Carbohydrate Accumulation and Differential Transcript Expression in Winter Wheat Lines with Different Levels of Snow Mold and Freezing Tolerance after Cold Treatment.

Winter wheat (Triticum aestivum L.) undergoes a period of cold acclimation in order to survive the ensuing winter, which can bring freezing temperatures and snow mold infection. Tolerance of these stresses is conferred in part by accumulation of carbohydrates in the crown region. This study investigates the contributions of carbohydrate accumulation during a cold treatment among wheat lines that differ in their snow mold tolerance (SMT) or susceptibility (SMS) and freezing tolerance (FrT) or susceptibility (FrS). Two parent varieties and eight recombinant inbred lines (RILs) were analyzed. The selected RILs represent four combinations of tolerance: SMT/FrT, SMT/FrS, SMS/FrT, and SMS/FrS. It is hypothesized that carbohydrate accumulation and transcript expression will differ between sets of RILs. Liquid chromatography with a refractive index detector was used to quantify carbohydrate content at eight time points over the cold treatment period. Polysaccharide and sucrose content differed between SMT and SMS RILs at various time points, although there were no significant differences in glucose or fructose content. Glucose and fructose content differed between FrT and FrS RILs in this study, but no significant differences in polysaccharide or sucrose content. RNAseq was used to investigate differential transcript expression, followed by modular enrichment analysis, to reveal potential candidates for other mechanisms of tolerance, which included expected pathways such as oxidative stress, chitinase activity, and unexpected transcriptional pathways. These differences in carbohydrate accumulation and differential transcript expression begin to give insight into the differences of wheat lines when exposed to cold temperatures.

Genotyping by multiplexed sequencing (GMS): A customizable platform for genomic selection.

As genotyping technologies continue to evolve, so have their throughput and multiplexing capabilities. In this study, we demonstrate a new PCR-based genotyping technology that multiplexes thousands of single nucleotide polymorphism (SNP) markers with high-throughput capabilities in a simple protocol using a two-step PCR approach. The bioinformatic pipeline is user friendly and yields results that are intuitive to interpret. This method was tested on two recombinant inbred line (RIL) populations that had previous genotyping data from the Illumina Infinium assay for Triticum aestivum L. and the two data sets were found to be 100% in agreement. The genotyping by multiplexed sequencing (GMS) protocol multiplexes 1,656 wheat SNP markers, 207 syntenic barley SNP markers, and 49 known informative markers, which generate a possible 2,433 data points (including homoeoalleles and paralogs). This genotyping approach has the flexibility of being sequenced on either the Ion Torrent or Illumina next generation sequencing (NGS) platforms. Products are the result of direct sequencing and are therefore more reliable than scatter plot analysis which is the output of other genotyping methods such as the Illumina Infinium assay, komeptitive allele specific PCR and other like technologies.

Exome sequencing of bulked segregants identified a novel TaMKK3-A allele linked to the wheat ERA8 ABA-hypersensitive germination phenotype.

KEY MESSAGE: Using bulked segregant analysis of exome sequence, we fine-mapped the ABA-hypersensitive mutant ERA8 in a wheat backcross population to the TaMKK3-A locus of chromosome 4A. Preharvest sprouting (PHS) is the germination of mature grain on the mother plant when it rains before harvest. The ENHANCED RESPONSE TO ABA8 (ERA8) mutant increases seed dormancy and, consequently, PHS tolerance in soft white wheat 'Zak.' ERA8 was mapped to chromosome 4A in a Zak/'ZakERA8' backcross population using bulked segregant analysis of exome sequenced DNA (BSA-exome-seq). ERA8 was fine-mapped relative to mutagen-induced SNPs to a 4.6 Mb region containing 70 genes. In the backcross population, the ERA8 ABA-hypersensitive phenotype was strongly linked to a missense mutation in TaMKK3-A-G1093A (LOD 16.5), a gene associated with natural PHS tolerance in barley and wheat. The map position of ERA8 was confirmed in an 'Otis'/ZakERA8 but not in a 'Louise'/ZakERA8 mapping population. This is likely because Otis carries the same natural PHS susceptible MKK3-A-A660S allele as Zak, whereas Louise carries the PHS-tolerant MKK3-A-C660R allele. Thus, the variation for grain dormancy and PHS tolerance in the Louise/ZakERA8 population likely resulted from segregation of other loci rather than segregation for PHS tolerance at the MKK3 locus. This inadvertent complementation test suggests that the MKK3-A-G1093A mutation causes the ERA8 phenotype. Moreover, MKK3 was a known ABA signaling gene in the 70-gene 4.6 Mb ERA8 interval. None of these 70 genes showed the differential regulation in wild-type Zak versus ERA8 expected of a promoter mutation. Thus, the working model is that the ERA8 phenotype results from the MKK3-A-G1093A mutation.

