly of E. sibiricus. [ABSTRACT FROM AUTHOR]

In practical of silage production, variations in the fermentation parameters of silage might be attributable to changes in weather data, especially for ambient temperature. The objective of this study was to investigate how the changed temperature of outside weather condition affects distributions of pH and fermentation products in large round bale silage. The bale silages (1.22 m × 1.25 m ø; density, about 168.9 kg dry matter [DM]/m3) were produced by BR6090 Combi from New Holland Agriculture, wrapped with six layers of stretch film, stored at ambient temperature (2–20°C) and constant temperature (10°C; 15°C; 25°C) for 3, 9, 15, 45 and 90 days. Compared with other silages, silage stored at 25°C had higher (p < 0.05) lactic acid content, lower (p < 0.05) pH, butyric acid content and ammonia‐nitrogen (N) ratio of total N. Fluctuant storage temperature limited lactic acid fermentation process with delayed pH decline and continuous butyric production and crude protein (CP) degradation. The differences of pH, lactic acid, propionic acid and ammonia‐N were considerably less evident in the centre of silages stored at different temperatures, and they increased towards the boundaries. The data shows that fluctuant storage temperature had undesirable effect on the fermentation quality of silage, especially for the external part of silo; relatively high and stable storage temperature for initial stage of ensiling process was necessary, but did not promote a positive response to the homogeneous distributions of pH and fermentation products in large round bale silage. [ABSTRACT FROM AUTHOR]

Background: Alfalfa (Medicago sativa) is a widely cultivated, essential commercial forage crop. The protein content in its leaves is the critical factor in determining the quality of alfalfa. Thus far, the understanding of the molecular mechanism of alfalfa defoliation traits remains unclear. The transcriptome database created by RNA-Seq is used to identify critical genes related to defoliation traits. Results: In this study, we sequenced the transcriptomes of the Zhungeer variety (with easy leaf abscission) and WL319HQ variety (without easy leaf abscission). Among the identified 66,734 unigenes, 706 differentially expressed genes (DEGs) upregulated, and 392 unigenes downregulated in the Zhungeer vs WL319HQ leaf. KEGG pathway annotations showed that 8,414 unigenes were annotated to 87 pathways and contained 281 DEGs. Six DEGs belonging to the "Carotenoid biosynthesis", "Plant hormone signal transduction" and "Circadian rhythm-plant" pathways involved in defoliation traits were identified and validated by RT-qPCR analyses. Conclusions: This study used RNA-Seq to discover genes associated with defoliation traits between two alfalfa varieties. Our transcriptome data dramatically enriches alfalfa functional genomic studies. In addition, these data provide theoretical guidance for field production practice and genetic breeding, as well as references for future study of defoliation traits in alfalfa. [ABSTRACT FROM AUTHOR]

Gastrointestinal bacteria are essential for host health, and only viable microorganisms contribute to gastrointestinal functions. When evaluating the gut microbiota by next generation sequencing method, dead bacteria, which compose a proportion of gut bacteria, may distort analysis of the live gut microbiota. We collected stomach, jejunum, ileum, cecum, and colon contents from Rex rabbits. A modified propidium monoazide (PMA) treatment protocol was used to exclude DNA from dead bacteria. Analysis of untreated samples yielded total bacteria, and analysis of PMA-treated samples yielded live bacteria. Quantitative polymerase chain reaction and 16S rRNA gene sequencing were performed to evaluate the live-to-total bacteria ratio and compare the difference between live and total microbiota in the entire digestive tract. A low proportion of live bacteria in the foregut (stomach 1.12%, jejunum 1.2%, ileum 2.84%) and a high proportion of live bacteria in the hindgut (cecum 24.66%, colon 19.08%) were observed. A significant difference existed between total and live microbiota. Clostridiales, Ruminococcaceae, and S24-7 dominated the hindgut of both groups, while Acinetobacter and Cupriavidus dominated only in live foregut microbiota. Clostridiales and Ruminococcaceae abundance decreased, while S24-7 increased in live hindgut microbiota. The alpha- and beta-diversities differed significantly between groups. Analysis of networks showed the mutual relationship between live bacteria differed vastly when compared with total bacteria. Our study revealed a large number of dead bacteria existed in the digestive tract of Rex rabbits and distorted the community profile of the live microbiota. Total bacteria is an improper representation of the live gut microbiota, particularly in the foregut. [ABSTRACT FROM AUTHOR]

