Genomic information on alfalfa adaptation to long-term grazing is useful for alfalfa genetic improvement. In this study, 14 alfalfa populations were collected from long-term grazing sites (> 25 years) across four soil zones in western Canada. Alfalfa cultivars released between 1926 and 1980 were used to compare degree of genetic variation of the 14 populations. Six agro-morphological and three nutritive value traits were evaluated from 2018 to 2020. The genotyping-by-sequencing (GBS) data of the alfalfa populations and environmental data were used for genotype-environment association (GEA). Both STRUCTURE and UPGMA based on 19,853 SNPs showed that the 14 alfalfa populations from long-term grazing sites had varying levels of parentages from alfalfa sub-species Medicago sativa and M. falcata . The linear regression of STRUCTURE membership probability on phenotypic data indicated genetic variations of forage dry matter yield, spring vigor and plant height were low, but genetic variations of regrowth, fall plant height, days to flower and crude protein were still high for the 14 alfalfa populations from long-term grazing sites. The GEA identified 31 SNPs associated with 13 candidate genes that were mainly associated with six environmental factors of. Candidate genes underlying environmental factors were associated with a variety of proteins, which were involved in plant responses to abiotic stresses, i.e., drought, cold and salinity-alkali stresses.

Background Alfalfa ( Medicago sativa L.) production decreases under salt stress. Identification of genes associated with salt tolerance in alfalfa is essential for the development of molecular markers used for breeding and genetic improvement. Result An RNA-Seq technique was applied to identify the differentially expressed genes (DEGs) associated with salt stress in two alfalfa cultivars: salt tolerant ‘Halo’ and salt intolerant ‘Vernal’. Leaf and root tissues were sampled for RNA extraction at 0 h, 3 h, and 27 h under 12 dS m − 1 salt stress maintained by NaCl. The sequencing generated a total of 381 million clean sequence reads and 84.8% were mapped on to the alfalfa reference genome. A total of 237 DEGs were identified in leaves and 295 DEGs in roots of the two alfalfa cultivars. In leaf tissue, the two cultivars had a similar number of DEGs at 3 h and 27 h of salt stress, with 31 and 49 DEGs for ‘Halo’, 34 and 50 for ‘Vernal’, respectively. In root tissue, ‘Halo’ maintained 55 and 56 DEGs at 3 h and 27 h, respectively, while the number of DEGs decreased from 42 to 10 for ‘Vernal’. This differential expression pattern highlights different genetic responses of the two cultivars to salt stress at different time points. Interestingly, 28 (leaf) and 31 (root) salt responsive candidate genes were highly expressed in ‘Halo’ compared to ‘Vernal’ under salt stress, of which 13 candidate genes were common for leaf and root tissues. About 60% of DEGs were assigned to known gene ontology (GO) categories. The genes were involved in transmembrane protein function, photosynthesis, carbohydrate metabolism, defense against oxidative damage, cell wall modification and protection against lipid peroxidation. Ion binding was found to be a key molecular activity for salt tolerance in alfalfa under salt stress. Conclusion The identified DEGs are significant for understanding the genetic basis of salt tolerance in alfalfa. The generated genomic information is useful for molecular marker development for alfalfa genetic improvement for salt tolerance.

Alfalfa (Medicago sativa L.) is one of the most important forage crops in the world. The objectives of this study were to assess alfalfa yield improvement in the Canadian prairies, and determine critical climatic factors influencing alfalfa yield in different soil zones. Forage yield data of alfalfa cultivars tested from 1997 to 2011 in the Western Forage Variety Testing System were used for the analysis. There was no significant trend of alfalfa yield increase in western Canada except at Saskatoon, SK. Regrowth yield of alfalfa cultivars released from 2000 to 2011, however, showed a significant (p <= 0.05) increase under irrigation. Based on structural equation modelling (SEM), at rain-fed sites, precipitation from April to June was the most important driver for the farm hay yield (1st-cut). Forage yield of alfalfa, however, was not associated with winter extreme temperatures, or number of days with freeze-thaw temperatures in April. Alfalfa yields were greatest under a 3-cut than 1- or 2-cut systems in the first production year, but this difference declined as stands became older. Alfalfa stands that were harvested more frequently were less responsive to growing season rain, but responded more strongly to increased snow cover, which may indicate reduced growth and less winder hardiness. At the irrigated sites, 1st-cut forage yield increased with accumulated temperatures above 5 degrees C from April to June. Development of alfalfa cultivars with tolerance to early season drought and improved regrowth, without reducing winter hardiness, would be necessary to stabilize alfalfa production under changing climate in the Canadian prairies.

