Identification and Validation of Reference Genes for RT-qPCR Analysis in Reed Canary Grass during Abiotic Stress.

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.

Integrating Full-Length Transcriptome and RNA Sequencing of Siberian Wildrye (Elymus sibiricus) to Reveal Molecular Mechanisms in Response to Drought Stress.

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.

Contribution of the bacterial community of poorly fermented oat silage to biogas emissions on the Qinghai Tibetan Plateau.

To better utilize poorly fermented oat silage on the Qinghai Tibetan Plateau, 239 samples of this biomass were collected from the plateau temperate zone (PTZ), plateau subboreal zone (PSBZ), and nonplateau climatic zone (NPCZ) in the region and analyzed for microbial community, chemical composition and in vitro gas production. Climatic factors affect the bacterial α-diversity and ß-diversity of poorly fermented oat silage, which led to the NPCZ having the highest relative abundance of Lactiplantibacillus plantarum. Furthermore, the gas production analysis showed that the NPCZ had the highest maximum cumulative gas emissions of methane. Through structural equation modeling analysis, environmental factors (solar radiation) affected methane emissions via the regulation of lactate production by L. plantarum. The enrichment of L. plantarum contributes to lactic acid production and thereby enhances methane emission from poorly fermented oat silage. Notably, there are many lactic acid bacteria detrimental to methane production in the PTZ. This knowledge will be helpful in revealing the mechanisms of environmental factors and microbial relationships influencing the metabolic processes of methane production, thereby providing a reference for the clean utilization of other poorly fermented silage.

Molecular Mechanism of Cold Tolerance of Centipedegrass Based on the Transcriptome.

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.

Effect of nitrogen fertilizer on seed yield and quality of Kengyilia melanthera (Triticeae, Poaceae).

Widely distributed in the alpine sandy grassland in east Qinghai-Tibet Plateau (QTP), Kengyilia melanthera is considered as an ideal pioneer grass for the restoration of degraded and desertification grassland in the region. Under the special ecological and climatic conditions in the northwest Sichuan plateau located in east QTP, it is of great significance to optimize the amount of nitrogen fertilizer for the seed production of this species. The impact of nitrogen (N) fertilizer application on seed yield and quality of K. melanthera 'Aba', the only domesticated variety in the Kengyilia genus of Poaceae, was investigated based on two-year field experiments in the northwestern Sichuan plateau. The results showed that with the increase of N fertilizer application, the number of tillers, number of fertile tillers, 1,000-seed weight and seed yield of this species increased likewise. The optimum N fertilizer rate deduced in the present study was 180 kg·hm-2, where the number of fertile tillers 1,000-seed weight and seed yield reached the peak values. Interestingly, the standard germination rate, germination energy, accelerated aging germination rate, dehydrogenase and acid phosphatase activity of seeds were not affected by the increasing the input of N fertilizer. The comprehensive evaluation of membership function showed that the optimal N fertilizer treatment was 180 kg·hm-2 both for 2016 and 2017. This study provided a certain practical suggestion for the improvement of seed production of K. melanthera in the northwest Sichuan plateau.

Full-length transcriptome sequencing analysis and characterization, development and validation of microsatellite markers in Kengyilia melanthera.

As a typical psammophyte of the Triticeae, Kengyilia melanthera possesses high feeding potential and great utilization values in desertification control in the Qinghai-Tibet Plateau. However, few gene function and genetic studies have been performed in K. melanthera. In this study, single-molecule real-time sequencing technology was used to obtain the full-length transcriptome sequence of K. melanthera, following the functional annotation of transcripts and prediction of coding sequences (CDSs), transcription factors (TFs), and long noncoding RNA (lncRNA) sequences. Meanwhile, a total of 42,433 SSR loci were detected, with 5'-UTRs having the most SSR loci and trinucleotide being the most abundant type. In total, 108,399 SSR markers were designed, and 300 SSR markers were randomly selected for diversity verification of K. melanthera. A total of 49 polymorphic SSR markers were used to construct the genetic relationships of 56 K. melanthera accessions, among which 21 SSR markers showed good cross-species transferability among the related species. In conclusion, the full-length transcriptome sequence of the K. melanthera will assist gene prediction and promote molecular biology and genomics research, and the polymorphic SSR markers will promote molecular-assisted breeding and related research of K. melanthera and its relatives.

