Unraveling the signaling pathways of phytohormones underlying salt tolerance in Elymus sibiricus: A transcriptomic and metabolomic approach.

Understanding phytohormonal signaling is crucial for elucidating plant defense mechanisms against environmental stressors. However, knowledge regarding phytohormone-mediated tolerance pathways under salt stress in Elymus sibiricus, an important species for forage and ecological restoration, remains limited. In this study, transcriptomic and metabolomic approaches uncover the dynamics of phytohormonal signaling in Elymus sibiricus under salt stress. Notably, four hours after exposure to salt, significant activity was observed in the ABA, JA, IAA, and CTK pathways, with ABA, JA, JA-L-Ile, and IAA identified as key mediators in the response of Elymus sibiricus' to salinity. Moreover, SAPK3, Os04g0167900-like, CAT1, MKK2, and MPK12 were identified as potential central regulators within these pathways. The complex interactions between phytohormones and DEGs are crucial for facilitating the adaptation of Elymus sibiricus to saline environments. These findings enhance our understanding of the salt tolerance mechanisms in Elymus sibiricus and provide a foundation for breeding salt-resistant varieties.

Single-gene FISH maps and major chromosomal rearrangements in Elymus sibiricus and E. nutans.

BACKGROUND: Chromosomal variations have been revealed in both E. sibiricus and E. nutans, but chromosomal structural variations, such as intra-genome translocations and inversions, are still not recognized due to the cytological limitations of previous studies. Furthermore, the syntenic relationship between both species and wheat chromosomes remains unknown. RESULTS: Fifty-nine single-gene fluorescence in situ hybridization (FISH) probes, including 22 single-gene probes previously mapped on wheat chromosomes and other newly developed probes from the cDNA of Elymus species, were used to characterize the chromosome homoeologous relationship and collinearity of both E. sibiricus and E. nutans with those of wheat. Eight species-specific chromosomal rearrangements (CRs) were exclusively identified in E. sibiricus, including five pericentric inversions in 1H, 2H, 3H, 6H and 2St; one possible pericentric inversion in 5St; one paracentric inversion in 4St; and one reciprocal 4H/6H translocation. Five species-specific CRs were identified in E. nutans, including one possible pericentric inversion in 2Y, three possible pericentric multiple-inversions in 1H, 2H and 4Y, and one reciprocal 4Y/5Y translocation. Polymorphic CRs were detected in three of the six materials in E. sibiricus, which were mainly represented by inter-genomic translocations. More polymorphic CRs were identified in E. nutans, including duplication and insertion, deletion, pericentric inversion, paracentric inversion, and intra- or inter-genomic translocation in different chromosomes. CONCLUSIONS: The study first identified the cross-species homoeology and the syntenic relationship between E. sibiricus, E. nutans and wheat chromosomes. There are distinct different species-specific CRs between E. sibiricus and E. nutans, which may be due to their different polyploidy processes. The frequencies of intra-species polymorphic CRs in E. nutans were higher than that in E. sibiricus. To conclude, the results provide new insights into genome structure and evolution and will facilitate the utilization of germplasm diversity in both E. sibiricus and E. nutans.

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.

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.

Construction of the first high-density genetic linkage map and identification of seed yield-related QTLs and candidate genes in Elymus sibiricus, an important forage grass in Qinghai-Tibet Plateau.

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.

Transcriptome profiling of Elymus sibiricus, an important forage grass in Qinghai-Tibet plateau, reveals novel insights into candidate genes that potentially connected to seed shattering.