Soil salinity is a global concern and often the primary factor contributing to land degradation, limiting crop growth and production. Alfalfa (Medicago sativa L.) is a low input high value forage legume with a wide adaptation. Examining the tissue-specific responses to salt stress will be important to understanding physiological changes of alfalfa. The responses of two alfalfa cultivars (salt tolerant 'Halo', salt intolerant 'Vernal') were studied for 12 weeks in five gradients of salt stress in a sand based hydroponic system in the greenhouse. The accumulation and localization of elements and organic compounds in different tissues of alfalfa under salt stress were evaluated using synchrotron beamlines. The pattern of chlorine accumulation for `Halo' was: root > stem similar or equal to leaf at 8 dSm(-1), and root similar or equal to leaf > stem at 12 dSm(-1), potentially preventing toxic ion accumulation in leaf tissues. In contrast, for 'Vernal', it was leaf > stem similar or equal to root at 8 dSm(-1) and leaf > root similar or equal to stem at 12 dSm(-1). The distribution of chlorine in 'Halo' was relatively uniform in the leaf surface and vascular bundles of the stem. Amide concentration in the leaf and stem tissues was greater for 'Halo' than 'Vernal' at all salt gradients. This study determined that low ion accumulation in the shoot was a common strategy in salt tolerant alfalfa up to 8 dSm 1 of salt stress, which was then replaced by shoot tissue tolerance at 12 dSm(-1).

Meadow bromegrass (Bromus riparius Rehmann) is an important forage crop in Canada and the Intermountain West, but it has not been extensively evaluated in the northern Great Plains (NGP).Our objectives were to 1) evaluate meadow, smooth, and hybrid bromegrasses for forage production under intensive management in eastern South Dakota, and 2) evaluate the effect of selection for vigor in multiple environments on forage production of meadow bromegrass.Thirteen populations (7 meadow bromegrass, 5 hybrid bromegrass, and 1 smooth bromegrass) were evaluated for 4 yrs (2005)(2006)(2007) 2009) at Brookings, SD.Biomass at anthesis was greater for smooth (6.75 Mg ha -1 ) than for meadow or hybrid brome (5.4 Mg ha -1 ) in 2005, but production at anthesis during 2006 and 2007 was similar for meadow and smooth bromegrass.Regrowth harvested during July and October 2005 and November 2006 was greater for meadow than smooth or hybrid bromegrass.Forage production at anthesis in 2009, after rest (i.e., no cutting) and fertilization in 2008, was 4.2 Mg ha -1 for meadow bromegrass compared with 3.3 Mg ha -1 and 2.6 Mg ha -1 for hybrid and smooth bromegrass, respectively.Smooth and hybrid bromegrasses had more leaves•tiller -1 than meadow bromegrass.After 6 yrs, meadow bromegrass had higher tiller density and greater potential for tolerating multiple harvests during a growing season than did smooth or hybrid bromegrass. Slection for vigor in multiple environments in North America resulted in experimental populations of meadow bromegrass with superior forage yield compared with 'Fleet' on marginal crop land in the northern Great Plains.

Crested wheatgrass [Agropyron cristatum (L.) Gaertn.] provides high quality, highly palatable forage for early season grazing. Genetic improvement of crested wheatgrass has been challenged by its complex genome, outcrossing nature, long breeding cycle, and lack of informative molecular markers. Genomic selection (GS) has potential for improving traits of perennial forage species, and genotyping-by-sequencing (GBS) has enabled the development of genome-wide markers in non-model polyploid plants. An attempt was made to explore the utility of GBS and GS in crested wheatgrass breeding. Sequencing and phenotyping 325 genotypes representing 10 diverse breeding lines were performed. Bioinformatics analysis identified 827, 3,616, 14,090 and 46,136 single nucleotide polymorphism markers at 20%, 30%, 40% and 50% missing marker levels, respectively. Four GS models (BayesA, BayesB, BayesCπ, and rrBLUP) were examined for the accuracy of predicting nine agro-morphological and three nutritive value traits. Moderate accuracy (0.20 to 0.32) was obtained for the prediction of heading days, leaf width, plant height, clump diameter, tillers per plant and early spring vigor for genotypes evaluated at Saskatoon, Canada. Similar accuracy (0.29 to 0.35) was obtained for predicting fall regrowth and plant height for genotypes evaluated at Swift Current, Canada. The Bayesian models displayed similar or higher accuracy than rrBLUP. These findings show the feasibility of GS application for a non-model species to advance plant breeding.