Evidence of cyclical light/dark-regulated expression of freezing tolerance in young winter wheat plants.

The ability of winter wheat (Triticum aestivum L.) plants to develop freezing tolerance through cold acclimation is a complex rait that responds to many environmental cues including day length and temperature. A large part of the freezing tolerance is conditioned by the C-repeat binding factor (CBF) gene regulon. We investigated whether the level of freezing tolerance of 12 winter wheat lines varied throughout the day and night in plants grown under a constant low temperature and a 12-hour photoperiod. Freezing tolerance was significantly greater (P<0.0001) when exposure to subfreezing temperatures began at the midpoint of the light period, or the midpoint of the dark period, compared to the end of either period, with an average of 21.3% improvement in survival. Thus, freezing survival was related to the photoperiod, but cycled from low, to high, to low within each 12-hour light period and within each 12-hour dark period, indicating ultradian cyclic variation of freezing tolerance. Quantitative real-time PCR analysis of expression levels of CBF genes 14 and 15 indicated that expression of these two genes also varied cyclically, but essentially 180° out of phase with each other. Proton nuclear magnetic resonance analysis (1H-NMR) showed that the chemical composition of the wheat plants' cellular fluid varied diurnally, with consistent separation of the light and dark phases of growth. A compound identified as glutamine was consistently found in greater concentration in a strongly freezing-tolerant wheat line, compared to moderately and poorly freezing-tolerant lines. The glutamine also varied in ultradian fashion in the freezing-tolerant wheat line, consistent with the ultradian variation in freezing tolerance, but did not vary in the less-tolerant lines. These results suggest at least two distinct signaling pathways, one conditioning freezing tolerance in the light, and one conditioning freezing tolerance in the dark; both are at least partially under the control of the CBF regulon.

Mapping genes for resistance to stripe rust in spring wheat landrace PI 480035.

Stripe rust caused by Puccinia striiformis Westend. f. sp. tritici Erikks. is an economically important disease of wheat (Triticum aestivum L.). Hexaploid spring wheat landrace PI 480035 was highly resistant to stripe rust in the field in Washington during 2011 and 2012. The objective of this research was to identify quantitative trait loci (QTL) for stripe rust resistance in PI 480035. A spring wheat, "Avocet Susceptible" (AvS), was crossed with PI 480035 to develop a biparental population of 110 recombinant inbred lines (RIL). The population was evaluated in the field in 2013 and 2014 and seedling reactions were examined against three races (PSTv-14, PSTv-37, and PSTv-40) of the pathogen under controlled conditions. The population was genotyped with genotyping-by-sequencing and microsatellite markers across the whole wheat genome. A major QTL, QYr.wrsggl1-1BS was identified on chromosome 1B. The closest flanking markers were Xgwm273, Xgwm11, and Xbarc187 1.01 cM distal to QYr.wrsggl1-1BS, Xcfd59 0.59 cM proximal and XA365 3.19 cM proximal to QYr.wrsggl1-1BS. Another QTL, QYr.wrsggl1-3B, was identified on 3B, which was significant only for PSTv-40 and was not significant in the field, indicating it confers a race-specific resistance. Comparison with markers associated with previously reported Yr genes on 1B (Yr64, Yr65, and YrH52) indicated that QYr.wrsggl1-1BS is potentially a novel stripe rust resistance gene that can be incorporated into modern breeding materials, along with other all-stage and adult-plant resistance genes to develop cultivars that can provide durable resistance.

Genomic Regions Associated with Tolerance to Freezing Stress and Snow Mold in Winter Wheat.

