Genome constitution and evolution of Elymus atratus (Poaceae: Triticeae) inferred from cytogenetic and phylogenetic analysis.

BACKGROUND: Elymus atratus (Nevski) Hand.-Mazz. is perennial hexaploid wheatgrass. It was assigned to the genus Elymus L. sensu stricto based on morphological characters. Its genome constitution has not been disentangled yet. OBJECTIVE: To identify the genome constitution and origin of E. atratus. METHODS: In this study, genomic in situ hybridization and fluorescence in situ hybridization, and phylogenetic analysis based on the Acc1, DMC1 and matK sequences were performed. RESULTS: Genomic in situ hybridization and fluorescence in situ hybridization results reveal that E. atratus 2n = 6x = 42 is composed of 14 St genome chromosomes, 14 H genome chromosomes, and 14 Y genome chromosomes including two H-Y type translocation chromosomes, suggesting that the genome formula of E. atratus is StStYYHH. The phylogenetic analysis based on Acc1 and DMC1 sequences not only shows that the Y genome originated in a separate diploid, but also suggests that Pseudoroegneria (St), Hordeum (H), and a diploid species with Y genome were the potential donors of E. atratus. Data from chloroplast DNA showed that the maternal donor of E. atratus contains the St genome. CONCLUSION: Elymus atratus is an allohexaploid species with StYH genome, which may have originated through the hybridization between an allotetraploid Roegneria (StY) species as the maternal donor and a diploid Hordeum (H) species as the paternal donor.

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.

Genetic Covariation Between the Vertically Transmitted Endophyte Epichloë canadensis and Its Host Canada Wildrye.

Symbiotic mutualisms are thought to be stabilized by correlations between the interacting genotypes which may be strengthened via vertical transmission and/or reduced genetic variability within each species. Vertical transmission, however, may weaken interactions over time as the endosymbionts would acquire mutations that could not be purged. Additionally, temporal variation in a conditional mutualism could create genetic variation and increased variation in the interaction outcome. In this study, we assessed genetic variation in both members of a symbiosis, the endosymbiotic fungal endophyte Epichloë canadensis and its grass host Canada wildrye (Elymus canadensis). Both species exhibited comparable levels of diversity, mostly within populations rather than between. There were significant differences between populations, although not in the same pattern for the two species, and the differences were not correlated with geographic distance for either species. Interindividual genetic distance matrices for the two species were significantly correlated, although all combinations of discriminant analysis of principle components (DAPC) defined multilocus genotype groups were found suggesting that strict genotype matching is not necessary. Variation in interaction outcome is common in grass/endophyte interactions, and our results suggest that the accumulation of mutations overtime combined with temporal variation in selection pressures increasing genetic variation in the symbiosis may be the cause.

Characterization and Application of EST-SSR Markers Developed from Transcriptome Sequences in Elymus breviaristatus (Poaceae: Triticeae).

BACKGROUND: Elymus L. is the largest genus in the Triticeae tribe. Most species in this genus are highly stress resistant, with excellent forage value. Elymus breviaristatus, a rare species endemic to the Qinghai-Tibet Plateau (QTP), is declining due to habitat fragmentation. However, genetic data for E. breviaristatus are limited, with expressed sequence tag (EST) markers being particularly rare, hampering genetic studies and protection measures. RESULTS: We obtained 9.06 Gb clean sequences from the transcriptome of E. breviaristatus, generating 171,522 unigenes, which were assembled and functionally annotated against five public databases. We identified 30,668 SSRs in the E. breviaristatus transcriptome, from which 103 EST-SSR primer pairs were randomly selected. Of these, 58 pairs of amplified products of the expected size, and 18 of the amplified products were polymorphic. Model-based Bayesian clustering, the unweighted pair group method with arithmetic average (UPGMA), and principal coordinate analysis (PCoA) of 179 wild E. breviaristatus in 12 populations using these EST-SSRs were generally consistent, grouping the 12 populations into two major clades. Analysis of molecular variance (AMOVA) found 70% of the genetic variation among the 12 populations and 30% within the populations, indicating a high level of genetic differentiation (or low gene exchange) among the 12 populations. The transferability of the 58 successful EST-SSR primers to 22 related hexaploid species was 86.2-98.3%. UPGMA analysis generally grouped species with similar genome types together. CONCLUSIONS: Here, we developed EST-SSR markers from the transcriptome of E. breviaristatus. The transferability of these markers was evaluated, and the genetic structure and diversity of E. breviaristatus were explored. Our results provide a basis for the conservation and management of this endangered species, and the obtained molecular markers represent valuable resources for the exploration of genetic relationships among species in the Elymus genus.