Breeding forage crops for high yields of digestible biomass along with improved resource-use efficiency and wide adaptation is essential to meet future challenges in forage production imposed by growing demand, declining resources, and changing climate. Bromegrasses (Bromus spp.) are economically important forage species in the temperate regions of world, but genetic gain in forage yield of bromegrass is relatively low. In particular, limited breeding efforts have been made in improving abiotic stress tolerance and resource-use efficiency. We conducted a literature review on bromegrass breeding achievements and challenges, global climate change impacts on bromegrass species, and explored the feasibility of applying high-throughput imaging phenotyping techniques and genomic selection for further advances in forage yield and quality selection. Overall genetic gain in forage yield of bromegrass has been low, but genetic improvement in forage yield of smooth bromegrass (Bromus inermis Leyss) is somewhat higher than that of meadow bromegrass (Bromus riparius Rehm). This low genetic gain in bromegrass yield is due to a few factors such as its genetic complexity, lack of long-term breeding effort, and inadequate plant adaptation to changing climate. Studies examining the impacts of global climate change on bromegrass species show that global warming, heat stress, and drought have negative effects on forage yield. A number of useful physiological traits have been identified for genetic improvement to minimize yield loss. Available reports suggest that high-throughput imaging phenotyping techniques, including visual and infrared thermal imaging, imaging hyperspectral spectroscopy, and imaging chlorophyll fluorescence, are capable of capturing images of morphological, physiological, and biochemical traits related to plant growth, yield, and adaptation to abiotic stresses at different scales of organization. The more complex traits such as photosynthetic radiation-use efficiency, water-use efficiency, and nitrogen-use efficiency can be effectively assessed by utilizing combinations of imaging hyperspectral spectroscopy, infrared thermal imaging, and imaging chlorophyll fluorescence techniques in a breeding program. Genomic selection has been applied in the breeding of forage species and the applications show its potential in high ploidy, outcrossing species like bromegrass to improve the accuracy of parental selection and improve genetic gain. Together, these new technologies hold promise for improved genetic gain and wide adaptation in future bromegrass breeding.

Annual companion crops are often used in new seedings of perennial forages in western Canada. Fast-establishing grass species that do not overwinter in this region have potential as companion crops to increase seeding year yields without reducing perennial forage establishment. Trials were seeded in two different years at Saskatoon and Melfort, SK, in the Dark Brown and Black soil zones, respectively. Crested wheatgrass [Agropyron cristatum (L.) Gaertn.], meadow bromegrass (Bromus riparius Rehm.), and alfalfa (Medicago sativa L.) were seeded in pure stands and mixtures with or without an annual ryegrass (Lolium multiflorum Lam.) or Festulolium (Lolium × Festuca) companion crop at two seeding rates. Forage dry matter yield in the establishment year was increased with annual ryegrass companion crops compared with no companion crop regardless of seeding rate, but was increased only with the higher rate of Festulolium at Saskatoon. Despite using low companion crop seeding rates, stand densities of most of the perennial forage stands were lower than with no companion crop at Saskatoon, but in Melfort, most stand densities did not differ. The annual ryegrass companion crop reduced yields the year after seeding at both sites, while the Festulolium also reduced yields, but not at Melfort for the lower companion crop seeding rate. Dry matter yields were usually not different between companion and no companion crop treatments in the second year after seeding and for 3-yr total yields. The use of these companion crops increased the percentage of the 3-yr total yield produced in the seeding year.

The objectives of this study were to characterize crested wheatgrass (Agropyron cristatum L.) germplasm for plant maturity and associated agronomic characteristics to identify germplasm with late maturity. A field trial was established in July 2014 at Saskatoon, Canada using 45 crested wheatgrass accessions representing materials from 18 countries in a randomized complete block design with four replications. Data were collected for days to heading, plant height, leaf-to-stem ratio, forage dry matter (DM) yield, crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), and plant vigor score in 2015, 2016 and 2017 at various developmental stages of crested wheatgrass. All measured traits differed significantly (P ≤ 0.05) among the accessions. Later maturing accessions showed positive associations with leaf-to-stem ratio (r = 0.23), NDF (r = 0.16) and ADF (r = 0.18). Forage DM yield was positively correlated with spring vigor (r = 0.77), leaf-to-stem ratio (r = 0.50), plant height (r = 0.56), regrowth (r = 0.67), and ADF (r = 0.11). There was a negative correlation between forage DM yield and CP concentration (r =  − 0.23). According to the unweighted pair group method with arithmetic mean and principal component analysis, the 45 crested wheatgrass accessions were grouped into three main clusters according to the agro-morphological and nutritive value data. Selection for late maturity in crested wheatgrass may lead to increase leaf-to-stem ratio and forage DM yield. Information obtained from this study on agro-morphological traits will be useful for future crested wheatgrass breeding in the region.

