Silicon nanoparticles improved the osmolyte production, antioxidant defense system, and phytohormone regulation in Elymus sibiricus (L.) under drought and salt stress.

Drought and salt stress negatively influence the growth and development of various plant species. Thus, it is crucial to overcome these stresses for sustainable agricultural production and the global food chain. Therefore, the present study investigated the potential effects of exogenous silicon nanoparticles (SiNPs) on the physiological and biochemical parameters, and endogenous phytohormone contents of Elymus sibiricus under drought and salt stress. Drought stress was given as 45% water holding capacity, and salt stress was given as 120 mM NaCl. The seed priming was done with different SiNP concentrations: SiNP1 (50 mg L-1), SiNP2 (100 mg L-1), SiNP3 (150 mg L-1), SiNP4 (200 mg L-1), and SiNP5 (250 mg L-1). Both stresses imposed harmful impacts on the analyzed parameters of plants. However, SiNP5 increased the chlorophylls and osmolyte accumulation such as total proteins by 96% and 110% under drought and salt stress, respectively. The SiNP5 significantly decreased the oxidative damage and improved the activities of SOD, CAT, POD, and APX by 10%, 54%, 104%, and 211% under drought and 42%, 75%, 72%, and 215% under salt stress, respectively. The SiNPs at all concentrations considerably improved the level of different phytohormones to respond to drought and salt stress and increased the tolerance of Elymus plants. Moreover, SiNPs decreased the Na+ and increased K+ concentrations in Elymus suggesting the reduction in salt ion accumulation under salinity stress. Overall, exogenous application (seed priming/dipping) of SiNPs considerably enhanced the physio-biochemical and metabolic responses, resulting in an increased tolerance to drought and salt stresses. Therefore, this study could be used as a reference to further explore the impacts of SiNPs at molecular and genetic level to mitigate abiotic stresses in forages and related plant species.

Pilot-scale two-phase anaerobic digestion of deoiled food waste and waste activated sludge: Effects of mixing ratios and functional analysis.

Anaerobic co-digestion of deoiled food waste (dFW) and waste activated sludge (WAS) can address the challenges derived from mono-digestion of FW. In the present study, a pilot-scale methanogenic bioreactor of a two-phase anaerobic digestion system was developed to explore the impact of dFW/WAS volatile solids ratios on the overall performance, microbial community, and metabolic pathways. Besides, the tech-economic of the system was analyzed. The results showed that the degradation efficiency of soluble chemical oxygen demand (SCOD) was more than 84.90% for all the dFW/WAS ratios (v/v) (1:0, 39:1, 29:1, 19:1 and 9:1). Moreover, the dominant genus of bacteria and archaea with different ratios were Lactobacillus (66.84-98.44%) and Methanosaeta (53.66-80.09%), respectively. Co-digestion of dFW and WAS (29: 1 in v/v ratios) obtained the highest yield of methane (0.41 L CH4/Ladded) with approximately 90% of SCOD being removed. In the pilot-scale experiment, the co-digestion of FW and WAS makes positive contribution to reusing solid waste for improving solid management.

Nanoparticle's uptake and translocation mechanisms in plants via seed priming, foliar treatment, and root exposure: a review.

Nanotechnology is one of the promising techniques and shares wide ranges of applications almost in every field of life. Nanomaterials are getting continuous attractions due to specific physical and chemical properties and being applied as multifunctional material. The use of nanomaterials/nanoparticles in agriculture sector for crop improvement and protection against various environmental threats have attained greater significance. Size and nature of nanoparticles, mode of application, environmental conditions, rhizospheric and phyllospheric environment, and plant species are major factors that influence the action of nanoparticles. The mode or method of nanoparticle applications to plants is attaining greater attentions. Recently, different methods for nanoparticle applications (seed priming, foliar, and root application) are being used to improve crop growth. It is of quite worth that which method is suitable for nanoparticle application, and how nanoparticles can possibly translocate to various plant tissues from root to shoot or vice versa. These information's are poorly understood and need more investigations to explore the comprehensive mechanism by which nanoparticles make their possible entry through different plant organs and how they transport to regulate various physiological and molecular functions in plant cells. Therefore, this study comprehensively provides the knowledge of nanoparticles uptake via seed priming, foliar exposure, and root application, and their possible translocation mechanism within plants influenced by various factors that has not clearly presented. This study will provide new insights to find out an actual uptake and translocation mechanism of nanoparticles that may help researchers to develop nanoparticle-based new strategies for plants to cope with various environmental challenges. This study also focuses on different soil factors or above ground factors that are involved in nanoparticles uptake and translocation and ultimately their functioning in plants.