Microbacterium elymi sp. nov., Isolated from the Rhizospheric Soil of Elymus tsukushiensis, a Plant Native to the Dokdo Islands, Republic of Korea.

Microbacterium elymi KUDC0405T was isolated from the rhizosphere of Elymus tsukushiensis from the Dokdo Islands. The KUDC0405T strain was Gram-stain-positive, non-spore forming, non-motile, and facultatively anaerobic bacteria. Strain KUDC0405T was a rod-shaped bacterium with size dimensions of 0.3-0.4 × 0.7-0.8 µm. Based on 16S rRNA gene sequences, KUDC0405T was most closely related to Microbacterium bovistercoris NEAU-LLET (97.8%) and Microbacterium pseudoresistens CC-5209T (97.6%). The dDDH (digital DNA-DNA hybridization) values between KUDC0405T and M. bovistercoris NEAU-LLET and M. pseudoresistens CC-5209T were below 17.3% and 17.5%, respectively. The ANI (average nucleotide identity) values among strains KUDC0405T, M. bovistercoris NEAU-LLET, and M. pseudoresistens CC-5209T were 86.6% and 80.7%, respectively. The AAI (average amino acid identity) values were 64.66% and 64.97%, respectively, between KUDC0405T and its closest related type strains. The genome contained 3,596 CDCs, three rRNAs, 46 tRNAs, and three non-coding RNAs (ncRNAs). The genomic DNA GC content was 70.4%. The polar lipids included diphosphatydilglycerol, glycolipid, phosphatydilglycerol, and unknown phospholipid, and the major fatty acids were anteiso-C17:0 and iso-C16:0. Strain KUDC0405T contained MK-12 as the major menaquinone. Based on genotypic, phylogenetic, and phenotypic properties, strain KUDC0405T should be considered a novel species within the genus Microbacterium, for which we propose the name M. elymi sp. nov., and the type strain as KUDC0405T (=KCTC 49411T, =CGMCC1.18472T).

Biosystematics studies on Elymus breviaristatus and Elymus sinosubmuticus (Poaceae: Triticeae).

BACKGROUND: Elymus breviaristatus and Elymus sinosubmuticus are perennial herbs, not only morphologically similar but also sympatric distribution. The genome composition of E. sinosubmuticus has not been reported, and the relationship between E. sinosubmuticus and E. breviaristatus is still controversial. We performed artificial hybridization, genomic in situ hybridization, and phylogenetic analyses to clarify whether the two taxa were the same species. RESULTS: The high frequency bivalent (with an average of 20.62 bivalents per cell) at metaphase I of pollen mother cells of the artificial hybrids of E. breviaristatus (StYH) × E. sinosubmuticus was observed. It illustrated that E. sinosubmuticus was closely related to E. breviaristatus. Based on genomic in situ hybridization results, we confirmed that E. sinosubmuticus was an allohexaploid, and the genomic constitution was StYH. Phylogenetic analysis results also supported that this species contained St, Y, and H genomes. In their F1 hybrids, pollen activity was 53.90%, and the seed setting rate was 22.46%. Those indicated that the relationship between E. sinosubmuticus and E. breviaristatus is intersubspecific rather than interspecific, and it is reasonable to treated E. sinosubmuticus as the subspecies of E. breviaristatus. CONCLUSIONS: In all, the genomic constitutions of E. sinosubmuticus and E. breviaristatus were StYH, and they are species in the genus Campeiostachys. Because E. breviaristatus was treated as Campeistachys breviaristata, Elymus sinosubmuticus should be renamed Campeiostachys breviaristata (Keng) Y. H. Zhou, H. Q. Zhang et C. R. Yang subsp. sinosubmuticus (S. L. Chen) Y. H. Zhou, H. Q. Zhang et L. Tan.