Sainfoin (Onobrychis viciifolia Scop.) is a perennial forage legume widely distributed in the northern temperate regions of the world. Its genetic improvement has been relatively slow due, in part, to the lack of genetic information including molecular characterization of sainfoin germplasm. An attempt was made to evaluate genetic diversity and relationships among 38 sainfoin accessions collected from different regions of the world using amplified fragment length polymorphism (AFLP) markers. Five AFLP primer pairs were used to assess 367 individual plants, which produced 1042 polymorphic AFLP bands. The frequencies of the scored bands in all assayed individuals ranged from 0.003 to 0.973, with a mean value of 0.165. The analysis of molecular variance revealed higher within-accession (84.3%) genetic variation than among accessions (15.7%). The genetic distance based on inter-accession distance matrices was significant for most accessions but was not significant for accessions sharing similar parents. A dendrogram of the collected accessions showed two clusters at an inter-accession genetic distance coefficient of 0.36. The revealed information on genetic distance and genetic diversity of the sainfoin accessions is useful for selecting genetically diverse germplasms for sainfoin genetic improvement efforts.

Recent advances in next generation sequencing technologies make genotyping-by-sequencing (GBS) more feasible for the molecular characterization of plant germplasm with complex and unsequenced genomes. This study represents the first preliminary effort using GBS to discover genome-wide genetic variants of northern wheatgrass (Elymus lanceolatus ssp. lanceolatus (Scribn. and J. G. Sm.) Gould) plants and to assess the genetic diversity present in four cultivated and six wild accessions. The effort generated the first novel set of genomic resources and 5659 single nucleotide polymorphism (SNP) markers for this tetraploid grass. The diversity analysis revealed 8.8% of SNP variation residing among the 10 accessions and 1.9% SNP variation present between cultivated and wild accessions. The Bayesian analysis identified three major clusters of the assayed samples, and the principal coordinates analysis revealed the genetic distinctness of the two accessions collected from Nevada and Wyoming. The flow cytometry analysis confirmed the tetraploid nature of some of the assayed samples and estimated the average genome size to be 9.3–9.4 Gb for this species. These findings are useful for the genetic improvement of this native grass species for forage production and rangeland reclamation. The findings are also encouraging for the broad application of genotyping-by-sequencing in the characterization of genome-wide genetic variability in non-model polyploid plants.

Molecular characterization of unsequenced plant species with complex genomes is now possible by genotyping-by-sequencing (GBS) using recent next generation sequencing technologies. This study represents the first use of GBS application to sample genome-wide variants of crested wheatgrass [Agropyron cristatum (L.) Gaertn.] and assess the genetic diversity present in 192 genotypes from 12 tetraploid lines. Bioinformatic analysis identified 45,507 single nucleotide polymorphism (SNP) markers in this outcrossing grass species. The model-based Bayesian analysis revealed four major clusters of the samples assayed. The diversity analysis revealed 15.8% of SNP variation residing among the 12 lines, and 12.1% SNP variation present among four genetic clusters identified by the Bayesian analysis. The principal coordinates analysis and dendrogram were able to distinguish four lines of Asian origin from Canadian cultivars and breeding lines. These results serve as a valuable resource for understanding genetic variability, and will aid in the genetic improvement of this outcrossing polyploid grass species for forage production. These findings illustrate the potential of GBS application in the characterization of non-model polyploid plants with complex genomes.

Sainfoin (Onobrychis viciifolia Scop.) is a bloat-free, perennial forage legume adapted to the northern temperate regions of the world. This study was carried out to evaluate phenotypic variation and nutritive value of 38 sainfoin accessions from 21 different countries. A field trial was established in 2014 using a randomized complete block design with four replications near Saskatoon, Canada, with data collected in 2015 and 2016. Analysis of variance revealed significant (p ) differences among sainfoin accessions for winter survival, dry-matter (DM) yield, regrowth per seed yield plant, 1,000-seed weight, plant height, growth rate, spring vigour, days to flower, stem number, crude protein (CP), neutral detergent fibre (NDF) and acid detergent fibre (ADF). Dry-matter yield of sainfoin accessions ranged from 74 to 239 g/plant, plant height ranged from 37 to 70 cm, winter survival ranged from 20% to 94%, and seed yield ranged from 5 to 64 g/plant. Sainfoin accessions from North America produced the highest DM yield. Dry-matter yield was positively correlated with plant height (r = 0.82), stem number (r = 0.75), growth rate (r = 0.72), spring vigour (r = 0.43), regrowth (r = 0.30), 1,000-seed weight (r = 0.29), NDF (r = 0.71) and ADF (r = 0.74). Dry-matter yield was negatively correlated with days to flower (r = -0.57) and CP concentration (r = -0.62). Sainfoin accessions were grouped into three main groups according to their agro-morphological traits and nutritive value. Several promising sainfoin accessions have been identified to select for high forage yield and high winter survival in the cold climatic zone of temperate grassland.