BACKGROUND: Elymus sibiricus is an important forage grass in semi-arid regions, but it is difficult to grow for commercial seed production due to high seed shattering. To better understand the underlying mechanism and explore the putative genes related to seed shattering, we conducted a combination of morphological, histological, physiochemical and transcriptome analysis on two E. sibiricus genotypes (XH09 and ZhN03) that have contrasting seed shattering. RESULTS: The results show that seed shattering is generally caused by a degradation of the abscission layer. Early degradation of abscission layers was associated with the increased seed shattering in high seed shattering genotype XH09. Two cell wall degrading enzymes, cellulase (CE) and polygalacturonase (PG), had different activity in the abscission zone, indicating their roles in differentiation of abscission layer. cDNA libraries from abscission zone tissue of XH09 and ZhN03 at 7 days, 21 days and 28 days after heading were constructed and sequenced. A total of 86,634 unigenes were annotated and 7110 differentially expressed transcripts (DETs) were predicted from "XH09-7 vs ZhN03-7", "XH09-21 vs ZhN03-21" and "XH09-28 vs ZhN03-28", corresponding to 2058 up-regulated and 5052 down-regulated unigenes. The expression profiles of 10 candidate transcripts involved in cell wall-degrading enzymes, lignin biosynthesis and phytohormone activity were validated using quantitative real-time PCR (qRT-PCR), 8 of which were up-regulated in low seed shattering genotype ZhN03, suggesting these genes may be associated with reduction of seed shattering. CONCLUSIONS: The expression data generated in this study provides an important resource for future molecular biological research in E. sibiricus.

Hybrid identification and genetic variation of Elymus sibiricus hybrid populations using EST-SSR markers.

BACKGROUND: Elymus sibiricus is an important native grass in Qinghai-Tibetan Plateau. Seed shattering is a serious problem for E. sibiricus, especially at harvest time. Cross breeding is an effective way to create new varieties with beneficial characteristic or improved traits, and to broaden genetic base. RESULTS: In this study, we created five hybrid populations by crossing seven E. sibiricus genotypes that have seed shattering variation. Then, nine EST-SSR primers were used for hybrid identification based on DNA fingerprinting, and genetic diversity analysis of hybrid populations and their respective parents. A total of 15 hybrids were identified. An analysis of amplified polymorphic bands among genuine hybrids and their respective parents revealed mainly two types of markers: 1) hybrids shared bands exclusively amplified in both parents; 2)hybrids shared bands exclusively amplified in male parents. For each hybrid population, the total number of amplified bands ranged from 37 to 57, the percentage of polymorphism varied from 65.12% to 75.68%, with an average of 70.51%. Novel bands found in each hybrid population varied from 0 to 9 bands, suggesting an occurrence of rearrangements in the hybrid population. The structure analysis revealed that all hybrid populations and parents were assigned to eight groups. The principal coordinate analysis (PCoA) showed similar results. CONCLUSIONS: In general, this study proved EST-SSR markers are efficient for hybrid identification, and suggested more genetic variation could be captured in hybrid populations by crossing breeding.

Assessing and Broadening Genetic Diversity of Elymus sibiricus Germplasm for the Improvement of Seed Shattering.

Siberian wild rye (Elymus sibiricus L.) is an important native grass in the Qinghai-Tibet Plateau of China. It is difficult to grow for commercial seed production, since seed shattering causes yield losses during harvest. Assessing the genetic diversity and relationships among germplasm from its primary distribution area contributes to evaluating the potential for its utilization as a gene pool to improve the desired agronomic traits. In the study, 40 EST-SSR primers were used to assess the genetic diversity and population structure of 36 E. sibiricus accessions with variation of seed shattering. A total of 380 bands were generated, with an average of 9.5 bands per primer. The polymorphic information content (PIC) ranged from 0.23 to 0.50. The percentage of polymorphic bands (P) for the species was 87.11%, suggesting a high degree of genetic diversity. Based on population structure analysis, four groups were formed, similar to results of principal coordinate analysis (PCoA). The molecular variance analysis (AMOVA) revealed the majority of genetic variation occurred within geographical regions (83.40%). Two genotypes from Y1005 and ZhN06 were used to generate seven F1 hybrids. The molecular and morphological diversity analysis of F1 population revealed rich genetic variation and high level of seed shattering variation in F1 population, resulting in significant improvement of the genetic base and desired agronomic traits.

Effects of plant age on antioxidant activity and endogenous hormones in Alpine Elymus sibiricus of the Tibetan Plateau.