Confirmation of Natural Hybridization between Kengyilia (StStYYPP) and Campeiostachys (StStYYHH) (Triticeae: Poaceae) Based on Morphological and Molecular Cytogenetic Analyses.

Natural hybridization has been frequently observed in Triticeae; however, few studies have investigated the origin of natural intergeneric Triticeae hybrids. In the present study, we discovered three putative hybrid Triticeae plants in the Western Sichuan Plateau of China. Morphologically, the putative hybrids were intermediate between Kengyilia melanthera (2n = 6x = 42; StStYYPP) and Campeiostachys dahurica var. tangutorum (2n = 6x = 42; StStYYHH) with greater plant height and tiller number. Cytological analyses demonstrated that the hybrids were hexaploid with 42 chromosomes (2n = 6x = 42). At metaphase I, 12.10-12.58 bivalents and 13.81-14.18 univalents per cell were observed in the hybrid plants. Genomic in situ hybridization demonstrated that the hybrids had StStYYHP genomes. Phylogenetic analysis of Acc1 sequences indicated that the hybrids were closely related to K. melanthera and C. dahurica var. tangutorum. Our morphological, cytological, and molecular analyses indicate that these hexaploid natural hybrid plants may be hybrids of K. melanthera and C. dahurica var. tangutorum.

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.

Characterization of Each St and Y Genome Chromosome of Roegneria grandis Based on Newly Developed FISH Markers.

The genera of the tribe Triticeae (family Poaceae), constituting many economically important plants with abundant genetic resources, carry genomes such as St, H, P, and Y. The genome symbol of Roegneria C. Koch (Triticeae) is StY. The St and Y genomes are crucial in Triticeae, and tetraploid StY species participate extensively in polyploid speciation. Characterization of St and Y nonhomologous chromosomes in StY-genome species could help understand variation in the chromosome structure and differentiation of StY-containing species. However, the high genetic affinity between St and Y genome and the deficiency of a complete set of StY nonhomologous probes limit the identification of St and Y genomes and variation of chromosome structures among Roegneria species. We aimed to identify St- and Y-enhanced repeat clusters and to study whether homoeologous chromosomes between St and Y genomes could be accurately identified due to high affinity. We employed comparative genome analyses to identify St- and Y-enhanced repeat clusters and generated a FISH-based karyotype of R. grandis (Keng), one of the taxonomically controversial StY species, for the first time. We explored 4 novel repeat clusters (StY_34, StY_107, StY_90, and StY_93), which could specifically identify individual St and Y nonhomologous chromosomes. The clusters StY_107 and StY_90 could identify St and Y addition/substitution chromosomes against common wheat genetic backgrounds. The chromosomes V_St, VII_St, I_Y, V_Y, and VII_Y displayed similar probe distribution patterns in the proximal region, indicating that the high affinity between St and Y genome might result from chromosome rearrangements or transposable element insertion among V_St/Y, VII_St/Y, and I_Y chromosomes during allopolyploidization. Our results can be used to employ FISH further to uncover the precise karyotype based on colinearity of Triticeae species by using the wheat karyotype as reference, to analyze diverse populations of the same species to understand the intraspecific structural changes, and to generate the karyotype of different StY-containing species to understand the interspecific chromosome variation.

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.

Transcriptome profiles identify the common responsive genes to drought stress in two Elymus species.