High-Altitude Genetic Selection and Genome-Wide Association Analysis of Yield-Related Traits in Elymus sibiricus L. Using SLAF Sequencing.

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.

Phylogeographical Pattern and Population Evolution History of Indigenous Elymus sibiricus L. on Qinghai-Tibetan Plateau.

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.

Molecular Phylogeography and Intraspecific Divergences in Siberian Wildrye (Elymus sibiricus L.) Wild Populations in China, Inferred From Chloroplast DNA Sequence and cpSSR Markers.

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.

Physiological and molecular responses of Phalaris arundinacea under salt stress on the Tibet plateau.

Phalaris arundinacea, with its characteristics of rapid growth and high biological yield, is regarded as an excellent forage grass in the Qinghai-Tibetan Plateau region of China. To explore the physiological and molecular response mechanism of Phalaris arundinacea under salt stress, we monitored the biomass and physiological indexes of two locally grown strains under conditions of exposure to 150 and 300 mM NaCl solution. Z0611 exhibited better salt stress tolerance than YS. Transcriptome sequencing analysis showed that YS and Z0611 had 1713 and 4290 differentially expressed genes (DEGs), respectively, including on metabolic processes, single-organism process, catalytic activity, and plant hormone signal transduction in the GO and KEGG databases. We also identified a large number of genes involved in hormone signaling, antioxidant systems, ion homeostasis, and photosynthetic systems. Our study provides physiological and molecular insight for establishing a salt resistance database and mining salt tolerance genes in Phalaris arundinacea, and also provides theoretical guidance for the restoration of saline-alkali land on the Qinghai-Tibet Plateau.

Down-regulation of PvSAMS impairs S-adenosyl-L-methionine and lignin biosynthesis, and improves cell wall digestibility in switchgrass.

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.

LATERAL BRANCHING OXIDOREDUCTASE, one novel target gene of Squamosa Promoter Binding Protein-like 2, regulates tillering in switchgrass.

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.

Effect of phenyllactic acid on silage fermentation and bacterial community of reed canary grass on the Qinghai Tibetan Plateau.

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.

Comparative Metabolomic Studies of Siberian Wildrye (Elymus sibiricus L.): A New Look at the Mechanism of Plant Drought Resistance.

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.

Mapping QTLs for spring green-up, plant vigor, and plant biomass in two lowland switchgrass populations.

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. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01296-7.

Physiological and Proteomic Responses of Contrasting Alfalfa (Medicago sativa L.) Varieties to High Temperature Stress.

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.

Study on the bacterial community structure and fermentation characteristics of fresh and ensiled paper mulberry.

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.

Physiological and transcriptional responses of Phalaris arundinacea under waterlogging conditions.

As a high-yielding forage grass, Phalaris arundinacea widely distributed in the Qinghai-Tibet Plateau region of China. To explore physiological and molecular response mechanism of Phalaris arundinacea under waterlogging, we analyzed the biomass and physiological indexes of three locally grown strains under the submerged condition of 10 cm. The material Z0611 showed the strongest waterlogging resistance while the YS showed the weakest performance. Transcriptome sequencing analysis demonstrated that the YS and Z0611 had 17010 and 7566 differently expression genes (DEGs), respectively, which were mainly concentrated in the metabolic process, cell, ribosome, phenylpropanoid biosynthesis pathway in GO and KEGG databases. We also identified a large number of genes involved in carbohydrate metabolism, hormone signaling regulation, transcription factors, antioxidant system, and ethylene signaling. Our research may provide a scientific basis for the restoration of wetland environment on the Qinghai-Tibet Plateau, and lay a foundation for further exploration of the waterlogging resistance genes of Phalaris arundinacea and breeding of new strains resistant with waterlogging stress.

Genomic survey sequencing, development and characterization of single- and multi-locus genomic SSR markers of Elymus sibiricus L.

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.

The Complete Mitogenome of Elymus sibiricus and Insights Into Its Evolutionary Pattern Based on Simple Repeat Sequences of Seed Plant Mitogenomes.

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.