Effects of lactic acid bacteria ( LAB) and formic acid ( FA) on silage quality of ultraviolet ( UV)-radiated Siberian wildrye ( Elymus sibiricus L.) were studied on the Qinghai-Tibetan plateau. Siberian wildrye seeds were sown in early April, 2012 and harvested in 2013, 2014 and 2016. Fresh forage was radiated for 0, 4 and 8 h under ambient/laboratory UV radiation conditions during the wilting process, and then mixed with LAB inoculant (105 cfu g−1 fresh matter [ FM]) and FA (3 mL kg−1 FM), respectively, no additives treatment as control. After additive treatments, chopped Siberian wildrye plants were packed in 30 cm × 40 cm polythene bags and stored in a dark room at ambient temperature (<15°C) for 90 days. Silages of UV-radiated Siberian wildrye had higher ( P < 0.05) pH and ammonia-nitrogen (N) content, lower ( P < 0.05) acetic and propionic acid content and in vitro ruminal dry matter digestibility. Compared with control, treatments with additives (especially for inoculant LAB) enhanced pH decline, increased ( P > 0.05) acetic acid content, decreased ( P < 0.05) butyric acid and ammonia-N content of silages of UV-radiated Siberian wildrye. It was concluded that the application of FA and inoculant LAB at ensiling could reduce the negative effects of extended UV radiation on the process of wilting. [ABSTRACT FROM AUTHOR]

The objective of this study was to evaluate the effect of grape pomace ( GP) with different adding levels (0%, 5%, 10% and 15%, fresh matter basis), alone ( GP- LAB) or in combine with an inoculant LAB ( GP+ LAB), on the fermentation quality and aerobic stability of sweet sorghum silage. After 90 days of ensiling in vacuumized mini-silos, silages were subject to a 7-day aerobic stability test, in which chemical, microbial and polyphenol composition were measured. In the GP- LAB group, adding GP decreased ( P < 0.05) concentrations of water-soluble carbohydrate ( WSC) and butyric acid in silage. In the GP+ LAB group, adding GP increased ( P < 0.05) concentrations of lactic acid, WSC and crude protein, decreased ( P < 0.05) final pH value, NH3-N ratio and butyric acid concentration in silage. Polyphenol level was reduced ( P < 0.05) after silage fermentation. During aerobic exposure, the fungi count, pH value and silage temperature increased ( P < 0.05), the levels of lactic acid, acetic acid and polyphenols (quercetin 3-O-glucoside and quercetin 3-O-glucuronid) decreased ( P < 0.05) in silage. GP+ LAB treated silage had a lag phase for aerobic spoilage. When the fermentation products, microbial counts, chemical and polyphenol composition were considered, the use of 10% GP+ LAB at ensiling could provide a valuable source for improved fermentation quality and aerobic stability of sweet sorghum silage. [ABSTRACT FROM AUTHOR]

Erianthus arundinaceus is not only a candidate plant for sugarcane breeding programs, but also a potential bioenergy grass. Genetic variation that is affected by geographic environments and ploidy level is very important for the utilization of Erianthus arundinaceus. In this study, effects of geographic environments and ploidy level on genetic variation were studied through analyzing the genetic diversity, genetic similarity and cluster analysis of 46 E. arundinaceus materials from natural habitats in China by using 7 ISSRs and 15 SSRs. Results showed that: 1) Seven ISSRs generated total 66 bands, of which 77% were polymorphic bands, the Nei's genetic similarity coefficient of tested materials ranged from 0.642 to 0.904 with an average value of 0.765. Fifteen SSRs generated 138 bands, of which 81% were polymorphic bands, the Nei's genetic similarity coefficient of tested materials ranged from 0.634 to 0.963 with an average value of 0.802. The results indicated great genetic diversity existed in the tested materials. 2)The tested materials were clustered into 3 groups and 7 subgroups, which demonstrated a strong geographic effect on variation of the local E. arundinaceus, and weak relationship was found between genetic distance and geographic distance. Five tetraploid materials were not clustered together, and were clustered together with materials from similar geographical location. 3) The genetic variation and cluster results were affected by geographic landforms and environments, the gene flow was blocked by Ocean and mountains, and promoted by river. The effect of ploidy level on genetic variation was little. [ABSTRACT FROM AUTHOR]

It is difficult to make good quality of silage from alpine gramineous from the Qinghai Tibetan plateau. The effects of lactic acid bacteria ( LAB) on the fermentation quality and aerobic stability of Siberian wildrye silage were studied in southeast of the Qinghai Tibetan plateau. Siberian wildrye materials were freshly cut at the sprouting stage, flowering stage, and milky stage. Silage was prepared by using a small-scale silage fermentation system (bag silos). Lactobacillus plantarum ( LP, 5 × 108 cfu/kg FM), Lactobacillus buchneri ( LB, 5 × 108 cfu/kg FM) and their mixture ( LP+ LB, 5 × 108 cfu/kg FM) as silage additives were separately added to ensiled forages, and no additive served as control ( CK). These bag silos were kept at room temperature (<15°C), and the silage qualities were analyzed after 60 days of ensiling. The number of indigenous LAB on fresh materials was less than that of yeasts and molds, and LAB species showed specification adapted to low temperature. LAB inoculated silages had lower ( P < 0.05) pH value, NH3-N/ TN and butyric acid content compared with control silage. Silage treated with LB had higher contents of acetic acid, propionic acid, WSC and CP. However, the aerobic stability of silages inoculated with LAB did not differ significantly between stages ( P > 0.05). When fermentation characteristics, chemical composition, and aerobic stability were considered, treatment with L. plantarum resulted in high quality of Siberian wildrye silage harvested at the flowering stage in the alpine region. [ABSTRACT FROM AUTHOR]

