Polycotyly: How Little Do We Know?

Polycotyly, an interesting characteristic of seed-bearing dicotyledonous plants with more than two cotyledons, represents one of the least explored plant characters for utilization, even though cotyledon number was used to classify flowering plants in 1682. Gymnosperm and angiosperm species are generally known to have one or two cotyledons, but scattered reports exist on irregular cotyledon numbers in many plant species, and little is known about the extent of polycotyly in plant taxa. Here, we attempt to update the documentation of reports on polycotyly in plant species and highlight some lines of research for a better understanding of polycotyly. This effort revealed 342 angiosperm species of 237 genera in 80 (out of 416) families and 160 gymnosperm species of 26 genera in 6 (out of 12) families with reported or cited polycotyly. The most advanced research included the molecular-based inference of the phylogeny of flowering plants, showing a significant departure from the cotyledon-based classification of angiosperm plants, and the application of genetic cotyledon mutants as tools to clone and characterize the genes regulating cotyledon development. However, there were no reports on breeding lines with a 100% frequency of polycotyly. Research is needed to discover plant species with polycotyly and to explore the nature, development, genetics, evolution, and potential use of polycotyly.

A Set of Yellow Mustard (Sinapis alba L.) Germplasm with Polycotyledony.

A world collection of 132 yellow mustard (Sinapis alba L.) accessions was characterized in a greenhouse to identify germplasm with polycotyledony and to assess the genetic segregation of tricot and tetracot seedlings in selfed S1 and S2 generations. The effort identified a set of 46 yellow mustard accessions with frequent occurrences of polycotyledony. The revealed genetic segregations seemed to suggest the development of tricot and tetracot seedlings in yellow mustard was largely controlled by a combination of genes at multiple diallelic loci. The identified tricot germplasm can be used to facilitate the genetic and/or genomic analysis of polycotyledonous germplasm for a better understanding of genetic and developmental mechanisms conditioning polycotyledony and the development of yellow mustard lines for explorable tricot breeding.

Deleterious and Adaptive Mutations in Plant Germplasm Conserved Ex Situ.

Conserving more than 7 million plant germplasm accessions in 1,750 genebanks worldwide raises the hope of securing the food supply for humanity for future generations. However, there is a genetic cost for such long-term germplasm conservation, which has been largely unaccounted for before. We investigated the extent and variation of deleterious and adaptive mutations in 490 individual plants representing barley, wheat, oat, soybean, maize, rapa, and sunflower collections in a seed genebank using RNA-Seq technology. These collections were found to have a range of deleterious mutations detected from 125 (maize) to 83,695 (oat) with a mean of 13,537 and of the averaged sample-wise mutation burden per deleterious locus from 0.069 to 0.357 with a mean of 0.200. Soybean and sunflower collections showed that accessions acquired earlier had increased mutation burdens. The germplasm with more years of storage in several collections carried more deleterious and fewer adaptive mutations. The samples with more cycles of germplasm regeneration revealed fewer deleterious and more adaptive mutations. These findings are significant for understanding mutational dynamics and genetic cost in conserved germplasm and have implications for long-term germplasm management and conservation.

Assessing Genetic Distinctness and Redundancy of Plant Germplasm Conserved Ex Situ Based on Published Genomic SNP Data.

Assessing genetic distinctness and redundancy is an important part of plant germplasm characterization. Over the last decade, such assessment has become more feasible and informative, thanks to the advances in genomic analysis. An attempt was made here to search for genebank germplasm with published genomic data and to assess their genetic distinctness and redundancy based on average pairwise dissimilarity (APD). The effort acquired 12 published genomic data sets from CIMMYT, IPK, USDA-ARS, IRRI, and ICRISAT genebanks. The characterized collections consisted of 661 to 55,879 accessions with up to 2.4 million genome-wide SNPs. The assessment generated an APD estimate for each sample. As a higher or lower APD is indicative of more genetic distinctness or redundance for an accession, respectively, these APD estimates helped to identify the most genetically distinct and redundant groups of 100 accessions each and a genetic outlier group with APD estimates larger than five standard deviations in each data set. An APD-based grouping of the conserved germplasm in each data set revealed among-group variances ranging from 1.5 to 53.4% across all data sets. Additional analyses showed that these APD estimations were more sensitive to SNP number, minor allele frequency, and missing data. Generally, 5000 to 10,000 genome-wide SNPs were required for an effective APD analysis. These findings together are encouraging and useful for germplasm management, utilization, and conservation, particularly in the genetic categorization of conserved germplasm.