Elymus sibiricus L. is a perennial forage species that has potential to serve as a forage source in livestock grazing systems. However, E. sibiricus has been shown to have a rapid and substantial reduction of aboveground biomass and seed yield after 3 or 4 years and an accelerated aging process. To determine possible aging mechanisms, we planted E. sibiricus seeds in triplicate blocks in 2012, 2015, and 2016, respectively, and harvested samples of leaves and roots at the jointing and heading stages in 2018 and 2019 to determine oxidative indices and endogenous hormones. The fresh aboveground biomass of 4- and 5-year old plants declined by 34.2% and 52.4% respectively compared with 3-year old plants, and the seed yield declined by 12.7% and 34.1%, respectively. The water content in leaves was 51.7%, 43.3%, and 35.6%, and net photosynthesis was 7.73, 6.35, and 2.08 µmol/m2·s in 3-, 4-, and 5-year old plants, respectively. The superoxide anion radical generation rate in leaves and roots did not show any aging pattern. There was a non-significant increase in malondialdehyde concentration with plant age, particularly in leaves and roots at the heading stage in 2019. The superoxide dismutase activity showed a declining trend with age of plant roots at the jointing stage in both 2018 and 2019. The peroxidase activity declined with plant age in both leaves and roots, for example, and the catalase activity in roots 4- and 7-year old plants declined by 13.8% and 0.85%, respectively, compared to 3-year old plants at the heading stage in 2018. Therefore, the reduced capacity of the antioxidant system may lead to oxidative stress during plant aging process. Overall, the concentrations of plant hormones, auxin (IAA), gibberellin (GA), zeatin (ZT), and abscisic acid (ABA) were significantly lower in roots than in leaves. The IAA concentration in leaves and roots exhibited different patterns with plant age. The ZT concentrations in leaves of 3-year old plants was 2.39- and 2.62-fold of those in 4- and 7-year old plants, respectively at the jointing stage, and in roots, the concentration declined with plant age. The changes in the GA concentration with plant age varied between the physiological stages and between years. The ABA concentrations appeared to increase with plant age, particularly in leaves. In conclusion, the aging process of E. sibiricus was apparently associated with an increase in oxidative stress, a decrease of ZT and an increase of ABA, particularly in roots. These findings highlight the effects of plant age on the antioxidant and endogenous hormone activity of E. sibiricus. However, these plant age-related trends showed variations between plant physiological stages and between harvest years that needs to be researched in the future to develop strategies to manage this forage species.

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.

Endodermal apoplastic barriers are linked to osmotic tolerance in meso-xerophytic grass Elymus sibiricus.

Drought is the most serious adversity faced by agriculture and animal husbandry industries. One strategy that plants use to adapt to water deficits is modifying the root growth and architecture. Root endodermis has cell walls reinforced with apoplastic barriers formed by the Casparian strip (CS) and suberin lamellae (SL) deposits, regulates radial nutrient transport and protects the vascular cylinder from abiotic threats. Elymus sibiricus is an economically important meso-xerophytic forage grass, characterized by high nutritional quality and strong environmental adaptability. The purpose of this study was to evaluate the drought tolerance of E. sibiricus genotypes and investigate the root structural adaptation mechanism of drought-tolerant genotypes' responding to drought. Specifically, a drought tolerant (DT) and drought sensitive (DS) genotype were screened out from 52 E. sibiricus genotypes. DT showed less apoplastic bypass flow of water and solutes than DS under control conditions, as determined with a hydraulic conductivity measurement system and an apoplastic fluorescent tracer, specifically PTS trisodium-8-hydroxy-1,3,6-pyrenetrisulphonic acid (PTS). In addition, DT accumulated less Na, Mg, Mn, and Zn and more Ni, Cu, and Al than DS, regardless of osmotic stress. Further study showed more suberin deposition in DT than in DS, which could be induced by osmotic stress in both. Accordingly, the CS and SL were deposited closer to the root tip in DT than in DS. However, osmotic stress induced their deposition closer to the root tips in DS, while likely increasing the thickness of the CS and SL in DT. The stronger and earlier formation of endodermal barriers may determine the radial transport pathways of water and solutes, and contribute to balance growth and drought response in E. sibiricus. These results could help us better understand how altered endodermal apoplastic barriers in roots regulate water and mineral nutrient transport in plants that have adapted to drought environments. Moreover, the current findings will aid in improving future breeding programs to develop drought-tolerant grass or crop cultivars.