Erianthus arundinaceus is not only an important germplasm resource for sugarcane breeding but also a potential bioenergy plant. Making clear the distribution of the chromosome ploidy of wild E. arundinaceus in china is the premise of the research and utilization of this species. Therefore, the objectives of this study were to determine the ploidy level and DNA content of the 55 E. arundinaceus accessions using flow cytometry and to identify the correlation between ploidy and phenotypic traits. Among the 55 accessions, four tetraploids and 51 hexaploids were identified. The four tetraploids originated from Mengma Yunnan, Shuangjiang Yunnan, Gaozhou Guangdong and Chengle Sichuan. The mean DNA content was 4.82 pg/2C for the tetraploid and 7.30 pg/2C for the hexaploid plants. The ploidy was negatively correlated with cellulose content and positively correlated (P<0.05) with plant height, stem diameter, leaf width, dry weight per plant, fresh weight per plant and hemicellulose content. However, ploidy was not correlated with leaf length, tiller number and the ratio of dry weight and fresh weight. This study will be useful for revealing the distribution of the ploidy of wild E. arundinaceus in Chin, traits markers analysis, and utilization of this species, such as cultivar improvement and sugarcane breeding in the future. [ABSTRACT FROM AUTHOR]

Background: E. arundinaceus (Retz.) Jeswiet is a warm-season, tall-growing perennial species native to much southern portion in China. The grass has been extensively used in sugarcane breeding and is recently targeted as a bioenergy feedstock crop. However, information on the genetic structure of the Chinese wild germplasm is limited. Knowledge of genetic variation within and among populations is essential for breeding new cultivars in the species. The major objective of this study was to quantify the magnitude of genetic variation among and within natural populations in China. Methodology/Principal Findings: In this experiment, we analyzed genetic variation of 164 individuals of 18 populations collected from natural habitats in six Chinese provinces using 20 sequence-related amplified polymorphism (SRAP) primer pairs generating 277 polymorphic bands. Among and within the populations, the percentage of polymorphic bands (PPB) was 80.00% and 27.07%, genetic diversity (HE) was 0.245 and 0.099, effective number of alleles (NE) was 1.350 and 1.170, and Shannon's information index (I) was 0.340 and 0.147, respectively. The populations were clustered into six groups exhibiting a high level of genetic differentiation, which was highly associated with geographic origins of respective germplasm populations, but was not significantly associated with geographic distances between the populations. Conclusions/Significance: This is the first report indicating that large genetic variation exists in the Chinese E. arundinaceus germplasm based on the SRAP molecular marker analysis of native populations. The genetic structure of populations in the species has been substantially affected by geographic landforms and environments. The diverse collection will be highly valuable in genetic improvement in the species per se and likely in sugarcane. [ABSTRACT FROM AUTHOR]

Elymus breviaristatus is a grass species only distributed in the southeast of Qinghai-Tibetan Plateau (QTP), which has suffered from serious habitat fragmentation. Therefore, understanding patterns of genetic diversity within and among natural E. breviaristatus populations could provide insight for future conservation strategies. In this study, sequence-related amplified polymorphism markers were employed to investigate the genetic diversity and hierarchical structure of seven E. breviaristatus populations from QTP, China. Multiple measures of genetic diversity indicated that there is low to moderate genetic variation within E. breviaristatus populations, consistent with its presumed mating system. In spite of its rarity, E. breviaristatus presented high genetic diversity that was equivalent to or even higher than that of widespread species. Bayesian clustering approaches, along with clustering analysis and principal coordinate analysis partitioned the studied populations of E. breviaristatus into five genetic clusters. Differentiation coefficients (Fst, GST, etc.) and AMOVA analysis revealed considerable genetic divergence among different populations. BARRIER analyses indicated that there were two potential barriers to gene flow among the E. breviaristatus populations. Despite these patterns of differentiation, genetic distances between populations were independent of geographic distances (r = 0.2197, p = 0.2534), indicating little isolation by distance. Moreover, despite detecting a common outlier by two methods, bioclimatic factors (altitude, annual mean temperature, and annual mean precipitation) were not related to diversity parameters, indicating little evidence for isolation caused by the environment. These patterns of diversity within and between populations are used to propose a conservation strategy for E. breviaristatus. [ABSTRACT FROM AUTHOR]