Plants grown through the winter are subject to selective pressures that vary with each year's unique conditions, necessitating tolerance of numerous abiotic and biotic stress factors. The objective of this study was to identify molecular markers in winter wheat (Triticum aestivum L.) associated with tolerance of two of these stresses, freezing temperatures and snow mold-a fungal disease complex active under snow cover. A population of 155 F2:5 recombinant inbred lines from a cross between soft white wheat cultivars "Finch" and "Eltan" was evaluated for snow mold tolerance in the field, and for freezing tolerance under controlled conditions. A total of 663 molecular markers was used to construct a genetic linkage map and identify marker-trait associations. One quantitative trait locus (QTL) associated with both freezing and snow mold tolerance was identified on chromosome 5A. A second, distinct, QTL associated with freezing tolerance also was found on 5A, and a third on 4B. A second QTL associated with snow mold tolerance was identified on chromosome 6B. The QTL on 5A associated with both traits was closely linked with the Fr-A2 (Frost-Resistance A2) locus; its significant association with both traits may have resulted from pleiotropic effects, or from greater low temperature tolerance enabling the plants to better defend against snow mold pathogens. The QTL on 4B associated with freezing tolerance, and the QTL on 6B associated with snow mold tolerance have not been reported previously, and may be useful in the identification of sources of tolerance for these traits.

Genes Upregulated in Winter Wheat (Triticum aestivum L.) during Mild Freezing and Subsequent Thawing Suggest Sequential Activation of Multiple Response Mechanisms.

Exposing fully cold-acclimated wheat plants to a mild freeze-thaw cycle of -3 °C for 24h followed by +3 °C for 24 or 48 h results in dramatically improved tolerance of subsequent exposure to sub-freezing temperatures. Gene enrichment analysis of crown tissue from plants collected before or after the -3 °C freeze or after thawing at +3 °C for 24 or 48 h revealed that many biological processes and molecular functions were activated during the freeze-thaw cycle in an increasing cascade of responses such that over 150 processes or functions were significantly enhanced by the end of the 48 h, post-freeze thaw. Nearly 2,000 individual genes were upregulated more than 2-fold over the 72 h course of freezing and thawing, but more than 70% of these genes were upregulated during only one of the time periods examined, suggesting a series of genes and gene functions were involved in activation of the processes that led to enhanced freezing tolerance. This series of functions appeared to include extensive cell signaling, activation of stress response mechanisms and the phenylpropanoid biosynthetic pathway, extensive modification of secondary metabolites, and physical restructuring of cell membranes. By identifying plant lines that are especially able to activate these multiple mechanisms it may be possible to develop lines with enhanced winterhardiness.

Measuring freezing tolerance: survival and regrowth assays.

Screening plants for freezing tolerance under tightly controlled conditions is an invaluable technique for studying freezing tolerance and selecting for improved winterhardiness. Artificial freezing tests of cereal plants historically have used isolated crown and stem tissue prepared by "removing all plant parts 3 cm above and 0.5 cm below the crown tissue" (Fowler et al., Crop Sci 21:896-901, 1981). Here, we describe a method of conducting freezing tolerance tests using intact plants grown in small horticultural containers, including suggested methods for collecting and analyzing the data.

Copy number and haplotype variation at the VRN-A1 and central FR-A2 loci are associated with frost tolerance in hexaploid wheat.

KEY MESSAGE: The interaction between VRN - A1 and FR - A2 largely affect the frost tolerance of hexaploid wheat. Frost tolerance is critical for wheat survival during cold winters. Natural variation for this trait is mainly associated with allelic differences at the VERNALIZATION 1 (VRN1) and FROST RESISTANCE 2 (FR2) loci. VRN1 regulates the transition between vegetative and reproductive stages and FR2, a locus including several tandemly duplicated C-REPEAT BINDING FACTOR (CBF) transcription factors, regulates the expression of Cold-regulated genes. We identified sequence and copy number variation at these two loci among winter and spring wheat varieties and characterized their association with frost tolerance. We identified two FR-A2 haplotypes-'FR-A2-S' and 'FR-A2-T'-distinguished by two insertion/deletions and ten single nucleotide polymorphisms within the CBF-A12 and CBF-A15 genes. Increased copy number of CBF-A14 was frequently associated with the FR-A2-T haplotype and with higher CBF14 transcript levels in response to cold. Factorial ANOVAs revealed significant interactions between VRN1 and FR-A2 for frost tolerance in both winter and spring panels suggesting a crosstalk between vernalization and cold acclimation pathways. The model including these two loci and their interaction explained 32.0 and 20.7 % of the variation in frost tolerance in the winter and spring panels, respectively. The interaction was validated in a winter wheat F 4:5 population segregating for both genes. Increased VRN-A1 copy number was associated with improved frost tolerance among varieties carrying the FR-A2-T allele but not among those carrying the FR-A2-S allele. These results suggest that selection of varieties carrying the FR-A2-T allele and three copies of the recessive vrn-A1 allele would be a good strategy to improve frost tolerance in wheat.