Elymus, the largest genus of the Triticeae Dumort, is a forage grass in the Qinghai-Tibetan Plateau, where the climate has gradually become increasingly dry in recent years. To understand the mechanisms of the response to drought stress in Elymus species, we first investigated physiological and biochemical responses to polyethylene glycol (PEG-6000) simulated drought stress in two Elymus species, Elymus nutans and Elymus sibiricus, and found that E. nutans was more tolerant to drought stress than E. sibiricus. De novo transcriptome analysis of these two Elymus species treated with or without 10 % PEG-6000 revealed that a total of 1695 unigenes were commonly regulated by drought treatment in these two Elymus species, with 1614 unigenes up-regulated and 81 unigenes down-regulated. The coexpressed differentially expressed genes (DEGs) were enriched in regulation of transcription and gene expression in the GO database. KEGG pathway analysis indicated plant hormone signaling transduction were mostly enriched in co-expressed DEGs. Furthermore, genes annotated in the plant hormone signaling transduction were screened from co-expressed DEGs, and found that abscisic acid plays the major role in the drought stress tolerance of Elymus. Meanwhile, transcription factors screened from co-expressed DEGs were mainly classified into the ERF subfamily and WRKY, DREB, and HSF family members. Our results provide further reference for studying the response mechanism and culturing highly tolerant grasses of the Elymus species under drought stress.

Overexpression of V-type H+ pyrophosphatase gene EdVP1 from Elymus dahuricus increases yield and potassium uptake of transgenic wheat under low potassium conditions.

Lack of potassium in soil limits crop yield. Increasing yield and conserving potassium ore requires improving K use efficiency (KUE). Many genes influence KUE in plants, but it is not clear how these genes function in the field. We identified the V-type H+-pyrophosphatase gene EdVP1 from Elymus dahurica. Gene expression analysis showed that EdVP1 was induced by low potassium stress. Protein subcellular localization analysis demonstrated that EdVP1 localized on the plasma membrane. We overexpressed EdVP1 in two wheat varieties and conducted K tolerance experiments across years. Yield per plant, grain number per spike, plant height, and K uptake of four transgenic wheat lines increased significantly compared with WT; results from two consecutive years showed that EdVP1 significantly increased yield and KUE of transgenic wheat. Pot experiments showed that transgenic plants had significantly longer shoots and roots, and higher K accumulation in shoots and roots and H+-PPase activity in shoots than WT under low K. A fluidity assay of potassium ion in EdVP1 transgenic plant roots showed that potassium ion influx and H+ outflow in transgenic plants were higher than WT. Overexpressing EdVP1 significantly improved yield and KUE of transgenic wheat and was related to higher K uptake capacity in root.

Molecular karyotyping of Siberian wild rye (Elymus sibiricus L.) with oligonucleotide fluorescence in situ hybridization (FISH) probes.

Siberian wild rye (Elymus sibiricus L.), an allotetraploid species, is a potentially high-quality perennial forage crop native to temperate regions. We used fluorescently conjugated oligonucleotides, representing ten repetitive sequences, including 6 microsatellite repeats, two satellite repeats, and two ribosomal DNAs, to characterize E. sibiricus chromosomes, using sequential fluorescence in situ hybridization and genomic in situ hybridization assays. Our results showed that microsatellite repeats (AAG)10 or (AGG)10, satellite repeats pAs1 and pSc119.2, and ribosomal 5S rDNA and 45S rDNA are specific markers for unique chromosomes. A referable karyotype ideogram was suggested, by further polymorphism screening, across different E. sibiricus cultivars with a probe mixture of (AAG)10, Oligo-pAs1, and Oligo-pSc119.2. Chromosomal polymorphisms vary between different genomes and between different individual chromosomes. In particular, two distinct forms of chromosome E in H genome were identified in intra- and inter-populations. Here, the significance of these results, for E. sibiricus genome research and breeding, and novel approaches to improve fluorescence in situ hybridization-based karyotyping are discussed.

Molecular cytogenetic characterization of wheat-Elymus repens chromosomal translocation lines with resistance to Fusarium head blight and stripe rust.