Variability in Maturity, Oil and Protein Concentration, and Genetic Distinctness among Soybean Accessions Conserved at Plant Gene Resources of Canada.

Soybean (Glycine max (L.) Merr.) is one of the important crops in Canada and has the potential to expand its production further north into the Canadian Prairies. Such expansion, however, requires the search for adapted soybean germplasm useful for the development of productive cultivars with earlier maturity and increased protein concentration. We initiated several research activities to characterize 848 accessions of the soybean collection conserved at Plant Gene Resources of Canada (PGRC) for maturity, oil and protein concentration, and genetic distinctness. The characterization revealed a wide range of variations present in each assessed trait among the PGRC soybean accessions. The trait variabilities allowed for the identification of four core subsets of 35 PGRC soybean accessions, each specifically targeted for early maturity for growing in Saskatoon and Ottawa, and for high oil and protein concentration. The two early maturity core subsets for Saskatoon and Ottawa displayed days to maturity ranging from 103 to 126 days and 94 to 102 days, respectively. The two core subsets for high oil and protein concentration showed the highest oil and protein concentration from 25.0 to 22.7% and from 52.8 to 46.7%, respectively. However, these core subsets did not differ significantly in genetic distinctness (as measured with 19,898 SNP markers across 20 soybean chromosomes) from the whole PGRC soybean collection. These findings are useful, particularly for the management and utilization of the conserved soybean germplasm.

The mosaic oat genome gives insights into a uniquely healthy cereal crop.

Cultivated oat (Avena sativa L.) is an allohexaploid (AACCDD, 2n = 6x = 42) thought to have been domesticated more than 3,000 years ago while growing as a weed in wheat, emmer and barley fields in Anatolia1,2. Oat has a low carbon footprint, substantial health benefits and the potential to replace animal-based food products. However, the lack of a fully annotated reference genome has hampered efforts to deconvolute its complex evolutionary history and functional gene dynamics. Here we present a high-quality reference genome of A. sativa and close relatives of its diploid (Avena longiglumis, AA, 2n = 14) and tetraploid (Avena insularis, CCDD, 2n = 4x = 28) progenitors. We reveal the mosaic structure of the oat genome, trace large-scale genomic reorganizations in the polyploidization history of oat and illustrate a breeding barrier associated with the genome architecture of oat. We showcase detailed analyses of gene families implicated in human health and nutrition, which adds to the evidence supporting oat safety in gluten-free diets, and we perform mapping-by-sequencing of an agronomic trait related to water-use efficiency. This resource for the Avena genus will help to leverage knowledge from other cereal genomes, improve understanding of basic oat biology and accelerate genomics-assisted breeding and reanalysis of quantitative trait studies.

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.

Patterns of Genetic Variation in a Soybean Germplasm Collection as Characterized with Genotyping-by-Sequencing.

Genomic characterization is playing an increasing role in plant germplasm conservation and utilization, as it can provide higher resolution with genome-wide SNP markers than before to identify and analyze genetic variation. A genotyping-by-sequencing technique was applied to genotype 541 soybean accessions conserved at Plant Gene Resources of Canada and 30 soybean cultivars and breeding lines developed by the Ottawa soybean breeding program of Agriculture and Agri-Food Canada. The sequencing generated an average of 952,074 raw sequence reads per sample. SNP calling identified 43,891 SNPs across 20 soybean chromosomes and 69 scaffolds with variable levels of missing values. Based on 19,898 SNPs with up to 50% missing values, three distinct genetic groups were found in the assayed samples. These groups were a mixture of the samples that originated from different countries and the samples of known maturity groups. The samples that originated from Canada were clustered into all three distinct groups, but 30 Ottawa breeding lines fell into two groups only. Based on the average pairwise dissimilarity estimates, 40 samples with the most genetic distinctness were identified from three genetic groups with diverse sample origin and known maturity. Additionally, 40 samples with the highest genetic redundancy were detected and they consisted of different sample origins and maturity groups, largely from one genetic group. Moreover, some genetically duplicated samples were identified, but the overall level of genetic duplication was relatively low in the collection. These findings are useful for soybean germplasm management and utilization.

Characterizing chloroplast genomes and inferring maternal divergence of the Triticum-Aegilops complex.