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.

Identification of Flowering Regulatory Networks and Hub Genes Expressed in the Leaves of Elymus sibiricus L. Using Comparative Transcriptome Analysis.

Flowering is a significant stage from vegetative growth to reproductive growth in higher plants, which impacts the biomass and seed yield. To reveal the flowering time variations and identify the flowering regulatory networks and hub genes in Elymus sibiricus, we measured the booting, heading, and flowering times of 66 E. sibiricus accessions. The booting, heading, and flowering times varied from 136 to 188, 142 to 194, and 148 to 201 days, respectively. The difference in flowering time between the earliest- and the last-flowering accessions was 53 days. Furthermore, transcriptome analyses were performed at the three developmental stages of six accessions with contrasting flowering times. A total of 3,526 differentially expressed genes (DEGs) were predicted and 72 candidate genes were identified, including transcription factors, known flowering genes, and plant hormone-related genes. Among them, four candidate genes (LATE, GA2OX6, FAR3, and MFT1) were significantly upregulated in late-flowering accessions. LIMYB, PEX19, GWD3, BOR7, PMEI28, LRR, and AIRP2 were identified as hub genes in the turquoise and blue modules which were related to the development time of flowering by weighted gene co-expression network analysis (WGCNA). A single-nucleotide polymorphism (SNP) of LIMYB found by multiple sequence alignment may cause late flowering. The expression pattern of flowering candidate genes was verified in eight flowering promoters (CRY, COL, FPF1, Hd3, GID1, FLK, VIN3, and FPA) and four flowering suppressors (CCA1, ELF3, Ghd7, and COL4) under drought and salt stress by qRT-PCR. The results suggested that drought and salt stress activated the flowering regulation pathways to some extent. The findings of the present study lay a foundation for the functional verification of flowering genes and breeding of new varieties of early- and late-flowering E. sibiricus.

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.

Unveiling the Actual Functions of Awns in Grasses: From Yield Potential to Quality Traits.

Awns, which are either bristles or hair-like outgrowths of lemmas in the florets, are one of the typical morphological characteristics of grass species. These stiff structures contribute to grain dispersal and burial and fend off animal predators. However, their phenotypic and genetic associations with traits deciding potential yield and quality are not fully understood. Awns appear to improve photosynthesis, provide assimilates for grain filling, thus contributing to the final grain yield, especially under temperature- and water-stress conditions. Long awns, however, represent a competing sink with developing kernels for photosynthates, which can reduce grain yield under favorable conditions. In addition, long awns can hamper postharvest handling, storage, and processing activities. Overall, little is known about the elusive role of awns, thus, this review summarizes what is known about the effect of awns on grain yield and biomass yield, grain nutritional value, and forage-quality attributes. The influence of awns on the agronomic performance of grasses seems to be associated with environmental and genetic factors and varies in different stages of plant development. The contribution of awns to yield traits and quality features previously documented in major cereal crops, such as rice, barley, and wheat, emphasizes that awns can be targeted for yield and quality improvement and may advance research aimed at identifying the phenotypic effects of morphological traits in grasses.

Genome assembly provides insights into the genome evolution and flowering regulation of orchardgrass.