Post-acclimation transcriptome adjustment is a major factor in freezing tolerance of winter wheat.

Cold-acclimated winter wheat plants were slowly frozen to -10 degrees C, and then the temperature was either maintained at -10 degrees C or was lowered further to -12 degrees C. Expression levels of a total of 423 genes were significantly altered in these treatments; genes upregulated outnumbered those downregulated by about a 9:1 ratio. Sixty-eight genes were upregulated at least fivefold in all freezing treatments; 17 of these 68 encoded transcription factors including C-repeat binding factor (Cbf), WRKY, or other Zn-finger proteins, indicating strong upregulation of genes involved in transcription regulation. Sixteen of the 68 highly upregulated genes encoded kinases, phosphatases, calcium trafficking-related proteins, or glycosyltransferases, indicating upregulation of genes involved in signal transduction. Six genes encoding chlorophyll a/b binding-like proteins were upregulated uniquely in response to the -12 degrees C treatment, suggesting a protective role of pigment-binding proteins in freezing stress response. Most genes responded similarly in the very freezing tolerant cultivar Norstar and in the moderately freezing tolerant Tiber, but some genes responded in opposite fashion in the two cultivars. These results showed that wheat crowns actively adapt as the temperature declines to potentially damaging levels, and genetic variation for this ability exists among cultivars.

Molecular structure and organization of the wheat genomic manganese superoxide dismutase gene.

The genomic structure of a manganese superoxide dismutase (MnSOD) gene in wheat was elucidated by sequencing a clone from a BAC library of a stripe rust resistant wheat line. The clone was identified by hybridization with a wheat MnSOD cDNA. The gene consisted of 6 exons interrupted by 5 introns with a total length of 4770 nucleotides from the start codon to the termination codon. The wheat MnSOD gene was the longest among those sequenced from plant species. The transcription initiation site was preceded by a G+C-rich promoter without a TATA or CAAT box. The promoter contained many putative cis-acting regulatory elements, including an abscisic acid (ABA)-responsive element, a stress-responsive element, and a GC-repeat, as well as several other structural features in common with the promoter of the rice MnSOD gene. A Stowaway-like transposable element was found in intron 5 of the wheat MnSOD gene, but further investigation revealed the transposable element was not present in all copies of the MnSOD genes.

Differential expression of manganese superoxide dismutase sequence variants in near isogenic lines of wheat during cold acclimation.

Numerous sequence variants of wheat (Triticum aestivum L.) manganese superoxide dismutase (MnSOD) genes have been found. Quantitative real-time PCR was used to measure the expression levels of three MnSOD genes distinguished by a variable amino acid, and three genes distinguished by sequence variation in the 3' untranslated region (3' UTR), in wheat plants grown at 20 degrees C and cold-acclimated for 1-4 weeks at 2 degrees C. The amino acid variants did not differ significantly in expression levels, however, differential expression of genes differing in the 3' UTR was observed. Diploid wheat-related species also carried sequence variants of MnSOD, with differing levels of expression, suggesting diversification of the MnSOD gene family occurred prior to the polyploidization events of hexaploid wheat.

Differential mRNA stability to endogenous ribonucleases of the coding region and 3' untranslated regions of wheat (Triticum aestivum L.) manganese superoxide dismutase genes.

The sequences of the 3' untranslated region (UTR) of the manganese superoxide dismutase (MnSOD) genes in wheat (Triticum aestivum) were found to be quite variable with different predicted thermostabilities. The degradation rates of the 3' UTR variants and the coding region were measured following exposure to endogenous nucleases. The degradation rates of the 3' UTR variants for 15 min were not significantly different, meaning the degradation rates of the 3' UTR variants were not directly related to the thermostabilities. However, the degradation rate of the coding region was significantly faster than those of the 3' UTR variants. Further investigation revealed the coding region seemed to have specific sites for degradation, indicating a possibility of increasing MnSOD expression by the degradation site alteration.