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.

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.

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.

Development of EST-SSR Markers Linked to Flowering Candidate Genes in Elymus sibiricus L. Based on RNA Sequencing.

Elymus sibiricus L. is an important cold-season grass with excellent cold and drought tolerance, good palatability, and nutrition. Flowering time is a key trait that affects forage and seed yield. Development of EST-SSR (expressed sequence tag simple sequence repeat) markers based on flowering genes contributes to the improvement of flowering traits. In the study, we detected 155 candidate genes related to flowering traits from 10,591 unigenes via transcriptome sequencing in early- and late-flowering genotypes. These candidate genes were mainly involved in the photoperiodic pathway, vernalization pathway, central integrator, and gibberellin pathway. A total of 125 candidate gene-based EST-SSRs were developed. Further, 15 polymorphic EST-SSRs closely associated to 13 candidate genes were used for genetic diversity and population structure analysis among 20 E. sibiricus accessions, including two contrasting panels (early-flowering and late-flowering). Among them, primer 28366, designed from heading date 3a (HD3a), effectively distinguished early- and late-flowering genotypes using a specifically amplified band of 175 bp. The polymorphic information content (PIC) value ranged from 0.12 to 0.48, with an average of 0.25. The unweighted pair group method analysis (UPGMA) cluster and structure analysis showed that the 20 E. sibiricus genotypes with similar flowering times tended to group together. These newly developed EST-SSR markers have the potential to be used for molecular markers assisted selection and germplasm evaluation of flowering traits in E. sibiricus.

Comparative Physiological and Proteomic Analysis Reveals Different Involvement of Proteins during Artificial Aging of Siberian Wildrye Seeds.

Seed aging has an important effect on the germplasm preservation and industrialized production of Siberian wildrye (Elymus sibiricus) in the Qinghai-Tibet Plateau. However, so far its underlying molecular mechanisms still largely remain unknown. To shed light on this topic, one-year stored seeds of E. sibiricus were exposed to artificial aging treatments (AAT), followed by seed vigor characteristics and physiological status monitoring. Then global proteomics analysis was undertaken by the tandem mass tags (TMT) technique, and the proteins were quantified with liquid chromatography-tandem mass spectrometry on three aging time points (0 h, 36 h and 72 h). Finally, we verified the expression of related proteins by parallel reaction monitoring (PRM). Our results demonstrated that the seed vigor decreased remarkably in response to artificial aging, but the relative ion-leakage and malondialdehyde content, superoxide anion and hydrogen peroxide showed the opposite situation. Proteomic results showed that a total of 4169 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 carbohydrate metabolism, lipid metabolism, and antioxidant activity were severely damaged by aging treatments. Numerous key proteins such as glyceraldehyde triphosphate glyceraldehyde dehydrogenase, succinate dehydrogenase, lipoxygenase, peroxidase, glutathione-s-transferase and late embryogenesis abundant proteins were significantly down-regulated. However, the up-regulation of the heat shock protein family has made a positive contribution to oxidative stress resistance in seeds. This study provides a useful catalog of the E. sibiricus proteomes with insights into the future genetic improvement of seed storability.

Selection of Suitable Reference Genes for RT-qPCR Gene Expression Analysis in Siberian Wild Rye (Elymus sibiricus) under Different Experimental Conditions.