Orchardgrass (Dactylis glomerata L.) is an important forage grass for cultivating livestock worldwide. Here, we report an ~1.84-Gb chromosome-scale diploid genome assembly of orchardgrass, with a contig N50 of 0.93 Mb, a scaffold N50 of 6.08 Mb and a super-scaffold N50 of 252.52 Mb, which is the first chromosome-scale assembled genome of a cool-season forage grass. The genome includes 40 088 protein-coding genes, and 69% of the assembled sequences are transposable elements, with long terminal repeats (LTRs) being the most abundant. The LTRretrotransposons may have been activated and expanded in the grass genome in response to environmental changes during the Pleistocene between 0 and 1 million years ago. Phylogenetic analysis reveals that orchardgrass diverged after rice but before three Triticeae species, and evolutionarily conserved chromosomes were detected by analysing ancient chromosome rearrangements in these grass species. We also resequenced the whole genome of 76 orchardgrass accessions and found that germplasm from Northern Europe and East Asia clustered together, likely due to the exchange of plants along the 'Silk Road' or other ancient trade routes connecting the East and West. Last, a combined transcriptome, quantitative genetic and bulk segregant analysis provided insights into the genetic network regulating flowering time in orchardgrass and revealed four main candidate genes controlling this trait. This chromosome-scale genome and the online database of orchardgrass developed here will facilitate the discovery of genes controlling agronomically important traits, stimulate genetic improvement of and functional genetic research on orchardgrass and provide comparative genetic resources for other forage grasses.

Potential Effects of Awn Length Variation on Seed Yield and Components, Seed Dispersal and Germination Performance in Siberian Wildrye (Elymus sibiricus L.).

: Awns, needle-like structures formed on the distal of the lemmas in the florets, are of interest because of their essential roles in seed dispersal, germination and photosynthesis. Previous research has reported the potential benefits of awns in major cereal grasses, yet reports on the agronomic and economic implications of awn length variation in forage grasses remain scarce. This study investigated the variation of awn length among 20 Siberian wildrye populations and the effect of awn length on seed yield and yield components. This work then studied the impact of awn length on seed dispersal and germination. The analyses indicated a high level of awn length variation among populations. Awn length showed a significant influence on harvested seed yield per plant (p < 0.05) mostly driven by interactions between awn length and the majority of seed yield components. Principal component analysis clearly revealed that the final impact of awn length on seed yield depends on the balance of its positive and negative effects on traits determining seed yield. Furthermore, awn length tended to increase seed dispersal distance, although little diversity in the nature of this progression was observed in some populations. Awn length exhibited a significant relationship (p < 0.05) with germination percentage. It also tended to shorten germination duration, although this interaction was not statistically significant. Collectively, these results provide vital information for breeding and agronomic programs aiming to maintain yield in grasses. This is the first report to demonstrate in Siberian wildrye the agronomic impacts of awn length variation.

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.

Morphological and Genetic Mechanisms Underlying Awn Development in Monocotyledonous Grasses.

The identification of biological mechanisms underlying the development of complex quantitative traits, including those that contribute to plant architecture, yield and quality potential, and seed dispersal, is a major focus in the evolutionary biology and plant breeding. The awn, a bristle-like extension from the lemma in the floret, is one of the distinct morphological and physiological traits in grass species. Awns are taught as an evolutionary trait assisting seed dispersal and germination and increasing photosynthesis. Awn development seems to be complex process, involving dramatic phenotypic and molecular changes. Although recent advances investigated the underlying morphological and molecular genetic factors of awn development, there is little agreement about how these factors interact during awn formation and how this interaction affects variation of awn morphology. Consequently, the developmental sequence of the awn is not yet well understood. Here, we review awn morphological and histological features, awn development pathways, and molecular processes of awn development. We argue that morphological and molecular genetic mechanisms of awn development previously studied in major cereal crops, such as barley, wheat, and rice, offered intriguing insights helping to characterize this process in a comparative approach. Applying such an approach will aid to deeply understand factors involved in awn development in grass species.

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.

EST-SSR marker development based on RNA-sequencing of E. sibiricus and its application for phylogenetic relationships analysis of seventeen Elymus species.