Long oligonucleotide microarrays in wheat: evaluation of hybridization signal amplification and an oligonucleotide-design computer script.

A computer script was written in the Perl language to design equal-length long oligonucleotides from DNA sequences. The script allows the user to specify G + C content, melting temperature, self-complementarity, the maximum number of contiguous duplicate bases, whether to start with the first start codon and whether to report reverse complements. Microarrays were fabricated with 95 oligonucleotides (60 mers) representing 41 genes. The microarray was interrogated with cDNA from roots and shoots of two near-isogenic lines and a commercial cultivar of Triticum aestivum L. (hexaploid wheat) challenged with cold temperature, hot temperature, or the biological control bacterium Pseudomonas fluorescens. Self-complementarity of the oligonucleotides was negatively correlated with signal intensity in 23 of 54 arrays (39%; P <0.01). Tyramide signal amplification was essential for signal generation and detection. Genes involved in signal transduction pathways responded similarly following exposure to cold, heat and P. fluorescens, suggesting intersection of the pathways involved in response to these disparate stress factors. Microarray results were corroborated by quantitative real-time PCR in 75% of samples assayed. We conclude that long oligonucleotide microarrays for interrogation with cDNA from hexaploid wheat should be constructed from oligonucleotides having minimal self complementarity that also meet user-specified requirements of length, G + C content and melting temperature; multiple oligonucleotides should be used to represent each gene; and Tyramide signal amplification is useful in wheat oligonucleotide microarray studies.

Quantitative real-time PCR method to detect changes in specific transcript and total RNA amounts

Quantitative real-time PCR (qRT-PCR), used in conjunction with reverse transcriptase, has been applied to the determination of the number of copies of a transcript per unit mass of RNA, but did not indicate any change in the amount of total RNA per mass of tissue. In the present work, we described a simple method to use qRT-PCR to estimate the change in the amount of total RNA per unit mass of wheat (Triticum aestivum L.) tissue in response to cold temperature. Three qRT-PCR templates, i.e. control, cold-exposed, and one of RNA extracted from a sample consisting of equal masses of control and cold-exposed tissue, were analyzed. The number of copies of target transcript per unit mass of RNA was estimated from the three samples using standard qRT-PCR techniques. Equations describing the number of copies of the target sequence in each of the tissue samples were solved simultaneously to describe the relative proportion of the target sequence that originated from each tissue sample in the mixture, thereby providing an estimate of relative amounts of total RNA in the two tissues.

Geographic Distribution and Genetic Diversity of Three Ophiosphaerella Species that Cause Spring Dead Spot of Bermudagrass.

The distribution of three Ophiosphaerella spp. that cause spring dead spot (SDS) of bermudagrass was studied by systematically sampling two golf courses in Oklahoma and one in Kansas. O. herpotricha was isolated from all three locations and was the most abundant species. It was the only SDS pathogen found at Jenks, Oklahoma. O. korrae was isolated from Afton, Oklahoma, and Independence, Kansas, whereas O. narmari was only detected in samples from Afton. This is the first report of all three Ophiosphaerella species on bermudagrass at the same location. Amplified fragment length polymorphism (AFLP) marker analysis was used to investigate inter- and intraspecific genetic diversity of Ophiosphaerella isolates from North America and Australia. A majority of the O. herpotricha and O. narmari isolates from Afton were distinct haplotypes, suggesting that sexual recombination was occurring within the population. Conversely, the presence of multiple isolates of O. herpotricha and O. narmari with the same haplotype also indicated that asexual propagation was occurring. The genetic diversity among O. herpotricha isolates from Afton was not distinctly different from that of isolates collected throughout the southern United States. In contrast, O. narmari isolates from Afton were distinct from those collected in Australia. The genetic diversity in O. korrae was markedly different than that in the other Ophiosphaerella spp. The population at Afton was dominated by just a few haplotypes, and these were nearly identical to isolates collected from bermudagrass and Kentucky bluegrass throughout western, central, and northern North America. However, O. korrae isolates collected in the southeastern United States were only distantly similar to other North American isolates.