The Triticum (wheat)-Aegilops (goatgrass) complex has been extensively studied, but the evolutionary history of polyploid wheats has not been fully elucidated. The chloroplast (cp) with maternal inheritance and homoplasy can simplify the sequence-based evolutionary inferences, but informative inferences would require a complete and accurate cp genome sequence. In this study, 16 cp genomes representing five Aegilops and 11 Triticum species and subspecies were sequenced, assembled and annotated, yielding five novel circular cp genome sequences. Analyzing the assembled cp genomes revealed no marked differences in genome structure and gene arrangement across the assayed species. A polymorphism analysis of 72 published cp genome sequences representing 10 Aegilops and 15 Triticum species and subspecies detected 1183 SNPs and 1881 SSRs. More than 80% SNPs detected resided on the downstream and upstream gene regions and only 2.78% or less SNPs were predicted to be deleterious. The largest nucleotide diversity was observed in the short single-copy genomic region. Relatively weak selection pressure on cp coding genes was detected. Different phylogenetic analyses confirmed that the maternal divergence of the Triticum-Aegilops complex had three deep lineages each representing a diploid species with nuclear A, B, or D genome. Dating the maternal divergence yielded age estimates of divergence that matched well with those reported previously. The divergence between emmer and bread wheats occurred at 8200-11,200 years ago. These findings are useful for further genomic studies, provide insight into cp genome evolvability and allow for better understanding of the maternal divergence of the Triticum-Aegilops complex.

Genome Assembly of the Canadian two-row Malting Barley cultivar AAC Synergy.

Barley (Hordeum vulgare L.) is one of the most important global crops. The six-row barley cultivar Morex reference genome has been used by the barley research community worldwide. However, this reference genome can have limitations when used for genomic and genetic diversity analysis studies, gene discovery, and marker development when working in two-row germplasm that is more common to Canadian barley. Here we assembled, for the first time, the genome sequence of a Canadian two-row malting barley, cultivar AAC Synergy. We applied deep Illumina paired-end reads, long mate-pair reads, PacBio sequences, 10X chromium linked read libraries, and chromosome conformation capture sequencing (Hi-C) to generate a contiguous assembly. The genome assembled from super-scaffolds had a size of 4.85 Gb, N50 of 2.32 Mb, and an estimated 93.9% of complete genes from a plant database (BUSCO, benchmarking universal single-copy orthologous genes). After removal of small scaffolds (< 300 Kb), the assembly was arranged into pseudomolecules of 4.14 Gb in size with seven chromosomes plus unanchored scaffolds. The completeness and annotation of the assembly were assessed by comparing it with the updated version of six-row Morex and recently released two-row Golden Promise genome assemblies.

Analysis of Stored mRNA Degradation in Acceleratedly Aged Seeds of Wheat and Canola in Comparison to Arabidopsis.

Seed aging has become a topic of renewed interest but its mechanism remains poorly understood. Our recent analysis of stored mRNA degradation in aged Arabidopsis seeds found that the stored mRNA degradation rates (estimated as the frequency of breakdown per nucleotide per day or ß value) were constant over aging time under stable conditions. However, little is known about the generality of this finding to other plant species. We expanded the analysis to aged seeds of wheat (Triticum aestivum) and canola (Brassica napus). It was found that wheat and canola seeds required much longer periods than Arabidopsis seeds to lose seed germination ability completely under the same aging conditions. As what had been observed for Arabidopsis, stored mRNA degradation (∆Ct value in qPCR) in wheat and canola seeds correlated linearly and tightly with seed aging time or mRNA fragment size, while the quality of total RNA showed little change during seed aging. The generated ß values reflecting the rate of stored mRNA degradation in wheat or canola seeds were similar for different stored mRNAs assayed and constant over seed aging time. The overall ß values for aged seeds of wheat and canola showed non-significant differences from that of Arabidopsis when aged under the same conditions. These results are significant, allowing for better understanding of controlled seed aging for different species at the molecular level and for exploring the potential of stored mRNAs as seed aging biomarkers.

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.

Arabidopsis UBC22, an E2 able to catalyze lysine-11 specific ubiquitin linkage formation, has multiple functions in plant growth and immunity.