BACKGROUND: Elymus L. is the largest genus in the tribe Triticeae Dumort., encompassing approximately 150 polyploid perennial species widely distributed in the temperate regions of the world. It is considered to be an important gene pool for improving cereal crops. However, a shortage of molecular marker limits the efficiency and accuracy of genetic breeding for Elymus species. High-throughput transcriptome sequencing data is essential for gene discovery and molecular marker development. RESULTS: We obtained the transcriptome dataset of E. sibiricus, the type species of the genus Elymus, and identified a total of 8871 putative EST-SSRs from 6685 unigenes. Trinucleotides were the dominant repeat motif (4760, 53.66%), followed by dinucleotides (1993, 22.47%) and mononucleotides (1876, 21.15%). The most dominant trinucleotide repeat motif was CCG/CGG (1119, 23.5%). Sequencing of PCR products showed that the sequenced alleles from different Elymus species were homologous to the original SSR locus from which the primer was designed. Different types of tri-repeats as abundant SSR motifs were observed in repeat regions. Two hundred EST-SSR primer pairs were designed and selected to amplify ten DNA samples of Elymus species. Eighty-seven pairs of primer (43.5%) generated clear and reproducible bands with expected size, and showed good transferability across different Elymus species. Finally, thirty primer pairs successfully amplified ninety-five accessions of seventeen Elymus species, and detected significant amounts of polymorphism. In general, hexaploid Elymus species with genomes StStHHYY had a relatively higher level of genetic diversity (H = 0.219, I = 0.330, %P = 63.7), while tetraploid Elymus species with genomes StStYY had low level of genetic diversity (H = 0.182, I = 0.272, %P = 50.4) in the study. The cluster analysis showed that all ninety-five accessions were clustered into three major clusters. The accessions were grouped mainly according to their genomic components and origins. CONCLUSIONS: This study demonstrated that transcriptome sequencing is a fast and cost-effective approach to molecular marker development. These EST-SSR markers developed in this study are valuable tools for genetic diversity, evolutionary, and molecular breeding in E. sibiricus, and other Elymus species.

Elymus nutans genes for seed shattering and candidate gene-derived EST-SSR markers for germplasm evaluation.

BACKGROUND: Elymus nutans and E. sibiricus are two important forage grasses of the genus Elymus. But they are difficult to grow for commercial seed production due to serious seed shattering. We conducted a comparative transcriptome analysis of abscission zone to find possible transcription changes associated with seed shattering, explore candidate genes involved in seed shattering and identify candidate gene-based EST-SSR markers for germplasm evaluation. RESULTS: cDNA libraries from abscission zone (AZ) and non-abscission zone (NAZ) tissues of E. nutans were constructed and sequenced. A total of 111,667 unigenes were annotated and 7644 differentially expressed transcripts (DETs) were predicted, corresponding to 6936 up-regulated in AZ and 708 down-regulated in NAZ. We identified 489 candidate genes related to transcription factor, cell wall hydrolysis or modification, hydrolase activity, phytohormone signaling and response, lignin biosynthesis, and signal transduction or protein turnover. Eleven similar candidate genes involved in polygalacturonase activity, hydrolase activity, and mitogen-activated protein kinase were up-regulated in the abscission zone of the two Elymus species, suggesting these genes may have specific function for abscission zone development and seed shattering. A total of 67 polymorphic EST-SSR markers were developed and characterized based on the sequences of these candidate genes. Fourteen polymorphic EST-SSR primers were finally used to study genetic diversity in 48 E. nutans genotypes with contrasting seed shattering habit. The dendrogram based on molecular data showed that most accessions with similar seed shattering degree tended to group together. CONCLUSIONS: The expression data generated from this study provides an important resource for future molecular biological research. Many DETs were associated with abscission zone development, and EST-SSR loci related to candidate genes may have potential application in identifying trait-associated markers in E. nutans in the future.

Integration of small RNAs and transcriptome sequencing uncovers a complex regulatory network during vernalization and heading stages of orchardgrass (Dactylis glomerata L.).