BACKGROUND: Fusarium head blight (FHB) caused by the fungus Fusarium graminearum Schwabe and stripe rust caused by Puccinia striiformis f. sp. tritici are devastating diseases that affect wheat production worldwide. The use of disease-resistant genes and cultivars is the most effective means of reducing fungicide applications to combat these diseases. Elymus repens (2n = 6x = 42, StStStStHH) is a potentially useful germplasm of FHB and stripe rust resistance for wheat improvement. RESULTS: Here, we report the development and characterization of two wheat-E. repens lines derived from the progeny of common wheat-E. repens hybrids. Cytological studies indicated that the mean chromosome configuration of K15-1192-2 and K15-1194-2 at meiosis were 2n = 42 = 0.86 I + 17.46 II (ring) + 3.11 II (rod) and 2n = 42 = 2.45 I + 14.17 II (ring) + 5.50 II (rod) + 0.07 III, respectively. Genomic and fluorescence in situ hybridization karyotyping and simple sequence repeats markers revealed that K15-1192-2 was a wheat-E. repens 3D/?St double terminal chromosomal translocation line. Line K15-1194-2 was identified as harboring a pair of 7DS/?StL Robertsonian translocations and one 3D/?St double terminal translocational chromosome. Further analyses using specific expressed sequence tag-SSR markers confirmed that the wheat-E. repens translocations involved the 3St chromatin in both lines. Furthermore, compared with the wheat parent Chuannong16, K15-1192-2 and K15-1194-2 expressed high levels of resistance to FHB and stripe rust pathogens prevalent in China. CONCLUSIONS: Thus, this study has determined that the chromosome 3St of E. repens harbors gene(s) highly resistant to FHB and stripe rust, and chromatin of 3St introgressed into wheat chromosomes completely presented the resistance, indicating the feasibility of using these translocation lines as novel material for breeding resistant wheat cultivars and alien gene mining.

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.

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.

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.

Genome constitution and evolution of Elytrigia lolioides inferred from Acc1, EF-G, ITS, TrnL-F sequences and GISH.

BACKGROUND: Elytrigia lolioides (Kar. et Kir.) Nevski, which is a perennial, cross-pollinating wheatgrass that is distributed in Russia and Kazakhstan, is classified into Elytrigia, Elymus, and Lophopyrum genera by taxonomists on the basis of different taxonomic classification systems. However, the genomic constitution of E. lolioides is still unknown. To identify the genome constitution and evolution of E. lolioides, we used single-copy nuclear genes acetyl-CoA carboxylase (Acc1) and elongation factor G (EF-G), multi-copy nuclear gene internal transcribed space (ITS), chloroplast gene trnL-F together with fluorescence and genomic in situ hybridization. RESULTS: Despite the widespread homogenization of ITS sequences, two distinct lineages (genera Pseudoroegneria and Hordeum) were identified. Acc1 and EF-G sequences suggested that in addition to Pseudoroegneria and Hordeum, unknown genome was the third potential donor of E. lolioides. Data from chloroplast DNA showed that Pseudoroegneria is the maternal donor of E. lolioides. Data from specific FISH marker for St genome indicated that E. lolioides has two sets of St genomes. Both genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH) results confirmed the presence of Hordeum genome in this species. When E genome was used as the probe, no signal was found in 42 chromosomes. The E-like copy of Acc1 sequences was detected in E. lolioides possibly due to the introgression from E genome species. One of the H chromosomes in the accession W6-26586 from Kazakhstan did not hybridize H genome signals but had St genome signals on the pericentromeric regions in the two-color GISH. CONCLUSIONS: Phylogenetic and in situ hybridization indicated the presence of two sets of Pseudoroegneria and one set of Hordeum genome in E. lolioides. The genome formula of E. lolioides was designed as StStStStHH. E. lolioides may have originated through the hybridization between tetraploid Elymus (StH) and diploid Pseudoroegneria species. E and unknown genomes may participate in the speciation of E. lolioides through introgression. According to the genome classification system, E. lolioides should be transferred into Elymus L. and renamed as Elymus lolioidus (Kar. er Kir.) Meld.

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.