Effects of SNP marker density and training population size on prediction accuracy in alfalfa (Medicago sativa L.) genomic selection.

Effects of individual single-nucleotide polymorphism (SNP) markers and the size of "training" and "test" populations affect prediction accuracy in genomic selection (GS). This study evaluated 11 subsets of 4932 SNPs using six genetic additive methods to understand marker density in GS prediction in alfalfa (Medicago sativa L.). In the GS methods, the effect of "training" to "test" population size was also evaluated. Fourteen alfalfa populations sampled from long-term grazing sites were genotyped using genotyping by sequencing for the identification of SNPs. These populations were also phenotyped for six agromorphological and three nutritive traits from 2018 to 2020. The accuracy of GS prediction improved across six GS methods when the ratio of "training" to "test" population size increased. However, the prediction accuracy of the six GS methods reduced to a range of -0.27 to 0.11 when random, uninformative SNPs were used. In this study, five Bayesian methods and ridge-regression best linear unbiased prediction (rrBLUP) method had similar GS accuracies for "training" sets, but rrBLUP tended to outperform Bayesian methods in independent "test" sets when SNP subsets with high mean-squared-estimated-marker effect were used. These findings can enhance the application of GS in alfalfa genetic improvement.

Evaluation of Rumen Degradation Kinetics of Low-Lignin Alfalfa 'Hi-Gest® 360' in Saskatchewan Canada.

The objective of this study was to determine rumen degradation kinetics of new low-lignin alfalfa (Medicago sativa L.) cv. Hi-Gest®360 (HiGest) in comparison with conventional alfalfa cv. AC Grazeland (Grazeland) in monoculture and binary mixtures at different maturity stages. Two cultivars of alfalfa (HiGest, and AC Grazeland) and their binary mixtures with hybrid bromegrass (HBG; cv. AC Success), grown in 2019 at two locations (Saskatoon and Lanigan), were cut at three maturity stages of alfalfa (1 = 10% bloom; 2 = 40% bloom; and 3 = 100% bloom). Rumen degradation characteristics, including rapidly degradable fraction (S), potentially degradable fraction (D), undegradable fraction (U), degradation rate (Kd), lag time (T0), and effective degradability (ED) of each component were determined using in situ technique and were analyzed by a first-order kinetic equation described by Ørskov and McDonald with lag time. Generally, in alfalfa monoculture, S or D were decreased and U was increased without affecting Kd and T0, resulting in decreased ED fraction with increasing stage of maturity. In binary mixtures, plant maturity stages have negligible effects on rumen degradation characteristics of CP. HiGest had higher effective degradability of DM (EDDM) as well as of NDF (EDNDF) than Grazeland. In conclusion, HiGest had greater DM and NDF rumen degradation potential relative to Grazeland. HiGest and Grazeland were different in DM and CP degradation patterns, with HiGest having higher EDDM and EDCP than Grazeland.

Genetic diversity and local adaption of alfalfa populations (Medicago sativa L.) under long-term grazing.

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.

Transcriptomic analysis of differentially expressed genes in leaves and roots of two alfalfa (Medicago sativa L.) cultivars with different salt tolerance.

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.

Tissue specific changes in elements and organic compounds of alfalfa (Medicago sativa L.) cultivars differing in salt tolerance under salt stress.

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 ~ leaf at 8 dSm-1, and root ~ leaf > stem at 12 dSm-1, potentially preventing toxic ion accumulation in leaf tissues. In contrast, for 'Vernal', it was leaf > stem ~ root at 8 dSm-1 and leaf > root ~ 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.

Advancing crested wheatgrass [Agropyron cristatum (L.) Gaertn.] breeding through genotyping-by-sequencing and genomic selection.

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.

Effect of stage of maturity at harvest for forage pea (Pisum sativum L.) on eating behavior, ruminal fermentation, and digestibility when fed as hay to yearling beef heifers.

The objective of this study was to evaluate the stage of maturity at harvest for pea hay (Pisum sativum L., c.v. CDC Horizon) on dry matter intake (DMI), eating behavior, ruminal fermentation, and digestibility when fed to beef heifers. Pea hay was cut at EARLY (defined to occur when flat pods were on one or more nodes), MID (when seeds filled the pods at one or more nodes and the leaves were changing from green to gold), and LATE (yellow dry seeds filled pods on most or all of the nodes and the pods and leaves had a yellow color) phases, and was cured in the field and baled. Six ruminally-cannulated Speckle Park heifers were used in a replicated 3 × 3 Latin square design with three 18-d periods including 12 d for adaptation, 2 d for measurement of ruminal pool sizes, and 4 d for the collection of eating behavior, ruminal pH, ruminal digesta, and feces. For all treatments, the respective pea hay was included at 40% of the dietary DM. Stage of maturity at harvest for pea hay did not affect total DMI, pea hay DMI, or the total short-chain fatty acid concentration in ruminal fluid with averages of 8.6 kg/d, 3.2 kg/d, and 96.55 mM, respectively. The duration of time spent ruminating decreased with advancing pea hay maturity when reported as min/d, min/kg DMI, and min/kg neutral detergent fiber (NDF) (P ≤ 0.01). Mean ruminal pH also decreased with advancing pea maturity (P < 0.01). The ruminal DM and undigested NDF corrected for OM pools were not affected by stage of maturity (P ≥ 0.55) nor was the rate of digestion for NDF. However, NDF passage rate decreased by 0.21%/h with advancing pea hay maturity (P = 0.02). Apparent total tract digestibility of NDF (average = 16.30%, P = 0.41) was not affected, but starch digestibility decreased from 96.10% to 93.08% with advancing pea hay maturity (P = 0.07). Overall, stage of maturity at harvest for pea hay does not appear to affect DMI or NDF digestibililty but decreases chewing activity, apparent total tract starch digestibility, ruminal pH, and ruminal NDF passage rate.

Developing Chloroplast Genomic Resources from 25 Avena Species for the Characterization of Oat Wild Relative Germplasm.

Chloroplast (cp) genomics will play an important role in the characterization of crop wild relative germplasm conserved in worldwide gene banks, thanks to the advances in genome sequencing. We applied a multiplexed shotgun sequencing procedure to sequence the cp genomes of 25 Avena species with variable ploidy levels. Bioinformatics analysis of the acquired sequences generated 25 de novo genome assemblies ranging from 135,557 to 136,006 bp. The gene annotations revealed 130 genes and their duplications, along with four to six pseudogenes, for each genome. Little differences in genome structure and gene arrangement were observed across the 25 species. Polymorphism analyses identified 1313 polymorphic sites and revealed an average of 277 microsatellites per genome. Greater nucleotide diversity was observed in the short single-copy region. Genome-wide scanning of selection signals suggested that six cp genes were under positive selection on some amino acids. These research outputs allow for a better understanding of oat cp genomes and evolution, and they form an essential set of cp genomic resources for the studies of oat evolutionary biology and for oat wild relative germplasm characterization.

Advancing Bromegrass Breeding Through Imaging Phenotyping and Genomic Selection: A Review.

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.

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.

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 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).

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.

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 gene expression for floral development in crested wheatgrass (Agropyron cristatum L.).

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.