BACKGROUND: Flowering is a critical reproductive process in higher plants. Timing of optimal flowering depends upon the coordination among seasonal environmental cues. For cool season grasses, such as Dactylis glomerata, vernalization induced by low temperature provides competence to initiate flowering after prolonged cold. We combined analyses of the transcriptome and microRNAs (miRNAs) to generate a comprehensive resource for regulatory miRNAs and their target circuits during vernalization and heading stages. RESULTS: A total of 3,846 differentially expressed genes (DEGs) and 69 differentially expressed miRNAs were identified across five flowering stages. The expression of miR395, miR530, miR167, miR396, miR528, novel_42, novel_72, novel_107, and novel_123 demonstrated significant variations during vernalization. These miRNA targeted genes were involved in phytohormones, transmembrane transport, and plant morphogenesis in response to vernalization. The expression patterns of DEGs related to plant hormones, stress responses, energy metabolism, and signal transduction changed significantly in the transition from vegetative to reproductive phases. CONCLUSIONS: Five hub genes, c136110_g1 (BRI1), c131375_g1 (BZR1), c133350_g1 (VRN1), c139830_g1 (VIN3), and c125792_g2 (FT), might play central roles in vernalization response. Our comprehensive analyses have provided a useful platform for investigating consecutive transcriptional and post-transcriptional regulation of critical phases in D. glomerata and provided insights into the genetic engineering of flowering-control in cereal crops.

Phenotype- and SSR-Based Estimates of Genetic Variation between and within Two Important Elymus Species in Western and Northern China.

Elymus nutans and Elymus sibiricus are two important perennial forage grasses of the genus Elymus, widely distributed in high altitude regions of Western and Northern China, especially on the Qinghai-Tibetan Plateau. Information on phenotypic and genetic diversity is limited, but necessary for Elymus germplasm collection, conservation, and utilization. In the present study, the phenotypic and genetic differentiation of 73 accessions of the two species were evaluated using 15 phenotypic traits and 40 expressed sequence tag derived simple sequence repeat markers (EST-SSRs). The results showed that only 7.23% phenotypic differentiation (Pst) existed between the two Elymus species based on fifteen quantitative traits. Principal component analysis (PCA) revealed that leaf traits, spike traits, and some seed traits were dominant factors in phenotypic variation. Moreover, 396 (97.8%) and 331 (87.1%) polymorphic bands were generated from 40 EST-SSR primers, suggesting high levels of genetic diversity for the two species. The highest genetic diversity was found in the Northeastern Qinghai-Tibetan Plateau groups. Clustering analysis based on molecular data showed that most accessions of each Elymus species tended to group together. Similar results were described by principal coordinates analysis (PCoA) and structure analysis. The molecular variance analysis (AMOVA) revealed that 81.47% and 89.32% variation existed within the geographical groups for the two species, respectively. Pearson's correlation analyses showed a strong positive correlation between Nei's genetic diversity and annual mean temperature. These results could facilitate Elymus germplasm collection, conservation, and future breeding.

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.

Transcriptome Analyses Reveal Candidate Pod Shattering-Associated Genes Involved in the Pod Ventral Sutures of Common Vetch (Vicia sativa L.).

The seed dispersion caused by pod shattering is a form of propagation used by many wild species. Loss of seeds from pod shattering is frequent in the common vetch (Vicia sativa L.), an important self-pollinating annual forage legume. However, pod shattering is one of the most important defects that limits the reproduction of the vetch in the field and the usage as a leguminous forage crop. To better understand the vetch pod shattering mechanism, we used high-throughput RNA sequencing to assess the global changes in the transcriptomes of the pod ventral sutures of shattering-susceptible and shattering-resistant vetch accessions screened from 541 vetch germplasms. A total of 1,285 significantly differentially expressed unigenes (DEGs) were detected, including 575 up-regulated unigenes and 710 down-regulated unigenes. Analyses of Gene Ontology and KEGG metabolic enrichment pathways of 1,285 DEGs indicated that 22 DEGs encoding cell wall modifications and hydrolases associated with pod shattering were highly expressed in shattering-susceptible accessions. These genes were mainly enriched in "hydrolase activity," "cytoplasm," and "carbohydrate metabolic process" systems. These cell wall modifications and hydrolases genes included ß-glucosidase and endo-polygalacturonase, which work together to break down the glycosidic bonds of pectin and cellulose, and to promote the dissolution and disappearance of the cell wall in the ventral suture of the pod and make the pod more susceptible to shattering. We demonstrated the differences in gene transcription levels between the shattering-susceptible and shattering-resistant vetch accessions for the first time and our results provided valuable information for the identifying and characterizing of pod shattering regulation networks in vetch. This information may facilitate the future identification of pod shattering-related genes and their underlying molecular mechanisms in the common vetch.

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.