Crested wheatgrass [Agropyron cristatum L. (Gaertn.)] is an important cool-season forage grass widely used for early spring grazing. However, the genomic resources for this non-model plant are still lacking. Our goal was to generate the first set of next generation sequencing data using the genotyping-by-sequencing technique. A total of 272 crested wheatgrass plants representing seven breeding lines, five cultivars and five geographically diverse accessions were sequenced with an Illumina MiSeq instrument. These sequence datasets were processed using different bioinformatics tools to generate contigs for diploid and tetraploid plants and SNPs for diploid plants. Together, these genomic resources form a fundamental basis for genomic studies of crested wheatgrass and other wheatgrass species. The raw reads were deposited into Sequence Read Archive (SRA) database under NCBI accession SRP115373 (https://www.ncbi.nlm.nih.gov/sra?term=SRP115373) and the supplementary datasets are accessible in Figshare (10.6084/m9.figshare.5345092).

Crested wheatgrass [Agropyron cristatum L. (Gaertn.)] is widely used for early spring grazing in western Canada and the development of late maturing cultivars which maintain forage quality for a longer period is desired. However, it is difficult to manipulate the timing of floral transition, as little is known about molecular mechanism of plant maturity in this species. In this study, RNA-Seq and differential gene expression analysis were performed to investigate gene expression for floral initiation and development in crested wheatgrass. Three cDNA libraries were generated and sequenced to represent three successive growth stages by sampling leaves at the stem elongation stage, spikes at boot and anthesis stages. The sequencing generated 25,568,846; 25,144,688 and 25,714,194 qualified Illumina reads for the three successive stages, respectively. De novo assembly of all the reads generated 311,671 transcripts with a mean length of 487 bp, and 152,849 genes with an average sequence length of 669 bp. A total of 48,574 (31.8%) and 105,222 (68.8%) genes were annotated in the Swiss-Prot and NCBI non-redundant (nr) protein databases, respectively. Based on the Kyoto Encyclopedia of Genes and Genome (KEGG) pathway database, 9,723 annotated sequences were mapped onto 298 pathways, including plant circadian clock pathway. Specifically, 113 flowering time-associated genes, 123 MADS-box genes and 22 CONSTANS-LIKE (COL) genes were identified. A COL homolog DN52048-c0-g4 which was clustered with the flowering time genes AtCO and OsHd1 in Arabidopsis (Arabidopsis thaliana L.) and rice (Oryza sativa L.), respectively, showed specific expression in leaves and could be a CONSTANS (CO) candidate gene. Taken together, this study has generated a new set of genomic resources for identifying and characterizing genes and pathways involved in floral transition and development in crested wheatgrass. These findings are significant for further understanding of the molecular basis for late maturity in this grass species.

Crested wheatgrass (Agropyron cristatum L.) breeding programs aim to develop later maturing cultivars for extending early spring grazing in Western Canada. Plant maturity is a complex genetic trait, and little is known about genes associated with late maturity in this species. An attempt was made using RNA-Seq to profile the transcriptome of crested wheatgrass maturity and to analyze differentially expressed genes (DEGs) between early and late maturing lines. Three cDNA libraries for each line were generated by sampling leaves at the stem elongation stage, spikes at the boot and anthesis stages. A total of 75,218,230 and 74,015,092 clean sequence reads were obtained for early and late maturing lines, respectively. De novo assembly of all sequence reads generated 401,587 transcripts with a mean length of 546 bp and N50 length of 691 bp. Out of 13,133 DEGs detected, 22, 17, and eight flowering related DEGs were identified for the three stages, respectively. Twelve DEGs, including nine flowering related DEGs at the stem elongation stage were further confirmed by qRT-PCR. The analysis of homologous genes of the photoperiod pathway revealed their lower expression in the late maturing line at the stem elongation stage, suggesting that their differential expression contributed to late maturity in crested wheatgrass.