Elymus sibiricus, which is a perennial and self-pollinated grass, is the typical species of the genus Elymus, which plays an important role in forage production and ecological restoration. No reports have, so far, systematically described the selection of optimal reference genes for reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) analysis in E. sibiricus. The goals of this study were to evaluate the expression stability of 13 candidate reference genes in different experimental conditions, and to determine the appropriate reference genes for gene expression analysis in E. sibiricus. Five methods including Delta Ct (ΔCt), BestKeeper, NormFinder, geNorm, and RefFinder were used to assess the expression stability of 13 potential reference genes. The results of the RefFinder analysis showed that TBP2 and HIS3 were the most stable reference genes in different genotypes. TUA2 and PP2A had the most stable expression in different developmental stages. TBP2 and PP2A were suitable reference genes in different tissues. Under salt stress, ACT2 and TBP2 were identified as the most stable reference genes. ACT2 and TUA2 showed the most stability under heat stress. For cold stress, PP2A and ACT2 presented the highest degree of expression stability. DNAJ and U2AF were considered as the most stable reference genes under osmotic stress. The optimal reference genes were selected to investigate the expression pattern of target gene CSLE6 in different conditions. This study provides suitable reference genes for further gene expression analysis using RT-qPCR in E. sibiricus.

Histological Characteristics, Cell Wall Hydrolytic Enzymes Activity and Candidate Genes Expression Associated with Seed Shattering of Elymus sibiricus Accessions.

Elymus sibiricus (siberian wildrye) is a perennial, cool-season, self-pollinating, and allotetraploid grass. As an economically important species, it has been widely grown and used for pasture and hay in northern China. Because of serious seed shattering (SS), however, E. sibiricus is difficult to grow for commercial seed production. To better understand the underlying mechanism of SS, we investigated the differences in SS of cultivars and wild accessions in relation to morphological and genetic diversity, histological characteristics, lignin staining, cell wall hydrolytic enzymes activity and candidate genes expressions. We found high level of morphological and genetic diversity among E. sibiricus accessions. In general, cultivars had higher average pedicel breaking tensile strength (BTS) value than wild accessions, of which PI655199 had the highest average BTS value (144.51 gf) and LQ04 had the lowest average BTS value (47.17 gf) during seed development. SS showed a significant correlation with seed length, awn length and 1000-seed weight. SS was caused by degradation of abscission layers that formed at early heading stage, and degradation of abscission layers occurred at 14 days after heading. Histological analysis of abscission zone (AZ) showed a smooth fracture surface on the rachilla in high SS genotype, suggesting higher degradation degree of abscission layers. This may resulted from the increased cellulase and polygalacturonase activity found in AZ at seed physiological maturity. Staining of pedicels of two contrasting genotypes suggested more lignin deposition in low SS genotype may play a role in resistance of SS. Furthermore, candidate genes that involved in cell wall-degrading enzyme and lignin biosynthesis were differentially expressed in AZ, indicating the involvement and role in SS. This study provided novel insights into the mechanism of SS in E. sibiricus.

The Effect of Seed-Borne Fungi and Epichloë Endophyte on Seed Germination and Biomass of Elymus sibiricus.

The interactive effects of asexual Epichloë (formerly known as Neotyphodium) endophytes isolated from Hordeum brevisubulatum, Elymus tangutorum and Achnatherum inebrians, and seed-borne fungi on Elymus sibiricus seeds, were determined by an in vitro study using supernatants from liquid cultures of the endophyte strains. In an 8 week greenhouse study, the effects on the seedlings growth was measured. The in vitro study was carried out with the seed-borne fungi Alternaria alternata, Bipolaris sorokiniana, Fusarium avenaceum, and a Fusarium sp. isolated from E. sibiricus. Different concentrations and combinations of the liquid cultures of endophytic fungi enhanced the interim germination, germination rate, length of coleoptile and radicle, and seedling dry weight of E. sibiricus under stress from seed-borne fungi. In the greenhouse study, different concentrations of the supernatant of the endophytes from H. brevisubulatum and E. tangutorum but not A. inebrians, signficantly (P < 0.05) enhanced E. sibiricus seed germination. There was no significant (P > 0.05) increase of the tiller numbers after 2 weeks. However, later on, there were significant (P < 0.05) increases in tiller number (4-8 weeks), seedling height (2-8 weeks) and dry weight (2-8 weeks). The application of Epichloë endophyte culture supernatants was an effective strategy to improve seed germination and growth under greenhouse conditions.