Protein ubiquitination is critical for various biological processes in eukaryotes. A ubiquitin (Ub) chain can be linked through one of the seven lysine (K) residues or the N-terminus methionine of the Ub, and the Ub-conjugating enzymes called E2s play a critical role in determining the linkage specificity of Ub chains. Further, while K48-linked polyubiquitin chain is important for protein degradation, much less is known about the functions of other types of polyubiquitin chains in plants. We showed previously that UBC22 is unique in its ability to catalyze K11-dependent Ub dimer formation in vitro and ubc22 knockout mutants had defects in megasporogenesis. In this study, further analyses of the Arabidopsis ubc22 mutants revealed four subtypes of plants based on the phenotypic changes in vegetative growth. These four subtypes appeared consistently in the plants of three independent ubc22 mutants. Transcriptomic analysis showed that transcript levels of genes related to several pathways were altered differently in different subtypes of mutant plants. In one subtype, the mutant plants had increased expression of genes related to plant defenses and showed enhanced resistance to a necrotrophic plant pathogen. These results suggest multiple functions of UBC22 during plant development and stress response.

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.

Elevated mutation and selection in wild emmer wheat in response to 28 years of global warming.

Global warming has been documented to threaten wild plants with strong selection pressures, but how plant populations respond genetically to the threats remains poorly understood. We characterized the genetic responses of 10 wild emmer wheat (Triticum dicoccoides Koern.; WEW) populations in Israel, sampling them in 1980 and again in 2008, through an exome capture analysis. It was found that these WEW populations were under elevated selection, displayed reduced diversity and temporal divergence, and carried increased mutational burdens forward. However, some populations still showed the ability to acquire beneficial alleles via selection or de novo mutation for future adaptation. Grouping populations with mean annual rainfall and temperature revealed significant differences in most of the 14 genetic estimates in either sampling year or over the 28 y. The patterns of genetic response to rainfall and temperature varied and were complex. In general, temperature groups displayed more temporal differences in genetic response than rainfall groups. The highest temperature group had more deleterious single nucleotide polymorphisms (dSNPs), higher nucleotide diversity, fewer selective sweeps, lower differentiation, and lower mutational burden. The least rainfall group had more dSNPs, higher nucleotide diversity, lower differentiation and higher mutational burden. These characterized genetic responses are significant, allowing not only for better understanding of evolutionary changes in the threatened populations, but also for realistic modeling of plant population adaptability and vulnerability to global warming.

Flax latitudinal adaptation at LuTFL1 altered architecture and promoted fiber production.

After domestication in the Near East around 10,000 years ago several founder crops, flax included, spread to European latitudes. On reaching northerly latitudes the architecture of domesticated flax became more suitable to fiber production over oil, with longer stems, smaller seeds and fewer axillary branches. Latitudinal adaptations in crops typically result in changes in flowering time, often involving the PEBP family of genes that also have the potential to influence plant architecture. Two PEBP family genes in the flax genome, LuTFL1 and LuTFL2, vary in wild and cultivated flax over latitudinal range with cultivated flax receiving LuTFL1 alleles from northerly wild flax populations. Compared to a background of population structure of flaxes over latitude, the LuTFL1 alleles display a level of differentiation that is consistent with selection for an allele III in the north. We demonstrate through heterologous expression in Arabidopsis thaliana that LuTFL1 is a functional homolog of TFL1 in A. thaliana capable of changing both flowering time and plant architecture. We conclude that specialized fiber flax types could have formed as a consequence of a natural adaptation of cultivated flax to higher latitudes.

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.

Arabidopsis Seed Stored mRNAs are Degraded Constantly over Aging Time, as Revealed by New Quantification Methods.

How plant seeds age remains poorly understood and effective tools for monitoring seed aging are lacking. Dry seeds contain various stored mRNAs which are believed to be required for protein synthesis during early stages of seed germination. We reasoned that seed stored mRNAs would undergo degradation during seed aging, based on the propensity of mRNAs to degrade. We performed RT-PCR and qPCR analyses to study the changes in stored mRNA levels of Arabidopsis seeds during aging. All stored mRNAs analyzed were gradually degraded in both naturally and artificially aged seeds. The difference in Ct values between aged and control seeds (ΔCt value) was highly correlated with the mRNA fragment size and seed aging time. We derived mathematical equations for estimating the relative amount of undamaged stored mRNAs and frequency of the breakdown at one nucleotide level for individual mRNAs. Stored mRNAs were found to break down randomly. The frequency of breaks per nucleotide per day, which we named ß value, remained fairly constant under the same aging conditions over aging time. This parameter should allow the effects of different conditions on the degradation of stored mRNAs to be quantitatively compared. Also, we showed that the change in stored mRNA levels could serve as a more precise biomarker for seed aging assessment than three existing methods. These methods and findings will advance the studies of stored mRNAs and seed ageing in plants, and likely slow RNA degradation in non-plant systems.

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.