Bill Biligetu

Sainfoin (Onobrychis viciifolia Scop.): Renewed interest as a forage legume for western Canada

Abstract: Sainfoin (Onobrychis viciifolia Scop.) is a perennial forage legume that has received renewed interest in western Canada because of its desirable forage characteristics. The objective of this review is to summarize previous studies on the agronomy, forage yield, nutritive value, seed characteristics, and diseases and pests of sainfoin. In addition, the review also focuses on the genetic diversity and registered cultivars of sainfoin and their potential use in temperate grasslands. Past studies have reported that sainfoin has a high nutritive value, and high voluntary intake and palatability to grazing animals. In western Canada, dry matter (DM) yield of sainfoin is 80%–95% that of alfalfa (Medicago sativa L.). Unlike other legumes such as alfalfa, sainfoin does not cause bloat in grazing animals due to the presence of condensed tannins. Sainfoin is suitable for monoculture or binary mixtures with either grasses or alfalfa. Genetic diversity studies revealed that sainfoin germplasm generally has high variation within populations. Genetic variation among plants provides an opportunity to develop improved cultivars with desirable characteristics. However, compared with the other forage legumes, sainfoin is still an underdeveloped forage crop with few cultivars being available in western Canada. New sainfoin cultivars with high DM yield and persistence under regional growing conditions are required.

Searching for an Accurate Marker-Based Prediction of an Individual Quantitative Trait in Molecular Plant Breeding

Molecular plant breeding with the aid of molecular markers has played an important role in modern plant breeding over the last two decades. Many marker-based predictions for quantitative traits have been made to enhance parental selection, but the trait prediction accuracy remains generally low, even with the aid of dense, genome-wide SNP markers. To search for more accurate trait-specific prediction with informative SNP markers, we conducted a literature review on the prediction issues in molecular plant breeding and on the applicability of an RNA-Seq technique for developing function-associated specific trait (FAST) SNP markers. To understand whether and how FAST SNP markers could enhance trait prediction, we also performed a theoretical reasoning on the effectiveness of these markers in a trait-specific prediction, and verified the reasoning through computer simulation. To the end, the search yielded an alternative to regular genomic selection with FAST SNP markers that could be explored to achieve more accurate trait-specific prediction. Continuous search for better alternatives is encouraged to enhance marker-based predictions for an individual quantitative trait in molecular plant breeding.

RNA-Seq Analysis of Plant Maturity in Crested Wheatgrass (Agropyron cristatum L.)

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.

Genotyping-by-sequencing data of 272 crested wheatgrass (Agropyron cristatum) genotypes

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).

Responses of three bromegrass (Bromus) species to defoliation under different growth conditions.

Bromegrass species are important forage crops in temperate regions of world. This study compared responses of three bromegrass species to defoliation in the greenhouse and field to determine if the former could predict responses in the latter. Experiments were conducted in 2006 and 2007 in Saskatoon ( N, W), Canada on meadow bromegrass (Bromus riparius Rehm.), smooth bromegrass (Bromus inermis Leyss.), and hybrid bromegrass (B. riparius X B. inermis) following defoliation to 5 cm stubble height. When defoliated at the vegetative stage, above-ground biomass was similar among the three species in the field, but meadow bromegrass produced greater above-ground biomass than smooth bromegrass in the greenhouse. When defoliated at the stem elongation stage, meadow bromegrass produced greater above-ground biomass than smooth bromegrass in both environments. In the field, for all defoliation treatments, tiller number was greatest in meadow bromegrass, intermediate in hybrid bromegrass, and least in smooth bromegrass. In the greenhouse, however, the three species did not differ in tiller number. Similar results were found for below-ground biomass. Thus, testing the effect of defoliation in the greenhouse environment did not accurately predict the effect in the field environment.

Effects of TT8 and HB12 Silencing on the Relations between the Molecular Structures of Alfalfa (Medicago sativa) Plants and Their Nutritional Profiles and In Vitro Gas Production

The objective of this study was to investigate the effects of silencing the TT8 and HB12 genes on the nutritive profiles and in vitro gas production of alfalfa in relation to the spectral molecular structures of alfalfa. TT8-silenced (TT8i, n = 5) and HB12-silenced (HB12i, n = 11) alfalfa were generated by RNA interference (RNAi) and grown with nontransgenic wild type controls (WT, n = 4) in a greenhouse. Alfalfa plants were harvested at early-to-mid vegetative stage. Samples were analyzed for their chemical compositions, CNCPS fractions, and in vitro gas production. Correlations and regressions of the nutritional profiles and in vitro gas production with the molecular spectral structures were also determined. The results showed that the transformed alfalfa had higher digestible fiber and lower crude protein with higher proportions of indigestible protein than WT. HB12 RNAi had lower gas production compared with those of the others. Some chemical, CNCPS, and gas-production profiles were closely correlated with spectral structures and could be well predicted from spectral parameters. In conclusion, the RNAi silencing of TT8 and HB12 in alfalfa altered the chemical, CNCPS and gas-production profiles of alfalfa, and such alterations were closely correlated with the inherent spectral structures of alfalfa.

Genotyping-by-Sequencing Enhances Genetic Diversity Analysis of Crested Wheatgrass [Agropyron cristatum (L.) Gaertn.]

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.